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The coagulation system is thought to play a pivotal role during the initial phase of wound healing, but mechanisms and cells involved are only partly understood. We have therefore examined human scars for the expression of thrombin, its precursor prothrombin and the thrombin receptors, thrombomodulin (TM) and protease-activated receptor-1 (PAR-1), compared with normal skin. Biopsies of scars were obtained from primary excision sites of melanoma patients ( n = 20) and were compared with normal skin distant from the scar ( n = 10), using immunohistochemistry. In addition, polymerase chain reaction analyses were performed on scar versus normal tissue and on cultured keratinocytes, fibroblasts and endothelial cells before and after stimulation with selected cytokines known to be active in wound healing. Normal epidermis was stained for prothrombin, thrombin, TM and PAR-1, and dermal tissue was stained only for TM and PAR-1. In scar tissue, thrombin and TM were upregulated in the epidermis and all four molecules in the dermis, independent of the age of the scars. In tissue extracts, mRNA expression of PAR-1 and prothrombin expression were, however, unchanged and TM even slightly decreased in scars, compared with normal skin. On analysis of cultured cells, keratinocytes expressed mRNA for PAR-1, TM and prothrombin, endothelial cells for PAR-1 and TM, and fibroblasts for PAR-1. An upregulation of PAR-1 mRNA was induced in fibroblasts on exposure to tumor necrosis factor- α (TNF- α), while it remained unchanged in endothelial cells in response to TNF- α. A downregulation of TM was induced in endothelial cells on exposure to TNF- α. These findings, showing a marked modulation of thrombin, PAR-1 and TM even in older human scar tissue, suggest that the coagulation system is not only involved during clotting, but also during the inflammatory and tissue remodelling phases of wound healing. [ABSTRACT FROM AUTHOR]

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryoktic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression to the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix in the HPV-negative cervical cancer line HT3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a β-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined of the cell extract prepared from the transfected cell with 14 C-labeled chloramphenicol. The CMV promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, was highly active in all the skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-. SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only The SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells, In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression. [ABSTRACT FROM AUTHOR]

Androgens are known to exert anti-inflammatory effects but their impact on mast cells (MCs) remains to be determined. Here, we show that MCs isolated from human foreskin samples (male) and those from breast skin (female) express the androgen receptor, albeit with a 10-fold difference between the subsets. While fundamental MC properties (FceRI, c-Kit, tryptase; histamine release upon FceRI cross-linking) were unaffected or slightly reduced (chymase) by testosterone, the hormone had a more profound impact on the production of cytokines, with IL-6 being a target (reduction by 53%). Interestingly, this effect was limited to breast skin MCs (15 of 16 donors displayed this phenomenon), but was not reproduced by foreskin MCs. Collectively, effector functions of human skin MCs are modulated by androgens in a gene-selective and MC subset-specific fashion. Possibly, MCs from women are more susceptible to testosterone. We also demonstrate that MC IL-6 production is highly variable among individuals. [ABSTRACT FROM AUTHOR]

The coincidence of skin tumors and elevated mast cell (MC) numbers has been known for many years. However, it has remained controversial whether, in this context, MCs promote or inhibit tumor growth. Addressing this problem, different melanoma and squamous cell carcinoma cell lines were co-cultivated with primary, dermal MC for 24 h and gene or protein expression of cytokines tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) estimated. Co-culture with MCs led to an increase in IL-8 gene expression and IL-8 protein release from melanoma cells and IL-6 and IL-8 gene expression and protein release from squamous cell carcinoma cells, respectively. Moreover induction of IL-6 and IL-8 was primarily regulated by MC-derived TNF-α. Our data suggest an interplay between MCs and tumor cells, which results in altered cytokine release and may, thus, have an impact on tumor growth, invasion and neovascularisation. [ABSTRACT FROM AUTHOR]

