A disintegrin and metalloprotease-10 is correlated with disease activity and mediates monocyte migration and adhesion in rheumatoid arthritis

A disintegrin and metalloprotease-10 is correlated with disease activity and mediates monocyte migration and adhesion in rheumatoid arthritis TAKEO ISOZAKI, SHO ISHII, SHINICHIRO NISHIMI, AIRI NISHIMI, NAO OGURO, SHINYA SEKI, YOKO MIURA, YUSUKE MIWA, KOEI OH, YOICHIRO TOYOSHIMA, MASANORI NAKAMURA, KATSUNORI INAGAKI, and TSUYOSHI KASAMA TOKYO, JAPAN

A disintegrin and metalloproteases (ADAMs) are a family of proteins that have been reported to be involved in several inflammatory conditions. We examined the secretion of ADAM-10 in biological fluids from patients with rheumatoid arthritis (RA) and the role it plays in monocyte migration. ADAM-10 levels were measured using enzyme-linked immunosorbent assays and immunofluorescence. To examine the role of ADAM-10 in RA synovial fluids (SFs), we studied THP-1 (human acute monocyte leukemia cell line) and monocyte chemotaxis. To determine whether ADAM-10 plays a role in cell proliferation in the RA synovium, we assayed the proliferation of ADAM-10 small interfering RNA (siRNA)-transfected RA fibroblast-like synoviocytes (FLSs). The ADAM-10 level in RA serum was significantly higher than that in normal serum and was correlated with a disease activity score of 28. ADAM-10– depleted RA SFs showed a decrease in THP-1 and monocyte migratory activity compared with that of sham-depleted controls. ADAM-10 siRNA inhibited monocyte adhesion to RA FLSs. Finally, blocking ADAM-10 secretion in RA FLSs resulted in decreased production of fractalkine/CX3CL1 and vascular endothelial cell growth factor. These data indicate that ADAM-10 plays a role in monocyte migration in RA and suggest that targeting ADAM-10 may provide a method of decreasing inflammation and potentially treating other inflammatory diseases. (Translational Research 2015;166:244–253) Abbreviations: ADAMs ¼ A disintegrin and metalloproteases; DAS28 ¼ disease activity score of 28; FLS ¼ fibroblast-like synoviocyte; OA ¼ osteoarthritis; RA ¼ rheumatoid arthritis; SFs ¼ synovial fluids; STs ¼ synovial tissues

INTRODUCTION

From the Department of Medicine, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan; Department of Orthopedics, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan. Submitted for publication October 8, 2014; revision submitted February 24, 2015; accepted for publication February 26, 2015. Reprint requests: Takeo Isozaki, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2015.02.005

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heumatoid arthritis (RA) is characterized by synovial inflammation in multiple joints and irreversible joint destruction in the absence of treatment.1 The inflammatory cells involved in the pathogenesis of RA are synovial tissue (ST) macrophages, which produce several cytokines and chemokines.2,3 Determining the cytokine and chemokine networks in RA poses an important challenge for furthering our understanding of the pathogenesisof RA.4-7 The therapeutic management of RA has changed dramatically in recent decades. One of the most effective current therapies is designed to block tumor

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MATERIALS AND METHODS

AT A GLANCE COMMENTARY Isozaki T, et al. Background

A disintegrin and metalloprotease 10 (ADAM-10) has been reported to be the enzyme responsible for the release of a number of cytokine. ADAM-10 was expressed in rheumatoid arthritis (RA) synovial tissues and mediated angiogenesis. Translational Significance

We demonstrate the expression of ADAM-10 in RA serum and synovial fluids. ADAM-10 was significantly correlated with disease activity. Blocking expression of ADAM-10 in RA synovial fibroblasts reduced cell adhesion and proliferation, because of inhibition of adhesion and proinflammatory mediator production. These results indicate the importance of ADAM-10 in RA and suggest that targeting ADAM-10 may be important in combating RA.

