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 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.     

Alterations in mast cells showing tryptase and chymase activity in epithelializating and chronic wounds

Mast cells can be found in contact with epidermis in certain circumstances; especially in chronic inflammatory skin diseases and chronic ulcers, but the significance of this association is obscure. In this study, the association of mast cells with wound healing was studied by counting mast cells in the wound edges at different stages after wounding the donor site skin for pinch-grafting. Chronic venous leg ulcers were biopsed for comparison. Tryptase- and chymase-positive mast cells were stained enzyme-histochemically for active proteinases. Both the number of tryptase-positive, i.e. total mast cells, and chymase-positive mast cells decreased during wound healing, but only the change in chymase-positive mast cells was statistically significant (P< or =0.03) the maximal decrease being 63% on day 7. No mast cells could be found in the vicinity of epithelialization margin. In venous leg ulcers, significantly more mast cells were present in the perilesional skin near the epithelium margin than in the wound bed (P=0.03), and mast cells were also seen in close contact with the basement membrane. Immunoreactivity for IL-4 and TNF-alpha in mast cells was studied to see if either of these molecules was associated with wound healing. In normally healing wounds, only a minority of mast cells were immunoreactive for these cytokines and no change in positive mast cell numbers could be seen during wound healing. In chronic wounds, IL-4 was absent in mast cells, and TNF-alpha positive mast cells were present only in perilesional skin and in small numbers. These results show that mast cells especially chymase-positive - decrease in number and can not be found in the epithelialization zone in normal wound healing, whereas tryptase-positive mast cells are associated with delayed wound healing and epithelialization in chronic wounds. Thus it seems, that mast cells attempt to control hyperproliferation of epidermis in chronic wounds.     

Mast cell survival and apoptosis in organ-cultured human skin

Mast cells accumulate and persist predominantly in the upper dermis of the skin but the mechanism for this is obscure. The skin is normally exposed to external air, which is essential for the maturation of the epidermis and probably also the dermis. In order to clarify the importance of air exposure on dermal mast cells, skin organ culture at the air-liquid interface (ALI) and submerged (SM) in medium (10% fetal calf serum and Dulbecco's modification of Eagle's medium) was used to study changes in tryptase-, chymase- and Kit-positive mast cell numbers during cultivation for up to 14 days. In addition, possible apoptosis (TACS TdT in situ apoptosis detection method) in chymase-positive mast cells was studied during the culture. In the less-physiologic SM culture, the number of Kit-positive mast cells decreased rapidly on day 1-2 and tryptase-positive cells decreased markedly on day 14. This decrease in mast cell numbers can be explained by the finding that a rapid increase in the apoptosis index of mast cells was induced on day 1-2. In contrast, in the more physiologic ALI culture, the number of Kit-positive cells was sustained over 1-2 days but then decreased on day 7. In addition, tryptase-positive cells decreased steadily in number but not to the same extent as those in the SM culture. Moreover, the increase in the apoptosis index of mast cells was delayed until day 7 in the ALI culture. Addition of exogenous stem cell factor (up to 200 ng/ml) to the SM culture could not prevent the decay in tryptase- and chymase-positive cells. However, stem cell factor reduced significantly the number of Kit-positive cells already on day 2 indicating that the cells had responded. Addition of histamine (0.25 or 1 mM) or tumor necrosis factor-alpha (500 or 2000 U/ml) caused a decrease in the number of tryptase- and Kit-positive cells in the SM culture. In conclusion, a novel finding was that air exposure in the ALI culture markedly delayed the rapid apoptosis and subsequent decrease in mast cell numbers noted to occur in the SM culture. Stem cell factor could not prevent the rapid decrease in mast cell numbers. Histamine and tumor necrosis factor-alpha are possible factors promoting the decline in mast cells.     

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.     

Low concentrations of formaldehyde induce DNA damage and delay DNA repair after UV irradiation in human skin cells

