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Elucidation of signaling molecules involved in macrophage activation by bacterial lipopolysaccharide

阐明细菌脂多糖参与巨噬细胞激活的信号分子

基本信息

批准号：
13670281
负责人：
SAITO Shinji
金额：
$ 1.98万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670281/
关键词：
macrophage lipopolysaccharide IL-12 MAPK ERK シグナル伝達 IL-1β

项目摘要

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.

响应 IPS，小鼠腹膜巨噬细胞产生 IL-12（p35 和 p40 亚基的异二聚体），但 RAW264.7 的海洋巨噬细胞系细胞不产生。在 LPS 刺激后，RAW264.7 不产生 IL-12p35，只产生少量 IL-12p40。对RAW264.7进行亚克隆并分离出IL-12p40的高产克隆（克隆Hi）。为了了解 LPS 诱导 IL-12 产生的潜在机制，研究了克隆 Hi 的 LPS 响应特征，并与其亲本 RAW264.7、低生产克隆（克隆 Lw）和另一个 IL-12p40 高生产细胞系 J774.1 进行比较。在 LPS 诱导的 TNF、IL-1、IL-6 和 NO 的产生或其 mRNA 的表达中，发现两个克隆之间没有显着差异。在用 BCG 刺激但用 LPS 刺激时，在克隆 Lw 和亲本 RAW264.7 中检测到 IL-12p40 的 mRNA 表达。这些结果表明LPS刺激RAW264.7和克隆Lw产生的IL-12p40的低产量并不是由于IL-12p40基因的缺陷或突变造成的。有人提出 LPS 诱导 IL-12p40 产生存在一些特定的信号传导过程。在 LPS 诱导的 MAP 激酶、ERK1/2、p38MAPK 和 SAPK/JNK 磷酸化方面，克隆 Hi 和 Lw 之间没有显着差异。然而，J774.1 中响应 LPS 的 ERK1/2 磷酸化显着低于 RAW264.7 及其亚克隆。在用 MEK1/2 抑制剂预处理的 RAW264.7 和克隆 Lw 中，IL-12p40 响应 LPS 的强 mRNA 表达缺陷。这些结果表明 ERK1/2 的过度激活会诱导 IL-12p40 表达的一些抑制信号。在克隆Hi中，在没有MEK1/2抑制剂预处理的情况下检测到响应LPS的IL-12p40 mRNA表达，尽管比J774.1弱。放线菌酮预处理抑制了除 TNF 之外的所有测试的 LPS 诱导的 mRNA 表达。这些结果表明这些基因表达需要从头合成蛋白质。