Decreased chymase activity is associated with increased levels of protease inhibitors in mast cells of psoriatic lesions

Mast cells contain large amounts of the powerful serine proteinases, tryptase and chymase, of which only chymase can be inactivated by serum protease inhibitors. In this study, 20 patients with psoriasis and a control group of 13 with atopic dermatitis were biopsied for lesional and non-lesional skin specimens. The presence of chymase inhibitor alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-AC), alpha 2-macroglobulin (alpha 2-MG) and C1-esterase inhibitor (C1-Inh) immunoreactivity in mast cells was verified using the sequential double-staining method. Tryptase- and chymase-positive mast cells were stained enzyme-histochemically. Tryptase-positive mast cells were increased in number in the upper dermis of the psoriatic lesion compared with lesion-free psoriatic skin (308 +/- 109 vs. 100 +/- 29 cells/mm2, respectively, mean +/- SD, p < 0.0005, t-test) while the percentage of mast cells showing chymase activity was decreased (76.8 +/- 22.1% vs. 28.6 +/- 14.4%, p < 0.0005). These findings are consistent with our previous ones. In contrast to the decreased percentage of chymase-positive mast cells, a novel finding was that the percentages of alpha 1-AC+ (86.9 +/- 7.2% vs. 59.5 +/- 12.6%, p < 0.0005), alpha 1-PI+ (72.2 +/- 14.9% vs. 33.4 +/- 18.6%, p < 0.0005) and alpha 2-MG+ (16.8 +/- 7.0% vs. 6.2 +/- 3.5%, p < 0.002) mast cells were significantly higher in the psoriatic lesion with the exception of the percentage of C1-Inh+ mast cells (13.7 +/- 10.0% vs. 11.0 +/- 6.1%, p < 0.7). The localization of these inhibitors in mast cells is not a characteristic feature of psoriasis, since mast cells in atopic dermatitis skin also showed immunoreactivity though in slightly lower percentages. Previously, we have shown that MCTC (tryptase+, chymase+) mast cells increase in number in the psoriatic lesion but chymase becomes inactive. The results of this study show again the decreased chymase activity, which could be due to increased levels of its inhibitors (alpha 1-AC, alpha 1-PI and alpha 2-MG) in the same mast cells. Thus, active tryptase could promote inflammation but chymase seems not to be an important mediator in the pathomechanism of psoriasis.     

Mast cells in developing subepidermal bullous diseases: emphasis on tryptase, chymase and protease inhibitors.

The possible involvement of mast cell tryptase and chymase in subepidermal bullous diseases was studied enzyme-histochemically in specimens from erythematous and vesicular skin and from non-involved skin of patients with dermatitis herpetiformis, bullous pemphigoid, erythema multiforme, infective bullous eruption and linear IgA dermatosis. Patients with pemphigus were biopsied for comparison. The immunoreactivity of chymase inhibitors, alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC), in mast cells was demonstrated using the sequential double staining method. Tryptase-positive mast cells were unchanged or only slightly increased in number in erythematous lesions and slightly decreased in blistering skin compared with healthy-looking skin. Only occasionally were mast cells seen in apparent contact with the basement membrane zone. Chymase-positive mast cells and the chymase/tryptase ratio steadily decreased during the development of the lesions in each subepidermal bullous disease. The percentage of alpha1-PI+ and/or alpha1-AC+ mast cells increased simultaneously, which could explain the disappearance of chymase activity. Similar results were obtained regardless of the bullous disease. The results were also similar in pemphigus, which is an intraepidermal bullous disease. In conclusion, these results show significant alterations in mast cell chymase and protease inhibitors in a range of different bullous diseases, suggesting mast cell involvement. The apparent inactivation of chymase could be due to the action of chymase inhibitors detected in numerous mast cells. However, these alterations probably reflect general inflammation rather than a specific reaction in a certain bullous disease.     

