Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 D. is online at: average * The Journal of Immunology published online 1 June 2011 from submission to initial decision 4 weeks from acceptance to publication Groshong, Gregory P. Cosgrove, Elizabeth F. Redente, Alison Bamberg, Kevin K. Brown, Nichole Reisdorph, Rebecca C. Keith, Stephen K. Frankel and David W. H. Riches http://www.jimmunol.org/content/early/2011/05/29/jimmun ol.1100447 Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis Murry W. Wynes, Benjamin L. Edelman, Amanda G. Kostyk, Michael G. Edwards, Christopher Coldren, Steve J Immunol Submit online. 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Published June 1, 2011, doi:10.4049/jimmunol.1100447 The Journal of Immunology Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis Murry W. Wynes,*,† Benjamin L. Edelman,* Amanda G. Kostyk,* Michael G. Edwards,† Christopher Coldren,† Steve D. Groshong,†,‡ Gregory P. Cosgrove,†,‡ Elizabeth F. Redente,* Alison Bamberg,*,x Kevin K. Brown,†,‡ Nichole Reisdorph,x Rebecca C. Keith,†,‡ Stephen K. Frankel,†,‡ and David W. H. Riches*,†,x Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the Downloaded from lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-a and IFN-g reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-a– and IFN-g–stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-kB– and p38mapk-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-a and IFN-g to sensitize fibroblasts to http://www.jimmunol.org/ Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal re- sistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF. The Journal of Immu- nology, 2011, 187: 000–000. rogressive pulmonary fibrosis, especially idiopathic pul- unrestrained accumulation of fibroblasts and myofibroblasts that by guest on September 23, 2021 monary fibrosis (IPF), is thought to arise following injury synthesize and deposit collagen fibrils within fibroblast foci lo- P to, and abnormal repair of, the distal alveolar-capillary cated in the pulmonary parenchyma (2, 3). Recent studies have units (1). Although little is known about how the alveolar epi- suggested that pulmonary fibroblasts arise by several routes thelium is injured, the ensuing fibrotic response is associated with including increased migration and proliferation of resident pul- monary fibroblasts, mesenchymal transition of the alveolar epithelium, and recruitment of bone marrow-derived progenitor *Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, cells (4–7). In the presence of TGF-b and other agonists, resident † CO 80206; Division of Pulmonary Sciences and Critical Care Medicine, Department fibroblast subsets transdifferentiate into a-smooth muscle actin- of Medicine, University of Colorado School of Medicine, Aurora, CO 80010; ‡Department of Medicine, National Jewish Health, Denver, CO 80206; and xDepart- positive myofibroblasts (8, 9), produce increased amounts of ment of Immunology, University of Colorado School of Medicine, Aurora, CO 80010 collagen, and, through their contractile activities, distort the pa- Received for publication February 11, 2011. Accepted for publication April 22, 2011. renchymal lung architecture (9, 10). Furthermore, in contrast to This work was supported by Public Health Service Grants HL068628 and HL055549 normal resolution of the repair process, in which fibroblasts and (to D.W.H.R.) and Grant T15 HL086386-01 (to N.R.) from the National Heart, Lung, myofibroblasts are eliminated by apoptosis (11), in situ studies and Blood Institute of the National Institutes of Health. M.W.W. was supported in part by a Parker B. Francis fellowship. A.B. was supported in part by T32 Training with fibrotic lung tissues from IPF patients and bleomycin-in- Grant AI07405 from the National Institute of Allergy and Infectious Diseases. E.F.R. duced pulmonary fibrosis in mice have shown that fibroblasts was supported by Ruth L. Kirschstein F32 National Research Service Award HL095274 from the National Heart, Lung, and Blood Institute and a Viola Vestal and myofibroblasts are resistant to apoptosis and accumulate in the Coulter scholarship from National Jewish Health. lung parenchyma (12–14). Remarkably, little is known about the The array data presented in this article have been submitted to the Gene Expression physiologic and pathologic mechanisms that contribute to fibro- Omnibus under accession number GSE26594. blast survival or to their susceptibility to apoptosis. Address correspondence and reprint requests to Dr. David W.H. Riches, Program in Fibroblast apoptosis can be induced by multiple agonists, cog- Cell Biology, Department of Pediatrics, Iris and Michael Smith Building Room nate receptors, and signaling pathways that converge to promote A549, National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] caspase activation. Conversely, resistance to apoptosis occurs fol- The online version of this article contains supplemental material. lowing exposure of fibroblasts to an array of prosurvival factors Abbreviations used in this article: DISC, death-inducing signaling complex; FasL, including TGF-b (15, 16). Recent studies have shown that Fas, Fas ligand; FDR, false discovery rate; ILD, Interstitial Lung Disease; IPF, idiopathic a cell-surface death receptor of the TNFR superfamily, paradox- pulmonary fibrosis; MOI, multiplicity of infection; qPCR, quantitative RT-PCR; ically initiates both survival/differentiation and apoptosis in a siRNA, small interfering RNA. context-specific fashion. Ligation of Fas alone activates ERK and Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 NF-kB signaling pathways, promotes neuronal and epithelial cell www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100447 2 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS survival (17–19), and induces minimal apoptosis in lung fibro- the Advancement of Medicine (Edison, NJ), in accordance with an ap- blasts (20). In contrast, prior exposure to TNF-a and IFN-g ren- proved Institutional Review Board protocol (HS-1539). All donors suffered ders fibroblasts and myofibroblasts exquisitely sensitive to Fas brain death and were evaluated for organ donation before research consent. All lung samples failed regional lung selection criteria for transplantation. ligation-induced apoptosis and allows them to overcome the pro- Individuals had no evidence of current systemic or pulmonary infection, survival effect of TGF-b (20). Two recent studies have shown a clear chest radiograph, and partial pressure for oxygen/fraction of in- . a similar sensitizing effect of PGE2 on Fas-induced fibroblast spired oxygen ratio 250 mm Hg O2. Patients were excluded if they had apoptosis (14, 21). Although the source(s) of Fas ligand (FasL) are any history of lung disease or a history of systemic disease that commonly affects the lungs (e.g., rheumatoid arthritis or systemic lupus eryth- not completely known, recent studies have suggested that myofi- ematosus). Fibrotic lung fibroblasts from IPF patients were derived from broblasts themselves may be an important source in the fibrotic surgical lung biopsy specimens obtained as part of a prospective, In- lung (22, 23). Taken together, these findings suggest that fibroblast stitutional Review Board-approved, longitudinal study of the pathobiology and myofibroblast accumulation in the lungs of IPF patients could of fibrotic lung disease