| Co-Investigator(Kenkyū-buntansha) |
HIROTA Seiichi Osaka University, Pathology, Assistant Professor, 医学部, 助手 (50218856)
JIPPO Tomoko Osaka University, Pathology, Assistant Professor, 医学部, 助手 (70252658)
KITAMURA Yukihiko Osaka University, Pathology, Professor, 医学部, 教授 (70028520)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
(1) Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c-kit gene, and practically no mast cells were detectable when the tissues were stained with alcian blue. Because alcian blue stains proteoglycans, there is a possibility that immature mast cells that do not contain a sufficient amount of proteoglycans are not detectable by this method. The histamine content in the skin of Ws/Ws rats was 0.3% that of control normal (+/+) rats. Because the number of alcian blue-positive mast cells in the skin of Ws/Ws rats was also 0.3% that of +/+ rats, histamine in the skin seemed to be concenrated to alcian blue-positive mast cells. Because mast cells in the skin of +/+ rats express messenger RNA (mRNA) of FcepsilonRI beta-subunit and c-kit, we examined the expression of FcepsilonRI beta-subunit and c-kit mRNA in the skin of Ws/Ws rats. No FcepsilonRI beta-subunit-expressing and no c-kit expressing cells were detectable in the skin of Ws/Ws rats by in situ hybridization histoch
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emistry. The present result suggests the absence of immature mast cells in tissues of Ws/Ws rats.
(2) Cultured mast cells (CMC) did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of CMC derived from Ws/Ws rats to Con A-SCM was comparable to that of CMC derived from +/+ rats, the proliferative response of Ws/Ws CMC to rat recombinant stem cell factor (rrSCF ; a ligand for the c-kit receptor) was much lower than that of +/+ CMC.Although CMC showed the phenotype of mucosal-type mast cells (MMC), +/+and Ws/Ws CMC acquired the phenotype of connecitive tissue-type mast cells (CTMC), when they cocultured with +/+ fibroblasts in the presence of ConA-SCM.Moreover, a fibroblast cell line derived from Sl/Sl mouse embryos that did mot produce SCF did not support the survival of both +/+ and Ws/Ws CMC but did induce the CTMC-like phenotype in about half of +/+ and Ws/Ws CMC when their survival was supported by the addition of ConA-SCM.The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of CTMC-like phenotype.
(3) Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtaineds from the bone marrow, peripheral blood, and small intestine of +/+ and Ws/Ws rats. In the culture containing ConA-SCM alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC.In the culture containing both ConA-SCM and SCF,the numbers of mast-cellcolonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC.Using this culture condition, we investigated changes in concentration of CFU-mast in the bone marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in s phase of the cell cycle remained at low levels in the bone marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site. Less
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