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TNF-α confers resistance to Fas-mediated apoptosis in rheumatoid arthritis through the induction of soluble Fas
Life Sciences 122 (2015) 37–41
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TNF-α confers resistance to Fas-mediated apoptosis in rheumatoid arthritis through the induction of soluble Fas Seokchan Hong, Eun-Jin Kim, Eun-Ju Lee, Bon San Koo 1, Soo Min Ahn, Seung-Hyeon Bae, Doo-Ho Lim, Yong-Gil Kim, Bin Yoo, Chang-Keun Lee ⁎ Division of Rheumatology, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 August 2014 Accepted 6 December 2014 Available online 19 December 2014 Keywords: Rheumatoid arthritis Apoptosis Fas Fas ligand
a b s t r a c t Aims: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that is characterized by hyperplastic synovial tissue containing activated synovial fibroblasts. Contradictory findings in the apoptosis of fibroblast-like synoviocytes (FLS) have been described elsewhere, showing that RA FLS have an enhanced susceptibility to Fas (also known as CD95)-mediated apoptosis in vitro in contrast to the observed lack of apoptosis in the RA synovium in vivo. However, the potential mechanisms responsible for this discrepancy remain under investigation. The soluble form of Fas (sFas) was found to inhibit Fas-induced apoptosis by binding to Fas ligand (FasL), thereby preventing the interaction between FasL and membrane-bound Fas. Main methods: We determined the levels of soluble FasL (sFasL) and sFas in patients with RA and the effects of proinflammatory mediators, including TNF-α, on the induction of apoptotic mediators in RA FLS. Key findings: The levels of sFasL and sFas were significantly elevated in the synovial fluids of RA patients compared with control subjects. In addition, we found that the sFas is substantially induced in RA FLS by TNF-α, which were abundantly present in the synovial fluid of RA. Significance: These findings suggest that TNF-α confers resistance to Fas-mediated apoptosis through sFas induction, which could explain the apparent resistance of RA synovial cells to apoptosis in vivo. © 2014 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that is characterized by inflammatory cell infiltration within the synovium [16]. While the exact mechanism of RA pathogenesis remains under investigation, synovial hyperplasia has been considered the major pathogenic finding in RA and could be associated with the persistent inflammation and joint destruction in RA [16,29]. Numerous studies have suggested the pathogenic mechanism underlying the hyperplasia of the fibroblast-like synoviocytes (FLS), which populate the lining layers of the synovium and are key to the induction and perpetuation of inflammation in RA [4]. Several lines of evidence show that FLS exhibit sensitivity to Fas (CD95)-mediated apoptotic signaling in vitro, in contrast to the observed lack of apoptotic cells in the RA synovium in vivo [14,23,26]. There have been substantial studies directed at this conflicting finding in order to address the underlying mechanism responsible for the resistance to apoptosis in RA synovial cells [10,15,17,22]. The cell surface Fas plays a pivotal role in the apoptosis of inflammatory
⁎ Corresponding author at: 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea. Tel.: +82 2 3010 3284; fax: +82 2 3010 6969. E-mail address: [email protected] (C.-K. Lee). 1 Current address: Division of Rheumatology, Department of Internal Medicine, Konkuk University College of Medicine, Chungju, Republic of Korea.
http://dx.doi.org/10.1016/j.lfs.2014.12.008 0024-3205/© 2014 Elsevier Inc. All rights reserved.
cells, including FLS, and there are intricate mechanisms regulating the Fas-mediated apoptotic pathway to maintain cellular homeostasis [13, 23,26]. The soluble form of Fas (sFas) inhibits Fas-induced apoptosis by binding to Fas ligand (FasL), thereby blocking interaction with the membrane Fas (mFas) receptor on the cell surface [6,7]. However, conflicting data have been reported regarding the levels of sFas and its relation with disease activity in RA [2,9,27]. In our present study, we determined the levels of soluble FasL (sFasL) and sFas in patients with RA and the effects of proinflammatory cytokines, including TNF-α, on the induction of apoptosis mediators in RA FLS. Materials and methods Patients and collection of plasma and synovial fluid The study included patients diagnosed with RA who were referred to a tertiary hospital in South Korea between January 2010 and December 2011. All patients fulfilled the 1987 revised American College of Rheumatology classification criteria for RA [1]. Plasma samples were obtained from 26 RA patients, and control samples were collected from 20 healthy volunteers and 20 patients with osteoarthritis (OA). Synovial fluid samples were collected from 15 patients with RA. Synovial fluids from 15 OA patients and 15 subjects suffering from sports injuries
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were included in the analysis. The samples were stored at −20 °C until use. All patients provided written informed consent, and the study protocol was approved by our institution's ethics committee.
total apoptotic cells (Annexin V+) was significantly increased by Fas stimulation in FLS from RA patients (Fig. 1B). Thus, we observed that RA synovial cells were sensitive to Fas-mediated apoptotic signaling, consistent with previous studies [8,18,21].
Measurement of sFasL, sFas and TNF-α by ELISA The levels of sFasL and sFas are elevated in patients with RA The levels of sFasL, sFas and TNF-α were measured by commercially available ELISA kits (R&D Systems, Minneapolis, MN).
