Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents

Please cite this paper as: Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents. Experimental Dermatology 2010; 19: 302–304. Abstract: Women predominate in the anaphylactic reactions to neuromuscular blocking agents (NMBA). The expression of oestrogen receptors has been demonstrated in mast cells and oestrogen treatment can enhance mast cell degranulation, but the influence of androgens remains largely unclear. Our immunocytochemical study showed the expression of androgen receptor (AR) in mast cells isolated from human foreskin as well as in two human mast cell lines, HMC‐1 and LAD2. The amount of AR was most abundant in human skin mast cells as determined by real‐time polymerase chain reaction analysis. Treatment of the HMC‐1 mast cells with testosterone or 17β‐oestradiol, alone or in combination with different NMBA, did not affect mast cell degranulation as measured by the release of β‐hexosaminidase. Our study shows for the first time the expression of AR in human skin mast cells. Further studies using primary human mast cell cultures are needed to understand whether and how sex hormones can influence mast cell activation.     

Dihydroavenanthramide D inhibits mast cell degranulation and exhibits anti‐inflammatory effects through the activation of neurokinin‐1 receptor

Chronic pruritus is difficult to treat. Current treatment options are frequently ineffective and new therapeutic approaches are urgently needed. Avenanthramides are active substances in oats that exhibit anti‐inflammatory effects. Their potential to interrupt pruritus mechanisms was investigated in this study. It was found that the synthetic analog dihydroavenanthramide D (DHAvD) can interact with the neurokinin‐1 receptor (NK1R) and inhibit mast cell degranulation. DHAvD also affects inflammatory processes and reduces secretion of the cytokine interleukin‐6. Our findings indicate that DHAvD may act as a NK1R inhibitor and could be a promising candidate for topical treatments of chronic pruritus.     

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.     

An efficient method for gene knock‐down by RNA interference in human skin mast cells

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 siRNA entities. The siRNA 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.     

Mouse mast cell cytokine production: rôle in cutaneous inflammatory and immunological responses

Abstract The rôle of mast cells in cutaneous physiology and pathology has long been a subject of intense speculation and great clinical interest. In this brief review, which is focused primarily on murine systems, we outline certain important aspects of the biology of the mast cell, including their ability to produce a variety of cytokines, describe the development of a model system (the “mast cell knock-in mouse”) for studying the roles of cutaneous mast cells in biological responses in vivo, and illustrate a few examples of how this approach has been used to investigate the contributions of mast cells and their cytokines to cutaneous inflammatory or immune responses.     

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.     

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.     

Regulation of mast cell characteristics by cytokines: divergent effects of interleukin-4 on immature mast cell lines versus mature human skin mast cells

Mast cells (MC) are of hematopoietic origin but complete their differentiation exclusively within tissues. The mediators that positively or negatively affect the maturation process are incompletely defined. Here, the human MC line HMC-1 (subclone 5C6) was used along with several treatments (IL-4, IL-6, NGF), either alone or in combination, and MC differentiation was monitored by flow-cytometric analysis of c-kit, tryptase, and FcRI expression. Of the different treatments, IL-4 displayed the clearest effects by suppressing the expression of the three markers and inhibiting cellular growth, while the other cytokines had no (NGF) or negligible (IL-6) effects only. The downregulating effects of IL-4 could not be overcome by any other treatment. There is some controversy in the literature as to the impact of IL-4 on the MC lineage. To determine whether the effects from IL-4 were differentiation stage dependent, two further human MC subsets (skin MC and LAD 2 cells) were investigated. No effects on c-kit and FcRI expression were noted when terminally differentiated skin MC were used as target cells, while a modest downregulation of c-kit was observed with intermediately matured LAD 2 cells. In sharp contrast to HMC-1 5C6 cells, the survival of skin MC was significantly enhanced by IL-4 treatment. Our data therefore imply that at a lower maturation stage, IL-4 acts as a negative regulator of the MC lineage, but that this property disappears or is even reversed upon terminal differentiation of the cell. Our study provides direct proof that the effects of IL-4 vary substantially in the course of MC maturation.     

