Marc E. Rothenberg, M.D., Ph.D Professor and Director. CHRF Rm

Transcription

1 Marc E. Rothenberg, M.D., Ph.D Professor and Director Cincinnati Children s Hospital Division of Allergy and Immunology CHRF Rm Rothenberg@cchmc.org

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5 Mast Cells Toluidine Blue Chloroacetate Esterase Tryptase 12 µm Exclusive tissue residents; never found in peripheral blood Perivascular and intraneural locations throughout the body Greatest concentration at interfaces with external environment Phenotypic heterogeneity reflects multiple physiologic functions

6 Divergent Pathways of Mast Cell Development

7 Products of Mast Cell Activation IgE Y Preformed Mediators (Minutes) Newly Formed Eicosanoids (Minutes) Induced Cytokines/ Chemokines (Hours) Histamine Proteases TNF-α Cysteinyl Leukotrienes PGD 2, LTB 4 IL-3 IL-4 IL-5 IL-6 IL-8 IL-9 IL-11 IL-13 MIP-1α MIP-1β MCP-1 TNF-α Early (Edema, Broncho- Constriction, Vasopermeability) Late (Inflammation, Cell Recruitment)

8 Cellular Membrane cpla 2 COOH Arachidonic acid FLAP 5-LO OOH COOH OH COOH OH 5-HPETE 5-HETE COOH OH O OH OH COOH COOH 6-trans-LTB 4 LTA 4 LTA 4 H LTB 4 OH LTC 4 S COOH OH OH COOH COOH S-Cys-Gly S-Cys-Gly S-Cys Glu γgt LTC 4 LTD DiP 4 LTE 4

9 Mast Cells Are Markedly Heterogeneous in CysLT-Generating Ability Lung, gut, and uterine mast cells: CysLTs = PGD 2 Lung MC Skin mast cells: PGD 2 >>>> CysLTs Skin MC

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11 Arachidonic Acid Pathway 5 HPETE Direct 5-Lipoxygenase or FLAP inhibitors Leukotriene A 4 LT C 4 LT B 4 Mediator of LT D 4 neutrophil chemotaxis LT E 4 Cys LT 1 Receptor Antagonists Mediators of allergy, bronchoconstriction, and mucous production

12 Mast Cells in Innate Immunity

13 Mast Cells Are Essential for Survival of Gram-Negative Peritonitis Treatment None Surviving Mice (%) Genotype +/+ 0 Genotype W/W v 0 Surviving W/W v Mice (%) BMMC + Anti-TNF BMMC + Control IgG Days Following CLP Days Following CLP Echtenacher B et al. Nature. 1996;381:75-77.

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17 MBP deposition in AD

18 Chronic Urticaria

19 Chronic Urticaria MBP staining

20 Rothenberg et al. Adv Immunol; 2001

21 Triggers Allergens Allografts Helminths Viruses Tissue Injury Rothenberg et al. Adv Immunol; 2001

22 Triggers Allergens Allografts Helminths Viruses Tissue Injury Cytotoxic Secretory Products ECP EDN EPO MBP Rothenberg et al. Adv Immunol; 2001

23 Triggers Allergens Allografts Helminths Viruses Tissue Injury Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Rothenberg et al. Adv Immunol; 2001

24 Triggers Allergens Allografts Helminths Viruses Tissue Injury Lipid Mediators Leukotrienes Platelet Activating Factor Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Rothenberg et al. Adv Immunol; 2001

25 Triggers Allergens Allografts Helminths Viruses Tissue Injury Lipid Mediators Leukotrienes Platelet Activating Factor Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IFN!,GM-CSF, TGF-"/#, TNF-" Rothenberg et al. Adv Immunol; 2001

26 Triggers Allergens Allografts Helminths Viruses Tissue Injury Chemokines Eotaxin MIP-1 RANTES Lipid Mediators Leukotrienes Platelet Activating Factor Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IFN!,GM-CSF, TGF-"/#, TNF-" Rothenberg et al. Adv Immunol; 2001

27 Triggers Allergens Allografts Helminths Viruses Tissue Injury Chemokines Eotaxin MIP-1 RANTES Neuro-mediators Substance P VIP Lipid Mediators Leukotrienes Platelet Activating Factor Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IFN!,GM-CSF, TGF-"/#, TNF-" Rothenberg et al. Adv Immunol; 2001