Mast cell chymase is present in uterine cervical carcinoma and it detaches viable and growing cervical squamous carcinoma cells from substratum in vitro

Increased numbers of mast cells is a typical feature of a variety of human cancers. The major mediators in the secretory granules of the MC_TC type of mast cells, serine proteinases tryptase and chymase, may be involved in squamous cell carcinoma (SCC) lesions by inducing matrix remodeling and epithelial cell detachment. The objective of this study was to analyze immunohistochemically whether MC_TC mast cells as well as protease inhibitors, squamous cell carcinoma antigens (SCCAs), are present in the uterine cervical SCC. In addition, the effect of tryptase and chymase on uterine cervical SCC cell lines was studied in vitro. Here we report that tryptase- and chymase-positive mast cells are present in significant numbers in the peritumoral stroma of SCC lesions. Also, weak SCCA-2 immunoreactivity is observed in the SCC lesions, but only SCCA-1 in uterine cervical specimens with nonspecific inflammation. In cell cultures, especially chymase, but not tryptase, was shown to induce effective detachment of viable, growing and non-apoptotic SiHa SCC cells from substratum. Chymase also detached viable ME-180 SCC cells from substratum as well as degraded fibronectin. In contrast, normal keratinocytes underwent apoptotic cell death after similar prolonged chymase treatment. No inhibition of chymase was detected by SiHa cell sonicates nor did these cells express marked SCCA immunopositivity. MC_TC mast cells containing tryptase and chymase are present in the peritumoral stroma of uterine cervical SCC and the malignant cells are only weakly immunoreactive for the chymase inhibitor SCCA-2. It is chymase that appears to be capable of inducing effective detachment of viable and growing SCC cells and therefore, it may release SCC cells from a tumor leading to spreading of malignant cells.     

Mast cell tryptase and chymase in developing and mature psoriatic lesions

The number and distribution of mast cells in nonlesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In nonlesional psoriatic skin, 70 ± 24% (mean ± SD) of the mast cells contained chymase enzyme activity, and 78 ± 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, ₁-antitrypsin and ₁-antichymotrypsin, localized immunohistochemically in mast cells of lesional and nonlesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27–38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis. Earliest alterations in tryptase-positive cells were observed on day 14 as increased mast cell contacts with the epidermis combined with only a slight increase in mast cell numbers in the upper dermis. During the development of a psoriatic lesion, TC mast cells (tryptase⁺, chymase⁺) increase in number in the upper dermis, but chymase becomes inactive at an early stage. The abundant presence of active trypase but inactive chymase in the upper dermis may have a potential role in psoriasis since both of these enzymes can process several biologically active peptides and proteins.     

Release of soluble tryptase but only minor amounts of chymase activity from cutaneous mast cells

Tryptase and chymase are the major serine proteinases of skin mast cells but their biologic significance depends on their activity. In this study, we demonstrate the release of soluble activity of tryptase, but not markedly that of chymase, into skin blister fluids induced by freezing with liquid nitrogen as well as into supernatant during incubation of 8 whole skin specimens with compound 48/80 for up to 2 days followed by sonication. Incubation of 3 other skin specimens in compound 48/80 for up to 2 days revealed that the number of mast cells displaying tryptase activity decreased significantly on day 2, and the number of mast cells showing chymase activity (but not those showing chymase immunoreactivity) decreased significantly on day 1 but not thereafter on day 2. The results of 3 skin organ cultures for up to 14 days showed steady decrease in the number of tryptase-positive cells but persistence of mast cells containing chymase activity. Chymase in solution was sensitively inhibited by 0.01 mg/ml alpha1-antichymotrypsin but higher concentrations (0.3-3.0 mg/ml) were needed for inhibiting chymase on skin sections. In conclusion, after mast cell degranulation tryptase activity is substantially solubilized and it may potentially affect both local and distant skin structures. Instead, chymase is partially inactivated and the remaining chymase activity persists at the site of degranulation having only local effects.     

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.     