necrosis factor a (TNF-a).8 However, despite the success of blocking TNF-a, not all patients with RA respond adequately to anti–TNF-a therapy.9-11 Trials using antibodies to other chemokines or chemokine inhibitors have been conducted in animal models of arthritis. Specific chemokine or chemokine receptor targeting may also be available in the near future, highlighting the possible importance of other molecules for RA treatment. A disintegrin and metalloproteases (ADAMs) are a family of proteases that liberate a variety of cell surface-expressed proteins.12,13 ADAM-10 has been reported to be responsible for the release of several chemokines, such as fractalkine/CX3CL1 and CXCL16.14,15 Yang et al16 reported the presence of ADAM-10 in human aortic smooth muscle cell supernatant. The researchers also demonstrated that overexpression of ADAM-10 in human aortic smooth muscle cells induced a significant stepwise increase in migration in a Boyden chamber assay. We previously showed that ADAM-10 was expressed in RA STs and mediated angiogenesis.17 However, the role of ADAM-10 in RA inflammation is not yet clear. Here, we demonstrate the secretion of ADAM10 in RA biological fluids and its role in inflammation and monocyte recruitment. We also show that blocking ADAM-10 in fibroblast-like synoviocytes (FLSs) reduced cell adhesion and cytokine production.

Patients. RA STs were obtained from patients undergoing arthroplasty or synovectomy. RA and osteoarthritis (OA) synovial fluids (SFs) were obtained from patients. All specimens were obtained with informed consent and collected following approval from the Showa University Institutional Review Board. Cell culture. Fresh STs were minced and digested in tissue enzyme digestion solution as described previously.18 The FLSs were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were seeded in 6-well plates (BD Biosciences, Bedford, Massachusetts) at a density of 1 3 105 cells per well and were maintained in complete medium. After overnight serum starvation, the cells were treated with TNF-a (R&D Systems, Minneapolis, Minnesota). THP-1 cells (human acute monocytic leukemia cell line) were purchased from the American Type Culture Collection (Manassas, Virginia). THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Enzyme-linked immunosorbent assay of ADAM-10, fractalkine/CX3CL1, and vascular endothelial growth factor. Enzyme-linked immunosorbent assays (ELISAs)

were performed as described previously.19 The level of ADAM-10 in serum and SFs was measured following the manufacturer’s protocol (My BioSource, San Diego, California). We used My BioSource ADAM-10 ELISA kit #MBS704324. The detection range was 7.8–500 pg/mL, and the minimum detectable dose of ADAM-10 was less than 1.95 pg/mL. Fractalkine/ CX3CL1 and vascular endothelial growth factor (VEGF) in RA FLS-conditioned medium were measured using an R&D Duo kit (R&D Systems). Immunofluorescence. To measure the secretion of ADAM-10 in RA ST synovial fibroblasts, mouse antihuman collagen-1, and rabbit anti–ADAM-10 were used as primary antibodies. RA FLSs were plated at a density of 20,000 per well in 8-well Labtek chamber slides. The next day, the cells were washed with phosphate-buffered saline (PBS) and were fixed. RA ST slides were fixed with cold acetone for 20 minutes and were washed with PBS. Then, the slides were blocked with 20% FBS and 5% donkey serum for 1 hour at 37 C. Mouse anti–collagen-1 antibody (1 mg/mL; Abcam #ab6308, Cambridge, Massachusetts) and rabbit anti-human ADAM-10 antibody (10 mg/mL; Abcam #ab1977) were used. Alexa Fluor 488–conjugated donkey anti-rabbit antibody and Alexa Fluor 555– conjugated donkey anti-mouse antibody were purchased from Life Technologies (Carlsbad, California). For nuclear staining, 40 ,6-diamidino-2-phenylindole was used. Images were taken at 3400 magnification.

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RNA extraction and quantitative polymerase chain reaction of RA FLSs. RNA extraction and quantitative po-

