Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-dependent nitric oxide production

Abstract

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.