Dimethylfumarate induces apoptosis in human mast cells

Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC‐1 and in primary cord blood‐derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca‐MF). Our data show that only DMF potently induces apoptotic cell death in HMC‐1 cells and CBMCs. DMF‐mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase‐9 and caspase‐6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL‐ and dexamethasone‐induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells.     

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.     

Evidence for altered mast cell proliferation and apoptosis in cutaneous mastocytosis

BACKGROUND: Mastocytosis presents as a focal or generalized increase of mast cells, particularly in the skin, but also in other organs. Activating mutations of KIT (formerly c-kit), the receptor of the mast cell growth factor stem cell factor (SCF), appear to play a key role in the pathogenesis of sporadic adult onset mastocytosis. However, these mutations are not present in childhood-onset and familial mastocytosis and also fail to explain the heterogeneity of adult-onset disease. Other factors such as prolonged survival of mast cells may therefore participate in causing and modulating the pathological increase of mast cells in mastocytosis. OBJECTIVES: To examine the expression of proliferation and apoptosis markers in the mast cells of cutaneous mastocytosis lesions in order to gain further insight into the pathogenesis of mastocytosis. METHODS: Lesional cutaneous biopsies from eight infants with solitary mastocytomas, five children with multiple mastocytomas, 11 children with generalized urticaria pigmentosa, 12 adults with urticaria pigmentosa, and skin from seven normal controls were used in this study. Serial sections were stained with toluidine blue to quantify mast cell numbers and with antibodies against the proliferation marker Ki67 protein, the tumour suppressor protein p53, and the inhibitor of cyclins and cyclin-dependent kinases p21WAF1/CIP1, using the alkaline phosphatase antialkaline phosphatase technique. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) method was used to assess apoptosis. RESULTS: Cutaneous mast cell counts were significantly increased in all patient sections, particularly in childhood lesions, and similarly, a small but significant increase of proliferation was found in the lesional mast cells of all patients. Enhanced mast cell numbers and proliferation was associated with a significant decrease of TUNEL staining, particularly in mastocytomas. p53 expression was highly variable, with an overall significant increase in all patient skin mast cells, whereas p21 expression was barely observed at all. CONCLUSIONS: These findings further support the concept that an imbalance of mast cell proliferation and apoptosis is prevalent in mastocytosis lesions that may account in part for the increased focal mast cell accumulation in this condition.     

SLAM family member 8 is involved in oncogenic KIT‐mediated signalling in human mastocytosis

The signalling lymphocytic activation molecule family member 8 (SLAMF8)/CD353 is a member of the CD2 family of proteins. Its ligand has not been identified. SLAMF8 is expressed by macrophages and suppresses cellular functions. No study has yet explored SLAMF8 expression or function in human mastocytosis, which features oncogenic KIT‐mediated proliferation of human mast cells. SLAMF8 protein was expressed in human mastocytosis cells, immunohistochemically. SLAMF8 expression was also evident in the human mast cell lines, HMC1.2 (expressing oncogenic KIT) and LAD2 (expressing wild‐type KIT) cells. SLAMF8 knock‐down significantly reduced the KIT‐mediated growth of HMC1.2 cells but not that of LAD2 cells. SLAMF8 knock‐down HMC1.2 cells exhibited significant attenuation of SHP‐2 activation and oncogenic KIT‐mediated RAS‐RAF‐ERK signalling. An interaction between SLAMF8 and SHP‐2 was confirmed in HMC1.2 cells and all pathological mastocytosis specimens examined (19 of 19 cases, 100%). Thus, SLAMF8 is involved in oncogenic KIT‐mediated RAS‐RAF‐ERK signalling and the subsequent growth of human neoplastic mast cells mediated by SHP‐2. SLAMF8 is a possible therapeutic target in human mastocytosis patients.     

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.     

Cytokine treatment of mast-cell-mediated skin diseases

Abstract Cytokines released by mast cells as well as their effects on mast cell functions appear to be of major importance in the pathogenesis of mast cell mediated skin diseases. In addition, the identification of some key mediators which were found to play a crucial in vivo rôle in certain disease states may allow the development of new therapeutie strategies using cytokines or cytokine antagonists for the treatment of inflammatory skin diseases. So far, encouraging results have been obtained when diseases with mast cell involvement such as atopic eczema or mastocytosis have been treated with different IFNs. Future trials using IFNs and other newly detected cytokines or their antagonists are required to establish effective therapy regimens.     