MAST CELL TRYPTASE AND CHYMASE ARE POTENTIAL REGULATORS OF NEUROGENIC INFLAMMATION IN PSORIATIC SKIN

Background. Tryptase and chymase are proteinases present only in mast cells. In psoriatic lesions, mast cells are increased in number. Certain neuropeptides are also more abundant in lesional than nonlesional psoriatic skin. Based on some earlier results as well as the results of the present study, a hypothesis is presented concerning the regulatory action of mast cell tryptase and chymase on neuropeptides in psoriatic inflammation. Methods. Forty patients were biopsied, 13 for a mature psoriatic plaque and 9 patients of 27 for a developing (1–3 weeks) psoriatic lesion induced by tape stripping (Koebner reaction). Each lesion had its nonlesional control from the same patient. Mast cell tryptase and chymase, and the neuropeptides Substance P (SP) vasoactive intestinal polypeptide (VIP), and calcitonin-gene-related peptide (CGRP) were stained by enzyme-and immunohistochemical methods. Morphological contacts between mast cells and neuropeptides were visualized using double stains and quantitated in the upper dermis. Results. As the lesion aged, MCTC mast cells displaying tryptase activity increased in number, whereas chymase activity in these cells decreased. All neuropeptides showed some increase along with the development of the lesion, but SP was most abundant in mature lesions. Substance P-positive nerves had also more contacts with mast cells compared to VIP- or CGRP-containing fibers, the contact count being highest in mature lesions. Conclusions. Tryptase is known to degrade VIP and CGRP, but not SP. Chymase is capable of cleaving both SP and VIP, but is rendered partially inactive in psoriatic skin. These data together with the results of the present study strongly suggest that SP has potency to act as an important mediator in different stages of the psoriatic inflammation.     

The production of collagen and the activity of mast-cell chymase increase in human skin after irradiation therapy

Fibrosis is a common complication of radiotherapy. The pathogenesis of radiation-induced fibrosis is not known in detail. There is increasing evidence to suggest that mast cells contribute to various fibrotic conditions. Several mast-cell mediators have been proposed to have a role in fibrogenesis. Tryptase and chymase, the predominant proteins in mast cells, have been shown to induce fibroblast proliferation and collagen synthesis in vitro. In order to explore the role of mast cells in irradiation-induced fibrosis, we analyzed skin biopsies and suction blister fluid (SBF) samples from the lesional and healthy-looking skin of 10 patients who had been treated for breast cancer with surgery and radiotherapy. The biopsies were analyzed histochemically for mast-cell tryptase, chymase, kit receptor, and tumor necrosis factor-alpha. Skin collagen synthesis was assessed by determining the levels of type I and III procollagen amino-terminal propeptides (PINP and PIIINP) in SBF and using immunohistochemical staining for PINP. Immunohistochemical stainings for prolyl-4-hydroxylase reflecting collagen synthesis and chymase immunoreactivity in irradiated and control skin were also performed. The mean level of procollagen propeptides in SBF, which reflects actual skin collagen synthesis in vivo, was markedly increased in irradiated skin compared to corresponding healthy control skin areas. The mean number of PINP-positive fibroblasts was also significantly increased in the upper dermis of radiotherapy-treated skin. The number of cells positive for tryptase, chymase and kit receptor was markedly increased in irradiated skin. In addition, using double-staining techniques, it was possible to demonstrate that in some areas of the dermis, tryptase-positive mast cells and fibroblasts are closely associated. These findings suggest a possible role of mast cells in enhanced skin collagen synthesis and fibrosis induced by radiotherapy.     

Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin

Summary Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymaseand tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72–73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activitiy in the upper dermis may lead to an imbalance in the biochemical regulatory systems.     