28 Antigen Presentation B7.2 MHC-II Neuro-mediators Substance P VIP Triggers Allergens Allografts Helminths Viruses Tissue Injury Chemokines Eotaxin MIP-1 RANTES Lipid Mediators Leukotrienes Platelet Activating Factor Cytotoxic Secretory Products ECP EDN EPO MBP Ribonucleases Cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IFN!,GM-CSF, TGF-"/#, TNF-" Rothenberg et al. Adv Immunol; 2001

29 Chemokine Family Zimmermann et al. JACI; 2003

30 BALF 3 hours after eotaxin administration to IL5 Tg mice

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32 Bone Marrow GATA-1 IL-3 GM-CSF IL-5

33 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1

34 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1 EOTAXIN Matthew et al; PNAS; 1998 Mishra et al. JCI; 1999 GI Tract

35 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1 EOTAXIN Thymus Matthew et al; PNAS; 1998 Mishra et al. JCI; 1999 GI Tract

36 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1 EOTAXIN Thymus Uterus Matthew et al; PNAS; 1998 Mishra et al. JCI; 1999 GI Tract

37 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1 EOTAXIN EOTAXIN Mammary Gland Thymus Uterus Matthew et al; PNAS; 1998 Mishra et al. JCI; 1999 Gouon-Evans et al. Development; 2002 GI Tract

38 Bone Marrow GATA-1 IL-3 GM-CSF IL-5 Blood IL-5 P-selectin CD18 ICAM-1 VLA-4 VCAM-1 EOTAXIN -1 The GI tract is the predominant reservoir of Eos. Eosinophil homing occurs early in development Eosinophil homing occurs independent of endogenous flora Matthew et al; PNAS; 1998 Mishra et al. JCI; 1999 Mishra et al. JBC; 2002 Humbles et al. PNAS 2002 Eosinophil homing is critically regulated by eotaxin-1/ccr3 Eotaxin-1 dependent eosinophil homing is regulated by β7-integrin

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40 Protective Immunity Against Parasites Parasite sige FcεRI Mediators: MBP ECP

41 Eosinophil beneficial and detrimental role Parasite Epithelium sige FcεRI sige FcεRI Inflammatory cells Protective immunity against parasites Allergic disorders

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43 Basophils vs Mast Cells Basophils are derived from GM-CFU whereas mast cells are distinct Basophils are CCR3+, IgEeR1+, but c-kit negative Basophils and mast cells release preformed histamine and de-novo synthesis LT and PGD2. Basophils are found in peripheral blood whereas mast cells are not Basophils are not circulating mast cells

44 Role of Basophils in the Immune System 1-Innate Immunity Several non antigenic specific stimuli derived form various organisms have been shown to induce mediator or cytokine release from basophils. 1-IgE binding to its receptor is necessary -HIV GP120 interact with IgE (V H 3). -Shistosoma mansoni egg stage antigen (SEA). -Plant Lectins 2-Independent of IgE binding to FcεRI Brugia malayi: Bm-tph-1 : homolog to histamine releasing factor.

45 Physiologic Role of Basophils in the Immune System Summary -Several pathogenic organisms may induce the activation of basophils by the release of superantigens, molecules with the ability to cross-link FcεRI either directly or via receptor bound IgE in non antigenic specific manner. - Molecules derived from pathogens or endogenous sources can directly activate basophils independently of the presence of specific IgE. IL-4 release Promoting T cell differentiation to the Th-2 phenotype Bridging the innate and specific immunity by skewing the differentiation of the Th-2 phenotype

46 Role of Basophils in the Immune System 2-Acquired immunity: 2-1: Late phase reaction (LPR) in allergic reaction *Basophils are recruited with Eos, T cells and PMNs during LPR into the lung and nose. How basophils are recruited to the inflammatory sites? Tissue recruitment by chemokines, LTs **Basophils are CCR3 positive. **Eotaxin/eotaxin-3 : an important role in basophils recruitment. **Respond to Eot2, MCP-1, MIP1α, Rantes, and MCP3. Activation by cytokines and other mediators ** IL-3,IL-5 and GM-CSF: facilitate basophils migration in vitro. ** PAF, C5a, C3a dependent activation: mediators release (LTC4, IL-4, IL-13 and histamine). LPR exacerbation by further influx of inflammatory cells VCAM-1up-regulation mediated mechanism

47 Role of Basophils in the Immune System IgE dependent activation (FcεRI dependent) ** Releasing performed IL-4&IL-13 >>IL-4&IL-13 produced by allergen activated unfractioned peripheral blood leukocytes of atopic patients: basophils derived. >> IL-4 and IL-13: are central to the recruitment of inflammatory cells by up-regulation of adhesion molecules and chemokines Example: Eotaxin (atopic dematitis) More inflammatory cells recruitment **Histamine and LTC4: induce muscle contraction Airway Hyperresponsiveness (AHR) Maintaining chronic allergic inflammation

48 Directly inducing the switch to the IgE isotype in B cells independently of T cells Provide necessary signals for IgE production in vitro: ** Basophils produce: IL-4/IL-13 and CD40L

49 Basophils play a central role in the orchestration of allergic inflammation, via IL-4 and IL-13, by providing: * a trigger to IL-4-mediated T helper 2 lymphocyte activation * B cell IgE switching * increased vascular adhesion molecule expression.

