Study of the Regulatory Mechanisms of Macrophage Apoptosis Induced by Lipopolysaccharides

在环己亚胺存在下，脂多糖（LPS）诱导巨噬细胞凋亡的最重要因素之一是p38 MAP激酶的持续磷酸化而非自身活化。尽管人们普遍认为该激酶的磷酸化与该酶的激活相对应，但我们发现SB202190，一种有效的特异性p38 MAP激酶抑制剂，在LPS存在下诱导巨噬细胞凋亡细胞死亡，完全抑制激酶活性，但持续磷酸化该激酶。这些结果表明，lps诱导的细胞凋亡与p38 MAP激酶磷酸化的调控密切相关，而不是通过下游激酶级联的激活，而是通过激酶磷酸化的延长和磷酸化激酶在细胞核中的存在。另一个重要的结果是凋亡和活化巨噬细胞之间信号通路的分离。在血清存在或不存在的情况下，将LPS脉冲暴露于巨噬细胞，随后对细胞进行清洗和再孵育，结果表明，只要1分钟就足以转导巨噬细胞凋亡和激活过程的信号，并且TLR4和CD14参与了这些过程，尽管在添加LPS后孵育超过30分钟，可以区分凋亡或激活的信号通路；前者需要通过添加环己亚胺或SB202190持续磷酸化p38 MAP激酶，如上所述。此外，CD14的表达程度被认为调节了信号转导的初始步骤。

Continuation of phosphorylation but not the activation itself of p38 MAP kinase was shown to be one of the most important factors for the induction of macrophage apoptosis induced by lipopolysaccharide (LPS) in the presence of cycloheximide. Although it has widely been believed that phosphorylation of the kinase corresponds to the activation of this enzyme, we showed that SB202190, a potent and specific inhibitor of p38 MAP kinase, induced apototic cell death of macrophages in the presence of LPS, with complete inhibition of the kinase activity but with sustained phosphorylation of the kinase. These results suggest that LPS-induced (nacrophage apoptosis is closely linked to the regulation of phosphorylation of p38 MAP kinase, not through activation of the down-stream kinase cascades but rather through prolonged phosphorylation of the kinase and the presence of the phosphorylated kinase in the nucleus.Another important result is the dissociation of the signaling pathways between apoptotic and activated macrophages. Refined procedures with pulse exposure of LPS to the macrophages in the presence or absence of serum and subsequent washing and reincubation of the cells revealed that as short as I min is enough to transduce the signals for the both apototic and activation processes of the macrophages, and that TLR4 and CD14 are involved in these processes, although later incubation than 30 min after LPS addition distinguish the signaling pathways either to the apoptosis or to the activation ; the former requires sustained phosphorylation of p38 MAP kinase by addition of cycloheximide or SB202190, as described above. Besides, the extents of CD14 expression was suggested to regulate the initial steps of the signal transduction.