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Mechanisms of Allergic Immunity crah1@le.ac.uk

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

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Mechanisms of Allergic Immunity crah1@le.ac.uk

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  1. Mechanisms of Allergic Immunity crah1@le.ac.uk

  2. Normal larynx Laryngeal oedema

  3. 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

  4. 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)

  5. Cellular culprits of allergy: T cells • 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

  6. Th2 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

  7. Lebman & Coffman 1988 J Exp Med 168, 853-862

  8. -ve Th2 Ig isotype switch -ve Differentiation and development MF Mast cell IgE Eosinophil B Th1 ‘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?

  9. The discovery of Th1 and Th2 subsets Journal of Immunology 136, 2348-2357 1986

  10. Enhances IgE & IgG1 IFN-g IL-4 T cell clones that make IFN-g, but not IL-4 Do not provide help to IgE and IgG1 secreting B cells T cell clones that make IL-4, but not IFN-g Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets

  11. Non-healing BALB/c Resistant C57BL/6 T Draining LN T cells express IL-4 mRNA Draining LN T cells express IFN-g mRNA Irradiated BALB/c recipient Resistance Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995

  12. IFN- / IL-12 or anti-IL-4 Relevance in vivo - Infection Pro-Th1 treatments or anti-Th2 treatments protect against infection

  13. IFN- Th1 Macrophage and Leishmania Inflammatory Th1 T cell Leishmania resistance - mechanism Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells

  14. Th1 Th2 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 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

  15. Tuberculoid leprosy

  16. Lepromatous Leprosy

  17. ‘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?

  18. Activation and migration of dendritic cells to site of inflammation Allergic immune responses are much like any other immune response and involves the same regulators Non self protein from allergen or pathogen Barrier: Skin, gut, lung, eye, nose etc Inflammation inc. MIP-1a, MCP-1 MIP-1b

  19. Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell

  20. [Ca2+]i Time (s) Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b [Ca2+]i [Ca2+]i Time (s) Time (s) Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 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.

  21. [Ca2+]i Time (s) Mature DC respond to the lymph node derived CCR7 ligand MIP-3b [Ca2+]i [Ca2+]i Time (s) Time (s) Migration of mature DC to 2º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b

  22. Anti OVA 323-329 TcR transgenic mouse Pulsed with AgOVA 323-329 DC labelled RED Splenic DC T cells labelled GREEN -18hr 0hr 2hr Anti-L selectin Ab DC – T cell interactions in the lymph node Mempel, T.R et al Nature 427: 154-159, 2004. Not pulsed with Ag Imaging at various timepoints

  23. Early entry of DC to the lymph nodeMempel, T.R et al Nature 427: 154-159, 2004. 1. DCs strategically cluster around HEV 18hr after entering the LN 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected

  24. 3. DC become highly migratory & change shape (20hr)

  25. 4. T cells cover large territories in LN

  26. 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

  27. T cells start to proliferate and produce cytokines 44hr after transfer 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again

  28. 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

  29. DC Th Signal 3 - pathogen polarised DC Signals 1, 2 and 3 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2?

  30. Signal 1 DC Th Signal 2 Signal 3 Th polarising signal Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3 Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells

  31. 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 • 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

  32. + + + Type 1PAMPSbind to PRR CD40 Class II T Type 2PAMPS bind to PRR Type 1 and 2 DC Polarising PAMPS CD80/CD86 Th1 polarisingfactor IL-12 Th2 polarisingfactor CCL2 (MCP-1)

  33. CD14 MD-2 TLR 2 TLR 1 TLR 6 TLR 2 TLR 3 TLR 4 TLR 9 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 Unmethylated CpG DNA dsDNA Low level IL-12p70 Some ligandsinduce IL-10or IL-12p35 HighIL-12p70IFN-a HighIL-12p70 HighIL-12p70IFN-a

  34. ? ? Type 2 PAMPS and their PRR

  35. 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

  36. 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 Indirect activation of DC by ‘modulatory tissue factors’ Allergen Activates the expression of costimulatory molecules on DC

  37. 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

  38. Viruses Fungi Parasites Bacteria IFN-a IL-18 Epithelium Viruses IFN-g Th1 Viruses Fungi Parasites NK Histamine Mast Th2 PGE2 CCR2L Viruses Fibroblast Sources of modulatory tissue factors

  39. 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? 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

  40. Th1 Immune response Th2 Th2 Age Balanced Th1/Th2 at ~2yr The intrauterine environment is powerfully Th2 – this imprints Th2 dominance upon the neonate Neonatal & infant immune systems Serial infections

  41. Immune response Th1 Th2 Age Unbalanced Th1/Th2 Th2 dominance at ~2yr Longer period of time in which to make and establish Th2 responses to environmental antigens (i.e. allergens) Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc

  42. Vaccinate with mycobacteria Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? Wheeze Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms?

  43. Vaccinate with mycobacteria Th CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 No asthma-like symptoms Do infections only reduce Th2 dominance by inducing Th1 responses? Wheeze Mycobacteria induced REGULATORY T cells

  44. Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated

  45. Journal of Immunology 1994 152 4755-4782 0 0 1 10 1 10 Factor increase over control Factor increase over control 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

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