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Darling Giyett Josefina Méndez V
Deutschen Nationalbibliografie;
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1. Auflage 2016
© 2016 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen
Printed in Germany
ISBN 978-3-86345-328-2
Verlag: DVG Service GmbH Friedrichstraße 17
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University of Veterinary Medicine Hannover
Density and subtypes of mast cells in the morphologically intact lower respiratory tract of subadult and adult cattle and in lungs of calves with pneumonia after experimental
infection with Mycoplasma bovis
INAUGURAL - DISERTATION
in partial fulfilment of the requirements for the degree of -Doctor of Veterinary Medicine-
Doctor medicinae veterinariae (Dr. med. vet.)
submitted by
Darling Giyett Josefina Méndez Vivas
Caracas, Venezuela
Hannover 2016
Academic supervision: 1. Univ.-Prof. Dr. Marion Hewicker-Trautwein
Department of Pathology, University of Veterinary Medicine Hannover
1. Referee: Univ.-Prof. Dr. med. vet. Marion Hewicker-Trautwein
2. Referee: Univ.-Prof. Dr. med. vet. Martina Hoedemaker
Day of the oral examination: May 09th, 2016
To my daughter, my husband and my beautiful land Venezuela
“If I have seen further than others, it is by standing upon the shoulders of giants”
Isaac Newton
Contents
Contents ... v
Abbreviations ... x
1 Introduction ... 1
2 Literature ... 3
2.1 Mast cell origins ... 3
2.2 Mast cells in different species ... 9
2.2.1 Rodent mast cells ... 9
2.2.2 Human mast cells ... 12
2.2.3 Canine mast cells ... 14
2.2.4 Feline mast cells ... 15
2.2.5 Bovine and ovine mast cells ... 16
2.3 Mast cell mediators ... 18
2.3.1 Preformed mediators of mast cells ... 18
2.4 Mast cell activation ... 28
2.4.1 Direct mast cell pathogen interactions ... 29
2.4.2 Indirect mast cell activation ... 30
2.4.3 Complement receptor-mediated activation ... 32
2.5 Biological functions of mast cells ... 32
2.5.1 Mast cell phagocytic and killing activity ... 33
2.5.2 Parasitic infections ... 36
2.5.3 Defence again venoms or other toxins ... 37
2.5.4 Fibrosis ... 38
2.5.5 Angiogenesis ... 39
2.5.6 Effects of mast cells on T and B cells... 40
2.5.7 Effects of mast cells on dendritic cells... 41
2.5.8 Mast cells and activation of the inflammatory response ... 42
2.6 Role of mast cells in bovine respiratory disease ... 44
2.7 Mycoplasma bovis ... 46
2.7.1 History ... 46
2.7.2 Taxonomy ... 47
2.7.3 Economic losses ... 48
2.7.4 Reservoir and the source of infection ... 49
2.7.5 Pathogenicity of M. bovis ... 50
2.7.6 Mycoplasma bovis infection in cattle ... 52
2.7.7 Pathology of Mycoplasma bovis pneumonia ... 53
2.7.8 Host immune responses in the progression of M. bovis infection ... 55
3 Material and Methods ... 59
3.1 Tissue samples ... 59
3.1.1 Tissue samples from animals without lesions of the respiratory tract (groups 1-3)... 59
3.1.2 Calves experimentally infected with M. bovis ... 65
3.2 Fixation of tissue specimens ... 66
3.3 Processing of formalin-fixed tissue samples ... 67
3.4 Haematoxylin and eosin (H&E) staining ... 67
3.5 Metachromatic staining technique ... 67
3.6 Double-labelling technique for tryptase and chymase ... 68
3.6.1 Enzyme histochemical staining of chymase ... 69
3.6.2 Immunohistochemical staining of tryptase ... 69
3.6.3 Controls for the double-labelling technique ... 70
3.6.4 Protocol for the double-labelling technique for detection of chymase and tryptase in mast cells... 70
3.7 Histopathological findings in trachea and lungs of M. bovis-infected animals . 72 3.8 Quantification of mast cells ... 72
3.8.1 Quantification of mast cells in tracheal and lung tissue of cattle without respiratory tract lesions ... 72
3.8.2 Quantification of mast cells in lung tissue of M. bovis-infected calves ... 73
3.9 Statistical analyses... 73
4 Results ... 75
4.1 Results of kresylecht-violet (KEV) positive mast cells in the trachea of animals without lesions of the respiratory tract (groups 1, 2, 3)... 75
4.2 Results of kresylecht-violet (KEV) positive mast cells in lungs of animals without lesions of the respiratory tract (groups 1, 2, 3) ... 80
4.3 Results of kresylecht-violet (KEV) positive mast cells in lungs of calves infected with M. bovis type strain PG45 (group 4) ... 86 4.4 Results of kresylecht-violet (KEV) positive mast cells in M. bovis-infected calves
with necrotising pneumonia (group 5) ... 87 4.5 Results of kresylecht-violet (KEV) positive mast cells in M. bovis-infected calves
with non-necrotising pneumonia (group 6) ... 87 4.6 Results of mast cell subtypes in the trachea of animals without lesions of the
respiratory tract (groups 1, 2, 3) ... 90 4.7 Results of mast cell subtypes in the lungs of animals without lesions of the
respiratory tract (groups 1, 2, 3) ... 95 4.8 Results of mast cell subtypes in calves infected with M. bovis type strain
PG45 (group 4) ... 101 4.9 Results of mast cell subtypes in calves with necrotising pneumonia (group 5) ... 102 4.10 Results of mast cell subtypes in calves with non-necrotising pneumonia (group
6) ... 103 5 Discussion ... 109 5.1 Density of metachromatically stained mast cells in tracheae and lungs of
subadult and adult cattle without morphological alterations ... 109 5.2 Density of double-labelled mast cells in tracheae and lungs of calves and cattle
without morphological alterations ... 112
5.3 Density of metachromatically stained mast cells in the lungs from calves infected
with M. bovis ... 115
5.4 Density of double-labelled mast cells in lungs of calves infected with M. bovis ... 117
6 Summary ... 120
7 Zusammenfassung ... 123
8 References ... 126
9 Acknowledgement ... 178
Abbreviations
ABC Avidin-biotin-peroxidase-complex AEC 3-amino-9-ethylcarbazole
AMP Antimicrobial peptide
BALT Bronchus-associated lymphoid tissue bFGF Basic fibroblast growth factor BMCPs Basophil mast cells progenitors BMMC Bone marrow-derived mast cells BRSV Bovine respiratory syncytial virus
BSA Bovine serum albumin
C/EBPα CCAAT/enhancer binding protein α CCL5 Chemokine (C-C motif) ligand 5
CLP Common lymphoid progenitor
CMPs Common myeloid progenitors
CR Complement receptor
CTMC Connective tissue mast cells CysLTs Cysteinyl leukotrienes
DCs Dendritic cells
EBL Embryonic bovine lung cells ECM Extracellular matrix
FC Fragment crystallizable
FcεRIα High-affinity receptor for immunoglobulin E FGF Fibroblast growth factor
GAG Glycosaminoglycan
GM-CSF Granulocyte-macrophage colony-stimulating factor GMPs Granulocyte/macrophage progenitors
GPCRs G-protein-coupled receptors GPI Glycosylphosphatidylinositol
H Histamine
HMCs Human mast cells HSP70 Heat-shock protein 70 IBD Inflammatory bowel disease ICAM-1 Intercellular adhesion molecule - 1
IFN-ƴ Interferon-gamma
IL Interleukin
kDa Kilodalton
KEV Kresylecht-violet
LAP Lingual antimicrobial peptide
LKT Leukotoxin
LT Leukotriene
LPS Lipopolysaccharide
LT-HSCs Long-term haematopoietic stem cells M. bovis Mycoplasma bovis
M. haemolytica Mannheimia haemolytica
MC Mast cells
MCC Chymase-positive mast cells MC-cpa Mast cell carboxypeptidase mMCP Mouse Mast cell protease MCPs Mast cell progenitors
MCTC Tryptase-chymase-positive mast cells MCT Tryptase-positive mast cells
MHC Major histocompatibility complex
MK Megakaryocyte
MMC Mucosal mast cells
MR Mannose receptor
NASDCA Naphthol-AS-D-chloroacetate NBF Neutral buffered formalin NETs Neutrophil extracellular traps