50 Mastocytosis Mastocytosis: a group of disorders characterized by aberrant mast cell proliferation and increased mast cell burden Cutaneous: accumulation of mast cells in skin only (urticaria pigmentosa, mastocytoma, diffuse cutaneous mastocytosis) most common variant in children Systemic: accumulation of mast cells in the skin and/or other organs Signs and symptoms of mastocytosis relate largely to mediator release (histamine, PGD 2, etc)

51 Urticaria Pigmentosa Defining feature of isolated cutaneous mastocytosis Present in most systemic mastocytosis Absence is a poor prognostic indicator

52 Skin Biopsy Urticaria Pigmentosa

53 Mutations in c-kit Most Cases of Systemic Mastocytosis Have D816V c-kit Mutation in Bone Marrow Extracellular Domain Exon 2 Exon 8 Exon 10 D52N (MPD) Δ (AML) V5301 (AML) Plasma Membrane Exon 11 Exon 17 Intracellular Domain Juxta-membrane Helix Δ (HMC1, GIST, K9MCD) Enzymatic pocket/activation loop D816V, E839K (MCD) V825A (Sinonasal NKTCL) Longley BJ et al. Leuk Res. 2001;25:

54 Hypereosinophilic Syndromes A group of disorders characterized by severely elevated levels of eosinophils in the blood and tissues for at least 6 months. Morbidity associated with end-organ damage caused by eosinophil products (e.g. cardiotoxicity).

55 A PHASE I/II STUDY OF THE EFFECT OF INTRAVENOUS ANTI-IL-5 (Mepolizumab) SB ON THE OUTCOME AND MANAGEMENT OF HYPEREOSINOPHILIC SYNDROMES Cohort C Cohort A/B Observation/ Stabilization Cohort D Observation weeks anti-il-5 anti-il-5 anti-il-5

56 Beneficial Effect of Anti-IL-5 on Patients with Hypereosinophlic Syndromes Garrett et al.; JACI; 2003

57 Genetic Event Involved in Hypereosinophilic Syndromes Microdeletion on Chromosome 4 Fusion of FIP1L1 with PDGFRA genes Generation of Imatinib sensitive activated tyrosine kinase Patients harboring this fusion gene have elevated mast cells and tryptase (systemic mastocytosis) Cools et al NEJM; 2003 Klion et al. Blood; 2003

58 Methods 5-FU injection Retroviral Constructs MSCV-IRES-EGFP 6 days Balb/c mice ITR IRES EGFP ITR MSCV-FIP1L1/PDGFRA-IRES-EGFP ITR FIP1L1 PDGFRA IRES EGFP ITR Harvest of bone marrow cells Culture with 1st. transduction (spin infection) o/n SCF (10ng/ml) IL-3 (6ng/ml) IL-6 (10ng/ml) o/n (MSCV-FIP1L1/PDGFRA-IRES-EGFP or MSCV-IRES-EGFP) 2nd. transduction (MSCV-FIP1L1/PDGFRA-IRES-EGFP or (spin infection) MSCV-IRES-EGFP) 3 hours Liquid culture with IL-3 (100ng/ml) Transplant into lethally irradiated mice 4.5Gy X2 In vitro Colony assay CFU-GM CFU-Mast cell CFU-Eosinophil HPP-CFC Yamada et al.

59 FIP1L1-PDGFRα induces intense leukocytosis (10 3 /ml) WBC P < MOCK Fusion (10 3 /ml) Neutrophils P < 0.01 (10 3 /ml) 3 2 Eosinophils P < MOCK Fusion 0 MOCK Fusion CML like disease with eosinophilia and neutrophilia Yamada et al.

60 Splenomegaly in FIP1L1-PDGFRα-induced disease mice (10 6 cells) (mg) Spleen Cell Counts P < 0.01 MOCK Fusion Spleen Weights P < 0.01 MOCK Fusion MOCK Fusion Yamada et al.

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