FLS were isolated from the synovial tissues obtained from patients affected by RA or OA who were undergoing joint replacement surgery. Briefly, the tissues were minced and incubated with collagenase (Invitrogen Life Technologies, Carlsbad, CA) for 5 h at 37 °C with gentle agitation. Isolated cells were collected by centrifugation and washed with Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen Life Technologies). The cell suspensions were then cultured at 37 °C in a humidified incubator. After overnight incubation, non-adherent cells were removed to yield the adherent synoviocytes. The adherent cells were further subcultured for three to four passages for use in the experiments. A purified population of FLS was identified by morphology showing elongated, often oval shaped cells.
To explore the relevance of the Fas-mediated apoptotic pathway in RA ex vivo, we measured the levels of sFasL in the plasma and synovial fluid of RA patients and control subjects. There was about 1.5-fold and 2-fold increases in sFasL in the plasma and synovial fluid, respectively, from RA patients compared to those of healthy controls or OA patients (Fig. 2A). Next, considering the discrepancy between in vitro susceptibility to Fas-mediated apoptosis and in vivo lack of apoptosis in synovial cells from RA patients, we attempted to discover the mechanistic basis of resistance to Fas-mediated apoptosis in RA. Since the sFas may act to inhibit Fas signaling through binding to FasL, the concentrations of sFas were measured in the plasma and synovial fluid of RA patients. While the concentration of sFas in the plasma was slightly higher in RA patients compared with that in healthy controls, the difference was not significant between RA patients and OA patients (Fig. 2B). In contrast, the concentration of sFas in the synovial fluid of RA patients was significantly higher than that of OA or control subjects.
Apoptosis assays
Membrane and soluble Fas are expressed in response to TNF-α
Cellular apoptosis was assessed using an Annexin V Apoptosis Detection Kit (Biolegend, San Diego, CA) as per the manufacturer's instructions. Briefly, cells were stimulated with or without anti-Fas activating antibody at a concentration of 2 μg/ml (CH11; Millipore, Billerica, MA). 2–4 × 105 cells were collected and re-suspended at 5–10 × 106 cells/ml in an Annexin V-binding buffer. The cells were then stained with Annexin V and 7-amino-actinomycin D (7-AAD) for 15 min at room temperature. Following staining, cells were acquired on a FACS Canto-II instrument (BD Biosciences, San Jose, CA), and the data were further analyzed using FlowJo software (TreeStar, Ashland, OR).
There was considerable expression of membrane-bound Fas on the surface of FLS although the expression levels of Fas were slightly lower in FLS from RA patients than those from OA patients (Fig. 3A). To determine the effects of inflammatory cytokines on Fas-mediated signaling, we determined the expression of mFas after stimulation with TNF-α, IL-1β or IL-17. The levels of mFas expression on the cell surface were slightly increased in RA FLS after stimulation with TNF-α and IL-1β, but not IL-17 (Fig. 3B). To further investigate the effects of inflammatory stimuli on the induction of sFas, which could oppose mFas-mediated apoptosis by binding to FasL in the synovium, we measured the levels of the sFas in the supernatant after stimulation with TNF-α, IL1β, or IL-17. As shown in Fig. 3C, there was a significant increase in sFas levels from TNF-α treated RA FLS. However, the expression of sFas was not different after stimulation with IL-1β or IL-17. Finally, we determined whether patients with RA had higher levels of TNF-α in the synovial fluid. As shown in Fig. 3D, the TNF-α levels were significantly higher in the synovial fluid of RA patients compared to those of OA patients or control subjects. Taken together, these data demonstrated that there was a significant increase of sFas as well as mFas expression in RA FLS after stimulation with inflammatory mediators such as TNF-α, which was present in the synovial cavity of RA.
Primary culture of FLS obtained from RA patients
Detection of membrane bound Fas in FLS FLS were incubated with TNF-α (50 ng/ml), IL-1β (50 ng/ml), or IL-17 (50 ng/ml). After 48 h, cells were harvested and washed twice with PBS containing 5% fetal bovine serum. Then, surface staining was performed for 30 min at 4 °C using anti-human CD95 (APO-1/Fas) antibody (eBioscience, San Diego, CA). Statistical analysis Data are expressed as the mean ± standard deviation (SD). Differences between experimental groups were tested using Student's t-test. The statistical significance in the level of sFasL, sFas and TNF-α between the groups was calculated by using the Kruskal–Wallis nonparametric ANOVA test, followed by Dunn's multiple comparison test. p values less than 0.05 were considered significant. Results FLS from patients with RA show a greater induction of Fas-mediated apoptosis than those from OA patients. To examine the apoptosis in FLS in vitro, we measured the number of cells in early apoptosis (Annexin V+, 7-AAD−) and late apoptosis/ necrosis (Annexin V+, 7-AAD+) using flow cytometry after incubation with or without stimulating Fas antibody. In the absence of Fas activation, the frequency of apoptotic cells in FLS from RA patients was lower compared to that from OA patients (Fig. 1A). However, after stimulation with Fas agonist antibody, FLS from RA patients underwent apoptosis more readily than those from OA patients. The proportion of
Discussion Cellular hyperplasia is one of the main pathological findings in the inflamed synovium of RA patients [16,29]. Increased proliferation and/ or impaired apoptosis might contribute to the expansion of synovial cells [13,23]. It is well known that there is contradictory evidence in the evaluation of the level of apoptosis in FLS, showing that RA FLS have an even greater susceptibility to Fas-mediated apoptosis in vitro in contrast to their apparent lack of apoptosis in vivo [23,26]. However, the mechanisms responsible for this discrepancy remain under investigation. In the present study, our findings suggest that the presence of sFas induced by TNF-α could account for the resistance to Fasmediated apoptosis in synovial cells of RA patients, although FLS exhibited high expression of mFas in RA. Synovial cells from patients with RA exhibited a unique phenotype of resistance to apoptotic signals. However, there are several lines of in vitro evidence demonstrating that RA FLS have an increased susceptibility to apoptosis in response to Fas stimulation [8,18,21]. Recent study
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Fig. 1. Fas-mediated apoptosis in FLS from patients with RA versus those with OA. FLS were incubated with (right) or without (left) Fas activating antibody at 2 μg/ml for 72 h, and then stained with Annexin V and 7-AAD. Cells in early apoptosis (Annexin V+, 7-AAD−) versus late apoptosis or necrosis (Annexin V+, 7-AAD+) were distinguished by flow cytometry. (A) Representative flow cytometry plots are presented. (B) Total apoptotic (Annexin V+) cells were compared with or without Fas stimulation. Error bars represent standard deviation. Statistical analysis was performed using Student's t-test. ***p b 0.001. Given are representative data of three independent experiments.