lymerase chain reaction (qPCR) were performed as previously described.17 Total RNA was isolated from human RA FLSs using RNeasy mini RNA isolation kits (Qiagen, Valencia, California) in accordance with the manufacturer’s protocol. Following isolation, the RNA was quantified and checked for purity using a spectrophotometer (Nanodrop Technologies, Wilmington, Delaware). Complementary DNA was prepared using a Reverse-IT MAX first-strand synthesis kit (Abgene, Rochester, New York) as per the manufacturer’s protocol. ADAM-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer pairs were purchased from Integrated DNA Technologies (Coralville, Iowa). The following primers were used: ADAM-10 forward, 50 -AATGGA TTGTGGCTCATTGGTGGG-30 ; ADAM-10 reverse, 50 -TGGAAGTGGTTTAGGAGGAGGCAA-30 ; GAPDH forward, 50 -GCTCACTGGCATGGCCTTCCG-30 ; and GAPDH reverse, 50 -GTGGGCCATGAGGTCCAC CAC-30 . ADAM-10 messenger RNA (mRNA) was measured by real-time qPCR with the use of SYBR Green/ROX master mix (SABiosciences, Frederick, Maryland) on an Mx3005P thermal cycler (Stratagene, Santa Clara, California). The ratio of each mRNA relative to the GAPDH mRNA was calculated using the DD threshold cycle method. All samples were run in duplicate and analyzed using Applied Biosystems software (Life Technologies). Neutralization of ADAM-10 in RA SFs. To determine the role of ADAM-10 in RA SFs, RA SFs were depleted using rabbit anti–ADAM-10. RA SFs (1:100 diluted with PBS) were preincubated either with rabbit antihuman ADAM-10 antibody (Abcam) or with an equivalent amount of a corresponding control antibody (nonspecific rabbit immunoglobulin G [IgG]) for 2 hours at 4 C. Samples were mixed with protein A/G agarose (Millipore, Billerica, Massachusetts) and rotated overnight at 4 C. Samples were centrifuged briefly to pellet the ADAM-10-antibody-protein A/G complex, and the ADAM-10–depleted SFs were collected. Isolation of peripheral blood monocytes. Peripheral blood monocytes were obtained from healthy individuals. The mononuclear cells were isolated by centrifugation on a Ficoll-Paque Plus (GE Healthcare Life Sciences, Piscataway, New Jersey) density gradient, after which the monocytes were separated by centrifugation on a density gradient (1.068 g/mL; Nycodenz, Nycomed AS Oslo, Norway). In vitro THP-1 and monocyte chemotaxis assay. To examine the bioactivity of ADAM-10 in RA SFs, we performed THP-1 and monocyte chemotaxis assays. Sham-depleted or ADAM-10–depleted RA SFs were

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added to the bottom wells of chambers, and a polycarbonate 5-mm membrane was placed over the reagents. THP-1 cells at 1 3 106 cells/mL or monocytes at 2.5 3 106 cells/mL were added to the top wells and incubated for 90 minutes. The membranes were fixed in methanol and stained with Protocol Hema 3 (Fisher Scientific, Pittsburgh, Pennsylvania). Each test group was assayed in quadruplicate. Three high-power (3400) fields were counted in each replicate well, and the results are expressed as cells per high-power fields. Transfection of RA FLSs with ADAM-10 small interfering RNA. RA FLSs were seeded in 6-well plates at a

density of 1 3 105 cells per well. Small interfering RNA (siRNA; 100 nM) against ADAM-10 or control siRNA was mixed with TransIT-TKO transfection reagent (Mirus, Madison, Wisconsin) according to the manufacturer’s instructions and overlaid on the cells. The cells were incubated with the siRNA-TransITTKO for 24 hours at 37 C. The ADAM-10 and control siRNAs were purchased from Life Technologies. Knockdown of ADAM-10 secretion was confirmed using Western blotting. Membranes were probed with rabbit anti-human ADAM-10 antibody (Abcam) and antiactin. In vitro cell adhesion assay. The adhesion of THP-1 cells to control siRNA-treated or ADAM-10 siRNAtreated RA FLSs grown to confluence in 96-well plates was examined. RA FLSs were serum-starved overnight. The next day, the cells were treated with TNF-a (10 ng/mL) for 24 hours. THP-1 cells were collected and labeled with Calcein AM fluorescent dye (5 mM; Life Technologies) for 30 minutes. After being washed twice, 1 3 105 THP-1 cells were added to each well and incubated for 30 minutes at room temperature. Nonadherent cells were washed away, and the fluorescence was measured using a Synergy HT fluorescence plate reader (BioTek Instruments). Cell surface ELISA for adhesion expression. Control siRNA-transfected or

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ADAM-10 siRNA-transfected RA FLSs (1 3 105/well) were seeded in 96-well plates. Confluent RA FLSs were serum-starved overnight before stimulation with TNFa (10 ng/mL) for 24 hours. The cells were fixed with 3.7% formalin in PBS for 30 minutes. Mouse antihuman antibodies specific for intercellular adhesion molecule 1 (ICAM-1, 10 mg/mL; R&D systems) or vascular cell adhesion molecule 1 (VCAM-1) were incubated for 1 hour. Subsequently, biotinylated anti-mouse antibody and streptavidin-horseradish peroxidase were added for 1 hour, and the concentration in the samples was measured at 450 nm after reaction with the tetramethylbenzidine substrate.

