Fällman M, Gullberg M, Hellberg C, Andersson T
Department of Cell Biology, University of Linköping, Sweden.
J Biol Chem. 1992 Feb 5;267(4):2656-63.
Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.
补体受体（CR）介导的吞噬作用与人中性粒细胞中甘油二酯（sn-1,2-二酰基甘油和/或1-O-烷基-2-酰基甘油）积累增加有关。当通过降低胞质游离Ca2+消除磷酸肌醇特异性磷脂酶C（PLC）活性时，C3bi介导的甘油二酯增加（5 - 20分钟）仅部分受损。然而，在早期时间点（1分钟），对照细胞中几乎检测不到甘油二酯的产生，而在胞质游离Ca2+浓度降低的细胞中产生量可观。用32P标记的中性粒细胞进行C3bi刺激导致[32P]磷脂酰胆碱（PC）迅速且显著分解，这不受Ca(2+)依赖性磷酸肌醇特异性PLC抑制的影响。因此，PC水解可能参与C3bi诱导的甘油二酯形成。用[3H]1-O-烷基溶血磷脂酰胆碱（[3H]烷基溶血磷脂酰胆碱）标记细胞进行刺激，导致[3H]1-O-烷基磷脂酸（[3H]烷基磷脂酸）形成增加，以及[3H]1-O-烷基甘油二酯（[3H]烷基甘油二酯）形成较晚且较慢；这表明磷脂酶D（PLD）被激活。当在0.5%乙醇存在下刺激这些标记细胞时，在对照细胞和钙降低细胞中均观察到[3H]1-O-烷基磷脂酰乙醇（[3H]烷基磷脂酰乙醇）显著积累，进一步支持了PLD活性参与的观点。与甘油二酯形成的持续增加同时，CR介导的吞噬作用也与细胞蛋白激酶C底物（MARCKS）的磷酸化有关。因此，似乎有理由认为C3bi诱导的PLD激活导致甘油二酯形成与蛋白激酶C激活之间存在因果关系。在不能摄取颗粒的电穿孔细胞中，C3bi颗粒仍能激活PLD并诱导甘油二酯形成。因此，这个信号事件必定是由颗粒与细胞的结合触发，而不是由吞噬过程触发。最重要的是，将蛋白激酶C抑制剂肽PKC(19 - 36)和PKC(19 - 31)引入这些通透细胞导致C3bi诱导的甘油二酯产生明显减少，表明CR介导的蛋白激酶C激活直接触发了额外甘油二酯形成的正反馈机制。综上所述，这些数据进一步阐明了CR介导的甘油二酯形成机制，并为蛋白激酶C在吞噬过程中起重要作用的概念提供了更多支持。
