30 Mast Cells in the Skin

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30 Mast Cells in the Skin

M.K. Church

Mast cells are a heterogeneous group of tissue-dwelling cells with roles in conditions as diverse as allergy, para- site infestation, inflammation, angiogenesis, and tissue remodeling. The cells were named Mastzellen in 1876 by Paul Ehrlich because they looked stuffed (German

„gemästet“, „Mast“) and he believed that the intracellular granules, which appeared purple in color when stained with aniline blue dyes, contained phagocytosed materials or nutrients [1]. This change in color, or metachromasia, we now know to represent the interaction of the dyes with the highly acidic heparin contained within mast cell granules.

The life of the human mast cells begins in the bone marrow as a pluripotent stem cell which enters the bloodstream early on in its development (Fig. 30.1).

Studies of culturing mast cells from cord blood suggest that the precursors are a CD34⁺/CD38⁺/HLA-DR^–population of cells [2]. In vivo in mastocytosis, immature mast cells have been recognized as mononuclear cells

Fig. 30.1. Development of mast cells and granulocytes. The right-hand side of the diagram represents a mucosal surface.

Note the close association of MCTCwith nerves and blood vessels and MCTwith mucosal epithelium

that both express mRNA for SCF and have SCF receptors (SCFR, CD 117) on their cell membranes [3]. From the blood the precursors migrate into the tissues where, under the influence of local microenvironmental factors, they undergo their final phases of differentiation and maturation into recognizable mast cells complete with cytoplasmic granules and receptors for IgE.

Again, studies of culturing mast cells from cord blood suggest that stem cell factor (SCF) and IL-6 are important for mast cell maturation [2, 4]. It is pertinent at this stage to distinguish mast cells from basophils, which were originally thought to be circulating mast cells, but are actually related more closely to eosinophils, devel- oping in the bone marrow from granulocyte precursors and entering the circulation only when fully mature [5].

Mast cells are distinguished immunocytochemically by their neutral protease content, the MCTphenotype containing only tryptase and the MCTCphenotype containing both tryptase and chymase [6]. Initially, these respective subtypes were suggested to be the equiva- lents of the „mucosal“ and „connective tissue“ previously described in experimental animals. However, it is now realized that variable amounts of both mast cell subtypes are present within any given tissue, their relative abundance changing with disease. For example, in allergy MCT, which appear to be „immune system- related“ mast cells with a primarily role in host defense, increase in numbers at mucosal surfaces and allergic foci. Conversely, increased numbers of MCTC, which appear to be „nonimmune system-related“ mast cells with functions in angiogenesis and tissue remodeling rather than immunological protection, are associated with fibrosis. However, it should be remembered that both phenotypes express Fc5 RI and may, therefore, participate fully in IgE-dependent allergic reactions.

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Mast cells are relatively abundant in human skin, being found in the greatest density in the papillary dermis and the superficial dermal zone immediately below the dermal-epidermal junction [7]. They are concen- trated particularly around dermal nerve endings and blood vessels [8, 9] and are, therefore, ideally situated to influence the function of both. Normal skin contains around 7,000 mast cells per mm³[10, 11] which equates to a histamine content of 12 – 20 ng/mg tissue [12, 13].

Skin mast cell numbers increase dramatically in sev- eral diseases. For example, the histamine content of the skin in Beh¸cet’s disease is reported to be twice that of normal skin [13] while mast cell numbers are 10-fold higher in urticarial lesional skin [14] and are even higher in urticaria pigmentosa [15]. In a study using antibodies to tryptase and chymase, the number of mast cells in the superficial dermis of mastocytosis lesions was 40,985 ± 21,772 /mm³(mean ± SD) com- pared with 7347 ± 2973 /mm³in normal skin. Further- more, the cells in skin lesions of mastocytosis were exclusively MC_TC [10]. Mast cell hyperplasia is also associated with skin tumors such as basal cell carcino- ma [16] and melanoma [17, 18].

