Population dynamics of inducible nitric oxide synthase production by LPSand LPS/Ifnγ-stimulated mouse macrophages

The reactive nitrogen intermediate, nitric oxide (NO) is important in host defense against both NO-sensitive microorganisms and tumor cells. Macrophages are one of the chief inflammatory sources, especially when stimulated with the combination of LPS and interferonγ (Ifnγ). It is not known, however, whether Ifnγ-mediated augmentation of LPS-induced production of NO is the result of greater production by all cells or to the recruitment of more producer macrophages within a given population. This question was addressed, first, by stimulating mouse macrophages (either bone marrow culture-derived, inflammatory peritoneal or those of the cell line, RAW 264.7) with up to 10 U/ml Ifnγ for as long as 24 h. Under these conditions, there was little or no production of NO and rare or no cells were immunocytochemically positive for the inducible form of nitric oxide synthase (iNOS), which catalyzes the production of NO. Populations similarly exposed to 1 ng/ml LPS were low producers of NO and contained somewhat more, but still only a few (< 15%), iNOS-positive cells. In contrast, as the concentration of Ifnγ was increased (≥ 1 U/ml) in the presence of a constant amount of LPS (1 ng/ml), the principal effect was to increase both the production of NO and the number of iNOS-positive macrophages. The amount of iNOS expressed by some cells also appeared to be increased. Two important conclusions can be drawn from these findings: (1) there is heterogeneity in mouse macrophage populations with respect to the production of iNOS; and (2) increasing concentrations of Ifnγ appear to augment LPS-induced secretion of NO by recruiting increasingly greater numbers of macrophages into the production of iNOS. Such results potentially provide important clues as to how Ifnγ may be acting at the subcellular level to enhance iNOS synthesis.