Down-regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 9, September 2004, pp 2803–2810 DOI 10.1002/art.20453 © 2004, American College of Rheumatology Down-Regulation of FLIP Sensitizes Rheumatoid Synovial Fibroblasts to Fas-Mediated Apoptosis Guillermo Palao, Begoña Santiago, Marı́a Galindo, Mónica Payá, Juan C. Ramirez, and José L. Pablos not detected. TNF␣ induced increases in FLIPL and FLIPS expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Downregulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas-mediated apoptosis. Conclusion. Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Downregulation of FLIP sensitizes RA FLS to Fas-mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia. Objective. Hyperplasia of fibroblast-like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICEinhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor ␣ (TNF␣) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS. Methods. We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti-Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNF␣, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility. Results. FLIPL was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas-induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were Inflammatory cell infiltration and the expansion of an aggressive population of fibroblast-like synoviocytes (FLS) in the synovial membrane are the pathologic hallmarks of rheumatoid arthritis (RA). FLS overgrowth may result from unbalanced proliferation and apoptosis, and both processes have been detected on tissue sections of rheumatoid synovium (1–3). Since proliferation is scarce in RA synovium (4), dysregulation of apoptosis has been proposed to explain the accumulation of FLS (5). In RA synovium, despite the expression of several death receptor ligands (such as tumor necrosis factor ␣ [TNF␣] and Fas ligand [FasL]) by infiltrating cells in close proximity to FLS, the net effect is toward hyperplasia rather than apoptotic elimination of FLS (5–7). Rheumatoid FLS display increased expression of several antiapoptotic proteins, but their specific function in these cells has not been completely elucidated (8–10). Whether cultured RA FLS are different from osteoarthritis (OA) FLS regarding susceptibility to Fasmediated apoptosis remains controversial (11–13). The response of different cell types to ligands of death receptors may vary from cell proliferation or activation to cell death, depending upon the different signaling pathways induced in each particular cell type (14). The first step induced by activation of death receptors is the recruitment of a complex of different proteins termed DISC (death-inducing signaling com- Supported by grants 02/0057 and G03/152 from Fondo de Investigación Sanitaria (Spain). Dr. Palao’s work was supported by Fondo de Investigación Sanitaria. Dr. Santiago’s work was supported by a grant from Fundación Española de Reumatologı́a–Abbott Laboratories. Guillermo Palao, MD, Begoña Santiago, PhD, Marı́a Galindo, MD, Mónica Payá, MSc, Juan C. Ramirez, PhD, José L. Pablos, MD: Hospital 12 de Octubre, Madrid, Spain. Address correspondence and reprint requests to José L. Pablos, MD, Servicio de Reumatologı́a, Hospital 12 de Octubre, Avenida Andalucı́a s.n., 28041 Madrid, Spain. E-mail: jlpablos@h12o. es or email@example.com. Submitted for publication January 26, 2004; accepted in revised form May 5, 2004. 2803 2804 PALAO ET AL plex) to the intracellular death domain of these receptors through the adapter FADD (15). DISC formation results in the homodimerization and proteolytic activation of caspase 8, and this step is counterbalanced by the parallel recruitment of FLICE-inhibitory protein (FLIP), which hetero-oligomerizes with caspase 8 and reduces its proteolytic activation (16,17). Caspase 8 may induce apoptosis in RA FLS through direct activation of terminal caspases or by prior activation of caspase 9 through the mitochondrial apoptotic pathway. Specific inhibition of Fas signaling by inhibitors of caspase 8, caspase 9, or terminal caspases efficiently restrains Fasmediated RA FLS apoptosis (12,18). However, although FLIP has been demonstrated to be a dominant inhibitor of caspase 8 activation and Fas-induced apoptosis in a large variety of human cells (19,20), its differential expression and potential in preventing Fas-mediated apoptosis of rheumatoid FLS are still matters of debate. On the one hand, studies of the expression of FLIP in cultured OA and RA FLS have shown variable expression in different cell lines, but not disease-specific significant differences (10,21). This may relate to FLIP heterogeneity among cells from different synovial regions, as suggested by in situ hybridization studies