Up-regulated transforming growth factor inducible gene h3 in rheumatoid arthritis mediates adhesion and migration of synoviocytes through ╨Ю┬▒v 3 integrinRegulation by cytokines.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 9, September 2006, pp 2734–2744 DOI 10.1002/art.22076 © 2006, American College of Rheumatology Up-Regulated Transforming Growth Factor ␤–Inducible Gene h3 in Rheumatoid Arthritis Mediates Adhesion and Migration of Synoviocytes Through ␣v␤3 Integrin Regulation by Cytokines Eon Jeong Nam,1 Keum Hee Sa,1 Dong Wan You,1 Jang Hee Cho,1 Jae Seok Seo,1 Seung Woo Han,1 Jae Yong Park,1 Sung Il Kim,2 Hee Soo Kyung,1 In San Kim,1 and Young Mo Kang1 Objective. To delineate the expression of transforming growth factor ␤–inducible gene h3 (␤IG-H3) in rheumatoid synovitis and to determine the regulatory role of ␤IG-H3 in the adhesion and migration of fibroblast-like synoviocytes (FLS). Methods. Synovial tissue was obtained from patients with rheumatoid arthritis (RA) during joint replacement surgery, and FLS were isolated using enzymatic treatment. Immunohistochemical staining was performed to show the expression of ␤IG-H3 within rheumatoid synovium. Synthesis of ␤IG-H3 from FLS was determined by semiquantitative reverse transcription–polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay. Cell adhesion and migration assays were performed using the YH18 peptide in the fourth fas-1 domain of ␤IG-H3 and function-blocking antibodies to integrins. Results. Expression of ␤IG-H3 was up-regulated in RA synovial tissue compared with synovial tissue from patients with osteoarthritis. FLS isolated from RA synovial tissue constitutively produced ␤IG-H3, which was up-regulated by transforming growth factor ␤1, interleukin-1␤, and tumor necrosis factor ␣. Although FLS expressed a variety of integrins, ␤IG-H3 mediated adhesion and migration of FLS through interaction with ␣v␤3 integrin. Cytokines failed to affect the ␤IG-H3– mediated adhesion. However, migration of FLS guided by ␤IG-H3 was enhanced by interferon-␥ and plateletderived growth factor type BB. The YH18 peptide in the fourth fas-1 domain of ␤IG-H3 inhibited adhesion and migration in a dose-dependent manner. Conclusion. The results suggest that ␤IG-H3, which is abundantly expressed in RA synovial tissue, plays a regulatory role in chronic destructive inflammation through the modulation of the adhesion and migration of FLS. This finding indicates the relevance of ␤IG-H3 and ␣v␤3 integrin–interacting motifs as potential therapeutic targets in this disease. Supported by a grant from the Korea Research Foundation (KRF-2004-041-E00169) funded by the Korean Government (MOERD). 1 Eon Jeong Nam, MD, Keum Hee Sa, MS, Dong Wan You, MD, Jang Hee Cho, MD, Jae Seok Seo, MD, Seung Woo Han, MD, Jae Yong Park, MD, Hee Soo Kyung, MD, PhD, In San Kim, MD, PhD, Young Mo Kang, MD, PhD: Kyungpook National University School of Medicine, Daegu, Republic of Korea; 2Sung Il Kim, MD: Pusan National University School of Medicine, Pusan, Republic of Korea. Dr. Nam and Ms. Sa contributed equally to this work. Address correspondence and reprint requests to Young Mo Kang, MD, PhD, Division of Rheumatology, Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2-Ga, Junggu, Daegu 700-721, Republic of Korea. E-mail: ymkang@ knu.ac.kr. Submitted for publication January 11, 2006; accepted in revised form June 9, 2006. Rheumatoid arthritis (RA) is a chronic inflammatory joint disease which is characterized by synovial hyperplasia and progressive destruction of cartilage and bone. Fibroblast-like synoviocytes (FLS) may mediate joint destruction as final effector cells in a cytokine and cell cascade in rheumatoid synovial tissue (1–3). Unlike the synovial tissue of healthy joints, RA synovium is characterized by hyperplasia of synoviocytes, which is the result of enhanced survival, proliferation, and guided migration (4). The extracellular matrix (ECM) provides the 2734 ␤IG-H3 IN RHEUMATOID ARTHRITIS mechanical scaffolding within which tissue is assembled. During inflammation, it is likely that a number of integrins expressed on the surface of activated FLS regulate critical adhesive interactions with a variety of ECM proteins. These adhesive interactions anchor cells and transduce signals that are critical for distinct biologic events such as adhesion, migration, proliferation, and differentiation (5). Inhibition of integrin–ECM interactions suppresses cellular growth or induces apoptotic cell death (6,7). Transforming growth factor ␤–inducible gene h3 (␤IG-H3) is a recently identified ECM protein that can be induced by transforming growth factor ␤ (TGF␤) in several cell types, including human melanoma cells, epithelial cells, keratinocytes, and lung fibroblasts (8,9). ␤IG-H3 consists of 683 amino acids containing 4 homologous internal repeat domains, denoted fas-1 domains. ␤IG-H3 has been suggested to function mainly in cellular growth (9), differentiation (10), adhesion, migration (8), and angiogenesis (11), which are mediated through interaction with integrins ␣3␤1, ␣v␤3, and ␣v␤5. The interactions with integrins ␣v␤3 and ␣v␤5 require at least 18 amino acids, including highly conserved tyrosine and leucine residues, which are denoted YH18 peptide (11,12). Previous reports have suggested that ␤IG-H3 is associated with diabetic nephropathy (13,14), cyclosporine-induced nephropathy (15,16), and astrocyte response to brain injury (17). To date, however, expression of ␤IG-H3 protein has not been demonstrated in RA synovial