Covalent Cross-Linking of Immune Complexes by Oxygen Radicals and Nitrite

Abstract

We have shown that polymorphonuclear neutrophils mediate the covalent cross-linking of immune complexes (ICs) using H2O2 and myeloperoxidase (MPO). Moreover, activated superficial chondrocytes produce large amounts of nitric oxide (NO), suggesting that high concentrations of these radicals may interact at the cartilage surface in rheumatoid arthritis. We describe the effects of the interaction of NO and its decay product, NO2, with H2O2 and MPO on IC cross-linking. Cross-linking was measured by resistance to the guanidine extraction of plastic-bound ICs. The combination of H2O2, MPO, and NO in the absence of O2 did not alter the magnitude of cross-linking. The addition of O2 resulted in a significant enhancement of cross-linking (p < 0.004), suggesting that nitrite was responsible for the increase observed. Indeed, NaNO2 greatly increased H2O2-dependent cross-linking (control: 29.2 ± 3.8; 1 mM NaNO2: 58.4 ± 9.9; 10 mM: 60.4 ± 4.2% cross-linking, p < 0.0002). Sodium azide, which is an inhibitor of MPO, completely inhibited cross-linking. These results indicated that the product of interaction of H2O2 and NO2 mediated by MPO may be responsible for the increase in cross-linking. The generation of nitrotyrosine was demonstrated when NO2 was added to the cross-linking system. Cross-linking was also shown with an O2−-generating system and NO. Peroxynitrite alone mediated cross-linking (100 μM ONOO−: 40.3 ± 1.9% cross-linking; p < 0.002), and the addition of MPO significantly enhanced this effect (100 μM: 57.7 ± 6.0%; p < 0.0002 with respect to no nitrite control). Oxygen radicals and NO are likely to interact at the cartilage surface in inflammatory arthritis, resulting in an increase in oxidative damage within the joint cavity.