Although histamine has been found in significant amounts in the epidermis [19, 20], mast cells are rarely observed in this layer in normal skin. Whether this indicates histamine synthesis by keratinocytes, as indi- cated by murine studies [21], or the ability of keratinocytes to take up histamine is not clear.

30.1

Mast Cell Activation

Mast cells may be activated by both immunological and nonimmunological mechanisms (Fig. 30.2). To facili- tate immunological activation, human mast cells have

Fig. 30.2. Activation sites of the human skin mast cell

10⁴to 10⁵high affinity (Ka ~ 10⁹/M) receptors (Fc5 RI) for immunoglobulin E (IgE) on the plasma-membrane [22, 23]. Mast cell Fc5 RI is composed of four subunits, an IgE-binding [ -chain, a q -chain, and two * -chains [24]. The presence of the signal-amplifying q -chain [25] in the heterotetrameric [ q * * mast cell and baso- phil receptor complex distinguishes it from the [ * * heterotrimeric receptor of dendritic cells and mono- cytes [23]. Cross linkage of these receptors by multiva- lent allergen stimulates phosphorylation of immunore- ceptor tyrosine activation motifs (ITAMs) [23] and initiation of the biochemical cascade which leads to the release of both preformed and newly generated mediators.

Nonimmunological activation, which appears to be unique among human mast cells, may be initiated in two ways, by complement fractions and by basic secretagogues. Human skin mast cells alone express on the plasma membrane CD88, the receptor for the anaphy- latoxin C5a, allowing them to be activated in complement-mediated disease [26, 27]. Also, skin mast cells alone respond to a variety of basic nonimmunological secretagogues, including neuropeptides, compound 48/80, and drugs such as morphine, codeine, and muscle relaxants [28, 29]. These agents stimulate a common activation site on the mast cell membrane which is associated with a pertussis toxin-sensitive G protein [30, 31]. The ability of human skin mast cells, but not those from other tissues, to respond to anaphylatoxins and basic nonimmunological secretagogues explains the flushing reactions observed in sensitive individuals in the absence of overt rhinorrhea or bronchoconstric- tion. Such responses may also be involved in physical urticarias.

In vitro studies of human isolated skin mast cells have shown distinct differences between immunological and nonimmunological mast cell activation. IgE- dependent activation is relatively slow, taking around 5 – 6 min to reach completion, and requires the presence of extracellular calcium. It is a „complete“ stimulus in that it causes release of preformed mediators and initiates the synthesis of the eicosanoids prostaglandin D2and leukotriene C4. In contrast, stimulation of mast cells with basic secretagogues and C5a causes a much more rapid release of histamine, being complete within 30 sec. This release mechanism in which G protein activation leads to subsequent activation of phospholipase C to increase in intracellular inositol triphosphate levels, proceeds in the absence of extracellular calcium,

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Histidine

Histamine Histidine decarboxylase

N-Methylhistamine Imidazole acetic acid Diamine oxidase (histaminase) Histamine N-

methyltransferase

(30%) (70%)

calcium mobilization from the endoplasmic reticulum being sufficient to support degranulation. Also, this is an „incomplete“ stimulus in that histamine release is accompanied by negligible eicosanoid generation [30].

Despite these biochemical and temporal differences, degranulation induced by both secretagogues is indis- tinguishable under the electron microscope, proceed- ing by compound exocytosis [32]. From these data it seems likely that IgE-dependent and neuropeptide stimulation of human skin mast cells activate distinct biochemical pathways which eventually merge to stimulate exocytosis of their preformed granule-associated mediators.

30.2

Mast Cell Mediators

30.2.1

Early Phase Mediators

The secretory granule of the human mast cell contains a crystalline complex of preformed inflammatory mediators within a matrix of heparin proteoglycan.

The granule-associated mediator most readily associated with the mast cell is the simple diamine histamine.

Histamine is synthesized in the Golgi apparatus of mast cells by decarboxylation of the amino acid, histidine, under the influence of histidine decarboxylase (Fig. 30.3). Following synthesis, histamine becomes ionically bound to acidic residues of the glycosaminoglycan (GAG) side chains of heparin proteoglycan [33].