tissue, and it is unclear whether ␤IG-H3 is produced by FLS and whether it mediates the functions of RA FLS. The aims of the present study were to delineate the expression of ␤IG-H3 within RA synovial tissue and to determine the regulatory role of ␤IG-H3 in the functions of an important stromal cell, the FLS. We found that ␤IG-H3 protein was expressed abundantly within RA synovial tissue, and the synthesis of ␤IG-H3 from FLS was enhanced by TGF␤1, interleukin-1␤ (IL-1␤), and tumor necrosis factor ␣ (TNF␣). ␤IG-H3 mediated the adhesion and migration of FLS through interactions with ␣v␤3 integrin, which were effectively blocked by the YH18 peptide. Together, these results indicate that ␤IG-H3, up-regulated within RA synovial tissue, may play a role in the pathogenesis of RA through the regulation of the functional activities of FLS. PATIENTS AND METHODS Patients. Synovial tissue was obtained during joint replacement surgery from patients with RA, defined according 2735 to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (18). Synovial tissue was also obtained from patients with osteoarthritis (OA) who were undergoing knee joint replacement surgery. The RA patients ranged in age from 32 years to 59 years (mean ⫾ SD 46.4 ⫾ 11.6 years). The OA patients ranged in age from 65 years to 71 years (mean ⫾ SD 69.2 ⫾ 2.8 years). All RA patients were taking nonsteroidal antiinflammatory drugs; 6 patients were taking methotrexate and 4 were taking low-dose steroids at the time of the operation. The RA patients had a mean ⫾ SD disease duration of 13.8 ⫾ 7.5 years. Cell isolation and culture. FLS were isolated by enzymatic dispersion of synovial tissue as previously described (19). Cells were cultivated in Dulbecco’s modified Eagle’s medium containing 4.5 gm/liter glucose (BioWhittaker, Walkersville, MD) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM ␥-glutamine, and 10% fetal calf serum, and were maintained in a humidified atmosphere of 5% CO2 at 37°C. Upon reaching confluence, cells were detached with trypsin/EDTA (Gibco BRL, Carlsbad, CA) and split. FLS between passages 3 and 8 were used for the experiments. Normal dermal fibroblasts were established from foreskin specimens (kindly provided by Dr. Ho Yun Chung, Kyungpook National University). Antibodies and recombinant products. The bacterial expression vector for wild-type ␤IG-H3 has been described previously. Recombinant human ␤IG-H3 protein was induced and purified as described previously (11). Monoclonal antibody to human ␤IG-H3 (clone 7A6) was prepared using recombinant human ␤IG-H3. Polyclonal antibody was generated by immunizing rabbits with recombinant human ␤IG-H3. YH18, YH18 control, RGD, and RGE peptides were synthesized at Peptron (Daejeon, Korea). YH18 peptide in the fourth fas-1 domain of wild-type ␤IG-H3 was selected. Monoclonal antibodies against CD68 (EBMII) and biotinylated rabbit anti-mouse Ig were purchased from Dako (Glostrup, Denmark), polyclonal antibody against fibronectin was purchased from Sigma (St. Louis, MO), and phycoerythrin-labeled antibodies against CD3 (SK7), CD14 (MP9), and CD20 (L27) were purchased from BD Biosciences (San Jose, CA). Neutralizing polyclonal anti-human TGF␤1 and recombinant human cytokines including interferon-␥ (IFN␥), TNF␣, IL-1␤, TGF␤1, IL-4, IL-10, basic fibroblast growth factor (bFGF), platelet-derived growth factor type BB (PDGF-BB), insulinlike growth factor 1, and epidermal growth factor were purchased from R&D Systems (Minneapolis, MN). Monoclonal function-blocking anti-␣2␤1 (BHA2.1), anti-␣3␤1 (M-KID2), anti-␣5␤1 (JBS5), anti-␣v␤3 (LM609), anti-␣v␤5 (P1F6), and anti-␤4 (ASC-3) antibodies were obtained from Chemicon International (Temecula, CA). Immunohistochemical staining. Sections of synovial tissue were prepared from frozen tissue embedded in OCT compound and fixed in acetone for 10 minutes. Tissue sections were incubated with primary antibodies after blocking endogenous peroxidase with 0.03% H2O2/NaN3 buffer and nonspecific protein binding with 5% normal goat serum. Biotinylated secondary antibodies, VECTASTAIN statin Elite ABC reagents (Vector Laboratories, Burlingame, CA), and 3,3⬘diaminobenzidine tetrahydrochloride (Dako) were used for 2736 development. Sections were mounted with VectaMount (Vector Laboratories) and analyzed under light microscopy. Measurement of ␤IG-H3 in the culture supernatant. The level of ␤IG-H3 was measured using the enzyme-linked immunosorbent assay (ELISA) method as described previously (13). Briefly, a 96-well flat-bottom microtiter plate was coated with wild-type ␤IG-H3 protein overnight at 4°C. Lyophilized culture media and standard ␤IG-H3 protein were preincubated with anti–␤IG-H3 antibody and transferred to the precoated plate and incubated for 30 minutes at room temperature. After reaction with horseradish peroxidase (HRP)–conjugated secondary antibody, substrate solution was added for the color reaction. The reaction was stopped with 8N H2SO4, and the absorbance was determined at 490 nm using a model 550 microplate reader (Bio-Rad, Hercules, CA). Flow cytometry analysis. FLS or dermal fibroblasts were incubated under adherent conditions in medium alone or in the presence of recombinant human TGF␤1 at 37°C, when indicated. Cells were detached and incubated with the primary antibodies at 4°C for 30 minutes. After washing in phosphate buffered saline (PBS) with 0.5% bovine serum albumin (BSA), cells were labeled with fluorescein isothiocyanate (FITC)– conjugated goat anti-mouse Ig (BD Biosciences) at 4°C