Elucidation of signaling molecules involved in macrophage activation by bacterial lipopolysaccharide

在IPS刺激下，小鼠腹腔巨噬细胞产生IL-12 （p35和p40亚基的异源二聚体），而RAW264.7的海洋巨噬细胞系细胞则不产生IL-12p35， LPS刺激下RAW264.7不产生IL-12p35，产生少量IL-12p40。对RAW264.7进行亚克隆，分离出IL-12p40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Hi克隆与其亲本RAW264.7、低产IL-12p40细胞系（克隆Lw）和另一个高产IL-12p40细胞系J774.1的LPS响应特性。在LPS诱导的TNF、IL-1、IL-6和NO的产生及其mRNA的表达中，两个克隆在BCG刺激下和亲本RAW264.7中检测到IL-12p40 mRNA的表达，而在LPS刺激下没有检测到IL-12p40 mRNA的表达。这些结果表明，LPS刺激RAW264.7和克隆Lw产生IL-12p40的低产量不是由于IL-12p40基因缺陷或突变所致。lps诱导IL-12p40产生的一些特定信号过程被认为存在。克隆Hi和克隆Lw在lps诱导的MAP激酶、ERK1/2、p38MAPK和SAPK/JNK磷酸化水平上无显著差异。然而，J774.1对LPS的ERK1/2磷酸化明显低于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的mRNA表达对LPS有强烈的响应。这些结果表明，ERK1/2的过度激活诱导了IL-12p40表达的一些抑制信号。在Hi克隆中，在没有MEK1/2抑制剂预处理的情况下，检测到IL-12p40 mRNA对LPS的响应，但比J774.1弱。预处理环己亚胺可抑制lps诱导的mRNA表达，TNF除外。这些结果表明，这些基因表达需要从头合成蛋白质。

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.