Fig. 2. The levels of sFasL and sFas in the plasma and synovial fluid. (A) sFasL in the plasma (left) and synovial fluid (right) measured from patients with RA and OA, or in control subjects. (B) Comparison of sFas levels in the plasma (left) and synovial fluid (right) from patients with RA and OA, and control subjects. Data are presented as the mean ± standard deviation. Statistical analysis was performed using the one-way ANOVA test with Dunn's multiple comparison test. **p b 0.01, ***p b 0.001, ns, not significant.
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Fig. 3. The effects of cytokine stimulation on mFas and sFas induction in FLS. (A) Surface staining of mFas was performed on FLS from patients with RA or OA. A representative histogram is presented. (B) Expression of mFas on the cell surface of FLS after incubation for 48 h with TNF-α (50 ng/ml; left), IL-1β (50 ng/ml; middle), or IL-17 (50 ng/ml; right). (C) sFas levels were measured in the supernatant of FLS from RA patients after stimulation for 48 h with the indicated concentrations of TNF-α, IL-1βand IL-17. Data are presented as the mean ± standard deviation. Statistical analysis was performed using Student's t-test. ***p b 0.001. The data are representative of at least three experiments. (D) TNF-α levels were determined in the synovial fluid from patients with RA and OA, or control subjects. Data are presented as the mean ± standard deviation. Statistical analysis was performed using the one-way ANOVA test followed by Dunn's multiple comparison test. ***p b 0.001.
showed that Fas-mediated signaling can exert pro-apoptotic effects in FLS from patients with hemophilic arthropathy as well as from patients with RA [28]. In the present study, we also observed enhanced apoptosis induction in RA FLS after stimulation with Fas activating antibody (Fig. 1). Thus, there should be mechanisms for lack of apoptosis in the synovial tissue of RA to explain the cellular hyperplasia of an inflamed joint [30]. Cellular and molecular mechanisms of decreased apoptosis in RA include high levels of anti-apoptotic molecules such as bcl-2 (B cell lymphoma gene 2) and FLIP (also known as CFLAR, CASP8 and FADD-like apoptosis regulator) [22,24]. In addition, certain factors present in the inflammatory milieu of the synovium appear to play a role in inhibition of apoptosis. TNF-α sensitizes FLS to Fas-mediated apoptosis as well as proliferation [11,12]. Besides the direct effects of TNF-α on FLS, TNF-α may exert an indirect influence on cellular proliferation. In this context, interestingly, we found that levels of sFas were strikingly elevated in the synovial fluid of RA patients relative to their peripheral blood. Further, we found in our current analyses that sFas was markedly induced by TNF-α, which was abundantly present in the synovial cavity of RA patients (Fig. 3C, D). A recent paper, however, showed that therapy with TNF-α blockers induced elevation of serum sFas levels with significant improvement in disease activity, suggesting that circulating sFas levels may negatively correlate with disease activity in RA upon anti-TNF-α treatment [27]. On the other hand, previous several studies have shown that increases in sFas levels were positively correlated with disease activity of RA, including an accumulation of sFas in the joints of active RA patients [2,9]. Thus we need to consider various factors affecting the effects of sFas as well as TNF-α on FLS apoptosis in RA, including different sample sources and types of therapy used for RA. Indeed, our and previous reports indicated that an alteration of sFas levels was more prominent in
the synovial fluid than in the peripheral blood of patients with RA (Fig. 2B) [9]. It is well known that TNF-α plays a pivotal role in the pathogenesis of RA. TNF-α influences diverse pathologic processes including joint destruction as well as inflammatory responses [16]. While TNF-α significantly increases the levels of sFas and mFas expression from FLS, IL-1β and IL-17 have minimal effects on sFas induction. The expression of decoy receptor 3 (DCR3), which binds to FasL and protects against Fas-mediated apoptosis, was reported previously to be increased by TNF-α [10]. It is interesting that the authors of that study noted a similar mechanism of resistance against Fas signaling in which FasL might be blocked by binding to sFas. To examine apoptosis in FLS in our present study, flow