for 30 minutes and analyzed with a FACSCalibur (BD Biosciences). RNA extraction and semiquantitative reverse transcription–polymerase chain reaction. Total RNA was extracted from FLS using TRIzol reagent (Gibco BRL) according to the manufacturer’s protocol. Complementary DNA was obtained from 5 g of total RNA using oligo(dT) and AMV reverse transcriptase (Roche, Basel, Switzerland) and amplified with primers (Bioneer, Daejeon, Korea) and LightCycler DNA master SYBR Green I (Roche). The amplification was conducted for 45 cycles at a denaturation temperature of 95°C for 1 second, an annealing temperature of 55°C for 3 seconds, and an extension temperature of 72°C for 8 seconds, in a LightCycler (Roche). After the final cycle, the numbers of transcripts were determined using melting curve analysis and an external standard curve. The following primers were used: for ␤IG-H3, 5⬘-AGATCGAGGACACCTTTGAG-3⬘ (sense) and 5⬘-TTGTTCAGCAGGTCTCTCAG-3⬘ (antisense); for GAPDH, 5⬘-GGAGTCAACGGATTTGGTCG-3⬘ (sense) and 5⬘-GACGGTGCCATGGAATTTGC-3⬘ (antisense). Immunofluorescence. FLS were mounted on a cover glass coated with poly-L-lysine (Sigma) and cultured in media alone or in the presence of TGF␤1. At the end of the incubation period, cells were fixed with 4% paraformaldehyde and permeabilized in PBS with 0.2% Triton X-100. After blocking nonspecific protein binding, cells were incubated serially with monoclonal anti-human ␤IG-H3 (7A6) and FITCconjugated goat anti-mouse Ig (Jackson ImmunoResearch, West Grove, PA), and nuclei were identified by 4⬘,6diamidino-2-phenylindole. Nonimmunized IgG was used as a negative control. The pattern of fluorescence was evaluated under a fluorescence microscope (Zeiss, Jena, Germany). Western blot analysis. Culture supernatants were concentrated by precipitation with trichloroacetic acid. Protein (10 g) from each sample was solubilized at 100°C for 5 minutes in sample buffer and separated by electrophoresis in 10% polyacrylamide gels, then transferred to a nitrocellulose membrane. The membranes were blocked with 10% nonfat dried milk in PBS containing 0.1% Tween 20 (PBST) and then incubated with the primary antibody at room temperature for NAM ET AL Figure 1. Immunohistochemical staining of transforming growth factor ␤–inducible gene h3 (␤IG-H3) in synovial tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Expression of ␤IG-H3, fibronectin, and CD68 was examined in serial sections of RA synovial tissue (A, C, and E, respectively) and OA synovial tissue (B, D, and F, respectively). As a negative control, nonimmunized isotypematched antibody was used for staining (G and H). (Hematoxylin counterstained; original magnification ⫻ 200.) 1 hour. Membranes were washed 3 times with PBST and probed with HRP-conjugated goat anti-rabbit Ig in 5% nonfat milk in PBST. The signals were developed using a Supersignal Immunodetection System (Pierce, Rockford, IL). Cell adhesion assay. The cell adhesion assay was performed as described previously (12). Briefly, flat-bottom 96-well ELISA plates (Costar, Cambridge, MA) were coated with recombinant human ␤IG-H3 proteins or purified human plasma fibronectin and then blocked for 1 hour at room temperature with PBS containing 2% BSA. Cells were suspended in culture media at a density of 3 ⫻ 105/ml, and 0.1 ml of the cell suspension was added to each well of the plate. For the inhibition assay, monoclonal antibodies to integrins and YH18, YH18 control, RGD, and RGE peptides were preincu- ␤IG-H3 IN RHEUMATOID ARTHRITIS 2737 Figure 2. Induction of transforming growth factor ␤–inducible gene h3 (␤IG-H3) synthesis in fibroblast-like synoviocytes (FLS) by transforming growth factor ␤1 (TGF␤1). A, Western blot, showing that ␤IG-H3 (68 kd) was secreted constitutively from FLS of 3 patients with rheumatoid arthritis (RA). B, Intracellular synthesis of ␤IG-H3 in RA FLS. FLS were cultured on poly-L-lysine–coated cover glass and stimulated with TGF␤1 (0.5 ng/ml) for 24 hours. After fixation of cells with paraformaldehyde and subsequent permeabilization with 0.2% Triton X-100, FLS were stained with anti–␤IG-H3 antibody and fluorescein isothiocyanate–conjugated goat anti-mouse IgG. Cells were examined under fluorescence microscopy. Results are representative of 3 independent experiments (original magnification ⫻ 200). C, The expression of ␤IG-H3 mRNA was quantified by reverse transcription–polymerase chain reaction. The number of ␤IG-H3 transcripts was divided by the number of GAPDH transcripts to normalize values between experiments. FLS were treated with TGF␤1 (0.5 ng/ml) for 2, 4, 12, 24, and 48 hours. Results are representative of 3 independent experiments. D, Concentrations of secreted ␤IG-H3 from FLS were measured by enzyme-linked immunosorbent assay with (solid bars) or without (open bars) TGF␤1 stimulation (0.5 ng/ml) for 12, 24, 48, and 96 hours. E, FLS were stimulated with different concentrations of TGF␤1 for 48 hours. Values are the mean ⫾ SEM of triplicate experiments. bated at 37°C for 30 minutes with FLS before FLS were added to the coated plates. After incubation for 1 hour at 37°C, unattached cells were removed by rinsing twice with PBS. Citrate buffer containing 3.75 mM p-nitrophenyl-N-acetyl ␤-Dglucosaminide (hexosaminidase substrate) and 0.25% Triton X-100 was added to each well and incubated for 1 hour at 37°C. Absorbance was measured at 405 nm in a model 550 microplate reader after blocking enzyme activity with 50 mM glycine buffer (pH 10.4) containing 5 mmoles/liter EDTA. Migration assay. Cell