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Fig 2. ADAM-10 in RA SFs has monocyte chemotactic activity. (A) THP-1 migration was measured in a chemotaxis assay using sham-depleted or ADAM-10–depleted RA SFs. ADAM-10 depletion resulted in a 56 6 9% (n 5 5) reduction in RA SF-induced THP-1 migration. (B) Monocyte migration was measured in a chemotaxis assay using sham-depleted or ADAM-10–depleted RA SFs. ADAM-10 depletion resulted in a 53 6 7% (n 5 6) reduction in RA SF-induced monocyte migration (n 5 number of RA patients). ADAM, A disintegrin and metalloprotease; hpfs, high-power fields; RA, rheumatoid arthritis; SFs, synovial fluids.

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Fig 3. ADAM-10 is expressed in RA synovial fibroblasts. (A) The upper-left panel shows ST stained with rabbit anti–ADAM-10. The upper-middle panel shows ST stained with mouse anti–collagen-1. The upper-right panel shows the merging of the upper-left panel and the upper-middle panel. The lower-left panel shows ST stained

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Statistical analysis. The data were analyzed using Student’s t test assuming equal variances. The relationship between ADAM-10 in RA serum and a disease activity score of 28 (DAS28) was evaluated using Spearman’s rank correlation. Data are reported as the mean 6 standard error of the mean (SEM). P values less than 0.05 were considered statistically significant.

RESULTS Levels of ADAM-10 in RA serum and SFs. We examined the levels of ADAM-10 in RA biological fluids. We measured ADAM-10 in normal (NL) and RA serum using ELISA. We found that ADAM-10 was expressed in NL serum (n 5 34) and that its expression was significantly increased in RA serum (n 5 90) (85 6 33 and 450 6 44 pg/mL, respectively, Fig 1, A). We also measured the ADAM-10 levels in OA and RA SFs. We found that the ADAM-10 concentration in RA SFs (n 5 10) was significantly increased compared with that in OA SFs (n 5 7) (727 6 144 and 255 6 42 pg/mL, respectively, Fig 1, B). To determine whether ADAM-10 contributes to the development of inflammation in RA, we examined the relationship between ADAM-10 and DAS28. DAS28 is a quantitative measure of disease activity and measures patient global health (patient self-assessment), tender joint-counts and swollen joint-counts (up to 28), and the erythrocyte sedimentation rate. We found that the level of ADAM-10 in RA serum showed a weak but significant positive correlation with DAS28 (r 5 0.33, P , 0.05, n 5 90 patients, Fig 1, C). The level of ADAM-10 in RA serum also showed a very weak positive correlation with erythrocyte sedimentation rate (r 5 0.28, P , 0.05). These results suggest that the level of ADAM-10 in RA serum is weakly correlated with disease activity. Function of ADAM-10 in RA SFs. To demonstrate the function of ADAM-10 in RA SFs, SFs depleted of ADAM-10 were assayed for their ability to induce monocyte chemotaxis. Because RA SF was potently chemotactic for monocytes, we performed in vitro THP-1 and monocyte chemotaxis assays using shamor ADAM-10–depleted RA SFs. We found that

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ADAM-10–depleted RA SFs showed a 56 6 9% (n 5 5 patients) decrease in THP-1 migratory activity compared with that of sham-depleted controls (number of THP-1 cells that migrated 6 SEM; 7 6 1 and 17 6 3 cells migrated, respectively, P , 0.05, Fig 2, A). In addition, ADAM-10–depleted RA SFs showed a 53 6 7% (n 5 6 patients) decrease in monocyte chemotactic activity compared with that of sham-depleted controls (number of monocytes migrated 6 SEM; 19 6 3 and 43 6 6, respectively, P , 0.05, Fig 2, B). Expression of ADAM-10 in RA ST lining cells and RA FLS. To determine whether ADAM-10 was expressed

in RA ST lining cells and FLS, immunofluorescence was performed. ADAM-10– and collagen-1–positive cells can be observed in RA ST (Fig 3, A), indicating that ADAM-10 was expressed in RA synovial lining cells. We then examined FLS isolated from STs, and immunofluorescence was performed. We found that ADAM-10 was expressed in RA FLS (Fig 3, B). TNFa upregulated ADAM-10 mRNA in RA FLS at 1 hour, and the mRNA levels were increased at 4 hours (Fig 3, C). Blocking ADAM-10 in RA FLS reduces THP-1 adhesion. To further examine the function of ADAM-

