Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct proox idant effect on macrophage-mediated LDL oxidation behind its antioxidant ef fect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophag es preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-ga mma /LPS) for 24 h, LDL oxidation was measured in terms of formation of thi obarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrop hages were measured by using Ellman's assay, the Griess reagent, and the SO D-inhibitable cytochrome c reduction method, respectively, IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite producti on in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages, TEARS format ion from LDL was suppressed in IFN-gamma- and IFN-gamma /LPS-treated iNOS(/+) macrophages but was increased in IFN-gamma -treated iNOS(-/-) macrophag es. In the presence of N-G-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma /LPS was abolished and T EARS formation was even increased to a level above that of untreated iNOS(/+) macrophage, NOC 18, an NO donor, dose dependently inhibited macrophage- mediated LDL oxidation. IFN-gamma increased superoxide and thiol production s in both types of macrophages. We conclude that IFN-gamma promotes macroph age-mediated LDL oxidation by stimulating superoxide and thiol production u nder conditions where iNOS-catalyzed NO release is restricted. (C) 2000 Aca demic Press.