which have shown preferential expression of FLIP in areas of bone and cartilage invasion (10). On the other hand, enforced FLIP expression has been shown to partially decrease Fas-induced apoptosis in RA FLS (22), but the function of constitutively expressed FLIP as an apoptosis inhibitor has not been evaluated in RA FLS. We have studied the expression of FLIP protein in synovial tissues and in cultured FLS from RA and OA patients, and we have examined its relevance in the susceptibility of these cells to Fas-induced apoptosis. We show that disease-specific differences in the expression of FLIP or in the susceptibility of FLS to Fas-mediated apoptosis do not occur; however, the constitutive expression of FLIP protein by FLS is an important survival factor that decreases the susceptibility of this cell type to Fas-induced apoptosis. PATIENTS AND METHODS Induction of apoptosis in FLS cultures. Synovial FLS were cultured by explant growth from synovial tissue obtained from RA or OA patients undergoing knee replacement surgery. All patients fulfilled the 1987 American College of Rheumatology (formerly, the American Rheumatism Association) revised criteria for the classification of RA (23). Cells were cultured in 10% fetal calf serum–Dulbecco’s modified Eagle’s medium in plastic flasks. FLS from 8 OA and 9 RA patients were used between passages 3 and 6. Fas stimulation was performed by exposing cells to the Fas-activating anti-Fas IgM monoclonal antibody (mAb) CH11 at 1 g/ml for 24 hours (MBL, Nagoya, Japan). This decavalent IgM antibody induces Fas receptor crosslinking similar to that of membrane FasL, in contrast to soluble FasL (24), which in our preliminary studies did not induce FLS apoptosis even in the presence of cycloheximide (CHX). Where indicated, 1.5 g/ml CHX was simultaneously added at the time of anti-Fas treatment. To inhibit caspase activation, cells were preincubated with the cell-permeable inhibitor Z-VAD-FMK at 20 M (Biomol, Plymouth Meeting, PA) 30 minutes before adding the anti-Fas CH11 mAb. Where indicated, cells were pretreated with human TNF␣ at 50 ng/ml (BD PharMingen, San Diego, CA) for 8 hours before adding anti-Fas mAb. Cell death and apoptosis assays. Cell death induced by exposure to anti-Fas mAb for 24 hours was quantified by direct counting of live and dead cells in Neubauer chambers, after staining with 0.2% trypan blue. All dead cells were quantified after recovery of both trypsinized adherent cells and previously detached cells, floating in the media, by centrifugation. To quantify apoptotic cell death, mono- and oligonucleosomal DNA was determined in FLS cytoplasmic extracts by enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). In preliminary experiments, apoptosis was confirmed by TUNEL fluorescent labeling of FLS grown on coverslips as previously reported (19), which showed a good correlation with results of nucleosomal release ELISAs. Western blot analysis. Protein from 106 FLS was extracted in ice-cold lysis buffer (10 mM Tris HCl [pH 8.0], 1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 10 g/ml leupeptin, 10 g/ml aprotinin, 2 g/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride). Protein extracts (30 g) were electrophoresed on 10% polyacrylamide gel and electrophoretically transferred to nitrocellulose filters. After blocking for 2 hours with 5% nonfat dried milk in Tris buffered saline containing 0.05% Tween 20 (TBST), the membranes were incubated overnight at 4°C with 1:500 anti-FLIP (Alexis, Lausen, Switzerland), 1:250 anti-FADD (Transduction Laboratories, Lexington, KY), 1:700 anti–caspase 8 (MBL), or 1:6,000 anti–␤-actin (clone AC-15; Sigma-Aldrich Quı́mica, Madrid, Spain) antibodies in 5% nonfat dried milk–TBST. The filters were washed and incubated for 1 hour with secondary antibodies linked to peroxidase at 1:8,000 dilution. Bands were visualized by an enhanced chemiluminescence system (Pierce, Rockford, IL) and analyzed by densitometry. Immunohistochemistry. Frozen sections from the above-described RA or OA synovial tissue samples were fixed in cold acetone for 10 minutes and, after blocking in 5% serum, were incubated overnight at 4°C with primary antibody. We used two different anti-FLIP antibodies, a polyclonal antibody (BD PharMingen) which preferentially recognized the FLIPS isoform in our Western blot studies, and mAb G-11 (Santa Cruz Biotechnology, Santa Cruz, CA), which preferentially recognized the FLIPL isoform. Negative controls with mouse IgG or nonimmune rabbit serum were included. Immunoperoxidase detection was performed by the ABC method according to the instructions of the manufacturer (Vector, Burlingame, CA). Peroxidase activity was developed EXPRESSION AND ANTIAPOPTOTIC