Fibrosis Accumulation in Idiopathic Pulmonary Apoptosis: Implications for Fibroblast Fibroblasts to Fas Ligation-Induced Necessa

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Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast This information is current as Accumulation in Idiopathic Pulmonary of October 1, 2021. 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, Kevin K. Brown, Nichole Reisdorph, Downloaded from Rebecca C. Keith, Stephen K. Frankel and David W. H. Riches J Immunol 2011; 187:527-537; Prepublished online 1 June 2011; doi: 10.4049/jimmunol.1100447 http://www.jimmunol.org/ http://www.jimmunol.org/content/187/1/527

Supplementary http://www.jimmunol.org/content/suppl/2011/06/01/jimmunol.110044 Material 7.DC1 References This article cites 62 articles, 18 of which you can access for free at: by guest on October 1, 2021 http://www.jimmunol.org/content/187/1/527.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 resistance 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: 527–537.

rogressive pulmonary fibrosis, especially idiopathic pul- unrestrained accumulation of fibroblasts and myofibroblasts that monary fibrosis (IPF), is thought to arise following injury synthesize and deposit collagen fibrils within fibroblast foci lo- by guest on October 1, 2021 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 pulmonary 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 528 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 within the Interstitial Lung Disease (ILD) Program occur as a result of impaired sensitization to Fas-induced apo- at National Jewish Health (HS1603). Pathologic diagnoses were determined via review by an expert pulmonary pathologist with the ILD ptosis. They are also consistent with the developing notion that Program, and a pathologic diagnosis of usual interstitial pneumonia was fibrosis can progress in the absence of a robust inflammatory re- required for a clinical diagnosis of IPF. All clinical diagnoses were de- sponse associated with reduced levels of proinflammatory sensi- termined by an ILD Program physician who was caring for the patient, tizing molecules such as TNF-a, IFN-g, and PGE (1, 24–26). adhering to established pathologic and clinical criteria for the diagnosis of 2 idiopathic pulmonary fibrosis (27, 28). Freshly harvested explants, surgical Seeking to understand how TNF-a and IFN-g sensitize fibro- lung biopsies, or murine lungs were minced into 1 to 2 mm3 sections and blasts to Fas-induced apoptosis, the goal of this study was to cultured in DMEM containing 10% heat-inactivated FCS media on scored investigate the mechanisms that couple cytokine-induced sensiti- tissue-culture dishes. After 3 to 4 d, fibroblasts derived from the tissue zation to the acquisition of susceptibility to Fas-induced apoptosis. were trypsinized and maintained in tissue culture. All experiments were Downloaded from Based on an initial analysis of the transcriptomes of unstimulated performed on early passage (2–4) cell cultures. and TNF-a– and IFN-g–stimulated lung fibroblasts, we addressed RNA isolation, Northern, microarray, and quantitative PCR the functional necessity of increased Fas expression in cytokine- analysis induced sensitization of fibroblasts to Fas ligation-induced apo- Total RNA was isolated using TRIzol (Life Technologies, Grand Island, ptosis. In this study, we show that exposure to TNF-a and IFN-g NY). Northern blot analysis was performed as previously described (29). mapk http://www.jimmunol.org/ increases cell surface Fas expression in an NF-kB– and p38 - Prior to quantitative RT-PCR (qPCR) analysis, total RNA was cleaned up dependent fashion. Furthermore, through the use of small in- using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA). Reverse tran- terfering RNA (siRNA)-mediated Fas knockdown and adenovirus- scription was performed on 1 mg total RNA with random hexamers in a 50- ml reaction using TaqMan RT reagents (Applied Biosystems). The single- mediated cell surface Fas expression, we show that among 603 plex qPCR reactions for Fas or GAPDH were performed using the probe/ genes for which expression was altered in response to TNF-a and primer sets Hs00531110_m1 and 4326317E, respectively, with 40 ng IFN-g, increased cell-surface expression of Fas is necessary and cDNA, 250 nM fluorogenic probe, and 900 nM each primer for Fas and sufficient to promote sensitization to Fas-induced apoptosis. Fur- 150 nM each primer for GAPDH. The specificity of PCR was verified by thermore, immunohistochemical staining of lung sections from the lack of signal in the no-template and no-reverse transcriptase controls. The threshold cycle was recorded for each sample. The relative Fas mRNA IPF patients revealed minimal expression of Fas by fibroblasts and expression levels were determined using the comparative threshold cycle by guest on October 1, 2021 myofibroblasts in fibroblastic foci. Collectively, these findings method, as previously described (30). Microarray analysis was conducted emphasize the importance of proinflammatory cytokines in the with 5 mg total RNA, which was labeled and hybridized to Affymetrix initiation of Fas-induced fibroblast apoptosis and suggest that the U133 Plus2.0 arrays (Affymetrix) using standard methods. The complete array data set and experimental details are available from the Gene Ex- underlying mechanism of their action involves raising Fas cell- pression Omnibus repository under accession number GSE26594 (http:// surface expression above a basal threshold. www.ncbi.nlm.nih.gov/geo/). Flow cytometry Materials and Methods Fas cell-surface expression was determined by flow cytometry. Lung fi- Materials broblasts were harvested by trypsinization and stained for 4 h on ice using Recombinant human and mouse TNF-a and IFN-g were purchased from either the APC-conjugated anti-Fas Ab or APC-conjugated nonimmune R&D Systems (Minneapolis, MN). Agonistic anti-human Fas (CH-11) and IgG. After washing, the cells were analyzed on an FACScalibur flow anti-mouse-Fas (Jo-2) Abs were from Upstate Biotechnology (Lake Placid, cytometer (BD Biosciences, San Jose, CA). For total Fas expression (cell NY) and BD Pharmingen (San Diego, CA), respectively. Fas probe and surface and intracellular), the cells were first fixed and permeabilized with primer sets (Hs00531110_m1) for quantitative real-time PCR were from 1.5% (w/v) paraformaldehyde and 0.1% (v/v) saponin, for 30 min, washed, Applied Biosystems (Foster City, CA). Human Genome U133 Plus 2.0 stained, and analyzed as above but with the inclusion of saponin in the microarray chips were purchased from Affymetrix (Foster City, CA). Fas stain and subsequent washes. The data were analyzed with FlowJo (Tree (sc715-R) and IkB-a (sc371R) Western Abs were from Santa Cruz Bio- Star, Ashland, OR) and Cell Quest software (BD Biosciences). technology (Santa Cruz, CA), the actin (mab1501) Western Ab was from Western blotting Millipore (Temecula, CA) and the phospho-specific HSP27 (PA1-005) Ab was from Affinity Bioreagents (Golden, CO). Conjugated Abs for flow Protein expression in cell lysates was determined by Western blot analysis. cytometry (allophycocyanin-labeled Fas Ab [558814] and control Ab Four hundred microliters lysis buffer (50 mM Tris-HCl [pH 7.4] containing mapk [555751]) were from BD Pharmingen. The inhibitors SB 203580 (p38 ) 150 mM NaCl, 1 mM Na2EDTA, 1% [v/v] Nonidet P-40, 0.1% [w/v] and Bay 11-7082 (NF-kB, PD98059 [MEK1] and LY294002 [PI3K]) were SDS, 1% [w/v] sodium deoxycholate, 20 mM b-glycerophosphate, 1 purchased from Calbiochem (San Diego, CA). mM Na3VO4,1mg/ml leupeptin, 2 mg/ml aprotinin, and 1 mM PMSF) were added to each well of a six-well plate. The cells were scraped off the Cell culture plate, and 10 mg protein from the postnuclear lysate was analyzed as Human primary lung fibroblasts from an IPF patient (FS087) and non- previously described (31). disease control subject (N78), the human fetal lung fibroblast cell lung Fas knockdown and adenovirus-mediated expression MRC-5 (American Tissue Culture Collection, Manassas, VA), and mouse primary lung fibroblasts were maintained in modified Eagle’s MEM, Fas knockdown was achieved in MRC5 cells using a pool of four Fas- supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% specific Accell SMARTpool siRNAs (E-003776-00-0010 human Fas, (v/v) heat-inactivated FBS under a 5% CO2 atmosphere as previously NM_152876; Dharmacon). Fas siRNAs or a nontargeting Accell control described (20). Primary cultures of human lung fibroblasts were derived siRNA (D-001910-10-20) were prepared according to the manufacturer’s from nondiseased human lungs obtained from the International Institute for instructions (Dharmacon) and incubated with MRC5 cells in six-well The Journal of Immunology 529 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Genome-wide mRNA expression responses to TNF-a and IFN-g stimulation in three human lung fibroblast cell lines. Two primary (FS087 and N78) and one immortalized (MRC5) cell line were stimulated with TNF-a and IFN-g, and transcriptional responses were measured using Affymetrix Human Genome U133 Plus 2.0 GeneChips. A, Principal components analysis plot representing global gene expression profile of control (red) and cytokine- stimulated (blue) fibroblast cell lines. B, Venn diagram showing commonality of transcripts with 2-fold or greater changes in expression between the stimulated and unstimulated states for the three different fibroblast cell lines. C, Distribution of p values generated from an unpaired t test of stimulated versus control cells. D, Immunological and apoptotic pathways are prevalent in genes differentially expressed with cytokine stimulation among the 10 most widely overrepresented pathways associated with the list of genes differentially expressed (FDR ,0.05) upon cytokine stimulation. The negative log of the p values for the probability of obtaining these genes associated with the given pathways by random chance is given at the top, whereas the bar line in the bottom graph is derived from the ratio of the number of genes on our list associated with a given pathway divided by the total number of genes that make up that pathway. The threshold line corresponds to a p value of 0.05. E, Detailed view of the canonical pathway death receptor signaling containing genes upregulated with cytokine stimulation. Genes colored red/pink indicate significant increases in expression (FDR ,0.05) upon cytokine stimulation. Ex- pression fold increases are indicated. A total of six arrays were analyzed. plates in delivery medium at a final concentration of 1 mM. Additional a further 36 h. The cells were then either stained for cell-surface Fas ex- plates of control cells were grown in parallel. After 36 h, TNF-a (10 ng/ pression or incubated with agonistic anti-Fas Ab (CH-11; 250 ng/ml) for ml) and IFN-g (50 U/ml) were added, and the incubations continued for 4 h and assessed for caspase-8 activation, as described above. 530 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

