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 vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc).


METHODS
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-p47phox-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 alpha1 and alpha2 type I collagen gene expression.


RESULTS
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, p47phox, 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-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, 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 TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, 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 alpha1(I) and alpha2(I) collagen messenger RNA.


CONCLUSIONS
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-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.
Sambo
P
P
Università di Ancona, Italy.
Baroni
S S
SS
Luchetti
M
M
Paroncini
P
P
Dusi
S
S
Orlandini
G
G
Gabrielli
A
A
D000111:Acetylcysteine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D018384:Oxidative Stress; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D012594:Scleroderma, Localized / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology; D012867:Skin / Q000473:pathology; D015854:Up-Regulation; D024502:alpha-Tocopherol / Q000494:pharmacology
D001713:Biphenyl Compounds; D016207:Cytokines; D006133:Growth Substances; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D012402:Rotenone; C031291:diphenyliodonium; D000493:Allopurinol; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D024502:alpha-Tocopherol; D000111:Acetylcysteine