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 the 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 13-acetate-stimulated monocytes and polymorphonucleate neutrophils of SSc patients to generate superoxide anion (O-2(.-)); and (ii) to investigate whether the O-2(.-) produced by these cells involved the activation of nicotinamide-adenine dinucleotide diphosphate oxidase biochemical pathway. Employing the superoxide dismutase-inhibitable reduction of cytochrome c to evaluate the generation of O-2(.-), unmanipulated monocytes of SSc patients generated more O-2(.-) than primary Raynaud's phenomenon patients and normal control monocytes (p = 0.0001), and the release was higher in patients with diffuse cutaneous involvement and 5 y or less disease duration (p = 0.02), The involvement of nicotinamide-adenine dinucleotide diphosphate oxidase in the enhanced O-2(.-) production was demonstrated by the finding that the cytosolic components of the enzyme, p47(phox) and p67(phox), Were both translocated to the plasma membrane of enriched but otherwise unmanipulated monocytes of SSc patients. The involvement of mitochondrial oxidases was excluded by the lack of inhibition of O-2(.-) production when monocytes were incubated in the presence of rotenone, a mitochondrial oxidase inhibitor. Upon stimulation with phorbol 12-myristate 13-acetate, monocytes of SSc patients produced more O-2(.-) than controls. In SSc patients untreated polymorphonucleate neutrophils generated significantly less O-2(.-) than monocytes (p = 0.0001) and only slightly more than polymorphonucleate neutrophils of primary Raynaud's phenomenon patients and normal controls (p = 0.03), In conclusion, we demonstrate that in patients with scleroderma, unmanipulated and phorbol 12-myristate 13-acetate-stimulated monocytes release in vitro increased amounts of superoxide anion through the activation of nicotinamide-adenine dinucleotide diphosphate oxidase and, thus, contribute to the oxidative stress found in this disease.