Enforced cell-surface expression of Fas was accomplished using an ml Eagle’s modified MEM containing 10% (v/v) heat-inactivated FCS were adenovirus expressing human Fas under the control of the chicken actin added, and 24 h later, the cells were placed in medium containing 0.1% (v/v) promoter. The adenoviral construct was created by ligating EcoRI and ClaI- heat-inactivated FCS and incubated for a further 36 h prior to analysis. digested full-length human Fas into EcoRI and ClaI pAd5 CMV GFP vector to create a bistronic construct in which Fas and GFP were separated by an Immunohistochemistry internal ribosome entry site. Next, the majority of the CMV enhancer was excised by digestion with NdeI and ClaI, and NdeI/ClaI-digested chicken Lung tissues were fixed with 10% (v/v) formalin and embedded in paraffin. b-actin promoter was ligated in place of the CMV promoter to yield the Each block was sequentially cut into 4-mm-thick serial sections on final construct in which Fas-GFP is transcribed under control of the Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA) and prepared for chicken b-actin promoter. The fidelity of the construct was confirmed by: immunohistochemical staining as previously described (13). Sections were 1) diagnostic restriction enzyme digestion; 2) nucleotide sequencing; and stained with: 1) anti-Fas (Santa Cruz Biotechnology; Ab SC 715) used at 3) expression of Fas and GPF in HEK293 cells as determined by Western a 1:100 dilution; or 2) anti-human a-smooth muscle actin Ab (Sigma- blotting and microscopy to visualize GFP-labeled cells. Adenoviruses Aldrich, St. Louis, MO; A2547) used at a 1:200 dilution. Negative con- expressing the Fas-GFP bistronic sequence were prepared by the Gene trols consisted of nonimmune mouse or rabbit IgG and were used at Transfer Vector Core at the University of Iowa (http://www.uiowa.edu/ the same concentration as the primary Abs. Immunohistochemistry was ∼gene/). MRC cells plated in six-well plates were transduced with Fas and performed either manually or using an automated immunohistochemical empty vector adenoviruses suspended in 1 ml Optimem (Invitrogen) for 1 h stainer (DakoCytomation, Carpinteria, CA) in which slides were incubated at 37˚C. Preliminary experiments were conducted to determine the level sequentially with each primary or control Ab (30 min, 25˚C) followed by of cell surface Fas expression achieved by transduction with the Fas- secondary Ab conjugated to polymerized HRP enzymatic domains (DAKO adenovirus at multiplicities of infection (MOI) of 10, 30, 100, and 300. Envision FLEX system; DakoCytomation), and chromogenic enzyme sub- Fas expression was found to be slightly higher than the level seen fol- strate (4 min, 25˚C) according to the manufacturer’s protocol. The slides lowing coincubation with TNF-a (10 ng/ml) and IFN-g (50 U/ml) fol- were counterstained with hematoxylin (4 min, 25˚C), rinsed in water, lowing transduction with the Fas adenovirus at an MOI of 300, and so this dehydrated with graded alcohols/xylene, and coverslipped with permanent MOI was used in all experiments. Following transduction, an additional 2 mounting solution. Downloaded from http://www.jimmunol.org/