NGF Nerve growth factor
NK Natural killer
NKT Natural killer T cells P. haemolytica Pasteurella haemolytica PAF Platelet activating factor
PAMPs Pathogen-associated molecular patterns PARs Protease-activated receptors
PBS Phosphate-buffered saline PDGF Platelet derived growth factor
PG Prostaglandins
PGD2 Prostaglandin D2
PGN Gram-positive bacterial cell-wall component peptidoglycan
PGs Proteoglycans
PRDS Paroxystic respiratory distress syndrome
Pro Progenitors
PRRs Pattern recognition receptors
Prss Protease serine members
RMCP-I Rat mast cell protease I RMCP-II Rat mast cell protease II
RT Room temperature
ST-HSC Short-term haematopoietic stem cell
TCR T cell receptor
TAP Tracheal antimicrobial peptide
TH2 T helper 2
TPO Thrombopoietin
TLRs Toll-like receptors TBS Tris-buffered saline
TGF-β Transforming growth factor-β
µm Micrometer
USA United States of America VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor
1 Introduction
In vertebrates, mast cells (MC) are widely distributed throughout the vascularised tissues, particularly near surfaces which are exposed to the environment including skin, airways and gastrointestinal tract, where pathogens, allergens and other environmental agents are frequently encountered (KITAMURA, 1989; KAWAKAMI and GALLI, 2002). MC can exert their effector functions through the direct or indirect actions of a wide spectrum of mast-cell-derived products, and such effects can be observed in both innate and acquired immune responses (MEKORI and METCALFE, 2000; GALLI et al., 2005b; BOYCE, 2007; DAWICKI and MARSHALL, 2007; GALLI et al., 2008; SAYED et al., 2008). In MC, different mediators, e.g. histamine, heparin, and proteases such as tryptase and chymase, are stored in cytoplasmic granules (METCALFE et al., 1997; WELLE, 1997). In tissue sections, MC can be visualised applying metachromatic stains such as kresylecht-violet or toluidine blue staining or using enzyme-histochemical or immunohistochemical techniques for demonstration of tryptase and chymase (IRANI et al., 1986; BÖCK et al., 1989; KÜTHER et al., 1998;
HARRIS et al., 1999).
For bovines, relatively few studies exist in which the distribution of MC and their numbers and subtypes in the respiratory tract or other tissues have been described (CHEN et al., 1990a,b; HUNT et al., 1991; KÜTHER et al., 1998; HARRIS et al., 1999;
JOLLY et al., 1999; JOLLY et al., 2000). In those investigations, using the different staining techniques named above, the numbers and subtypes of MC in normal bovine lungs and trachea were characterised. Those studies revealed that in bovines, as in humans and other species, three mast cell subtypes are distinguished in the respiratory tract: tryptase-positive MC (MCT), chymase-positive MC (MTC) and MC containing both tryptase and chymase (MCTC) (KÜTHER et al., 1998; JOLLY et al., 1999; JOLLY et al., 2000).
It has been postulated that MC play an important role as modulator of host defence in the lung (ABRAHAM et al., 1997). In calves infected with bovine respiratory syncytial
virus (BRSV) evidence for the involvement of MC degranulation and MC mediators in the pathogenesis of BRSV-induced lung lesions was reported (KIMMAN et al., 1989;
JOLLY et al., 2004). Furthermore, studies in calves with experimental respiratory infection with Mannheimia haemolytica have shown that MC contribute to the acute inflammatory response (RAMÍREZ-ROMERO et al., 2000).
Mycoplasma (M.) bovis is the most pathogenic mycoplasma for bovines. It is an important cause of respiratory disease and arthritis in feedlot cattle, young dairy and veal calves (NICHOLAS and AYLING, 2003). In the respiratory tract of cattle M. bovis is certainly capable of causing acute respiratory disease, although it is more accepted as a cause of chronic bronchopneumonia with caseous and sometimes coagulative necrosis characterised by a persistent infection (CASWELL and ARCHAMBAULT, 2007). To date, the possible role of MC in the lung of cattle infected with M. bovis has not been studied.
One aim of this study was to carry out a systematic characterisation of MC density and MC subtypes in formalin-fixed and paraffin-embedded tissue sections from trachea and lungs from bovines without pathomorphological changes of three different age groups (calves, 6-7 months-old animals, adult animals) using both a metachromatic staining method (kresylecht-violet staining) and a combined immunohistochemical and enzyme-histochemical double-labelling technique for demonstration of tryptase- and chymase-positive MC. The second aim was to investigate the MC numbers and subtypes in formalin-fixed and paraffin-embedded lung tissue samples from calves with different types of pneumonia (acute pneumonia, chronic necrotising pneumonia, chronic non-necrotising pneumonia) induced by experimental infection with M. bovis applying the same methods as used for lung samples of normal bovines.
2 Literature
2.1 Mast cell origins
Mast cells (MC) are present in all classes of vertebrates including amphibian reptiles, birds and mammals (BRADDING, 2009). The storage of histamine in vertebrate mast cells and the use as an inflammatory mediator was established in primitive reptilias as Lepidosauria an estimated 276 million years ago (BRADDING, 2009) MC persistence throughout the evolution of vertebrates indicates a strong selective pressure favouring their survival suggesting that these cells have beneficial and important roles (BRADDING, 2009). MC were first observed in 1863 by F. von Recklinghausen.
However, in those studies, the cells that in retrospect probably were MC were not described in a way that would permit their clear distinction from other cells that exhibited overlapping light microscopic features (METZ et al., 2007).
It was Paul Ehrlich in the late 1870s that provided the first description of some of the distinctive histochemical characteristics as their peculiar metachromatic staining properties (METZ et al., 2007). The monomorphic cytoplasmic granules of the round to long-stretched cells with an oval to reniform nucleus show a strong purple-red metachromasia after staining with basic dyes such as methylene or toluidine blue (WELLE, 1997). In German, the term “Mastzellen”, meaning “well fed cells” or
“fattened" is a misnomer as it is now fully appreciated that MC cytoplasmic granules are not phagocytosed, but are synthesised (SCHULMAN, 1993). The “Metachromasie”
or metachromasia was applied to describe the unusual alterations in colour that occur after staining of these cells with standard aniline dyes such as toluidine blue and Alcian blue (SCHULMAN, 1993).
The tissue origin of MC was unclear for a long time; initially the investigators believed that these cells were derived from T cells, fibroblasts or macrophages (CZARNETZKI et al., 1982; STERRY and CZARNETZKI, 1982). Nevertheless, KITAMURA et al.
(1978) demonstrated that murine MC arise from multipotent haematopoietic progenitors in bone marrow. More recently, a cell population as mast cell progenitors
(MCPs) was identified in adult mouse bone marrow, suggesting that these MCPs are derived from multipotential progenitors (MPPs), but not from common myeloid progenitors (CMPs) or granulocyte/macrophage progenitors (GMPs) (OKAYAMA and KAWAKAMI, 2006). Two models of mast cell-related haematopoiesis are suggested (Figure 2-1).
