FIG. 2.

(A) Synergistic effect of TNF-α and IFN-γ on the induction of IDO activity in HBMEC. Aliquots of 3 × 10⁴ HBMEC were stimulated with IFN-γ (0 to 1,000 U/ml) in the absence or in the presence of TNF-α (200 U/ml) for 3 days. Thereafter, culture supernatants were harvested and the kynurenine content was determined by the use of Ehrlich's reagent. Data are given as mean optical density at 492 nm (OD₄₉₂) ± the standard error of triplicate cultures. (B) Detection of IDO mRNA in IFN-γ-activated HBMEC. HBMEC were stimulated with or without IFN-γ (200 U/ml), and RT-PCR was performed as described in the text, 18 h after cytokine stimulation. As a control, GAPDH mRNA was analyzed in parallel. The data shown indicate that IFN-γ is a strong inducer of IDO mRNA in HBMEC. (C) Synergistic effect of TNF-α on IFN-γ-induced IDO protein production. HBMEC were stimulated in the absence or presence of IFN-γ (800 U/ml) or TNF-α (200 U/ml). After 3 days cells were harvested and subjected to Western blot analysis. As a control, GAPDH was analyzed in parallel. The data shown indicate that TNF-α is unable to induce IDO activity in HBMEC, but it enhances IDO expression in cells costimulated with IFN-γ.