http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; 24(5): 766–769 © 2014 Japan College of Rheumatology DOI: 10.3109/14397595.2013.879413
ORIGINAL ARTICLE
Identification of citrullinated cellular fibronectin in synovial fluid from patients with rheumatoid arthritis
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Eri Kimura1, Takeyuki Kanzaki2, Koichiro Tahara1, Haeru Hayashi1, Shiori Hashimoto3, Akari Suzuki4, Ryo Yamada5, Kazuhiko Yamamoto6, and Tetsuji Sawada1 1Department of Rheumatology, Tokyo Medical University Hospital, Tokyo, Japan, 2Department of Internal Medicine, Yamanashi Prefectural Central Hospital, Kofu, Japan, 3Department of Neurology, Tokyo Women’s Medical University, Tokyo, Japan, 4Laboratory for Autoimmune Diseases, Center for Genomic Medicine, The Institute of Physical and Chemical Research (RIKEN), Wako, Japan, 5Center for Genomic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan, and 6Department of Allergy and Rheumatology, University of Tokyo School of Medicine, Tokyo, Japan
Abstract
Keywords
Objectives. Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients. Methods. Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification. Results. Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF. Conclusions. Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.
Citrullinated antigen, Cellular fibronectin, Rheumatoid arthritis, Synovial fluid
Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the chronic inflammation of synovial tissue, resulting in bone erosion and joint destruction. Using linkage disequilibrium and single nucleotide polymorphism analysis, we previously identified peptidylarginine deiminase (PADI) Type 4 as an RA-susceptible gene [1]. This is one of five PADI genes that encode enzymes which convert peptide arginine residues to citrulline residues. Furthermore, peptidyl citrulline is targeted by anti-citrullinated peptide antibodies that are highly specific to RA [2]. Citrullination plays an important role in the pathogenesis of RA. Several citrullinated peptides, such as fibrin(ogen) [3], vimentin [4] and cellular fibronectin (cFn) [5], were reported to be present in the synovial tissue of RA patients. Moreover, some citrullinated peptides were retrieved from the sera of RA patients, indicating that they may be involved in the pathogenesis of RA through their autoantigen activity. Several reports have described the
Correspondence to: Tetsuji Sawada, MD, PhD, Third Department of Internal Medicine, Department of Rheumatology, Tokyo Medical University, 7-6-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan. E-mail: [email protected]
History Received 18 July 2013 Accepted 25 December 2013 Published online 4 February 2014
citrullinated peptides contained in rheumatoid arthritis synovial fluid (RASF), including fibrinogen [6], vimentin [7], fibronectin [7–9], exosome-associated peptides and Sp-α [10], α-enolase [11], apolipoprotein E, myeloid nuclear differentiation antigen and β-actin [9]. However, those that are pathognomonic for RA remain to be elucidated. Fibronectins are glycoproteins found in plasma, extracellular matrix and on cell surfaces that play an important role in cell– cell and cell–matrix interactions [12]. Alternative splicing generates different isoforms of plasma and cFn. Plasma fibronectin is secreted by hepatocytes and distributed in the plasma, while cFn is produced locally by various cell types, such as endothelial cells and fibroblasts, and is characterized by the presence of alternatively spliced exons, including extra domains A and B (EDA and EDB). Although cFn is water-insoluble and deposited as an extracellular matrix protein, it constitutes less than 1–2% of the total plasma fibronectin [13]. cFn has been detected in inflamed synovium as well as in synovial fluid obtained from active RA patients and is a possible indicator of synovial inflammation in RA [14,15]. We previously demonstrated the presence of the citrullinated form of total fibronectin in plasma and that of cFn in synovial tissues in RA, and we showed that both cellular and plasma fibronectin citrullinated in vitro by PADI were targeted by RA serum,
Citrullinated cellular fibronectin in RASF 767
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DOI 10.3109/14397595.2013.879413
demonstrating an altered capacity to bind to their receptors and growth factors [5]. The presence of citrullinated forms of fibronectin in synovial fluid from RA patients was also reported by others based on a proteomics-based analysis [7–9]. In addition, Chang et al. showed that citrullination of fibronectin reduced the binding and inhibition of the aggrecan-degrading enzyme ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) by a 40-kDa fibronectin fragment, which suggests that citrullination of fibronectin might contribute to cartilage destruction in RA [16]. These observations suggest that cFn is involved in the pathogenesis of RA, especially in its citrullinated form. In the present study, we investigated whether the citrullinated form of cFn could be detected in the plasma and synovial fluid of patients with RA and osteoarthritis by western blotting and enzyme-linked immunosorbent assay (ELISA). We also discuss the role of cFn citrullination in the pathogenesis of RA.
