Inducible Nitric Oxide Synthase Knockout Mouse Macrophages Disclose Prooxidant Effect of Interferon-γ on Low-Density Lipoprotein Oxidation

To test our hypothesis that interferon-γ (IFN-γ) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS+/+) or iNOS knockout mouse (iNOS−/−) macrophages preincubated with IFN-γ or IFN-γ plus lipopolysaccharide (IFN-γ/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-γ alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS+/+ macrophages, but not in iNOS−/− macrophages. TBARS formation from LDL was suppressed in IFN-γ- and IFN-γ/LPS-treated iNOS+/+ macrophages but was increased in IFN-γ-treated iNOS−/− macrophages. In the presence of NG-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-γ and IFN-γ/LPS was abolished and TBARS 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-γ increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-γ promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.