FUNCTION OF FLIP 2805 Figure 1. Expression of FLICE-inhibitory protein (FLIP) by immunohistochemistry in synovial tissue sections from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Frozen sections were immunostained with antibodies preferentially recognizing FLIPL (a and b) or FLIPS (d and e) isoforms. RA (a and d) and OA (b and e) tissue sections are representative of 9 and 8 synovial tissue samples, respectively. Controls were performed by incubating sections with mouse (c) or rabbit (f) IgG instead of anti-FLIP antibodies. Immunoperoxidase-labeled cells appear brown, and sections are counterstained with hematoxylin. (Original magnification ⫻ 400 in a–f; ⫻ 1,000 in insets.) by diaminobenzidine substrate, and slides were counterstained in Gills hematoxylin. Antisense (AS) oligodeoxynucleotide transfection. A phosphorothioate-modified and fluorescein isothiocyanate (FITC)–conjugated single-stranded oligodeoxynucleotide directed against the human FLIP translation initiation codon (FLIP-AS: 5⬘-GACTTCAGCAGACATCCTAC-3⬘) and a similarly modified control nonsense (NS) oligodeoxynucleotide (FLIP-NS: 5⬘-TGGATCC GACATGTCAGA-3⬘) were synthesized as previously described by Perlman et al (20). RA FLS were grown to ⬃50% confluence and incubated with 5 M FITC-conjugated oligodeoxynucleotides premixed with Oligofectamine (Life Technologies, Gaithersburg, MD). Transfection efficiency was analyzed 24 hours later by flow cytometry. After 4 hours of incubation with the transfection mixture, anti-Fas antibody CH11 was added and cells were analyzed for apoptosis at 24 hours. FLIP protein expression in transfected cells was analyzed by Western blot in parallel cultures 24 hours after transfection. RESULTS Expression of FLIP protein in OA and RA FLS. FLIP protein was uniformly detected in the synovial lining and in rare scattered cells of the sublining of RA and OA synovial tissue sections by anti-FLIPL antibody, and differences between groups were not observed (Figures 1a–c). Since previous studies have focused on macrophage staining of FLIP, and this cell type has been shown to mainly express FLIPS (25) (in contrast to fibroblasts, which mainly express FLIPL ), we also immunostained with an anti-FLIP antibody which in our laboratory preferentially recognizes FLIPS by Western blot. Immunostaining with this antibody showed a different pattern, consisting of abundant immunostained cells with large mononuclear, macrophage-like morphology in the sublining of both RA and OA tissues (Figures 1d–f). In RA tissue sections, a higher number of FLIP-positive macrophage-like cells was detected (results not shown). In contrast, lining synoviocytes were not immunostained by this antibody. To further analyze FLIP isoforms expressed by FLS and potential differences between RA and OA FLS, we performed Western blot studies in cultured cells. Cultured FLS from either RA or OA synovium displayed abundant expression of FLIPL isoform in Western blots (Figure 2a), whereas FLIPS was only barely detected (results not shown). Expression of FLIPL was variable in different OA and RA cell lines, but a densitometric analysis corrected by ␤-actin expression did not detect significant differences between OA and RA groups (Figure 2b). Since caspase 8 levels may also modify apoptotic susceptibility in response to Fas activation in other human fibroblasts (19), we also examined caspase 8 protein expression by OA and RA FLS lines. Similar to 2806 PALAO ET AL did not modify caspase 8 or FADD protein expression (Figure 3a). We also analyzed the effect of TNF␣ treatment on FLIP protein expression in RA FLS. Treatment with TNF␣ increased the expression levels of FLIPL and FLIPS isoforms, with a maximal increase at 8 hours (Figure 3b). Fas-mediated apoptosis in OA and RA FLS. Fas triggering by exposure to CH11 mAb for 24 hours induced detectable apoptosis in all RA and OA FLS lines, as evaluated by cytotoxicity or nucleosomal release assays (Figure 4). Treatment with CHX significantly sensitized cultured RA and OA FLS to Fas-induced apoptosis (P ⬍ 0.005). Although Fas- and Fas ⫹ CHX– mediated apoptosis were variable in different FLS lines, statistically significant differences between OA and RA groups were not detected (Figure 4). Pretreatment of FLS with the caspase inhibitor Z-VAD-FMK prevented apoptosis in response to either anti-Fas alone or anti-Fas ⫹ CHX (Figure 4c). Pretreatment of RA FLS with TNF␣ for 8 hours did not induce detectable apoptosis, but it significantly decreased the apoptotic response to anti-Fas alone or to anti-Fas ⫹ CHX (Figure 4d). Apoptosis and down-regulation of FLIP by antisense oligonucleotide. Since CHX significantly increased the susceptibility to apoptosis of cultured FLS