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.     

Substance P-induced histamine release in human cutaneous mast cells

Substance P is an undecapeptide found in multiple sites throughout the central and peripheral nervous systems including small unmyelinated (type C) cutaneous nerve fibers. Previous studies demonstrated that antidromic stimulation results in substance P (SP) release from nerve endings, SP stimulates histamine release (HR) from rat mast cells in vitro, and intradermal SP in humans produces wheals identical to those induced by histamine. These studies suggest a possible role for SP as a link between neurologic events and cutaneous mast cell-mediated reactions. We therefore investigated SP-induced HR in an in vitro preparation of human skin mast cells. Human foreskin sections were incubated with varying concentrations of SP. Histamine was assayed using automated fluorimetry and release was calculated as a percentage of total tissue histamine. Substance P caused dose-dependent HR over a range from 10(-5) M (1.3%) to 5 X 10(-4) M (25.1%). Histamine release was optimal at 3 mM calcium and was blocked by pretreatment with calcium chelation. Naloxone failed to block HR. These studies suggest that HR from skin mast cells by SP may play a role in neural modulation of poorly understood inflammatory skin conditions.     

Corticotropin-releasing hormone receptor-1 and histidine decarboxylase expression in chronic urticaria

Certain skin disorders, such as contact dermatitis and chronic urticaria, are characterized by inflammation involving mast cells and worsen by stress. The underlying mechanism of this effect, however, is not known. The skin appears to have the equivalent of a hypothalamic-pituitary-adrenal (HPA) axis, including local expression of corticotropin-releasing hormone (CRH) and its receptors (CRH-R). We have reported that acute stress and intradermal administration of CRH stimulate skin mast cells and increase vascular permeability through CRH-R1 activation. In this study, we investigated the expression of CRH-R1, the main CRH-R subtype in human skin, and the mast cell related gene histidine decarboxylase (HDC), which regulates the production of histamine, in normal and pathological skin biopsies. Quantitative real time PCR revealed that chronic urticaria expresses high levels of CRH-R1 and HDC as compared to normal foreskin, breast skin and cultured human keratinocytes. The lichen simplex samples had high expression of CRH-R1, but low HDC. These results implicate CRH-R in chronic urticaria, which is often exacerbated by stress.     

Substance P induces the expression of an endothelial-leukocyte adhesion molecule by microvascular endothelium.

Leukocyte trafficking in normal and diseased skin appears to be initially governed by endothelial surface glycoproteins that promote adhesive interactions with circulating leukocytes. In a separate study, we have demonstrated that one of these glycoproteins, endothelial-leukocyte adhesion molecule-1 (ELAM-1), is rapidly induced on postcapillary dermal venules as a direct consequence of experimentally-elicited degranulation of adjacent mast cells (Proc Natl Acad Sci USA 86:8972-8976, 1989). A principle endogenous mediator of mast cell degranulation is the neuropeptide substance P. In this study, we exposed organ cultures of neonatal human foreskins for 45 min to substance P or to a substance P analogue (D-pro4, D-trp7,9)SP(4-11) that binds to the identical mast cell surface receptor but which does not provoke histamine release. Dermal mast cells were uniformly degranulated only in explants exposed to substance P, as judged by ultrastructural analysis. After subsequent culture in medium alone for 6 h, superficial venules of explants exposed to substance P showed evidence of ELAM-1 induction, as documented histochemically using H4/18 monoclonal antibody. ELAM-1 was not induced by substance P analogue. Furthermore, preincubation of explants with analogue or with the mast cell inhibitor, cromolyn sodium, abrogated the ability of substance P to induce ELAM-1. From these results we suggest that substance P endogenously released by dermal nerve fibers upon physiologic or electrical stimulation may be important in the regulation of endothelial-leukocyte interactions in vivo. This concept provides further evidence for a neurogenic and psychogenic modulation of the immune response, and may be relevant to the course of naturally occurring dermatoses (e.g., psoriasis) that are commonly exacerbated by emotional stress.     

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.     

Ultraviolet irradiation induces apoptosis in human immature, but not in skin mast cells

As diverse pruritic cutaneous diseases respond to ultraviolet treatment, we have examined whether ultraviolet light is capable of inducing apoptosis in mast cells. Human mast cell line 1 (HMC1) derived from a patient with malignant mastocytosis and purified skin mast cells were irradiated with single doses of ultraviolet B or ultraviolet A1, or pretreated with 8-methoxypsoralen prior to ultraviolet A1 exposure. After 0 to 48 h of incubation, the percentage of apoptotic and dead cells was assessed. In HMC1 cells, morphologic features of apoptosis were further evaluated by electron microscopy. All ultraviolet treatment induced apoptosis of HMC1 cells in a time- and dose-dependent manner. Apoptosis was associated with activation of caspase-3, release of cytochrome C, cleavage of poly(ADP-ribose)-polymerase, and nuclear accumulation of p53. In contrast, resting skin mast cells were resistant to ultraviolet light induced apoptosis. After incubation with stem cell factor and interleukin-4 for 2 wk, however, slowly proliferating skin mast cells also underwent apoptosis in response to ultraviolet light. In conclusion, these data demonstrate that ultraviolet light directly affects mast cells, but mainly aims at the proliferating mast cells as found in mastocytosis and mast cell dependent pruritic diseases, where increased numbers are observed due to the recruitment mast cell precursors from the blood.     