Figure 2-1 Two models of mast cell-related haematopoiesis (modified from CHEN et al., 2005 and ARINOBU et al., 2005)
(A) The model by CHEN et al. (2005) proposes that MCPs derive mainly from MPPs. (B) Another model described by ARINOBU et al. (2005) proposes that integrin β7-expressing GMPs in the bone marrow are the major source of these basophil mast cell progenitors (BMCPs), a population of bipotent progenitors for basophils and mast cells, which are supposed to go through the circulation to the spleen.
BMCPs expressing CCAAT/enhancer binding protein α or (C/EBPα) will differentiate into basophils whereas BMCPs without C/EBPα expression become MCPs.
Long-term hematopoietic stem cell (LT-HSC), short-term haematopoietic stem cell (ST-HSC), multipotential progenitor (MPP), common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte/erythrocyte progenitor (MEP), mast cell progenitors (MCP), granulocyte/macrophage progenitors (GMP), progenitors (Pro) megakaryocyte (MK).
Human tissue mast cells originate from cells generated in the bone marrow compartment as MCPs from pluripotent haematopoietic progenitor cells (KIRSHENBAUM et al., 1991; ISHIZAKA et al., 1993; FÖDINGER et al., 1994;
METCALFE et al., 1997). These progenitor cells circulate as mononuclear leukocytes lacking characteristic secretory granules and they have been isolated from peripheral blood as c-kit+, CD34+, CD33+, CD13+, CD117+, but rarely HLA-DR+ cells (ROTTEM et al., 1994; KEMPURAJ et al.,1999; KIRSHENBAUM et al.,1999).
Contrary to other haematopoietic stem cells, MC continue to express c-kit also when they are mature cells; an explanation for this fact is the great plasticity of these cells, i.e. the ability to change phenotype depending on the local environment (GURISH and BOYCE, 2002). Whereas most descendants of pluripotent stem cells like erythrocytes, neutrophils, basophils, or eosinophils do not leave the haematopoietic tissue until their differentiation is complete, MC in the vertebrates are widely distributed throughout the vascularised tissues and invade the connective or mucosal tissue as morphologically unidentifiable MCPs (KITAMURA, 1989; WELLE, 1997; KAWAKAMI and GALLI, 2002;
CHEN et al., 2005), especially adjacent to the surfaces which are exposed to the environment, such as skin, airways and gastrointestinal tract, where pathogens, allergens and other environmental agents are commonly found (KITAMURA, 1989;
METCALFE et al., 1997; KAWAKAMI and GALLI, 2002).
The MC, toguether with the dendritic cells (DCs), are well-positioned to be one of the first cells of the immune system to interact with antigens and allergens, invading pathogens or environmentally derived toxins (KITAMURA, 1989; GALLI et al., 2005a;
RYAN et al., 2007; GALLI and TSAI 2010). Once MC reach their destination, they have the ability to re-enter into the cell cycle and proliferate following an appropriate stimulation (KITAMURA, 1989; METCALFE et al., 1997; KAWAKAMI and GALLI, 2002; RYAN et al., 2007). The mechanisms for homing or recruitment of progenitor mast cells to peripheral tissues during physiological and inflammatory states are not well understood (DA SILVA et al., 2014). However, some investigations highlight the importance of some integrins, adhesion molecules, chemokines and their receptors, as well as cytokines and growth factors as important players in directed migration of MC to specific locations under physiological and inflammatory states (DA SILVA et al., 2014).
The expansion of the mature mast cells in tissues could theoretically be due to cell division of mature mast cells or maturation of MCPs that either are newly recruited or a result of cell division from a pool of MCPs. However, mature mast cells are generally not thought to undergo cell division and are considered terminally differentiated (DAHLIN and HALLGREN, 2015). Nevertheless, mature connective tissue mast cells in mice may have an ability to proliferate in vivo (DAHLIN and HALLGREN, 2015)
The development of mast cell numbers, as well as local changes in their tissue distribution and/or phenotypic characteristics, can occur during T helper (TH2) cell response, cronic inflammation and/or tissue remodelling (KITAMURA, 1989; RYAN et al., 2007; GALLI and TSAI, 2010). The magnitude and nature of the secretory responses of MC also occur under the influence of growth factors as stem cell factor (SCF) that is most important for MC development, survival as well as adhesion to the extracellular matrix (GURISH and BOYCE, 2002; OKAYAMA and KAWAKAMI, 2006;
BISCHOFF, 2007; GALLI et al., 2008). SCF binds to c-kit on the mast cell surface and especially in humans also has been shown to enhance IgE-dependent mediator release and suppression of mast cell apoptosis (BISCHOFF and SELLGE, 2002). But many growth factors, cytokines and chemokines can influence mast cell numbers and phenotype, including interleukin (IL)-3 which is especially important in mice; thus the character and responsiveness of MC at a given site in the body is highly dependent on unique blend of grown factors found in the immediate microenvironment (Table 2-1) (RODEWALD et al., 1996; ANDERSSON et al., 2009).
This ability of the MC lineage to generate individual subpopulations that differ in morphology, histochemical fixation reaction, functional properties and mediator contents (BIENENSTOCK et al., 1982; KITAMURA, 1989; METCALFE et al., 1997) probably confer to the MC a potentially complex repertory which can be activated with specific functions and can vary according to the requirements of individual physiological, immunological, inflammatory or other biological responses (METZ et al., 2007).
Table 2-1 Factors that affect mast cell development (OKAYAMA and KAWAKAMI, 2006)
Cytokine Receptor Effects on mouse MC Effects on human MC
SCF Kit
1- Directly stimulates proliferation of uncommitted progenitors 2- 2- Induces granulation and connective tissue proteases
1- Directly stimulates proliferation of uncommitted progenitors 2- Induces granulation and connective tissue proteases
IL-3 IL-3R
1- 1- Directly stimulates proliferation 2- of uncommitted progenitors
2- Directly promotes granule assembly
1- 1- Directly stimulates proliferation of uncommitted progenitors 2- 2- Directly promotes granule
assembly
IL-4 IL-4R
1- Cofactor for proliferation 2- 2- Promotes connective tissue 3- phenotype
Depends on the MC subtype or cytokine milieu
IL-5 IL-5R Not clearty characterised Cofactor for proliferation IL-6 IL-6R Induces MC development in
combination with TNF-α Cofactor for proliferation or inhibition
IL-9 IL-9R 1- Cofactor for proliferation
2- May induce mucosal proteases Cofactor for proliferation IL-10 IL-10R 1- Cofactor for proliferation
2- May induce mucosal proteases Not clearly characterised IFN-γ IFN-γR Inhibits proliferations Inhibits proliferations
NGF NGFR
1- Cofactor for proliferation 2- Promotes connective tissue
phenotype
Inhibits apoptosis in the presence of SCF TGF-β TGF-βR Inhibits proliferation Inhibits proliferation GM-CSF GM-SFR Inhibits proliferation Inhibits proliferation
TPO TPOR Not characterised Induces MC development
Interleukin (IL), stem cell factor (SCF), interferon-gamma (IFN-γ), nerve growth factor (NGF), transforming growth factor- β (TGF-β), recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO).
2.2 Mast cells in different species
2.2.1 Rodent mast cells
The variation in the phenotype of MC at different anatomical sites or during the course of an individual response has been called “mast cell heterogeneity” (KITAMURA, 1989;
METCALFE et al., 1997). ENERBÄCK (1966) reports that Hardy and Westbrook described tissue-dependent morphological differences in rat MCs as long ago as 1895, but that it was not until 1966 that these findings were further substantiated. The classification of “MC “heterogeneity” was based on phenotypic differences as connective tissue mast cells (CTMC), particularly of the skin and peritoneal cavity, and mucosa mast cells (MMC), particularly in the intestinal lamina propria (METCALFE et al., 1997; WELLE, 1997).
