The correlation of syndecan-4 with disease activity and serological characteristics of rheumatoid arthritis

[Objectives] To investigate the expression of syndecan-4 in serum, synovial uid (SF) and synovium in rheumatoid arthritis (RA) by comparing with osteoarthritis (OA) patients, and to analyze the correlation of syndecan-4 with disease activity of RA . [Methods] Syndecan-4 in sera of 60 RA patients, 20 OA patients, 20 healthy controls, and in paired SF of 23 RA patients were tested by enzyme linked immunosorbant assay (ELISA). The expressions of syndecan-4 in synovium of 5 RA patients and 5 OA patients were detected by immunohistochemistry. The expressions of syndecan-4 of cultured synovial broblasts from RA and OA patients were detected by immunouorescence. The correlation between serum syndecan-4 concentration and disease activity were analyzed in 60 RA patients.


Introduction
Rheumatoid arthritis (RA), with morbidity of 0.36% in China, is a chronic systemic autoimmune disease characterized as articular synovitis and bone erosion that ultimately results in joint destruction. The aberrant B-cells activation, synovial broblasts proliferation and cartilage degradation are central features of RA. However, the pathogenesis of RA has not been clari ed.
Syndecan-4, one of the members of transmembrane heparan sulphate proteoglycans (HSPGs), is the receptor of various cytokines and chemokines that can regulate a series of signal transductions [1]. It could be expressed in several cells under in ammation and involved in regulation of in ammation, angiogenesis, focal adhesion and cell migration [2]. Syndecan-4 ectodomains are constitutively shed and replaced under physiological conditions to maintain balance. However, in response to certain stimuli, such as extracellular cytokines, metalloprotease (MMP) and oxidative stress, syndecan-4 shedding increases dramatically, and the soluble syndecan-4 can promote in ammation and the synthesis of new syndecan-4 [3]. The cleavage and shedding of syndecans, which modulates the in ammatory response, was reported in lungs and heart [4,5].
The role of syndecan-4 in in ammatory arthritis has been proved in animal models. Syndecan-4 de cient mice were resistant to the induction of collagen-induced arthritis (CIA) due to loss of the migration ability of B cells [6]. The tumour necrosis factor transgenic (hTNFtg) mouse model demonstrated that syndecan-4 was prominently involved in synovial broblasts activation and its attachment to the cartilage in the early stage of arthritis [7]. Syndecan-4 de cient mice exhibited signi cantly reduced cartilage destruction compared with hTNFtg mice carrying syndecan-4 [7]. Intra-articular injection of anti-syndecan-4 antibodies into wild-type mice prevented cartilage damage to a similar extent as seen in syndecan-4de cient mice [8].
Several members of HSPG family, including syndecan-1, 2, 3, 4 and glypican-1, can be highly expressed by the broblast-like synoviocyte (FLS) of RA, but only syndecan-4 can interact with receptor protein tyrosine phosphatase sigma (RPTPσ), a highly-expressed phosphatase in arthritic FLS which can regulate the RA FLS migration [9]. Syndecan-4 could up-regulate the levels of MMP with thrombospondin motifs (ADAMTS)-5 to promote the in ammation and cartilage degeneration [10]. A previous study reported little staining for syndecan-4 in synovium of early RA [12]. A most recent study demonstrated that highly expressed syndecan-4 was associated with decreased apoptosis in RA-FLS [11].
However, the levels of syndecan-4 in sera and synovial uid (SF) of RA has not been reported. The expression of syndencan-4 in synovial tissue of RA is still controversial. The correlation of syndencan-4 with clinical features of RA, such as disease activity and the autoantibody, still remain unclear. The aim of the current study was to pro le the expression of syndecan-4 in sera, SF and synovium in RA by comparing with OA patients and healthy controls, and analyze the correlation of serum syndecan-4 concentration with RA disease activity.