cytometric analysis was performed (Fig. 1). Early events during apoptosis were detected by Annexin V staining. Annexin V binds preferentially to phosphatidylserine and other negatively-charged phospholipids, which are normally present in the inner leaflet of the plasma membrane and are exposed to the outer leaflet during early apoptosis [3,31]. Thus, early apoptosis and late apoptosis/necrosis could be discriminated by Annexin V+, 7-AAD− and Annexin V+, 7-AAD+, respectively. Since most earlier studies used the TUNEL assay or lactate dehydrogenase (LDH) assay to detect apoptosis [11,12,18], some portions of early apoptotic cells might have been underestimated previously. This is a particularly important consideration because we here observed that the response to Fas triggering was more pronounced during early apoptosis than late apoptosis/necrosis in RA FLS (Fig. 1). There is insufficient evidence at this time to conclude that sFas is the principal factor for resistance to Fas-mediated apoptosis in vivo. A recent study investigating the role of membrane-bound FasL (mFasL) versus secreted FasL in apoptosis showed that only mFasL, but not the secreted
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form of FasL, triggered a Fas-dependent apoptotic pathway [20]. In addition, TNF-α could have direct effects on apoptosis and on cellular proliferation [32], and TNF-α also can affect cellular homeostasis via a number of pro-inflammatory responses, including cellular differentiation, adhesion and migration of inflammatory cells into the inflamed tissue [5,19]. Indeed, cellular homeostasis is complex, and apoptosis can be influenced by various factors including interaction with neighboring cells expressing FasL on their cell surface [25]. Surely, it will be necessary in the future to address the potential effects of TNF-α on the apoptosis of cells other than FLS, including inflammatory cells. Conclusion In conclusion, we have here demonstrated that high levels of sFas and sFasL are present in the synovial fluid of RA patients. Furthermore, TNF-α, which was readily detected in the synovial cavity of RA, significantly induces sFas from FLS. Hence, sFas induction by TNF-α in vivo could account for the imbalance in apoptosis in FLS, resulting in the synovial hyperplasia of RA. Conflict of interest statement The authors have no conflicts of interest to declare.
Acknowledgment This study was supported by a grant (2014–330) from the Asan Institute for Life Sciences, Seoul, Korea. References [1] F.C. Arnett, S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper, et al., The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis, Arthritis Rheum. 31 (1988) 315–324. [2] A. Ates, G. Kinikli, M. Turgay, M. Duman, The levels of serum-soluble Fas in patients with rheumatoid arthritis and systemic sclerosis, Clin. Rheumatol. 23 (2004) 421–425. [3] J.P. Aubry, A. Blaecke, S. Lecoanet-Henchoz, P. Jeannin, N. Herbault, G. Caron, et al., Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity, Cytometry 37 (1999) 197–204. [4] N. Bottini, G.S. Firestein, Duality of fibroblast-like synoviocytes in RA: passive responders and imprinted aggressors, Nat. Rev. Rheumatol. 9 (2013) 24–33. [5] J.R. Bradley, TNF-mediated inflammatory disease, J. Pathol. 214 (2008) 149–160. [6] I. Cascino, G. Fiucci, G. Papoff, G. Ruberti, Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing, J. Immunol. 154 (1995) 2706–2713. [7] J. Cheng, T. Zhou, C. Liu, J.P. Shapiro, M.J. Brauer, M.C. Kiefer, et al., Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule, Science 263 (1994) 1759–1762. [8] H. Hashimoto, M. Tanaka, T. Suda, T. Tomita, K. Hayashida, E. Takeuchi, et al., Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis, Arthritis Rheum. 41 (1998) 657–662. [9] T. Hasunuma, N. Kayagaki, H. Asahara, S. Motokawa, T. Kobata, H. Yagita, et al., Accumulation of soluble Fas in inflamed joints of patients with rheumatoid arthritis, Arthritis Rheum. 40 (1997) 80–86. [10] S. Hayashi, Y. Miura, T. Nishiyama, M. Mitani, K. Tateishi, Y. Sakai, et al., Decoy receptor 3 expressed in rheumatoid synovial fibroblasts protects the cells against Fasinduced apoptosis, Arthritis Rheum. 56 (2007) 1067–1075. [11] T. Kobayashi, K. Okamoto, T. Kobata, T. Hasunuma, T. Sumida, K. Nishioka, Tumor necrosis factor alpha regulation of the FAS-mediated apoptosis-signaling pathway in synovial cells, Arthritis Rheum. 42 (1999) 519–526.
