Oxidative stress in scleroderma - Maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway

Objective. To explore the role of reactive oxygen species (ROS) in the in v itro activation of skin fibroblasts from patients with systemic sclerosis ( SSc). Methods. Fibroblasts were obtained from involved skin of patients with limi ted or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels Of O-2(-) and H2O2 released from fibr oblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytoch rome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted wi th an anti-p47(phox)-specific antibody. Fibroblasts were stimulated with va rious cytokines and growth factors to determine whether any of these factor s modulate ROS generation. Cell proliferation was estimated by H-3-thymidin e incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. Results. Unstimulated skin fibroblasts from SSc patients released more O-2( -) and H2O2 in vitro through the NADPH oxidase complex pathway than did nor mal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodon ium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppr ession was not seen with rotenone, a mitochondrial oxidase inhibitor, or al lopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic compone nt of NADPH oxidase, p47(phox), was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1 beta (IL-1 beta), pl atelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGF beta1), and H2O2. Treatment of normal and SSc fibroblasts with t umor necrosis factor alpha (TNF alpha), IL-2, IL-4, IL-6, IL-10, interferon -alpha (IFN alpha), IFN gamma, granulocyte-macrophage colony-stimulating fa ctor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effe ct on ROS generation. Constitutive ROS production by SSc fibroblasts was no t inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGF beta1, PDGF-BB, and o ther agonists (IL-4, IL-6, TNF alpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhi bited ROS production, and this was accompanied by decreased proliferation o f these cells and down-regulation of alpha1(I) and alpha2(I) collagen messe nger RNA. Conclusion. The constitutive intracellular production of ROS by SSc fibrobl asts derives from the activation of an NADPH oxidase-like system and is ess ential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O-2(-), H2O2, IL-1 beta, TGF beta1, PDGF-BB, IL-4, IL-6, TNF alpha, or CTGF as mediators of a positive, autocri ne feedback mechanism of ROS generation.