Biology of mast cells

1. Biology of Mast Cell By Wat Mitthamsiri, MD. Allergy and Clinical Immunology Fellow King Chulalongkorn Memorial Hospital

2. Outline • Introduction • Development • Heterogeneity • Homing mechanism • Ultrastructure and mediators • Activation mechanism • Roles in allergen sensitization • Roles in allergic diseases

3. Introduction

4. History 276 million years ago Mast cells already present in primitive reptiles 1863, RECKLINGHAUSEN Granulated cells found 1878/1879 EHRLICH Mast cells and basophils U Blank, et al., Allergy 2013; 68: 1093–1101. P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

5. Some milestones U Blank, et al., Allergy 2013; 68: 1093–1101.

6. Development

7. Development J Douaiher, et al., Advances in Immunology, Volume 122, 2014: 211-252

8. Development J Douaiher, et al., Advances in Immunology, Volume 122, 2014: 211-252

9. Obligate growth factor Langley KE, et al., Blood 81 (3): 656–60. Zhang, et al., Proc.Natl.Acad.Sci.USA, 2000, 97: 7732-7737. P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251. • Stem cell factor (SCF) – Other names: • Kit ligand • Steel factor – 141 residues – Molecular weight • 18.5 KDa – Level in normal human blood serum • 3.3 ng/mL

10. Obligate growth factor Geissler EN, et al., Somat. Cell Mol. Genet. 17 (2): 207–14. P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251. • Stem cell factor (SCF) – Gene locus: • Chromosome 12q22-12q24 in humans – Ligand for Kit (CD117) encoded by the proto-oncogene c-kit – Derived from many cellular sources • Epithelial cells • Mesenchymal cells – Soluble and membrane-bound forms

11. Other factors P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251. • Factors that enhance mast cell growth and survival – Nerve growth factor (NGF) – IL-3 – IL-6 – IL-9 – IL-10 – Lysophosphatidic acid (LPA) – Silencing of MS4A2 gene – TRPM7 ion channel

12. Other factors P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251. • Factors that inhibit mast cell growth – Granulocyte-macrophage colony- stimulating factor (GM-CSF) – Retinoids – Transforming growth factor-β (TGF-β)

13. Other factors P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251. • Factors that can either enhance or inhibit mast cell growth and survival – IL-4 • Inhibits proliferation of immature human peripheral blood–derived mast cells (HPBMCs) • Potentiates proliferation of more mature HPBMCs, – IL-5 and interferon-gamma (IFN-γ) • Prolong HCBMC survival on SCF withdrawal • Inhibit immature HPBMC proliferation

14. Heterogeneity

15. Factors leading to heterogeneity P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Interactions with the tissue matrix and resident cells such as fibroblasts • Progenitors are possibly committed to a particular phenotype early in their development

16. Main mast cell subsets P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

17. Other mast cell subsets P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Mast cells expressing tryptase and carboxypeptidase A, but not chymase – Found in the airway epithelium in asthmatic airways • Mast cells containing chymase and carboxypeptidase without tryptase (MCC) – Found in the lung, nose, gut, and kidney – Unknown function

18. Intra-tissue heterogeneity P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Marked differences in: – Size and shape – Expression of • Tryptase • Chymase • FcεRIα • IL-9 receptor • Histidine decarboxylase • 5-lipoxygenase • Leukotriene C4 (LTC4) synthase • Renin • Vascular endothelial growth factor (VEGF) • Basic fibroblast growth factor (FGF)

19. Plasticity Gurish MF, Austen KF: The diverse roles of mast cells. J Exp Med 194:F1, 2001

20. Distinct mast cell phenotypes in different tissues P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

21. Homing mechanism

22. Chemoattractants P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251.

25. Ultrastructure and mediators

26. Ultrastructure Figure from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.html P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Cell membranes contain fingerlike projections: microplicae

27. Ultrastructure P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Immature mast cells – May have a multilobed nucleus • Mature mast cells – Monolobed nucleus – No apparent nucleoli – Little condensed chromatin – Prominent cytoplasmic structures are the electron-dense granules = membrane- bound and contain preformed mediators

28. Ultrastructure P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Crystalline structures pattern of membrane-bound secretory granules: – Scrolls

29. Ultrastructure P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Crystalline structures pattern of membrane-bound secretory granules: – Grating