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[12] T. Kobayashi, K. Okamoto, T. Kobata, T. Hasunuma, T. Kato, H. Hamada, et al., Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules, Arthritis Rheum. 43 (2000) 1106–1114. [13] A. Korb, H. Pavenstadt, T. Pap, Cell death in rheumatoid arthritis, Apoptosis 14 (2009) 447–454. [14] H. Liu, R.M. Pope, Apoptosis in rheumatoid arthritis: friend or foe, Rheum. Dis. Clin. N. Am. 30 (2004) 603–625 x. [15] H. Liu, P. Eksarko, V. Temkin, G.K. Haines 3rd, H. Perlman, A.E. Koch, et al., Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis, J. Immunol. 175 (2005) 8337–8345. [16] I.B. McInnes, G. Schett, The pathogenesis of rheumatoid arthritis, N. Engl. J. Med. 365 (2011) 2205–2219. [17] I. Meinecke, A. Cinski, A. Baier, M.A. Peters, B. Dankbar, A. Wille, et al., Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 5073–5078. [18] T. Nakajima, H. Aono, T. Hasunuma, K. Yamamoto, T. Shirai, K. Hirohata, et al., Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes, Arthritis Rheum. 38 (1995) 485–491. [19] C.A. Notley, J.J. Inglis, S. Alzabin, F.E. McCann, K.E. McNamee, R.O. Williams, Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells, J. Exp. Med. 205 (2008) 2491–2497. [20] L.A. O'Reilly, L. Tai, L. Lee, E.A. Kruse, S. Grabow, W.D. Fairlie, et al., Membrane-bound Fas ligand only is essential for Fas-induced apoptosis, Nature 461 (2009) 659–663. [21] S. Ohshima, T. Mima, M. Sasai, K. Nishioka, M. Shimizu, N. Murata, et al., Tumour necrosis factor alpha (TNF-alpha) interferes with Fas-mediated apoptotic cell death on rheumatoid arthritis (RA) synovial cells: a possible mechanism of rheumatoid synovial hyperplasia and a clinical benefit of anti-TNF-alpha therapy for RA, Cytokine 12 (2000) 281–288. [22] G. Palao, B. Santiago, M. Galindo, M. Paya, J.C. Ramirez, J.L. Pablos, Down-regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis, Arthritis Rheum. 50 (2004) 2803–2810. [23] S.L. Peng, Fas (CD95)-related apoptosis and rheumatoid arthritis, Rheumatology (Oxford). 45 (2006) 26–30. [24] H. Perlman, C. Georganas, L.J. Pagliari, A.E. Koch, K. Haines 3rd, R.M. Pope, Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability, J. Immunol. 164 (2000) 5227–5235. [25] H. Perlman, L.J. Pagliari, H. Liu, A.E. Koch, G.K. Haines 3rd, R.M. Pope, Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fasmediated apoptosis, Arthritis Rheum. 44 (2001) 21–30. [26] R.M. Pope, Apoptosis as a therapeutic tool in rheumatoid arthritis, Nat. Rev. Immunol. 2 (2002) 527–535. [27] E. Romano, R. Terenzi, M. Manetti, F. Peruzzi, G. Fiori, F. Nacci, et al., Disease activity improvement in rheumatoid arthritis treated with tumor necrosis factor-alpha inhibitors correlates with increased soluble Fas levels, J. Rheumatol. 41 (2014) 1961–1965. [28] E. Romano, M. Manetti, F. Peruzzi, D. Melchiorre, A.F. Milia, S. Bellando-Randone, et al., Agonistic anti-human Fas monoclonal antibody induces fibroblast-like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications, Haemophilia 20 (2014) e32–e39. [29] D.L. Scott, F. Wolfe, T.W. Huizinga, Rheumatoid arthritis, Lancet 376 (2010) 1094–1108. [30] M. Sugiyama, T. Tsukazaki, A. Yonekura, S. Matsuzaki, S. Yamashita, K. Iwasaki, Localisation of apoptosis and expression of apoptosis related proteins in the synovium of patients with rheumatoid arthritis, Ann. Rheum. Dis. 55 (1996) 442–449. [31] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V, J. Immunol. Methods 184 (1995) 39–51. [32] H.G. Zhang, N. Huang, D. Liu, L. Bilbao, X. Zhang, P. Yang, et al., Gene therapy that inhibits nuclear translocation of nuclear factor kappaB results in tumor necrosis factor alpha-induced apoptosis of human synovial fibroblasts, Arthritis Rheum. 43 (2000) 1094–1105.
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
TNF-α confers resistance to Fas-mediated apoptosis in rheumatoid arthritis through the induction of soluble Fas Seokchan Hong, Eun-Jin Kim, Eun-Ju Lee, Bon San Koo 1, Soo Min Ahn, Seung-Hyeon Bae, Doo-Ho Lim, Yong-Gil Kim, Bin Yoo, Chang-Keun Lee ⁎ Division of Rheumatology, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 August 2014 Accepted 6 December 2014 Available online 19 December 2014 Keywords: Rheumatoid arthritis Apoptosis Fas Fas ligand
a b s t r a c t Aims: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that is characterized by hyperplastic synovial tissue containing activated synovial fibroblasts. Contradictory findings in the apoptosis of fibroblast-like synoviocytes (FLS) have been described elsewhere, showing that RA FLS have an enhanced susceptibility to Fas (also known as CD95)-mediated apoptosis in vitro in contrast to the observed lack of apoptosis in the RA synovium in vivo. However, the potential mechanisms responsible for this discrepancy remain under investigation. The soluble form of Fas (sFas) was found to inhibit Fas-induced apoptosis by binding to Fas ligand (FasL), thereby preventing the interaction between FasL and membrane-bound Fas. Main methods: We determined the levels of soluble FasL (sFasL) and sFas in patients with RA and the effects of proinflammatory mediators, including TNF-α, on the induction of apoptotic mediators in RA FLS. Key findings: The levels of sFasL and sFas were significantly elevated in the synovial fluids of RA patients compared with control subjects. In addition, we found that the sFas is substantially induced in RA FLS by TNF-α, which were abundantly present in the synovial fluid of RA. Significance: These findings suggest that TNF-α confers resistance to Fas-mediated apoptosis through sFas induction, which could explain the apparent resistance of RA synovial cells to apoptosis in vivo. © 2014 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that is characterized by inflammatory cell infiltration within the synovium [16]. While the exact mechanism of RA pathogenesis remains under investigation, synovial hyperplasia has been considered the major pathogenic finding in RA and could be associated with the persistent inflammation and joint destruction in RA [16,29]. Numerous studies have suggested the pathogenic mechanism underlying the hyperplasia of the fibroblast-like synoviocytes (FLS), which populate the lining layers of the synovium and are key to the induction and perpetuation of inflammation in RA [4]. Several lines of evidence show that FLS exhibit sensitivity to Fas (CD95)-mediated apoptotic signaling in vitro, in contrast to the observed lack of apoptotic cells in the RA synovium in vivo [14,23,26]. There have been substantial studies directed at this conflicting finding in order to address the underlying mechanism responsible for the resistance to apoptosis in RA synovial cells [10,15,17,22]. The cell surface Fas plays a pivotal role in the apoptosis of inflammatory
⁎ Corresponding author at: 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea. Tel.: +82 2 3010 3284; fax: +82 2 3010 6969. E-mail address: [email protected] (C.-K. Lee). 1 Current address: Division of Rheumatology, Department of Internal Medicine, Konkuk University College of Medicine, Chungju, Republic of Korea.