MRGPRX2 was recently uncovered as the "missing link" in clinically relevant mast cell (MC) activation explaining previously puzzling phenomena. It is the receptor for various endogenous ligands and exogenous compounds alike, whose binding evokes rapid degranulation much like allergen‐mediated exocytosis. While the perceivable outcomes are similar, the two activation routes differ regarding mechanism and regulation. We recently reported that acute SCF administration curbs responses evoked by MRGPRX2 in human skin MCs. Maintenance of MCs in culture requires the presence of MC supportive factors and renders the cells functionally and molecularly unequal to ex vivo counterparts. Here, we asked whether expansion in culture impacts the pseudo‐allergic route, and if so, what contribution SCF and IL‐4 play in this scenario. We report that the in vitro micromilieu dampens (but does not erase) pseudo‐allergic responses and that this is accompanied by strongly reduced MRGPRX2 expression. Withdrawal of SCF or IL‐4 individually, but most potently of both collectively, partially reinstates the MRGPRX2 pathway, revealing that SCF and IL‐4 make negative adjustments to the pseudo‐allergic pathway. Under all conditions, the FcεRI‐triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo‐allergic MC activation can obey opposite rules, hinting at possible competition between them. [ABSTRACT FROM AUTHOR]

Mast cells ( MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock-down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several 'difficult-to-transfect' cells. In the present work, we therefore tested this method on skin-derived MCs using different si RNA entities. The si RNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability. [ABSTRACT FROM AUTHOR]

Mast cells ( MCs) are unique constituents of the human body. While inter-individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c-kit, Fc ε RI α, Fc ε RI β, Fc ε RI γ, histidine decarboxylase, tryptase, and chymase; surface expression of c-Kit, Fc ε RI α; activity of tryptase, and chymase; histamine content and release triggered by Fc ε RI and Ca2+ ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33-386%). Surface expression of Fc ε RI was positively associated with Fc ε RI α mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co-regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. Fc ε RI triggered histamine release was highly variable and was unrelated to Fc ε RI expression but unexpectedly tightly correlated with histamine release elicited by Ca2+ ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter-individual variability in MCs, but also uncovers unexpected patterns of co-regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike. [ABSTRACT FROM AUTHOR]

The article offers information on the characteristics of mast cells in skin tissues and discusses the variability in them in different cohorts through an analysis. Topics discussed include the morphology of the cells, the granules in mast cells, and the differences in mast cells based on the location of the skin on the body.

Despite their hematopoietic origin, mast cells ( MCs) develop exclusively in tissues, hampering their ample use in research. To circumvent this problem, tissue-derived MCs are typically first expanded in culture, but the changes MCs may undergo in the novel micromilieu are poorly defined. Here, we monitor skin MCs from a number of donors over time, revealing profound yet non-synchronized modulations in culture. While tryptase and chymase, the most specific markers, strongly decline, Fc ε RI surface expression, and Fc ε RI-mediated histamine release steeply increase (from ≈15.5% to ≈60%), replicated by similar increments in TNF- α secretion. Interestingly, the modulations are independent of cell cycle progression, as they are comparable in the growth and postgrowth phase, implying they primarily result from microenvironmental conditioning. The data highlight a high degree of MC versatility, but also advise that results based on cultured MCs should be viewed with some caution, as they may not accurately reflect their counterparts in situ. [ABSTRACT FROM AUTHOR]

Since its first description around the year 2000, the local renin‐angiotensin system (RAS) in skin has been subject of an increasing number of studies with many additions over the last two to three years. A focus of research has been investigations on the role of cutaneous angiotensin receptors and locally synthesised angiotensin II in wound healing, in dermatoses associated with skin fibrosis and in melanoma. This review will provide an introduction into the RAS with emphasis on information relevant for the cutaneous RAS. It will discuss the role of the RAS in skin physiology, followed by a detailed review of the existing literature addressing the role of local angiotensin II and angiotensin AT1 and AT2 receptors in wound healing and in various skin diseases such as hypertrophic scars/keloids, scleroderma, dystrophic epidermolysis bullosa, Dupuytren's disease, squamous cell carcinoma, melanoma and psoriasis. In a final section, the potential relevance of drugs, which interfere with the RAS, for future therapy of dermatological disorders is discussed. Collectively, research about the RAS in skin can currently be described as an area, which has gained increasing attention by basic researchers, thus resulting in a multitude of preclinical studies pointing to the potential relevance of components of the RAS as drug targets in dermatological diseases. With a few small clinical studies already performed successfully for indications such as hypertrophic scars and keloids, it can be said that the skin RAS is now in the critical phase of translation from preclinical evidence to clinical use. [ABSTRACT FROM AUTHOR]

