This site uses cookies to deliver our services and to show you relevant ads and presentations. By clicking on "Accept", you acknowledge that you have read and understand our Cookie Policy , Privacy Policy , and our Terms of Use.
X

Download Allergy Disease PowerPoint Presentation


Login   OR  Register
X


Iframe embed code :



Presentation url :

X

Description :

Available Allergy Disease powerpoint presentation for free download which is uploaded by honey an active user in belonging ppt presentation Health & Wellness category.

Tags :

Allergy Disease

Home / Health & Wellness / Health & Wellness Presentations / Allergy Disease PowerPoint Presentation

Allergy Disease PowerPoint Presentation

Ppt Presentation Embed Code   Zoom Ppt Presentation

About This Presentation


Description : Available Allergy Disease powerpoint presentation for free download which is uploaded by honey an ac... Read More

Tags : Allergy Disease

Published on : Mar 14, 2014
Views : 403 | Downloads : 1


Download Now

Share on Social Media

             

PowerPoint is the world's most popular presentation software; you can create professional Allergy Disease powerpoint presentation with this powerful software easly. And give your presentation on Allergy Disease in conference, a school lecture, a business proposal or in webinar.

Uploader spend their valuable time to create this Allergy Disease powerpoint presentation slides, to share their knowledgable content with the world. This ppt presentation uploaded by slidesfinder in their relavent Health & Wellness category is available for free download and use according to your industries likefinance,marketing,education,health and many more.

SlidesFinder.com provides a platform for marketers, presenters and educationists along with being the preferred search engine for professional PowerPoint presentations on the Internet to upload your Allergy Disease ppt presentation slides to BUILD YOUR CROWD!!

