Abstract • 70

Adhesion of leukocytes to endothelial cells during the immune response to pathogens is a multistep process involving selectin-mediated rolling and integrin-dependent firm adhesion. In the present study we stimulated Jurkat T lymphocytes and p561ck deficient Jurkat cells (JCaM1.6) with monoclonal antibodies of the Dreg series and glycomimetics, respectively, in order to identify intracellular molecules involved in signal transduction.

Recently, we have demonstrated an L-selectin triggered activation of the Ras/MAP-kinase signaling pathway in Jurkat cells, but not in JCaM cells.

Here, we present evidence for the activation of Jurkat cells by L-selectin via an alternative pathway: L-selectin triggering results in a release of ceramide, a lipid second messenger which has been recently shown to have multiple signaling functions. The release of ceramide correlated with consumption of cellular sphingomyelin. Cellular L-selectin triggering resulted in activation of the neutral sphingomyelinase, but not the acidic. Stimulation of JCaM or Jurkat cells preincubated with Herbimycin A, a src-tyrosine kinase inhibitor, did not prevent activation of the neutral sphingomyelinase (nSMase), pointing to a src-tyrosine kinase independent pathway. Inhibition of the nSMase by glutathione abolished L-selectin induced capping, suggesting a role for the nSMase in this process.

These data point to the involvement of at least two independent intracellular signaling cascades upon L-selectin triggering: The activation of the neutral sphingomyelinase by L-selectin seems to be independent of tyrosine kinases whereas the activation of the small G-proteins Ras, Rac and stress-activated protein kinases, cytoskeletal changes and release of oxygen radicals is critically dependent on the activation of the p561ck kinase. These signaling events might be involved in adhesion and the immunomodulatory functions of leukocytes during inflammatory processes.