migration assays were performed as described previously (11). Briefly, the undersurface of the membrane in a transwell plate of 8-m pore size (Costar) was coated overnight at 4°C with recombinant ␤IG-H3 proteins and then blocked for 1 hour at room temperature with PBS containing 2% BSA. FLS were suspended in medium at a density of 5 ⫻ 105/ml, and 100 l of the cell suspension was added to the upper compartment of the filter. In some experiments, FLS were preincubated for 30 minutes at 37°C with function-blocking monoclonal antibodies or YH18, YH18 control, RGD, or RGE peptides. After allowing cells to migrate for 6 hours at 37°C, migration was terminated by removing cells from the upper chamber. FLS on the lower surface of the membrane were fixed and stained with crystal violet. The number of cells was determined in 9 randomly selected high-power fields by using light microscopy. Statistical analysis. Differences between groups were examined for statistical significance using the Mann-Whitney U test. P values less than 0.05 were considered significant. 2738 NAM ET AL Figure 3. Regulation of ␤IG-H3 production in FLS by cytokines. FLS were stimulated with cytokines in the presence or absence of TGF␤1 (0.5 ng/ml) for 48 hours. Cell-free supernatants were collected, and the concentrations of ␤IG-H3 were determined using enzyme-linked immunosorbent assay. A, Effect of neutralizing antibody (Ab) against TGF␤1 on the production of ␤IG-H3 induced by 0.5 ng/ml TGF␤1. ⴱ ⫽ P ⬍ 0.05 versus TGF␤1-stimulated FLS in the absence of neutralizing antibody. B and C, RA FLS were stimulated with interferon-␥ (IFN␥) (B) or with interleukin-1␤ (IL-1␤) or tumor necrosis factor ␣ (TNF␣) (C). D, FLS were treated with dexamethasone (Dexam; 1.0 and 100 M) or cyclosporin A (CSA; 10 and 1,000 ng/ml) for 48 hours in the presence of 0.5 ng/ml TGF␤1. ⴱ ⫽ P ⬍ 0.05 versus TGF␤1-stimulated FLS. Values are the mean and SEM. Results are representative of 3 independent experiments. See Figure 2 for other definitions. RESULTS Up-regulated expression of ␤IG-H3 in RA synovial tissue compared with that in OA synovial tissue. Synovial tissue sections obtained from 7 patients with RA and 5 patients with OA were stained with monoclonal anti–␤IG-H3 antibody or with isotype-matched nonimmunized IgG antibodies as a negative control. ␤IG-H3 was highly up-regulated in all RA synovial tissue (Figure 1A). While fibronectin showed a diffuse distribution within both synovial lining and sublining layers (Figures 1C and D), ␤IG-H3 showed a more intense staining within the lining layer. Endothelial cells within RA synovial tissue also expressed ␤IG-H3. In contrast, the expression of ␤IG-H3 was markedly lower in OA synovial tissue, in which the lining layer was the main source of ␤IG-H3 (Figure 1B). The distribution of ␤IG-H3 was similar to that of CD68-positive cells within both RA and OA synovial tissue (Figures 1E and F). Tonsil tissue was stained with anti–␤IG-H3 antibody as a positive control and showed well-demarcated interfollicular distribution (results not shown). Regulation of ␤IG-H3 production in FLS by cytokines. In an attempt to identify the cellular source of ␤IG-H3, which was abundantly expressed within RA synovial tissue, we selected FLS as the primary candidate because of their capacity to produce other ECM proteins such as fibronectin, type IV and VI collagens, and laminin (20). As the first step, we used Western blot analysis to examine the presence of ␤IG-H3 protein in cultured unstimulated FLS. FLS from RA patients con- ␤IG-H3 IN RHEUMATOID ARTHRITIS 2739 Figure 4. Role of ␣v␤3 integrin as a functional receptor for ␤IG-H3–mediated adhesion of FLS. A and B, Adhesion of FLS on surfaces coated with bovine serum albumin (BSA) and different concentrations of ␤IG-H3. A, Different concentrations of ␤IG-H3 were used for coating plates. The cells were fixed with 8% paraformaldehyde, stained with crystal violet, and observed under the inverted microscope (original magnification ⫻ 200). B, Ninety-six–well enzyme-linked immunosorbent assay plates coated with BSA or ␤IG-H3 were incubated with FLS at 37°C for 2 hours. Cells attached to the surfaces were quantified by hexosaminidase assay. Values are the mean and SD of triplicate experiments. C, Flow cytometric analysis of the surface expression of ␤4, ␣2␤1, ␣3␤1, ␣5␤1, ␣v␤3, and ␣v␤5 integrins by unstimulated FLS. The data are expressed as cell number plotted as a function of fluorescence intensity and are representative of 3 independent experiments. Dotted histograms represent the negative control, and shaded histograms represent the surface expression of each integrin. D, FLS were preincubated with function-blocking antibodies (Ab) to integrins at 10 g/ml for 30 minutes at 37°C and then added to the precoated wells with 10 g/ml ␤IG-H3. Values are the mean and SEM of at least 3 independent experiments. ⴱ ⫽ P ⬍ 0.05 versus ␤IG-H3 alone. See Figure 2 for other definitions. stitutively expressed ␤IG-H3 protein (Figure 2A). With stimulation by TGF␤1 (0.5 ng/ml), FLS showed more intense intracellular ␤IG-H3 staining with concomitant changes in size and shape (Figure 2B). Stimulation of FLS with TGF␤1 (0.5 ng/ml) up-regulated the expression of ␤IG-H3 transcript and protein in a timedependent manner (Figure 2C). ␤IG-H3 protein was secreted constitutively when cultured in serum-free media. Synthesis of ␤IG-H3 protein was increased in a dose-dependent manner after stimulation with TGF␤1 and peaked at 0.5 ng/ml of TGF␤1 (Figure 2E). Rheumatoid synovium is a