Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) re presents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected w ith Leishmania infantum. Macrophages obtained from peripheral blood of heal thy dogs were activated with recombinant human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) and then infected with L. infantum prom astigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophage s were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F (ab ')(2) goat anti-rabbit IgG. For immunoblotting, cell lysates were submi tted to SDS-PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Resul ts demonstrated that L. infantum-infected cells, after stimulation with rhI FN-gamma and LPS, displayed high levels of fluorescence for the NOS2 in the ir cytoplasm, unlike unstimulated uninfected macrophages. In western blotti ng, polyclonal anti-NOS2 reacted specifically with a protein band correspon ding to 130 kDa. The signal produced in Leishmania-infected cells stimulate d with rhIFN-gamma and LPS was higher than that produced in Leishmania-infe cted unstimulated cells. No band was detected in cellular lysates from unin fected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-gamma and LPS in NOS2 induction al so in this animal model. (C) 2001 Elsevier Science Ltd. All rights reserved .