吞噬作用,宿主防御的基本过程,需要多种信号反应的协调。MT-II,无酶活性的Lys49磷脂酶A2(PLA2)同源物,和MT-III,已知具有催化活性的Asp49PLA2激活巨噬细胞中的吞噬作用。在这项研究中,介导吞噬作用的信号通路,专注于蛋白激酶,被调查了。腹膜内注射巯基乙酸盐96小时后,从雄性瑞士小鼠腹膜中获得巨噬细胞。在存在或不存在特异性抑制剂的情况下,使用未调理的酵母聚糖颗粒评估吞噬作用。以及PKC和PKC-α的共聚焦显微镜定位。此外,通过两种PLA2刺激的巨噬细胞中的γP32ATP评估蛋白激酶C(PKC)活性。数据显示两种sPLA2都增加了吞噬作用。细胞松弛素D,星形孢菌素/H7,Wortmannin,和除比霉素,肌动蛋白聚合抑制剂,PKC,磷酸肌醇3-激酶(PI3K),和蛋白酪氨酸激酶(PTK),分别,显著降低两种PLA2s诱导的吞噬作用。两种PLA2刺激的巨噬细胞中PKC活性均增加。免疫荧光证明了肌动蛋白聚合和距蛋白,并且在两个PLA2s刺激后5分钟募集了距蛋白。MT-II和MT-III刺激60分钟后,细胞内的PKC和PKC-α定位增加。这些数据表明,两种PLA2的作用取决于肌动蛋白细胞骨架重排和PKC的激活,PI3K,和吞噬作用所需的PTK信号事件。
Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.