FIGURE 2.

Nitric oxide production and iNOS levels in T. cruzi-infected macrophages. A, nitrite production. Macrophages were exposed to IFN-γ/LPS for 24 h in the absence and presence of epimastigotes (epimastigotes:macrophages, 5:1 and 20:1) or trypomastigotes (20:1). The effect of apocynin (apo) and l-NMMA (2 mm) was also evaluated. Nitrite accumulation was determined using the Griess reaction. epis, epimastigotes; CTL, control; trypos, trypomastigotes. B, DAF-2DA oxidation. Macrophages were exposed to IFN-γ/LPS in the presence of epimastigotes (20:1), trypomastigotes (20:1), or l-NMMA (2 mm) in DMEM at 37 °C. Control experiments were performed in the absence of cytokine stimulation. After 5 h, the medium was replaced with dPBS containing 5 μm DAF-2DA, and probe oxidation was followed in a fluorescence plate reader at 37 °C. RFU, relative fluorescence units. C, iNOS mRNA levels of macrophages exposed to IFN-γ/LPS, IFN-γ/LPS + trypomastigotes (10:1), IFN-γ, and IFN-γ + trypomastigotes (10:1) were evaluated by RT-PCR. Total RNA from 5 × 10⁶ macrophages under all conditions was extracted after 3 h of infection. Host iNOS and GAPDH cDNAs were amplified using primers defined under “Experimental Procedures.” D, iNOS expression was evaluated by immunocytochemistry. Macrophages plated in slides were stimulated for 5 h with IFN-γ/LPS in the presence or absence of T. cruzi epimastigotes or trypomastigotes. Immunocytochemical analysis of iNOS expression was performed using polyclonal anti-NOS antibody and visualized with Alexa-594-conjugated anti-rabbit antibody (magnification, ×400). Data shown are representative of at least three independent experiments performed on separate days.