User Presentation
Related Presentation
Free PowerPoint Templates
Slide 1 - Mechanisms of Allergic Immunity crah1@le.ac.uk
Slide 2 - Mechanisms of Allergic Immunity crah1@le.ac.uk
Slide 3 - Mechanisms of Allergic Immunity crah1@le.ac.uk
Slide 4 - Mechanisms of Allergic Immunity crah1@le.ac.uk
Slide 5 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema
Slide 6 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked
Slide 7 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present)
Slide 8 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells
Slide 9 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells
Slide 10 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation
Slide 11 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862
Slide 12 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for?
Slide 13 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets
Slide 14 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets
Slide 15 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995
Slide 16 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection
Slide 17 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism
Slide 18 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses
Slide 19 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy
Slide 20 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy
Slide 21 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy
Slide 22 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion?
Slide 23 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b
Slide 24 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell
Slide 25 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i
Slide 26 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769
Slide 27 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints
Slide 28 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004.
Slide 29 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr)
Slide 30 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN
Slide 31 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells
Slide 32 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer
Slide 33 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops
Slide 34 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2?
Slide 35 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2
Slide 36 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA
Slide 37 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1)
Slide 38 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a
Slide 39 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ?
Slide 40 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins
Slide 41 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC
Slide 42 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18
Slide 43 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors
Slide 44 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances?
Slide 45 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections
Slide 46 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc
Slide 47 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms?
Slide 48 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells
Slide 49 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated
Slide 50 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245
Slide 51 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system
Slide 52 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma
Slide 53 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described?
Slide 54 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae
Slide 55 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae
Slide 56 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein
Slide 57 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen
Slide 58 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators
Slide 59 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone
Slide 60 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells
Slide 61 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171
Slide 62 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils
Slide 63 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812
Slide 64 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812
Slide 65 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812
Slide 66 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils
Slide 67 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils
Slide 68 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812
Slide 69 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils
Slide 70 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE
Slide 71 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862
Slide 72 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions
Slide 73 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells
Slide 74 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40
Slide 75 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells
Slide 76 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus
Slide 77 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells
Slide 78 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus
Slide 79 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter
Slide 80 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts
Slide 81 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA
Slide 82 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination
Slide 83 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype
Slide 84 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase
Slide 85 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs
Slide 86 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase?
Slide 87 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact
Slide 88 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact GGGUTGA CCCGACT P P P P P P P P P P P P G - U mismatch repair APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P P P P P P P P P P P P OH DNA is now nicked to produce a single strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1 APE1 GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break
Slide 89 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact GGGUTGA CCCGACT P P P P P P P P P P P P G - U mismatch repair APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P P P P P P P P P P P P OH DNA is now nicked to produce a single strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1 APE1 GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break Processing of staggered ends GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Exonuclease activity Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm
Slide 90 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact GGGUTGA CCCGACT P P P P P P P P P P P P G - U mismatch repair APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P P P P P P P P P P P P OH DNA is now nicked to produce a single strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1 APE1 GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break Processing of staggered ends GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Exonuclease activity Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Cm Cd Cg3 VDJ Cg1 Ca1 Cg2 Cg4 Ce Ca2 VDJ Ce Ca2 Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Excised episomal circle of intervening DNA Activation of Im & Ie promoter by Ag, IL-4/13 and CD40L Production of germline transcripts and splicing of Sm and Se Deamination of ssDNA in Sm and Se by AID Base excision and mismatch repair Blunt-ended ds breaks and synapsis of Sm to Se by non-homologous end joining Process occurs in two S regions simultaneously
Slide 91 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact GGGUTGA CCCGACT P P P P P P P P P P P P G - U mismatch repair APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P P P P P P P P P P P P OH DNA is now nicked to produce a single strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1 APE1 GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break Processing of staggered ends GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Exonuclease activity Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Cm Cd Cg3 VDJ Cg1 Ca1 Cg2 Cg4 Ce Ca2 VDJ Ce Ca2 Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Excised episomal circle of intervening DNA Activation of Im & Ie promoter by Ag, IL-4/13 and CD40L Production of germline transcripts and splicing of Sm and Se Deamination of ssDNA in Sm and Se by AID Base excision and mismatch repair Blunt-ended ds breaks and synapsis of Sm to Se by non-homologous end joining Process occurs in two S regions simultaneously 7 23 9 7 12 9 Non-homologous end joining in class switch V D J Closely resembles another B cell Ig gene mechanism Defects in NHEJ proteins impair class switch
Slide 92 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P P P P P P P P P P P P GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG Base is removed, but backbone remains intact GGGUTGA CCCGACT P P P P P P P P P P P P G - U mismatch repair APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P P P P P P P P P P P P OH DNA is now nicked to produce a single strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1 APE1 GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break Processing of staggered ends GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Exonuclease activity Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Cm Cd Cg3 VDJ Cg1 Ca1 Cg2 Cg4 Ce Ca2 VDJ Ce Ca2 Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Excised episomal circle of intervening DNA Activation of Im & Ie promoter by Ag, IL-4/13 and CD40L Production of germline transcripts and splicing of Sm and Se Deamination of ssDNA in Sm and Se by AID Base excision and mismatch repair Blunt-ended ds breaks and synapsis of Sm to Se by non-homologous end joining Process occurs in two S regions simultaneously 7 23 9 7 12 9 Non-homologous end joining in class switch V D J Closely resembles another B cell Ig gene mechanism Defects in NHEJ proteins impair class switch NFkB BCL-6 BCL-6 BCL-6 binds to the Stat-6 binding site and represses switching Stat6 is involved in Th2 cell differentiation, the expression of CD23 (the low affinity IgE receptor) and VCAM expression BCL-6 may exert it’s anti/pro-allergic activities via these genes Stat6 Transcription blocked BCL-6 -/- mice have enhanced IgE isotype switching BCL-6 -/- Stat6 -/- mice have no IgE An RFLP has been mapped to the first intron of the BCL-6 gene that is significantly associated with atopy - but not IgE levels
Slide 93 - Mechanisms of Allergic Immunity crah1@le.ac.uk Normal larynx Laryngeal oedema Cellular culprits of allergy: Mast cells Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked Cellular culprits of allergy: Mast cells?? Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present) The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells Cellular culprits of allergy: T cells Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Lebman & Coffman 1988 J Exp Med 168, 853-862 ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals?What are they really for? Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Lepromatous Leprosy ‘Textbook’ scheme of allergic immunity is centred around polarised Th cells Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion? Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1a, MCP-1 MIP-1b Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell [Ca2+]i Time (s) [Ca2+]i Time (s) Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. 3. DC become highly migratory & change shape (20hr) 4. T cells cover large territories in LN 6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Signal 1 Signal 2 Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216 Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1) Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a Type 2 PAMPS and their PRR ? ? Endogenous molecular patterns Endogenous molecular patterns Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Include: Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Indirect activation of DC by ‘modulatory tissue factors’ Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expressionof PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Allergen Activates the expression of costimulatory molecules on DC Could be argued that the development of Th2 cells is the default pathway DC polarisation by modulatory tissue factors DC polarising factorsIFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factorsCCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 NK Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Sources of modulatory tissue factors The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… …does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? Neonatal & infant immune systems Serial infections Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Do infections only reduce Th2 dominance by inducing Th1 responses? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Wheeze Vaccinate with mycobacteria No asthma-like symptoms Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Wheeze No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Mycobacteria induced REGULATORY T cells Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782 Priming conditions IFNg U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFNg Ab 348 1245 Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2mm sections from a mucosal biopsy of a patient with asthma What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described? L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators Journal of Immunology 2001 167 1014-1021 PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a IL-4, Do protease allergens induce IL-4 release by Mast cells Journal of Leukocyte Biology 2003, 73 165-171 Constitutive & Induced CytokineExpression by KU812 Basophils b-actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - - b-actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812 516bp b-actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812 Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils 516bp 516bp 516bp 516bp 516bp b-actin IL-4 IL-5 IL-13 IFN-g - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812 Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils The switch to IgE Lebman & Coffman 1988 J Exp Med 168, 853-862 Switch regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions Switch recombination to IgE A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Y Y Y T cell help to B cells B Antigen Th IL-4 and IL-13 CD40 Ligand CD40 Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Stat-6 P Stat-6 P Soluble help via IL-4 or IL-13 from T helper cells IL-4Ra IL-4Ra gC IL-13Ra1/2 IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R JAK1 JAK3 TYK1 JAK1 TYK2 P P Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P P Dimerised Stat-6 translocates to nucleus Switch recombination to IgE A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells Ligation promotes aggregation in lipid rafts Cognate help via CD40 L from T helper cells CD40 2 3 5 6 TNF receptor associated factors IkB NF kB IkB NF kB Uninhibited NFkB translocates to the nucleus Stat6 Ie Ce1 Ce2 Ce3 Ce4 Se Ie NFkB C/EBP PU.1 BSAP AP-1 BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Induced by IL-4/IL-13 and CD40 ligation Activation of the Ie promoter Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie Germline IgE transcripts Transcription Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts What do germline transcripts do? Ce Ie Ce1 Ce2 Ce3 Ce4 Se Ie RNA Spliced RNA Se RNA S region RNA hybridises with template DNA Single stranded DNA Ie Ce1 Se Se 5’ 3’ R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination NFkB Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C Activation-induced cytidine deaminase AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs Preferred Se region target sequence for AID GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Activation induced cytidine deaminase NH2 N N O Cytidine O N HN O Uridine AID AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase AID may also deaminate C on the template strand ?RNAase? GGGUTGA CCCGACT GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA G - U mismatch repair GGGUTGGGUTGAGUTGRGUTGAGUTGRGUT