Histamine is present within the granules at ~100 mM, equivalent to about ~4 pg/cell in skin mast cells [34]. In

Fig. 30.3. Synthesis and catabolism of histamine

the extracellular environment, the effects of histamine are normally of relatively short duration as it is rapidly metabolized, usually within 1 – 2 min, by histamine-N- methyltransferase (~70 %), and by diamine oxidase (histaminase) (~30 %) (Fig. 30.3). Interestingly, reduced diamine oxidase activity has been associated with recurrent urticaria [35].

Histamine exerts many effects pertinent to the immediate phase of allergic responses, including initiation of the wheal and flare response initially described by Thomas Lewis in 1927 [36] (Fig. 30.4). The wheal reaction is a direct effect of histamine acting on H₁- receptors firstly on local vascular smooth muscle [37]

to cause vasodilatation and then on endothelial cells [38] of postcapillary venules to allow the exudation of plasma proteins [39]. The action of histamine on sen- sory C-fibers also initiates the flare and itch responses [40 – 42].

The other „early phase“ mediators released from the skin mast cell are PGD₂and LTC₄[43]. While there is little evidence for a role of the former in skin inflammation, the success, particularly in individuals with a variant LTC₄synthase allele [44], of leukotriene receptor antagonists in atopic dermatitis [45] and urticaria [46] suggests that this eicosanoid may be more important in skin disease than considered previously. It should be emphasized at this point that eosinophils are probably the major producers of LTC4in asthma. The findings that there are large deposits of extracellular eosinophil granule proteins in the skin in both urticaria [47] and atopic eczema [48, 49] and raised circulating levels of eosinophil major basic protein in atopic eczema [50] suggest that eosinophils may also make

Fig. 30.4. Wheal and flare in human skin

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Mast Cell

MC_T

Tryptase Tryptase

MC_TC

Chymase Carboxypeptidase

Cathepsin G a significant contribution to allergic inflammation,

and probably to LTC₄generation, in the skin.

30.2.2 Proteoglycans

Proteoglycans are macromolecules which comprise a protein core to which glycosaminoglycan (GAG) side chains are covalently bound (Fig. 30.5). The diverse biological roles of proteoglycans range from simple mechanical support functions to effects on cell differentiation and proliferation, cell adhesion and motility, and tissue morphogenesis [51]. In the human mast cell the dominant proteoglycans is a relatively low molecular mass species of heparin (~ 60 kDa) with small amounts of chondroitin E [52, 53]. The highly anionic nature of the GAG side chains is responsible for many of the functions of proteoglycans, such as the noncova- lent binding of the mast cell proteases, tryptase, chymase, and carboxypeptidase A, tryptase being in a complex distinct from that of the other two enzymes [54].

A wide variety of biological functions have been attributed to heparin as an extracellular mast cell mediator. Probably the most widely recognized is its capacity to interfere with blood coagulation by enhancing the ability of antithrombin 3 (AT-III) to inhibit the serine proteases involved in the coagulation cascade. Heparin neutralizes the cytotoxic actions of the eosinophil-derived basic proteins [55] and has anticomplementary activity [56]. Heparin also modu- lates the function of the mast cell proteases. It stabilizes

Fig. 30.5. Heparin and chondroitin sulfate proteoglycans

tryptase in its biologically active tetrameric form at neutral pH [57]. As there appear to be no endogenous inhibitors of tryptase, it is likely that it is restricted to having only very local effects because, as it diffuses away from heparin, it rapidly dissociates into inactive monomers. Heparin also enhances the activity of chymase [58].

30.2.3

Neutral Proteases

Neutral proteases comprise the majority of the protein of the secretory granules of human mast cells and represent the major mediators of this cell type on a weight basis, MC_TCcontaining up to 60 pg proteases per cell (Fig. 30.6). Until recently, the study of these abundant mast cell products has been neglected, but they are now attracting attention as important mediators of allergic disease and as potential targets for therapeutic inter- vention.

The major mast cell protease, tryptase, originally identified by Schwartz in 1981 [59], is a ~130-kDa tetrameric serine protease which is stored in a fully active form complexed with heparin in the granule [60, 61].