10 in RA FLS, we used RA FLSs that were transfected with siRNA directed against ADAM-10. Specific knockdown of ADAM-10 was confirmed by Western blotting, and the ADAM-10 protein levels were decreased (Fig 4, A). To determine whether ADAM-10 mediates leukocyte adhesion to RA FLS, we performed in vitro adhesion assays. We found that the adhesion of THP-1 cells to ADAM-10 siRNAtransfected RA FLS in response to TNF-a was significantly decreased compared with that in control siRNA-transfected RA FLS (Fig 4, B). In addition, we performed a cell surface ELISA to determine whether cell adhesion molecules were decreased on the cell surface of ADAM-10 siRNA-transfected RA FLS. We found that the VCAM-1 on TNF-a–stimulated ADAM-10 siRNA-transfected RA FLS was decreased compared with that on the control siRNA-transfected RA FLS (Fig 4, C). However, ICAM-1 on TNF-a– stimulated ADAM-10 siRNA-transfected RA FLS was

with rabbit IgG. The lower-middle panel shows ST stained with mouse IgG. The lower-right panel shows the merging of the lower-left panel and the lower-middle panel. The yellow colour indicates ADAM-10 associated with ST lining cells (original magnification 3400). The arrows indicate ADAM-10–positive ST lining cells. (B) The left panel shows RA FLS stained with rabbit anti–ADAM-10. The middle panel shows RA FLS stained with rabbit IgG. The right panel shows RA FLS stained with rabbit anti–smooth muscle actin (original magnification 3400). (C) TNF-a upregulated ADAM-10 mRNA in RA FLS at 1 hour, and the mRNA levels were increased at 4 hours (n 5 number of replicates). ADAM, A disintegrin and metalloprotease; FLS, fibroblast-like synoviocyte; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IgG, immunoglobulin G; RA, rheumatoid arthritis; mRNA, messenger RNA; NS, nonstimulated; ST, synovial tissue; TNF-a, tumor necrosis factor a.

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Fig 4. ADAM-10 expression was decreased by the application of siRNA against ADAM-10 in RA FLS. Cells were stimulated with TNF-a (10 ng/mL) for 24 hours. (A) ADAM-10 expression in ADAM-10 siRNA-treated RA FLS and control siRNA-treated RA FLS. (B) ADAM-10 mediates the adhesion of THP-1 cells to RA FLS. The adhesion of THP-1 cells to NS- or TNF-a–stimulated ADAM-10 siRNA-transfected RA FLS was significantly decreased compared with the adhesion of THP-1 cells to NS- or TNF-a–stimulated control siRNAtransfected cells. (C) The VCAM-1 expression on TNF-a–stimulated ADAM-10 siRNA-transfected RA FLS was significantly decreased compared with that on TNF-a–stimulated control siRNA-treated RA FLS (n 5 number of RA patients). ADAM, A disintegrin and metalloprotease; FLS, fibroblast-like synoviocyte; NS, nonstimulated; RA, rheumatoid arthritis; siRNA, small interfering RNA; TNF-a, tumor necrosis factor a; VCAM-1, vascular cell adhesion molecule 1.

not decreased compared with that on control siRNAtransfected RA FLS (data not shown). These results confirm that ADAM-10 inhibition regulates TNF-ainduced fibroblast adhesion and VCAM-1 but not ICAM-1 expression. Blocking ADAM-10 in RA FLS inhibits the expression of proinflammatory mediators. We found that the secretion

of fractalkine/CX3CL1 and VEGF in medium conditioned by TNF-a–stimulated, ADAM-10 siRNAtransfected RA FLSs was significantly decreased compared with the secretion in medium conditioned by control siRNA-transfected RA FLSs (Fig 5, A and B). These results indicate that ADAM-10 in RA FLS regulates cell adhesion and proliferation by secreting proinflammatory cytokines. DISCUSSION

