Prolonged culture of mast cells with high-glucose medium enhances the Fc epsilon RI-mediated degranulation response and leukotriene C4 production.

BACKGROUND: Mast cell-released chemical mediators such as histamine, leukotriene (LT) C(4) and prostaglandin (PG) D(2) lead to the onset of allergic disorders. ATP provided from glycolysis is essential for histamine release and LTC(4) secretion from mast cells upon Fc epsilon RI cross-linking, indicating that glucose is a primary environmental factor for mast cell activation. In this study, we investigated whether increases in concentrations of glucose in culture media affect the activation of bone marrow-derived mouse mast cells (BMMCs) upon Fc epsilon RI cross-linking. METHODS: BMMCs were cultured in RPMI-1640 supplemented with varying concentrations (5.5, 11, 16.5, 22, 27.5 and 33 mM) of D-glucose for 3 h, or 1, 3 or 7 days. D-Mannitol was added to the medium containing 5.5 mMD-glucose for osmotic control. After culturing, these cells were sensitized with anti-TNP IgE and then stimulated with TNP-BSA. RESULTS: We found that long-term culture (7 days) of BMMCs with 33 mMD-glucose increases the Fc epsilon RI-dependent release of beta-hexosaminidase and LTC(4) without affecting surface expression levels of Fc epsilon RI, intracellular ATP levels or calcium signaling. Biochemical analyses demonstrated that Fc epsilon RI-dependent phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) at the Ser505 residue was significantly increased by culturing with 33 mM glucose. CONCLUSIONS: Taken together, our data suggest that glucose can augment Fc epsilon RI-mediated mast cell activation, particularly the degranulation response and LTC(4) secretion after prolonged culture of mast cells with high-glucose medium. Moreover, it is suggested that increased phosphorylation of cPLA(2) at the Ser505 residue contributes to the enhancement of LTC(4) secretion.

Selinidin suppresses IgE-mediated mast cell activation by inhibiting multiple steps of Fc epsilonRI signaling.

IgE-mediated mast cell activation is critical for development of allergic inflammation. We have recently found that selinidin, one of the coumarin derivatives isolated from Angelica keiskei, attenuates mast cell degranulation following engagement of the high-affinity receptor for IgE (Fc epsilonRI) with IgE and antigen. In the present study, we investigated the effects of selinidin on intracellular signaling and mast cell activation employing bone marrow-derived mast cells. Here, we report that selinidin attenuates the release of beta-hexosaminidase, synthesis of leukotriene C4, and production of tumor necrosis factor-alpha without affecting IgE-Fc epsilonRI binding. Furthermore, biochemical analyses of the Fc epsilonRI-mediated signaling pathway demonstrated that selinidin decreases phosphorylation of phospholipase C-gamma1, p38 mitogen-activated protein kinase, and IkappaB-alpha upon FcepsilonRI stimulation. These results suggest that this compound suppresses IgE-mediated mast cell activation by inhibiting multiple steps of FcepsilonRI-dependent signaling pathways and would be beneficial for the prevention of allergic inflammation.

FHL3 negatively regulates human high-affinity IgE receptor beta-chain gene expression by acting as a transcriptional co-repressor of MZF-1.

The high-affinity IgE receptor FcepsilonRI plays a key role in triggering allergic reactions. We recently reported that human FcepsilonRI beta-chain gene expression was down-regulated by a transcription factor, MZF-1, through an element in the fourth intron. In the present study, we found that this transcriptional repression by MZF-1 required FHL3 (four and a half LIM domain protein 3) as a cofactor. Yeast two-hybrid and immunoprecipitation assays demonstrated that FHL3 bound MZF-1 in vitro and in vivo. Overexpression of FHL3 in KU812 cells suppressed the beta-chain promoter activity through the element in the fourth intron in an MZF-1-dependent manner. Furthermore, results from pull-down assays and gel-filtration chromatography employing nuclear extracts indicated that MZF-1 and FHL3 formed a complex of high molecular mass with some additional proteins in the nucleus. Granulocyte-macrophage colony-stimulating factor, which was reported to decrease FcepsilonRI expression, induced the accumulation of FHL3 in the nucleus, in accordance with the repressive role of FHL3 in beta-chain gene expression.

Molecular cloning of rat transcription factor YY1.

YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6% identical to that of mouse YY1 and 97.8% identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114-193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56-113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1-78/100-106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.

Regulation of the human Fc epsilon RI alpha-chain distal promoter.

The alpha-chain of the high affinity receptor for IgE (Fc epsilon RI) is essential for cell surface expression of Fc epsilon RI and binding of the IgE Ab. The human alpha-chain gene possesses two promoters: the proximal promoter, which is highly conserved with that of rodent; and the distal promoter, the structure and role of which are largely unknown. Transcriptional regulation of the alpha-chain distal promoter was investigated in this study. Transient reporter assay revealed critical region for transcription activity located within -27/-17. EMSA identified Elf-1, YY1, and PU.1 as transcription factors binding to this region. In contrast to the proximal promoter, which was trans-activated by YY1 and PU.1, these transcription factors exhibited repressive function on this promoter. Addition of IL-4 caused a marked increase in transcription from the distal promoter and subsequently increased the intracellular production of the alpha-chain. These results indicate that IL-4-dependent up-regulation of the human alpha-chain was due to enhancement of distal promoter activity and suggests that the two promoters have different regulatory mechanisms for alpha-chain expression.

Preferential expression of T(h)2-type chemokine and its receptor in atopic dermatitis.

Lesional skin of patients with atopic dermatitis (AD) is histologically characterized by hypertrophy of the skin, and the infiltration of a large number of eosinophils and T cells into the dermis. Recent studies have indicated that Th2 cells play a crucial role in the pathogenesis of AD skin. Chemokines and their receptors are implicated in the development of symptoms of various skin diseases such as AD and psoriasis vulgaris (psoriasis). We have examined the in situ expression of a typical Th2-type chemokine, thymus- and activation-regulated chemokine (TARC), and its receptor (CCR4) using immunohistochemical techniques. TARC was found to be highly expressed in the basal epidermis of the lesional skin of AD patients and only slightly in the non-lesional skin. On the other hand, no positive cells were seen in the lesional skin of psoriasis. Consistently, CCR4+ cells were present predominantly in the lesional skin of AD patients, but not in the non-lesional skin. In contrast, in the lesional skin of psoriasis patients, cells positive for CCR5, which is expressed on Th1 cells, were abundantly present. Interestingly, psoralen plus ultraviolet A therapy reduced the number of CCR4+ cells in the AD skin lesions. These results suggest that Th2-type cytokines such as TARC are involved in the pathogenesis of skin lesions in AD patients through the preferential recruitment of Th2 cells.

Regulation of human Fc epsilon RI alpha-chain gene expression by multiple transcription factors.

Transcriptional regulation of the gene-encoding human Fc epsilon RI alpha-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The alpha-chain promoter activity was up-regulated approximately 2-fold by exogenously expressed YY1 or PU.1 and approximately 7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter approximately 41- or approximately 27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the alpha-chain gene expression. Transcriptional regulation of the alpha-chain gene through four transcriptional factors is discussed.