Study of the Regulatory Mechanisms of Macrophage Apoptosis Induced by Lipopolysaccharides

p38 MAP激酶磷酸化的持续而非激活本身被证明是放线菌酮存在下脂多糖（LPS）诱导的巨噬细胞凋亡诱导的最重要因素之一。虽然人们普遍认为，磷酸化的激酶对应于这种酶的激活，我们表明，SB 202190，一个有效的和特异性的抑制剂p38 MAP激酶，诱导细胞凋亡的巨噬细胞在LPS的存在下，与激酶活性的完全抑制，但持续磷酸化的激酶。这些结果表明，LPS诱导的巨噬细胞凋亡与p38 MAP激酶磷酸化的调节密切相关，不是通过激活下游激酶级联，而是通过激酶的长期磷酸化和磷酸化激酶在核中的存在。另一个重要的结果是凋亡和活化的巨噬细胞之间的信号通路的解离。在存在或不存在血清的情况下将LPS脉冲暴露于巨噬细胞以及随后洗涤和再孵育细胞的精细程序揭示，短至1分钟足以消除巨噬细胞凋亡和活化过程的信号，并且TLR 4和CD 14参与这些过程，尽管在加入LPS后30 min以后孵育区分了凋亡或激活的信号通路;前者需要通过加入放线菌酮或SB 202190来持续磷酸化p38 MAP激酶，如上所述。此外，CD 14表达的程度可能对信号转导的起始步骤起着调节作用。

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.