Fractalkine/CX3CL1 and CXCL16 are strong chemotactic chemokines, and these molecules mediate

inflammatory cell infiltration, cell adhesion, and angiogenesis.20-23 Fractalkine/CX3CL1 and CXCL16 are membrane-spanning molecules expressed on the cell surface and exist in both soluble and membranebound forms. Both chemokines can be cleaved by ADAM-10 and ADAM-17.15,24,25 ADAM-10 is a protease with a range of substrates that plays roles in cancer proliferation and inflammation. We previously reported for the first time that ADAM-10 was overexpressed in RA ST ECs compared with the expression in OA or NL STs.17 On the basis of this, we sought to investigate the expression and regulation of ADAM-10 in RA inflammation. The present study demonstrates that ADAM-10 is expressed in RA serum and is highly increased compared with the level in NL serum. Wang et al26 reported that the expression of ADAM-10 in gastric cancer was significantly related to lymph node and distant metastasis and suggested that ADAM-10 may be a useful marker to predict tumor progression

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Fig 5. ADAM-10 expression was decreased by the application of siRNA against ADAM-10 in RA FLS. Cells were stimulated with TNF-a (10 ng/mL) for 24 hours. (A) Fractalkine/CX3CL1 expression in ADAM-10 or control siRNA-treated RA FLS. (B) VEGF expression in ADAM-10 or control siRNA-treated RA FLS. Mean values are given together with the standard error of the mean (n 5 number of replicates). ADAM, A disintegrin and metalloprotease; FLS, fibroblastlike synoviocyte; RA, rheumatoid arthritis; siRNA, small interfering RNA; TNF-a, tumor necrosis factor a; VEGF, vascular endothelial growth factor.

and to provide a prognosis. We show that the level of ADAM-10 in RA serum is significantly positively correlated with DAS28. These results indicate that the level of ADAM-10 in serum may be a biomarker of disease activity in RA. In addition, we show that ADAM10 is present in RA SFs and its level is significantly increased compared with that in OA SFs. Our findings support the notion that ADAM-10 may play an important role in RA pathophysiology.

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Oh et al27 reported highly increased expression of ADAM-10 in psoriatic skin compared with NL skin. Schulz et al28 showed that ADAM-10 actively plays a role in the thrombin-induced decrease in EC-EC interactions. We previously reported an indirect role of ADAM-10 in EC chemotaxis but not a direct role. We found that ADAM-10 was overexpressed in RA serum and SFs. Because of these results, we sought to determine whether ADAM-10 in RA SFs has chemotactic activity. We showed that RA SFs depleted of ADAM-10 induced less THP-1 cell migration compared with that in sham-depleted RA SFs. We also showed that RA SFs depleted of ADAM-10 induced less monocyte migration compared with that in sham-depleted RA SFs, suggesting that ADAM-10 may have an important function in RA inflammation. Compared with previous results, the ADAM-10 in RA SFs had greater monocyte chemotactic ability. These results suggested that ADAM-10 in RA SF may differ from recombinant ADAM-10. We next focused on ADAM-10 in RA synovial fibroblasts. We found that ADAM-10 was expressed on RA ST lining cells and FLS. ADAM-10 transcripts detected by real-time qPCR were promptly upregulated by TNFa stimulation. These results indicate that ADAM-10 has a role in inflammation associated with RA. We then hypothesized that ADAM-10 mediates monocyte adhesion to RA FLS and fibroblast proliferation in RA. We and others previously reported that ADAM-10 plays a role in angiogenesis in RA and some cancers.17,29,30 In this study, we clearly demonstrate that the adhesion of myeloid THP-1 cells to ADAM-10 siRNA-transfected RA FLS was significantly decreased compared with that in control siRNA-transfected RA FLS. The THP1 cell adhesion index was decreased after the ADAM10 production was blocked in RA FLS. However, the number of adhered cells was low. These results may suggest that other molecules are correlated with monocyte adhesion in FLS. These findings are consistent with those of Nagara et al,31 who showed that the shedding of cell adhesion molecule 1, one of the multifunctional cell adhesion molecules, was catalyzed by ADAM-10. Grabowska et al32 also showed that endotherial growth factor-associated shedding appears to be mediated by ADAM-10 because knockdown of ADAM-10 results in reduced shedding of soluble E-cadherin. However, these groups did not examine which adhesion molecules were differentially expressed during ADAM-10 inhibition in RA FLS. Here, we demonstrate that expression of the cell surface adhesion molecule VCAM-1, but not ICAM-1, was decreased on ADAM-10 siRNA-transfected RA FLS compared with that on control siRNA-transfected FLS. These findings indicate not only that ADAM-10 in RA FLS is important for cell