Differential expression of mast cell characteristics in human myeloid cell lines

In order to better understand the mechanisms governing display of mast cell characteristics in human myeloid cells, we have studied the mast cell phenotype in human promyelocytic (HL-60) and myelocytic (U-937, TPH-1) vs. basophilic (KU-812) and mast cell (HMC-1) lines, in part also in skin mast cells and blood monocytes, at mRNA and protein level before and after stimulation with mast cell growth factors. In unstimulated cells, mRNA for the stem cell factor (SCF) receptor c-kit and the gamma chain of the high-affinity IgE receptor (FcepsilonRI) was noted in all cells studied. Like mast and basophilic cells, THP-1 cells expressed the FcepsilonRIalpha and beta chains and weakly histidine decarboxylase (HDC), but they lacked mRNA for mast cell-specific proteases [tryptase, chymase, carboxypeptidase A (CPA)]. In contrast, HL-60 and U-937 cells lacked FcepsilonRIalpha, but expressed tryptase and chymase, HL-60 cells also CPA. KU-812 cells failed to express the basophil-specific marker 2D7. After a 10-day culture with SCF or fibroblast supernatants, baseline mRNA expression of most mast cell characteristics was upregulated, whereas c-kit mRNA expression decreased in all but THP-1 cells. Differential mRNA expression of FcepsilonRI vs. protease (tryptase) was confirmed at protein level by immunocytochemistry and enzymatic activity. KU-812 cells are thus closest to skin mast cells in that they express all molecules studied, except for chymase, followed by THP-1 cells that lack all mast cell proteases. In contrast, HL-60 and U-937 cells fail to express the FcepsilonRIalpha and beta chains but express most mast cell proteases. The selective and differential expression of mast cell characteristics in human myeloid cell lines suggests that induction of the mast cell phenotype is regulated by several independent genes and that mast cells and basophils branch off at early and distinct points of myeloid development.     

Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue

In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.     

Purification of mast cell proteases from murine skin

Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.     

Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneous scars

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.     

Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation

Please cite this paper as: Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation. Experimental Dermatology 2010; 19: 117–122. Abstract: Mast cells are increasingly present in the lesional skin of chronic skin inflammatory diseases including psoriasis and basal cell carcinoma (BCC). It has previously been shown that proteinase‐activated receptor (PAR)‐2 is expressed by mast cells, and tryptase is a potent activator of this receptor. In this study, skin biopsies from both healthy‐looking and lesional skin of patients with psoriasis and superficial spreading BCC were collected and the expression of PAR‐2 immunoreactivity in tryptase‐positive mast cells was analysed. PAR‐2 expression was confirmed in vitro in different mast cell populations. Cord‐blood derived mast cells (CBMC) were stimulated with a PAR‐2 activating peptide, 2‐furoyl‐LIGRLO‐NH₂. Consequently, IL‐8 and histamine production was analysed in the supernatants. We observed a significant increase in the percentage of mast cells expressing PAR‐2 in the lesional skin of psoriasis and BCC patients compared with the healthy‐looking skin. HMC‐1.2, LAD‐2 and CBMC mast cells all expressed PAR‐2 both intracellularly and on the cell surface. CBMC activation with the PAR‐2 activating peptide resulted in an increased secretion of IL‐8, but no histamine release was observed. Furthermore, both PAR‐2 and IL‐8 were co‐localized to the same tryptase‐positive mast cells in the lesional BCC skin. These results show that mast cells express increased levels of PAR‐2 in chronic skin inflammation. Also, mast cells can be activated by a PAR‐2 agonist to secrete IL‐8, a chemokine which can contribute to the progress of inflammation.     

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the FcRI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.     

Acute exposure of human skin to ultraviolet or infrared radiation or heat stimuli increases mast cell numbers and tryptase expression in human skin in vivo

Background  Mast cells are key effector cells in diverse immunological and pathological processes. It is still unclear why there are more mast cells at peripheral and sun-exposed skin sites than at sun-protected sites.
Objectives  To investigate changes in mast cell numbers associated with natural ageing and photoageing, and to observe the effects of ultraviolet (UV) and infrared (IR) radiation and heat on the prevalence of mast cells and tryptase expression in human skin in vivo .
Methods  Sun-exposed and sun-protected skin samples were taken from individuals in four different age groups. UV, IR or heat-treated buttock skin of young volunteers was also obtained. Mast cells were quantified by immunohistochemical staining of mast cell-specific tryptase and chymase. The expression of tryptase was determined by Western blotting.
Results  Both sun-exposed and sun-protected skin showed a gradual decrease in total mast cells (MC_Total) number with ageing. The number of mast cells in sun-exposed skin was significantly higher than that in sun-protected skin. After UV irradiation (2 minimal erythema doses), MC_Total and mast cells expressing tryptase and chymase were significantly increased at 24 and 48 h postirradiation. After IR irradiation (3 minimal heating doses) and heat treatment (43 °C for 90 min), MC_Total reached peak induction at 8 and 48 h after stimulation, respectively. Tryptase expression was also clearly upregulated by UV, IR and heat.
Conclusions  Our data demonstrate that mast cell numbers decreased with ageing in human skin. Also, mast cells may be activated and recruited by UV, IR and heat. These findings should further our understanding of the reason for the high prevalence of mast cells at peripheral sun-exposed skin sites.     