FIGURE 2. Stimulation with TNF-a and IFN-g increases Fas mRNA, protein, and cell-surface expression in human ﬁbroblasts. A, Exposure of MRC-5 cells to TNF-a 10 ng/ml) plus IFN-g (50 U/ml) increases Fas mRNA abundance as determined by qPCR (left panel) and Northern analysis (center panel). The right panel shows quantiﬁcation of Fas mRNA levels as determined by densitometry of

Northern blots. B, Increased expression of total Fas by guest on October 1, 2021 protein by MRC-5 cells exposed to TNF-a 10 ng/ml) plus IFN-g (50 U/ml). Upper panel shows Western blots probed for Fas and b-actin; lower panels show the fold increase in total Fas protein compared with unstimulated cells. C, TNF-a 10 ng/ml) plus IFN-g (50 U/ml) induced increased to cell-surface Fas in MRC-5 cells. Upper panel shows a representative flow cytometry histogram illustrating the patterns of Fas staining. Lower panels show the increase (fold over unstimulated) in cell-surface Fas expression. D, Fas is not stored in an intracellular pool prior to translocation to the cell surface. Left panel, Repre- sentative flow cytometry histograms of MRC5 cells cultured in medium alone and then stained with an allophycocyanin-labeled Fas-specific Ab under conditions that maintain plasma membrane integrity (surface) or after permeabilization and fixation (total). Center panel, Representative flow cytometry histograms of unstimulated and TNF-a (10 ng/ml) plus IFN-g (50 U/ml)-stimulated MRC-5 cells stained with APC-labeled anti-Fas Ab under conditions that maintain cell integrity (surface) or after permeabilization and fixation (total). Right panel, Quantification of surface and total Fas staining in unstimulated and TNF-a plus IFN-g–stimulated MRC-5 cells. Data represent the mean 6 SEM of three independent experiments. The Journal of Immunology 531

Statistical analysis (10 ng/ml) and IFN-g (50 U/ml) for up to 48 h and measured All experiments were conducted a minimum of three times. Statistical Fas mRNA levels by qPCR and Northern analysis. qPCR analysis analyses using GraphPad InStat version 3.0b for Macintosh (GraphPad, San showed that Fas mRNA expression peaked at ∼6-fold above basal Diego CA) were performed by one-way ANOVA, and comparisons among levels by 24 h and declined to ∼4-fold above basal levels at 48 h groups were performed with a Newman-Keul multiple comparison test. A p (Fig. 2A). Similarly, Northern analysis indicated that Fas mRNA , value 0.05 was considered to be significant. All microarray data met the was increased ∼3.5 fold following 36 h of stimulation with TNF-a quality control criteria established by the Tumor Analysis Best Practices Working Group (32). Microarray data were analyzed in Partek Genomics and IFN-g (Fig. 2A). Suite (Partek, St. Louis, MO), using GC robust multiarray average probe set summarization. Transcripts that were differentially expressed between cytokine-stimulated and control fibroblasts were identified using a Student t test, with a significance threshold defined by the 5% false discovery rate (FDR). Biological themes differentially affected by cytokine stimulation were identified among the 603 differentially expressed transcripts using a modified Fischer’s exact test as implemented in Ingenuity Pathway Analysis (http://www.ingenuity.com).

Results Changes in normal and IPF lung fibroblast transcriptomes induced by TNF-a and IFN-g To investigate the mechanism by which TNF-a and IFN-g re- Downloaded from program fibroblasts from resistance to sensitivity to Fas ligation- induced apoptosis, we exposed representative normal and fibrotic human lung fibroblasts and the human lung fibroblast cell line MRC5 to the combination of TNF-a (10 ng/ml) and IFN-g (50 U/ ml) for 36 h and analyzed changes in their transcriptomes using Affymetrix microarrays. We observed that the transcriptional responses to cytokine stimulation were large and consistent within http://www.jimmunol.org/ the set of fibroblastic cells examined. Approximately 50% of the transcripts with 2-fold or greater expression change upon stimulation within a single fibroblast line were likewise changed in all three fibroblast groups (Fig. 1B). This consistent pattern of gene expression change is visible in an unsupervised principal components analysis (Fig. 1A). In this plot, each experimental sample is represented as a solid shape for which the position in space