Based on these discoveries the terminology of the MMC and CTMC has been based on observations in rat tissues, but the same criteria can be applied to mast cell populations of mice (KITAMURA, 1989). These two mast cell populations can be differentiated using morphological, biochemical, and physiological criteria (BEFUS et al., 1987). MMC are smaller, variable in shape, may have a uni- or bilobed nucleus (PEARCE, 1988), have many but small granules and consist of a relatively soluble matrix containing chondroitinsulfate and little histamine (WELLE, 1997). MMC have also lightly stained granules which have glycosaminoglycans of lower sulfation with low evidence of heparin, and low levels or lack of serotonin (BIENENSTOCK et al., 1982) MMC contain rat mast cell protease II whereas CTMC contain rat mast cell protease I (PEARCE, 1988; WELLE, 1997).
In addition, MMC have a short life span. T cell-dependent proliferation of MMC has been shown to be mediated by IL-3, -4, -9, and -10 (MAYRHOFER, 1980; KITAMURA et al., 1993). Their anionic site is blocked by aldehyde (PEARCE, 1988) and therefore, special conditions of fixation and staining are required to reveal this cell type (WELLE, 1997). After fixation in some common formalin-based fixatives, the granules of the MMC may become resistant to metachromatic staining which acts through a diffusion barrier of a protein nature (PEARCE, 1988; KITAMURA, 1989; METCALFE et al., 1997; WELLE, 1997). However, when appropriately fixed sections are stained, rat MMC stain blue with alcian blue, while the granules of rat CTMC stain red with safranin (KITAMURA, 1989). Additionally, these MMC appear to have the remarkable property of storing IgE within their cytoplasm (MAYRHOFER and FISHER, 1979; GUY-GRAND et al., 1984; BEFUS et al., 1985; PEARCE, 1988; METACALFE et al., 1997).
On the other hand, CTMC contain considerably larger amounts of histamine and heparin which favour the metachromatic staining characteristic of the MC when they are coloured with a basic dye (WELLE, 1997). CTMC exhibit small or no T cell- dependent proliferation (MAYRHOFER and FISHER, 1979) and they are found mostly in skin, peritoneal cavity, and muscularis propria of the digestive canal, among other sites (KITAMURA,1989). Fibroblast-derived factors, e.g. SCF, appear to mediate the development of this subtype (KITAMURA et al., 1993). CTMC are larger than MMC, have few granules and they have a long life span and the IgE is only present on their surface (Table 2-2) (ENERBÄCK and WINGREN, 1980; BIENENSTOCK et al., 1982;
PEARCE, 1988).
A heterogeneous population of MC has been also reported in other species. These cells have been isolated from different sites in the same organ and same individual, presenting differences regarding to dye binding, granule component, type of protease, sensibility to degranulating agents and dependency to T lymphocytes (BEFUS et al., 1988; DVORAK et al., 1991; SCHULMAN, 1993; METCALFE et al., 1997).
Table 2-2 Rodent mast cell characteristics (METCALFE et al., 1997; WELLE, 1997)
Characteristics Peritoneal cavity mast cell Intestinal mucosa mast cell
Alternative names Connective tissue mast cell, typical mast cell
Mucosal mast cell, atypical mast cell
Size 10-20 µm 5-10 µm
Appearance Large, uniform shape, uniform-size granules
Small, variable shape, variable-size granules
Behaviour Non-migratory Migratory
Lifespan Long: half life >6 months Short: half life <40 days
Formaldehyde fixation Resistant Sensitive
Staining Safranin Alcian blue
T-cell dependence
in development No Yes
Protease content Chymase (RMCP-I) Chymase (RMCP- II) Proteoglycans
molecular mass Heparin 750–1000 kDa Chondroitinsulfate di B chain: 100-150 kDa
Histamine 10-20 pg/cell 1 pg/cell
5-Hydroxytryptamine 1-2 pg/cell < 0.5 pg/cell
Prostaglandin D2 + +
Leukotriene C4 - +
Activated by:
Fc,RI aggregation Yes Yes
Compound 48/80 Yes No
Substance P Yes No
Inhibited by sodium
romoglycate Yes No
Micrometers (µm), rat mast cell protease I (RMCP-I), rat mast cell protease II (RMCP-II), Kilodalton (kDa) Picogram per cell (pg/cell), receptor type I (Fc,RI), positive (+), negative (-)
2.2.2 Human mast cells
In contrast to mast cells of rats and mice, staining with dyes is not so useful for discriminating between human mast cell (HMC) subpopulations (IRANI et al., 1986;
KITAMURA, 1989). The classification of the different subtypes of HMC based only on their histochemical heterogeneity or on the presence or absence of heparin, result in formalin resistance and hence does not appear to be a useful marker for distinguishing different subtypes of human MC (WELLE, 1997). Nevertheless, it is possible to discriminate HMC based on morphological and biochemical determinations (IRANI et al., 1986). Most of the MC granules from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa were rimmed by incomplete scrolls forming parallel lamellae, centrally, amorphous granular material and/or grating/lattice-like structures occurred (scroll-poor morphology) (WEIDNER and AUSTEN, 1991). Conversely, MC in lung and intestinal mucosa have predominantly scroll-like structures (scroll-rich morphology) (WEIDNER and AUSTEN, 1991) (Table 2-3).
Some biochemical determinations demonstrated a contrast between these two mast cell groups (IRANI et al., 1986). One MC population contains only tryptase (MCT) and is also called tryptase-positive mucosa-associated MC and a second population containing tryptase, chymase, carboxypeptidase A and cathepsin G (MCTC), also called chymase- and tryptase-positive connective tissue-associated MC (BRADDING et al., 1995; IRANI and SCHWARTZ, 1994; IRANI et al., 1986). A rare third population of MC that contains only chymase (MCC) has also been reported, they are especially present in lung and bowel (WEIDNER and AUSTEN, 1991).
In human respiratory tract, the MCT phenotype is typically found on the mucosal surface, nasal and bronchial epithelium in rhinitis and asthma, respectively, the bronchial lamina propria in health and disease (BENTLEY et al., 1992). In gut, MCT
can also be found in small intestinal mucosa (CRAIG et al., 1988). On the other hand, the MCTC phenotype favours connective tissue such as normal skin (IRANI et al., 1986), the small intestinal submucosa (IRANI et al., 1987), the bundles of airway smooth muscle in patients with asthma (BRIGHTLING et al., 2002), and
atherosclerosis (JEZIORSKA et al., 1997). In axillary lymph nodes and lung as well as bowel mucosa and submucosa it has been found MC containing only chymase (WEIDNER and AUSTEN, 1993). In the respiratory tract, it has been reported that there was a marked variability in the distribution of MCTC and MCT and their content (WEIDNER and AUSTEN, 1993).
MC subtypes develop in the presence of specific factors and show a high degree of plasticity and a rapid conversion of cultured MCTC cells to MCT in coculture with human airway epithelium (HSIEH et al., 2005). There is a marked variability regarding the size and shape of cells from different sites, with large MCTC and small MCT evident particularly in pulmonary vessels. Except in alveoli, the shape MCTC was more circular than MCT in all compartments of the lungs (ANDERSSON et al., 2009). More than 90% of MC found in the epithelium and lumen of bronchioles and bronchi, as well as in the alveolar walls are of MCT phenotype, whereas MCTC are found in the subepithelial regions of the bronchi and in the connective tissue (HSIEH et al. 2005;
ANDERSSON et al., 2009).
Regarding the expression of the high-affinity IgE receptor and histidine decarboxylase has been shown that was relatively high in airway MCT and MCTC, but virtually absent in alveolar MC (ANDERSSON et al., 2009). Rennin was highly expressed in pulmonary vascular MCTC but not elsewhere (BRADDING et al., 2009). FGF and VEGF were expressed in airway, alveolar and pulmonary vascular MCT as well as in large airway and pulmonary vascular MCTC (IRANI et al., 1986; BRADDING et al., 1995).
