DIACYLGLYCEROL LIPASE ACTIVIATION AND 5-LIPOXYGENASE ACTIVATION AND TRANSLOCATION FOLLOWING TCR CD3 TRIGGERING IN T-CELLS/

Arachidonic acid (AA) release was observed following T cell receptor ( TCR)/CD3 complex cross-linking in different tumor T cell lines as well as on purified peripheral T cells in vivo. Direct measurement of enzy matic activity in vitro of TCR/CD3-stimulated Jurkat cell extracts on labeled vesicle substrates showed that TCR/CD3 cross-linking resulted in AA release from sn-1,2-diacylglycerol (DAG) vesicles, as detected b y TLC analysis, suggesting that DAG lipase was activated following TCR /CD3 stimulation and DAG generation. On the contrary, no phospholipase A2 activation was observed in response to TCR/CD3 stimulation, since no lyso-phospholipids were generated in vitro from either phosphatidyl choline or phosphatidylinositol-3,4-bisphosphate, or from phosphatidic acid vesicles. Moreover, the 1-DAG lipase inhibitor RHC80267 complete ly blocked TCR/CD3-dependent AA release in vitro and in vivo, without effect upon TCR/CD3-dependent inositol-1,4,5-trisphosphate (IP3) gener ation. Importantly, evidence for further metabolism of released AA was obtained, since synthesis and release of cysteinyl leukotrienes (CLT) , but not of leukotriene B4 or cyclooxygenase products, could be detec ted by radioimmunoassay in different T cell lines and peripheral blood T cells following TCR/CD3 cross-linking. Moreover, HPLC analysis reve aled an accumulation of leukotriene E4 in TCR/CD3 stimulated Jurkat ce lls. This was associated with translocation of 5-lipoxygenase from the cytosol to the cell membranes. Finally, TCR/CD3-mediated CLT producti on was blocked by MK886, a specific inhibitor of 5-LO translocation an d activation. Our data help define a further level in the fate of seco nd messengers generated after TCR/CD3 triggering and suggest that addi tional mediators can play a role in the context of T cell activation.