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adhesion but also that these interactions may lead to the activation of inflammatory cells and the perpetuation of inflammation in the RA synovium. Fractalkine/CX3CL1 and VEGF are well-known proinflammatory molecules in RA and induce angiogenesis and leukocyte recruitment.20,33,34 We found that fractalkine/CX3CL1 and VEGF secretion were upregulated by TNF-a in RA FLS. We also found that the fractalkine/CX3CL1 and VEGF levels were moderately decreased in medium conditioned by TNF-a–stimulated ADAM-10 siRNA-transfected RA FLS compared with that in medium conditioned by control siRNA-transfected FLS. We measured the production of these cytokines in the cell supernatant but not the total cytokine production. ADAM-10 may not only have shedding activity but also produce cytokines. We plan to address this issue in future work. These findings suggest that ADAM-10 expressed in FLS plays an important role in RA inflammation by contributing to the production of proinflammatory mediators. Our study demonstrated that ADAM-10 is overexpressed in RA biological fluids and its level was correlated with RA disease activity. In addition, the ADAM-10 in RA SFs has monocyte chemotactic properties. Blocking the expression of ADAM-10 in RA FLS reduced the cell adhesion because of the inhibition of adhesion and proinflammatory mediator production. We propose that ADAM-10 plays roles in mediating arthritis through this multistep process. Taken together, these results indicate the importance of ADAM-10 in RA and suggest that targeting ADAM-10 may be important for combating RA. ACKNOWLEDGMENTS

Conflicts of Interest: All authors have read the journal’s policy on the disclosure of potential conflicts of interest and signed. The authors declare that they have no conflict of interest. All authors have read the journal’s authorship agreement. We thank Mrs Takeuchi for performing ELISAs. REFERENCES

1. Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 2001;344:907–16. 2. Koch AE, Kunkel SL, Harlow LA, et al. Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. J Clin Invest 1994;94:1012–8. 3. Koch AE, Kunkel SL, Harlow LA, et al. Macrophage inflammatory protein-1 alpha. A novel chemotactic cytokine for macrophages in rheumatoid arthritis. J Clin Invest 1994;93:921–8. 4. Brennan FM, Chantry D, Jackson A, Maini R, Feldmann M. Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis. Lancet 1989;2: 244–7.

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5. Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford) 2006;45:669–75. 6. Tanida S, Yoshitomi H, Nishitani K, et al. CCL20 produced in the cytokine network of rheumatoid arthritis recruits CCR61 mononuclear cells and enhances the production of IL-6. Cytokine 2009; 47:112–8. 7. Badolato R, Oppenheim JJ. Role of cytokines, acute-phase proteins, and chemokines in the progression of rheumatoid arthritis. Semin Arthritis Rheum 1996;26:526–38. 8. Weinblatt ME, Keystone EC, Furst DE, et al. Adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial. Arthritis Rheum 2003; 48:35–45. 9. Gomez-Reino JJ, Rodriguez-Lozano C, Campos-Fernandez C, et al. Change in the discontinuation pattern of tumour necrosis factor antagonists in rheumatoid arthritis over 10 years: data from the Spanish registry BIOBADASER 2.0. Ann Rheum Dis 2012;71:382–5. 10. Plasencia C, Pascual-Salcedo D, Garcia-Carazo S, et al. The immunogenicity to the first anti-TNF therapy determines the outcome of switching to a second anti-TNF therapy in spondyloarthritis patients. Arthritis Res Ther 2013;15:R79. 11. Chatzidionysiou K, van Vollenhoven RF. Rituximab versus antiTNF in patients who previously failed one TNF inhibitor in an observational cohort. Scand J Rheumatol 2013;42:190–5. 12. Edwards DR, Handsley MM, Pennington CJ. The ADAM metalloproteinases. Mol Aspects Med 2008;29:258–89. 13. Rocks N, Paulissen G, El Hour M, et al. Emerging roles of ADAM and ADAMTS metalloproteinases in cancer. Biochimie 2008;90: 369–79. 14. Hurst LA, Bunning RA, Sharrack B, Woodroofe MN. siRNA knockdown of ADAM-10, but not ADAM-17, significantly reduces fractalkine shedding following pro-inflammatory cytokine treatment in a human adult brain endothelial cell line. Neurosci Lett 2012;521:52–6. 15. Abel S, Hundhausen C, Mentlein R, et al. The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNFalpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10. J Immunol 2004;172:6362–72. 16. Yang K, Lu L, Liu Y, et al. Increase of ADAM10 level in coronary artery in-stent restenosis segments in diabetic minipigs: high ADAM10 expression promoting growth and migration in human vascular smooth muscle cells via Notch 1 and 3. PLoS One 2013;8:e83853. 17. Isozaki T, Rabquer BJ, Ruth JH, Haines GK 3rd, Koch AE. ADAM-10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis. Arthritis Rheum 2013;65: 98–108. 18. Hanyuda M, Kasama T, Isozaki T, et al. Activated leucocytes express and secrete macrophage inflammatory protein-1alpha upon interaction with synovial fibroblasts of rheumatoid arthritis via a beta2-integrin/ICAM-1 mechanism. Rheumatology (Oxford) 2003;42:1390–7. 19. Matsunawa M, Isozaki T, Odai T, et al. Increased serum levels of soluble fractalkine (CX3CL1) correlate with disease activity in rheumatoid vasculitis. Arthritis Rheum 2006;54:3408–16. 20. Ruth JH, Volin MV, Haines GK 3rd, et al. Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis. Arthritis Rheum 2001;44:1568–81. 21. Volin MV, Woods JM, Amin MA, Connors MA, Harlow LA, Koch AE. Fractalkine: a novel angiogenic chemokine in rheumatoid arthritis. Am J Pathol 2001;159:1521–30.