Mast cells protect from skin tumor development and limit tumor growth during cutaneous de novo carcinogenesis in a Kit‐dependent mouse model

Epidermal tumors belong to the most frequent type of neoplasms, and tumor‐associated accumulation of mast cells (MCs) has first been observed more than a century ago. Therefore, MCs have been implicated in tumor development and growth; however, the results regarding the role of MC in cutaneous de novo carcinogenesis are still controversially discussed. Here, we subjected MC‐deficient Kit^W/Kit^W?v mice to chemical skin carcinogenesis. Tumors were induced using the carcinogen 7,12‐dimethylbenz[a]‐anthracene and subsequent treatment with the tumor promoter 12‐tetradecanoyl‐phorbol‐13‐acetat. The treatment resulted in pronounced inflammatory cell infiltrates that were diminished in MC‐deficient animals. Unexpectedly, tumor development and growth was significantly increased in MC‐deficient Kit^W/Kit^W?v mice. The repair of their MC deficiency by local adoptive transfer of MCs normalized tumor incidence and growth. The recruitment of skin‐infiltrating immune cells, particularly of F4/80+ monocytes, Gr‐1+ granulocytes, B220+ B cells and CD8+ T lymphocytes, to sites of tumor development was, in part, also controlled by MCs. Recent evidence indicated the importance of local antitumor tissue immunity which prevents tumor development. These findings suggest a critical role for MCs in mediating these host antitumor immune responses in the skin.     

Absence of MHC class II antigen on mast cells at sites of inflammation in human skin

Abstract Since the presence of major histocompatibility complex (MHC) antigens has recently been reported on murine and human mast cells under various conditions, we have investigated their expression on mast cells in different types of cutaneous inflammation. Cryostat sections from lesional biopsies of patients with psoriasis, atopic eczema, chronic urticaria, lichen planus, bullous pemphigoid and urticaria pigmentosa were immunohistochemically stained with monoclonal antibodies against MHC class I and class II antigens using a double staining APAAP/toluidine blue methodology. While strongly positive staining with the antibody directed against MHC class I antigens was found on nearly all mast cells in normal skin and in inflammatory dermatoses, reactivity for HLA-DR and HLA-DQ antigens on mast cells could not be detected, except for less than 2% of cells with doubtful staining. Human mast cells therefore probably play no significant rôle as antigen-presenting cells in the conditions studied.     

Interleukin‐1 from epithelial cells fosters T cell‐dependent skin inflammation

Background Chronic inflammatory skin diseases such as atopic dermatitis and psoriasis are characterized by the infiltration of lymphocytes into the epidermal compartment. Several studies point to an active role of skin epithelial cells in the pathophysiology of such diseases. Objectives In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes. Methods We used a human coculture model with keratinocytes grown from epidermal stem cells of the outer root sheath of human hair follicles and autologous T cells. Results In our coculture system we observed a high production of interferon (IFN)‐γ, but not Th2 cytokines, in the presence of superantigen or antigen‐pulsed autologous monocytes. Critical parameters for this effect were: (i) T‐cell receptor activation, (ii) an intercellular adhesion molecule‐1/lymphocyte function‐associated antigen (LFA)‐1‐dependent interaction between keratinocytes and T cells, and (iii) secretion of interleukin (IL)‐1β. Remarkably, in the presence of activated T cells, epithelial cells seemed to be a more significant source of IL‐1β than monocytes. Application of the LFA‐1 blocker efalizumab or IL‐1 receptor antagonist anakinra enabled us to suppress completely the production of IFN‐γ by T cells in the coculture. Conclusions IL‐1 secretion and the physical contact between keratinocytes and activated, infiltrating T cells may be central for the development of chronic inflammatory skin conditions.     

Ablation of human skin mast cells in situ by lysosomotropic agents

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell‐mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu‐Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.     