Long-term occupational exposure to formaldehyde (FA) increases the risk for nasopharyngeal squamous cell carcinoma. As the skin is also in contact with FA by environmental exposure, we tested the genotoxic properties of appropriate low concentrations (<100 microM) of FA on cultured keratinocytes and fibroblasts of human skin. The initial DNA damage was assessed by comet assay. The induction of DNA protein crosslinks was measured by the ability of FA to reduce DNA migration induced by methyl-methane-sulfonate. Upon 4 h of exposure to FA, significant (P < 0.05) crosslink formations were observed in fibroblasts (50 microM FA) and in keratinocytes (25 microM FA). Upon 8 h of exposure to FA (25 microM FA), significant crosslink formations were observed in both the cell types. FA is known to inhibit different DNA repair pathways. Therefore, we studied the effect of FA on UV-induced repair. Human keratinocytes and fibroblasts exposed to 10 microM FA prior to UV irradiation showed disturbed repair kinetics after UVC and UVB, but not after UVA irradiation. Single-strand breaks (SSBs) derived from nucleotide excision repair disappeared 6 h after solely UVC (3 mJ/cm2) or 3 h solely UVB (30 mJ/cm2) exposure in both the cell types. In the presence of FA, SSBs were still present at these time points containing a reference to a delay in DNA resynthesis/ligation. FA at a concentration not inducing micronuclei (12.5 microM) caused significant increase of UVC-induced (4 mJ/cm2) chromosomal damage. Proliferation of keratinocytes and fibroblasts was in parallel to observed DNA damages. In conclusion, our data suggest that environmental exposure to FA may contribute to UV-induced skin carcinogenesis.     

Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin

Radiation-induced fibrosis is a common side-effect of cancer treatment. The pathophysiological events leading to fibrosis are not known in detail. We analysed the effect of therapeutic irradiation on human skin collagen synthesis, skin thickness, gelatinases and their inhibitors. Twenty randomly chosen women who had been treated for breast cancer with surgery and radiation therapy participated in the study. In each patient, the irradiated skin area was compared with a corresponding non-treated skin area. Suction blister fluid (SBF) and serum samples were analysed for the aminoterminal propeptides of type I and type III procollagens (PINP and PIIINP), tissue inhibitors of matrix metalloproteinases (MMPs) 1 and 2 (TIMP-1 and TIMP-2) and MMP-9 and MMP-2/TIMP-2 complex. Skin biopsies were analysed for PINP and immunohistochemical staining was used for PIIINP. In irradiated skin, PINP, PIIINP, TIMP-1 and MMP-2/TIMP-2 complex levels in SBF and the number of PINP-positive fibroblasts in tissue sections were significantly higher in comparison with non-treated skin. The levels of TIMP-2 in irradiated and non-irradiated skin were similar. MMP-9 could not be detected in SBF with the assay used. The serum levels of MMP-9 were higher in the treated subjects than the reference values. The serum values of PINP, PIIINP, TIMP-1, TIMP-2 and MMP-2/TIMP-2 complex were not significantly affected. These results indicate increased local collagen synthesis and accumulation of connective tissue in irradiated skin. The marked upregulation of collagen synthesis as a result of irradiation offers a possibility to treat this complication with compounds such as topical steroids which downregulate collagen synthesis.     

A study of Q-switched Nd:YAG laser irradiation and paracrine function in human skin cells

BACKGROUND AND OBJECTIVES: This preliminary laboratory-based study looks at the paracrine release from human skin cells subject to sublethal Q-switched Nd:YAG 532 nm laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Human dermal fibroblast and keratinocyte cultures were exposed to sublethal energy using the Nd:YAG 532 nm laser. Altered gene expression was then screened using RT-PCR for a range of paracrine factors known to affect melanogenesis, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), stem cell factor (SCF), melanocyte stimulating hormone (MSH), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and protease-activated receptor-2 (PAR-2). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein production. Conditioned medium was used to assess altered melanogenesis in a melanoma cell line. Results: Fibroblasts exposed to sublethal radiation showed upregulation of b-FGF, HGF and SCF. This contrasts with keratinocytes which showed upregulation of IL-6. Elevated protein levels of b-FGF and SCF were confirmed by ELISA assay. Conditioned fibroblast medium was shown to stimulate melanogenesis in a melanoma cell line. CONCLUSIONS: This preliminary laboratory study reports, for the first time, specific gene upregulation using the Q-switched Nd:YAG 532 nm laser.     

Effect of parathyroid hormone-related protein on fibroblast proliferation and collagen metabolism in human skin

The parathyroid hormone-related protein (PTHrp), structurally similar to the parathyroid hormone (PTH) in its NH(2)-terminal part, was first identified as a tumour-derived peptide responsible for a paraneoplastic syndrome known as humoral hypercalcemia of malignancy. The PTHrp gene is expressed not only in cancer but also in normal tissues during adult and/or fetal life, where it plays predominantly paracrine and/or autocrine roles. In the skin PTHrp produced by keratinocytes acts on fibroblasts by complex cooperative circuits involving cytokines and growth factors. In this report, we studied the direct effects of synthetic PTHrp 1-40 on proliferation and collagen synthesis and matrix metalloproteinase-2 (MMP-2) activity in cultures of fibroblasts isolated from normal human skin. Fibroblasts exposure to varying doses of PTHrp for 48 h, significantly and dose-dependently inhibited proliferation evaluated by [(3)H]-thymidine incorporation into DNA. A dose-dependent stimulation of cAMP released into the medium was concomitantly observed. In contrast, PTHrp had no effect on collagen synthesis evaluated either by [(3)H]-proline incorporation or by radioimmunoassay (RIA) of the carboxyterminal fragment of type I procollagen (PICP). MMP-2 activity, evaluated by quantitative zymographic analysis, was significantly increased by PTHrp treatment at doses of 160 and 320 nM. These findings indicate that PTHrp may play a role in normal dermal physiology by controlling both fibroblast proliferation and extracellular matrix degradation.     