Table 2-3 Characteristics of human mast cell subsets (METCALFE et al., 1997; WELLE, 1997)
Characteristic MCT MCTC
Neutral protease
(cellular content pg/cell) tryptase
tryptase, chymase, carboxypeptidase and cathepsin G
Granule morphology complete scroll- rich
grating/lattice complete scroll-poor
T-cell dependence Yes No
Cytokine profile IL-5, IL-6 IL-4
Distribution %:
Skin <1 >99
Lung
Alveolar tissue 91 8
Bronchi 78 10
Bronchial epithelium 100 0
Nasal mucosa 66 34
Tonsils 40 0
Small intestine
Mucosa 81 19
Submucosa 23 77
2.2.3 Canine mast cells
In contrast to humans and rodents, only few data exist regarding mast cells in dogs.
Canine MC heterogeneity based on formalin sensitivity has been identified in the skin, trachea, lung, intestine and mastocytomas (BECKER et al., 1985; KUBE et al., 1998).
Other authors found the classification system, which is useful in rodents, not to be appropriate for subtyping MC in dogs (OSBORNE et al., 1989; SOMMERHOFF et al., 1990b). After histochemical and biochemical evidence that canine MC contain trypsin- and chymotrypsin-like proteinases (GLENNER and COHEN, 1960; POWERS et al., 1985), the expression of different mast cell-specific serine proteases was used as a
further criterion for mast cell heterogeneity in dogs. SCHECHTER et al. (1988) demonstrated that canine skin MC contain tryptase and chymase, which have immunological and biochemical properties similar to human tryptase and chymase. In studies realised by KUBE et al. (1998) three subtypes were identified according to their content of the mast cell-specific proteases: MCT, MCC and MCTC. It was also observed that the predominant MC type differs within one location depending on the fixation technique. In general, a higher percentage of MCTC and MCC can be detected after fixation in Carnoy’s fluid (KUBE et al., 1998).
2.2.4 Feline mast cells
MC in healthy felines have not been extensively investigated so far, principal investigations have focussed on MC tumours in cats. In one study realised by NOVIANA et al. (2001), MC were examined in terms of distribution and protease activity in samples from stomach, duodenum, jejunum, ileum, colon, and rectum, as well as in samples of different other feline organs. NOVIANA et al. (2001) found that the number of mast cells vary, in each location, when the MC are visualised by metachromatic staining using Alcian Blue. Enzyme histochemical analysis revealed the existence of two MC subtypes where MCT were clearly identifiable in every organ examined, while MCC were predominantly observed in the ear (skin), tongue, spleen, and submucosa of the stomach and rectum (NOVIANA et al., 2001).
HARDER et al. (2008) examined MC of the gastrointestinal tract based on their protease content and their histochemical properties in healthy controls and cats with inflammatory bowel disease (IBD). The authors described that regarding all IBD cases higher counts of MCC, especially in the inflamed intestinal regions were present, whereas in unaffected areas an increase of the numbers of MCT was detected (HARDER, 2008).
2.2.5 Bovine and ovine mast cells
In contrast to the extended investigations of MC in humans and rodents, only few studies on MC exist for ruminants. Studies realised by HUNT et al. (1991) in skin and lung of bovines have demonstrated that the majority of the MC were formalin-sensitive.
HARRIS et al. (1999) found a significantly lower number of MC in the trachea of cattle when formalin fixation instead of Carnoy´s fluid and staining with toluidine blue and safranine O was used.
In the lower respiratory tract, i.e. trachea, right major bronchus and lung, of calves and cows that were fixed in either isotonic formal-acetic-acid or neutral buffered formalin MC were found at all levels of the tract, with the highest density in the major bronchi (CHEN et al., 1990a). In that study, the tissues from cows had significantly more MC than those from calves. It was also found that in tissue from calf airways fixed with isotonic formal acetic-acid significantly more mast cells were present compared with the same tissues fixed with neutral buffered formalin (CHEN et al., 1990a). Regardless of age and fixation methods, MC were located predominantly in the alveolar septa and in the lamina propria of the airway mucosae. In the trachea MC were also commonly encountered within the mucosa (CHEN et al., 1990a).
It has been proposed that the different requirements of mucosal mast cells (MMC) and connective tissue mast cells (CTMC) for fixation are related to cell maturity (FLINT, 1987). The MMC lying in the mucosal positions may never reach maturity due to constant exposure to antigens and resultant degranulation, while CTMC may shift gradually towards formalin resistance (CHEN et al., 1990a). MMC and their sensitivity to formalin fixation were also demonstrated in multiple organs from several species (ALDENBORG and ENERBÄCK, 1994; BEFUS et al., 1985; IRANI et al., 1986;
STROBEL et al., 1981). CHEN et al. (1990a) found a statistically significant higher density of MC in calf airways fixed with isotonic formal-acetic-acid fixative (pH 2.9) compared with the density of MC in the same tissues fixed with NBF. KÜTHER et al.
(1998) also reported that in the lungs of cattle there is a mixed population of both formalin-sensitive and -resistant MC although they did not observe a statistically
significant formalin sensitivity of MC in all tissue sites examined. This suggests that MC of various tissues exhibit different sensitivities to formalin fixation (KÜTHER et al., 1998).
Formalin-sensitive MC were observed in bovine and canine lungs (KUBE et al., 1998) and in ovine lungs (CHEN et al., 1990b). In cattle, the studies performed by HUNT et al. (1991) and HARRIS et al. (1999), based on different effects such as type of fixative, staining characteristics and histamine content, revealed a mix of MC subtypes in lung and trachea of adult animals. Those authors discussed that these differences are due to the presence of two subtypes of MC, one analogous to the classical CTMC and the other similar to MMC as described in the intestine of rats (WELLE et al., 1995). Like in humans and dogs, MC heterogeneity in cattle has been established based on their protease content. MCT were identified in cutaneous tissue and mast cell tumours in bovines (WELLE et al., 1995). Using a polyclonal rabbit anti-human tryptase antibody, it was demonstrated that there is a striking antigenic similarity of bovine tryptase to canine and human equivalents (WELLE et al., 1995).
Studies in different tissues of cattle such as forestomach, small bowel, uterus, lung, skin, and mesenterial lymph node using histochemical and immunohistochemical staining three mast cell subtypes were identified: MCT, MCTC and MCC (KÜTHER et al., 1998). Specifically in the lung the predominant subtype was MCT. Additionally it was found that formalin fixation affected the staining of MC granules (KÜTHER et al., 1998). Studies in skin, lung and intestine of bovines determined the presence of MCT
(JOLLY et al., 1999) and MCC (JOLLY et al., 2000). These two studies confirmed that the distribution of bovine MC more resembles that in human and dog tissues than that observed in mice and rats.
In studies of the respiratory tract of sheep, two subtypes of MC were found (CHEN et al., 1990b). One type was histochemically similar to connective tissue MC and formalin-resistant, the other type, which was similar to mucosal MC as found in the intestine of rats, was formalin-sensitive, and both types were similar to human lung
mast cells (CHEN et al., 1990b). Chymase is present in MC of the ovine gastrointestinal tract (HUNTLEY et al., 1986), and the number of chymase-positive MC increases after infection with nematodes (HUNTLEY et al., 1987).
2.3 Mast cell mediators
MC produce and release a heterogeneous group of mediators that differ in their potency and biological activities. They are both pleiotropic and redundant; that means each mediator has more than one function, and mediators may overlap in their biological effects (METCALFE et al., 1997). These mediators qualify of the following form:
-preformed mediators principally vasoactive amines, neutral proteases, proteoglycans and some cytokines and growth factors which are stored in the cytoplasmic granules and are released by fusion and exocytosis, by a procedure called degranulation.
