Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 11, November 2004, pp 3495–3503 DOI 10.1002/art.20581 © 2004, American College of Rheumatology Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Mediates Matrix Metalloproteinase 3 Synthesis Enhanced by Oxidized Low-Density Lipoprotein in Rheumatoid Arthritis Cartilage Takumi Kakinuma,1 Tadashi Yasuda,2 Takefumi Nakagawa,3 Teruko Hiramitsu,3 Miki Akiyoshi,3 Masao Akagi,4 Tatsuya Sawamura,5 and Takashi Nakamura3 bation with neutralizing anti–LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. Results. The majority of the RA chondrocytes stained positively with both anti–LOX-1 and anti–oxLDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti–LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti–LOX-1 antibody reversed ox-LDL–stimulated MMP-3 synthesis to control levels. Conclusion. Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor–ligand interaction in cartilage pathology in RA. Objective. To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. Methods. Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincuDr. Yasuda’s work was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science. Dr. Sawamura’s work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Senri Life Science Foundation; Mitsubishi Pharma Foundation; and Suzuki Memorial Foundation. 1 Takumi Kakinuma, MD: Kyoto University Graduate School of Medicine, Kyoto, Japan, and National Cardiovascular Center Research Institute, Osaka, Japan; 2Tadashi Yasuda, MD, PhD: Kyoto University Graduate School of Medicine, Kyoto, Japan, and Faculty of Health, Budo, and Sports Studies, Tenri University, Tenri, Japan; 3 Takefumi Nakagawa, MD, PhD, Teruko Hiramitsu, MD, Miki Akiyoshi, MD, Takashi Nakamura, MD, PhD: Kyoto University Graduate School of Medicine, Kyoto, Japan; 4Masao Akagi, MD, PhD: Kinki University School of Medicine, Osaka, Japan; 5Tatsuya Sawamura, MD, PhD: National Cardiovascular Center Research Institute, Osaka, Japan. Address correspondence and reprint requests to Tadashi Yasuda, MD, PhD, Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: firstname.lastname@example.org. Submitted for publication October 20, 2003; accepted in revised form July 13, 2004. Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and destruction of cartilage and bone around the inflamed joint. Lipid metabolism in RA patients has been reported to differ from that in healthy subjects (1), and it might be one of the causes of a high incidence of cardiovascular disease in such patients. The lipids and lipoproteins in synovial fluid from inflamed joints of RA patients are oxidized (2–4), as is fatty acid in oxidized serum (5). It has also been shown that oxidized lowdensity lipoprotein (ox-LDL) accumulates in inflamed 3495 3496 joints as foam cells (6) similar to those found in vascular atherosclerotic lesions. In the formation of atherosclerotic plaques, inflammatory cytokines, including interleukin-1 (IL-1), control immune-mediated inflammatory processes, which result in the accumulation of ox-LDL at the lesions (7). Lipid peroxidation and oxLDL affect gene expression in endothelial cells and atherogenic changes in vivo. Recent studies have implicated the involvement of lipid peroxides in cartilage degradation (8) because lipid peroxides cause structural destabilization of cartilage matrix (9). In addition, reactive oxygen species (ROS) mediate collagenase expression by IL-1␤ in chondrocytes (10). Thus, the relationship between RA and atherosclerosis has been recognized with regard to immunologic aspects as well as lipid metabolism and redox status (8). Although ox-LDL apparently plays a central role in the pathogenesis of atherosclerosis (11,12), there are no clear data on the pathologic roles of ox-LDL in RA. Lectin-like ox-LDL receptor 1 (LOX-1), originally cloned from vascular endothelial cells, is one of the ox-LDL receptors. This 273–amino acid molecule has been named OLR1 (oxidized LDL) by the Human Genome Organisation Gene Nomenclature Committee at the University College London. It is a singletransmembrane protein with the carboxyl-terminal end containing an extracytoplasmic C-type lectin domain (13). LOX-1 is expressed in various cells, including endothelial cells, macrophages, vascular smooth muscle cells, and dendritic cells, and its expression is enhanced by oxidative stress, stimulation by mediators of inflammation such as IL-1␤ and tumor necrosis factor ␣ (TNF␣) (13–16), and fluid shear stress (17). LOX-1 has been implicated in the initiation and development of atherosclerosis, the promotion of phagocytosis of aged or apoptotic cells by endothelium, and antigen processing in dendritic cells. We have recently demonstrated that ox-LDL up-regulates LOX-1 messenger RNA (mRNA) in rat articular chondrocytes in vitro (18). Using a rat zymosan-induced arthritis (ZIA) model, we have also shown that LOX-1 promotes joint inflammation and cartilage destruction with the accumulation of ox-LDL in articular chondrocytes (19). Currently, however, the presence of LOX-1 in human RA cartilage remains unclear. Matrix metalloproteinases (MMPs) are the critical