After washing, reagents A and B were applied and incubated overnight at 37°C. After further washing, the wells were blocked with 5% bovine serum albumin for 30 min, then incubated with anti-modified citrulline antibody diluted at 1:2,500 for 3 h at 37°C, and with goat F(ab’)2 anti-rabbit immunoglobulin-HRP conjugate diluted at 1:5,000 for 2 h at 37°C. After a final wash, the 3,3′,5,5′tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD) was applied and the color development was stopped with TMB stop solution. Optical density values at a wavelength of 450 nm (OD450) were then measured. Anti-CCP antibodies and matrix metalloproteinase-3 (MMP-3) in SFs were measured using the second-generation anti-CCP ELISA kit (DIASTAT Anti-CCP; Medical & Biological Laboratories (MBL), Nagoya, Japan) and SensoLyte MMP-3 ELISA kit (Fremont, CA), respectively, according to the manufacturers’ instructions.
Materials and methods
Statistical analysis
Samples
A comparison of mean values between the two groups was made using the Mann–Whitney U test. Differences were considered significant at p ⬍ 0.05.
Twenty-five RASF and seven osteoarthritis synovial fluid (OASF) samples were collected in heparinized tubes at the time of therapeutic arthrocentesis, centrifuged at 450 ⫻ g for 10 min to remove debris, and the supernatants were collected. Plasma samples were obtained from 12 patients with RA and 12 healthy subjects after informed consent was obtained. This study was approved by the appropriate ethics committee. Immunoprecipitation-western blotting Immunoprecipitation (IP) of cFn was performed using an agaroseconjugated anti-cFn monoclonal antibody (IgG1) (sc-8422 AC, Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes extracellular and matrix fibronectin and is non-cross-reactive with soluble, dimeric plasma fibronectin. Synovial fluid (SF) and plasma diluted at 1:10 were incubated with agarose-conjugated anti-cFn monoclonal antibody (mAb) overnight at 4°C. After thorough washing, purified cFn was eluted with 1 ⫻ NuPage LDS sample reducing buffer (Life Technologies, Foster City, CA) at 70°C for 10 min and was subsequently separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transblotted onto polyvinylidene fluoride membranes. To detect cFn, blots were incubated in blocking buffer (phosphate-buffered saline (PBS) containing 5% nonfat dried milk and 0.1% NP-40) for 1 h at room temperature, and subsequently with anti-cFn mAb for 1–3 h. After washing the blot, bound antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by chemiluminescence. For the detection of citrullinated proteins using the anti-citrulline (modified) detection kit (Merck Millipore, Darmstadt, Germany), the blot was chemically treated before immunostaining according to the manufacturer’s instructions.
Results ELISA detection of Cit-cFn in RASF Figure 1A shows that the levels of total cFn in SFs were significantly higher in RASF than in OASF. Levels of Cit-cFn were then measured by ELISA and quantitated by an anti-modified citrulline antibody. These levels were significantly higher in RASF than in OASF (Figure 1B), while cFn citrullination was not detected by ELISA in plasma or serum from RA patients (data not shown). Detection of Cit-cFn in RASF by IP-western blotting IP/western blotting was subsequently performed to demonstrate the presence of the citrullinated form of cFn in RASF. Representative data are shown in Figure 2, in which IP using anti-cFn antibody was carried out on RASF and OASF samples. After SDS-PAGE, anti-cFn antibody immunoprecipitates were shown to contain cFn with a molecular weight of approximately 200 kDa (Figure 2A, upper). The corresponding bands were stained with anti-modified citrulline antibody after chemical modification (Figure 2A, lower). Although the use of IP/western blotting was not intended to quantitate the Cit-cFn in synovial fluid, it appears that RASF contained more Cit-cFn than OASF. Furthermore, while cFn was detected in plasma samples, no citrullinated cFn was detected by IP/western blotting (Figure 2B).
ELISA Detection of citrullination associated with cFn (Cit-cFn) was performed by sandwich ELISA using a human cFn-specific mAb adsorbed on a microplate in combination with an anti-modified citrulline antibody (rabbit polyclonal IgG) according to the manufacturer’s instructions (cFn ELISA kit; Biohit Oyj, Helsinki, Finland). First, 100 μL of RASFs and control SFs diluted 1:25 with a dilution buffer were added in duplicate to wells pre-coated with anti-human cFn mAb and incubated for 1 h at 37°C. The wells were washed, then incubated with 1% glutaraldehyde/PBS for 1 h, and subsequently with 0.2 M Tris-Cl, pH 7.8 for 30 min.
Figure 1. ELISA detection of cFn and Cit-cFn in RASF and OASF samples. (A) Levels of total (citrullinated and non-citrullinated) cFn were determined by sandwich ELISA. (B) Levels of Cit-cFn were determined by ELISA using a combination of anti-cFn mAb and anti-modified citrulline (AMC) antibody with chemical modifications. Data are expressed as optical density values measured at 450 nm (OD450).
