| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
In response to IPS, murine peritoneal macrophages produced IL-12 (heterodimer of p35 and p40 subunits) but marine macrophage line cells of RAW264.7 did not RAW264.7 produced no IL-12p35 and a small amount of IL-12p40 upon LPS stimulation. Subcloning of RAW264.7 was carried out and isolated high producer clones of IL-12p40 (clone Hi). To understand the underlying mechanisms of LPS-induced IL-12 production, clone Hi was investigated its LPS responsive features in comparison to those of its parent RAW264.7, low producer clone (clone Lw), and another IL-12p40 high producer cell line, J774.1. In LPS-induced productions of TNF, IL-1, IL-6, and NO, or in expressions of their mRNA, no significant differences between the two clones were found mRNA expression of IL-12p40 was detected in clone Lw and the parent RAW264.7 by stimulation with BCG but not with LPS. These results indicated that low production of IL-12p40 by LPS stimulated RAW264.7 and clone Lw was not due to the defect or mutation on IL-12p40 gene. Presence of some specific signaling processes for LPS-induced IL-12p40 production was suggested. There was no significant difference between clone Hi and Lw in LPS-induced phosphorylations of MAP kinases, ERK1/2, p38MAPK, and SAPK/JNK. However, ERK1/2 phosphorylation in J774.1 in response to LPS was remarkably lower than that in RAW264.7 and its subclones. Strong mRNA expression of IL-12p40 in response to LPS was defected in RAW264.7 and clone Lw pretreated with an inhibitor of MEK1/2. These results suggest that over activation of ERK1/2 induce some suppressive signals on IL-12p40 expression. In clone Hi, expression of IL-12p40 mRNA in response to LPS was detected without the MEK1/2 inhibitor pretreatment, although weaker than that of J774.1. Pretreatment of cycloheximide suppressed all of LPS-induced mRNA expressions tested except for TNF. These results suggest that these gene expressions require de novo protein synthesis.
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