enzymes involved in the destruction of articular cartilage in various arthritic diseases, including RA and osteoarthritis (OA). Among the MMPs, MMP-3 is active against cartilage matrix components such as proteoglycan and fibronectin, and can activate proMMPs (20). KAKINUMA ET AL Higher levels of MMP-3 (stromelysin 1) are found in the synovial fluid of RA patients than in that of OA patients (21). Serum levels of MMP-3 have been shown to correlate with the severity of RA (22,23). The present study was designed to identify LOX-1 expression in chondrocytes in RA cartilage and elucidate the pathogenic role of ox-LDL binding to LOX-1 in MMP-3 production by chondrocytes. We showed that the association between ox-LDL and LOX-1, both of which were elevated in RA cartilage, enhanced MMP-3 production in articular cartilage explant culture. MATERIALS AND METHODS Reagents and antibodies. Phosphate buffered saline (PBS), 10% neutralized formalin solution, and ionophore monensin were purchased from Sigma-Aldrich (Tokyo, Japan). Biotin-blocking reagent for immunofluorescence study was obtained from Dako (Kyoto, Japan). Block Ace for immunostaining and immunoblotting was obtained from Dainippon (Osaka, Japan). Rabbit anti-mouse IgG antibody conjugated with Alexa Fluor 488, streptavidin–Alexa Fluor 488, and DiIC18(3) were purchased from Molecular Probes (Eugene, OR). Mouse monoclonal anti–ox-LDL antibody (clone OXL 41.1) was obtained from NeoMarkers (Fremont, CA). Control mouse IgG, rabbit anti–MMP-3 antibody, and goat anti-rabbit IgG antibody conjugated with alkaline phosphatase were purchased from Dako. Human recombinant IL-1␤ was obtained from R&D Systems (Minneapolis, MN). Preparation of LDL and ox-LDL. Human LDL (density 1.019–1.063 gm/ml) was isolated from fresh donated plasma by sequential ultracentrifugation, as previously described (13). Briefly, the fresh plasma was centrifuged twice in a KBr gradient solution for 16 hours at 4°C to obtain native LDL. After dialysis in PBS overnight at 4°C, the native LDL solution was reacted with 7.5 M CuSO4 for 20 hours at 37°C for oxidation. The oxidative state was confirmed by measuring the amount of thiobarbituric acid–resistant substances (⬃10 nmoles malondialdehyde equivalent per mg protein in oxLDL). Agarose gel electrophoresis showed increased electrophoretic mobility and minimal aggregation of ox-LDL particles. DiI was conjugated with ox-LDL according to the manufacturer’s instructions (Molecular Probes). Our previous study showed that the DiI-labeled ox-LDL prepared with CuSO4 is actively taken up by Chinese hamster ovary cells that express LOX-1 (13). Articular cartilage explant culture. Human articular cartilage with no significant arthritic changes was obtained from non–weight-bearing regions of the femoral head from patients undergoing replacement surgery for femoral neck fracture. OA cartilage specimens were obtained from non– weight-bearing areas of the distal femur and the proximal tibia from patients undergoing total knee replacement surgery who were diagnosed as having OA based on the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) criteria (24). RA cartilage specimens were obtained from non–weight-bearing regions of the posterior con- ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA dyles of the distal femur during total knee replacement surgery in patients who fulfilled the ACR 1987 revised criteria (25). The cartilage was transferred to 24-well plates (⬃100 mg/well; Corning, Corning, NY) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 mM HEPES buffer, 100 units/ml penicillin, 100 units/ml streptomycin (all from Gibco BRL, Grand Island, NY), and 3.7 gm/liter NaHCO3. The cartilage was precultured in serum-free DMEM for 2 days at 37°C in a humidified atmosphere of 5% CO2/95% air, with or without pretreatment of cartilage explant with 15 g/ml neutralizing anti–LOX-1 antibody (JTX-92) or with 15 g/ml normal mouse IgG for 24 hours. After preculture, the cartilage pieces were incubated for 2 days in serum-free DMEM supplemented with DiI-labeled ox-LDL (10 g/ml). To measure MMP-3 in culture medium, articular cartilage was preincubated in the presence or absence of 15 g/ml neutralizing anti–LOX-1 antibody or control mouse IgG for 24 hours, followed by coincubation with ox-LDL (10, 40, or 100 g/ml) or native LDL (150 g/ml) for 5 days. The conditioned medium was collected and stored at ⫺20°C. Measurement of the activity of lactate dehydrogenase (LDH). The effects of ox-LDL on cell viability were determined by measuring LDH activity released into the culture medium (LDH C II test kit; Wako, Osaka, Japan). The assay was performed in accordance with the manufacturer’s protocol using cartilage explant cultures in the presence or absence of ox-LDL for 5 days. Immunofluorescence study. Articular cartilage specimens were obtained as described above. The cartilage samples were fixed immediately after harvest in 10% neutralized formalin and frozen in Tissue-Tek OCT compound (Sakura, Westhaven, CA). Frozen sections of 8 m thickness were prepared with a cryostat (CM1850; Leica Microsystems, Wetzlar, Germany) on