http://dx.doi.org/10.1016/j.lfs.2014.12.008 0024-3205/© 2014 Elsevier Inc. All rights reserved.
cells, including FLS, and there are intricate mechanisms regulating the Fas-mediated apoptotic pathway to maintain cellular homeostasis [13, 23,26]. The soluble form of Fas (sFas) inhibits Fas-induced apoptosis by binding to Fas ligand (FasL), thereby blocking interaction with the membrane Fas (mFas) receptor on the cell surface [6,7]. However, conflicting data have been reported regarding the levels of sFas and its relation with disease activity in RA [2,9,27]. In our present study, we determined the levels of soluble FasL (sFasL) and sFas in patients with RA and the effects of proinflammatory cytokines, including TNF-α, on the induction of apoptosis mediators in RA FLS. Materials and methods Patients and collection of plasma and synovial fluid The study included patients diagnosed with RA who were referred to a tertiary hospital in South Korea between January 2010 and December 2011. All patients fulfilled the 1987 revised American College of Rheumatology classification criteria for RA [1]. Plasma samples were obtained from 26 RA patients, and control samples were collected from 20 healthy volunteers and 20 patients with osteoarthritis (OA). Synovial fluid samples were collected from 15 patients with RA. Synovial fluids from 15 OA patients and 15 subjects suffering from sports injuries
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S. Hong et al. / Life Sciences 122 (2015) 37–41
were included in the analysis. The samples were stored at −20 °C until use. All patients provided written informed consent, and the study protocol was approved by our institution's ethics committee.
total apoptotic cells (Annexin V+) was significantly increased by Fas stimulation in FLS from RA patients (Fig. 1B). Thus, we observed that RA synovial cells were sensitive to Fas-mediated apoptotic signaling, consistent with previous studies [8,18,21].
Measurement of sFasL, sFas and TNF-α by ELISA The levels of sFasL and sFas are elevated in patients with RA The levels of sFasL, sFas and TNF-α were measured by commercially available ELISA kits (R&D Systems, Minneapolis, MN).
FLS were isolated from the synovial tissues obtained from patients affected by RA or OA who were undergoing joint replacement surgery. Briefly, the tissues were minced and incubated with collagenase (Invitrogen Life Technologies, Carlsbad, CA) for 5 h at 37 °C with gentle agitation. Isolated cells were collected by centrifugation and washed with Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen Life Technologies). The cell suspensions were then cultured at 37 °C in a humidified incubator. After overnight incubation, non-adherent cells were removed to yield the adherent synoviocytes. The adherent cells were further subcultured for three to four passages for use in the experiments. A purified population of FLS was identified by morphology showing elongated, often oval shaped cells.
To explore the relevance of the Fas-mediated apoptotic pathway in RA ex vivo, we measured the levels of sFasL in the plasma and synovial fluid of RA patients and control subjects. There was about 1.5-fold and 2-fold increases in sFasL in the plasma and synovial fluid, respectively, from RA patients compared to those of healthy controls or OA patients (Fig. 2A). Next, considering the discrepancy between in vitro susceptibility to Fas-mediated apoptosis and in vivo lack of apoptosis in synovial cells from RA patients, we attempted to discover the mechanistic basis of resistance to Fas-mediated apoptosis in RA. Since the sFas may act to inhibit Fas signaling through binding to FasL, the concentrations of sFas were measured in the plasma and synovial fluid of RA patients. While the concentration of sFas in the plasma was slightly higher in RA patients compared with that in healthy controls, the difference was not significant between RA patients and OA patients (Fig. 2B). In contrast, the concentration of sFas in the synovial fluid of RA patients was significantly higher than that of OA or control subjects.
Apoptosis assays
Membrane and soluble Fas are expressed in response to TNF-α
Cellular apoptosis was assessed using an Annexin V Apoptosis Detection Kit (Biolegend, San Diego, CA) as per the manufacturer's instructions. Briefly, cells were stimulated with or without anti-Fas activating antibody at a concentration of 2 μg/ml (CH11; Millipore, Billerica, MA). 2–4 × 105 cells were collected and re-suspended at 5–10 × 106 cells/ml in an Annexin V-binding buffer. The cells were then stained with Annexin V and 7-amino-actinomycin D (7-AAD) for 15 min at room temperature. Following staining, cells were acquired on a FACS Canto-II instrument (BD Biosciences, San Jose, CA), and the data were further analyzed using FlowJo software (TreeStar, Ashland, OR).