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Figure 4. Relationship between MMP-3 and levels of Cit-cFn and total cFn in RASF samples. (A) Correlation between MMP-3 and Cit-cFn (expressed as OD450). (B) Correlation between MMP-3 and total cFn. r, correlation coefficient.
Figure 2. Citrullinated form of cFn in synovial fluid and plasma. Western blot of anti-cFn mAb immunoprecipitates stained with anti-cFn antibody (upper) and AMC antibody after chemical modification (lower). (A) Representative IP/western blotting data of RASF (lanes 1–5) and OASF (lanes 6–8). The values of Cit-cFn in each sample (as detected by ELISA; OD450) were as follows. RASF (lanes 1–5): 0.20, 0.07, 0, 0.04, 0.07; OASF (lanes 6–8): 0, 0. 0.01. (B) Representative IP/western blotting data of plasma from RA patients (lanes 1–5) and healthy controls (lanes 6–8). RA synovial fluid was used as a positive control.
Relationship of cFn with cFn and anti-CCP antibody positivity Anti-CCP antibody expression was detected in 24 out of 25 RASF samples. No significant correlation was observed in RASF between cFn citrullination (as determined by ELISA) and the anti-CCP antibody. However, there was a slight, but non-significant, positive correlation between cFn citrullination and total cFn (r ⫽ 0.25, p ⫽ 0.11) (Figure 3). Relationship between cFn citrullination with MMP-3 A significant positive correlation was observed between the levels of MMP-3 and cFn citrullination in RASF (Figure 4). On the other hand, a positive correlation was noted between MMP-3 and total cFn in RASF, although this did not reach significance.
Discussion Although cFn is located on the surface of many different cell types where it forms insoluble fibrillar matrices, it is also secreted as a soluble molecule. We previously demonstrated that cFn in synovial tissues was citrullinated in RA [5]. Fibronectin citrullination in RASF has been demonstrated by van Beers et al. [8], although their mass spectrometry failed to identify EDA/EDB-containing cFn, presumably because of a limited sample size or technical limitations, and by Tabushi et al. [7], who used proteomics-based
Figure 3. Relationship between citrullinated (Cit)-cFn and levels of anti-CCP antibody and total cFn in RASF samples. (A) Correlation between Cit-cFn (expressed as OD450) and levels of anti-CCP antibody. (B) Correlation between Cit-cFn and total cFn. r, correlation coefficient.
analysis but no appropriate control samples, so the significance of their results is unclear. In the present study, we clearly demonstrated for the first time the presence of citrullinated cFn in synovial fluid, but not in the plasma or serum, of RA patients by ELISA and IP/western blotting. We also showed that the degree of cFn citrullination was significantly higher in RASF than in OASF and correlated significantly with MMP-3 levels in RASF. It should be noted, however, that sandwich ELISA data do not preclude the possibility that cFn forms a complex with other citrullinated peptide(s) in RASF, which is responsible for the peptidyl-citrulline ELISA signal. For this reason, we performed IP/western blotting to demonstrate the presence of the citrullinated form of cFn observed in RASF. It should also be noted that the multiple bands of cFn in IP/western blotting (Figure 2A) are consistent with the results of a previous report demonstrating a large range of fibronectin polypeptide lengths in RASF samples caused by the presence of different isoforms [8]. We previously demonstrated the presence of the citrullinated form of total fibronectin in the plasma of RA patients [5], which is presumed to contain mainly citrullinated plasma fibronectin since Cit-cFn was not detected in the RA plasma in the present study. It should be noted, however, that the amount of cFn contained in plasma is small, so we cannot preclude the possibility that CitcFn may be detected in the plasma of RA patients when a higher sensitivity assay is used. Since no citrullinated cFn was detected in the plasma of RA patients, it is conceivable that citrullination occurred within the affected joint. However, this is unclear, and the PADI isoforms involved remain unknown. Although PADI2 and PADI4 are usually localized in the cytoplasm and nucleus, respectively, both enzymes have previously been detected in RASF [11]. As the extracellular calcium concentration is sufficiently high for PADI to function, it is possible that secreted cFn is citrullinated by extracellular PADI. Another possibility is that activated cells, such as synoviocytes and endothelial cells, citrullinate intracellular cFn and subsequently express citrullinated cFn on their cell surfaces. MMP-3, which is produced within the joints, is considered to play a role in cartilage destruction, since it can degrade a wide range of substrates, including the main components of joint cartilage such as aggrecan and Type-II collagen. In RA, overexpression of MMP-3 has been demonstrated in the rheumatoid synovium, and serum levels of MMP-3, which reflect the severity of rheumatoid synovitis, can be used as a disease marker of RA [17]. In the present study, we demonstrated that Cit-cFn, as measured by ELISA, correlates significantly with MMP-3 in RASF, suggesting that cFn citrullination is relevant to the pathophysiology of RA. Regarding the pathogenetic role of citrullinated cFn in RA, we previously demonstrated that, following citrullination by PADI in vitro, fibronectin increased its binding activity to vascular
Citrullinated cellular fibronectin in RASF 769
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DOI 10.3109/14397595.2013.879413
