Mechanisms of phospholipase A_2-dependent killing of microorganisms on macrophages

本研究旨在观察结核分枝杆菌（MTB）和鸟分枝杆菌复合体（MAC）感染巨噬细胞（Mφ）后胞浆型磷脂酶A_2（cPLA_2）的胞内转位和磷酸化变化。我们还研究了与cPLA_2转位和磷酸化有关的巨噬细胞内信号转导模式。结果表明：（1）ATP能显著增强Mφ抗MTB或抗MAC的杀菌活性; ATP的这种作用可被cPLA_2抑制剂花生四烯酸三氟甲基酮（a-TFMK）特异性阻断。（2）α-TFMK还能特异性阻断巨噬细胞内膜性花生四烯酸（AA）分子向含MAC的吞噬体的转运。(3)By用共聚焦显微镜观察感染MC的Mφ，发现ATP可促进cPLA_2向含MC吞噬体的胞内转运。（4）cPLA_2易位与感染诱导的Mφ凋亡一致。（5）用MAC感染CHO细胞和转染GFP标记的cPLA_2（GFP-cPLA_2）的CD 36 ^+CHO细胞，可观察到细胞内GFP-cPLA_2移位到含MAC的吞噬体中。由于cPLA_2分子的某些丝氨酸残基的磷酸化密切调控cPLA_2的活性和转运，因此研究cPLA_2的丝氨酸残基（包括Ser 505、Ser 515和Ser 727）在MAC感染的Mφ细胞内cPLA_2的转运和活化中的作用具有重要意义。在此背景下，我们目前正试图产生编码具有适当磷酸化位点突变的GFP标记的cPLA_2的cDNA的载体。

The purpose of the present study is to investigate the profiles of intracellular translocation and phosphorylation of cytosolic phospholipase A_2(cPLA_2) in macrophage (Mφ) infected with Mycobacterium tuberculosis (MTB) or Mycobacterium avium complex (MAC). We also examined the mode of intramacrophage signal transduction related to the cPLA_2 translocation and phosphorylation. The present study gave the following findings, (1)ATP significantly potentiated Mφ anti-MTB or anti-MAC bactericidal activity. This effects of ATP was specifically blocked by a cPLA_2 inhibitor, arachidonyl trifluoromethylketone (a-TFMK). (2)Intramacrophage translocation of membranous arachidonic acid (AA) molecules to MAC-containing phagosomes was also specifically blocked by a-TFMK. (3)By confocal microscopic observation of MAC-infected Mφs, it was found that ATP enhanced intracellular translocation of cPLA_2 into MAC-containing phagosomes. (4)This cPLA_2 translocation was found to coincide with infection-induced Mφ apoptosis. (5)When CHO cells and CD36^+CHO cells transfected with GFP tagged cPLA_2 (GFP-cPLA_2) were infected with MAC, intracellular GFP-cPLA_2 was observed to translocate to the MAC-containing phagosomes. Since cellular cPLA_2 activities and translocation are tightly regulated by phosphorylation of certain serine residues of cPLA_2 molecules, it is of interest to examine the roles of cPLA_2's serine residues, including Ser505,Ser515 and Ser727,in intracellular translocation and activation cPLA_2 in MAC-infected Mφs. In this context, we are currently attempting to generate vectors encoding the cDNAs of GFP-tagged cPLA_2 with appropriate phosphorylation site mutations.