The receptor tyrosine kinase c-Kit is fundamental to mast cell (MC) development and maintenance. Its regulation can occur at various levels, but nothing is known about how this is accomplished in normal human tissue MC. Likewise, the baseline turnover of c-Kit has not been addressed yet. We used mature MC from human skin, along with the MC lines LAD-2 and HMC-1 and treated them with stem cell factor (SCF), cycloheximide, actinomycin D (AD) and combinations thereof, and determined expression levels of c-Kit and other surface receptors by flow cytometry. Ligand-induced internalization of c-Kit was found to be a universal mechanism and detectable in all MC subtypes. By Western blot analysis of LAD-2 cells, c-Kit was found to nearly disappear 3 h after the addition of SCF to slowly recover thereafter. Investigations into the baseline turnover of c-Kit expression revealed that c-Kit is strongly affected by the inhibition of de novo translation in all MC subsets, while a suppression of transcription had a weaker effect and displayed greater cell-to-cell variation. Only a minor impact on other cell surface receptors (CD29, CD50 and CD54) was noted. On combined treatment, cycloheximide, AD and SCF displayed additive effects, resulting in a complete disappearance of c-Kit from the cell surface. In conclusion, c-Kit represents a rapidly cycling cell surface receptor. It is not only immediately internalized upon binding of its ligand, but it is also heavily affected by the inhibition of translation or transcription when viewed against an average background. Interestingly, c-Kit regulation seems largely independent of the MC subtype. [ABSTRACT FROM AUTHOR]

Steckelings UM, Wollschläger T, Peters J, Henz BM, Hermes B, Artuc M. Human skin: source of and target organ for angiotensin II. The present study examined the expression of angiotensin receptors in human skin, the potential synthesis of angiotensin II (Ang II) in this location and looked for a first insight into physiological functions. AT1 and AT2 receptors were found within the epidermis and in dermal vessel walls. The same expression pattern was found for angiotensinogen, renin and angiotensin-converting enzyme (ACE). All components could additionally be demonstrated at mRNA level in cultured primary keratinocytes, melanocytes, dermal fibroblasts and dermal microvascular endothelial cells, except for AT2 receptors in melanocytes. The ability of cutaneous cells to synthesize Ang II was proved by identifying the molecule in cultured keratinocytes. Furthermore, in artificially wounded keratinocyte monolayers, ACE-mRNA expression was rapidly increased, and enhanced ACE expression was still found in cutaneous human scars 3 months after wounding. These findings suggest that the complete renin–angiotensin system is present in human skin and plays a role in normal cutaneous homeostasis as well as in human cutaneous wound healing. [ABSTRACT FROM AUTHOR]

Ständer S, Moormann C, Schumacher M, Buddenkotte J, Artuc M, Shpacovitch V, Brzoska T, Lippert U, Henz BM, Luger TA, Metze D, Steinhoff M. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells and epithelial cells of appendage structures. The vanilloid receptor subtype 1 (VR1)/(TRPV1), binding capsaicin, is a non-selective cation channel that recently has been shown in human keratinocytes in vitro and in vivo. However, a description of VR1 localization in other cutaneous compartments in particular cutaneous nerve fibers is still lacking. We therefore investigated VR1 immunoreactivity as well as mRNA and protein expression in a series ( n = 26) of normal ( n = 7), diseased ( n = 13) [prurigo nodularis (PN) ( n = 10), generalized pruritus ( n = 1), and mastocytosis ( n = 2)], and capsaicin-treated human skin ( n = 6). VR1 immunoreactivity could be observed in cutaneous sensory nerve fibers, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands. Upon reverse transcriptase-polymerase chain reaction and Western blot analysis, VR1 was detected in mast cells and keratinocytes from human skin. In pruritic skin of PN, VR1 expression was highly increased in epidermal keratinocytes and nerve fibers, which was normalized after capsaicin application. During capsaicin therapy, a reduction of neuropeptides (substance P, calcitonin gene-related peptide) was observed. After cessation of capsaicin therapy, neuropeptides re-accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a major role for this receptor, e.g. in nociception and neurogenic inflammation. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]