There was considerable expression of membrane-bound Fas on the surface of FLS although the expression levels of Fas were slightly lower in FLS from RA patients than those from OA patients (Fig. 3A). To determine the effects of inflammatory cytokines on Fas-mediated signaling, we determined the expression of mFas after stimulation with TNF-α, IL-1β or IL-17. The levels of mFas expression on the cell surface were slightly increased in RA FLS after stimulation with TNF-α and IL-1β, but not IL-17 (Fig. 3B). To further investigate the effects of inflammatory stimuli on the induction of sFas, which could oppose mFas-mediated apoptosis by binding to FasL in the synovium, we measured the levels of the sFas in the supernatant after stimulation with TNF-α, IL1β, or IL-17. As shown in Fig. 3C, there was a significant increase in sFas levels from TNF-α treated RA FLS. However, the expression of sFas was not different after stimulation with IL-1β or IL-17. Finally, we determined whether patients with RA had higher levels of TNF-α in the synovial fluid. As shown in Fig. 3D, the TNF-α levels were significantly higher in the synovial fluid of RA patients compared to those of OA patients or control subjects. Taken together, these data demonstrated that there was a significant increase of sFas as well as mFas expression in RA FLS after stimulation with inflammatory mediators such as TNF-α, which was present in the synovial cavity of RA.
Primary culture of FLS obtained from RA patients
Detection of membrane bound Fas in FLS FLS were incubated with TNF-α (50 ng/ml), IL-1β (50 ng/ml), or IL-17 (50 ng/ml). After 48 h, cells were harvested and washed twice with PBS containing 5% fetal bovine serum. Then, surface staining was performed for 30 min at 4 °C using anti-human CD95 (APO-1/Fas) antibody (eBioscience, San Diego, CA). Statistical analysis Data are expressed as the mean ± standard deviation (SD). Differences between experimental groups were tested using Student's t-test. The statistical significance in the level of sFasL, sFas and TNF-α between the groups was calculated by using the Kruskal–Wallis nonparametric ANOVA test, followed by Dunn's multiple comparison test. p values less than 0.05 were considered significant. Results FLS from patients with RA show a greater induction of Fas-mediated apoptosis than those from OA patients. To examine the apoptosis in FLS in vitro, we measured the number of cells in early apoptosis (Annexin V+, 7-AAD−) and late apoptosis/ necrosis (Annexin V+, 7-AAD+) using flow cytometry after incubation with or without stimulating Fas antibody. In the absence of Fas activation, the frequency of apoptotic cells in FLS from RA patients was lower compared to that from OA patients (Fig. 1A). However, after stimulation with Fas agonist antibody, FLS from RA patients underwent apoptosis more readily than those from OA patients. The proportion of
Discussion Cellular hyperplasia is one of the main pathological findings in the inflamed synovium of RA patients [16,29]. Increased proliferation and/ or impaired apoptosis might contribute to the expansion of synovial cells [13,23]. It is well known that there is contradictory evidence in the evaluation of the level of apoptosis in FLS, showing that RA FLS have an even greater susceptibility to Fas-mediated apoptosis in vitro in contrast to their apparent lack of apoptosis in vivo [23,26]. However, the mechanisms responsible for this discrepancy remain under investigation. In the present study, our findings suggest that the presence of sFas induced by TNF-α could account for the resistance to Fasmediated apoptosis in synovial cells of RA patients, although FLS exhibited high expression of mFas in RA. Synovial cells from patients with RA exhibited a unique phenotype of resistance to apoptotic signals. However, there are several lines of in vitro evidence demonstrating that RA FLS have an increased susceptibility to apoptosis in response to Fas stimulation [8,18,21]. Recent study
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Fig. 1. Fas-mediated apoptosis in FLS from patients with RA versus those with OA. FLS were incubated with (right) or without (left) Fas activating antibody at 2 μg/ml for 72 h, and then stained with Annexin V and 7-AAD. Cells in early apoptosis (Annexin V+, 7-AAD−) versus late apoptosis or necrosis (Annexin V+, 7-AAD+) were distinguished by flow cytometry. (A) Representative flow cytometry plots are presented. (B) Total apoptotic (Annexin V+) cells were compared with or without Fas stimulation. Error bars represent standard deviation. Statistical analysis was performed using Student's t-test. ***p b 0.001. Given are representative data of three independent experiments.
Fig. 2. The levels of sFasL and sFas in the plasma and synovial fluid. (A) sFasL in the plasma (left) and synovial fluid (right) measured from patients with RA and OA, or in control subjects. (B) Comparison of sFas levels in the plasma (left) and synovial fluid (right) from patients with RA and OA, and control subjects. Data are presented as the mean ± standard deviation. Statistical analysis was performed using the one-way ANOVA test with Dunn's multiple comparison test. **p b 0.01, ***p b 0.001, ns, not significant.
