Biology of mast cells

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

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

• 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

• Factors that inhibit mast cell growth– Granulocyte-macrophage colony-

stimulating factor (GM-CSF)– Retinoids– Transforming growth factor-β (TGF-β)

Other factors

• 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

Other mast cell subsets

• 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

• 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

Homing mechanism

Chemoattractants

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

• 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

• Crystalline structures pattern of membrane-bound secretory granules:– Scrolls

Ultrastructure

• Crystalline structures pattern of membrane-bound secretory granules:– Grating

Ultrastructure

• 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

• 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

Release of mediators

• Degranulation from activation– Energy-dependent– After almost complete degranulation,

HLMCs are able to survive and undergo regranulation over a period of 48 hours

• Piecemeal degranulation– Poorly understood mechanism– Variable loss of granule contents– Granules and their membranes remain

intact

• Synthesis of new mediators

• Synthesis and secretion of cytokines

• Synthesis and secretion of chemokines – CCL1-7– CCL12– CCL17– CCL19– CCL20– CCL22– CXCL5– CXCL8

Image from: http://www.biochemj.org/csb/011/Fig11_mast_cell_signallinga.jpg

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

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

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

• Microscopic process

• 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

• 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

• 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

• 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

• Activation via TLR-2 induces Ca2+ mobilization, degranulation and LTC4 production

• Activation via TLR3 can deteriorate airway physiology

Roles in allergen sensitization

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

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

• 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

• 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).

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

Interaction with epithelium

Interaction with fibroblasts

• 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

• 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

Biology of mast cells

Health & Medicine

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