Diacylglycerol and ceramide kinetics in primary cultures of activated T-lymphocytes

T cell activation results in the generation of diacylglycerol (DAG), the 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 mitogen induction of DAG and ceramide in T cells have been conducted in cell lines over short periods of time (0-30 min) relative to the 2-3-h time frame required for commitment to proliferation. Therefore, we examined T cell mitogen-induced DAG and ceramide kinetics under physiologically relevant conditions during the initial 2 h of culture. Freshly isolated murine splenic lymphocytes were stimulated with the T cell-specific mitogen, concanavalin A (Con A). Our results show that Con A induced a multiphasic DAG response with significant peaks in DAG mass occurring at 2, 20 and 120 min. Concomitantly, ceramide mass was significantly increased 2 min following Con A addition and remained elevated until 120 min. Addition of C8-ceramide (10 microM) to lymphocyte cultures significantly enhanced mitogen-induced proliferation. These results demonstrate that DAG is continuously produced by activated T lymphocytes in a multiphasic fashion, and that ceramide is a positive effector molecule with respect to murine T cell proliferation. These results establish a foundation for further examination of the relationship between DAG, ceramide and T cell activation.