endothelial growth factor but decreased that to integrin β1 as well as its ability to stimulate apoptosis. This suggests that citrullinated cFn alters the immunological micro-environment of the synovium, leading to the perpetuation of inflammation [5]. Fan et al. previously found that citrullinated fibronectin could inhibit the apoptosis of synoviocytes, which may promote their survival and the secretion of proinflammatory cytokines from rheumatoid synoviocytes in a pathogenic role in RA [18]. Shelef et al. recently demonstrated that citrullinated fibronectin impaired the adhesion and migration of synoviocytes, which might be associated with the metastasis of synovial cells and the spread of arthritis [19]. Another possibility is that citrullinated cFn is involved in the pathogenesis of RA by acting as an autoantigen in the RA cycle, as proposed by van Venrooij et al. [20]. Thus, the expression of CitcFn within the joint, as demonstrated in the present study, together with the reported presence of the anti-citrullinated fibronectin antibody [5,8], implies that Cit-cFn is a candidate autoantigen that drives the chronic inflammatory response through the formation of immune complexes which lead to joint destruction in RA. In the present study, the relationship between cFn citrullination and levels of anti-CCP antibody or those of total cFn was not statistically significant. This could be explained as a result of the production mechanisms of the anti-CCP antibody and cFn being unlinked to cFn citrullination. Based on the association between PADI4 polymorphism and RA, we previously hypothesized that increased PADI4 mRNA stability in RA leads to higher PADI4 production following the increased amounts of citrullinated peptides that serve as autoantigens, thus breaking the tolerance threshold [1]. However, no significant correlation was observed between the levels of Cit-cFn and those of anti-CCP antibodies in RASF in the present study. Although the reason for this discrepancy is unknown, one possibility is that the amount of autoantigens, which are necessary to elicit and maintain autoantibody production, may differ between susceptible individuals. Another possibility is the small number of RA patients tested. Therefore, further study is warranted to examine a greater number of RASF samples, including those from anti-CCP-negative RA patients, to explore the role of Cit-cFn in the pathogenesis of RA. In conclusion, we demonstrated the presence of the citrullinated form of cFn in RASF. To better understand the pathogenesis of RA, further studies are necessary to elucidate the location and responsible PADI subtype involved in cFn citrullination and to investigate its arthrogenic properties in appropriate animal arthritis models.
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Acknowledgement
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This work was supported in part by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) Number 22591086. The authors thank Professor Masato Odawara, MD, chairman of Internal Medicine III, Tokyo Medical University for his continuous encouragement and guidance.
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Conflict of interest None. 19.
References 1. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, et al. Functional haplotypes of PADI4, encoding citrullinating enzyme
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peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet. 2003;34(4):395–402. Suwannalai P, Trouw LA, Toes RE, Huizinga TW. Anti-citrullinated protein antibodies (ACPA) in early rheumatoid arthritis. Mod Rheumatol. 2012;22(1):15–20. Masson-Bessiere C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T, Serre G. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin. J Immunol. 2001;166(6):4177–84. Tilleman K, Van Steendam K, Cantaert T, De Keyser F, Elewaut D, Deforce D. Synovial detection and autoantibody reactivity of processed citrullinated isoforms of vimentin in inflammatory arthritides. Rheumatology (Oxford). 2008;47(5):597–604. Chang X, Yamada R, Suzuki A, Kochi Y, Sawada T, Yamamoto K. Citrullination of fibronectin in rheumatoid arthritis synovial tissue. Rheumatology (Oxford). 2005;44(11):1374–82. Takizawa Y, Suzuki A, Sawada T, Ohsaka M, Inoue T, Yamada R, Yamamoto K. Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids. Ann Rheum Dis. 2006;65(8):1013–20. Tabushi Y, Nakanishi T, Takeuchi T, Nakajima M, Ueda K, Kotani T, et al. Detection of citrullinated proteins in synovial fluids derived from patients with rheumatoid arthritis by proteomics-based analysis. Ann Clin Biochem. 2008;45(Pt 4): 413–7. van Beers JJ, Willemze A, Stammen-Vogelzangs J, Drijfhout JW, Toes RE, Pruijn GJ. Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigenDRB1 shared epitope alleles. Arthritis Res Ther. 2012;14(1):R35. van Beers JJ, Schwarte CM, Stammen-Vogelzangs J, Oosterink E, Božič B, Pruijn GJ. The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and beta-actin. Arthritis Rheum. 2013;65(1):69–80. Skriner K, Adolph K, Jungblut PR, Burmester GR. Association of citrullinated proteins with synovial exosomes. Arthritis Rheum. 2006;54(12):3809–14. Kinloch A, Lundberg K, Wait R, Wegner N, Lim NH, Zendman AJ, et al. Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis. Arthritis Rheum. 2008;58(8):2287–95. To WS, Midwood KS. Plasma and cellular fibronectin: distinct and independent functions during tissue repair. Fibrogenesis Tissue Repair. 