Guinea-pig lymphotoxin-mediated cytotoxicity and activation of endogenous phospholipase A2.

The effect of cations on lysis of target cells (L . P3 cells) by guinea-pig lymphotoxin (GLT) was examined. Depletion of Ca2+ ions from the extracellular environment caused limited cytolysis, whereas addition of Ca2+ led to enhanced lysis. By applying the GLT pulse technique, it was shown that Ca2+ was not required for the initial step of GLT-mediated cytolysis, the step of binding of GLT molecules to target cell membranes. Among other cations tested, La3+ was found to interfere with the cytolytic action. Neither Mg2+ nor Mn2+ was effective. When target cells whose phospholipids were specifically radiolabelled at the C-2 position with [14C]-arachidonic acid were treated with GLT, radioactivity in the phospholipid fraction decreased. On the contrary, that in the free fatty acid, triglyceride and diglyceride fractions increased. This indicates the existence of endogenous phospholipase A2 that can be activated by GLT. Activation of the enzyme was partially inhibited by addition of EGTA with a concomitant decrease of cytolysis. When GLT was added to a crude membrane fraction of the radiolabelled L . P3 cells, liberation of free fatty acids from phospholipids was readily observed, but the increase of triglyceride-associated radioactivity was negligible. In this case, Ca2+ was found to be prerequisite for the GLT-induced activation of the enzyme. The results suggest that GLT-induced cytolysis includes a substantially Ca2+-dependent rate-limiting stage which comes after the binding of GLT to the target cells. Activation of membrane phospholipase A2 may be at least one of the biochemical changes that constitutes the Ca2+-dependent stage, although it remains to be clarified whether the activation of phospholipase A2 is directly responsible for target cell destruction or not.