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.     

Mast cell and myeloid marker expression during early in vitro mast cell differentiation from human peripheral blood mononuclear cells

In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, < 10% expressed FcepsilonRIalpha, rare cells were CD34 + ( < 0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14% - 43% positive cells), tryptase (26% - 79%), CD33 (57%), and CD64 (64%), an upregulation of FcepsilonRIalpha (23% - 52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcepsilonRIalpha and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcepsilonRIalpha-positive cells were mostly CD14 + (90%), CD64 + (82%), and CD68 + (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcepsilonRIalpha expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcepsilonRIalpha, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. Keywords: c-kit/FcepsilonRI/SCF/tryptase.     

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.     

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.     

Alterations in ganglioside expression during the differentiation of human mast cells

Gangliosides are physiological components of the outer cell membrane. In the present study, the role of ganglioside expression during differentiation of human mast cells was evaluated. After 11 days of culture in medium known to induce mast cell differentiation, 70% of peripheral blood mononuclear cells (PBMC) showed positive staining for the high affinity IgE receptor and tryptase on immunocytochemistry and an associated 20-fold increase of ganglioside GM3 expression. Furthermore, exogenous addition of GM3 during cultivation of PBMC in medium containing low levels of growth factors induced an increase of mast cell specific tryptase. The association of ganglioside expression with mast cell differentiation was confirmed by experiments with the human mast cell line HMC-1. FcepsilonRI-positive cultured cells enriched with immunobeads exhibited a 3-fold higher expression of GM3, compared to FcepsilonRI negative HMC-1 cells. Furthermore, measurable amounts of the gangliosides GM2, GM1 and GD1a were found only in the FcepsilonRI positive cells. A corresponding transient increase of mRNA for GalNAcT, the key enzyme in the production of these latter gangliosides, could be detected preceding the expression of these gangliosides and the FcepsilonRI by RT-PCR. Taken together, these data point to a functional role of gangliosides in the differentiation of human mast cells.     

Effect of granulocyte macrophage colony-stimulating factor in a patient with benign systemic mastocytosis

We report the in vitro and in vivo effects of granulocyte macrophage colony stimulating factor (GM-CSF), a known inhibitor of in vitro mast cell differentiation, in a patient with benign, adult-onset systemic mastocytosis. In vitro effects of GM-CSF on bone marrow cultures before the start of treatment showed a marked inhibition of mast cell marker expression [tryptase, Kit, and high-affinity IgE receptor (FcepsilonRIalpha)] at both protein and mRNA levels. Therefore, the patient was treated with daily injections of GM-CSF for 10 weeks. After an initial improvement, increasing worsening of clinical symptoms was noted, and the patient refused further treatment. Lesional skin biopsies showed an increase of toluidine blue-positive mast cells, compared with uninvolved skin, with further significant increase after treatment. Similar results were obtained on staining for mast cell-specific tryptase and Kit, as well as for CD1a and FcepsilonRIalpha. These findings show that GM-CSF inhibits human bone marrow mast cell differentiation in vitro, and also in mastocytosis. However, GM-CSF apparently enhances recruitment of mast cell as well as dendritic cell precursors into the tissue during systemic treatment. These findings and the observed adverse clinical effects in the present patient make it unlikely that GM-CSF monotherapy will be beneficial for the treatment of mastocytosis.     

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.     

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.     

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.     

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.     

