Fibroblasts support IL-23 release from DCs. (A) Monocyte-derived DCs were preactivated with LPS for 3 hours. Subsequently, LPS-stimulated DCs (termed DCact in the entire manuscript) were washed extensively and then cocultured with fibroblasts (DCact + Fb) overnight. As control, LPS-stimulated DCs were cultured alone (DCact). Furthermore, we cultured immature DCs without (DC) and with fibroblasts (DC + Fb) or fibroblasts (Fb) alone. IL-23 protein levels were measured by ELISA. *P < .001 compared with DCact (n = 5 independent experiments). (B) DCact and fibroblasts were cocultured for 3 hours; afterward, DCs (DCact separated after coculture) and fibroblasts (Fb separated after coculture) were separated from the coculture by anti–Thy-1–coupled magnetic beads. For comparison, DCact and fibroblasts (Fb) were cultured alone. Subsequently, RNA preparation and real-time PCR of IL-23p19 mRNA were performed. IL-23p19 mRNA expression values were normalized to the unregulated housekeeping gene RPS26 and are given as percentage of IL-23p19 mRNA expression in DCact. *P < .001 compared with DCact (n = 3 independent experiments).
