IMMUNE REGULATION BY THE P38 MAP KINASE PATHWAYS

为了分析p38 MAP激酶途径的功能意义，对mkk 3和mkk 6敲除小鼠进行分析。为了检查激酶在先天免疫中的作用，从mkk 3-/-mkk 6 +/-小鼠和对照小鼠制备骨髓衍生的巨噬细胞（Mφ）。当LPS刺激mkk 3-/-mkk 6 +/-来源的Mφ时，TNFα和IL-12的分泌严重受损。此外，还检查了炎症化学介质。通过真实的时间RT-PCR判断，iNOS表达显著降低。提示p38 MAP激酶通路在天然免疫、辅助性T细胞极化和炎症反应中起重要作用。为了观察p38 MAP激酶途径是否也参与细胞凋亡，产生了具有mkk 3-/-mkk 6-/-基因型的成神经细胞样细胞系。该细胞系在TNFα刺激后表现出p38活化受损，但在UV暴露后则没有，这表明mkk 4可能在p38的残留活化中发挥作用。饥饿后的细胞凋亡明显受到抑制，这与皮下注射给裸鼠时细胞的致瘤活性很好地对应。这些结果表明，p38 MAP激酶不仅在免疫调节中起作用，而且在细胞凋亡和肿瘤生长中起作用。最后，我确定了被各种细胞因子磷酸化的分子，这些细胞因子具有与p38 MAP激酶相似的动力学，即STAM 1和Hrs，这两种分子参与囊泡转运。STAM和Hrs形成紧密复合物，并且STAM 1的降解被控制，但Hrs的存在。由于Hrs在T细胞中表达并调节T细胞存活，因此我得出结论，不仅p38，而且Hrs和STAM也参与免疫调节，包括T细胞存活/凋亡。

In order to analyze the functional significance of the p38 MAP kinase pathways, mkk3 and mkk6 knockout mice were subjected to analysis. To examine the role of the kinases in innate immunity, bone marrow derived macrophages (Mφ) were prepared from mkk3-/-mkk6+/-mice and control mice. When mkk3-/-mkk6+/-derived Mφ were stimulated by LPS, secretion of TNFα as well as IL-12 was severely impaired. Further, inflammatory chemical mediators were also examined. iNOS expression, as judged by the real time RT-PCR, was significantly reduced. These results suggest that p38 MAP kinas pathways play significant roles in innate immunity, polarization of T helper T cells and inflammations. To see if p38 MAP kinase pathways are also involved in cellular apoptosis, a fibloblastoid cell line with mkk3-/-mkk6-/-genotype was generated. This cell line manifested impaired p38 activation upon TNFα stimulation but not by UV exposure, suggesting the possible roles for mkk4 for the residual activation of p38. Apoptosis upon starvation was clearly inhibited in the cells, which corresponded well with the tumorigenic activity of the cell when injected subcutaneously to nude mice. Collectively these results indicated that p38 MAP kinases not only plays role in immune regulation but also in apoptosis and tumor growth. Finally I identified molecules that are phosphorylated by various cytokines with similar kinetics to the p38 MAP kinases, namely STAM1 and Hrs, two molecules involved in vesicular transport. STAM and Hrs forms a tight complex and the degradation of STAM1 was controlled but the presence of Hrs. Since Hrs is expressed in T cells and regulate T cell survival, I concluded that not only p38 but also Hrs and STAMs are involved in immune regulation including T cell survival/apoptosis.