The increase in tryptase- and chymase-positive mast cells is associated with partial inactivation of chymase and increase in protease inhibitors in basal cell carcinoma

BACKGROUND: In basal cell carcinoma (BCC), mast cells accumulate in the peritumoral stroma. The serine proteinases tryptase and chymase are the major mediators in mast cell granules and they may exert their enzymatic activity in the BCC lesion by inducing matrix remodeling and epithelial cell detachment. OBJECTIVE: To analyse the numbers of mast cells showing tryptase enzyme activity, chymase enzyme activity and chymase immunoreactivity as well as the presence of chymase inhibitors alpha(1)-antichymotrypsin (alpha(1)-AC), alpha(1)-proteinase inhibitor (alpha(1)-PI) and squamous cell carcinoma antigen-2 (SCCA-2) in BCC. METHODS: Eleven biopsies were taken from the lesion and healthy-looking skin of 10 patients with superficial spreading BCC. The frozen biopsies were analysed enzyme- and immunohistochemically, and a sequential double-staining method was applied. RESULTS: In the BCC lesion, the number of mast cells with tryptase activity and chymase immunoreactivity was significantly increased by 2.2- to 2.3-fold. Practically all tryptase-immunopositive cells contained tryptase activity although occasional tryptase-immunopositive cells (about 1% of total) revealed no activity. However, the ratio of cells with chymase activity to those with chymase immunoreactivity was significantly decreased from 49 +/- 19% in the healthy skin to 33 +/- 19% in the BCC lesion. Instead, the percentage of mast cells displaying alpha(1)-AC or alpha(1)-PI immunoreactivity was significantly increased by 1.7-fold in the BCC lesion. SCCA-2 expression was strongly increased in the malignant BCC epithelium but mostly in the suprabasal layers. CONCLUSIONS: Tryptase- and chymase-positive mast cells (MC(TC)) increased in the BCC lesion. However, chymase is partially inactivated, possibly by the effective chymase inhibitors alpha(1)-AC and alpha(1)-PI. SCCA-2 increased in BCC, but was localized mostly to the suprabasal layers, and thus it seems not to be crucial in inhibiting chymase.     

Cytokines involved in growth and differentiation of human basophils and mast cells

Abstract Mast cells and basophils are multifunctional effector cells of the immune system. Both are myeloid cells and originate from multipotent hemopoietic progenitor cells. Usually, human basophils complete their differentiation in the bone marrow. In contrast, mast cells usually undergo differentiation in extramedullary organs. During the past few years, growth factors for human basophils and a growth factor for human mast cells have been identified. Interleukin-3 is the most potent differentiation factor for human basophils and activates mature basophils via high affinity binding sites. Other basophil agonists are GM-CSF. IL-5. NGF and certain chemokines (IL-8, MCP-1). Mast cells apparently loose cytokine binding sites during mastopoiesis and as mature cells, do not express detectable amounts of IL-3R, GM-CSFR or 1L-8R. However, in contrast to other myeloid cells, mast cells express SCF receptor/c-kit during mastopoiesis and on mature cells. Furthermore, the ligand of c-kit, SCF, induces differentiation of human mast cells from their progenitor cells and upreglates effector functions in mature mast cells.     

A possible mechanism of mast cell proliferation in mastocytosis

The abnormality in mastocytosis is the excessive accumulation of mast cells in the affected tissue. The growth and differentiation of human mast cells are quite dependent on stem cell factor (SCF), the ligand for the protein products of c-kit. Recent studies have demonstrated that all adult patients examined so far carry c-kit point mutations, leading to SCF-independent autophosphorylation of the receptor and autonomous cell growth. On the other hand, typical pediatric patients have been found to bear no activating Asp816Val mutation in c-kit. Although most mastocytosis patients are children, the mechanism by which mast cells proliferate in these pediatric patients remains unclear. Recently, were reported that human mast cells obtained from adult skin could dramatically proliferate when cultured with SCF. From these experimental results, it is speculated that local excessive production of SCF results in the mast cell proliferation in pediatric patients.     

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.     

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.     

