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

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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.