is determined by genome-wide transcript levels. The consistent by guest on October 1, 2021 pattern of expression changes upon stimulation appears as a vector of similar orientation and length connecting the control and stimulated samples from each line. The most robustly induced family of transcripts was chemokine receptor ligands (e.g., CCL20, CCL5, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL3, and CXCL9), which exhibited nominal increases from 18.2- to 5930- fold. A statistically defined set of 603 differentially expressed transcripts was identified using a Student t test, with a significance threshold determined by the 5% FDR (Supplemental Table I). Biological themes underlying this expression pattern were identified based on the overrepresentation of predefined groups of transcripts within the statistically defined set. As expected, the most overrepresented canonical pathways (Fig. 1D) include pathways associated with IFN signaling, the inflammatory re- FIGURE 3. Increased Fas expression and sensitization to Fas ligation- sponse, and death receptor signaling (Fig. 1E). Among the latter, induced apoptosis by TNF-a and IFN-g is dependent on signaling by TNF- Trail (APO2L) mRNA expression was increased 1051-fold, R1, p38mapk, and NF-kB. A, TNF-a and IFN-g induce Fas expression whereas expression of the antiapoptotic genes cIAP and c-FLIP by primary mouse lung fibroblasts from wild-type mice and TNF-R22/2 2 2 was increased 132-fold and 9.1-fold, respectively. Notably, within mice, but not TNF-R1 / mice. B, TNF-a and IFN-g sensitize primary 2 2 the Fas signaling pathway, Fas expression was increased 6.1-fold, mouse lung fibroblasts from wild-type and TNF-R2 / mice, but not TNF- 2/2 mapk whereas transcripts encoding other members of the Fas-signaling R1 mice, to Fas ligation-induced apoptosis. C, The p38 inhibitor cascade were not affected by cytokine stimulation. Together, these SB203580 (20 mM) blocks TNF-a and IFN-g induction of Fas expression in human MRC-5 lung fibroblastic cells. D, Inhibition of TNF-a and IFN-g findings suggest that the level of Fas expression might play induce Fas expression by MRC fibroblastic cells in the presence of the NF- a dominant role in the sensitization to Fas ligation-induced apo- kB inhibitor Bay 11-7082 (3 mM). E, The p38mapk inhibitor SB203580 (20 ptosis. mM) inhibits TNF-a and IFN-g sensitization of MRC-5 lung fibroblastic Fas expression is increased in response to TNF-a and IFN-g cells to Fas ligation-induced apoptosis. F, TNF-a and IFN-g sensitization to Fas-induced apoptosis is inhibited by the NF-kB inhibitor Bay 11-7082 Based on the findings from the microarray experiments, we con- (3 mM). The data shown in the upper panels of C and D are representative firmed the effect of TNF-a and IFN-g on Fas mRNA levels by data from a single experiment. The lower panels show the mean 6 SEM stimulating human lung fibroblastic MRC5 cells with TNF-a from three independent experiments. 532 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

We next measured the effect of TNF-a and IFN-g on expression cellular Fas staining was the same as cell-surface staining in of Fas protein. MRC5 cells were incubated in either medium alone unstimulated cells. In addition, the TNF-a– and IFN-g–induced or with TNF-a (10 ng/ml) and IFN-g (50 U/ml) for up to 36 h. The increase in total Fas and cell-surface Fas staining was identical cells were then lysed and evaluated for Fas expression by Western (Fig. 2D, center and right panels), suggesting that Fas is expressed blotting (Fig. 2B) or harvested and subjected to ﬂow cytometry at the cell surface and not associated with an intracellular pool. analysis following staining with allophycocyanin-conjugated anti- Together, these ﬁndings indicate that TNF-a and IFN-g increase Fas Ab (Fig. 2C). Both approaches indicated that stimulation with Fas transcription and translation and lead to increased cell-surface TNF-a and IFN-g for 36 h resulted in an ∼6-fold increase in total expression of Fas.

Fas protein levels and cell-surface Fas expression. In addition, Fig. mapk 2C (upper panel) shows that the increase in Fas expression in Signaling via TNF-R1, p38 and NF-kB are required for response to TNF-a and IFN-g was seen in a single population of increased Fas expression and sensitization to Fas cells. ligation-induced apoptosis Previous studies have suggested that Fas is localized both at the TNF-a–initiated signaling involves two receptors (TNF-R1, TNF- cell surface and within intracellular organelles, including the Golgi R2) and multiple signaling pathways (35). To investigate the role complex (33, 34). Although the data shown in Fig. 2A–C suggest of TNF-R1 and TNF-R2 in TNF-a– and IFN-g–induced fibroblast that the increase in cell-surface Fas expression is most likely due sensitization to Fas-induced apoptosis, primary cultures of lung 2 2 2 2 to a coordinated increase in Fas transcription, translation, and fibroblasts from TNF-R1 / , TNF-R2 / , and C57BL/6 mice translocation to the cell surface, they do not completely rule out were stimulated with TNF-a (10 ng/ml), IFN-g (50 U/ml), or both the possibility that translocation of a pre-existing intracellular cytokines for 48 h and analyzed for cell-surface Fas expression by pool contributes to the TNF-a– and IFN-g–dependent increase in flow cytometry. Fig. 3A shows that stimulation with TNF-a alone Downloaded from cell-surface Fas. To address this possibility, we compared total and TNF-a plus IFN-g significantly increased Fas expression on 2 2 cellular Fas levels (intracellular and cell-surface) and cell-surface fibroblasts from TNF-R2 / mice (p , 0.01) and C57BL/6 mice Fas levels by flow cytometry in detergent-permeabilized and non- (p , 0.01), but failed to increase in Fas expression on lung 2 2 permeabilized cells. Fig. 2D (left and right panels) shows that total fibroblasts from TNF-R1 / mice (p . 0.05). Similarly, Fig. 3B http://www.jimmunol.org/

