Oxidative modification of inflammatory synovial fluid immunoglobulin G.

Abstract

The release of highly reactive oxygen-derived products by activated phagocytic cells in inflammatory foci plays a major role in defense mechanisms against infection and in the generation of tissue injury. Oxidative modification of proteins in inflammatory foci may give rise to products that contribute to the perpetuation of inflammation. The present studies analyze the oxidative alterations of inflammatory synovial fluid immunoglobulin G (IgG). IgG was purified from the synovial fluids of five patients with rheumatoid arthritis and two patients with acute gouty arthritis by three sequential steps: gel filtration chromatography, immunoaffinity chromatography, and a final gel filtration chromatography step under dissociative conditions (4 M guanidine). The resulting protein peaks of > 150 kDa (pool I-1), 150 kDa (pools I-2 and II-2), and < 150 kDa (pools I-3 and II-3) were tested for the presence of a fluorescence profile distinctive of oxidized proteins, the peak corresponding to monomer IgG (pool II-2) was subjected to quantitative amino acid analysis, and the results were compared with a standard preparation of normal IgG oxidized with HOCl. In addition, the protein pools were tested for the presence of lipid peroxide products. The results indicate that a portion of the affinity-purified IgG formed high-molecular-mass covalently cross-linked aggregates as evidenced by its presence in the > 150-kDa pool after dissociatve gel filtration chromatography. Moreover, this fraction and the pool corresponding to monomer IgG (pool II-2) exhibited the fluorescence profile characteristic of oxidized proteins. Amino acid analysis of the monomer IgG fraction revealed decreases in the content of histidine, methionine, tyrosine, and cysteine, which were similar to the alterations measured in normal IgG oxidized by HOCl. The synovial fluid and standard oxidized IgG showed the presence of oxidative by-products of tyrosine (monochlorotyrosine) and cysteine (cysteic acid). The synovial fluid IgG yielded a novel component that was not present in the standard control or oxidized IgG. This component was partially identified by mass spectrometry. Finally, the smaller peptide fraction isolated from affinity-purified synovial fluid IgG (pools I-3 and II-3) only after the gel filtration chromatography step under dissociative conditions exhibited evidence of oxidative damage and the presence of high concentrations of thiobarbituric acid-reactive material (TBAR). These observations suggest that oxidative processes in inflammatory foci generate products derived from protein and lipids that may contribute to the self-perpetuation of inflammation.