30. Ultrastructure P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Crystalline structures pattern of membrane-bound secretory granules: – Lattices

31. Appearance in tissue Images from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.html P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Can be round or spindle-shaped • Most effective way to identify the location and subtype histologically = immunohistochemical analysis using Ab against mast cell–specific proteases

32. Granule matrix P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Formed from proteoglycans, with glycosaminoglycan (GAG) side chains • Heparin = main proteoglycans in human mast cells – Stabilizes the β-tryptase tetramer – Neutral proteases, acid hydrolases, and histamine molecules are attached to heparin by ionic linkage to the sulfate groups on the GAGs. • Some chondroitin E also present

33. Preform mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

34. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Degranulation from activation – Energy-dependent – After almost complete degranulation, HLMCs are able to survive and undergo regranulation over a period of 48 hours

35. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Piecemeal degranulation – Poorly understood mechanism – Variable loss of granule contents – Granules and their membranes remain intact

36. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Synthesis of new mediators

37. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Synthesis and secretion of cytokines

38. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Synthesis and secretion of cytokines

39. Release of mediators P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Synthesis and secretion of chemokines – CCL1-7 – CCL12 – CCL17 – CCL19 – CCL20 – CCL22 – CXCL5 – CXCL8

40. Release of mediators Image from: http://www.biochemj.org/csb/011/Fig11_mast_cell_signallinga.jpg

41. Release of mediators F I Hsu, J A Boyce, Middleton’s Allergy 7th edition, 2009, 311-328.

42. Activation mechanisms

43. IgE-dependent activation Monomeric IgE-dependent activation Non-immunologic mast cell activation Activation mechanisms P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

44. IgE-dependent activation Toshiaki Kawakami & Stephen J. Galli, Nature Reviews Immunology 2, 2002, 773-786 P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Initiate through the high-affinity IgE receptor FcεRI

45. IgE-dependent activation Stephen J. Galli, Mindy Tsai & Adrian M. Piliponsky, Nature 454 (2008), 445-454

46. Granule swelling Crystal dissolution Granule fusion with surrounding granules and cell membrane Exocytosis + release of mediators into the extracellular space IgE-dependent activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Microscopic process

47. IgE-dependent activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Inhibition measures – “Mast cell stablilizer” • Target: 1 of LPA receptors, GPR35 • Poor efficacy in vivo • Rapid tachyphylaxis – β2-adrenoceptor agonists • Poor efficacy in vivo • Rapid tachyphylaxis – Syk inhibitor • Poor outcome

48. Monomeric IgE activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Binding of monomeric IgE alone to FcεRI initiates intracellular signaling events and Ca2+ influx • In HCBMCs, monomeric IgE binding induces the release of CCL1, CCL3, and GM-CSF without histamine release • In HLMCs, IgE binding induces secretion of histamine, LTC4, and CXCL8, which is markedly enhanced in the presence of SCF

49. Monomeric IgE activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • In HLMCs – Ongoing signaling is dependent on the presence of “free” IgE – Signaling ceases immediately when free IgE is removed • SCF and free IgE concentrations are elevated in asthmatic airways • Good correlation has been found between total serum IgE and presence of asthma and bronchial hyperresponsiveness

50. Non-immunologic activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

51. Non-immunologic activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • SCF inhibits β2-adrenoceptor (β2- AR) signaling in HLMCs and HMC-1 within minutes of exposure,… followed by internalization. • => Impaired β2-AR–dependent inhibition of • Histamine and LTC4 release • Ion channel modulation

52. Non-immunologic activation P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Activation via TLR-2 induces Ca2+ mobilization, degranulation and LTC4 production • Activation via TLR3 can deteriorate airway physiology

53. Roles in allergen sensitization

54. Roles in allergen sensitization P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • From mouse model and in vitro study, mast cells could contribute to Th2 differentiation at the onset of an immune response – Bee venom phospholipase (PL)A2 and Der p 1 induce the release of histamine and IL-4 from HLMCs in the absence of cell-bound IgE – Cockroaches, fungal spores, pollens, and cats can induce the release of phospholipases and proteases

55. Roles in allergen sensitization P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Why allergen sensitization does not occur in everyone? – Environmental factors • Level of allergen exposure – Genetic factors • Mast cell releasability • Epithelial integrity and permeability • Local antiprotease activity • Regulation of cytokine production