FIGURE 4. The TNF-a– and IFN-g–induced in- by guest on October 1, 2021 crease in Fas expression is necessary to sensitize lung fibroblastic cells to Fas ligation-induced apoptosis. A, Fas siRNA inhibits the TNF-a plus IFN-g–induced increase in Fas expression in human MRC-5 fibroblastic cells without affecting basal Fas expression. The cells were incubated with Fas-specific or control siRNAs (1 mM) for 36 h prior to stimulation with TNF-a (10 ng/ml) plus IFN-g (50 U/ml) for 36 h. The left panel shows a representative flow cytometry histogram. The right panel shows the fold changes in Fas expression. B, Prevention of the TNF-a plus IFN-g–induced increase in cell-surface Fas expression using a Fas-specific siRNA inhibits sensitization to Fas ligation-dependent apoptosis. Upper panels show representative flow cytometry histograms for active caspase-8 in cells treated with cytokines and Fas ligation in the absence of siRNAs (left panels) or in the presence of a control siRNA (right panels). The lower left panel shows representative flow histograms for cells treated with Fas siRNA prior to and during exposure to cytokines 6 Fas ligation. The lower right panel shows the quantification of apoptosis in cells treated with no siRNA, control siRNA, and Fas siRNA. The bars represent the means 6 SEM of three experiments. The Journal of Immunology 533 shows that lung fibroblasts from TNF-R12/2 mice were not sen- though incompletely, inhibited cytokine-induced sensitization to sitized to Fas-induced apoptosis (p . 0.05). Interestingly, com- Fas-induced apoptosis (p , 0.01 and p , 0.01, respectively; Fig. pared with fibroblasts from wild-type C57BL/6 mice, lung fi- 3E,3F). In control experiments, the concentrations of SB203580 broblasts from TNF-R22/2 mice exhibited a significant increase in and Bay11-7082 were found to completely block p38mapk and caspase-8 activation following stimulation with TNF-a alone (p , NF-kB activation as reflected by inhibition of the phosphorylation 0.05) and trended toward a significant increase in response to of IkBa and Hsp-27, respectively (Supplemental Fig. 1). Collec- TNF-a plus IFN-g (p = 0.08). Together, these data indicate that tively, these findings suggest that the TNF-a– and IFN-g–induced signaling via TNF-R1 is required for cytokine-induced upregula- increases in Fas expression and sensitization to Fas ligation- tion of Fas and sensitization to Fas-induced apoptosis. In addition, induced apoptosis require occupation of TNF-R1 and signaling signaling via TNF-R2, although not being required for Fas ex- via p38mapk and NF-kB. pression, delivers an antiapoptotic signal that reduces the level of apoptosis induced by ligation of TNF-R1. Increased Fas expression is necessary and sufficient to sensitize Next, we investigated the role of NF-kB, Akt, ERK, and p38mapk fibroblasts to Fas-induced apoptosis signaling in the TNF-a– and IFN-g–induced increase in Fas ex- To determine if the increase in Fas expression was necessary for pression and Fas-induced apoptosis using pharmacologic inhib- cytokine-induced sensitization to Fas-induced apoptosis, we used itors. Incubation of MRC5 cells with TNF-a and IFN-g in the siRNA knockdown to block the TNF-a and IFN-g increase in presence of the PI3K and MEK1 inhibitors LY294002 (20 mM) cell-surface Fas expression. We then determined the effect of and PD98059 (20 mM) failed to block the cytokine-induced in- Fas knockdown on cell-surface Fas expression and Fas ligation- crease Fas expression (data not shown). In contrast, pharmaco- induced caspase-8 activation. MRC5 cells were incubated with logic inhibition of p38mapk and NF-kB with SB203580 (20 mM, Fas or control siRNAs (1 mM) or medium alone for 36 h. Next, Downloaded from Fig. 3C) and Bay 11-7082 (3 mM, Fig. 3D), respectively, signifi- TNF-a (10 ng/ml) and IFN-g (50 U/ml), or medium alone, were cantly reduced the TNF-a– and IFN-g–induced increase in Fas added to the cells and the incubations continued for an additional expression (SB203580, p , 0.001; Bay11-7082, p , 0.001), 36 h. The cells were then evaluated for cell-surface Fas expres- suggesting that both p38mapk and NF-kB are required for the sion. Fig. 4A shows that neither Fas siRNA nor control siRNA TNF-a– and IFN-g–induced increase in Fas expression. Similarly, significantly affected basal cell-surface Fas expression (p . 0.05 pharmacologic inhibition of p38mapk and NF-kB significantly, and p . 0.05, respectively). However, whereas the control siRNA http://www.jimmunol.org/