silane-coated glass plates (Matsunami Glass, Osaka, Japan). The specimens were air-dried and nonspecific reactivity was blocked by treatment with Block Ace. The sections were reacted with mouse monoclonal anti–ox-LDL antibody (1:100 in PBS–10% Block Ace) at room temperature for 1 hour, and thereafter with secondary anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1:1,000 in PBS) at room temperature for 1 hour. To visualize LOX-1 in chondrocytes, the specimens were treated with a biotin-blocking reagent before reacting with biotinylated anti-human LOX-1 monoclonal antibody or control mouse IgG (15 g/ml in PBS–10% Block Ace), followed by streptavidin–Alexa Fluor 488 (1:1,000 in PBS). To detect LOX-1 and MMP-3 by immunofluorescence analysis, the cartilage was incubated with ox-LDL or native LDL for 48 hours in a 24-well plate (⬃100 mg/well) in DMEM after preculture for 2 days. The ionophore monensin was added to the cultures at 5 M for the last 12 hours to prevent the secretion of newly synthesized proteins. Cryostat sections were permeabilized for 10 minutes at room temperature with 0.15% Triton X-100 in PBS, and then incubated with biotinylated anti-human LOX-1 monoclonal antibody (15 g/ml in PBS–10% Block Ace) followed by streptavidin–Alexa Fluor 488 (1:1,000 in PBS), or alternatively with rabbit anti–MMP-3 antibody (1:200 in PBS–10% Block Ace) followed by secondary anti-rabbit antibody conjugated with Alexa Fluor 488 (1:1,000 in PBS). The specimens were washed with PBS–0.05% 3497 Tween 20 for 20 minutes between each procedure. Images were obtained with a FluoView confocal microscope (Olympus, Tokyo, Japan) and prepared with Photoshop software (Adobe Systems, San Jose, CA). Evaluation of MMP-3. The collected medium, normalized by the wet weight of the cartilage piece in each well for culture, was separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis with 4–20% gradient gel (Daiichi Pharmaceutical, Tokyo, Japan). The proteins were electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA), which were immersed in methanol and air-dried to block nonspecific reactivity, followed by reacting with rabbit anti–MMP-3 antibody (1 g/ml) at 4°C overnight. After an extensive wash with PBS– Tween 20, the membranes were incubated with goat anti-rabbit antibody conjugated with alkaline phosphatase (0.5 g/ml), followed by the addition of nitroblue tetrazolium/BCIP to visualize MMP-3. The membranes containing MMP-3 were scanned and the intensity of the protein band was quantified using Photoshop and Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The experiments were repeated 4 times. In each experiment, the signal intensity of the samples was divided by that of the normal control to yield a normalized value for the relative signal intensity. MMP-3 levels in the conditioned medium were also evaluated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (The Binding Site, Birmingham, UK) for proMMP-3 according to the manufacturer’s instructions. Statistical analysis. Comparisons of the relative protein band intensities of immunoblotting for MMP-3 and the determinations of MMP-3 by ELISA were analyzed using Student’s t-tests with StatView software (SAS Institute, Cary, NC). Data are expressed as the mean ⫾ SD. P values less than 0.05 were considered significant. RESULTS Expression of LOX-1 in chondrocytes in RA cartilage. Initially, the level of LOX-1 expression was determined in chondrocytes in RA cartilage. Articular cartilage specimens that were obtained from 3 RA patients were incubated with anti–LOX-1 antibody and streptavidin–Alexa Fluor 488. Immunofluorescence microscopic analyses demonstrated intense immunolocalization of LOX-1 in association with chondrocytes in the 2 of 3 RA cartilage specimens (Figure 1). LOX-1 was localized in chondrocytes throughout the RA cartilage, while the signal intensity of LOX-1 appeared to be strong near the articular surface. The remaining specimen showed weak but clear localization of LOX-1 in some chondrocytes, whereas when OA and normal cartilage specimens were examined, no immunoreactive staining was found with anti–LOX-1 antibody. Control nonspecific mouse IgG yielded no positive staining in chondrocytes. 3498 Figure 1. Identification of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) on chondrocytes in rheumatoid arthritis (RA) cartilage. Cryostat sections of RA, osteoarthritis (OA), and normal cartilage were stained with biotinylated mouse monoclonal anti– LOX-1 antibody and streptavidin–Alexa Fluor 488. Articular surfaces are at the top. Shown are RA and OA cartilage specimens obtained from 3 patients and a representative of 3 normal specimens. Nonspecific mouse IgG was used as a negative control. Note that the fluorescent signals around the chondrocyte lacunae in the deep layer were artifactual. Bar ⫽ 200 m. Association of ox-LDL with chondrocytes in RA cartilage. The cartilage specimens used for LOX-1 staining were also examined for ox-LDL by immunofluorescence microscopic studies with mouse monoclonal anti– ox-LDL antibody and secondary anti-mouse IgG conjugated with Alexa Fluor 488. In all 3 RA