Tryptase is secreted in proteoglycan complexes with molecular weights of 200 – 250 and 400 – 560 kDa [54].

The large size of these active complexes will severely limit diffusion away from sites of mast cell activation and helps to explain why increases in circulating levels of tryptase seem to occur only following the massive mast cell activation of anaphylactic shock. In the extracellular environment, the neutral pH allows tryptase to

Fig. 30.6.

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become enzymatically functional. The properties of tryptase pertinent to the skin include: cleavage of the vasodilator neuropeptide, calcitonin gene-related pep- tide (CGRP) [62]; a kallikrein-like activity [63]; and, cleavage of matrix components, including 75-kDa gela- tinase/type IV collagen, fibronectin and type VI collagen [64, 65], which would be in keeping with participa- tion in processes of tissue destruction and remodeling.

Tryptase also has mitogenic activity for fibroblasts [66].

Chymase is a 30-kDa monomeric protease stored in the same secretory granules as tryptase in the MC_TC subset of mast cells [67]. Like tryptase, chymase is present in a catalytically active form in the granule [68].

Chymase is inhibited by the circulating inhibitors -an- tichymotrypsin and ₁-antitrypsin [69], an absolute necessity when it is realized that this protease can cleave angiotensin I to angiotensin II more effectively than angiotensin-converting enzyme [70]. Chymase degrades the neuropeptide neurotensin [71], but not substance P or VIP [70]. A role for chymase in tissue destruction and remodeling is also suggested from its ability to activate stromelysin and interstitial collage- nase, to convert procollagen I to collagen-sized frag- ments [72], and to degrade type IV collagen [73]. Fur- thermore, incubation of fresh human skin with human chymase can result in extensive separation of the epidermal-dermal layers [74].

Two other proteinases, carboxypeptidase and cathepsin G, have been associated with the MCTCsub- set of human mast cells [75, 76]. Carboxypeptidase is a unique 34.5-kDa metalloproteinase which removes the carboxyl terminal residues from a range of peptides, including angiotensin, leu⁵-enkephalin, kinetensin, neuromedin N, and neurotensin. Cathepsin G is a chy- motryptic enzyme with a structure seemingly identical to neutrophil cathepsin G. When mast cells are activated, chymase, carboxypeptidase, and cathepsin G are released together in a 400 to 500-kDa complex with proteoglycan and are likely to act in concert with the other enzymes to degrade proteins.

Although few formal studies have been performed on the part played by mast cell proteases in inflammatory diseases of the skin, it is clear that they have a potential role. As early as 1978, Wintroub and colleagues [77] reported evidence of mast cell degranulation in lesions of bullous pemphigoid and suggested a causal relationship. This suggestion was reinforced by the studies of Briggman and colleagues [74], who

showed that the epidermal-dermal junction is highly susceptible to neutral serine proteases located in mast cells and neutrophils. Finally, the finding of IgE autoantibodies in bullous pemphigoid suggested a way in which mast cells may be activated in the disease [78].

Another area of research which may involve mast cell proteases is the recently made association between Netherton’s syndrome, a congenital skin disorder whose symptoms include severe ichthyosis, and polymorphisms in SPINK5, a gene encoding for the serine protease inhibitor LEKTI [79, 80]. In vitro, LEKTI has been shown to inhibit the serine proteases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin [81]. Whether or not LEKTI inhibits mast cell tryptase is not yet known.

However, the findings that polymorphisms of SPINK5 are also associated with atopic dermatitis, asthma, and high IgE levels [82 – 84] suggests an axial role for proteases in allergic disease.

30.2.4 Cytokines

The concept that the mast cell, by the generation of proinflammatory cytokines, plays a pivotal role in the stimulation and maintenance of allergic inflammation is now well established. In addition, human purified lung mast cells incubated with SCF and anti-IgE express mRNA for IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, GM-CSF, and TNF[ but not IL-2 or IFN * [85].