reservoir of abundant cytokines, both proinflammatory and antiinflammatory. To test whether these cytokines regulate ␤IG-H3 production, FLS were stimulated with a single cytokine or with a combination of cytokines. Among proinflammatory cytokines, IFN␥ at a lower concentration (10 ng/ml) up-regulated the production of ␤IG-H3 induced by TGF␤1, while IFN␥ at a higher concentration (100 ng/ml) down-regulated this production. Stimulation with IFN␥ alone did not change the production of ␤IG-H3 (Figure 3B). TNF␣ (10 ng/ml) and IL-1␤ (10 ng/ml), which are known to be the most influential in the generation and maintenance of RA, up-regulated 2740 NAM ET AL Figure 5. Regulation of ␤IG-H3–mediated migration by cytokines. Migration of FLS was assayed using 2-compartment transwells in which the undersurface of the filter was coated with ␤IG-H3. FLS (5 ⫻ 104) in 0.1 ml medium were added to the upper chamber of transwells. A, The undersurfaces of the transwell membrane were coated with different concentrations of ␤IG-H3. B, FLS were suspended in medium containing interferon-␥ (IFN␥) or platelet-derived growth factor type BB (PDGF-BB) and added into the upper compartments of the transwell plate. BSA ⫽ bovine serum albumin. C and D, FLS were preincubated with function-blocking antibodies (Ab) to integrins at the concentration indicated in Figure 4 and added to the upper chambers of transwells. Cell migration was quantified by counting migrated cells in 9 microscope fields. Values are the mean and SEM of at least 3 independent experiments. ⴱ ⫽ P ⬍ 0.01 versus ␤IG-H3 alone. See Figure 2 for other definitions. (Original magnification ⫻ 200 in A and C.) ␤IG-H3 synthesis (Figure 3C). Treatment with neutralizing antibody against TGF␤1 at a concentration that could block the concentration of TGF␤1 effective for inducing ␤IG-H3 production (Figure 3A) did not inhibit the effect of TNF␣ (10 ng/ml) and IL-1␤ (10 ng/ml), showing that these cytokines stimulate the production of ␤IG-H3 in a TGF␤1-independent pathway (data not shown). ␤IG-H3 synthesis induced by TGF␤1 was inhibited efficiently with a high dose of dexamethasone (100 M), but not with cyclosporin A (Figure 3D). Mediation of FLS adhesion by ␤IG-H3 through interaction with ␣v␤3 integrin. ␤IG-H3 has been reported to mediate cellular adhesion through interaction with integrins. Interestingly, the integrins responsible for ␤IG-H3–mediated cellular adhesion differ with different cell types. The ␣3␤1 integrin is a functional receptor in epithelial cells, ␣v␤5 integrin in fibroblasts, and ␣v␤3 integrin in endothelial cells (8,11,12). In the present study, we found that ␤IG-H3 mediated adhesion of FLS in a dose-dependent manner (Figures 4A and B). Adhesion of FLS on coated ␤IG-H3 at a concentration of 10 g/ml was similar to that on coated fibronectin at a concentration of 5 g/ml (data not shown). To identify the integrin responsible for the ␤IG-H3–mediated adhesion, we tested several functionblocking antibodies against integrins. On flow cytometric ␤IG-H3 IN RHEUMATOID ARTHRITIS Figure 6. YH18 peptide inhibits adhesion and migration of FLS mediated by ␤IG-H3. A, YH18 peptide (spanning amino acids 563–580 in the fourth fas-1 domain [D-IV] of ␤IG-H3) (see ref. 12), YH18 control peptide (YH18-con), and RGD (GRGDSP) and RGE (GRGESP) peptides were prepared as described in Patients and Methods. B, Inhibition of adhesion by YH18 peptide. FLS were preincubated with RGE, RGD, YH18 control, or YH18 peptides at 500 M for 30 minutes at 37°C and added to the ␤IG-H3–coated wells. Values are the mean and SEM of at least 3 independent experiments. ⴱ ⫽ P ⬍ 0.01 versus YH18 control peptide; ⴱⴱ ⫽ P ⬍ 0.01 versus RGE peptide. C, Effect of the YH18 and RGD peptides on ␤IG-H3–mediated migration of FLS. Cells were preincubated with the RGE, RGD, YH18, or YH18 control peptides for 30 minutes before being added into the upper compartments of the transwell plate. Cells that migrated through the filter were stained and quantified by counting migrated cells. Values are the mean and SEM; unstimulated migration has been normalized to 100. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, YH18 peptide versus YH18 control peptide and RGD peptide versus RGE peptide. See Figure 2 for other definitions. analysis, unstimulated FLS expressed ␣2␤1, ␣3␤1, ␣5␤1, ␣v␤3, and ␣v␤5, but not ␤4 (Figure 4C). The adhesion of FLS on ␤IG-H3 was specifically blocked by function- 2741 blocking antibody against ␣v␤3, but not by functionblocking antibodies against ␣3␤1 and ␣v␤5 (Figure 4D). ␤IG-H3–mediated adhesion was not blocked by other types of function-blocking antibodies against integrins such as ␣2␤1, ␣5␤1, and ␤4 (data not shown). In contrast, adhesion of normal dermal fibroblasts, which also express ␣3␤1, ␣v␤3, and ␣v␤5 integrins, was efficiently blocked by antibody to ␣3␤1 integrin instead of antibody to ␣v␤3 integrin (data not shown). To determine whether cytokines modulate ␤IG-H3–mediated adhesion, FLS were incubated with the cytokines TNF␣, IL-1␤, TGF␤1, bFGF, and PDGF-BB. None of these mediators affected ␤IG-H3–mediated adhesion of FLS (data not shown). Mediation of FLS migration by ␤IG-H3 through the ␣v␤3 integrin. Integrins, the major receptors connecting cells to the surrounding ECM, not only support cell attachment but also act to regulate survival, differentiation, proliferation, and migration (21,22). To test whether ␤IG-H3 mediates directed migration in FLS, we performed a transmigration assay using a 2-compartment transwell system. ␤IG-H3 mediated