FIGURE 4. Effect of SM4s on phagocytosis of apoptotic C26 cells by macrophages.

A and B, For ex vivo phagocytosis assay, murine peritoneal macrophages were coincubated with DiO-labeled SM4s- positive or SM4s-free apoptotic C26 at 37°C for 1 h, washed, stained with anti-mouse CD11b-PE Ab, and analyzed by flow cytometry. Alternatively, macrophages were directly exposed to SM4s before coincubation with SM4s-free apoptotic C26 cells. Double-positive population represented macrophages that took part in tethering and/or uptake of apoptotic C26. C, FACS phagocytosis index was calculated by dividing mean relative DiO fluorescence intensities of macrophages (upper quadrants of density plots) and apoptotic C26 (lower right quadrant). D–F, FACS analysis of in vivo uptake experiments. Number of macrophages per mouse was counted to ascertain constant ratio macrophages:apoptotic cells (1:2). G and H, Macrophages were cocultured with apoptotic cells preincubated with 10 µM SM4s (SM4), galactosylceramide (GC), cholesterol sulfate (CS), seminolipid (SM4g), ceramide-1-phosphate (C1P), or vehicle at 37°C for 1 h. Phagocytosis was analyzed by flow cytometry. Results represent mean ± SEM of three experiments in duplicates. Statistical differences were evaluated by paired t test (ex vivo data) or Mann-Whitney U test (in vivo data). *, p < 0,05; and ***, p < 0,001.