2011;4:21. Peters JH, Maunder RJ, Woolf AD, Cochrane CG, Ginsberg MH. Elevated plasma levels of ED1⫹ (“cellular”) fibronectin in patients with vascular injury. J Lab Clin Med. 1989;113(5):586–97. Walle TK, Vartio T, Helve T, Virtanen I, Kurki P. Cellular fibronectin in rheumatoid synovium and synovial fluid: a possible factor contributing to lymphocytic infiltration. Scand J Immunol. 1990;31(4):535–40. Shiozawa K, Hino K, Shiozawa S. Alternatively spliced EDAcontaining fibronectin in synovial fluid as a predictor of rheumatoid joint destruction. Rheumatology (Oxford). 2001;40(7):739–42. Yan X, Yin L, Wang Y, Zhao Y, Chang X. The low binding affinity of ADAMTS4 for citrullinated fibronectin may contribute to the destruction of joint cartilage in rheumatoid arthritis. Clin Exp Rheumatol. 2012;31(2):201–206. Yoshihara Y, Obata K, Fujimoto N, Yamashita K, Hayakawa T, Shimmei M. Increased levels of stromelysin-1 and tissue inhibitor of metalloproteinases-1 in sera from patients with rheumatoid arthritis. Arthritis Rheum. 1995;38(7):969–75. Fan L, Wang Q, Liu R, Zong M, He D, Zhang H, et al. Citrullinated fibronectin inhibits apoptosis and promotes the secretion of proinflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis. Arthritis Res Ther. 2012;14(6):R266. Shelef MA, Bennin DA, Mosher DF, Huttenlocher A. Citrullination of fibronectin modulates synovial fibroblast behavior. Arthritis Res Ther. 2012;14(6):R240. van Venrooij WJ, Pruijn GJ. An important step towards completing the rheumatoid arthritis cycle. Arthritis Res Ther. 2008;10(5):117.
ORIGINAL ARTICLE
Identification of citrullinated cellular fibronectin in synovial fluid from patients with rheumatoid arthritis
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Eri Kimura1, Takeyuki Kanzaki2, Koichiro Tahara1, Haeru Hayashi1, Shiori Hashimoto3, Akari Suzuki4, Ryo Yamada5, Kazuhiko Yamamoto6, and Tetsuji Sawada1 1Department of Rheumatology, Tokyo Medical University Hospital, Tokyo, Japan, 2Department of Internal Medicine, Yamanashi Prefectural Central Hospital, Kofu, Japan, 3Department of Neurology, Tokyo Women’s Medical University, Tokyo, Japan, 4Laboratory for Autoimmune Diseases, Center for Genomic Medicine, The Institute of Physical and Chemical Research (RIKEN), Wako, Japan, 5Center for Genomic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan, and 6Department of Allergy and Rheumatology, University of Tokyo School of Medicine, Tokyo, Japan
Abstract
Keywords
Objectives. Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients. Methods. Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification. Results. Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF. Conclusions. Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.
Citrullinated antigen, Cellular fibronectin, Rheumatoid arthritis, Synovial fluid
Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the chronic inflammation of synovial tissue, resulting in bone erosion and joint destruction. Using linkage disequilibrium and single nucleotide polymorphism analysis, we previously identified peptidylarginine deiminase (PADI) Type 4 as an RA-susceptible gene [1]. This is one of five PADI genes that encode enzymes which convert peptide arginine residues to citrulline residues. Furthermore, peptidyl citrulline is targeted by anti-citrullinated peptide antibodies that are highly specific to RA [2]. Citrullination plays an important role in the pathogenesis of RA. Several citrullinated peptides, such as fibrin(ogen) [3], vimentin [4] and cellular fibronectin (cFn) [5], were reported to be present in the synovial tissue of RA patients. Moreover, some citrullinated peptides were retrieved from the sera of RA patients, indicating that they may be involved in the pathogenesis of RA through their autoantigen activity. Several reports have described the
Correspondence to: Tetsuji Sawada, MD, PhD, Third Department of Internal Medicine, Department of Rheumatology, Tokyo Medical University, 7-6-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan. E-mail: [email protected]
History Received 18 July 2013 Accepted 25 December 2013 Published online 4 February 2014
citrullinated peptides contained in rheumatoid arthritis synovial fluid (RASF), including fibrinogen [6], vimentin [7], fibronectin [7–9], exosome-associated peptides and Sp-α [10], α-enolase [11], apolipoprotein E, myeloid nuclear differentiation antigen and β-actin [9]. However, those that are pathognomonic for RA remain to be elucidated. Fibronectins are glycoproteins found in plasma, extracellular matrix and on cell surfaces that play an important role in cell– cell and cell–matrix interactions [12]. Alternative splicing generates different isoforms of plasma and cFn. Plasma fibronectin is secreted by hepatocytes and distributed in the plasma, while cFn is produced locally by various cell types, such as endothelial cells and fibroblasts, and is characterized by the presence of alternatively spliced exons, including extra domains A and B (EDA and EDB). Although cFn is water-insoluble and deposited as an extracellular matrix protein, it constitutes less than 1–2% of the total plasma fibronectin [13]. cFn has been detected in inflamed synovium as well as in synovial fluid obtained from active RA patients and is a possible indicator of synovial inflammation in RA [14,15]. We previously demonstrated the presence of the citrullinated form of total fibronectin in plasma and that of cFn in synovial tissues in RA, and we showed that both cellular and plasma fibronectin citrullinated in vitro by PADI were targeted by RA serum,
Citrullinated cellular fibronectin in RASF 767
Mod Rheumatol Downloaded from informahealthcare.com by Korea University on 01/05/15 For personal use only.