and Figure 2. Expression of FLIP and caspase 8 in RA and OA fibroblastlike synoviocytes (FLS). Protein extracts from FLS lines obtained from different patients were analyzed by Western blot. The immunoblot shown is representative of 3 independent experiments including all FLS lines from 9 different RA and 8 different OA donors (a). Results of densitometric analysis are shown (b), including mean and SD levels of FLIP, caspase 8, or FLIP:caspase 8 ratio normalized to ␤-actin protein expression in 8 OA and 9 RA FLS lines. See Figure 1 for other definitions. the findings for FLIP, different cell lines displayed variable levels of caspase 8 expression, but significant differences between RA and OA FLS lines were not detected (Figure 2). The FLIP:caspase 8 ratio was also similar in both groups. To analyze whether the main intracellular proteins involved in Fas signaling were specifically modulated by low concentrations of CHX in RA FLS, we treated RA FLS with CHX at a 10-fold lower concentration than that required to inhibit protein synthesis nonspecifically. Treatment of RA FLS with CHX abrogated FLIP protein expression, whereas this treatment Figure 3. Effect of cycloheximide (CHX) and tumor necrosis factor ␣ (TNF␣) on the expression of Fas pathway proteins by RA fibroblastlike synoviocytes (FLS). RA FLS were incubated for 24 hours with either 1.5 g/ml CHX or medium alone, and FLIP, FADD, caspase 8, and ␤-actin protein expression levels were analyzed by Western blot (a). RA FLS were incubated with TNF␣ for different time periods, and FLIP and ␤-actin protein expression levels were analyzed by Western blot (b). Results are representative of 3 independent experiments using different RA FLS lines. Unst. ⫽ unstimulated cells (see Figure 1 for other definitions). EXPRESSION AND ANTIAPOPTOTIC FUNCTION OF FLIP 2807 Figure 4. Apoptosis of FLS in response to Fas activation. Cultured FLS were activated by anti-Fas CH11 monoclonal antibody for 24 hours in the presence or absence of CHX as indicated. Apoptosis was quantified by cytotoxicity (a) or by nucleosomal release (b) assays in 8 OA and 9 RA FLS lines. The effect of pretreatment with the caspase inhibitor Z-VAD-FMK (zVAD) on Fas-mediated apoptosis of RA FLS is also shown (c). Data are representative of 3 independent experiments including 5 different RA FLS lines. Also shown is the effect of TNF␣ pretreatment of FLS on their susceptibility to Fas-mediated apoptosis in the presence or absence of CHX (d). Data are representative of 3 independent experiments performed in 5 different RA FLS lines. Values are the mean and SD. Abs ⫽ absorbance (see Figures 1 and 3 for other definitions). down-regulated FLIP expression, whereas the opposite effects were observed by treatment with TNF␣, we evaluated whether specific down-regulation of FLIP protein by FLIP-AS oligonucleotide modulates FLS susceptibility to Fas. Transfection of RA FLS with FLIP-AS or NS oligonucleotide was monitored by flow cytometric studies of transfected cultures performed in parallel to those used for analysis of FLIP protein expression by Western blot and for Fas-induced apoptosis. This analysis demonstrated similar rates of FITCconjugated FLIP-AS or NS oligonucleotide transfection (Figure 5a). By densitometric analysis of Western blots, FLIP protein expression was down-regulated by 60% in RA FLS transfected with FLIP-AS oligonucleotide compared with those transfected with NS oligonucleotide (Figure 5b). Fas-induced apoptosis was increased 3-fold in FLIP-AS–transfected RA FLS compared with NStransfected RA FLS (P ⬍ 0.001) (Figure 5c). DISCUSSION Our results confirm that FLIP protein is constitutively and similarly expressed by OA and RA FLS, in which it operates as an inhibitor of Fas-mediated apoptosis, which explains the relative resistance of this cell type to Fas activation. The kinetics of Fas-mediated cell death in human fibroblasts are slow and inefficient compared with those of other Fas-expressing cells, such as lymphoid cells (19,26). This resistance to death receptor–mediated apoptosis has also been observed in other normal and tumor cells, and in many cases it is abrogated by down-regulating FLIP protein, which is very sensitive to low concentrations of CHX (27,28). Fibroblasts from FLIP-knockout mice are more sensitive to Fas-mediated apoptosis (29), and human dermal fibroblasts are also sensitized to Fas activation by CHX or FLIP antisense oligonucleotide transfection (19), 2808 Figure 5. Effect of FLIP antisense (AS-FLIP) oligonucleotide transfection on FLIP expression and Fas-induced apoptosis. RA FLS were transfected with fluorescein isothiocyanate (FITC)–conjugated ASFLIP or nonsense (NS) oligonucleotide, and uptake was analyzed by flow