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Biology of mast cells Presented by Wat Mitthamsiri, MD. June6, 2014
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Biology of Mast Cell
ByWat Mitthamsiri, MD.
Allergy and Clinical Immunology FellowKing Chulalongkorn Memorial Hospital
Outline• Introduction• Development• Heterogeneity• Homing mechanism• Ultrastructure and mediators• Activation mechanism• Roles in allergen sensitization• Roles in allergic diseases
Introduction
History276 million years ago
Mast cells already present in primitive reptiles
1863, RECKLINGHAUSENGranulated cells found
1878/1879 EHRLICHMast cells and basophils
U Blank, et al., Allergy 2013; 68: 1093–1101.P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
Some milestones
U Blank, et al., Allergy 2013; 68: 1093–1101.
Development
Development
J Douaiher, et al., Advances in Immunology, Volume 122, 2014: 211-252
Development
J Douaiher, et al., Advances in Immunology, Volume 122, 2014: 211-252
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
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
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
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)
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
Heterogeneity
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
Main mast cell subsets
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
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
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)
Plasticity
Gurish MF, Austen KF: The diverse roles of mast cells. J Exp Med 194:F1, 2001
Distinct mast cell phenotypes in different tissues
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
Homing mechanism
Chemoattractants
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251.
Chemoattractants
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251.
Chemoattractants
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013: 228-251.
Ultrastructure and mediators
Ultrastructure
Figure from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.htmlP Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Cell membranes contain fingerlike projections: microplicae
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
Ultrastructure
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Crystalline structures pattern of membrane-bound secretory granules:– Scrolls
Ultrastructure
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Crystalline structures pattern of membrane-bound secretory granules:– Grating
Ultrastructure
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Crystalline structures pattern of membrane-bound secretory granules:– Lattices
Appearance in tissue
Images from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.htmlP 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
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
Preform mediators
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
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
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
Release of mediators
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Synthesis of new mediators
Release of mediators
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Synthesis and secretion of cytokines
Release of mediators
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Synthesis and secretion of cytokines
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
Release of mediators
Image from: http://www.biochemj.org/csb/011/Fig11_mast_cell_signallinga.jpg
Release of mediators
F I Hsu, J A Boyce, Middleton’s Allergy 7th edition, 2009, 311-328.
Activation mechanisms
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.
IgE-dependent activation
Toshiaki Kawakami & Stephen J. Galli, Nature Reviews Immunology 2, 2002, 773-786P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
• Initiate through the high-affinity IgE receptor FcεRI
IgE-dependent activation
Stephen J. Galli, Mindy Tsai & Adrian M. Piliponsky, Nature 454 (2008), 445-454
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
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
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
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
Non-immunologic activation
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
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
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
Roles in allergen sensitization
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
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
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
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.
Roles in allergic diseases:Anaphylaxis
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
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
Roles in allergic diseases:Allergic rhinitis
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
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
Roles in allergic diseases:Allergic conjunctivitis
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
Roles in allergic diseases:Atopic dermatitis and urticaria
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
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
Roles in allergic diseases:Asthma
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
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
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).
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,
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
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
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
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
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
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
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
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
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)
Interaction with ASM
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
Interaction with epithelium
P Bradding, H Saito., Middleton’s Allergy 8th edition, 2013, 228-251.
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
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
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
Thank you