Patients and samples
The syndecan-4 concentrations in sera were tested in 60 RA patients, and 20 OA patients and 20 healthy controls by enzyme linked immunosorbant assay (ELISA). The OA patients and healthy controls were matched with the RA patients by age and gender. The correlation of syndecan-4 concentration in sera and disease activity of RA patients were analyzed in the 60 RA patients. 23 RA patients had paired sera and SF samples. The sera and SF samples of one individual patient were collected at the same time point. Syndecan-4 concentrations in SF of the 23 RA patients and another 23 OA patients were also tested by ELISA. The syndecan-4 concentrations in SF of RA and OA patients were compared with the syndecan-4 concentrations in sera, respectively.
Expressions of syndecan-4 in synovial tissues of 5 RA patients and 5 OA patients were tested by immunohistochemistry. All the synovial tissues were derived from patients undergoing knee replacement. The RA patients and OA patients were also matched by age and gender.
All the above RA patients ful lled the 2010 American College of Rheumatology/The European League Against Rheumatism (ACR/EULAR) classi cation criteria with various disease activity [from remission to high disease activity (HDA)]. All the enrolled OA patients ful lled the OA classi cation criteria [13,14]. Patients with malignancies and other in ammatory diseases, including other in ammatory arthritis, connective tissue diseases and infections were excluded.

Syndecan-4 concentrations in sera and synovial uid
Blood and SF were collected into tubes free of anticoagulants, centrifuged 3000 rpm for 10 min to remove cellular debris and stored at -80°C until use. Syndecan-4 concentrations in sera and SF were measured by the human syndecan-4 ELISA kit (R&D systems, the USA), following the manufacturer's protocols. Goat anti human syndecan-4 capture antibody was diluted to a terminal concentration of 0.8 ug/mL using PBS, coated onto the wells of a polystyrene microtiter plate and incubated at room temperature overnight. Test sera without dilution were added to the microwells and incubated at room temperature for 2 hours. After washed for 3 times, a detective antibody with a terminal concentration of 100 ng/mL was then added and incubated at room temperature for 2 hours. Streptavidin-HRP was then added after washed for 3 times. After incubation for 20 minutes, substrate solution was added to the microwells. The reaction was terminated by the addition of stop solution and absorbance was measured at 450 nm and 540 nm. Finally, sample concentration of syndecan-4 was determined by extrapolation in a standard curve prepared from seven geometric syndecan-4 standards.

Immunohistochemistry
The protocol of previous study was followed [15] with minor modi cations by anti-syndecan-4 antibodies.
Brie y, 10 mm thick serial cryostat sections of synovia were dried for 1 hour and stored at liquid nitrogen (-196℃) until required. Prior to immunohistochemical analysis, slides were left to equilibrate to room temperature for 30 minutes, xed in acetone (4℃) for 10 minutes, air dried, and then sections were rehydrated in phosphate buffered saline (PBS) for 5 minutes. All primary antibodies were used at 5 mg/ml since initial experiments showed that this concentration gave the optimum speci c staining over the range of 2.5-10 mg/ml. Antibody binding was detected using DAB staining kit (Vector Labs, Burlingame, California, USA). Sections were counterstained with Mayer Haematoxylin and mounted. In control experiments, isotype matched control mouse Ig or rabbit Ig were added instead of primary antibodies and stained with the DAB kit as above.
Synovial broblast culture Synovial tissues were obtained from RA and OA patients during joint replacement surgery of knees. Tissues were minced and treated with 4 mg/ml type I collagenase (Gibco, USA) in serum free Dulbecco's modi ed Eagle's medium (DMEM; Gibco, USA) at 37 °C for 1 h. Primary cultured synovial cell lines were established and maintained in DMEM including 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA), 1% penicillin-streptomycin (100 U/ml; Gibco, USA), and 1% L-glutamine (2 mM; Gibco, USA), in a humidi ed incubator at 37 °C in the presence of 5% CO2. Harvested cells were continuously cultured to obtain synovial broblasts, and cells of passages 3-6 were used in the next experiments.

Statistical analysis
All statistical analyses were performed using SPSS 20.0 (IBM Company, the USA) and all charts were performed by GraphPad Prism 5.0 (CA Company, the USA). The results of immunohistochemistry were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics Company, the USA). Quantitative variables were expressed as mean ± SD or median (min-max) for normal distribution or non normal distribution data, respectively. Statistical analysis was performed by the χ 2 test (frequencies), nonparametric Mann-Whitney U test (non normal distribution data) and Student's t test (normal distribution data), as appropriate. Correlation analysis was performed by Spearman correlation analysis. P value less than 0.05 was considered signi cant.

