Effect of CD40 activation on VEGF production and migration is inhibited by 5C8 blocking anti-CD40L mAb.

MM cells were cultured in the presence or absence of sCD40L (10 μg/mL [μM]) and a 5C8 blocking anti-CD40L mAb (0-10 μg/mL [μM]) to confirm the specificity of sCD40L-triggered VEGF mRNA expression and cell migration. Blocking mAbs were added to the wells at the initiation of cultures at 37°C. (A) Twenty-four hours after indicated treatments, cells were collected and total RNA was prepared for Northern blotting (upper panel, RPMI 8226; lower panel, SV). Total RNA (5 μg) was used for analysis, and 18 rRNA served as an internal control. All autoradiographs are representative of 2 experiments with similar results. (B) MM cells were subjected to transmigration assay with the addition of sCD40L in the presence or absence of 5C8 blocking anti-CD40L mAb in the lower chamber of the transwell. (■) RPMI 8226 at 37°C; (▪) SV at 37°C. Identical effects of CD40 activation on the induction of VEGF were obtained with anti-CD40 mAb G28.5 (1 to 10 μg/mL [μM]) (data not shown).