specimens, strong staining of ox-LDL was observed in chondrocytes (Figure 2). In contrast, no detectable ox-LDL was found in OA or normal cartilage specimens. Control mouse IgG showed no positive staining (results not shown). Effects of neutralizing anti–LOX-1 antibody on ox-LDL binding to chondrocytes. Further experiments were performed to clarify the involvement of LOX-1 in binding of ox-LDL to chondrocytes. When normal cartilage explants were incubated with 10 g/ml DiI-labeled ox-LDL for 48 hours following preculture in serum-free DMEM, confocal microscopic images showed that the DiI-labeled ox-LDL penetrated the cartilage matrix and associated with chondrocytes in a punctated pattern (Figure 3A). The association of ox-LDL was more apparent in chondrocytes near the surface of the cartilage explants. Preincubation of articular cartilage with KAKINUMA ET AL Figure 2. Identification of oxidized low-density lipoprotein (ox-LDL) in rheumatoid arthritis (RA) cartilage. Cryostat sections of RA, osteoarthritis (OA), and normal cartilage were stained with mouse monoclonal anti–ox-LDL antibody and Alexa Fluor 488–conjugated anti-mouse secondary antibody. Shown are RA and OA cartilage specimens obtained from 3 patients and a representative of 3 normal specimens. Bar ⫽ 100 m. neutralizing anti–LOX-1 antibody at 15 g/ml for 24 hours before addition of DiI-labeled ox-LDL completely blocked the accumulation of DiI-labeled ox-LDL in chondrocytes (Figure 3B). In contrast, control IgG failed to inhibit ox-LDL accumulation (Figure 3C). These findings suggest that LOX-1 principally mediates the binding of ox-LDL to articular chondrocytes, which is consistent with our previous observation using rat articular chondrocytes (18). Figure 3. Association of oxidized low-density lipoprotein (ox-LDL) with chondrocytes via lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1). Normal cartilage was incubated with DiI-labeled ox-LDL at 10 g/ml under serum-free conditions for 48 hours, fixed, and examined by confocal microscopy (A). Following preincubation with neutralizing anti–LOX-1 antibody at 15 g/ml (B) or control mouse IgG at 15 g/ml (C) for 24 hours, cartilage was incubated for 48 hours with DiI-labeled ox-LDL at 10 g/ml in the presence of the antibody at the same concentration. Bars ⫽ 20 m. ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA Figure 4. Induction of LOX-1 in ox-LDL–stimulated chondrocytes in cartilage explant culture. Normal cartilage specimens were incubated for 2 days with no additives (A), with native LDL at 150 g/ml (B), or with ox-LDL at 40 g/ml (C). The ionophore monensin was added for the final 12 hours. Cryostat sections were stained with anti–LOX-1 monoclonal antibody and streptavidin–Alexa Fluor 488. Bar ⫽ 50 m. See Figure 3 for definitions. Induction of LOX-1 in ox-LDL–stimulated chondrocytes. To examine the effects of ox-LDL on LOX-1 in explant culture, normal cartilage was incubated with or without ox-LDL at 40 g/ml or with native LDL at 40 or 150 g/ml for 2 days. The ionophore monensin was used to prevent the secretion of newly synthesized proteins. Immunofluorescence analysis with anti–LOX-1 monoclonal antibody revealed that LOX-1 was rarely found in untreated chondrocytes (Figure 4A) or in those treated with native LDL (Figure 4B). In contrast, ox-LDL enhanced LOX-1 accumulation in chondrocytes in normal cartilage explant culture (Figure 4C). Enhanced production of MMP-3 in ox-LDL– stimulated chondrocytes. Finally, the functional role of the interaction between ox-LDL and LOX-1 was investigated. Normal articular cartilage was incubated with ox-LDL at 40 g/ml or with native LDL at 40 or 150 g/ml, and the ionophore monensin was added 12 hours before fixation. IL-1␤ (2 ng/ml) was used as a positive control to detect MMP-3. Since monensin caused intracellular accumulation of newly synthesized MMP-3 by blocking the secretion of proteins, immunofluorescence microscopic analyses with anti–MMP-3 antibody demonstrated that IL-1␤ treatment resulted in a marked increase in MMP-3 in chondrocytes (Figure 5A). Control cultures with no additives showed no positive staining of MMP-3 (Figure 5E). Ox-LDL at 40 g/ml also stimulated the intracellular accumulation of MMP-3 (Figure 5C). In contrast, native LDL at 40 or 150 g/ml failed to induce MMP-3 (Figure 5D). When articular cartilage was pretreated with neutralizing anti–LOX-1 antibody at 15 g/ml, we found no enhanced accumulation of MMP-3 induced by ox-LDL at 40 g/ml (Figure 5B). Control mouse IgG failed to alter the effect of ox-LDL on enhanced synthesis of MMP-3 (results not shown). Levels of secreted MMP-3 after treatment with 3499 ox-LDL were also evaluated by immunoblotting with anti–MMP-3 antibody. When normal cartilage was incubated with ox-LDL at 10, 40, or 100 g/ml, we found that 40 g/ml of ox-LDL was sufficient to induce increased secretion of MMP-3 into conditioned medium (Figure 6A). Levels of secreted MMP-3 induced by ox-LDL at 40 and 100 g/ml (mean ⫾ SD 2.07 ⫾ 1.17 and 2.22 ⫾ 1.18, respectively) (Figure 6B, lanes 4 and 5) were significantly (P ⬍ 0.05) higher than those induced by native LDL (0.91 ⫾ 