Biology of mast cells

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

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

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.

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

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.
• 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

Factors leading to
heterogeneity
• 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

Chemoattractants

Ultrastructure
Figure from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.html
• 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
– Grating

Ultrastructure
– Lattices

Appearance in tissue
Images from: http://www.pathologyoutlines.com/topic/bonemarrowmastcells.html
• 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.

IgE-dependent activation
Monomeric IgE-dependent
activation
Non-immunologic mast cell
activation
Activation mechanisms

Toshiaki Kawakami & Stephen J. Galli, Nature Reviews Immunology 2, 2002, 773-786
• 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

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

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

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

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

Biology of mast cells

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