FIGURE 5. Increased cell-surface Fas expression

is necessary to sensitize human lung MRC-5 fibro- by guest on October 1, 2021 blastic cells to Fas ligation-induced apoptosis. A, Representative flow histogram illustrating increased cell-surface Fas expression following transduction of human MRC-5 fibroblastic cells with an adenovirus encoding human Fas. The induction of Fas by TNF-a and IFN-g is also shown as a control. B, Represen- tative flow cytometry histogram showing that ligation of adenovirus Fas-transduced MRC-5 cells was sufficient to permit Fas ligation-induced apoptosis, though less efficiently than TNF-a plus IFN-g. Quantification of Fas ligation-induced apoptosis (measured by Annexin V staining) in adenovirus Fas and empty adenovirus-transduced MRC-5 cells in the absence of TNF-a plus IFN-g. Sensitization to Fas-induced apoptosis by TNF-a plus IFN-g is shown as a comparison. C, A graph illustrating the relationship between increasing quartiles of adenovirus-mediated Fas expression (0–50 [1st quartile], 50–100 [2nd quartile], 100–150 [3rd quartile], and 150–200 [4th quartile]) and Fas ligation-induced apoptosis suggests that the ability to undergo Fas- induced apoptosis is directly correlated with the level of cell-surface Fas expression. The data represent the mean 6 SEM of three independent experiments. *p . 0.05 between the 1st and 2nd quartiles, **p , 0.05 between the 1st and 3rd quartiles, ***p , 0.01 between the 1st and 4th quartiles. 534 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS had no effect on cytokine-induced Fas expression (p . 0.05), overcomes the basal resistance of lung fibroblasts to Fas ligation- incubation with Fas siRNA almost completely blocked the in- induced apoptosis, we next investigated Fas expression in the fi- crease seen in response to TNF-a and IFN-g (p , 0.01) (Fig. 4A). broblastic foci of lung tissues from IPF patients by immunostaining Next, we investigated the effect of Fas knockdown on Fas ligation- paraffin sections of lung tissues from IPF patients with an anti-Fas induced caspase-8 activation. Fig. 4B shows that compared with Ab. As shown in Fig. 6A,6C, and 6D, we observed low to minimal cells incubated in the absence of siRNAs or incubated with control Fas staining by fibroblasts and myofibroblasts in the fibroblastic siRNA, specific Fas knockdown resulted in a complete inhibi- foci of IPF patients, with only occasional positive Fas staining by tion of Fas-induced caspase-8 activation in TNF-a– and IFN-g– interstitial macrophages. In contrast, Fas was abundantly detected exposed cells. These data indicate that the cytokine-induced in- in type I and type II cells overlying the fibroblastic foci, as well as crease in cell-surface Fas expression is required for Fas ligation- in alveolar macrophages (Fig. 6A,6C) and lymphocytes clustered induced caspase-8 activation and apoptosis in fibroblasts. in lymphoid aggregates (Fig. 6A). Fas was detected primarily in To determine if increased Fas expression was sufficient to alveolar macrophages in the lung parenchyma of nondiseased sensitize fibroblasts to Fas ligation-induced apoptosis, we con- control subjects (Fig. 6E). structed an adenovirus expressing human Fas under the control of the chicken b-actin promoter and transduced MRC5 cells with Discussion Fas-expressing or empty vector adenoviruses at an MOI of 300 Building on earlier work showing that the proinflammatory Th1 for 36 h. Fig. 5A shows that transduction with the Fas adenovirus cytokines TNF-a and IFN-g reprogram lung fibroblasts and myo- led to an ∼8-fold increase in cell-surface Fas expression, where- fibroblasts from resistance to sensitivity to Fas-induced apoptosis, as transduction with the empty vector adenovirus had no effect the goal of the current study was to investigate the mechanisms Downloaded from on Fas expression. We next determined the effect of adenovirus- underlying this phenotypic change. In this study, we show for the mediated increased Fas expression on Fas ligation-induced ap- first time, to our knowledge, that among 603 transcripts for which optosis. In these experiments, apoptosis was quantified by mea- expression was reprogrammed by TNF-a and IFN-g, increased suring exposure of phosphatidylserine using allophycocyanin- conjugated Annexin V because the intrinsic fluorescence of adenovirus-encoded GFP was found to interfere with the caspase- 8 activation assay. Fig. 5B shows that following transduction with http://www.jimmunol.org/ the Fas adenovirus, MRC5 cells exhibited increased Fas ligation- induced Annexin V binding compared with cells transduced with empty vector adenovirus (p , 0.01). Of interest, we noticed that whereas the level of cell-surface Fas expression achieved by transduction with Fas-expressing adenovirus was greater than that induced by TNF-a and IFN-g (Fig. 5A), the level of Fas ligation- induced apoptosis was lower in cells transduced with the Fas adenovirus compared with fibroblasts exposed to TNF-a and IFN- by guest on October 1, 2021 g (Fig. 5B). These findings suggest that although increased cell- surface Fas expression is sufficient to render the cells susceptible to Fas-induced apoptosis, additional mechanisms induced by TNF-a and IFN-g may contribute to the efficiency of the sensitization. To explore this notion further, we investigated the relationship between the level of adenoviral-transduced cell-surface Fas expression and sensitivity to Fas ligation-induced apoptosis by quantifying the sensitivity to Fas ligation-induced apoptosis at incremental quartiles of Fas expression. Fig. 5C shows that the induction of apoptosis following Fas ligation increased linearly with increasing cell-surface Fas expression, and the slopes of the lines obtained in the presence and absence of Fas ligation were significantly different (p , 0.001). However, significant increases in Fas ligation-induced apoptosis were only detected in the third and fourth quartiles of Fas expression (p , 0.05 and p , 0.01, respectively). Taken together, these data suggest that Fas expression must exceed a threshold level before the cells can undergo Fas ligation-induced apoptosis. In addition, because the level of FIGURE 6. Minimal Fas expression by the fibroblastic cells of fibroblast Fas expression seen following transduction with the Fas adeno- foci in IPF. A–D show lung tissues from patients with IPF, whereas E and F virus was higher than that induced by cytokine stimulation, the are from nondiseased human control subjects. A, C, D, and E were data provide further support to the notion that exposure to TNF-a immunohistochemically stained for Fas, whereas B was stained for a- and IFN-g also reduces the threshold for Fas ligation-induced smooth muscle actin to aid in the localization of Fas in the serial section apoptosis. shown in A. F illustrates lung tissue from a human nondisease control subject stained with nonimmune IgG as a negative control. Fas was Fas is minimally expressed by fibroblastic cells in lung tissues detected in alveolar epithelial cells (A, C), alveolar macrophages (A, D), from IPF patients and lymphocytes forming lymphoid aggregates (C). Fas was minimally detected or absent from the fibroblastic cells of fibroblast foci (A, C, and at Our overall rationale for investigating the mechanisms of sensi- higher magnification in D). Fas was only detected in alveolar macrophages tization of fibroblasts to Fas ligation-induced apoptosis was to learn in lung tissues to human nondisease control subjects (E). The images more about how this process is impaired in the fibroblastic foci of shown are representative Fas staining from 20 IPF patients (original IPF patients. Given our data showing that increased Fas expression magnification 3200). The Journal of Immunology 535 cell-surface expression of Fas was necessary and sufficient to other cell types have suggested that activation of NF-kB, c-Jun, overcome the basal resistance of lung fibroblastic cells to Fas and p53 (36–38) contribute to increased Fas expression. In addi- ligation-induced apoptosis. In addition, we show that the tion, p38mapk has been implicated in arachidonic acid-induced Fas apoptosis-resistant phenotype of fibroblastic cells in the fibroblast expression in U937 cells (39). However, although activation of foci of IPF/usual interstitial pneumonia patients is associated with NF-kB and p38mapk are key steps in increased Fas expression and low Fas expression. Together, these findings suggest that thera- sensitization to apoptosis induced by TNF-a and IFN-g, additional peutic interventions aimed at increasing Fas expression by fibrotic signaling mechanisms are likely to be involved because IL-1b and lung fibroblasts may reverse their state of basal resistance and may IL-6 both activate NF-kB and p38mapk yet fail to induce sensiti- potentially provide a new avenue to reduce fibroblast accumula- zation to Fas ligation-dependent apoptosis (S.K. Frankel and tion in the lungs of IPF patients. D.W.H. Riches, unpublished observations). Interestingly, although The dramatic and durable phenotypic changes in fibroblasts our studies with TNFR-deficient lung fibroblasts revealed that li- exposed to TNF-a and IFN-g are reflected in the large number of gation of TNF-R1 was required for the increase in Fas expression transcriptional changes this exposure elicits. These expression and susceptibility to Fas ligation-induced apoptosis, they also changes are similar in direction and magnitude in each of the three suggest a role for TNF-R2 in protecting the cells from apoptosis as cell lines for a majority of transcripts, even though the cell lines previously reported (40). Furthermore, because ligation of TNF- originate from vastly different lungs, suggesting that reprogram- R2 has also been shown to activate NF-kB and p38mapk signaling ming by TNF-a and IFN-g is a fundamental pathway open to lung (41), it is plausible that the incomplete inhibition of caspase-8 fibroblasts in multiple contexts. The biological themes that these activation seen in the presence of Bay11-7082 and SB203580 expression changes represent include those that are likely to be the may be related to loss of antiapoptotic signaling initiated by li- direct result of cytokine stimulation (i.e., the activation of IFN gation of TNF-R2. Collectively, our findings suggest that although Downloaded from signaling and inflammatory responses). In contrast, changes as- p38mapk and NF-kB activation are necessary in TNF-a– and IFN- sociated with genes involved in death receptor signaling appear to g–induced Fas expression and sensitization to apoptosis, their reflect a change in the poise of the cell with respect to signaling activation is unlikely to be sufficient to induce these responses. by these receptors rather than activation of this pathway. In- Thus, additional, and as yet unknown, mechanisms likely con- terestingly, expression of transcripts encoding the antiapoptotic tribute to the control of fibroblast apoptosis. molecules cIAP and c-FLIP was found to increase in response to Based on in vivo studies in mice (42, 43), we used an siRNA http://www.jimmunol.org/ TNF-a and IFN-g and may afford protection against the induction approach to show that increased Fas expression was necessary for of apoptosis by cytokines during the sensitization process. How- TNF-a– and IFN-g–induced sensitization to Fas ligation-induced ever, their expression is clearly not sufficient to protect the cells apoptosis. In addition, we showed that adenovirus-mediated over- from apoptosis following Fas ligation. Taken together, the expression of Fas was sufficient to render fibroblasts sensitive to microarray data suggest that fibroblast programming by TNF-a Fas ligation-induced apoptosis. However, although the level of Fas and IFN-g does not result in autocrine activation of death receptor expression achieved following transduction with the Fas- signaling, but reflects changes that may underlie their increased expressing adenovirus was higher than that induced by TNF-a sensitivity to Fas ligation. and IFN-g, the level of apoptosis detected in cells expressing the by guest on October 1, 2021 Among the genes for which expression was increased by TNF-a Fas adenovirus was lower than that induced by cytokines. These and IFN-g, Fas mRNA and cell-surface protein expression in- findings suggest that although increased Fas expression is neces- creased ∼5- to 6-fold. Both the increase in Fas expression and sary and sufficient to enable fibroblasts to undergo Fas ligation- sensitivity to Fas ligation-induced apoptosis were found to be induced apoptosis, other genes for which expression are differ- dependent on ligation of the TNFR TNF-R1 and activation of entially regulated by TNF-a and IFN-g may also contribute to p38mapk and NF-kB. Although little is known about transcriptional reprogramming fibroblast sensitivity to Fas ligation. In particular, regulation of Fas expression in lung fibroblasts, previous studies in as has been reported for several receptors including T cell and