-the de novo synthesis of proinflammatory lipid mediators, such as prostaglandins (PG), leukotrienes (LT).
-the synthesis and secretion of many growth factors, cytokines and chemokines (DVORAK et al.,1991; DVORAK et al.,1994; METCALFE et al.,1997; WILLIAMS and GALLI, 2000; SAYAMA et al., 2002; SIRAGANIAN, 2003; KINET, 1999; KAWAKAMI and GALLI, 2002; RIVERA, 2002; MARSHALL, 2004; GALLI et al., 2005a; METZ et al., 2007).
2.3.1 Preformed mediators of mast cells
Histamine
Histamine (H) is the single amine known to be stored in human mast cells (DVORAK et al., 1994), although mast cells of other species are known to store additional amines.
For instance, rodent MC also store serotonin (RINGVALL et al., 2008). In bovines, it has been demonstrated that skin MC contain twice as much histamine than lung MC
(HUNT et al., 1991). In humans, several pathogenic bacteria have been shown to increase the histamine release from human pulmonary and tonsil MC (BRZEZIŃSKA- BLASZCZYK et al., 1988; CHURCH et al., 1987). Similarly, bovine pathogens such as parainfluenza-3 virus and Pasteurella (P.) haemolytica have been shown to induce an increase of histamine release by bovine mast cell (OGUNBIYI et al., 1988; ADUSU et al., 1994; STAHL et al., 1996). The decarboxylation process of the amino acid histidine to form histamine takes place in the Golgi apparatus of MC and basophils (MACGLASHAN, 2003). Secretory granule exocytosis and release of histamine occurs rapidly after either immunological or non-immunological stimuli (LOWMAN et al., 1988). Within minutes of release, histamine is metabolised into methylhistamine, methylimidazole acetic acid, or imidazole acetic acid. It is thus likely that histamine usually influences events locally at or near the site of release (METCALFE et al., 1997).
Histamine exerts many pathological and physiological effects through its interaction with four histamine receptor subtypes: H1, H2, H3, and H4 receptors located on target cells that all belong to the family of G-protein-coupled receptors (LI et al., 2003).
Histamine binds to H1R that activates inositol-1, -4, -5 pathways, mobilising intracellular calcium, which induces the vascular endothelium to release nitric oxide and stimulate guanyl cyclase to increase the production of cyclic GMP in vascular endothelial cells (LI et al., 2003). H1-mediated actions include increased venular permeability, bronchial and intestinal smooth muscle contraction, increment of the nasal mucus production, widened pulse pressure, increased heart rate and cardiac output, flushing, and T cell neutrophil and eosinophil chemotaxis (MARSCHAL and JAWDAT, 2004; BRYCE et al., 2006). Other cells such as basophils and platelets also have some limited capacity to produce histamine (XU et al., 2006).
2.3.1.1 Proteases
The most abundant proteins stored in MC secretory granules are endopeptidases, which are released outside of the cell during exocytosis (CAUGHEY, 2007). The principal peptidases are tryptases, chymase, carboxypeptidase A3, and dipeptidylpeptidase I (cathepsin C). Tryptase is stored with chymase, carboxypeptidase, and a cathepsin G-like proteinase in the same granules, but resides in distinct macromolecular complexes (GOLDSTEIN et al., 1992). Although proteases (tryptase, chymase) are not classified as cytokines, they have many cytokine-like effects (KLEIJ and BIENENSTOCK, 2005). These cytokine-like activities often activate cells via protease-activated receptors (PARs), cleavage of which results in signal transduction (KLEIJ and BIENENSTOCK, 2005). The contribution of MC to host defence has been devoted to peptidases, and numerous studies suggest that these enzymes are important and even critical for host defence and homeostasis (CAUGHEY, 2007; TRIVEDI and CAUGHEY, 2010). Studies in rodents also established that the contribution of MC and their products, including peptidases, can be pro- or anti-inflammatory, depending on timing and context (TRIVEDI and CAUGHEY, 2010).
2.3.1.2 Tryptases
The tryptases are a subgroup of trypsin-family serine peptidases with shared enzymatic, structural, and phylogenetic features (CAUGHEY, 2006; CAUGHEY, 2007). They are trypsin-like target preferences, which is to say that they cleave peptide and protein substrates after lysine and arginine (CAUGHEY, 2007). Compared to trypsin, the tryptases can show major physical and behavioural contrasts such as intracellular pre-activation prior to secretion, membrane anchorage, and formation of toroidal, proteasome-like oligomers that resist circulating anti-peptidases (CAUGHEY, 2007). Therefore, they are able to hydrolyse extracellular peptides and proteins such as peptides like calcitonin gene-related peptide and vasoactive intestinal peptide (TAM and CAUGHEY, 1990; CAUGHEY, 2006; CAUGHEY, 2007). Most of the human MC, regardless of tissue location, make and store tryptases, which are the major proteins
in their secretory granules. It is the primary protease of MC of the lung, skin, and gastrointestinal tract (METCALFE et al., 1997).
Tryptases are packed so densely into secretory granules with other biomolecular parts that they form semi-crystalline structures (CAUGHEY, 2007). When MC degranulate in response to allergen-bound IgE or to non-immunological stimuli, tryptases are released toguether with histamine, heparin, proteoglycan and other granule constituents of mast cells (SAKAI et al., 1996; METCALFE et al., 1997; WELLE, 1997;
CAUGHEY, 2006). The release can be local as in bronchi in acute asthma or widespread as in systemic mastocytosis or anaphylaxis (CAUGHEY, 2007). Systemic release often is followed by large increases in blood levels of immunoreactive tryptase (CAUGHEY, 2006). These characteristic of the tryptase, especially of the β tryptase makes them useful as markers of mast cell activation in anaphylaxis and anaphylactoid reactions (CAUGHEY, 2006).
The tryptases have been described only in mammal mast cells, in which they show significant variability regarding to their form, activity and in some cases redundancy (CAUGHEY, 2007). In humans MCexpressing tryptase fall into two major groups:
membrane-anchored and soluble. The sole known member of the membrane anchored group is γ, also known as transmembrane tryptase (CAUGHEY, 2007). In humans, the soluble group includes α and β tryptase which are likely to contribute to circulating tryptase levels and to systemic features of anaphylaxis (CAUGHEY, 2006). The β tryptase making the major contribution, given that levels of immunoreactive tryptase change little in individuals without α genes (CAUGHEY, 2006).
The increase in total tryptase levels often, but not always, correlates with an increase in histamine levels (CAUGHEY, 2006). Mouse MC expresses at least 15 serine proteases designated as mouse MC protease (mMCP): mMCP-6, mMCP-7, mMCP- 11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MC (STEVENS and ADACHI, 2007), of which MCP-6 and MCP-7 are of the soluble group (CAUGHEY, 2006). In rat MC the
tryptase RMCP-6 and RMCP-7 have been reported (LÜTZELSCHWAB et al., 1997).
Bovine MC also express two tryptase types (GAMBACURTA et al., 2003), while dogs, pigs and mice express an additional MC tryptase-like enzyme named mastin (VANDERSLICE et al., 1989), which is not expressed in humans because the gene appears to have mutated into a pseudogene (CAUGHEY, 2007). β-tryptases may induce a responses from respiratory epithelial cells, including adhesion molecule expression and IL-8 release, thus attracting leucocytes and other inflammatory cells to the airways (CAUGHEY, 2007). Human tryptase βI plays a critical role in the antibacterial host defenses of the lung (HUANG et al., 2001).