56. Roles in allergen sensitization P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Influence on development of dendritic cells and their ability to activate T cells – Histamine and PGD2 increases IL-10 and decreases IL-12 production by mature dendritic cells -> naive T cells become polarized toward Th2 phenotype – Mast cell dependence for the generation of Th2-promoting dendritic cells is evident in mice

57. Roles in allergen sensitization P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Influence on development of dendritic cells and their ability to activate T cells – Mast cell exosomes induce immature dendritic cells to become mature plasmacytoid dendritic cells capable of antigen presentation by upregulating MHC class II, CD80, CD86, and CD40 molecules – Mast cell–derived TNF-α is important for dendritic cell migration during immune responses.

58. Roles in allergic diseases: Anaphylaxis

59. Roles in anaphylaxis P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Anaphylaxis is mediated predominantly by mast cells tryptase – α-tryptase • Released by mast cells constitutively • Increased baseline release in mastocytosis – β-tryptase • Stored in mast cell granules • Released after IgE-dependent activation • > More specific marker than total tryptase. • > BEST marker of systemic mast cell activation in anaphylaxis

60. Roles in anaphylaxis P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Why does systemic activation of mast cell occurred? – Systemic diffusion of allergen? Unlikely – Amplification mechanisms? • Neurologic reflexes • Platelet-activating factor (PAF) – Can activate human mast cells – Can cause mast cells to release histamine – Induces the release of CXCL8 – Transiently upregulates mRNA expression for several other chemokines – Enhances IgE-dependent mediator release

61. Roles in allergic diseases: Allergic rhinitis

62. Roles in AR P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. •  No. of mast cells in the epithelium •  Expression of Th2 cytokines in mast cells •  No. of CD34+, tryptase-negative cells (mast cell progenitor) in the nasal epithelium •  Expression of IL-4, which is reversed by the application of topical corticosteroids

63. Roles in AR P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • AR = IgE-driven, mast cell–dependent disease – Histamine are not elevated, but antihistamine therapy is highly effective at ameliorating symptoms – Anti-IgE therapy also is effective – Ongoing mast cell activation in nasal mucosa + Biologic effects of mast cell products can explain much of the symptomatology and pathology of AR

64. Roles in allergic diseases: Allergic conjunctivitis

65. Roles in AC P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. •  No. of mast cells and evidence of degranulation in all types of AC •  Levels of histamine, tryptase, and LTC4 are found in tears after allergen exposure •  No. Of MCT cell found in conjunctival epithelium and subepithelial layers of PAC, SAC, and VKC patient •  No. Of MCTC cells AKC and ABC patient

66. Roles in allergic diseases: Atopic dermatitis and urticaria

67. Roles in atopic dermatitis P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. •  No. of MCT increases in the skin of patients with atopic dermatitis •  Expression of IL-4 of skin mast cells in atopic dermatitis patient

68. Roles in urticaria P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • In acute urticaria – Mast cell degranulation is evident – Antihistamines is useful treatment, suggesting that the skin lesions result from mast cell activation • In CIU, mast cell activation is a factor –  Constitutive histamine release compared with control subjects – 30% of patients have autoAb to FcεRI or IgE – Anti-IgE (omalizumab) is highly effective treatment

69. Roles in allergic diseases: Asthma

70. Experimentally induced asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. Late asthmatic reaction (LAR): 4 - 12 hr Early asthmatic reaction (EAR): 10 min – 2 hr Bronchial allergen challenge Then, check the fall of FEV1

71. Early asthmatic reaction P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Mediator release from HLMCs in vitro: – Half-maximal release occurring • Similar pattern found in bronchoalveolar lavage Histamine PGD2 LTC4 2 min 5 min 10 min

72. Early asthmatic reaction P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Histamine, PGD2, and LTC4/LTD4 induce bronchoconstriction, mucosal edema, and mucus secretion • EAR was markedly attenuated by inhibitors of – Histamine (H1 receptor) – LTC4/LTD4 (cysteinyl LTRl) – To a lesser extent, PGD2 (thromboxane TP receptor).

