Synergistic and p38-dependent induction of IFN-γ mRNA and protein by IL-12 and IL-18. (A) Freshly isolated human PBLs (4 × 106/mL) were treated with 1 μM SB203580 (SB; ▪) or vehicle (0.1% DMSO; □), IL-12 (20 ng/mL) and IL-18 (20 ng/mL) as indicated for 7 hours. Supernatants were collected and IFN-γ protein content was measured by ELISA (top). Error bars indicate SD of triplicate measurements. Total RNA was purified and subjected to ribonuclease protection assay to quantitate IFN-γ mRNA, plus L32 and GAPDH mRNAs as loading controls (bottom). (B) Freshly isolated human PBLs were treated with 1 μM SB203580 (SB; ▪) or vehicle (0.1% DMSO; □) and then stimulated with IL-12 and IL-18 (20 ng/mL each) for 7 hours. IFN-γ protein and mRNA were quantified as described for panel A, and calculated as percentage of expression in the absence of SB203580. Mean values from 7 different donors are plotted. Error bars indicate SEM. (C) Freshly isolated human PBLs (7 × 106/mL) were treated with the indicated doses of SB203580 or vehicle (0.1% DMSO) and stimulated with IL-12 and IL-18 (20 ng/mL each) for 7 hours. IFN-γ protein content of supernatants was measured by ELISA (top). Error bars indicate SD of triplicate measurements. Total RNA was purified; IFN-γ mRNA was quantified by Northern blotting and 28S rRNA was quantified as a loading control (bottom). This experiment was performed twice with very similar results.