2.3.1.2.1 Chymase and cathepsin G
MC produce two major types of peptidases with chymotrypsin-like activity, defined as a propensity for cleaving peptide and protein targets after aromatic amino acids, especially tyrosine and phenylalanine. These are the chymases and cathepsin G (CAUGHEY, 2007). Human chymase and cathepsin G belong to a family of immune serine peptidases whose human members include granzymes A, B, H, and M of cytolytic lymphocytes and natural killer cells, elastase and proteinase 3 of neutrophils and monocytes, and factor D of the complement system (TRIVEDI and CAUGHEY, 2010). Human chymase is a monomer of 30 kDa, is not affected by heparin, and is inhibited by biological inhibitors of serine proteases such as α1-antichymotrypsin and α2-macroglobulin (METCALFE et al., 1997). Chymases are expressed fairly selectively by mast cells. Only MC appear capable of accumulating chymases in secretory granules (CAUGHEY, 2007).
Chymases have various isoforms for which interspecies variations exist. Although not all of the chymases have been characterised chemically and catalytically, they demonstrate wide variations in their properties as charge, solubility, proteoglycan binding, glycosylation, catalytic efficiency, and regulated expression in mast cell subsets (WELLE, 1997). In humans, mast cell expression of chymase and cathepsin G is confined largely to MCC and MCTC subsets, which tend to be most abundant in the dermis of the skin (CAUGHEY, 2007). They are expressed little or not at all in MCT
cells, which are over-represented in mast cells populating mucosa, such as the gut (CAUGHEY, 2007).
Alveolar interstitium of human contain little chymase MC (IRANI et al., 1989), while in the pleura and vessel walls of airway they are relatively abundant (CAUGHEY, 2007).
Human cathepsin G is expressed in the same subset of cells that express chymase such as MCTC, however it is still not clear of its MC-specific roles because it is expressed in a variety of cell as dendritic cells and leukocytes including neutrophils, monocytes (RAPTIS et al., 2005; TRIVEDI and CAUGHEY, 2010). Studies in mice lacking cathepsin G suggest that it is important for host defense against injected bacteria and fungi (RAPTIS et al., 2005; TRIVEDI and CAUGHEY, 2010). Cathepsin G is generally a weaker enzyme and at the same time broader in peptidase specificity (CAUGHEY, 2007). In dogs and humans, MCC is the product of a single gene, CMA1 (CAUGHEY et al., 1993; CAUGHEY et al., 1997). In humans and rodents alike, cathepsin G is the product also of a single gene, CTSG (CAUGHEY, 2007).
It has been described that the mouse chymase MCP-1 is involved in the expulsion of intestinal worms (KNIGHT et al., 2000). MCP-4 supports in vitro evidence that chymases are principal activators of pro–matrix metalloproteinase 9 and help to limit colletion of extracellular matrix (TCHOUGOUNOVA et al., 2005). Studies releved that the use of chymase-selective inhibidor in a variety of animals suggest that chymases have also roles in promoting atherosclerosis, postinjury vascular stenosis, ventricular hypertrophy, myocardial infarction and fibrosis, and peritoneal adhesions (TRIVEDI and CAUGHEY, 2010). It has been reported that both enzymes, chymase and cathepsin G, also can be pro-inflammatory (CAUGHEY, 2011) and that they can stimulate secretion by cultured serous cell gland cells (SOMMERHOFF et al., 1990a).
2.3.1.2.2 Carboxypeptidase
Carboxypeptidase A is a specialised metallo-exopeptidase that appears to be exclusive for MC (GOLDSTEIN et al., 1987). It is a protein of 35 kDa that acts as a hydrolytic enzyme at neutral pH to cleave the peptide and ester bonds of the amino end of the COOH-terminal of aromatic amino acids in a manner similar to that of chymase (METCALFE et al., 1997). In humans, carboxypeptidase A3 not only is expressed in the same MCTC that express tryptases and chymase, but also appears to co-segregate with chymase within granules and to be released as a proteoglycan- bound complex (GOLDSTEIN et al., 1992). Mouse MCTC express carboxypeptidase A (MC-cpa) and additionally MC protease 2 (MCp-2), MCp-4, MCp-5, MCp-6, and MCp-7.
Both MC-cpa and Mcp-5 are bound to heparin, which itself is an essential component in these complexes as shown in mast cells lacking sulfated heparin (FEYERABEND et al., 2005). Despite the close linkage of these enzymes, studies in mice suggest important independent MC carboxypeptidase functions, such as hydrolytic inactivation of potentially lethal endogenous peptides such as neurotensin and endothelin (TRIVEDI and CAUGHEY, 2010) and detoxification of sarafotoxin-class snake venoms. Toxic doses of these peptidases are more likely to be lethal in the absence of MC carboxypeptidase. The roles played by this enzyme in lung disease remain to be determined (TRIVEDI and CAUGHEY, 2010).
2.3.1.3 Proteoglycans
Proteoglycans (PGs) conform a heterogeneous group of glycoproteins that all contain one or more glycosaminoglycan (GAG) chains attached to their respective “core”
protein (DAVID and BERNFIELD, 1998; BERNFIELD et al., 1999). PGs are present in various extracellular locations including cartilage (WATANABE et al., 1998) and basement membranes (TIMPL, 1993). PGs that are found inside cells can either be
“part time” intracellular PGs, i.e. being present in the intracellular space (e.g. during uptake or secretion processes) or be destined for an intracellular compartment, i.e.
being “committed” intracellular PGs (ÅBRINK et al., 2004). The committed intracellular PGs are predominantly found in the secretory granule of haematopoietic cells such as MC, cytotoxic T lymphocytes, neutrophils, and platelets, where they have an important function in binding to various secretory granule compounds, thus facilitating their storage (ÅBRINK et al., 2004).
Proteoglycans heparin and chondroitin sulfate E have been associated with human MC. Additional proteoglycans have been identified in mast cells of other species, such as chondroitin sulfate di-B in the rat (METCALFE et al., 1997). The connective tissue- type MC predominantly synthesises GAG chains of heparin type, and the mucosal MC subtype synthesises chondroitin sulfate chains (ÅBRINK et al., 2004). The metachromatic staining of mast cell granules is due to sulfated, anionic proteoglycans such as heparin and chondroitin sulfate (AVRAHAM et al., 1989; STEVENS et al., 1987). Heparin may serve to stabilise the multimeric complex of histamine, proteoglycan, and activate neutral proteases within the secretory granule (PEJLER et al., 2010). With granule exocytosis, heparin retains many of the proteases in the macromolecular complex (SCHWARTZ et al., 1981; GOLDSTEIN et al., 1992;
GHILDYAL et al., 1996). Heparin also functions as an anticoagulant, inhibits the complement cascade and markedly potentiates the action of angiogenic factors such as FGF (OSCARSSON et al., 1989; GOSPODAROWICZ and CHENG, 1986).
Chondroitinsulfate-E, a highly sulfated proteoglycan like heparin, has kinin pathway activation effects and protease-stabilising functions (THOMPSON et al., 1988).
2.3.1.4 Newly synthesised lipid mediators
The activation of MC not only causes the release of preformed granule-associated mediators, but initiates de novo synthesis of lipid-derived substances (METCALFE et al., 1997). When activated by specific antigen, complement, or other transmembrane stimuli, MC generate three eicosanoids: prostaglandin D2 (PGD2) (MURRAY et al., 1986), leukotriene (LT) B4 (MACGLASHAN et al., 1982), and LTC4 the parent molecule of the cysteinyl leukotrienes (cysLTs) (PETERS et al., 1984). MC are using membrane phospholipids as a source of arachidonic acid for the synthesis of PG, LT,
and platelet activating factor (PAF). For that reason these mediators are referred to as eicosanoids (BOYCE, 2007).
PGD2 is the principal prostaglandin produced by MC and it has been recognised in bronchial or nasal secretions after allergen challenge (MURRAY et al., 1986; KNANI et al., 1992). PGD2 requires the conversion of arachidonic acid to the intermediate PGH2 by prostaglandin H synthase. After PGD2 is synthesised, a PG transporter protein mediates its export to the extracellular space where it exerts diverse receptor- mediated effects in allergic and immune responses (BOYCE, 2007).
