ARTHRITIS & RHEUMATISM Vol. 44, No. 7, July 2001, pp 1667–1676 © 2001, American College of Rheumatology Published by Wiley-Liss, Inc. Decreased Susceptibility to Fas-Induced Apoptosis of Systemic Sclerosis Dermal Fibroblasts Begon ˜a Santiago, Marı ´a Galindo, Miguel Rivero, and Jose ´ Luis Pablos Objective. To determine whether dysregulated ap- optosis of systemic sclerosis (SSc) fibroblasts contrib- utes to progressive fibrosis by promoting fibroblast longevity. Methods. We examined the pattern of fibroblast proliferation and apoptosis in SSc skin lesions and the susceptibility of cultured SSc dermal fibroblasts to apoptosis. Skin biopsy samples from SSc patients and control subjects were used to establish fibroblast cul- tures and were examined histologically. In skin sections, apoptosis was examined by TUNEL, and proliferation by immunostaining for proliferating cell nuclear anti- gen. Susceptibility of fibroblasts to apoptosis induced in vitro by different stimuli was studied by TUNEL. Ex- pression of Bcl-2, Bcl-x, and Bax proteins in cultured fibroblasts was studied by Western blotting. Results. Proliferation of dermal fibroblasts was not observed in normal skin but was present in skin from patients with SSc and other inflammatory skin diseases. Apoptosis of fibroblasts in SSc fibrotic skin lesions was not observed. In vitro, SSc fibroblasts were specifically resistant to apoptosis induced by Fas recep- tor stimulation but had normal susceptibility to apopto- sis induced by nonspecific stimuli (protein kinase inhi- bition or serum withdrawal). Decreased susceptibility to Fas stimulation was not caused by decreased levels of surface Fas receptor. In SSc fibroblasts, quiescence induced by confluence and serum starvation was fol- lowed by an abnormal down-regulation of proapoptotic Bax protein. Up-regulation of the Bax:Bcl-2 ratio in SSc fibroblasts by Bcl-2 antisense oligonucleotides restored their susceptibility to Fas-mediated apoptosis. Conclusion. Our findings suggest that abnormal apoptotic regulation in fibroblasts can contribute to the pathogenesis of progressive fibrosis in SSc. Modulation of Bcl-2–related proteins appears to be a potential target for the development of apoptosis-based antifi- brotic strategies. The pathogenesis of systemic sclerosis (SSc) in- volves vascular injury, inflammatory cell infiltration, and the presence of an excessive number of fibroblasts with an activated phenotype. These alterations ultimately result in an excessive accumulation of extracellular matrix components, mainly fibrillar types I and III collagens, and a severe and progressive fibrosis of the skin and internal organs (1,2). The transcription levels of types I and III procollagen genes by normal or SSc dermal fibroblasts is highly heterogeneous, as demon- strated by in situ hybridization or in vitro cloning of fibroblasts (3–8). The hallmark of SSc skin fibrosis is the presence of an expanded pool of fibroblasts containing high levels of matrix gene messenger RNA (mRNA), which continue to overexpress matrix genes for many passages in culture. Selective growth of high collagen mRNA–producing fibroblasts has been proposed to explain this alteration (7,8). Increased amounts of cytokines and growth fac- tors with the potential to modify fibroblast behavior are detected in fibrotic skin (9–11). Among these factors, transforming growth factor  (TGF) is a potent tran- scriptional activator of procollagen genes in fibroblasts (12,13). In vitro, TGF is not mitogenic, but it can induce the expression of the fibroblast mitogen platelet- derived growth factor and its  receptor, and this autocrine loop seems to operate in SSc (14–16). There- fore, both transcriptional activation and excessive growth of activated fibroblasts have been proposed to explain the observed phenotype. Supported by grant FIS 98/0356 from the Fondo de Investi- gaciones Sanitarias and by grant PM 96/0028 from the Ministerio de Educacio ´n y Cultura, Spain. Begon ˜a Santiago, MSc, Marı ´a Galindo, MD, Miguel Rivero, PhD, Jose ´ Luis Pablos, MD: Hospital 12 de Octubre, Madrid, Spain. Address correspondence and reprint requests to Jose ´ Luis Pablos, MD, Unidad de Investigacio ´n, Hospital 12 de Octubre, 28041 Madrid, Spain. Submitted for publication July 18, 2000; accepted in revised form February 26, 2001. 1667