UVA1 phototherapy: a concise and practical review

High intensity long-wavelength ultraviolet A (340-400 nm; UVA1) lamps were initially developed as skin research tools; over time they have proven to be useful for treating a number of chronic dermatoses. UVA1 units and dosimetry are strikingly different from conventional UV phototherapy. The therapeutic effect of UVA1 is related to the fact that its long wavelength penetrates the dermis more deeply than UVB. UVA1 radiation induces collagenase (matrix metalloproteinase-1) expression, T-cell apoptosis, and depletes Langerhans and mast cells in the dermis. UVA1 exposure stimulates endothelial cells to undergo neovascularization. Ultraviolet A1 exerts significant therapeutic effects in atopic dermatitis and morphea; there is also evidence for its use in other skin diseases, including cutaneous T-cell lymphoma and mastocytosis.     

Atypical maculopapular cutaneous mastocytosis showing a nevus spilus‐like lesion

A 13‐year‐old Korean girl presented with a 7‐year history of a pruritic, light‐brown patch containing multiple 0.2‐ to 0.5‐cm brownish‐to‐reddish maculopapules on the left anterior chest. When her skin was rubbed, the lesion became itchy and red. Histopathologic evaluation demonstrated marked dense dermal infiltration of mast cells. We report a rare case of atypical maculopapular cutaneous mastocytosis with clinical features similar to those of nevus spilus.     

Urinary N-methylhistamine as an indicator of bone marrow involvement in mastocytosis

Thirty-seven patients with mastocytosis and unexplained elevated levels of urinary N-methylhistamine who were undergoing bone marrow biopsy were studied with respect to the diagnosis of mastocytosis and the manifestations of the disease. These patients were from a group of 66 patients from whom a bone marrow biopsy was obtained and urinary N-methylhistamine levels were measured in the period 1990-1998. In seven (19%) of the 37 patients, mastocytosis was limited to the skin. Five (14%) of the 37 patients showed accumulation of mast cells in the bone marrow without characteristic skin lesions, whereas seven (19%) of the 37 patients showed increased numbers of mast cells both in the skin and the bone marrow. Eighteen (49%) of the 37 patients with elevated N-methylhistamine did not have mast cell accumulation in either the skin or the bone marrow biopsy. The median level of N-methylhistamine in the urine of patients with mastocytosis limited to the skin was 245 micro mol/mol creatinine. The average level of N-methylhistamine was 509 micro mol/mol creatinine in patients with mast cell accumulation in the bone marrow and cutaneous mastocytosis. There was a significant difference in the levels of N-methylhistamine in patients with mast cell accumulation in the bone marrow biopsy compared with those without. The likelihood of mastocytosis with mast cell accumulation in the bone marrow biopsy at a given level of N-methylhistamine was calculated. It was established that an N-methylhistamine level of 297 micro mol/mol creatinine or higher may be considered as a threshold indicator for obtaining a bone marrow biopsy in patients suspected of mastocytosis with mast cell accumulation in the bone marrow. For practical purposes, we propose to consider the cut-off level of approximately 300 micro mol/mol N-methylhistamine creatinine for this assay.     

Ultraviolet radiation and skin mast cells: Effects,mechanisms and relevance for skin diseases

Mast cells (MCs) are well known as versatile effector cells in allergic reactions and several other immune responses. Skin MCs and cutaneous MC responses are subject to the effects of environmental factors including ultraviolet radiation (UVR). Numerous studies have assessed the effects of UVR on MCs, in vitro and in vivo. Interestingly, UVR seems to have variable effects on non‐activated and activated mast cells. In general, UV therapy is beneficial in the treatment of urticaria and mastocytosis, but the effects are variable depending on treatment regimen and type of UVR. Here, we review and summarise key reports from the older and current literature on the crosstalk of UVR and skin MCs. Specifically, we present the literature and discuss published reports on the effects of UVR on skin MCs in rodents and humans. In addition, we review the role of MCs in UVR‐driven skin diseases and the influence of UV light on MC‐mediated skin diseases. This summary of our current understanding of the interplay of skin MCs and UVR may help to improve the management of patients with urticaria and other MC disorders, to identify current gaps of knowledge, and to guide further research.     