0.07) (Figure 6B, lane 2). There was a tendency for ox-LDL at 10 g/ml to increase MMP-3 production (1.44 ⫾ 1.41) (Figure 6B, lane 3), although it was not statistically significant compared with native LDL. Preincubation with neutralizing anti–LOX-1 antibody at 15 g/ml almost completely blocked enhanced MMP-3 secretion by ox-LDL at 40 g/ml (0.92 ⫾ 0.41; P ⬍ 0.05 versus ox-LDL at 40 g/ml) (Figure 6B, lane 6). Control IgG had no effect on increased MMP-3 secretion induced by ox-LDL at 40 g/ml (mean ⫾ SD 2.13 ⫾ 1.85; data not shown). IL-1␤ at 2 ng/ml caused a stronger secretion of MMP-3 (5.62 ⫾ 1.92) (Figure 6B, lane 7) compared with the levels induced by ox-LDL treatment and controls (P ⬍ 0.01). MMP-3 levels in the conditioned medium determined by ELISA (Figure 6C) were consistent with the results of immunoblot analysis (Figure 6B). Compared with the level in LDL-treated cultures (5.12 ⫾ 1.16 ng/ml/mg of cartilage), treatment with ox-LDL at 40 Figure 5. Identification of enhanced matrix metalloproteinase 3 (MMP-3) production mediated by LOX-1 in ox-LDL–stimulated cartilage explant culture. Normal cartilage specimens were incubated with interleukin-1␤ (IL-1␤) at 2 ng/ml (A), with 40 g/ml ox-LDL with (B) or without (C) anti–LOX-1 antibody at 15 g/ml, with 150 g/ml native LDL (D), or with no additives (control) (E) for 2 days. The ionophore monensin was added for the final 12 hours. Cryostat sections were stained with anti–MMP-3 antibody. Bar ⫽ 100 m. See Figure 3 for other definitions. 3500 KAKINUMA ET AL Figure 6. Evaluation of enhanced matrix metalloproteinase 3 (MMP-3) production in ox-LDL–stimulated cartilage explant culture. A and B, Normal cartilage was incubated for 5 days with no additives (control, lane 1), with 150 g/ml native LDL (lane 2), with 10, 40, or 100 g/ml ox-LDL (lanes 3, 4, and 5, respectively), with 40 g/ml ox-LDL with 15 g/ml anti–LOX-1 antibody (lane 6), or with 2 ng/ml interleukin-1␤ (IL-1␤) (lane 7). Conditioned medium was subjected to immunoblotting with anti–MMP-3 antibody. The amount of sample applied for sodium dodecyl sulfate–polyacrylamide gel electrophoresis was normalized with the wet weight of the cartilage specimen in each well. Four separate experiments were performed, with similar results. A representative result is shown in A. The relative signal intensity of the protein band for MMP-3 was determined against a control. Values in B are the mean ⫾ SD of 4 determinations. ⴱ ⫽ P ⬍ 0.05 versus lanes 1, 2, and 6; # ⫽ P ⬍ 0.05 versus lane 4; ⴱⴱ ⫽ P ⬍ 0.01 versus all the other lanes. C, Normal cartilage was incubated for 5 days with no additives (control), with 150 g/ml native LDL, 40 g/ml ox-LDL, 40 g/ml ox-LDL with 15 g/ml anti–LOX-1 antibody, or 2 ng/ml IL-1␤. MMP-3 protein levels in the conditioned medium were determined by enzyme-liked immunosorbent assay. Values are the mean ⫾ SD of 4 determinations. ⴱ ⫽ P ⬍ 0.05 versus native LDL–treated cultures; ⴱⴱ ⫽ P ⬍ 0.05 versus ox-LDL–treated cultures, by t-test. See Figure 3 for other definitions. g/ml resulted in a significant increase in MMP-3 (13.6 ⫾ 3.90 ng/ml/mg cartilage; P ⬍ 0.05), which was significantly reversed with preincubation with anti– LOX-1 antibody at 15 g/ml (8.12 ⫾ 2.56 ng/ml/mg cartilage; P ⬍ 0.05). When LDH levels in the conditioned medium were assayed after treatment with or without ox-LDL at 40 or 100 g/ml for 5 days, there was no significant difference between the cultures with and without oxLDL treatment (data not shown). DISCUSSION It has been suggested that lipid peroxidation is involved in the pathogenesis of arthritis. In contrast to synovial fluid from healthy subjects, RA synovial fluid contains large amounts of lipoproteins (26). The concentration of lipoproteins in synovial fluid from an RA patient increases by ⬃50% over that in the serum from the same patient, in contrast to ⬍10% in healthy synovial fluid (27,28), possibly because lipoproteins can easily permeate the synovial membrane in RA joints (28). In addition, cholesterol crystals in synovial fluid are occasionally found in RA patients (29,30). LDL can be oxidatively modified under inflammatory conditions in vivo (31). Accordingly, the lipids and lipoproteins in RA synovial fluid can be oxidized. Indeed, ox-LDL has been identified in RA synovial fluid (2–4), although ox-LDL concentration has not yet been determined because no standard quantitative assay has been developed for ox-LDL generated in vivo. ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA There are no reports in the current literature showing the presence of ox-LDL in RA cartilage. Consistent with our previous findings in articular cartilage of rats with ZIA (19), this is the first study to demonstrate that ox-LDL associates with articular chondrocytes in human RA cartilage. Because even such a large molecule as hyaluronan (HA) can penetrate cartilage matrix after IL-1 treatment (32), ox-LDL penetration could occur in degraded RA cartilage. We have also shown that fluorescent dye–labeled ox-LDL permeates the cartilage matrix and binds to chondrocytes. Similarly, HA penetrates normal cartilage explants and binds to the cells (33), although there is no clear explanation as to why HA can penetrate articular cartilage. Thus, it is likely that LDL diffusing from serum into joint fluid could be modified oxidatively in the inflamed joint cavity and the resultant ox-LDL could penetrate cartilage matrix and associate with chondrocytes. The ox-LDL receptor, LOX-1, is mainly expressed in vivo in vascular endothelial cells and vascularrich organs such as the placenta and lungs (13). Although we have already shown that ox-LDL induces LOX-1 expression in rat chondrocytes in monolayer culture (18), the presence of LOX-1 in cartilage, which is an avascular tissue, has never been examined. The current study is the first to identify LOX-1 protein in chondrocytes in RA cartilage. Various factors are known to regulate the expression of LOX-1 in endothelial cells (34). Shear stress can up-regulate LOX-1 protein and mRNA in the cells (17). In addition, mechanical strain such as cyclic tensile stretch enhances ROS generation in chondrocytes (35), which may lead to cartilage degradation. Thus, it is possible that mechanical stress may induce LOX-1 in chondrocytes in the weight-bearing regions of cartilage. In contrast to normal and OA cartilage from non– weight-bearing regions, we found that chondrocytes expressed LOX-1 in non–weight-bearing areas in RA cartilage (Figure 1). This indicates that LOX-1 could be up-regulated in RA cartilage through other mechanism(s). The present study demonstrated that LOX-1 can be induced by its ligand, ox-LDL, in chondrocytes (Figure 4), consistent with previous findings using rat primary chondrocytes (18) and human coronary artery endothelial cells (36). Therefore, increased expression of LOX-1 in RA cartilage may involve such an induction mechanism. There is evidence that elevated MMP-3 levels in the serum and synovium from RA patients are an indicator of inflammation and are associated with joint damage. In RA synovial fluid, MMP-3 levels are signif- 3501 icantly higher than those in OA (21). Furthermore, the serum levels of MMP-3 are related to the activity of RA and predict the outcome of early RA (22,23,37,38). In human articular chondrocytes, MMP-3 can be induced by IL-1␤, TNF␣, phorbol myristate acetate, and histamine (39,40). Recent studies have demonstrated that ox-LDL enhances the expression of MMP-1 and MMP-3 via LOX-1 in human coronary artery endothelial cells in vitro (41). This study is the first to show that ox-LDL can up-regulate MMP-3 production in normal articular chondrocytes (Figure 5). While LOX-1 in chondrocytes was undetectable with immunofluorescence staining in normal cartilage immediately after harvest (Figure 1) or in explant culture (Figure 4), LOX-1 can be induced by ox-LDL stimulation in normal cartilage explant culture (Figure 4). Our previous study also showed LOX-1 upregulation by ox-LDL in monolayer chondrocytes that constitutively express the receptor without ox-LDL stimulation (18). Pretreatment with anti–LOX-1 antibody resulted in significant suppression of ox-LDL– stimulated MMP-3 production (Figures 5 and 6). Taken together, these findings suggest that LOX-1 could mediate ox-LDL action on MMP-3, even in normal cartilage. Thus, intense localization of LOX-1 and ox-LDL in RA cartilage (Figures 1 and 2) indicates that ox-LDL may contribute to MMP-3 induction through LOX-1 in RA chondrocytes. In addition to MMP-3, MMP-13 is thought to play a major role in cartilage destruction through type II collagen cleavage in diseased cartilage (42,43). Immunoblot analysis using the same samples as with MMP-3 revealed that MMP-13 from ox-LDL– stimulated cartilage explants was below detectable levels (data not shown). Further studies are required to elucidate the effects of ox-LDL on MMP-13 as well as other MMPs in cartilage. Information regarding the pathophysiologic roles of LOX-1 is accumulating. Physiologically, LOX-1 may work as a scavenger or remove cellular debris and other related materials (44,45). Under pathologic conditions, the binding of LOX-1 to ox-LDL and cellular ligands may result in the activation of endothelial cells, transformation of smooth muscle cells, and accumulation of lipids in macrophages, which are all involved in the promotion of atherosclerosis (45–47). In addition, the receptor–ligand interaction may cause cell death. OxLDL at 10–100 g/ml, the concentrations that stimulate MMP-1 and MMP-3 expression in human coronary artery endothelial cells (41), induces apoptosis of cells (36) in association with NF-B activation (48). Ox-LDL 3502 KAKINUMA ET AL at 40–100 g/ml, the concentrations used in the present study, which induce MMP-3 (Figure 5), causes nonapoptotic cell death through the Akt pathway in rat articular chondrocytes in monolayer culture (18). In contrast, the present finding that ox-LDL failed to alter LDH activity suggests that ox-LDL ligation with LOX-1 caused little cytotoxic effect on human chondrocytes in cartilage explant culture. While ox-LDL can penetrate cartilage matrix (Figure 3), ox-LDL added in the culture medium could not reach chondrocytes surrounded by cartilage matrix in explants as readily as in