NKp46 regulates the production of serine proteases and IL‐22 in human mast cells in urticaria pigmentosa

NKp46 (natural cytotoxic receptor 1/CD335) is expressed on natural killer cells and Th2‐type innate lymphocytes. However, NKp46 expression in human mast cells has not yet been reported. Here, we explored the expression of, and possible role played by, NKp46 in such cells. NKp46 protein was expressed in human mast cells in urticaria pigmentosa principally of the tryptase‐positive/chymase‐negative type (MCT), but not in human non‐neoplastic skin mast cells of the tryptase‐positive/chymase‐positive (MCTC) type. NKp46 expression was also evident in the human neoplastic mast cell line HMC1.2. NKp46 knockdown changed the phenotype of this cell line from MCT to MCTC and downregulated GrB production, but did not influence IL‐22 production. An agonistic anti‐NKp46 antibody upregulated production of GrB and IL‐22, but did not change the MCT‐like phenotype of HMC1.2 cells. NKp46 was thus involved in the production of serine proteases and IL‐22 in human mast cells.     

Ultrastructural and immunohistochemical characterization of normal mast cells at multiple body sites

This article reviews the ultrastructural and immunohistochemical features of normal human mast cells (MC) at multiple tissue sites. Current literature indicates that granules containing discrete scrolls (scroll-rich morphology) are frequent in MC from bowel mucosa and lung, locations where the majority of MC show only tryptase immunoreactivity (MCT). In contrast, most MC from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa are characterized by scroll-poor morphology (that is, granules are rimmed by incomplete scrolls forming parallel lamellae and containing central, amorphous granular material or grating/lattice-like structures) and show both tryptase and chymase immunoreactivity (MCTC). MC having granules with both scroll-rich and scroll-poor features can occur in all tissue sites, and an occasional MC, especially in lung and bowel, may show only chymase immunoreactivity (MCC). Chymase immunoreactivity in MC also is closely associated with avidin binding and carboxypeptidase reactivity. We conclude that there is ultrastructural and immunophenotypic diversity among normal human MC, although certain forms predominate in specific tissue environments. In skin, breast tissue, axillary lymph nodes, and bowel submucosa MC tend to have scroll-poor granules and stain for avidin, chymase, tryptase, and carboxypeptidase, whereas, in lung and bowel mucosa MC granules tend to be scroll-rich and stain only for tryptase with currently available reagents.     

Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines

Please cite this paper as: Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines. Experimental Dermatology 2010; 19 : 845–847. Abstract: To circumvent the costly isolation procedure associated with tissue mast cells (MC), two human MC lines, i.e. HMC‐1 and LAD2, are frequently employed, but their relation to mature MC is unknown. Here, we quantitatively assessed their expression of MC markers in direct comparison to skin MC (sMC). sMC expressed all lineage markers at highest and HMC‐1 cells at lowest levels. LAD2 cells expressed comparable high‐affinity IgE receptor α (FcεRIα) and FcεRIγ but less FcεRIβ than sMC and displayed slightly reduced, but robust FcεRI‐mediated histamine release. Only minor differences were found for total histamine content and c‐Kit expression. Huge, and to this level unexpected, differences were found for MC tryptase and chymase, with sMC >>> LAD2 > HMC‐1. Taken together, HMC‐1 cells represent very immature malignantly transformed MC, whereas LAD2 cells can be considered intermediately differentiated. Because of the minute levels of MC proteases, MC lines can serve as surrogates of tissue MC to a limited degree only.