DIACYLGLYCEROL AND CERAMIDE KINETICS IN PRIMARY CULTURES OF ACTIVATEDT-LYMPHOCYTES

T cell activation results in the generation of diacylglycerol (DAG), t he physiological activator of protein kinase C. Recently, ceramide, a bioactive lipid intracellular second messenger, has been shown to play a positive role in T cell proliferation. Most studies examining mitog en induction of DAG and ceramide in T cells have been conducted in cel l lines over short periods of time (0-30 min) relative to the 2-3-h ti me frame required for commitment to proliferation. Therefore, we exami ned T cell mitogen-induced DAG and ceramide kinetics under physiologic ally relevant conditions during the initial 2 h of culture. Freshly is olated murine splenic lymphocytes were stimulated with the T cell-spec ific mitogen, concanavalin A (Con A). Our results show that Con A indu ced a multiphasic DAG response with significant peaks in DAG mass occu rring at 2, 20 and 120 min. Concomitantly, ceramide mass was significa ntly increased 2 min following Con A addition and remained elevated un til 120 min. Addition of C-8-ceramide (10 mu M) to lymphocyte cultures significantly enhanced mitogen-induced proliferation. These results d emonstrate that DAG is continuously produced by activated T lymphocyte s in a multiphasic fashion, and that ceramide is a positive effector m olecule with respect to murine T cell proliferation. These results est ablish a foundation for further examination of the relationship betwee n DAG, ceramide and T cell activation.