Antineutrophil cytoplasmic antibodies induce human monocytes to produce oxygen radicals in vitro

Objective. Antineutrophil cytoplasmic antibodies (ANCA) are believed to pla y a pathogenetic role in necrotizing small-vessel vasculitis. While the inv olvement of neutrophils in this disease has been extensively studied in vit ro, we undertook to analyze thoroughly the contribution of monocytes to tis sue destruction in systemic vasculitis. Methods. Monocytes obtained from normal human individuals were stimulated b y ANCA isolated from patients with active vasculitis. The formation of oxyg en radicals was measured by a fluorometric assay using 2',7'-dichlorofluore scin diacetate. Results. ANCA induced monocytes to produce oxygen radicals, resulting in a mean 43% increase (range 21-84%) in oxygen radical formation compared with normal IgG. The formation of reactive oxygen species was time and concentra tion dependent and was also induced by, ANCA F(ab')(2) fragments. Normal no nspecific IgG or their corresponding F(ab')(2) fragments induced no release or very little release of oxygen radicals. Preincubation of monocytes with the Fc gamma receptor type II-blocking monoclonal antibody IV.3 before add ition of ANCA greatly reduced formation of oxygen radicals. Using ligand af finity chromatography with proteinase 3 (PR3) and myeloperoxidase (MPO), AN CA were further purified by depletion of patient IgG. The stimulation of mo nocytes with these pure PR3- and MPO-ANCA confirmed that cellular activatio n was specifically induced by ANCA. Conclusion. These results show that ANCA induce the formation of reactive o xygen species in human monocytes. These findings support the notion that AN CA specifically activate monocytes by several mechanisms to participate in the inflammatory process of ANCA-associated vasculitis.