Results
Demographic and clinical features of RA patients 60 RA patients as well as 20 age and gender matched OA patients and 20 healthy controls were enrolled in the study. Among 60 RA patietns, there were 12 (20%) males and 48 (80%) females with mean age of 54.8±11.6 years and median disease duration of 6.3 (0.5-34) years. 37 (61.7%) of them were rheumatoid factor (RF) positive and 53 (88.3%) were anti-cyclic citrullinated protein (anti-CCP) positive (Table 1). All the 60 patients were distributed in four different disease activity groups, including remission, low disease activity (LDA), moderate disease activity (MDA) and high disease activity (HDA), evaluated by disease activity score based on 28 joint count-C reactive protein (DAS28-CRP), DAS28-erythrocyte sedimentation rate (DAS28-ESR), simpli ed disease activity index (SDAI) and clinical disease activity index (CDAI).
The comparison of syndecan-4 in serum, synovial uid and synovial tissue among RA patients, OA patients and healthy controls The syndecan-4 concentrations in sera of 60 RA patients, 20 OA patients and 20 healthy controls were 509.3 (345.2-769.8) pg/mL, 363.6±137.6 pg/mL and 204.4±59.1pg/mL, respectively. The serum syndecan-4 concentration was signi cantly higher in RA patients than in OA patients and healthy controls (p<0.001, p<0.001, respectively). It was also higher in OA patients than in healthy controls (p<0.001). (Figure 1) In the 23 paired serum and SF samples of RA patients, the syndecan-4 concentrations was signi cantly lower in SF than in sera (234.3±106.3 pg/mL vs 85.3±61.2 pg/mL, p<0.001).( Figure S1) The syndecan-4 concentrations was also signi cantly lower in SF than in sera of OA patients [363.6±137.6 pg/mL vs 51.0 (27.2-163.6) pg/mL, p<0.001], although the sera and SF samples were not paired.
No signi cant difference of syndecan-4 concentrations in SF was found between the RA and OA patients (p=0.733), although it was slightly higher in RA patients.
Syndecan-4 expression was detected by immunohistochemistry in the lining and sublining layers of all the 5 RA and 5 OA patents ( Figure 2). The mean area, mean diameter, mean density and mean integrated optical density (IOD) were comparable between RA and OA patients (Table S1).
Immuno uorescence demonstrated that Syndecan-4 was positively stained in cultured synovial broblasts from both of RA and OA patients. The uorescence intensity was similar between RA and OA patients. (Figure S2 Then we de ned patients with at least one of the above two antibodies positive as "sera positive", and there were 53 sera positive and 7 sera negative patients. The result was the same as that when strati ed by anti-CCP antibody. The correlation between Syndecan-4 concentration with disease activity of RA patients Among the 60 RA patients, the syndecan-4 concentration in sera was positively correlated with TJC, SJC, PGA, EGA, and the disease activity scores, including DAS28-ESR, DAS28-CRP, SDAI and CDAI ( Table 2). The correlation coe cents (r value) were mild to moderate ( Table 2). Based on any of these disease activity scores, the patients in HDA had the highest serum syndecan-4 concentration, compared with those in remission, LDA or MDA (Table 3, Figure 3) Among the 23 RA patients, the syndecan-4 concentration in SF was positively correlated with PGA, EGA, CRP, DAS28-ESR, DAS28-CRP and SDAI (Table S2). All of the correlation coe cents (r value) were higher than 0.5.

