DC-derived TNF-α and IL-1β are involved in a feedback loop mechanism that causes increased IL-23 production in cocultures of DCs and fibroblasts. (A) DC_act were cultured in the presence of fibroblast supernatants (DCact + [Fb-sn]) or PFA-fixated fibroblasts (DCact + Fb-fix). As controls, DC_act either cultured alone (DCact) or cocultured with fibroblasts (DCact + Fb) were used. IL-23 was detected by ELISA. *P < .001 compared with DCact (n = 3 independent experiments). (B) Neutralizing anti–TNF-α or anti–IL-1β antibodies were added to coculture of DC_act and fibroblasts separately (DCact + Fb + aTNF-α and DCact + Fb +aIL-1beta) or in combination (DCact + Fb + aTNFalpha/aIL-1beta). IL-23 levels were compared with coculture with isotype control (DCact + Fb + iso) and preactivated DCs cultured alone (DCact). #,*P < .05 (n = 4 independent experiments). (C-D) DC_act and fibroblasts were cocultured for 3 hours and then separated (DCact separated after coculture and Fb separated after coculture). For comparison, preactivated DCs (DCact) and fibroblasts (Fb) were cultured alone. Subsequently, RNA preparations and real-time PCR were performed. (C) Relative level of TNF-α mRNA was calculated on the basis of ΔCt values. mRNA expression was normalized to the unregulated housekeeping gene RPS26 and computed as percentage of TNF-α mRNA expression in DC_act. *P < .01 compared with DC_act (n = 3 independent experiments). (D) IL-1β mRNA was quantified through a standard curve, normalized to the unregulated housekeeping gene RPS26, and computed as percentage of mRNA expression in DC_act (n = 3 independent experiments).