Monocytes of patients with systemic sclerosis (scleroderma) spontaneously release in vitro increased amounts of superoxide anion

It has been suggested that toxic oxygen free radicals can be involved in th e pathogenesis of systemic sclerosis (scleroderma) (SSc). Because the cells that contribute to the generation of free radicals are not known, our aim was (i) to evaluate the ability of unmanipulated and phorbol 12-myristate 1 3-acetate-stimulated monocytes and polymorphonucleate neutrophils of SSc pa tients to generate superoxide anion (O-2(.-)); and (ii) to investigate whet her the O-2(.-) produced by these cells involved the activation of nicotina mide-adenine dinucleotide diphosphate oxidase biochemical pathway. Employin g the superoxide dismutase-inhibitable reduction of cytochrome c to evaluat e the generation of O-2(.-), unmanipulated monocytes of SSc patients genera ted more O-2(.-) than primary Raynaud's phenomenon patients and normal cont rol monocytes (p = 0.0001), and the release was higher in patients with dif fuse cutaneous involvement and 5 y or less disease duration (p = 0.02), The involvement of nicotinamide-adenine dinucleotide diphosphate oxidase in th e enhanced O-2(.-) production was demonstrated by the finding that the cyto solic components of the enzyme, p47(phox) and p67(phox), Were both transloc ated to the plasma membrane of enriched but otherwise unmanipulated monocyt es of SSc patients. The involvement of mitochondrial oxidases was excluded by the lack of inhibition of O-2(.-) production when monocytes were incubat ed in the presence of rotenone, a mitochondrial oxidase inhibitor. Upon sti mulation with phorbol 12-myristate 13-acetate, monocytes of SSc patients pr oduced more O-2(.-) than controls. In SSc patients untreated polymorphonucl eate neutrophils generated significantly less O-2(.-) than monocytes (p = 0 .0001) and only slightly more than polymorphonucleate neutrophils of primar y Raynaud's phenomenon patients and normal controls (p = 0.03), In conclusi on, we demonstrate that in patients with scleroderma, unmanipulated and pho rbol 12-myristate 13-acetate-stimulated monocytes release in vitro increase d amounts of superoxide anion through the activation of nicotinamide-adenin e dinucleotide diphosphate oxidase and, thus, contribute to the oxidative s tress found in this disease.