73. Early asthmatic reaction P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Evidences supporting mast cell origin: – Kinetics of IgE-dependent mediator release in vivo parallels that of HLMC in vitro – Rapidly increased concentration of mast cell– specific tryptase in BAL occurs after local bronchial allergen challenge – β-agonists, when applied acutely in vitro, completely abolish EAR and associated increase in plasma histamine levels – EAR is almost completely ablated after 12 to 16 weeks of pretreatment with omalizumab,

74. Late asthmatic reaction P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Associated with inflammatory cell accumulation and activation •  Concentrations of histamine, PGD2, and LTC4 – But in different ratios than during the EAR • Tryptase levels fall • GM-CSF – Released after allergen provocation – Inhibits expression of tryptase in HMC-1 cells but does not attenuate histamine release –  IgE-dependent histamine release in HLMCs • LAR is attenuated markedly by omalizumab

75. Role in chronic allergic asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Mast cells present in the bronchial mucosal are in an activated state • Degranulation is continuous •  No.of mast cells in BAL fluid •  Histamine and tryptase •  Expression of IL-4 and IL-5 mRNA in mast cells •  Expression of mast cell–associated IL-4 and TNF-α

76. Role in chronic allergic asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Enhanced IgE-dependent release • Higher IgE concentrations • Upregulation of FcεRI • Enhanced IgE-related signaling • Enhanced allergen-dependent mediator release • In conclusion: – Atopic asthmatic phenotype = interaction among allergens, IgE, and hyperreactive mast cells

77. Role in non-allergic asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. •  Mast cell FcεRI+ expression in bronchial mucosa, may be due to –  Epsilon germline gene (Iε) and mature epsilon heavy chain (Cε) mRNA+ B cells in the bronchial mucosa – So…  local IgE synthesis •  Expression of Th2 cytokines IL-4 and IL- 5 occurs at both mRNA and protein levels • Accordingly, anti-IgE therapy may potentially be very effective, too

78. Role in occupational asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Pathology of occupational asthma (with the exception of irritant-induced asthma) is virtually identical to that seen in atopic and intrinsic asthma

79. Role in exercise-induced asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • This is not a distinct disease entity, but a marker of poor asthma control and ongoing airway inflammation • Agents that might help: – Histamine H1 receptor antagonists – Cyclooxygenase (COX) inhibitors – LTRA – Cromolyn sodium

80. Role in ASA-induced asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • It is associated with  LTC4 in nasal secretions and  LTE4 in urine • Mast cell LT generation may be involved –  No.of mast cells in the airways –  Proportion of these mast cells express COX-2 – Mast cells are the predominant cells expressing LTC4 synthase

81. Role in asthma exacerbations P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • RSV can induce mast cell degranulation • Evidence that mast cells contribute directly to asthma exacerbations – Activation of mast cells via TLR3, induces secretion of both IFN-α and IFN- β, – Dual stimulation through TLR3 and FcεRI enhances the release of IL-1β, TNF-α, IL-5, and cysteinyl leukotrienes – Omalizumab significantly reduces the rate of severe exacerbations

82. Mast cell location in asthma P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • No.of mast cells in the lamina propria is not increased in asthmatic airway • But in asthmatic patient, mast cells infiltrate 3 key structures – Airway epithelium – Airway mucosal glands – Airway smooth muscle (ASM)

83. Interaction with ASM P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

84. Interaction with epithelium P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.

85. Interaction with fibroblasts P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • Mast cells have the potential to activate subepithelial myofibroblasts • Mast cells and fibroblasts interact intimately through several mechanisms • Histamine, basic FGF, and IL-4 promote fibroblast proliferation in humans • IL-4 is a chemoattractant for human fibroblasts and also induces fibroblasts to secrete collagen type I, III and fibronectin

86. Interaction with fibroblasts P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251. • IL-13 increases CCL11 release from human airway fibroblasts • Heparin stabilizes basic FGF structurally and preserves its bioactivity by protecting it from degradation – Thereby potentiating fibroblast activation and proliferation indirectly

87. Take Home Messages • Mast cells are tissue-resident immune cells, with wide array of function in response to various stimuli • They are capable to secrete numerous multifunctional substances (autocoid, protease, cytokines, chemokines) • They play important roles in host defense and in allergic diseases • They have complex interactions with other immunologic and structural cells

88. Thank you

Biology of mast cells

Chulalongkorn Allergy and Clinical Immunology Research Group

Biology of mast cells