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Accumulating evidence suggests that several IgE clones can activate mast cells during the sensitization phase even in the absence of antigen. They were found to induce pro-inflammatory cytokine release, histamine synthesis, chemotaxis, adhesion, and accelerated maturation of mast cells, although it remains unknown whether antigen-induced responses can be affected by differences of IgE clones. We compared two IgE clones, which were different in the capacity to activate mast cells during sensitization, in terms of potentials to affect antigen-induced degranulation and cytokine releases using IL-3-dependent murine bone marrow-derived cultured mast cells (BMMCs). Antigen-induced degranulation and pro-inflammatory cytokine release were augmented, when BMMCs were sensitized with elevated concentrations of a clone IgE-3, which did not induce phosphorylation of JNK and cytokine release in the absence of antigen, whereas those were significantly rather decreased, when BMMCs were sensitized with elevated concentrations of a clone SPE-7, one of the most potent cytokinergic IgE clones, which intensively induced phosphorylation of JNK. This attenuated response with SPE-7 was accompanied by decreased tyrosine phosphorylation of the cellular proteins including Syk upon antigen stimulation. SP600125, which is known to inhibit JNK, restored the levels of antigen-induced degranulation and phosphorylation of Syk in BMMCs sensitized with higher concentrations of a clone SPE-7 when it was added before sensitization. Treatment with anisomycin, a potent activator of JNK, before IgE sensitization significantly suppressed antigen-induced degranulation. These findings suggest that differences of sensitizing IgE clones can affect antigen-induced responses and activation of JNK during sensitization might suppress antigen-induced activation of mast cells.

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell–deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS–ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3–L-PGDS–DP1 loop that drives mast cell maturation.

We investigated IgE-mediated allergic responses in a metabolic syndrome model rat strain, SHRSP.Z, which develops obesity and hypertension to cast light on the relationship between metabolic disturbances and allergic responses. IgE-mediated cutaneous anaphylactic responses were severely attenuated in this strain regardless of the presence of fa / fa mutation, compared with the parental WKY/Izm strain. Furthermore, in the peritoneal mast cells of both the SHRSP.Z and SHRSP/Izm strains, IgE-mediated activation, such as degranulation and protein tyrosine phosphorylation, was severely impaired whereas no significant differences were found in morphology and number of peritoneal mast cells. Immunoblot analyses revealed that phosphorylation levels of Syk upon IgE-mediated antigen stimulation were significantly decreased and basal expression of linker for activation of T cells (LAT) was down-regulated in peritoneal mast cells of the SHRSP strains. These results suggest that attenuated cutaneous allergic responses in the SHRSP.Z strain might be attributed to impaired FcɛRI-mediated signal transduction in mast cells.

Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E2stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE2receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977–17981, 2003). We here investigated PGE2-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca2+mobilization plays a pivotal role in PGE2-induced adhesion of BMMCs. Adhesion and Ca2+mobilization induced by PGE2were abolished in the Ptger3−/−BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca2+channel inhibitor, SKF 36965, indicating the involvement of Gi-mediated Ca2+influx. We then investigated PGE2-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE2induced adhesion of the peritoneal mast cells of the Ptger3+/+mice, but not that of the Ptger3−/−mice. In rat peritoneal mast cells, PGE2or an EP3 agonist stimulated both Ca2+mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE2-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca2+mobilization.

Obesity is a condition in which excess body fat accumulates due to lipids producing adipocytes and an increased number of differentiated mature cells. Recently, new findings have shown that macrophages infiltrate into adipose tissues and produce various pro-inflammatory cytokines in obese subjects. The inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus of metabolic syndrome. Caffeic acid phenethyl ester (CAPE) is known to have many functions, including antibacterial, anticancer and anti-inflammatory properties, but there is no evidence of its effect on the inflammatory responses in hypertrophic adipocytes through stimulation by macrophages. We investigated the effect of CAPE on macrophages and hypertrophic adipocytes in this study. CAPE significantly suppressed the levels of lipopolysaccharide (LPS)-induced interleukin (IL)-1-beta, tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 from a macrophage cell line, RAW264.7. Supernatants of stimulated RAW264.7 macrophages drastically increased mRNA levels of pro-inflammatory cytokines such as IL-6, MCP-1 and TNF-alpha in 3T3-L1 hypertrophic adipocytes. CAPE also significantly and dose-dependently reduced the gene expression of these cytokines. Our findings indicate that CAPE has inhibitory effects on the production of pro-inflammatory cytokines from LPS-stimulated RAW264.7 macrophages. In addition, CAPE suppressed gene expressions of cytokines under inflammatory conditions of hypertrophic adipocytes, suggesting that it may have the potential to suppress inflammation by macrophage infiltration into adipose tissue in obese patients.

Recent studies have demonstrated that a variety of chemokine receptors are expressed in mast cells. We investigated the changes in mRNA expression of CXCRs in murine IL-3-dependent bone marrow-derived mast cells (BMMCs) to clarify how the CXCR expression is regulated in mast cells.Expression of CXCR mRNA was measured by RNase protection assay. Functional expression of CXCRs was confirmed by monitoring intracellular Ca(2+) mobilization.CXCR4 mRNA expression was transiently induced in BMMCs in serum-dependent fashion and was completely suppressed upon IgE-mediated antigen stimulation. In contrast, CXCR5 mRNA expression was induced upon IgE-mediated antigen stimulation. Changes in the intracellular Ca(2+) mobilization induced by CXCL12 strongly indicated the functional expression of CXCR4. The decrease in CXCR4 and the increase in CXCR5 mRNA expression was also observed in BMMCs stimulated with thapsigargin, a phorbol ester, and stem cell factor.The mRNA expression of CXCR4 is differentially regulated in BMMCs upon various stimuli including IgE-mediated antigen stimulation.

It remains to be fully clarified how adhesion of mast cells is regulated in vivo. We previously reported that PGE2-receptor EP4 stimulated the adhesion of mouse mastocytoma P-815 cells to plate-bound fibronectin. Our purpose in this study is to evaluate the adhesion using a system, which can mimic the in vivo adhesion.P-815 cells were transplanted in an air pouch produced in the transplantable mice, CDF1. The number of cells that adhere to the subcutaneous tissues overlaying the inside cavity surface was determined.The number of adhered cells was decreased in mice administered with ibuprofen or an EP4 antagonist, ONO AE3-208. A local administration of PGE(2) or a phorbol ester, PMA, increased the number of adhered cells, which was also suppressed in the mice treated with ONO AE3-208.Our results suggest that PGE(2)-mediated adhesion of P-815 cells in the subcutaneous tissues of the air pouch is mediated by the EP4 subtype.

Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.