cytometry (a). The open histogram depicts the fluorescence of untransfected cells, the shaded histogram represents AS-FLIP– transfected cells, and the superimposed thick-line histogram represents the fluorescence of NS-transfected cells. FLIP protein expression was analyzed by Western blot after 24 hours of transfection (b). After transfection, RA FLS were treated with anti-Fas CH11 monoclonal antibody for 24 hours, and apoptosis was quantified by nucleosomal release assay (c). Data are representative of 3 independent experiments using 3 different RA FLS lines. Values are the mean and SD. Abs ⫽ absorbance (see Figures 1 and 3 for other definitions). which points to constitutive FLIP as a relevant antiapoptotic factor in this cell type. In TNF␣-treated RA FLS, adenoviral transfer of FLIP gene decreases their susceptibility to Fas-mediated apoptosis (22). However, the effect of enforced FLIP expression does not reflect a physiologic situation, and such expression may either protect against or induce apoptosis depending upon the level of expression achieved (30). Our data demonstrate that most RA FLS survive after 24 hours of Fas challenge, reflecting a relative resistance compared with other Fas receptor–expressing cell types. Down-regulation of constitutively expressed FLIP by CHX or specific FLIP antisense oligonucleotide strongly sensitizes these cells to Fas-mediated apoptosis. This suggests that constitutively expressed FLIP oper- PALAO ET AL ates as a dominant inhibitor of Fas-mediated apoptosis in rheumatoid FLS. The pattern of expression of FLIP proteins in synovial tissues reveals differential expression of FLIPL and FLIPS isoforms by synoviocytes and sublining macrophages. Whereas FLIPL is the main isoform observed in cultured FLS and lining synoviocytes, we found FLIPS in sublining large mononuclear cells, consistent with previous observations that identified mainly the FLIPS isoform in rheumatoid macrophages (25). We did not find differences in the levels of expression of FLIPL between cultured OA and RA FLS or in lining synoviocytes in tissue sections. A previous study of synovial tissue sections by Catrina et al identified higher levels of FLIP in early RA than in late RA; however, these observations seemed to apply to sublining macrophages, in which these investigators preferentially detected FLIP protein expression (31). Investigators in another study failed to detect FLIP protein in protein extracts from either OA or RA FLS, in contrast to findings in FLS obtained from joint fractures; in that study, however, the isoform detected was not specified (21). Schedel et al detected variable FLIP expression in cultured OA and RA FLS lines obtained at synovectomy or arthroplastic surgery, without significant differences between groups (10). Interestingly, using in situ hybridization, these investigators identified higher FLIP messenger RNA expression in fibroblasts located in areas of bone and cartilage erosion. Collectively, these data suggest that FLIP expression is similar in RA FLS and OA FLS, although different FLS lines or fibroblasts located in different areas can be heterogeneous. Previous studies in other cell types have shown that FLIP behaves as an NF-B–inducible factor (32,33). Therefore, activation of NF-B by local exposure to TNF␣ may increase FLIP expression and consequently protect RA synoviocytes from Fas-mediated apoptosis. Previous studies in cultured RA FLS have shown either down- or up-regulation of FLIP after variable periods of exposure to TNF␣ (10,22). Our data show that, consistent with the antiapoptotic role of FLIP, exposure to TNF␣ increases FLIP expression and decreases Fasmediated apoptosis, suggesting another pathogenetic mechanism for this pleiotropic cytokine in RA. TNF␣ and Fas share several intracellular signaling pathways (14,15). On the one hand, both can induce the assembling of the intracellular DISC, leading to caspase 8 activation and apoptosis. FLIP protein can also protect cells from TNF␣-mediated apoptosis (33), and, consistently, RA FLS are completely resistant to EXPRESSION AND ANTIAPOPTOTIC FUNCTION OF FLIP TNF␣-mediated apoptosis unless NF-B activation is blocked (34). On the other hand, TNF␣ and Fas receptor activation induces NF-B translocation, which leads to increased FLIP expression (35,36). This NF-B loop of Fas signaling may protect cells from Fas-mediated cell death, resulting in proinflammatory and survival, rather than cytotoxic, effects of death receptor stimulation (36–38). Permissivity to death receptor–induced apoptosis in other cell types has been suggested to depend upon low NF-B and FLIP induction by death ligand activation (39). 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