The skin in mastocytosis.

The most frequent site of organ involvement in patients with any form of mastocytosis is the skin. Cutaneous expressions include urticaria pigmentosa, mastocytoma, diffuse and erythrodermic cutaneous mastocytosis, and telangiectasia macularis eruptiva perstans. The cutaneous lesions tend to appear early in life. Although urticaria pigmentosa has been reported in 12 pairs of twins and one set of triplets, the majority of affected individuals have no familial association. Most patients with systemic mastocytosis have skin lesions; however, an occasional patient will have systemic disease with no other skin features than flushing. In lesional cutaneous sites and in non-lesional skin, there is an increase in the number of mast cells. Electron microscopy shows quantitative differences between lesional skin mast cells from patients with and without systemic disease. The mast cells from adult patients with systemic disease have a larger mean cytoplasmic area, nuclear size, and granule diameter. The granules contain predominantly grating/lattice structures. The cutaneous mast cells contain tryptase and chymase. They retain their functional reactivities to relevant secretory stimuli, such as C3a, morphine sulfate, and calcium ionophore A23187. Lesional skin contains histamine, leukotriene B4, prostaglandin D2, 5-hydroxyeicosatetraenoic acid, platelet-activating factor, and heparin. Treatment of the cutaneous manifestations includes the use of H1 and H2 antihistamines, oral disodium cromoglycate, psoralens plus ultraviolet A photochemotherapy, and potent topical corticosteroid preparations.     

"Smoldering systemic mastocytosis. "Successful therapy with cladribine

Mastocytoses are a heterogenous group of diseases characterized by proliferation and accumulation of mast cells in the skin and other organs. They are subdivided into cutaneous mastocytoses; systemic forms, which may appear with or without skin lesions; mast cell sarcomas and extracutaneous, localized, benign mastocytomas. Systemic mastocytoses apart from the skin mainly involve bone marrow, gastrointestinal tract, bones, lymph nodes, spleen and liver. Whereas indolent forms of systemic mastocytosis are mainly treated with antihistamines, glucocorticosteroids and PUVA therapy, the more aggressive forms, including mast cell leukemia, often require cytostatic chemotherapy. A 53-year old patient with beginning "smoldering systemic mastocytosis" failed to respond to high-dose systemic glucocorticosteroids and interferon-alpha. Treatment with cladribine led to an impressive improvement of skin lesions, a significant decrease in tryptase serum levels and stabilization of bone marrow infiltrates.     

Mast cell quantitation by image analysis in adult mastocytosis and inflammatory skin disorders

Mast cell numbers were quantitated in adult cases of mastocytosis demonstrating non-diffuse perivascular and upper dermal concentrations of mast cells. Using the Leder stain and computerised video image analysis, a mean of 382 (+/- 28 SE) mast cell per mm2 were counted in the superficial dermis in skin biopsies from 30 adult cases of mastocytosis, in contrast to a mean of 43 (+/- 5 SE) mast cells per mm2 in skin biopsies from 50 inflammatory dermatoses represented by subacute dermatitis, pigmented purpuric dermatosis, erythema multiforme, lichen planus and granuloma annulare. Ten skin biopsies showing no significant inflammation had a mean of 54 (+/- 7 SE) mast cells per mm2 in the upper dermis. The mean area of individual mast cells as assessed by image analysis in the mastocytosis group was 47.40 microns 2 (+/- 2.26 microns 2, SE) which was significantly different (P < 0.01) than the mast cell area (32.34 microns 2 +/- 2.22 microns 2, SE) in all other groups combined. Computerised video image analysis represents an alternative technique which is useful in assessing mast cell numbers and particularly mast cell size in adult cases of macular mastocytosis and in other dermatoses.     