migration of FLS in a dosedependent manner (Figure 5A). The number of migrating FLS was sharply increased up to a ␤IG-H3 concentration of 10 g/ml, which was used in subsequent experiments. To determine whether inflammatory mediators, which are known to be involved in the pathogenesis of RA, regulate ␤IG-H3–mediated migrations of FLS, we studied the role of cytokines and growth factors. As shown in Figure 5B, IFN␥ and PDGF-BB substantially enhanced the migratory response of FLS, which was increased 1.5–3-fold compared with basal transmigration on a ␤IG-H3–coated filter. In contrast, IL-1␤ (10 ng/ml) slightly inhibited ␤IG-H3–mediated migration of FLS (data not shown). Other cytokines such as TNF␣, TGF␤1, and bFGF did not exert a significant effect (data not shown). Enhanced migration mediated by ␤IG-H3 was effectively blocked by the functionblocking antibody to the ␣v␤3 integrin, but not by antibodies to the ␣3␤1 and ␣v␤5 integrins (Figures 5C and D). These results demonstrate that ␤IG-H3 mediates FLS migration through the ␣v␤3 integrin, and that this migration is synergistically enhanced by IFN␥ and PDGF-BB. Inhibition of ␤IG-H3–mediated FLS adhesion and migration by YH18 peptide. Previous reports (11,12) noted that the 18–amino-acid peptide YH18, which includes tyrosine, histidine, and flanking leucine/ isoleucine residues within a fragment corresponding to 2742 NAM ET AL amino acids 548–614 of the fourth fas-1 domain, effectively inhibited cell adhesion to the ␤IG-H3 protein. The YH18 peptide inhibited ␤IG-H3–mediated adhesion of both MRC-5 fibroblasts (12) and endothelial cells (11), although the integrins involved in the cell adhesion are ␣v␤5 and ␣v␤3, respectively. To examine whether the YH18 peptide blocks ␤IG-H3–mediated FLS adhesion, we used YH18 synthetic peptides of the fourth fas-1 domain in a cell adhesion assay (Figure 6A). This peptide inhibited FLS adhesion to ␤IG-H3 at a concentration of 500 M. Cell adhesion to ␤IG-H3 was also inhibited by RGD peptide in a dose-dependent manner, but not by RGE peptide (Figure 6B). These results indicate that the YH18 peptide contains a sequence essential for FLS adhesion to ␤IG-H3. Using a transwell system, we then tested whether the RGD and YH18 peptides are required for ␤IG-H3–mediated migrations of FLS. Both the RGD and YH18 peptides inhibited FLS migration toward ␤IG-H3 in a dose-dependent manner (Figure 6C). DISCUSSION In the present study, we demonstrated that ␤IG-H3 is abundantly expressed within RA synovial tissue and produced by RA FLS. We also found that ␤IG-H3 mediates adhesion and migration of RA FLS through ␣v␤3 integrin, which can be efficiently blocked by YH18 peptide. These findings indicate that ␤IG-H3 is involved in the pathogenesis of RA by mediating cell trafficking, and that it may be a potential therapeutic target in RA. In RA synovial tissue, there is hyperplasia of resident stromal cells such as FLS, which are the main effector cells of joint-destroying pannus (23). FLS are the major resident cells producing an abundance of ECM proteins such as collagen, fibronectin, hyaluronic acid, and laminin. In previous studies that have observed ECM expression in RA synovium, fibronectin and vitronectin have been reported to be present at increased levels (24–26). We showed that the expression of ␤IG-H3 is substantially increased in the synovial lining and sublining layers in RA synovial tissue compared with OA synovial tissue. In our study, FLS isolated from RA patients produced ␤IG-H3 constitutively. The production of ␤IG-H3 from FLS was most efficiently induced by TGF␤1 and was also induced by other cytokines such as TNF␣, IL-1␤, and IFN␥. The source of abundant ␤IG-H3 within RA synovial tissue may be at least partially ex- plained by the proliferated FLS and the up-regulation of ␤IG-H3 production by cytokines from these cells. In RA, TGF␤1 was found predominantly in the thickened synovial lining layer and was also detected at the cartilage–pannus junction (27,28). Although systemically administered TGF␤1 suppresses development of experimental arthritis (29), localized injections of TGF␤1 into the articular space induced apparent arthritis which was characterized by pronounced synovial hyperplasia (30,31). Antagonism of TGF␤1 with a neutralizing antibody blocked pathologic development in the tissue in an experimental model of chronic erosive arthritis (32). The role of TGF␤1 in inflammation is only partially known and involves recruitment and activation of immature leukocyte populations (33). Considering that TGF␤1 is one of the main stimuli for ECM production, accumulation of ECM may be involved in regulating inflammation through interactions with stromal resident cells as well as with infiltrating inflammatory leukocytes. As a TGF␤1-inducible ECM protein, ␤IG-H3 may also mediate inflammation by directly or indirectly participating in the regulation of cellular adhesion, migration, and activation of FLS. In addition, ECM–FLS interactions are important because the synovial lining cells and the pannus are susceptible to shear stress (34). We demonstrate that ␤IG-H3 mediates the adhesion of FLS as effectively as fibronectin. The ECM– cell interactions, coupled to cytoskeletal elements, provide a potent anoikis resistance factor (35). The interaction between FLS and ␤IG-H3 may provide an additional signal against apoptosis, and needs further study. The present study revealed that ␤IG-H3 mediates the adhesion of RA FLS via interaction with ␣v␤3 integrin. ␤IG-H3 has multiple cell adhesion