The first association of TNF[ with mast cells was made in 1980 [86] while examining the antitumor effects of murine mast cells. Initial studies relating to human mature mast cells showed that skin explants in vitro stimulated with mast cell secretagogues produced a factor that stimulated endothelial-leukocytic adhesion molecule-1 (ELAM-1) expression on endothelial cells. Inhibition of this response by specific antibodies strongly suggested that it was TNF[ [87]. TNF [ is stored preformed within the granules of human skin mast cells and is released rapidly after initiation of an allergic response [88]. In contrast, macrophages and lymphocytes, which also produce large amounts of TNF[ , have little or no capacity to store it and thus only generate it slowly after transcription. Functionally, TNF[ is a key cytokine in allergic inflammation, being required to activate NF-κB, a transcription factor that upregulates the expression of mRNA for TNF[ , GM- CSF, IL-8, IL-2, IL-6, E selectin, ICAM-1, and VCAM-1

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in a variety of cells including endothelial, epithelial, and mast cells [89, 90].

Of the other NF-κB-associated both IL-8 and GM- CSF have also been demonstrated to be synthesized and stored by mast cells [91, 92].

The other group of cytokines are the so-called TH2 cytokines, including IL-4, IL-5, and IL-13 which are responsible for promoting and maintaining many aspects of the allergic response [93, 94].

IL-4, the cytokine largely responsible for stimulating and maintaining Th2 cell proliferation and switch- ing the B cell to IgE synthesis [95], is seen in approxi- mately 80 % of mast cells, both MC_Tand MC_TC,of the bronchial mucosa [96, 97]. Furthermore, immunogold electron microscopy applied to ultrathin sections of human purified dispersed mast cells and biopsies has shown that IL-4 is localized to the secretory granules [98, 99]. The ability of mast cells to store IL-4 and release it upon stimulation has been suggested to be an important initiating event in allergic inflammation by stimulating the expansion of the repertoire of TH₂ cytokine-producing cells in the local microenviron- ment [100].

IL-13, a cytokine with many properties in common with IL-4, has been demonstrated to be associated with mast cells in conjunctival biopsies from patients with seasonal allergic conjunctivitis [101] and be synthesized by mast cells in vitro [102, 103].

IL-5, which is crucial for the maturation, activation, and survival of eosinophils, has also been localized to human mast cell [93, 104]. Unlike IL-4, IL-5 is present only in 10 % of mast cells found to be IL-5 positive and is restricted to MCT[105], the subset of mast cells that are under T cell control and that increase in numbers in parasitic infestation and at sites of chronic allergic inflammation. Furthermore, in vitro studies have shown that IgE-dependent stimulation of human lung mast cells induces IL-5 production [106, 107] which may be suppressed by dexamethasone [108].

30.3 Conclusions

Mast cells possess the armory to participate actively in many forms of inflammation in the skin. Perhaps the most obvious associations are with urticaria, which, in terms of appearance, is the clinical counterpart of the wheal and flare response induced by the intradermal

injection of allergen, codeine, or histamine [40]. Per- haps the most direct evidence for mast cell involve- ment in urticaria is the study in cold-induced urticaria performed by Anderson and colleagues [109]. They inserted microdialysis fibers in the upper dermis below a site of challenge with an ice cube. During the warm- ing-up period following challenge, the development of the wheal was paralleled by an up to 80-fold increase of the levels of histamine in the dialysis effluent. Also, in chronic idiopathic urticaria, circulating autoantibodies against Fc5 RI, IgE, or both, occur in approxi- mately one third of patients (Fig. 30.7). Injection of autologous serum containing these antibodies causes a wheal and flare response, suggesting degranulation of cutaneous mast cells to be the cause of the urticarial condition [110, 111]. The picture in atopic eczema is far less clear. Clearly, IgE-bearing cells are axial in the disease mechanism, but in the skin these include cells of the Langerhans’ cell/dendritic cell lineage as well as mast cells [112]. Thus, while the dermal mast cell plays a role in many cutaneous conditions, it is not necessari- ly a major player in them all.

Fig. 30.7. Auto antibodies in chronic urticaria

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