PGE₂, which is produced because of coculturing of activated DCs and fibroblasts, is also involved in IL-23 up-regulation. (A) Indomethacin's PGE₂ inhibiting efficiency was verified by detection of PGE₂ levels in DC_act cultured alone (DCact) and in coculture of DC_act and fibroblasts with and without 2μM indomethacin (DCact + Fb and DCact + Fb + indomethacin, respectively). *P < .01 (n = 7 independent experiments). (B) DCs were preactivated with LPS and subsequently cocultured with fibroblasts (DCact + Fb) or without fibroblasts (DCact). Indomethacin (2μM) was added to the cocultures (DCact + Fb + indomethacin) and IL-23 was measured by ELISA. *P < .001 (n = 7 independent experiments). (C) DCs and fibroblasts were separated using anti–Thy-1 coated microbeads after a coculture time of 3 hours (DCact separated after coculture and Fb separated after coculture). Controls were again LPS-stimulated DC (DCact) and fibroblasts (Fb) cultured alone. Then, RNA was prepared and quantitative RT-PCR was performed. Cox-2 mRNA expression values were normalized to the unregulated housekeeping gene RPS26 and are given as percentage of Cox-2 mRNA expression in DC_act. *P < .01 (n = 3 independent experiments).