Alopecia in IL‐10‐deficient mouse pups is c‐kit‐dependent and can be triggered by iron deficiency

Please cite this paper as: Alopecia in IL‐10‐deficient mouse pups is c‐kit‐dependent and can be triggered by iron deficiency. Experimental Dermatology 2010; 19: 518–526. Abstract: Hair loss (alopecia) can result from a variety of metabolic, endocrine, immunologic, and environmental causes. This investigation was undertaken to determine the mechanisms underlying the sporadic development of alopecia in litters from C57BL/6 interleukin‐10‐deficient (Il10^?/?) mice. All pups in affected litters demonstrated alopecia by postnatal days 17–19, with hair loss from their trunks but not from their head, base of tail, or feet. Histopathology revealed distorted hair follicles containing broken hair shafts and prominent dermal infiltrates containing increased numbers of activated mast cells. Hair re‐growth began soon after weaning, suggesting that the alopecia was triggered by factors transmitted during lactation. Milk from Il10^?/? dams induced macrophage secretion of pro‐inflammatory cytokines in vitro regardless of whether or not their pups developed alopecia. Feeding dams a diet containing 3–6 ppm iron increased the percentage of litters with alopecia to 100% for pups with mast cells, with 0% alopecia in mast cell‐deficient pups. When dams were fed a diet containing 131 ppm iron, significantly lower haemoglobin and hematocrit values were observed in pups from litters with alopecia (71%; 5 of 7 litters) compared to litters without alopecia. Genetic or pharmacologic inhibition of c‐kit that resulted in depletion of mast cells in pups prevented hair loss in at‐risk litters. These studies demonstrate that maternal iron‐restricted diets enhance the incidence of alopecia in IL‐10‐deficient mouse pups and suggest mast cells as potential effector cells. Further studies are indicated to further explore the mechanisms involved and to determine how mast cells may contribute to alopecia in humans.     

Effects of basophil-priming and stimulating cytokines on histamine release from isolated human skin mast cells

Cell priming and stimulation of different cytokines (which include chemokines and growth factors) are typical features of human basophils. Recently, it has been shown that the macrophage chemotactic protein-1 (MCP-1), RANTES and macrophage inflammatory protein-1α (MIP-1α) are potent direct secretagogues for human basophils and that interleukin-3 (IL-3), IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are priming factors for subsequent potentiation of mediator release from basophils induced by different stimuli. This observation may be clinically important for the activation and recruitment of inflammatory cells in different immune responses of the skin (e.g. late-phase reactions). The aim of the present study was to investigate whether cytokines and chemokines are also capable of priming or stimulating isolated human skin mast cells (SMC). SMC were either stimulated directly with the cytokines alone or preincubated with these factors for 10 min before being activated with suboptimal concentrations of anti-IgE, A23187 or substance P. IL-3, IL-5, GM-CSF, platelet factor-4 (PF-4), IL-8, MCP-1 and MIP-1α (each at concentrations of 1 ng/ml to 1 μg/ml, log steps) did not significantly modulate histamine release from SMC induced by the three different secretagogues. RANTES exhibited a weak but significant potentiating effect on IgE-mediated activation. Stem cell factor (SCF) as a positive control was able to prime mast cell histamine release strongly. In addition, PF-4, MCP-1, RANTES and MIP-1α were incapable of inducing direct histamine release from SMC. In experiments with isolated human peripheral basophils, however, we observed potent Fc _ε RI-mediated priming effects evoked through IL-3, IL-5, and GM-CSF. We conclude that SMC derived from healthy donors are not targets of (immuno)modulatory factors that prime or stimulate basophils. Received: 21 June 1995     