DOI 10.3109/14397595.2013.879413
demonstrating an altered capacity to bind to their receptors and growth factors [5]. The presence of citrullinated forms of fibronectin in synovial fluid from RA patients was also reported by others based on a proteomics-based analysis [7–9]. In addition, Chang et al. showed that citrullination of fibronectin reduced the binding and inhibition of the aggrecan-degrading enzyme ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) by a 40-kDa fibronectin fragment, which suggests that citrullination of fibronectin might contribute to cartilage destruction in RA [16]. These observations suggest that cFn is involved in the pathogenesis of RA, especially in its citrullinated form. In the present study, we investigated whether the citrullinated form of cFn could be detected in the plasma and synovial fluid of patients with RA and osteoarthritis by western blotting and enzyme-linked immunosorbent assay (ELISA). We also discuss the role of cFn citrullination in the pathogenesis of RA.
After washing, reagents A and B were applied and incubated overnight at 37°C. After further washing, the wells were blocked with 5% bovine serum albumin for 30 min, then incubated with anti-modified citrulline antibody diluted at 1:2,500 for 3 h at 37°C, and with goat F(ab’)2 anti-rabbit immunoglobulin-HRP conjugate diluted at 1:5,000 for 2 h at 37°C. After a final wash, the 3,3′,5,5′tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD) was applied and the color development was stopped with TMB stop solution. Optical density values at a wavelength of 450 nm (OD450) were then measured. Anti-CCP antibodies and matrix metalloproteinase-3 (MMP-3) in SFs were measured using the second-generation anti-CCP ELISA kit (DIASTAT Anti-CCP; Medical & Biological Laboratories (MBL), Nagoya, Japan) and SensoLyte MMP-3 ELISA kit (Fremont, CA), respectively, according to the manufacturers’ instructions.
Materials and methods
Statistical analysis
Samples
A comparison of mean values between the two groups was made using the Mann–Whitney U test. Differences were considered significant at p ⬍ 0.05.
Twenty-five RASF and seven osteoarthritis synovial fluid (OASF) samples were collected in heparinized tubes at the time of therapeutic arthrocentesis, centrifuged at 450 ⫻ g for 10 min to remove debris, and the supernatants were collected. Plasma samples were obtained from 12 patients with RA and 12 healthy subjects after informed consent was obtained. This study was approved by the appropriate ethics committee. Immunoprecipitation-western blotting Immunoprecipitation (IP) of cFn was performed using an agaroseconjugated anti-cFn monoclonal antibody (IgG1) (sc-8422 AC, Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes extracellular and matrix fibronectin and is non-cross-reactive with soluble, dimeric plasma fibronectin. Synovial fluid (SF) and plasma diluted at 1:10 were incubated with agarose-conjugated anti-cFn monoclonal antibody (mAb) overnight at 4°C. After thorough washing, purified cFn was eluted with 1 ⫻ NuPage LDS sample reducing buffer (Life Technologies, Foster City, CA) at 70°C for 10 min and was subsequently separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transblotted onto polyvinylidene fluoride membranes. To detect cFn, blots were incubated in blocking buffer (phosphate-buffered saline (PBS) containing 5% nonfat dried milk and 0.1% NP-40) for 1 h at room temperature, and subsequently with anti-cFn mAb for 1–3 h. After washing the blot, bound antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by chemiluminescence. For the detection of citrullinated proteins using the anti-citrulline (modified) detection kit (Merck Millipore, Darmstadt, Germany), the blot was chemically treated before immunostaining according to the manufacturer’s instructions.