FIGURE 7. Schematic illustrating the proposed mechanisms of sensitization of lung fibroblasts to apoptosis. A, Proposed mechanism controlling TNF-a– and IFN-g–induced Fas expression by lung fibroblasts. TNF-R1 ligation is proposed to induce sensitization to Fas ligation-induced apoptosis in two steps. Step 1 involves dissociation and/or reduced expression of a Fas-associated inhibitor of DISC assembly. Step 2 involves TNF-R1–induced increased cell- surface Fas expression via p38mapk and NF-kB signaling. Step 2 is bypassed by adenovirus-induced Fas expression, whereas step 1 is not. Step 1 is proposed to control threshold sensitivity to Fas-induced apoptosis. B, Proposed mechanisms underlying the persistence (resistance) and sensitivity of lung fibroblasts to Fas ligation-induced apoptosis. Basal (low) fibroblast Fas expression fails to induce Fas ligation-induced apoptosis (upper left panel), and fibroblastic cells persist in the fibroblastic foci of IPF patients (upper right panel). TNF-a and IFN-g increase Fas expression and enable Fas-induced apoptosis (lower left panel), thereby promoting a reduction in the burden of fibroblastic cells in fibroblastic foci (lower right panel). Left panels, Blue rectangles, Fas; red squares, soluble or cell-surface FasL. 536 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

B cell Ag receptors (44, 45), our findings suggest that until Fas growth, differentiation, and chemokine production (57–62). In expression exceeds a certain threshold, its ability to signal apo- contrast, when Fas is expressed at comparatively high levels (e.g., ptosis remains blocked. As illustrated in Fig. 7A, an intriguing in hepatocytes and TNF-a– and IFN-g–stimulated lung fibro- possibility is that one of these putative additional gene(s) may blasts) (20, 42), its ligation results in DISC assembly and in- contribute to the control of this threshold by preventing death- duction of apoptosis. Thus, as illustrated schematically in Fig. 7B, inducing signaling complex (DISC) assembly at low levels of we speculate that resistance or sensitivity of lung fibroblasts to Fas Fas expression. ligation-induced apoptosis is controlled by the level of cell-surface Because apoptotic fibroblastic cells are rarely seen in the fi- Fas expression, even if FasL is ubiquitously present. It is also broblast foci of IPF patients (12, 13), and lung fibroblasts are possible that expression of FasL on the surface of myofibroblasts largely resistant to Fas-induced apoptosis when isolated and may allow the cells to interact with Fas on adjacent fibroblasts and studied in vitro (14, 20, 46), we investigated the level of Fas ex- other cell types in trans, suggesting that cell–cell contact and the pression in lung tissue from patients with IPF. Consistent with three-dimensional structure of the fibroblastic foci may play an previous reports, Fas was strongly expressed by alveolar epithelial important role in the regulation of Fas signaling and function. cells and alveolar macrophages (47). We also noted abundant Fas Clearly, understanding the events that control fibroblast and myo- expression by cells located in lymphoid aggregates in IPF patients. fibroblast sensitivity to apoptosis should provide insights into how In contrast, Fas was rarely detected in fibroblastic cells located in these cells persist in the lungs of IPF patients and, desirably, how fibroblast foci. Recently, we also showed that unlike the overlying they may be therapeutically eliminated. Approaches aimed at in- alveolar epithelium, fibroblastic cells within fibroblast foci exhibit creasing fibroblast Fas expression might therefore be therapeuti- minimal nuclear translocation of NF-kB (13). Taken together with cally relevant. the present finding that increased Fas expression by fibroblasts is Downloaded from dependent on NF-kB activation, it is possible that the failure of Acknowledgments fibroblastic cells to undergo apoptosis in fibroblast foci is due in We thank Linda Remigio and Jane Parr for outstanding technical assistance. part to reduced NF-kB activation. In turn, reduced NF-kB acti- We also thank Dr. Doug Everett-Curran (Division of Biostatistics and Bio- vation may contribute to the low level of Fas expression, thereby informatics, National Jewish Health) for advice on the statistical analysis of preventing the ability of these cells to undergo apoptosis. Al- the data. though many mechanisms could contribute to reduced NF-kB http://www.jimmunol.org/ activation, the microarray data presented in this study revealed Disclosures similar patterns of cytokine-induced increases in NF-kB–de- The authors have no financial conflicts of interest. pendent gene expression in primary cultures of normal and fibrotic lung fibroblasts. Furthermore, we previously showed that normal References and fibrotic lung fibroblasts respond to TNF-a and IFN-g in vitro 1. Selman, M., T. E. King, and A. Pardo; American Thoracic Society; European with similar increases in Fas expression (20). In addition, we have Respiratory Society; American College of Chest Physicians. 2001. Idiopathic shown that TGF-b has no effect on the TNF-a and IFN-g in- pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134: 136–151. duction of Fas expression (20), suggesting that the TGF-b–rich

