Histamine synthesis is required for granule maturation in murine 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.

Restriction of mast cell proliferation through hyaluronan synthesis by co-cultured fibroblasts.

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.

Prostaglandin E2-EP4 signaling suppresses adipocyte differentiation in mouse embryonic fibroblasts via an autocrine mechanism.

The prostaglandin (PG) receptors EP4 and FP have the potential to exert negative effects on adipogenesis, but the exact contribution of endogenous PG-driven receptor signaling to this process is not fully understood. In this study, we employed an adipocyte differentiation system from mouse embryonic fibroblasts (MEF) and compared the effects of each PG receptor-deficiency on adipocyte differentiation. In wild-type (WT) MEF cells, inhibition of endogenous PG synthesis by indomethacin augmented the differentiation, whereas exogenous PGE2, as well as an FP agonist, reversed the effect of indomethacin. In EP4-deficient cells, basal differentiation was upregulated to the levels in indomethacin-treated WT cells, and indomethacin did not further enhance differentiation. Differentiation in FP-deficient cells was equivalent to WT and was still sensitive to indomethacin. PGE2 or indomethacin treatment of WT MEF cells for the first two days was enough to suppress or enhance transcription of the Pparg2 gene as well as the subsequent differentiation, respectively. Differentiation stimuli induced COX-2 gene and protein expression, as well as PGE2 production, in WT MEF cells. These results suggest that PGE2-EP4 signaling suppresses adipocyte differentiation by affecting Pparg2 expression in an autocrine manner and that FP-mediated inhibition is not directly involved in adipocyte differentiation in the MEF system.

Involvement of CD44 in mast cell proliferation during terminal differentiation.

By using the recently established culture system that reproduces the terminal differentiation process of connective tissue-type mast cells, we found significant transcriptional induction of CD44. As CD44 is a primary receptor for hyaluronan (HA), which is one of the major extracellular matrix components, we investigated the role of CD44 in cutaneous mast cells. When co-cultured with fibroblasts, mouse bone marrow-derived cultured mast cells (BMMCs) were found to form clusters in an HA-dependent manner. As compared with BMMCs derived from the wild-type mice, those from the CD44(-/-) mice exhibited impaired growth during the co-cultured period. Furthermore, in the peritoneal cavities and ear tissues, mature mast cells were fewer in number in the CD44(-/-) mice than in the wild-type mice. We investigated roles of CD44 in mast cell proliferation by reconstituting BMMCs into the tissues of mast cell-deficient, Kit(W)/Kit(W-v) mice, and found that the number of metachromatic cells upon acidic toluidine blue staining in the tissues transplanted with CD44(-/-) BMMCs was not significantly changed for 10 weeks, whereas that in the tissues transplanted with the CD44(+/+) BMMCs was significantly increased. These results suggest that CD44 plays a crucial role in the regulation of the cutaneous mast cell number.

Establishment of the culture model system that reflects the process of terminal differentiation of connective tissue-type mast cells.

To understand physiological roles of tissue mast cells, we established a culture system where bone marrow-derived immature mast cells differentiate into the connective tissue-type mast cell (CTMC)-like cells through modifying the previous co-culture system with Swiss 3T3 fibroblasts. Our system was found to reproducibly mimic the differentiation of CTMCs on the basis of several criteria, such as granule maturation and sensitivity to cationic secretagogues. The gene expression profile obtained by the microarray analyses was found to reflect many aspects of the differentiation. Our system is thus helpful to gain deeper insights into terminal differentiation of CTMCs.

Long-term outcomes of add-on adefovir dipivoxil therapy to ongoing lamivudine in patients with lamivudine-resistant chronic hepatitis B.

AIM: Add-on adefovir dipivoxil (ADV) therapy has been a standard rescue treatment for patients with lamivudine (LAM)-resistant chronic hepatitis B, but the overall benefits of long-term add-on ADV therapy are still limited. The aim of this study was to evaluate the long-term efficiency of add-on ADV treatment and to explore predictive factors associated with it. METHODS: A total of 158 patients with LAM-resistant chronic hepatitis B were included in this retrospective, multicenter, nationwide study in Japan. After confirming LAM resistance, ADV was added to LAM treatment. Three types of events were considered as outcomes: virological response, hepatitis B e antigen (HBeAg) clearance and alanine aminotransferase (ALT) normalization. Virological response was defined as serum hepatitis B virus (HBV) DNA levels of less than 3 log copies/mL. Baseline factors contributing to these outcomes were examined by univariate and multivariate analyses. RESULTS: The median total duration of ADV treatment was 41 months (range, 6-84). The rate of virological response was 90.8% at 4 years of treatment; HBeAg clearance and ALT normalization were achieved by 34.0% and 82.7%, respectively, at the end of follow up. Each outcome had different predictive factors: baseline HBV DNA and albumin level were predictive factors for virological response, history of interferon therapy and ALT level for HBeAg clearance, and sex and baseline albumin level for ALT normalization. CONCLUSION: Long-term add-on ADV treatment was highly effective in LAM-resistant chronic hepatitis B patients in terms of virological and biochemical responses. Lower HBV replication and lower albumin level at baseline led to better outcomes.