motifs within the fas-1 domains that can mediate interactions with several cell types through different integrins, including ␣3␤1 (8,36), ␣v␤3 (11), and ␣v␤5 (12). Although FLS expressed all these integrins, only ␣v␤3 was used for adhesion on ␤IG-H3. In contrast, adhesion of normal dermal fibroblasts, which also expressed all 3 integrins, was blocked mainly by antibody to ␣3␤1 integrin. The choice of the integrin may depend on the activation state of the integrins in each cell type (12). Intracellular molecular events may modulate the affinity and avidity of integrins for their ECM ligand (37,38). In FLS, ␣v␤3, but not ␣3␤1 or ␣v␤5, may have an appropriate conformation to engage the binding motif on ␤IG-H3. The ability of extracellular domains of integrin to bind ligands can be activated on a time scale of less than 1 second by signals within the cell, which is particularly ␤IG-H3 IN RHEUMATOID ARTHRITIS evident with integrins on leukocytes (39). In the present study, pretreatment of FLS with cytokines and growth factors did not influence the ␤IG-H3–mediated adhesion. The conserved consensus sequence of ␤IG-H3 that binds to both ␣v␤3 and ␣v␤5 integrins contains highly conserved tyrosine and histidine residues that are designated YH motif (11,12). At least 18 amino acids including these conserved residues are required to mediate fibroblast and endothelial cell adhesion. We demonstrated that adhesion of FLS to ␤IG-H3 was effectively blocked by both RGD peptide and YH18 peptide. Because the ␣v␤3 and ␣v␤5 integrins are known to share ligands (40), and because their interactions with the YH18 peptide have been shown to be RGD dependent (11,12), the YH18 and RGD peptides may interact with the ␣v␤3 and ␣v␤5 integrins in a similar manner. We have shown that migration of FLS induced by ␤IG-H3 is markedly enhanced by IFN␥ and PDGF-BB in a dose-dependent manner. In addition to supporting cell adhesion to the ECM, integrins act in concert with receptors for soluble factors to regulate cell migration, survival, proliferation, and gene transcription (21,41,42). This cooperation occurs at many levels, ranging from membrane-proximal interactions, in which the different types of receptors influence each other’s activity, to multiple inputs into common pathways (43). In contrast, TGF␤1 and bFGF, which can drive FLS proliferation (31,44,45), had no effect on ␤IG-H3–mediated migration of FLS. These results indicate that ␤IG-H3 synthesis is not directly linked to migratory stimulation of FLS. Within RA synovial tissue, a large pool of ECM supports the architecture and also regulates the functions of a variety of cell types. ␤IG-H3 has been isolated from articular cartilage and has been suggested to play a role in osteogenesis, since ␤IG-H3 transcripts were observed in proliferating chondrocytes and areas of endochondral ossification (46). There is a possibility that ␤IG-H3 expressed in the bone may interact with ␣v␤3expressing osteoclasts; if this is true, such an interaction could be blocked by YH18 peptides. Although the overall relevance of ␤IG-H3 in the pathogenesis of RA is still not clear, it might be important in the hierarchy of synovial inflammation considering its abundance within rheumatoid synovial tissue and its regulatory effects on FLS. Therefore, further investigation is required to understand the functions of ␤IG-H3 in rheumatoid synovial inflammation and to define the therapeutic potential of ␤IG-H3–derived peptides. 2743 REFERENCES 1. Sarkissian M, Lafyatis R. Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts. J Immunol 1999;162:1772–9. 2. Firestein GS. Invasive fibroblast-like synoviocytes in rheumatoid arthritis: passive responders or transformed aggressors? [review]. Arthritis Rheum 1996;39:1781–90. 3. Weyand CM, Kang YM, Kurtin PJ, Goronzy JJ. The power of the third dimension: tissue architecture and autoimmunity in rheumatoid arthritis. Curr Opin Rheumatol 2003;15:259–66. 4. Shiozawa K, Shiozawa S, Shimizu S, Fujita T. Fibronectin on the surface of articular cartilage in rheumatoid arthritis. Arthritis Rheum 1984;27:615–22. 5. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673–87. 6. Montgomery AM, Reisfeld RA, Cheresh DA. Integrin ␣v␤3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen. Proc Natl Acad Sci U S A 1994;91:8856–60. 7. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, et al. Integrin ␣v␤3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994;79: 1157–64. 8. Kim JE, Kim SJ, Lee BH, Park RW, Kim KS, Kim IS. Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-␤-induced gene, ␤ig-h3. J Biol Chem 2000;275:30907–15. 9. Skonier J, Bennett K, Rothwell V, Kosowski S, Plowman G, Wallace P, et al. ␤ ig-h3: a transforming growth factor-␤-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice. DNA Cell Biol 1994;13:571–84. 10. Kim JE, Kim EH, Han EH, Park RW, Park IH, Jun SH, et al. A TGF-␤-inducible cell adhesion molecule, ␤ig-h3, is downregulated in melorheostosis and involved in osteogenesis. J Cell Biochem 2000;77:169–78. 11. Nam JO, Kim JE, Jeong HW, Lee SJ, Lee BH, Choi JY, et al. Identification of the ␣v␤3 integrin-interacting motif of ␤ig-h3 and its anti-angiogenic effect. J Biol Chem 2003;278:25902–9. 12. Kim JE, Jeong HW, Nam JO, Lee BH, Choi JY, Park RW, et al. Identification of motifs in the fasciclin domains of the transforming growth factor-␤-induced matrix protein ␤ig-h3 that interact with the ␣v␤5 integrin. J Biol Chem 2002;277:46159–65. 