Steroidal anti-inflammatory drugs are widely used for the treatment of chronic cutaneous inflammation, such as atopic dermatitis, although it remains unknown how they modulate cutaneous mast cell functions. We investigated the effects of prolonged treatment with a synthetic glucocorticoid, dexamethasone, on murine connective tissue-type mast cells using in vitro and in vivo models. Our connective tissue-type bone marrow-derived cultured mast cell model was found to be sensitive to mast cell secretagogues, such as compound 48/80 and substance P, and higher expression levels of α subunit of a trimeric G protein, Gi1, and several Mas-related G protein-coupled receptor (Mrgpr) subtypes were observed in comparison with immature cultured mast cells. Secretagogue-induced degranulation and up-regulation of these genes was suppressed when cultured in the presence of dexamethasone. The profiles of granule constituents were drastically altered by dexamethasone. Topical application of dexamethasone down-modulated secretagogue-induced degranulation and the expression levels of several Mrgpr subtypes in cutaneous tissue. These results suggest that mast cell-mediated IgE-independent cutaneous inflammation could be suppressed by steroidal anti-inflammatory drugs through the down-regulation of G αi1 and several Mrgpr subtypes in mast cells.
Accumulating evidence suggests that activated mast cells are involved in contact hypersensitivity, although the precise mechanisms of their activation are still not completely understood. We investigated the potential of common experimental allergens to induce mast cell activation using murine bone marrow-derived cultured mast cells and rat peritoneal mast cells. Among these allergens, 1-chloro-2,4-dinitrobenzene and 1-fluoro-2,4-dinirobenzene (DNFB) were found to induce degranulation of rat peritoneal mast cells. DNFB-induced degranulation is accompanied by cytosolic Ca 2+ mobilization and is significantly inhibited by pertussis toxin, U73122 (a phospholipase C inhibitor), and BAPTA (a Ca 2+ chelator), raising the possibility that DNFB acts on the G protein-coupled receptors and activates G i , which induces activation of phospholipase C, as well as known mast cell secretagogues, such as compound 48/80. DNFB could induce mast cell degranulation in the absence of serum proteins and IgE. Structure-activity relationship analyses revealed an inverse correlation between the degree of degranulation and the electron density of the C1 carbon of the DNFB derivatives. These findings raise a possibility that DNFB functions as a potent contact allergen through induction of cutaneous mast cell degranulation.Keywords: Contact hypersensitivity r 1-Fluoro-2,4-dinitrobenzene r Histamine r Inflammation r Mast cells Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Satoshi Tanaka e-mail: tanaka-s@okayama-u.ac.jp IntroductionA series of recent studies have enriched our understanding about the pathology of contact hypersensitivity [1]. Contact hypersensitivity was initially recognized as an allergic responses induced C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2017. 47: 60-67 Innate immunity 61 mainly by activated T cells but now is regarded as the complex process involving many kinds of immune cells in addition to T cells. Chemical compounds, such as 1-fluoro-2,4-dinitrobenzene (DNFB), oxazolone, and picryl chloride, have been used for induction of contact hypersensitivity [2]. These models have revealed that self-proteins conjugated with these compounds induced Tcell-mediated immune responses, which required dendritic cell migration. Although it has long been controversial whether mast cells are involved in contact hypersensitivity, a series of the transfer experiments has indicated that IgE and mast cells could initiate immune responses in several models [3,4]. Recent studies have expanded the pathological roles of IgE and cutaneous mast cells in murine contact hypersensitivity models [5,6]. Bryce et al. demonstrated that successful contact hypersensitivity with oxazolone should require mast cells equipped with IgE [7]. Cutaneous mast cells were found to promote dendritic cell migration into the draining lymph node, which is regarded as the primary step of contact hypersensitivity, through the release...
Steroidal anti-inflammatory drugs are widely used for treatment of chronic cutaneous inflammation, such as atopic dermatitis, although it remains unknown how they modulate cutaneous mast cell functions. Murine connective tissue-type mast cells, which were sensitive to mast cell secretagogues, such as compound 48/80 and substance P, were generated by co-culture of bone marrow-derived mast cells with Swiss 3T3 fibroblasts in the presence of stem cell factor. This process was accompanied by up-regulation of a subunit of a trimeric G protein, Gi1, and several Mas-related G protein-coupled receptor (Mrgpr) subtypes. Secretagogue-induced degranulation and up-regulation of these genes were suppressed when they were cultured in the presence of a synthetic glucocorticoid, dexamethasone. The profiles of granule constituents were drastically altered by dexamethasone. Several Mrgpr subtypes were found to be expressed in the cutaneous tissues and their expression levels were decreased in response to topical application of dexamethasone. The numbers of degranulated cutaneous mast cells in response to compound 48/80 were decreased in mice treated with dexamethasone. These results suggest that mast cell-mediated IgE-independent cutaneous inflammation could be suppressed by steroidal anti-inflammatory drugs through down-regulation of Gai1 and several Mrgpr subtypes in mast cells.
Background: Steroidal anti-inflammatory drugs have been often used for therapy of chronic inflammatory diseases, such as atopic dermatitis, although the mechanism of their actions remains to be clarified in detail. We investigated the effects of one of steroidal anti-inflammatory drugs, dexamethasone, on the process of murine mast cell maturation and degranulation in response to various mast cell secretagogues. Methods: A mature mast cell model was established by performing co-culture of IL-3-dependent murine bone marrowderived cultured mast cells with Swiss 3T3 fibroblasts in the presence of c-kit ligand. Various indices related to mast cell maturation, such as granule protease expression, histamine content, and degranulation in response to various secretagogues, were compared between in the absence and presence of dexamethasone. Dexamethasone was applied on the surface of ear pinna of BALB/c mice for 6 days and degranulation of cutaneous mast cells stimulated with secretagogues was investigated. Results: Prolonged treatment with dexamethasone (1 μM) during the co-culture period suppressed the proliferation, upregulation of chymase, and degranulation induced by compound 48/80 or substance P, but augmented the granule storage of histamine, carboxypeptidase A and β-hexosaminidase. Expression levels of α subunit of the trimeric G protein, Gα i1 and several members of Mas-related G protein-coupled receptors (Mrgpr) were up-regulated during the co-culture period and returned to the basal levels in the presence of dexamethasone. Although 6 days of cutaneous application of dexamethasone did not change the number of mast cells, it augmented cutaneous histamine content and significantly suppressed degranulation in response to compound 48/80 and IgE-mediated antigen stimulation. Conclusion: Dexamethasone affected the granule contents and lowered the sensitivity to secretagogues of murine mature cultured mast cells. Down-regulation of Mrgpr family genes and Gα i1 in mast cells by dexamethasone might be involved in the therapeutic effects on chronic dermatitis.
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