CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent

Objective: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE(2) and PGI(2) secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. Materials and Methods: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE(2), PGI(2) and IL-6 synthesis. Results: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154(+) D1.1 cells but not CD154(-) 132.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE(2) and PGI(2) secretion triggered by CD154(+) D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE(2) triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. Conclusions: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.