The eicosanoids LTC4, PGD2 and LTB4 act through specific cognate G-protein- coupled receptors (GPCRs) and, in concert with other mediators, induce changes in vascular permeability and smooth muscle constriction, patterns of antigen presentation, effector cell recruitment, and stromal cell activation (BOYCE, 2007). In contrast to the preformed MC mediators, which are released immediately by exocytosis, lipid mediators are de novo synthesised and released from activated MCs rather than stored, and exert autocrine and paracrine functions (MOON et al., 2014).
Lipid mediators also enhance lysosomal enzyme release, and augment superoxide anion production. LTB4 has also been suggested as a modifier of lymphocyte function by inducing specific suppressor lymphocytes and augmenting human natural cytotoxic cell activity (METCALFE et al., 1997).
PAF has been detected after activation of mouse bone marrow-derived MC, rabbit basophils, and human MC through the action of the enzyme phospholipase A2
(TRIGGIANI et al., 1990; METCALFE et al., 1997). PAF acts through a specific receptor to chemoattract eosinophils, neutrophils, monocytes, and macrophages (CZARNETZKI et al., 1982; PRPIC, et al., 1988; OKADA et al., 1997) and to stimulate macrophage cytokine production (THIVIERGE and ROLA-PLESZCZYNSKI, 1992).
The PAF is able to induce bronchoconstriction and vasopermeability at tissue level (SMITH, 1991). Endothelial PAF interacts with neutrophils leading to changes in their
integrin expression with binding to ICAM-1 on the endothelial cell, thereby promoting neutrophil attachment and transmigration (ZIMMERMAN et al., 1996).
2.3.1.5 Synthesis and secretion of growth factors, cytokines and chemokines
The mast cell is increasingly recognised as a source of multifunctional cytokines that may participate in the recruitment and activation of other cells in the inflammatory microenvironment (GORDON and GALLI, 1990). MC with different secretory granule protease phenotypes exhibit differences in their cytokine profiles, thereby indicating further heterogeneity depending on tissue localization (BRADDING et al., 1995). The mast cell is a source of TH2-type cytokines, and MC numbers as well as local changes in their tissue distribution and/or phenotypic characteristics can occur during TH2 reactions (KITAMURA, 1989; RYAN et al., 2007).
These TH2 responses are associated with cytokines such as IL-4 (BISCHOFF et al., 1999), IL-9 (HÜLTNER et al., 1990), IL-10 (RENNICK et al., 1995) and IL-13 which is also named mast cell growth factor (HU et al., 2007). IL-10 is associated with the transforming growth factor beta 1 (TGF-β1) (KITAMURA, 1989; AUSTEN and BOYCE, 2001). It has also been reported the association of stem-cell factor (SCF) and IL-4 (TORU et al., 1998). IL-4 is also implicated in the development and up-regulation of TH2 cells, is required for the biosynthesis of IgE, and stimulates the production of cysteinyl LT in a positive feedback loop that results in reactive mast cell hyperplasia (JIANG et al., 2006).
Human lung MC also release IL-5 when activated ex vivo through the high affinity IgE receptor, Fc (fragment crystallisable) epsilon RI (FcεRI). IL-5 is a potent eosinophil maturation and cytoprotective factor (JAFFE et al., 1995). IL-3 is especially important in mice because it stimulates proliferation of BMMC and survival of CTMC (TSUJI et al., 1991). (NILSSON et al., 1995; NIGROVIC et al., 2007). Tumour necrosis factor- alpha (TNF-α,) was the first cytokine localised in human MC (GORDON and GALLI, 1990). Some TNF-α may be constitutively stored in the granules, but the vast majority is induced with immunologic activation (GORDON and GALLI, 1991).
Studies using mast cell deficient mice have demonstrated the importance of the mast cell-derived TNF-α in neutrophil recruitment in bacterial peritonitis, in protection from endotoxic shock, and in driving the initial immune response by directing antigen presenting cell (dendritic cell) migration to local draining lymph nodes (ECHTENACHER et al., 1996; MALAVIYA et al., 1996b; SUTO et al., 2006). MC derived TNF-α also up-regulates expression of endothelial adhesion molecules such as ELAM-1 and ICAM-1 facilitating adhesion and ingress of eosinophils and T cells to the inflammatory locus (KLEIN et al., 1989; WEGNER et al., 1990). TNF-α derived from mast cells also may underly lymph node hypertrophy in response to bacterial inflammation (MCLACHLAN et al., 2003). Transcripts for transforming growth factor (TGF)-β, a potent fibroblast activator and proliferation factor, are present in human MC from fibrotic lung and rheumatoid synovium (QU et al., 1995). MC release also a number of chemokines, including those that have two N-terminal cysteine residues separated by one non conserved residue (ALAM, 1997; GURISH and BOYCE, 2006).
2.4 Mast cell activation
It has been recognised for long time the role of MC as key effector cells in IgE- associated immediate hypersensitivity and allergic reactions, as well as their activation during certain types of parasitic infections (MALAVIYA et al., 1994; METCALFE et al., 1997; KAWAKAMI and GALLI, 2002; MARSHALL, 2004). However, due to remarkable variety of mediators derived from these cells and recognition that MC release these mediators in response to stimulation by different stimuli, and changes in MC numbers in various anatomic sites, led to infer that they have an important contribution in host defence as regulation of the innate and adaptive immune responses and other aspects of modulation of many physiological processes (METCALFE et al., 1997; MEKORI and METCALFE, 2000; KAWAKAMI and GALLI, 2002; MARONE et al., 2002; MARSHALL, 2004; GALLI et al., 2005b).
2.4.1 Direct mast cell pathogen interactions
The capacity of MC to recognise and react to pathogens depends on a combination of direct and indirect receptors that detect the presence of harmful pathogens or other
“danger” signals and trigger the subsequent rapid and particular reaction (MARSHALL, 2004). Direct interactions between pathogens and immune effector cells are essential for the generation of early innate immune responses, as well as the generation of an appropriate acquired immunity (GALLI and TSAI, 2010).They are thought to be most significant in the context of primary infection. Consistent with their role as sentinels, MC have a wide variety of cell-surface receptors that can interact directly with pathogens (MARSHALL, 2004).
Toll-like receptors (TLRs): It has been demonstrated that HMCs can distinguish between different pathogen-associated signals and generate highly selective responses to bacterial, fungal and viral infections (MARSHALL, 2004). Treatment of mice with Gram-positive bacterial cell-wall component peptidoglycan (PGN) leads to the production of a different range of cytokines, including IL-4, IL-5, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as the induction of degranulation (MARSHALL, 2004). Lipopolysaccharides (LPS) stimulate the selective production of inflammatory cytokines, such as TNF and IL-6 without concurrent degranulation (MARSHALL, 2004).
Toll-like receptor (TLR) families are transmembrane proteins containing repeated leucine-rich motifs in their extracellular portions similar to other pattern recognition proteins of the innate immune system, and a cytoplasmic domain that is homologous to the signaling domain of the IL-1R (ANDERSON, 2000). The TLRs can recognise pathogen-associated molecular patterns (PAMPs) (VARADARADJALOU et al., 2003).
It has been demonstrated that HMCs express TLR1, TLR2, TLR5, TLR4, TLR6, TLR7 and TLR9 (KULKA et al., 2004) and that the response is specific for each PAMP.
Activation through different TLRs leads to individual patterns of cytokine production (e.g. TNF-α, IL-6, IL-13, IL-1β) with or without degranulation (MARSHALL, 2004;
SUPAJATURA et al., 2002; MCCURDYet al., 2003;VARADARADJALOU et al., 2003).
