Kinetics of sphingomyelinase activation and ceramide formation during PMN phagocytosis of EIgG. PMNs (2 × 106/mL) were activated with FMLP (100 nmol/L) for 10 minutes at 37°C. EIgG (1 × 108/mL) were added and aliquots of cells removed at the indicated time points. EIgG not ingested were lysed and PMNs resuspended in buffer. Samples were probe sonicated and assayed for ceramide and sphingomyelinase activity. (A) Kinetics of phagocytosis. Inset: Rate of phagocytosis was determined by removing aliquots of PMNs at 30-second intervals for the first 2.5 minutes after addition of EIgG. Subsequent time points were at 60-second intervals for the next 3.5 minutes and at 4-minute intervals thereafter. Rate of ingestion was calculated by dividing the number of EIgG ingested in a given time interval by the number of minutes in that interval. The rates were plotted over the midpoint for each interval. (B) Kinetics of ceramide formation and sphingomyelinase activation. Values represent the mean ± SEM of three experiments. * = Significantly different from the zero time point, P < .05.