Results ELISA detection of Cit-cFn in RASF Figure 1A shows that the levels of total cFn in SFs were significantly higher in RASF than in OASF. Levels of Cit-cFn were then measured by ELISA and quantitated by an anti-modified citrulline antibody. These levels were significantly higher in RASF than in OASF (Figure 1B), while cFn citrullination was not detected by ELISA in plasma or serum from RA patients (data not shown). Detection of Cit-cFn in RASF by IP-western blotting IP/western blotting was subsequently performed to demonstrate the presence of the citrullinated form of cFn in RASF. Representative data are shown in Figure 2, in which IP using anti-cFn antibody was carried out on RASF and OASF samples. After SDS-PAGE, anti-cFn antibody immunoprecipitates were shown to contain cFn with a molecular weight of approximately 200 kDa (Figure 2A, upper). The corresponding bands were stained with anti-modified citrulline antibody after chemical modification (Figure 2A, lower). Although the use of IP/western blotting was not intended to quantitate the Cit-cFn in synovial fluid, it appears that RASF contained more Cit-cFn than OASF. Furthermore, while cFn was detected in plasma samples, no citrullinated cFn was detected by IP/western blotting (Figure 2B).
ELISA Detection of citrullination associated with cFn (Cit-cFn) was performed by sandwich ELISA using a human cFn-specific mAb adsorbed on a microplate in combination with an anti-modified citrulline antibody (rabbit polyclonal IgG) according to the manufacturer’s instructions (cFn ELISA kit; Biohit Oyj, Helsinki, Finland). First, 100 μL of RASFs and control SFs diluted 1:25 with a dilution buffer were added in duplicate to wells pre-coated with anti-human cFn mAb and incubated for 1 h at 37°C. The wells were washed, then incubated with 1% glutaraldehyde/PBS for 1 h, and subsequently with 0.2 M Tris-Cl, pH 7.8 for 30 min.
Figure 1. ELISA detection of cFn and Cit-cFn in RASF and OASF samples. (A) Levels of total (citrullinated and non-citrullinated) cFn were determined by sandwich ELISA. (B) Levels of Cit-cFn were determined by ELISA using a combination of anti-cFn mAb and anti-modified citrulline (AMC) antibody with chemical modifications. Data are expressed as optical density values measured at 450 nm (OD450).
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Figure 4. Relationship between MMP-3 and levels of Cit-cFn and total cFn in RASF samples. (A) Correlation between MMP-3 and Cit-cFn (expressed as OD450). (B) Correlation between MMP-3 and total cFn. r, correlation coefficient.
Figure 2. Citrullinated form of cFn in synovial fluid and plasma. Western blot of anti-cFn mAb immunoprecipitates stained with anti-cFn antibody (upper) and AMC antibody after chemical modification (lower). (A) Representative IP/western blotting data of RASF (lanes 1–5) and OASF (lanes 6–8). The values of Cit-cFn in each sample (as detected by ELISA; OD450) were as follows. RASF (lanes 1–5): 0.20, 0.07, 0, 0.04, 0.07; OASF (lanes 6–8): 0, 0. 0.01. (B) Representative IP/western blotting data of plasma from RA patients (lanes 1–5) and healthy controls (lanes 6–8). RA synovial fluid was used as a positive control.
Relationship of cFn with cFn and anti-CCP antibody positivity Anti-CCP antibody expression was detected in 24 out of 25 RASF samples. No significant correlation was observed in RASF between cFn citrullination (as determined by ELISA) and the anti-CCP antibody. However, there was a slight, but non-significant, positive correlation between cFn citrullination and total cFn (r ⫽ 0.25, p ⫽ 0.11) (Figure 3). Relationship between cFn citrullination with MMP-3 A significant positive correlation was observed between the levels of MMP-3 and cFn citrullination in RASF (Figure 4). On the other hand, a positive correlation was noted between MMP-3 and total cFn in RASF, although this did not reach significance.
Discussion Although cFn is located on the surface of many different cell types where it forms insoluble fibrillar matrices, it is also secreted as a soluble molecule. We previously demonstrated that cFn in synovial tissues was citrullinated in RA [5]. Fibronectin citrullination in RASF has been demonstrated by van Beers et al. [8], although their mass spectrometry failed to identify EDA/EDB-containing cFn, presumably because of a limited sample size or technical limitations, and by Tabushi et al. [7], who used proteomics-based
Figure 3. Relationship between citrullinated (Cit)-cFn and levels of anti-CCP antibody and total cFn in RASF samples. (A) Correlation between Cit-cFn (expressed as OD450) and levels of anti-CCP antibody. (B) Correlation between Cit-cFn and total cFn. r, correlation coefficient.
