Purification of B cell specific immunosuppressive factors from fetal calf serum and the approach to characterize their biological significance in vitro
| Project/Area Number |
03557112
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
YAMAMOTO Itaru FACULTY OF PHARMACEUTICAL SCIENCES,OKAYAMA UNIVERSITY PROFESSOR, 薬学部, 教授 (20028848)
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| Co-Investigator(Kenkyū-buntansha) |
MUTO Norio FACULTY OF PHARMACEUTICAL SCIENCES,OKAYAMA UNIVERSITY PROFESSOR, 薬学部, 助手 (30112642)
GOHDA Eiichi FACULTY OF PHARMACEUTICAL SCIENCES,OKAYAMA UNIVERSITY ASSOCIATE PROFESSOR, 薬学部, 助教授 (20028814)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1991: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Keywords | IMMUNE REGULATION / B CELL SUPPRESSION / beta-CYCLODEXRIN / AUTOIMMUNE DISEASE / CHOLESTEROL / 自己抗体 / ポリマーβ-シクロデキストリン / 免疫抑制物質 / 抗SRBCPFC応答 / 低分子免疫抑制因子 |
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| Research Abstract |
In order to induce an optimal plaque-forming cell response against sheep red blood cells in vitro, murine splenocytes have been cultured in RPMI-1640 containing 10% FCS.At that time, the magnitude of the response has been known to be dependent on the lot of fetal calf serum. In previous papers, we reported that the PFC response supported by 2% FCS preteated with polymer-beta-cyclodextrin was the same response as that by 10% intact FCS,which can be inactivate serum immunosuppressive factors. This result suggested that immunosuppressive factors are present in the some kind of FCS and they are inactivated by binding to beta-cyclodextrin. In this paper, we demonstrate that these suppressive factors affected only B cell activation and antibody formation but didn't affect T cell proliferation. THerefore, we had to purify and identify B cell specific suppressive factors from deficient FCS using polymer beta-cyclodextrin. The immunosuppressive factors were further purified by silica gel column
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chromatography, TLC and HPLC from deficient FCS pre-treated polymer beta-cyclodextrin. The specific activity of these compounds were then characterized by the anti-SRBC PFC assay and by the LPS- or Con A-induced proliferation. One of the immunosuppressive factors, analyzed by MNR,is cholesterol, other factors are still under investigation. Cholesterol siginificantly inhibited only B cell-related immune responses but had no effect on the Con A-induced T cell proliferation or non specific cytotoxicity. The serum level of cholesterol is tightly related to immune mechanisms and appears to be an important cause of atherosclerosis in vivo. This study suggestes that cholesterol has some immunene regulating functions mediated by cholesterol enriched macrophages, namely, formyl macrophages. Furthermore, the modulation of the intracellular cholesterol metabolism influences many types of immune responses. In order to study the direct effect of cholesterol on immune mechanisms, we modulated the intracellular cholesterol metabolism in various cell types by the addition of the exogenous high concentration of cholesterol. The cholesterol enriched splenocytes displayd a marked decrease in DNA synthesis in response to LPS but not to Con A,as evidenced by Brdu incorporation. These differences may be caused by formyl macrophages which undergo inflammatory response and give inflammatory signals to peripheral lymphocytes. In order to provide better explanations, we examined the effect of cholesterol on the mixed lymphocyte reaction (MLR), murine IL-1 production and autoantibody production in serum of MRL/1 mice. MLR showed a marked increase and seems probably to be caused by the upregulation of IL-1. These findings indicated that an increase in the cholesterol content in the extracellular milieu may augment T cells actvation by modulating the production of IL-1 on macrophages. This study strongly suggests cholesterol synthesis inhibitors as immunosuppressive drugs with a higher selecti Less
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