Discussion
In the current study, we found that syndecan-4 concentration in sera was positively related with the disease activity of RA. There were plenty of evidence proved the correlation between syndecan-4 and in ammation. The ectodomain of syndecan-4 could shed from the membrane under oxidative stress and in ammation [17,18]. Syndecan-4 can be up-regulated by TNFα, IL-1β and lipopolysaccharide (LPS) through a functional nuclear factor (NF)-κB site in its promoter [19]. TNFα can promote the shedding and decrease the attenuation of syndecan-4, both of which can amplify the effect of syndecan-4 [20]. Sheded heparan sulfate-substituted syndecan-4 ectodomains in turn increased ICAM-1, VCAM1, TNFα and IL-1β expression and NF-κB activation which all participate in the progression of RA, suggesting positive feedback regulation of in ammatory pathways [5].
Tenascin-C was considered essential in RA as high levels of its large-splice variant has been found in RA SF [21]. An immunodominant peptide from citrullinated tenascin-C was identi ed as a major target of autoantibodies in RA [22]. Tenascin-C might regulate the pannus invasion into cartilage. Syndecan-4 was required for tenascin-C action. Inhibition of syndecan-4 suppressed tenascin-C activity and overexpression of syndecan-4 enhanced the effects of tenascin-C. Tenascin-C and syndecan-4 might work together to control broblast morphology and regulate matrix contraction [23]. This may one of the possible mechanisms by which Syndecan-4 participates in RA pathogenesis.
The exact pathways and mechanisms by which syndecan-4 contribute to in ammation and joint destruction in RA just began to emerge. A most recent published study demonstrated syndecan-4 silencing signi cantly suppressed the levels of reactive oxygen species (ROS), nitric oxide (NO) and in ammation and promoted the apoptosis of RA-FLSs. The apoptosis of RA-broblast-like synoviocytes (FLSs) regulated by syndecan-4 was mainly through the intrinsic pathway in which the P53, Caspase-3, B-cell leukemia/lymphoma 2 (Bcl2), and Bcl2-associated X protein (Bax) were involved. It provided evidence that Syndecan-4 mediated the in ammation and apoptosis of RA-FLSs by mediating the ROS and NO release [11].
We also found that the serum syndecan-4 level was signi cantly higher in RF positive RA patients than in RF negative ones. This might suggest that syndecan-4 participates in the activation of B cells and the formation of autoantibodies of RA. Endo T et al. found that syndecan-4 de cient mice had reduced numbers and migration function of B cells, causing de cient germinal center (GC) formation in draining lymph nodes, suggesting that syndecan-4 may contributed to the development of CIA by promoting GC formation [6]. Unfortunately, we did not nd difference between anti-CCP antibody positive and negative RA patients. This might be due to limited samples in the anti-CCP antibody negative group. B cells have been believed to play important roles in the pathogenesis of RA [24]. Syndecan-4 is one of the receptors of a proliferation inducing ligand (APRIL), which has been proved to be a critical signal for plasmablasts survival and an important molecular in the regulation of maturation and differentiation of B cells [25]. It role of APRIL on the pathogenesis of RA had been revealed [26]. Our previous study also demonstrated that the soluble APRIL in sera was higher in RA than in OA patients, higher in sero-positive RA patients than in sero-negative ones, and more in ltration of APRIL positive cells in RA synovium than in OA patients [27]. The interaction between APRIL and syndecan-4 may contribute to the process of RA. Further study is needed.
Interestingly, we found the syndecan-4 concentration in sera was higher in OA patients than in healthy controls. Syndecan-4 levels in both of SF and synovium were similar in RA and OA, suggesting striking similarities in the pathogenesis of these two diseases. They are both degenerative joint diseases characterized by progressive loss of cartilage matrix and cartilage clefts. Increased MMP-3, MMP-13 and decreased bone morphogenetic protein-5 (BMP-5) have been found in both of RA and OA [28,29].
Syndecan-4 was closely related to cartilage destruction. The de ciency of syndecan-4 in mice and intraarticular injections of syndecan-4 speci c antibodies into wild-type mice can both protect from proteoglycan loss and thereby prevent OA cartilage damage [8]. Syndecan-4 regulates ADAMTS-5 activation which is crucial for the breakdown of cartilage matrix during OA [8,10].
Syndecan-4 can be produced by a variety of different cells. In this study, it was observed that the concentration of syndecan-4 was signi cantly lower in SF than in sera either in RA or OA patients, suggesting that serum syndecan-4 is not derived from syndecan-4 produced in the joint. However, the syndecan-4 concentration in SF was also positively related with disease activity of RA. The correlation coe cents (r value) of syndecan-4 in SF with disease activity was even higher than that of syndecan-4 in sera with disease activity. It suggested that syndecan-4 played an important role in the in ammation of arthritis.
The correlations between syndecan-4 and serological feature and disease activity of RA suggested syndecan-4 may participate in the pathogenesis of RA. It provided preliminary evidence to support syndecan-4 as a potential target for the prevention and treatment of RA.

Conclusions
The serum syndecan-4 concentration is higher in RA patients than OA patients and healthy controls, and signi cantly higher in RF-positive RA patients than in RF-negative ones. Serum and SF syndecan-4 concentrations are both positively correlated with the disease activity of RA patients. It may suggest that syndecan-4 may participate in the pathogenesis of RA and provided a novel therapeutic target for RA.

Declarations
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