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Fig. 3. The effects of cytokine stimulation on mFas and sFas induction in FLS. (A) Surface staining of mFas was performed on FLS from patients with RA or OA. A representative histogram is presented. (B) Expression of mFas on the cell surface of FLS after incubation for 48 h with TNF-α (50 ng/ml; left), IL-1β (50 ng/ml; middle), or IL-17 (50 ng/ml; right). (C) sFas levels were measured in the supernatant of FLS from RA patients after stimulation for 48 h with the indicated concentrations of TNF-α, IL-1βand IL-17. Data are presented as the mean ± standard deviation. Statistical analysis was performed using Student's t-test. ***p b 0.001. The data are representative of at least three experiments. (D) TNF-α levels were determined in the synovial fluid from patients with RA and OA, or control subjects. Data are presented as the mean ± standard deviation. Statistical analysis was performed using the one-way ANOVA test followed by Dunn's multiple comparison test. ***p b 0.001.
showed that Fas-mediated signaling can exert pro-apoptotic effects in FLS from patients with hemophilic arthropathy as well as from patients with RA [28]. In the present study, we also observed enhanced apoptosis induction in RA FLS after stimulation with Fas activating antibody (Fig. 1). Thus, there should be mechanisms for lack of apoptosis in the synovial tissue of RA to explain the cellular hyperplasia of an inflamed joint [30]. Cellular and molecular mechanisms of decreased apoptosis in RA include high levels of anti-apoptotic molecules such as bcl-2 (B cell lymphoma gene 2) and FLIP (also known as CFLAR, CASP8 and FADD-like apoptosis regulator) [22,24]. In addition, certain factors present in the inflammatory milieu of the synovium appear to play a role in inhibition of apoptosis. TNF-α sensitizes FLS to Fas-mediated apoptosis as well as proliferation [11,12]. Besides the direct effects of TNF-α on FLS, TNF-α may exert an indirect influence on cellular proliferation. In this context, interestingly, we found that levels of sFas were strikingly elevated in the synovial fluid of RA patients relative to their peripheral blood. Further, we found in our current analyses that sFas was markedly induced by TNF-α, which was abundantly present in the synovial cavity of RA patients (Fig. 3C, D). A recent paper, however, showed that therapy with TNF-α blockers induced elevation of serum sFas levels with significant improvement in disease activity, suggesting that circulating sFas levels may negatively correlate with disease activity in RA upon anti-TNF-α treatment [27]. On the other hand, previous several studies have shown that increases in sFas levels were positively correlated with disease activity of RA, including an accumulation of sFas in the joints of active RA patients [2,9]. Thus we need to consider various factors affecting the effects of sFas as well as TNF-α on FLS apoptosis in RA, including different sample sources and types of therapy used for RA. Indeed, our and previous reports indicated that an alteration of sFas levels was more prominent in
the synovial fluid than in the peripheral blood of patients with RA (Fig. 2B) [9]. It is well known that TNF-α plays a pivotal role in the pathogenesis of RA. TNF-α influences diverse pathologic processes including joint destruction as well as inflammatory responses [16]. While TNF-α significantly increases the levels of sFas and mFas expression from FLS, IL-1β and IL-17 have minimal effects on sFas induction. The expression of decoy receptor 3 (DCR3), which binds to FasL and protects against Fas-mediated apoptosis, was reported previously to be increased by TNF-α [10]. It is interesting that the authors of that study noted a similar mechanism of resistance against Fas signaling in which FasL might be blocked by binding to sFas. To examine apoptosis in FLS in our present study, flow cytometric analysis was performed (Fig. 1). Early events during apoptosis were detected by Annexin V staining. Annexin V binds preferentially to phosphatidylserine and other negatively-charged phospholipids, which are normally present in the inner leaflet of the plasma membrane and are exposed to the outer leaflet during early apoptosis [3,31]. Thus, early apoptosis and late apoptosis/necrosis could be discriminated by Annexin V+, 7-AAD− and Annexin V+, 7-AAD+, respectively. Since most earlier studies used the TUNEL assay or lactate dehydrogenase (LDH) assay to detect apoptosis [11,12,18], some portions of early apoptotic cells might have been underestimated previously. This is a particularly important consideration because we here observed that the response to Fas triggering was more pronounced during early apoptosis than late apoptosis/necrosis in RA FLS (Fig. 1). There is insufficient evidence at this time to conclude that sFas is the principal factor for resistance to Fas-mediated apoptosis in vivo. A recent study investigating the role of membrane-bound FasL (mFasL) versus secreted FasL in apoptosis showed that only mFasL, but not the secreted
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form of FasL, triggered a Fas-dependent apoptotic pathway [20]. In addition, TNF-α could have direct effects on apoptosis and on cellular proliferation [32], and TNF-α also can affect cellular homeostasis via a number of pro-inflammatory responses, including cellular differentiation, adhesion and migration of inflammatory cells into the inflamed tissue [5,19]. Indeed, cellular homeostasis is complex, and apoptosis can be influenced by various factors including interaction with neighboring cells expressing FasL on their cell surface [25]. Surely, it will be necessary in the future to address the potential effects of TNF-α on the apoptosis of cells other than FLS, including inflammatory cells. Conclusion In conclusion, we have here demonstrated that high levels of sFas and sFasL are present in the synovial fluid of RA patients. Furthermore, TNF-α, which was readily detected in the synovial cavity of RA, significantly induces sFas from FLS. Hence, sFas induction by TNF-α in vivo could account for the imbalance in apoptosis in FLS, resulting in the synovial hyperplasia of RA. Conflict of interest statement The authors have no conflicts of interest to declare.
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