Fig. 5. Interaction of activated lymphocytes with brain endothelial cells induces formation of ceramide-enriched membrane platforms, activation of the acid sphingomyelinase and release of ceramide. (a) Confocal microscopy images of lymphocytes from immunized wildtype mice after 5 min incubation with mouse brain endothelial bEnd.3 cells show formation of ceramide-enriched membrane platforms. Acid sphingomyelinase activity (b) and ceramide concentrations (c) increase only upon co-incubation of lymphocytes from immunized mice with bEnd.3 cells, but not if wt lymphocytes from non-immunized mice were co-incubated with bEnd.3 cells. The confocal microscopy studies indicated that only a small fraction of immunized T lymphocytes interacted with endothelial cells and showed clustering of ceramide upon contact with the endothelial cells. Shown are representative confocal microscopy images or means ± SD of 5 independent experiments; *p, 0.05 Scale bar, 5 mm in a.