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.

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 H 4 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. [6,7]. In a mouse mastocytoma P-815 cells, caspase-9-mediated cleavage results in enzymatic activation of HDC [8]. KeywordsWe have explored novel functions of histamine using a mouse strain lacking the Hdc gene. In the first report of Hdc −/− mice, we noticed that peritoneal mast cells (pMCs) and skin mast cells exhibited an aberrant morphology with severely decreased granule contents [9]. Although several studies indicated that mast cells express specific histamine receptors, it remains unknown how histamine affects the nature of mast cells. On the other hand, positive modulators for growth and differentiation of mast cells, such as IL-3 and c-kit ligand (Kitl), were found to induce a transient increase in histamine synthesis [10,11], raising a possibility that histamine is involved in the process of mast cell maturation. We hypothesized that newly synthesized histamine may play a critical role in granule maturation of mast cells in an autocrine fashion. We, here, took two different approaches to investigate the mechanism underlying the histamine-mediated promotion of granule maturation of mast cells; an in vitro approach using a mast cell fibroblast coculture model and an in vivo one using the mast cell reconstitution system with mast cell deficient Kit W /Kit W-v mice. This study proposes a novel function of histamine in mast cell biology. Results Characterization of the pMCs in Hdc −/− miceWe previously reported that the cutaneous and pMCs of Hdc −/− mice demonstrate aberrant granules with severely decreased contents [9]. pMCs in Hdc −/− mice were poorly-stained with Safranin-O and acidic Toluidine blue, compared with those in WT mice (Fi...

By using the recently established culture system that reproduces the terminal differentiation process of connective tissuetype 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 À/À 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 À/À 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 À/À BMMCs was not significantly changed for 10 weeks, whereas that in the tissues transplanted with the CD44 þ / þ BMMCs was significantly increased. These results suggest that CD44 plays a crucial role in the regulation of the cutaneous mast cell number. As heterogeneity of tissue mast cells arise as a result of differences in the microenvironment that affects the process of the terminal differentiation, extensive investigation of the maturation process is required for precise understanding of the roles of tissue mast cells. Although several mast cell lines and IL-3-dependent bone marrow-derived cultured mast cells (BMMCs) have been used as models to investigate the process of mast cell activation and subsequent production of proinflammatory mediators, these models have limitations in analyzing functions specific to mature mast cells. Hence, recent studies have often employed the mast cell knock-in models using the mast cell-deficient mice, such as WBB6F1-) and C57BL/6-Kit), to investigate the roles of mature mast cells in vivo.1 By modifying the co-culturing method of BMMCs together with fibroblasts in the presence of stem cell factor (SCF), we have recently established a culture system for development of mature mast cells, which exhibit properties characteristic of connective tissue-type mast cells (CTMCs), such as degranulation induced by cationic secretagogues, Safranin-positive granules, and high levels of mast cell protease expression. 4 We then investigated the gene expression profile during the culture period and identified a cluster of candidate genes, which have potentials to modulate mast cell