Figure 6

SpA-induced CXCR4 downregulation is concomitant with BCR internalization and depends on PKC. (A) B cells were incubated with medium, 2 mmol/L MβCD or 0.4 mol/L sucrose for 1 h at 37°C before treatment with medium (empty histogram) or 100 ng/mL CXCL12 (black histogram) for 2 h at 37°C. Then, expression of CXCR4 on B cells was assessed by flow cytometry. (B) B cells were incubated with medium, 2 mmol/L MβCD or 0.4 mol/L sucrose for 1 h at 37°C before treatment with medium (empty histogram) or 10 µg/mL Mod-SpA (gray histogram) for 2 h at 37°C. Then expression of CXCR4 (upper panel) and SIgD (lower panel) on B cells was assessed by flow cytometry. (C, E) B cells from three to six independent donors were first cultured for 30 min at 37°C with medium alone (white bar), 20 µmol/L PP2, 5 µmol/L Herb, 5 µmol/L CC, 2 µmol/L U73122 or 5 µmol/L WMN. Cells were then incubated with 10 ng/mL anti-IgM Ab (C, hatched bars), 10 µg/mL Mod-SpA (D, black bars) or 100 nmol/L CXCL12 (E, dotted bars) for 16 h (C, D) or 2 h (E) at 37°C. After staining with CD20-PEcy7 and CXCR4-PE mAbs or IgG1-PE control, surface expression of CXCR4 was assessed by flow cytometry. Results are expressed as the mean ± SEM percentage of MFI values for residual surface CXCR4 expression. Two-tailed Wilcoxon test was used to determine statistical significance, and p values are indicated as *p < 0.05.