Substance P induces inositol 1,4,5-trisphosphate and intracellular free calcium increase in cultured normal human epidermal keratinocytes

Abstract Substance P is a neuropeptide which is present in peripheral C nerve endings and released from them. Free nerve endings of C nerve are present in human epidermis. The effects of substance P on the transmembrane signaling system of pig epidermal sheets were previously reported. In these studies, a small amount of cells other than keratinocytes contaminated the epidermal sheets and the species difference from human was also noticed. Therefore we investigated the effects of substance P on cultured normal human epidermal keratinocytes. Alteration of intracellular free calcium (Ca²⁺) in single living keratinocytes was studied using an inverted fluorescence microscope and Ca²⁺ -sensitive dye, Fura 2-AM. Treatment of normal human epidermal kertinocytes with substance P resulted in an increase in inositol 1,4,5-trisphosphate and in intracellular Ca²⁺. Substance P inhibited DNA synthesis of the keratinocytes in a dose-dependent manner. These results are consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate hydrolysis of human keratinocytes, resulting in inositol 1,4,5-trisphosphate-Ca²⁺ signal.     

Protease-activated receptor-1 (thrombin receptor) is expressed in mesenchymal portions of human hair follicle

Protease nexin-1, a serine protease inhibitor, is expressed specifically in the dermal papilla (DP) of anagen hair follicles and is suggested to be one of the modulators of the cyclic growth of hair follicles. Accumulating evidence has shown that protease nexin-1 plays its biologic role by inhibiting thrombin action in various systems other than the hair follicle. Thrombin has various physiologic functions including blood coagulation cascade, mostly via activation of protease-activated receptors (PAR). In this study, we investigated the expression of PAR mRNA using RT-PCR in dissected human hair follicles. We showed that PAR-1 mRNA was expressed specifically in the mesenchymal portions, including DP and connective tissue sheath, of anagen hair follicles. Furthermore, immunoreactivity for PAR-1 was detected in the DP and lower portion of connective tissue sheath in the anagen and catagen phases and in the DP of telogen hair follicles. Because only a pharmacologic level (100 nM) of thrombin significantly stimulated cell proliferation and DNA synthesis of the cultured dermal papilla cells, thrombin does not seem to have a mitogenic effect on dermal papilla cells physiologically. These results raise the possibility that thrombin is involved in the cyclic hair growth through its receptor of PAR-1.     

Topical tretinoin increases dermal mast cells,induces epidermal mast cell growth factor (c-kit ligand) and modulates its distribution in hairless mice

In previous studies we have noted that mast cells are increased in tretinoin-treated photoaged hairless mouse skin. Because UV radiation is known to increase mast cell numbers, we were interested in whether tretinoin alone would modulate the mast cell population in unirradiated mice. Animals were treated topically with 0.05% tretinoin, 5 days a week, for 2, 4, 6, 8 and 10 weeks. Untreated and vehicle controls were included. Biopsies were processed for light microscopy and stained with toluidine blue. Mast cells in the upper and lower dermis were scored separately under high magnification. After 2 weeks of tretinoin, mast cells in the upper dermis were significantly increased, as indicated by the appearance of small, moderately metachromatically granulated cells near the dermal-epidermal junction. Mast cells in the lower dermis, the site of a granulomatous reaction, were large, densely granular and significantly increased after 6 weeks of treatment. Immunohistochemical evaluation for mast cell growth factor (MGF) revealed a marked increase in keratinocyte cytoplasmic staining by week 2. After 4–6 weeks, membrane-associated or intercellular staining was evident. Cells in the upper dermis also showed membrane reactivity for MGF. By 8–10 weeks, epidermal MGF reactivity had dissipated in the more basal keratinocytes. These findings show that topical tretinoin can induce epidermal MGF along with an associated mast cell hyperplasia. It is suggested that the two populations of dermal mast cells may have different functions.     

扁平苔藓皮损中肥大细胞数和P物质的表达及其意义

目的:探讨肥大细胞和P物质在扁平苔藓发病中的作用。方法:采用组织化学和免疫组化的染色方法,检测扁平苔藓皮损中未脱颗粒的肥大细胞数和P物质的表达情况。结果:甲苯胺蓝特殊染色发现,扁平苔藓皮损中未脱颗粒的肥大细胞数(3.56±1.52)明显低于正常对照组(5.22±1.28),其差异具有统计学意义(P<0.01);免疫组化染色发现,扁平苔藓皮损中P物质的表达强度明显高于正常对照组,其差异具有统计学意义(P<0.01);采用Spearman等级相关分析,二者呈一定的负相关(rs=-0.397,P<0.05)。结论:肥大细胞和P物质在扁平苔藓的发病中可能起一定的作用。     

Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis

Substance P is a neuropeptide present in, and released from, peripheral C nerve endings. The presence of substance P-positive nerve fibres in the epidermis has been reported. We investigated the effect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5-trisphosphate (IP3), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the beta-adrenergic- or histamine (H2)- adenylate cyclase responses of the epidermis. Neither forskolin-induced, nor cholera toxin-induced cyclic AMP accumulation were affected by substance P treatment. These results consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis of keratinocytes, resulting in IP3-Ca2+ and diacylglycerol-protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyclase system, no evidence for such 'cross-talk regulation' was detected in keratinocytes by substance P treatment.