analysis but no appropriate control samples, so the significance of their results is unclear. In the present study, we clearly demonstrated for the first time the presence of citrullinated cFn in synovial fluid, but not in the plasma or serum, of RA patients by ELISA and IP/western blotting. We also showed that the degree of cFn citrullination was significantly higher in RASF than in OASF and correlated significantly with MMP-3 levels in RASF. It should be noted, however, that sandwich ELISA data do not preclude the possibility that cFn forms a complex with other citrullinated peptide(s) in RASF, which is responsible for the peptidyl-citrulline ELISA signal. For this reason, we performed IP/western blotting to demonstrate the presence of the citrullinated form of cFn observed in RASF. It should also be noted that the multiple bands of cFn in IP/western blotting (Figure 2A) are consistent with the results of a previous report demonstrating a large range of fibronectin polypeptide lengths in RASF samples caused by the presence of different isoforms [8]. We previously demonstrated the presence of the citrullinated form of total fibronectin in the plasma of RA patients [5], which is presumed to contain mainly citrullinated plasma fibronectin since Cit-cFn was not detected in the RA plasma in the present study. It should be noted, however, that the amount of cFn contained in plasma is small, so we cannot preclude the possibility that CitcFn may be detected in the plasma of RA patients when a higher sensitivity assay is used. Since no citrullinated cFn was detected in the plasma of RA patients, it is conceivable that citrullination occurred within the affected joint. However, this is unclear, and the PADI isoforms involved remain unknown. Although PADI2 and PADI4 are usually localized in the cytoplasm and nucleus, respectively, both enzymes have previously been detected in RASF [11]. As the extracellular calcium concentration is sufficiently high for PADI to function, it is possible that secreted cFn is citrullinated by extracellular PADI. Another possibility is that activated cells, such as synoviocytes and endothelial cells, citrullinate intracellular cFn and subsequently express citrullinated cFn on their cell surfaces. MMP-3, which is produced within the joints, is considered to play a role in cartilage destruction, since it can degrade a wide range of substrates, including the main components of joint cartilage such as aggrecan and Type-II collagen. In RA, overexpression of MMP-3 has been demonstrated in the rheumatoid synovium, and serum levels of MMP-3, which reflect the severity of rheumatoid synovitis, can be used as a disease marker of RA [17]. In the present study, we demonstrated that Cit-cFn, as measured by ELISA, correlates significantly with MMP-3 in RASF, suggesting that cFn citrullination is relevant to the pathophysiology of RA. Regarding the pathogenetic role of citrullinated cFn in RA, we previously demonstrated that, following citrullination by PADI in vitro, fibronectin increased its binding activity to vascular
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DOI 10.3109/14397595.2013.879413
endothelial growth factor but decreased that to integrin β1 as well as its ability to stimulate apoptosis. This suggests that citrullinated cFn alters the immunological micro-environment of the synovium, leading to the perpetuation of inflammation [5]. Fan et al. previously found that citrullinated fibronectin could inhibit the apoptosis of synoviocytes, which may promote their survival and the secretion of proinflammatory cytokines from rheumatoid synoviocytes in a pathogenic role in RA [18]. Shelef et al. recently demonstrated that citrullinated fibronectin impaired the adhesion and migration of synoviocytes, which might be associated with the metastasis of synovial cells and the spread of arthritis [19]. Another possibility is that citrullinated cFn is involved in the pathogenesis of RA by acting as an autoantigen in the RA cycle, as proposed by van Venrooij et al. [20]. Thus, the expression of CitcFn within the joint, as demonstrated in the present study, together with the reported presence of the anti-citrullinated fibronectin antibody [5,8], implies that Cit-cFn is a candidate autoantigen that drives the chronic inflammatory response through the formation of immune complexes which lead to joint destruction in RA. In the present study, the relationship between cFn citrullination and levels of anti-CCP antibody or those of total cFn was not statistically significant. This could be explained as a result of the production mechanisms of the anti-CCP antibody and cFn being unlinked to cFn citrullination. Based on the association between PADI4 polymorphism and RA, we previously hypothesized that increased PADI4 mRNA stability in RA leads to higher PADI4 production following the increased amounts of citrullinated peptides that serve as autoantigens, thus breaking the tolerance threshold [1]. However, no significant correlation was observed between the levels of Cit-cFn and those of anti-CCP antibodies in RASF in the present study. Although the reason for this discrepancy is unknown, one possibility is that the amount of autoantigens, which are necessary to elicit and maintain autoantibody production, may differ between susceptible individuals. Another possibility is the small number of RA patients tested. Therefore, further study is warranted to examine a greater number of RASF samples, including those from anti-CCP-negative RA patients, to explore the role of Cit-cFn in the pathogenesis of RA. In conclusion, we demonstrated the presence of the citrullinated form of cFn in RASF. To better understand the pathogenesis of RA, further studies are necessary to elucidate the location and responsible PADI subtype involved in cFn citrullination and to investigate its arthrogenic properties in appropriate animal arthritis models.
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Acknowledgement
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This work was supported in part by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) Number 22591086. The authors thank Professor Masato Odawara, MD, chairman of Internal Medicine III, Tokyo Medical University for his continuous encouragement and guidance.
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Conflict of interest None. 19.
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