Mast cells are critical for the limitation of thrombin‐induced skin inflammation

Thrombin, a key player in coagulation, is widely held to induce and promote inflammation. As of now, the features, kinetics and control of thrombin's proinflammatory effects on the skin remain to be characterized in detail. We, therefore, injected thrombin into the ear skin of mice and observed strong, dose‐dependent and transient ear swelling responses as well as mast cell (MC) degranulation. Unexpectedly, thrombin induced even stronger, not reduced, ear swelling in MC‐deficient Kit^{W‐sh/W‐sh} mice. Prior local reconstitution of Kit^{W‐sh/W‐sh} mice with MCs inhibited this effect, indicating that MCs may contribute to the control of thrombin‐induced skin inflammation. In line with previous studies, we found that MCs express the thrombin receptors PAR1, PAR3 and PAR4, thrombin induces direct and dose‐dependent MC degranulation, and that degranulated MCs inactivate thrombin. Further findings suggested that MC‐mediated protection from thrombin‐induced inflammation is likely to rely on the effects of MC proteases. We show for the first time that MC‐deficient mice and MC protease 4‐deficient mice with normal numbers of MCs show markedly increased ear swelling in response to thrombin as compared to wild‐type mice. Taken together, these results suggest that thrombin‐induced skin inflammation is controlled, in part, by MC protease 4 released from activated MCs. For MC‐driven diseases such as chronic spontaneous urticaria, which has been linked to increased thrombin generation, this might mean that MCs may contribute to the resolution of skin inflammatory responses.     

Extracellular superoxide dismutase ameliorates house dust mite‐induced atopic dermatitis‐like skin inflammation and inhibits mast cell activation in mice

Extracellular superoxide dismutase (EC‐SOD) is an enzyme that catalyses the dismutation of superoxide anions. It has multiple functions, such as reactive oxygen species scavenging, anti‐angiogenic, anti‐inflammatory, antichemotatic and antitumor activities. Recently, we demonstrated that EC‐SOD inhibits ovalbumin‐induced allergic airway inflammation in mice. However, the anti‐allergic effect of EC‐SOD on skin tissue and the role of EC‐SOD in mast cells, which are important for allergic responses, have not been well studied. In this study, we investigated whether EC‐SOD can alleviate atopic dermatitis in mice and inhibit mast cell activation. Treatment with human recombinant EC‐SOD ameliorated house dust mite‐induced atopic dermatitis in mice. Furthermore, the levels of pro‐allergic cytokine gene expression and histamine release increased in EC‐SOD KO mast cells and decreased in EC‐SOD overexpressing mast cells, suggesting that EC‐SOD inhibits mast cell activation. Consistently, a passive cutaneous anaphylaxis experiment showed more blood leakage from EC‐SOD KO mouse ear skin, implying that the lack of EC‐SOD increases allergic responses. These results suggest that EC‐SOD inhibits mast cell activation and atopic dermatitis and that the loss of EC‐SOD causes more severe allergic responses, implying that EC‐SOD might be a good drug candidate for treatment of allergic disorders, such as atopic dermatitis.     

Murine mast cells secrete a unique profile of cytokines and prostaglandins in response to distinct TLR2 ligands

Abstract:  Mast cells (MCs) are important effector cells in host defense against bacteria. In the course of a bacterial infection, MCs can be activated by various mechanisms, i.e. bacterial toxins, endogenously produced infection-associated peptides or via complement receptors, fimbrial adhesion molecules and toll-like receptors (TLRs). While some of these mechanisms are well established, the effects of TLR2 ligand-driven MC activation are far less understood. Here, we show that murine mature connective tissue-type MCs, but not immature bone marrow-derived cultured mast cells, express significant amounts of full length TLR2 on their surface. Activation by various TLR2 ligands only induces the selective release of cytokines in peritoneum-derived cultured mast cells (PCMCs) with preferential secretion of pro-inflammatory cytokines (IL-6 > IL-17 > IFN-γ TNF > IL-1 > GM-CSF) upon stimulation with lipoteichoic acid (LTA). This response is much lower in PCMCs stimulated with the TLR2/6 agonist macrophage-activating lipopeptide-2 (MALP-2), which most prominently triggers the release of the immunomodulatory cytokine IL-10. Furthermore, only LTA but not MALP-2 induces prostaglandin D2 secretion which is again restricted to the mature MC phenotype. These findings suggest that TLR2 ligand-mediated activation of mature MCs, i.e. tissue-residing cells, which most likely occurs during infection, can selectively raise a potent inflammatory or anti-inflammatory response, depending on TLRs which are engaged.