Studies on the Mechanism of Macrophage Activation by Using Variant Cells in Phospholipase A_2 Activities.

本研究旨在阐明在巨噬细胞活化早期观察到的花生四烯酸及其代谢物前列腺素等释放机制。从巨噬细胞样细胞系RAW264.7中分离到4个磷脂酶A2活性缺陷的变异体。其中两个分别在2-花生四烯基磷脂酰胆碱(PC)或磷脂酰乙醇胺(PE)中具有较低的分解活性。另有两株菌株对两种磷脂的分解活性均降低。从体内和体外对亲本细胞的研究中，我们还发现PC而不是PE分解代谢活性需要Ca~(2+)&Gt；这些结果表明，PC和PE的2-花生四烯酸的裂解基本上是由至少两种不同的磷脂酶A2催化的，但在脂多糖(LPS)处理后的花生四烯酸释放方面，这两种变异体与亲本细胞株之间的差异很小。我们认为，一种不同细胞中一种磷脂酶活性的降低被另一种磷脂酶活性的增加所补偿，巨噬细胞中花生四烯酸及其代谢产物的释放不仅受到一种特定磷脂酶活性的调节，而且还受到调节整个花生四烯酸释放系统的某些因素的控制。我们发现这些蛋白质性质的因素之一是在内毒素激活巨噬细胞的早期阶段诱导的。该因子通过DEAE纤维素柱层析得到部分纯化，表明这是一个新的因子，与以前报道的不同。此外，在耐高浓度脂多糖的变异细胞中，我们发现了一株磷脂酶A2活性比亲本高5倍的菌株。在该菌株中，还观察到较高的花生四烯酸释放。这一结果可以通过调节整个花生四烯酸释放系统的一个因子的增加来解释。这方面的研究目前正在进行中。

The aim of this research is to clarify the mechanisms of the release of arachidonate and its metabolites, prostaglandins etc., observed in the earlier stage of macrophage activation.From macrophage-like cell line, RAW264.7, four variants defective in phospholipase A_2 activities were obtained. Two of them had a decreased catabolizing activity exclusively in 2-arachidonyl phosphatidylcholine (PC) or phosphatidylethanolamine (PE), respectively. Another two strains had decreased activities in both phospholipids catabolization. From the studies of parent cells in vivo and in vitro, we also found that PC but not PE catabolizing activity required Ca^<2+>. These results suggested that the cleavage of 2-arachidonate from PC and PE was essentially catalyzed at least two different phospholipases A_2.However, in arachidonate release after lipopolysaccharide (LPS) treatment very small difference was detected between these variants and the parent cell line. We think the decrease in a phospholipase activity in a variant cells is compensated with an increase in another phospholipase activity and the release of arachidonate and its metabolites from macrophage cells are controlled not only by the regulation of one particular phospholipase activity but also by some factors which regulate whole arachidonate releasing system. We found one of these protein-natured factors induced in the earlier stage of macrophage activation with LPS. This factor was partially purified by using DEAE cellulose column chromatography, and was suggested that this was a new factor and was different from those reported previously.In addition, in the variant cells resistant to high concentrations of LPS we found a strain with five times higher phospholipase A_2 activity than the parent. In this strain higher arachidonate release was also observed. It is possible to explain this result by an increase in a factor regulating whole arachidonate releasing system. Studies along this line are now in progress.