The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 7, July 2006, pp 2152–2163 DOI 10.1002/art.21951 © 2006, American College of Rheumatology The Catabolic Pathway Mediated by Toll-like Receptors in Human Osteoarthritic Chondrocytes Hyun Ah Kim,1 Mi-La Cho,2 Hye Young Choi,1 Chang Sik Yoon,1 Joo Yeon Jhun,2 Hey Jwa Oh,2 and Ho-Youn Kim2 Objective. To examine the catabolic pathways mediated by Toll-like receptor (TLR) ligands in human osteoarthritic (OA) chondrocytes. Methods. The presence of TLRs in OA and non-OA articular cartilage was analyzed by immunohistochemistry. The regulation of TLR messenger RNA (mRNA) by interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) was analyzed by reverse transcription– polymerase chain reaction. For stimulation of TLR-2 and TLR-4, chondrocytes were treated with Staphylococcus aureus peptidoglycan and lipopolysaccharides (LPS), respectively. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. Production of nitric oxide (NO) was analyzed by the Griess reaction. Regulation of cyclooxygenase 2 protein and phosphorylation of MAPKs (p38, ERK, and JNK) were evaluated by Western blotting or solid-phase kinase assay. NF-B activation was evaluated by electrophoretic mobility shift assay. Results. Expression of TLRs 2 and 4 was upregulated in lesional areas of OA cartilage. Treatment with IL-1, TNF␣, peptidoglycan, and LPS all significantly up-regulated TLR-2 mRNA expression in cultured chondrocytes. Production of MMPs 1, 3, and 13 and of NO and PGE2 was significantly increased after treating chondrocytes with either of the TLR ligands. Prolonged culture of cartilage explants with TLR ligands also led to a significant increase in the release of proteoglycan and type II collagen degradation product. Treatment with TLR ligands led to phosphorylation of all 3 MAPKs and activation of NF-B. Conclusion. We found that TLRs are increased in OA cartilage lesions. TLR-2 and TLR-4 ligands strongly induce catabolic responses in chondrocytes. Modulation of TLR-mediated signaling as a therapeutic strategy would require detailed elucidation of the signaling pathways involved. Degradation of extracellular matrix in articular cartilage is a central event that leads to joint destruction in many arthritic conditions, including rheumatoid arthritis (RA), osteoarthritis (OA), and septic arthritis. Chondrocytes respond to a variety of stimuli, such as proinflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators. Whether the initiation of matrix degradation is cytokine driven or biomechanical, it is likely that the downstream degradative pathways are similar, involving specific matrix metalloproteinases (MMPs) (1). Degradation of cartilage matrix macromolecules is also associated with increased expression of mediators of inflammation, such as nitric oxide (NO) and prostaglandin E2 (PGE2). NO is involved in the stimulation of MMP messenger RNA (mRNA) expression and activity (2,3), inhibition of matrix component production (4,5), and, in the presence of superoxide anions, induction of chondrocyte apoptosis (6). PGE2 produced at high levels at sites of inflammation mediates cartilage resorption by decreasing proliferation, enhancing MMP activity, and inhibiting aggrecan synthesis in chondrocytes, and it can also potentiate the effects of other mediators of inflammation (7,8). It is well accepted that interleukin-1 (IL-1) Supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare (01-PJ3-PG6-01GN11-0002) and by a grant from the Korea Science and Engineering Foundation (R11-2002098-05001-0). 1 Hyun Ah Kim, MD, PhD, Hye Young Choi, MS, Chang Sik Yoon, MS: Hallym University Sacred Heart Hospital, Anyang, Republic of Korea; 2Mi-La Cho, PhD, Joo Yeon Jhun, MS, Hey Jwa Oh, MS, Ho-Youn Kim, MD, PhD: Catholic University, Seoul, Republic of Korea. Address correspondence and reprint requests to Hyun Ah Kim, MD, PhD, Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchondong, Dongan-gu, Anyang, Kyunggi-do 431-070, Republic of Korea. E-mail: email@example.com. Submitted for publication October 13, 2005; accepted in revised form March 30, 2006. 