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22. Ruth JH, Haas CS, Park CC, et al. CXCL16-mediated cell recruitment to rheumatoid arthritis synovial tissue and murine lymph nodes is dependent upon the MAPK pathway. Arthritis Rheum 2006; 54:765–78. 23. Isozaki T, Arbab AS, Haas CS, et al. Evidence that CXCL16 is a potent mediator of angiogenesis and is involved in endothelial progenitor cell chemotaxis: studies in mice with K/BxN seruminduced arthritis. Arthritis Rheum 2013;65:1736–46. 24. Ludwig A, Hundhausen C, Lambert MH, et al. Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb Chem High Throughput Screen 2005;8:161–71. 25. Tsou CL, Haskell CA, Charo IF. Tumor necrosis factor-alphaconverting enzyme mediates the inducible cleavage of fractalkine. J Biol Chem 2001;276:44622–6. 26. Wang YY, Ye ZY, Li L, Zhao ZS, Shao QS, Tao HQ. ADAM 10 is associated with gastric cancer progression and prognosis of patients. J Surg Oncol 2011;103:116–23. 27. Oh ST, Schramme A, Stark A, Tilgen W, Gutwein P, Reichrath J. Overexpression of ADAM 10 and ADAM 12 in lesional psoriatic skin. Br J Dermatol 2008;158:1371–3. 28. Schulz B, Pruessmeyer J, Maretzky T, et al. ADAM10 regulates endothelial permeability and T-cell transmigration by

Isozaki et al

29.

30.

31.

32.

33.

34.

253

proteolysis of vascular endothelial cadherin. Circ Res 2008;102: 1192–201. Rose AA, Annis MG, Dong Z, et al. ADAM10 releases a soluble form of the GPNMB/Osteoactivin extracellular domain with angiogenic properties. PLoS One 2010;5:e12093. Hong KJ, Wu DC, Cheng KH, Chen LT, Hung WC. RECK inhibits stemness gene expression and tumorigenicity of gastric cancer cells by suppressing ADAM-mediated Notch1 activation. J Cell Physiol 2014;229:191–201. Nagara Y, Hagiyama M, Hatano N, et al. Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by gamma-secretase complex. Biochem Biophys Res Commun 2012;417:462–7. Grabowska MM, Sandhu B, Day ML. EGF promotes the shedding of soluble E-cadherin in an ADAM10-dependent manner in prostate epithelial cells. Cell Signal 2012;24:532–8. Lee A, Qiao Y, Grigoriev G, et al. Tumor necrosis factor alpha induces sustained signaling and a prolonged and unremitting inflammatory response in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum 2013;65:928–38. Ruth JH, Park CC, Amin MA, et al. Interleukin-18 as an in vivo mediator of monocyte recruitment in rodent models of rheumatoid arthritis. Arthritis Res Ther 2010;12:R118.