13. Lee SH, Bae JS, Park SH, Lee BH, Park RW, Choi JY, et al. Expression of TGF-␤-induced matrix protein ␤ig-h3 is up-regulated in the diabetic rat kidney and human proximal tubular epithelial cells treated with high glucose. Kidney Int 2003;64: 1012–21. 14. Ha SW, Kim HJ, Bae JS, Jeong GH, Chung SC, Kim JG, et al. Elevation of urinary ␤ig-h3, transforming growth factor-␤-induced protein in patients with type 2 diabetes and nephropathy. Diabetes Res Clin Pract 2004;65:167–73. 15. Langham RG, Egan MK, Dowling JP, Gilbert RE, Thomson NM. Transforming growth factor-␤1 and tumor growth factor-␤-inducible gene-H3 in nonrenal transplant cyclosporine nephropathy. Transplantation 2001;72:1826–9. 16. Sun BK, Li C, Lim SW, Jung JY, Lee SH, Kim IS, et al. Expression of transforming growth factor-␤-inducible gene-h3 in normal and cyclosporine-treated rat kidney. J Lab Clin Med 2004;143:175–83. 17. Yun SJ, Kim MO, Kim SO, Park J, Kwon YK, Kim IS, et al. Induction of TGF-␤-inducible gene-h3 (␤ig-h3) by TGF-␤1 in astrocytes: implications for astrocyte response to brain injury. Brain Res Mol Brain Res 2002;107:57–64. 18. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 2744 19. Braun A, Takemura S, Vallejo AN, Goronzy JJ, Weyand CM. Lymphotoxin ␤–mediated stimulation of synoviocytes in rheumatoid arthritis. Arthritis Rheum 2004;50:2140–50. 20. Iwanaga T, Shikichi M, Kitamura H, Yanase H, Nozawa-Inoue K. Morphology and functional roles of synoviocytes in the joint. Arch Histol Cytol 2000;63:17–31. 21. Danen EH, Yamada KM. Fibronectin, integrins, and growth control. J Cell Physiol 2001;189:1–13. 22. Eliceiri BP. Integrin and growth factor receptor crosstalk. Circ Res 2001;89:1104–10. 23. Pap T, Muller-Ladner U, Gay RE, Gay S. Fibroblast biology: role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res 2000;2:361–7. 24. Clemmensen I, Holund B, Andersen RB. Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid. Arthritis Rheum 1983;26:479–85. 25. Vartio T, Vaheri A, Von Essen R, Isomaki H, Stenman S. Fibronectin in synovial fluid and tissue in rheumatoid arthritis. Eur J Clin Invest 1981;11:207–12. 26. Nikkari L, Haapasalmi K, Aho H, Torvinen A, Sheppard D, Larjava H, et al. Localization of the ␣v subfamily of integrins and their putative ligands in synovial lining cell layer. J Rheumatol 1995;22:16–23. 27. Lafyatis R, Thompson NL, Remmers EF, Flanders KC, Roche NS, Kim SJ, et al. Transforming growth factor-␤ production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats: studies on secretion by synovial fibroblast-like cells and immunohistologic localization. J Immunol 1989;143:1142–8. 28. Chu CQ, Field M, Abney E, Zheng RQ, Allard S, Feldmann M, et al. Transforming growth factor-␤1 in rheumatoid synovial membrane and cartilage/pannus junction. Clin Exp Immunol 1991;86: 380–6. 29. Brandes ME, Allen JB, Ogawa Y, Wahl SM. Transforming growth factor ␤1 suppresses acute and chronic arthritis in experimental animals. J Clin Invest 1991;87:1108–13. 30. Fava RA, Olsen NJ, Postlethwaite AE, Broadley KN, Davidson JM, Nanney LB, et al. Transforming growth factor ␤1 (TGF-␤1) induced neutrophil recruitment to synovial tissues: implications for TGF-␤-driven synovial inflammation and hyperplasia. J Exp Med 1991;173:1121–32. 31. Allen JB, Manthey CL, Hand AR, Ohura K, Ellingsworth L, Wahl SM. Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor ␤. J Exp Med 1990;171:231–47. 32. Wahl SM, Allen JB, Costa GL, Wong HL, Dasch JR. Reversal of NAM ET AL 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. acute and chronic synovial inflammation by anti-transforming growth factor ␤. J Exp Med 1993;177:225–30. Brandes ME, Wakefield LM, Wahl SM. Modulation of monocyte type I transforming growth factor-␤ receptors by inflammatory stimuli. J Biol Chem 1991;266:19697–703. Konttinen YT, Li TF, Hukkanen M, Ma J, Xu JW, Virtanen I. Fibroblast biology: signals targeting the synovial fibroblast in arthritis. Arthritis Res 2000;2:348–55. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 1996;134:793–9. Bae JS, Lee SH, Kim JE, Choi JY, Park RW, Yong Park J, et al. ␤ig-h3 supports keratinocyte adhesion, migration, and proliferation through ␣3␤1 integrin. Biochem Biophys Res Commun 2002;294:940–8. Zhang Z, Vuori K, Wang H, Reed JC, Ruoslahti E. Integrin activation by R-ras. Cell 1996;85:61–9. Hughes PE, Renshaw MW, Pfaff M, Forsyth J, Keivens VM, Schwartz MA, et al. Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 1997; 88:521–30. Takagi J, Springer TA. Integrin activation and structural rearrangement. Immunol Rev 2002;186:141–63. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct ␣v integrins. Science 1995;270:1500–2. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer 2002;2:91–100. Aplin AE, Howe AK, Juliano RL. Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol 1999;11:737–44. Hynes RO. Cell adhesion: old and new questions. Trends Cell Biol 1999;9:M33–7. Kobayashi T, Okamoto K, Kobata T, Hasunuma T, Kato T, Hamada H, et al. Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor ␣ and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules. Arthritis Rheum 2000;43:1106–14. Lafyatis R, Remmers EF, Roberts AB, Yocum DE, Sporn MB, Wilder RL. Anchorage-independent growth of synoviocytes from arthritic and normal joints: stimulation by exogenous plateletderived growth factor and inhibition by transforming growth factor-␤ and retinoids. J Clin Invest 1989;83:1267–76. Ferguson JW, Mikesh MF, Wheeler EF, LeBaron RG. Developmental expression patterns of ␤-ig (␤IG-H3) and its function as a cell adhesion protein. Mech Dev 2003;120:851–64.