The effect of ultraviolet radiation exposure on the prevalence of mast cells in human skin

BACKGROUND: Dermal mast cells have been implicated as important effector cells in innate immunity, hypersensitivity responses and ultraviolet (UV)B-induced suppression of cell-mediated immune responses to contact allergens. Humans, like mouse strains, display variations in dermal mast cell prevalence. The factors determining these differences are yet to be fully elucidated. In mice, expression of the receptor for stem cell factor, c-kit, on dermal mast cells correlates with prevalence. OBJECTIVES: To evaluate dermal mast cell prevalence and mast cell c-kit expression in non-sun-exposed and sun-exposed skin in the same donor. METHODS: In 14 subjects, biopsies of skin (4 mm) were sampled from the skin sites of buttock, inner arm, shoulder and back of hand skin and dermal mast cell prevalence quantified. Non-sun-exposed buttock and chronically sun-exposed hand skin were evaluated for mast cell expression of c-kit and elastin content, a feature of photoageing and surrogate marker of UV exposure. RESULTS: The prevalence of dermal mast cells was significantly higher in hand skin than in the three other anatomically different skin sites. Significant correlations were observed in hand but not buttock skin between increasing dermal mast cell densities, extent of elastin content in the papillary dermis and age of the subject. Cellular expression of c-kit correlated with mast cell prevalence in hand skin. However, no relationship was observed in hand skin between c-kit expression, elastin content and age. CONCLUSIONS: The prevalence of mast cells in human skin is altered by factors that are intrinsic (mechanisms regulating c-kit expression) and extrinsic (chronic sun exposure), and the fact that the associations of mast cell prevalence with age is explained by the latter being a correlate of cumulative sun exposure.     

Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas

In order to identify possible cellular abnormalities in human mastocytosis, sections from 13 urticaria pigmentosa lesions and 5 mastocytomas were compared with 5 normal skin specimens using histochemical, enzyme histochemical and immunohistochemical techniques. All toluidine blue-positive mast cells also reacted with FcRI and c-kit antibodies, almost all stained for tryptase, many for chymase and the myeloid workshop mast cell antibodies, few for FcRII and none for the proliferation marker Ki-67. Urticaria pigmentosa lesions contained fewer epidermal Langerhans cells and a lower percentage of avidin-positive mast cells than mastocytomas and normal skin. Mastocytomas exhibited generally weaker staining for mast cell markers and mostly lacked FcRI-bound IgE on mast cells and Langerhans cells, although the receptor was able to bind IgE in tissue sections. Most of the mast cell antibodies also reacted with other cell types. Only toluidine blue, avidin, tryptase and chymase stains were mast cell specific. Mast cells in mastocytosis thus differed only to a minor degree from normal mast cells, although distinct pathomechanisms may play a role in urticaria pigmentosa and mastocytosis.     

Mast cell chymase in experimentally induced psoriasis

Mast cell chymase can have a pro‐inflammatory or an immunosuppressive function in psoriasis, but the outcome may depend on the level of chymase activity. Therefore, mast cells showing chymase activity (Chy_act) and immunoreactivity (Chy_prot) were studied during the Köbner reaction (0 days, 2 h, 1 day, 3 days and 7 days) of psoriasis induced by the tape‐stripping technique. Also, the effect of recombinant human chymase (rh‐chymase) or human LAD2 mast cells (LAD2) on the ³H‐thymidine uptake of psoriatic peripheral blood mononuclear cells (PBMC) or total T cells was studied. The Chy_act/Chy_prot ratio tended to be higher in all time‐point biopsies in the Köbner‐negative (n = 10) than ‐positive (n = 8) group (P = 0.073), although chymase activity decreased significantly at 2 h to 1 day only in the Köbner‐negative group. rh‐chymase (0.05–0.5 μg/mL) stimulated to a varying extent PBMC in eight out of nine cultures, but in all cultures 5 μg/mL rh‐chymase turned the stimulation towards inhibition. The effect of rh‐chymase on T cells varied from stimulation to inhibition, but in 11 of 15 cultures rh‐chymase, at least at 5 μg/mL, produced a change to inhibition. In co‐cultures, LAD2 inhibited PBMC in the absence of soybean trypsin inhibitor (SBTI). In the presence of SBTI, LAD2 stimulated PBMC in the majority of seven cultures. In summary, the psoriatic immunopathogenesis may be promoted at low, but controlled at high, activity status of chymase.