title: Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages.
abstract: Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents 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 with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes. zymodeme MONI. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab')2 goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS-PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-gamma and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-gamma and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-gamma and LPS in NOS2 induction also in this animal model.
authors.affiliation: Dipartimento di Clinica Medica, Immunologia e Malattie Infettiva e Sezione di Microbiologia e Immunologia, University of Bari, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Sisto
authors.forename: O
authors.initials: O
authors.lastname: Brandonisio
authors.forename: M A
authors.initials: MA
authors.lastname: Panaro
authors.forename: A
authors.initials: A
authors.lastname: Acquafredda
authors.forename: D
authors.initials: D
authors.lastname: Leogrande
authors.forename: A
authors.initials: A
authors.lastname: Fasanella
authors.forename: T
authors.initials: T
authors.lastname: Trotta
authors.forename: L
authors.initials: L
authors.lastname: Fumarola
authors.forename: V
authors.initials: V
authors.lastname: Mitolo
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004283:Dog Diseases / Q000097:blood / Q000276:immunology / Q000469:parasitology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D015151:Immunoblotting / Q000662:veterinary; D007371:Interferon-gamma / Q000494:pharmacology; D018314:Leishmania infantum / Q000276:immunology* / Q000472:pathogenicity; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000276:immunology / Q000662:veterinary; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000201:enzymology* / Q000276:immunology / Q000469:parasitology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D011994:Recombinant Proteins
chemical_list: D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II