Alginate oligosaccharides modulate cell morphology, cell proliferation and collagen expression in human skin fibroblasts in vitro

Abstract Effects of alginate oligosaccharides on cell proliferation and expression of collagen in cultured skin fibroblasts were studied. The oligosaccharides were found to suppress fibroblast proliferation to half the level in control cultures at a dose of 10 mg/ml during a period of 5 days. The inhibition was accompanied by a change in cell shape. The inhibition of cell proliferation was reversible, since depletion of these oligosaccharides led to a recovery of cell motility. Treatment of confluent cells with 10 mg/ml oligosaccharides for 5 days resulted in a reduction in collagen synthesis to one half of that in control cultures and inhibition of steady state levels of α₁(I), α₂(I), α₁(III) and α₁(VI) collagen mRNAs. These results suggest that alginate oligosaccharides are potential modulators of dermal fibroblasts and may provide a useful tool for the treatment of disorders related to abnormal collagen metabolism. Received: 16 October 1998 / Received after revision: 8 February 1999 / Accepted: 12 February 1999     

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin

Summary Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzemidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-antitrypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15,30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefullnes of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.     

Effect of histamine on collagen and collagen m-RNA production in human skin fibroblasts.

Direct effects of histamine on collagenous and non-collagenous protein synthesis by human skin fibroblasts were studied. Fibroblasts derived from human skin were incubated with various concentrations of histamine. Collagen and non-collagenous protein synthesis were measured by incorporation of 3H-proline. Both collagen synthesis measured as protein-bound hydroxyproline and non-collagenous protein synthesis measured as protein-bound proline increased in the presence of histamine at concentrations of 10(1)-10(2) micrograms/ml. Total RNA was extracted and m-RNA levels of various proteins were estimated by dot blot analysis, and densitometrically quantified. The levels of alpha 1(I) collagen and beta-actin m-RNA were clearly increased at the same concentrations. m-RNA levels of alpha 1(III) collagen were also increased but the rate was lower than that of alpha 1(I) collagen. No alteration of beta-tubulin m-RNA level was observed at the same concentrations. These results demonstrate that stimulation of collagen synthesis by histamine is pretranslationally controlled.     

Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation

Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II-III skin were irradiated at 100-600 J/m(2) every 2-7 days over a 4- to 5-week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m(2)) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana-Masson staining, melanocyte function by expression of melanocyte-specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46-phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53-positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV-induced DNA damage but not as effectively as constitutive pigmentation.     

UVB irradiation induces melanocyte increase in both exposed and shielded human skin

This study demonstrates for the first time in humans that UV light induces an increase of the melanocyte population in exposed skin as well as in shielded areas. Because an increased mitotic activity could promote tumor development, UV exposure might play a role in melanoma development not only in exposed but also in covered skin. In addition, it was found that subjects who initially had a small melanocyte population showed a larger increase in both exposed and covered skin compared to those with a high initial density. Individuals with a low density might therefore constitute a risk group for the development of malignant melanoma. These findings support the view that infrequent periods of intensive UV irradiation might be more harmful than regular exposure.     

Synthesis of glycosaminoglycans and collagen in skin fibroblasts cultured from a patient with lichen myxedematosus

Summary A patient is described with typical skin lesions of lichen myxedematosus and IgG-type lambda paraproteinemia. Fibroblasts cultured from the skin of the patient and from the skin of control persons were used to study glycosaminoglycan and collagen synthesis; the cultures were labelled with ³H-glucosamine and ³H-proline, respectively. Fibroblasts from the patient grew to a cell density which was lower than that of the control fibroblasts. The production of glycosaminoglycans was increased in lichen myxedematosus cultures, so that the ratio of hyaluronic acid to sulphated glucosaminoglycans was higher in the patient's cultures than in control cultures. Collagen production in the patient's cultures was about half of that in control cultures, whereas the ratio of type III to type I collagen was normal.