Pleomorphic mastocytoma in an adult

Cutaneous mastocytoma (CM) is a localized variant of mastocytosis, characterized by an over‐accumulation of mast cells in the skin, without extra‐cutaneous organ involvement. It is defined as the presence of up to 3 isolated mast‐cell skin lesions and commonly develops in newborns and children. We report the case of a 35‐year‐old healthy Caucasian woman presenting with a 4‐year history of a pruritic brown plaque on her left breast. Hematoxylin‐eosin staining showed a dense dermal infiltrate of atypical mast cells extending to the subcutis. The cells presented a marked nuclear pleomorphism with bilobed and multilobed nuclei. Immunohistochemical studies revealed strongly expressed KIT (CD117) and CD25 proteins. Serum tryptase levels and bone marrow biopsy were normal. The diagnosis was a solitary cutaneous pleomorphic mastocytoma. This case can be added to 17 other cases of adult mastocytoma documented in the literature, although, unlike other reported cases, and as far as we are aware, this is the first case of pleomorphic mastocytoma in an adult.     

„Smouldering systemic mastocytosis“

Mastocytoses are a heterogenous group of diseases characterized by proliferation and accumulation of mast cells in the skin and other organs. They are subdivided into cutaneous mastocytoses; systemic forms , which may appear with or without skin lesions; mast cell sarcomas and extracutaneous, localized, benign mastocytomas. Systemic mastocytoses apart from the skin mainly involve bone marrow, gastrointestinal tract, bones, lymph nodes, spleen and liver. Whereas indolent forms of systemic mastocytosis are mainly treated with antihistamines, glucocorticosteroids and PUVA therapy, the more aggressive forms, including mast cell leukemia, often require cytostatic chemotherapy. A 53-year old patient with beginning “smoldering systemic mastocytosis” failed to respond to high-dose systemic glucocorticosteroids and interferon-α. Treatment with cladribine led to an impressive improvement of skin lesions, a significant decrease in tryptase serum levels and stabilization of bone marrow infiltrates.     

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

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

Histamine release from rat peritoneal mast cells exposed to ultraviolet light

After irradiation with ultraviolet light, rat peritoneal mast cells incubated with the histamine liberator compound 48/80 were found to have reduced capacity to release histamine. The action spectrum for histamine release inhibition seemed to be in the wavelength region of UVB. UV-light per se did not induce histamine liberation other than for dosages greater than or equal to 1.45 J/cm2. No such release was observed with the doses of UVA studied (3.4-20.5 J/cm2). The metabolic inhibitor 2.4-dinitrophenol (DNP), which almost completely inhibited compound 48/80-elicited histamine release, did not influence the UV-induced histamine release, indicating that the latter was not a secretory process but due to cytotoxic leakage of histamine from the cells. These results suggest that inhibition of histamine-releasing capacity or reduction of skin histamine due to photolysis of mast cells may explain the beneficial effect of UV in pruritic disorders where histamine release from mast cells is involved.     

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.     

In vitro functional reactivities of cutaneous mast cells from patients with mastocytosis

Cutaneous mast cells from 3 patients with mastocytosis were evaluated for their morphologic characteristics and in vitro functional reactivities to different secretory agonists. By electron microscopy, mastocytosis mast cells appeared larger than normal skin mast cells, frequently had atypical, highly indented or bilobed nuclei, and each contained numerous, elongated cytoplasmic projections. Suspensions of mastocytosis mast cells were obtained from lesional skin biopsy specimens, and their response to both immunologic and nonimmunologic secretagogues was compared with mast cells from normal skin. Lesional skin mast cells had a net histamine release of 12.3% (+/- 1.3 SEM) and 31.1% (+/- 6.0 SEM) following stimulation with the purified human anaphylotoxin C3a and mouse monoclonal antihuman IgE antibodies, respectively. This specific release was similar to the responses observed in normal skin mast cells (11.5% +/- 4.5 SEM and 16.7% +/- 2.1 SEM, respectively). Mast cells from cutaneous lesions of mastocytosis also responded to the nonimmunologic secretagogues, morphine sulfate and calcium ionophore A23187 with a specific histamine release of 15.1% (+/- 1.2 SEM) and 39.8% (+/- 8.7 SEM), respectively. The results of this study demonstrate that mast cells from lesions of mastocytosis are morphologically atypical, but have a histamine content similar to normal skin mast cells and retain their functional reactivities to clinically relevant secretory stimuli.