To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc).

Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O₂⁻ and H₂O₂ released from fibroblasts were estimated by the superoxide dismutase (SOD)–inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti‐p47^phox–specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by ³H‐thymidine incorporation. Northern blot analysis was used to study α1 and α2 type I collagen gene expression.

Unstimulated skin fibroblasts from SSc patients released more O₂⁻ and H₂O₂ in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component 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‐1β (IL‐1β), platelet‐derived growth factor type BB (PDGF‐BB), transforming growth factor β1 (TGFβ1), and H₂O₂. Treatment of normal and SSc fibroblasts with tumor necrosis factor α (TNFα), IL‐2, IL‐4, IL‐6, IL‐10, interferon‐α (IFNα), IFNγ, granulocyte–macrophage colony‐stimulating factor (GM‐CSP), G‐CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL‐1 receptor antagonist, or antibodies blocking the effect of TGFβ1, PDGF‐BB, and other agonists (IL‐4, IL‐6, TNFα, CTGF). In contrast, treatment of SSc fibroblasts with the membrane‐permeant antioxidant N‐acetyl‐L‐cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down‐regulation of α1(I) and α2(I) collagen messenger RNA.

The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase–like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O₂⁻, H₂O₂, IL‐1β, TGFβ1, PDGF‐BB, IL‐4, IL‐6, TNFα, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.