2. Katzenstein, A. L., and J. L. Myers. 1998. Idiopathic pulmonary fibrosis: clinical by guest on October 1, 2021 environment of the lung parenchyma in IPF has little or no impact relevance of pathologic classification. Am. J. Respir. Crit. Care Med. 157: 1301– on the ability of fibroblasts to increase Fas expression upon ex- 1315. 3. Myers, J. L., and A. L. Katzenstein. 1988. Epithelial necrosis and alveolar posure to the appropriate stimuli. Thus, it is plausible that the collapse in the pathogenesis of usual interstitial pneumonia. Chest 94: 1309– failure to activate NF-kB and increase Fas expression in fibroblast 1311. 4. Hashimoto, N., H. Jin, T. Liu, S. W. Chensue, and S. H. Phan. 2004. Bone foci is due to the absence or reduced presence of the appropriate marrow-derived progenitor cells in pulmonary fibrosis. J. Clin. Invest. 113: 243– NF-kB–activating or other sensitizing stimuli within the fibro- 252. blastic foci. Consistent with this notion, previous studies have 5. Willis, B. C., J. M. Liebler, K. Luby-Phelps, A. G. Nicholson, E. D. Crandall, R. M. du Bois, and Z. Borok. 2005. Induction of epithelial-mesenchymal tran- suggested relative deficiencies of key sensitizing molecules in- sition in alveolar epithelial cells by transforming growth factor-beta1: potential cluding TNF-a, IFN-g, and PGE2 in lung tissues and bron- role in idiopathic pulmonary fibrosis. Am. J. Pathol. 166: 1321–1332. choalveolar lavage specimens from IPF patients (24, 25, 48, 49). 6. Wu, Z., L. Yang, L. Cai, M. Zhang, X. Cheng, X. Yang, and J. Xu. 2007. De- tection of epithelial to mesenchymal transition in airways of a bleomycin in- Taken together, our findings suggest a previously unrecognized duced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre role for increased cell-surface Fas expression in promoting the transgenic mouse. Respir. Res. 8: 1. 7. Kim, K. K., M. C. Kugler, P. J. Wolters, L. Robillard, M. G. Galvez, sensitization of lung fibroblasts to apoptosis. This finding obviously A. N. Brumwell, D. Sheppard, and H. A. Chapman. 2006. Alveolar epithelial cell raises the question of the relevant source(s) of FasL. Constitutive mesenchymal transition develops in vivo during pulmonary fibrosis and is reg- cell-surface FasL expression has been detected in a variety of ulated by the extracellular matrix. Proc. Natl. Acad. Sci. USA 103: 13180–13185. 8. Tanjore, H., X. C. Xu, V. V. Polosukhin, A. L. Degryse, B. Li, W. Han, airway epithelial cells including Clara cells (50, 51). Soluble FasL T. P. Sherrill, D. Plieth, E. G. Neilson, T. S. Blackwell, and W. E. Lawson. 2009. has also been detected in the bronchoalveolar lavage fluid of Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. patients with a variety of interstitial lung diseases including IPF Am. J. Respir. Crit. Care Med. 180: 657–665. 9. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier, and R. A. Brown. 2002. (52, 53). Previous studies have shown FasL to be expressed Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. by monocytes, macrophages, and NK cells (54, 55), and recent Rev. Mol. Cell Biol. 3: 349–363. 10. Zhang, K., M. D. Rekhter, D. Gordon, and S. H. Phan. 1994. Myofibroblasts and studies have suggested that myofibroblasts themselves are an their role in lung collagen gene expression during pulmonary fibrosis. A com- important source of cell-surface FasL (22, 23). Myofibroblast bined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145: expression of FasL has been implicated in epithelial injury in mice 114–125. 11. Gabbiani, G. 2003. The myofibroblast in wound healing and fibrocontractive following intratracheal instillation with bleomycin (22, 56), and diseases. J. Pathol. 200: 500–503. epithelial cell apoptosis in IPF patients is often seen in areas 12. Lappi-Blanco, E., Y. Soini, and P. Paä¨kko¨. 1999. Apoptotic activity is increased overlying fibroblastic foci (56). Thus, it is conceivable that FasL in the newly formed fibromyxoid connective tissue in bronchiolitis obliterans organizing pneumonia. Lung 177: 367–376. may be constitutively available to engage Fas on fibroblasts and 13. Cha, S. I., S. D. Groshong, S. K. Frankel, B. L. Edelman, G. P. Cosgrove, myofibroblasts. When Fas is expressed at comparatively low J. L. Terry-Powers, L. K. Remigio, D. Curran-Everett, K. K. Brown, C. D. Cool, and D. W. Riches. 2010. Compartmentalized expression of c-FLIP in lung tissues levels, Fas ligation has been shown to stimulate a variety of of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 42: nonapoptotic responses including proliferation, survival, tumor 140–148. The Journal of Immunology 537

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