monolayer culture. In RA and OA, loss of chondrocytes is found initially at the articular surface and later throughout the cartilage (49). Degraded cartilage matrix in RA and OA could provide easier access of ox-LDL to LOX-1. Thus, reduced cellularity in diseased cartilage may involve the interaction between ox-LDL and LOX-1. This is currently being investigated. In rat ZIA, intravenous administration of anti– LOX-1 antibody suppressed cartilage destruction, possibly by blocking LOX-1 on the endothelium of synovial vessels, resulting in a decrease in leukocyte infiltration into the arthritic joints (19). Foam cells containing ox-LDL are found in the synovium in RA (6), and macrophage-like type A synoviocytes from RA patients could take up acetylated LDL (50). Lipoproteins that are increased in RA synovial fluid are assumed to have some role in the development of synovitis (51). However, LOX-1 expression in RA synovium has not yet been investigated. Further study of the levels of LOX-1 in RA synovium is needed for increased understanding of the pathologic roles of ox-LDL and LOX-1 in RA joints. ACKNOWLEDGMENTS The authors thank Naoko Honda for technical assistance, and Lisbeth Stewart for critical reading. We thank the Japanese Red Cross Society for providing us with fresh plasma for the experiments. REFERENCES 1. Park YB, Lee SK, Lee WK, Suh CH, Lee CW, Lee CH, et al. Lipid profiles in untreated patients with rheumatoid arthritis. J Rheumatol 1999;26:1701–4. 2. James MJ, van Reyk D, Rye KA, Dean RT, Cleland LG, Barter PJ, et al. Low density lipoprotein of synovial fluid in inflammatory joint disease is mildly oxidized. Lipids 1998;33:1115–21. 3. Dai L, Zhang Z, Winyard PG, Gaffney K, Jones H, Blake DR, et al. A modified form of low-density lipoprotein with increased electronegative charge is present in rheumatoid arthritis synovial fluid. Free Radic Biol Med 1997;22:705–10. 4. Dai L, Lamb DJ, Leake DS, Kus ML, Jones HW, Morris CJ, et al. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients. Free Radic Res 2000;32: 479–86. Jira W, Spiteller G, Richter A. Increased levels of lipid oxidation products in low density lipoproteins of patients suffering from rheumatoid arthritis. Chem Phys Lipids 1997;87:81–9. Winyard PG, Tatzber F, Esterbauer H, Kus ML, Blake DR, Morris CJ. Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis. Ann Rheum Dis 1993;52:677–80. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance [review]. J Biol Chem 1997;272:20963–6. Henrotin YE, Bruckner P, Pujol JP. The role of reactive oxygen species in homeostasis and degradation of cartilage [review]. Osteoarthritis Cartilage 2003;11:747–55. Tiku ML, Shah R, Allison GT. Evidence linking chondrocyte lipid peroxidation to cartilage matrix protein degradation: possible role in cartilage aging and the pathogenesis of osteoarthritis. J Biol Chem 2000;275:20069–76. Lo YY, Conquer JA, Grinstein S, Cruz TF. Interleukin-1␤ induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species. J Cell Biochem 1998;69: 19–29. Klatt P, Esterbauer H. Oxidative hypothesis of atherogenesis [review]. J Cardiovasc Risk 1996;3:346–51. Yla-Herttuala S. Oxidized LDL and atherogenesis [review]. Ann N Y Acad Sci 1999;874:134–7. Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, et al. An endothelial receptor for oxidized low-density lipoprotein. Nature 1997;386:73–7. Yoshida H, Kondratenko N, Green S, Steinberg D, Quehenberger O. Identification of the lectin-like receptor for oxidized lowdensity lipoprotein in human macrophages and its potential role as a scavenger receptor. Biochem J 1998;334:9–13. Kataoka H, Kume N, Miyamoto S, Minami M, Morimoto M, Hayashida K, et al. Oxidized LDL modulates Bax/Bcl-2 through the lectinlike Ox-LDL receptor-1 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2001;21:955–60. Delneste Y, Magistrelli G, Gauchat J, Haeuw J, Aubry J, Nakamura K, et al. Involvement of LOX-1 in dendritic cell-mediated antigen cross-presentation. Immunity 2002;17:353–62. Murase T, Kume N, Korenaga R, Ando J, Sawamura T, Masaki T, et al. Fluid shear stress transcriptionally induces lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res 1998;83:328–33. Nakagawa T, Yasuda T, Hoshikawa H, Shimizu M, Kakinuma T, Chen M, et al. LOX-1 expressed in cultured rat chondrocytes mediates oxidized LDL-induced cell death-possible role of dephosphorylation of Akt. Biochem Biophys Res Commun 2002;299: 91–7. Nakagawa T, Akagi M, Hoshikawa H, Chen M, Yasuda T, Mukai S, et al. Lectin-like oxidized low-density lipoprotein receptor 1 mediates leukocyte infiltration and articular cartilage destruction in rat zymosan-induced arthritis. Arthritis Rheum 2002;46: 2486–94. Nagase H. Activation mechanisms of matrix metalloproteinases [review]. Biol Chem 1997;378:151–60. Yoshihara Y, Nakamura H, Obata K, Yamada H, Hayakawa T, Fujikawa K, et al. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Ann Rheum Dis 2000;59:455–61. Catrina AI, Lampa J, Ernestam S, af Klint E, Bratt J, Klareskog L, et al. Anti-tumour necrosis factor (TNF)-␣ therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis. Rheumatology (Oxford) 2002;41: 484–9. Green MJ, Gough AK, Devlin J, Smith J, Astin P, Taylor D, et al. ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Serum MMP-3 and MMP-1 and progression of joint damage in early rheumatoid arthritis. Rheumatology (Oxford) 2003;42:83–8. Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheum 1986;29:1039–49. 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. Prete PE, Gurakar-Osborne A, Kashyap ML. Synovial fluid lipoproteins: review of current concepts and new directions [review]. Semin Arthritis Rheum 1993;23:79–89. Ananth L, Prete PE, Kashyap ML. Apolipoproteins A-I and B and cholesterol in synovial fluid of patients with rheumatoid arthritis. Metabolism 1993;42:803–6. Fairburn K, Grootveld M, Ward RJ, Abiuka C, Kus M, Williams RB, et al. Alpha-tocopherol, lipids and lipoproteins in knee-joint synovial fluid and serum from patients with inflammatory joint disease. Clin Sci (Lond) 1992;83:657–64. Ettlinger RE, Hunder GG. Synovial effusions containing cholesterol crystals report of 12 patients and review. Mayo Clin Proc 1979;54:366–74. Lazarevic MB, Skosey JL, Vitic J, Mladenovic V, Myones BL, Popovic J, et al. Cholesterol crystals in synovial and bursal fluid. Semin Arthritis Rheum 1993;23:99–103. Memon RA, Staprans I, Noor M, Holleran WM, Uchida Y, Moser AH, et al. Infection and inflammation induce LDL oxidation in vivo. Arterioscler Thromb Vasc Biol 2000;20:1536–42. Fukuda K, Dan H, Takayama M, Kumano F, Saitoh M, Tanaka S. Hyaluronic acid increases proteoglycan synthesis in bovine articular cartilage in the presence of interleukin-1. J Pharmacol Exp Ther 1996;277:1672–5. Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T. Inhibition of interleukin-1␤–stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum 2004;50:516–25. Chen M, Masaki T, Sawamura T. LOX-1, the receptor for oxidized low-density lipoprotein identified from endothelial cells: implications in endothelial dysfunction and atherosclerosis [review]. Pharmacol Ther 2002;95:89–100. Yamazaki K, Fukuda K, Matsukawa M, Hara F, Matsushita T, Yamamoto N, et al. Cyclic tensile stretch loaded on bovine chondrocytes causes depolymerization of hyaluronan: involvement of reactive oxygen species. Arthritis Rheum 2003;48:3151–8. Li D, Mehta JL. Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors. Arterioscler Thromb Vasc Biol 2000;20:1116–22. Ichikawa Y, Yamada C, Horiki T, Hoshina Y, Uchiyama M. Serum matrix metalloproteinase-3 and fibrin degradation product levels correlate with clinical disease activity in rheumatoid arthritis. Clin Exp Rheumatol 1998;16:533–40. Yoshihara Y, Obata K, Fujimoto N, Yamashita K, Hayakawa T, 3503 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. Shimmei M. Increased levels of stromelysin-1 and tissue inhibitor of metalloproteinases-1 in sera from patients with rheumatoid arthritis. Arthritis Rheum 1995;38:969–75. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum 2001;44:585–94. Tetlow LC, Woolley DE. Histamine stimulates matrix metalloproteinase-3 and -13 production by human articular chondrocytes in vitro. Ann Rheum Dis 2002;61:737–40. Li D, Liu L, Chen H, Sawamura T, Ranganathan S, Mehta JL. LOX-1 mediates oxidized low-density lipoprotein-induced expression of matrix metalloproteinases in human coronary artery endothelial cells. Circulation 2003;107:612–7. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 1997;99:1534–45. Dahlberg L, Billinghurst RC, Manner P, Nelson F, Webb G, Ionescu M, et al. Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1). Arthritis Rheum 2000;43: 673–82. Oka K, Sawamura T, Kikuta K, Itokawa S, Kume N, Kita T, et al. Lectin-like oxidized low-density lipoprotein receptor 1 mediates phagocytosis of aged/apoptotic cells in endothelial cells. Proc Natl Acad Sci U S A 1998;95:9535–40. Kakutani M, Masaki T, Sawamura T. A platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1. Proc Natl Acad Sci U S A 2000;97:360–4. Chen M, Kakutani M, Minami M, Kataoka H, Kume N, Narumiya S, et al. Increased expression of lectin-like oxidized low density lipoprotein receptor-1 in initial atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits. Arterioscler Thromb Vasc Biol 2000;20:1107–15. Nagase M, Kaname S, Nagase T, Wang G, Ando K, Sawamura T, et al. Expression of LOX-1, an oxidized low-density lipoprotein receptor, in experimental hypertensive glomerulosclerosis. J Am Soc Nephrol 2000;11:1826–36. Cominacini L, Pasini AF, Garbin U, Davoli A, Tosetti ML, Campagnola M, et al. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-B through an increased production of intracellular reactive oxygen species. J Biol Chem 2000;275:12633–8. Poole AR. Cartilage in health and disease. In: McCarty DJ, Koopman WJ, editors. Arthritis and allied conditions: a textbook of rheumatology. 3rd ed. Baltimore: Williams & Wilkins; 1997. p. 255–308. Higaki M, Sato K, Miyasaka N, Nishioka K. Uptake of acetylated low density lipoprotein (ac-LDL) by synovial cells. Scand J Rheumatol 1993;22:102–6. Prete PE, Gurakar-Osborne A. The contribution of synovial fluid lipoproteins to the chronic synovitis of rheumatoid arthritis. Prostaglandins 1997;54:689–98.