Figure 2.
C2-ceramide inhibits both fluid phase and mannose receptor mediated endocytosis by DCs. (A, C, E) 2 × 105 DCs were incubated with different concentrations of C2-ceramide (open circles), C2-dihydroceramide (closed circles), or diacylglycerol (closed triangles) for 10 min on ice, then transferred at 37°C and LY (1 mg/ml) (A), FITC–DX (1 mg/ml) (C), or HRP (0.1 μg/ml) (E) were added for 30 min. Results are expressed as percent of maximum uptake. The background (cells pulsed at 0°C) was less than 1% of the uptake at 37°C in all the experiments. (B, D, F) 2 × 105 DCs were pretreated with 80 μM C2-ceramide (open circles) or medium (closed squares) for 10 min on ice, then transferred at 37°C and LY (B), FITC–DX (D) or HRP (F) accumulation was measured at different times. Results are expressed as mean fluorescence intensity (B, D) or as amount of cell-associated HRP (F). Comparable results were obtained using five different DC preparations. The vehicle did not affect endocytosis (data not shown).