2152 TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES and tumor necrosis factor ␣ (TNF␣) are key cytokines involved in articular cartilage destruction as well as in the inflammatory response in arthritis. Biologics that inhibit the signaling cascade mediated by both of these cytokines have been effective in the treatment of RA, resulting in alleviation of both inflammation and joint destruction. However, blocking of IL-1 and/or TNF␣ does not lead to complete protection of the joint structure, indicating that other signaling pathways that mediate joint catabolism remain to be elucidated. Toll-like receptors (TLRs) are phylogenetically conserved receptors involved in the innate immune response, and they recognize pathogen-associated molecular patterns (9). The mammalian homologs of TLRs belong to a family that currently consists of 10 members in humans (10). The ligands for several of the TLRs (TLRs 2–6 and 9) have been identified and include nonbacterial products, such as Hsp70 and fatty acids, as well as microbial constituents (11). Because ligand recognition by TLRs elicits strong activation of proinflammatory cytokines and up-regulation of costimulatory molecules (12), the role of TLRs in the exacerbation of the inflammatory response and joint destruction in arthritis has been postulated. Synovial tissues from RA joints express TLR-2 predominantly at sites of cartilage and bone destruction, and expression of TLR-2 in RA synovial fibroblasts was shown to be increased after treatment with proinflammatory cytokines (13). Bacterial peptidoglycan, a TLR-2 ligand, activates synovial fibroblasts to express integrins, MMPs, proinflammatory cytokines, and chemokines, suggesting that signaling through TLR can elicit a proinflammatory response in synoviocytes (14,15). Mice deficient in myeloid differentiation factor 88 (MyD88), a Toll/IL-1 receptor (IL-1R) domain–containing adaptor molecule known to have a central role in TLR signaling, do not develop a streptococcal cell wall (SCW)–induced arthritis and show significant amelioration of both joint swelling and cartilage degradation (16). This underscores the importance of the TLR-mediated signaling pathway in the regulation of inflammation and joint destruction of arthritis. Recently, human articular chondrocytes were shown to express TLRs (17,18). Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate monohydrate (MSU) were found to trigger NO generation via TLR-2–mediated signaling in chondrocytes, indicating the potential of TLR-mediated signaling to directly contribute to degradative tissue reactions associated with arthritis. In the current study, we examined the catabolic signaling pathway mediated by TLR 2153 ligands, bacterial peptidoglycan, and lipopolysaccharides (LPS), as well as the role played by MAPKs and NF-B in TLR-mediated catabolic signaling, in human articular chondrocytes. MATERIALS AND METHODS Reagents. Staphylococcus aureus peptidoglycan was purchased from Fluka (Buchs, Switzerland) and was tested for endotoxin using the Limulus amebocyte cell lysate assay (BioWhittaker, Walkersville, MD). Endotoxin levels did not exceed 0.06 endotoxin units/ml in all tested lots. LPS from Escherichia coli 026:B6 was purchased from Sigma (St. Louis, MO). A nitrate/nitrite colorimetric assay kit was purchased from Cayman Chemical (Ann Arbor, MI). SB202190 (a p38 MAPK inhibitor) and SN50 (a peptide NF-B inhibitor) were purchased from Alexis (Carlsbad, CA). PD98059 (a MEK-1/2 inhibitor), hypoestoxide (a direct inhibitor of IB kinase), and SP600125 (a JNK inhibitor) were purchased from Calbiochem (San Diego, CA). Anti–phospho–ERK-1/2, anti–phospho-p38, anti–ERK-1/2, and anti-p38 were purchased from New England Biolabs (Beverly, MA). Anti-human TLR-2 (H-175), anti-human TLR-4 (H-80), and anti–JNK-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti– cyclooxygenase 2 (anti–COX-2) was purchased from Cayman Chemical, anti–MMP-1 and anti–MMP-3 were purchased from R&D Systems (Minneapolis, MN), and anti–MMP-13 was purchased from Chemicon International (Temecula, CA). Blocking anti–TLR-2 and anti–TLR-4 (clones TL2.1 and HTA125) were obtained from eBioscience (San Diego, CA). MMP-1, MMP-3, MMP-13, and PGE2 enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems. All other reagents were obtained from Sigma unless specified otherwise. Chondrocyte monolayer and explant culture. Cartilage samples were obtained from the femoral condyles and tibial plateaus of the knees of OA patients at the time of joint replacement surgery. Full-thickness cartilage slices were obtained from above the subchondral bone from a relatively lesion-free area. For monolayer cultures, slices were minced and incubated sequentially with Pronase and collagenase in Dulbecco’s modified Eagle’s medium (DMEM) until the fragments were digested. Released cells were seeded at 5 ⫻ 106/plate in 10-cm culture plates in DMEM supplemented with 10% fetal calf serum (FCS), 1% L-glutamine, 1% Fungizone (Gibco, Grand Island, NY), and penicillin/streptomycin (150 units/ml and 50 mg/ml each). After ⬃7 days, confluent chondrocytes were split once and seeded at high density, and these first-passage chondrocytes were used within 2 days in subsequent experiments. For explant cultures, full-thickness slices were obtained from the femoral condyle. Each slice was cut further, and a piece measuring ⬃2 mm wide ⫻ 5 mm long ⫻ full thickness was weighed and cultured at 200 l/well in a 48-well culture plate in the same medium described above for the monolayer culture. Explants were incubated in the medium for 3 days before experiments were performed to allow them to stabilize in the in vitro conditions. Monolayer and explant cultures were incubated with DMEM containing 0.5% FCS for 16 hours 2154 prior to treatment with peptidoglycan or LPS. For the blocking experiments, anti–TLR-2 or anti–TLR-4 antibodies were added to the chondrocytes at 20 g/ml, 2 hours before stimulation with peptidoglycan or LPS, respectively. The concentrations of blocking antibodies and the duration of preincubation were determined in our preliminary inhibition experiments of MMP stimulation by TLR ligands (data not shown). For experiments with MAPK or NF-B inhibitors, SB202190 (0.5–2 M), PD98059 (5–25 M), SP600125 (5 or 10 M), hypoestoxide (50 M), or SN50 (20 M) was added to the chondrocytes 1 hour before stimulation with peptidoglycan or LPS. Immunohistochemistry. Cartilage tissues obtained from OA patients at the time of joint replacement surgery were cut to the subchondral bone from both lesional and lesion-free areas, immediately fixed with 4% paraformaldehyde, and embedded in paraffin. For non-OA cartilage, we used macroscopically and microscopically normal cartilage samples obtained from the femoral head of patients with femoral neck fracture. After dewaxing and dehydration, sections were blocked with normal goat serum for 60 minutes. TLR-2 and TLR-4 were detected with polyclonal antibodies against human TLR-2 and TLR-4 at 4°C for overnight. The bound antibodies were probed with biotinylated anti-rabbit immunoglobulin and peroxidase-conjugated streptavidin. Slides were stained with 3,3⬘-diaminobenzidine solution (Dako, Glostrup, Denmark) and counterstained with hematoxylin for 30 seconds. Western blotting. For MMPs 1 and 3, first-passage chondrocytes were seeded (30,000/well) in 96-well culture plates, serum-starved, and subsequently treated with peptidoglycan (0.05–1 g/ml) or LPS (0.05–1 g/ml) for 24 hours. Culture supernatants were collected and electrophoresed on a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel, and transferred. Blots were blocked with Tris buffered saline containing 5% nonfat milk at room temperature for 1 hour and incubated with respective antibodies overnight at 4°C. The blots were then incubated with 1:5,000 peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, Hercules, CA) for 1 hour. Bound immunoglobulin was detected with an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). For MMP-13, chondrocytes were seeded (100,000/500 l/well) in 24-well culture plates and treated with peptidoglycan or LPS for 24 hours. Culture supernatants were collected and concentrated to a volume of 50 l using Centricon YM 10 filters (Millipore, Bedford, MA), electrophoresed, and blotted. For COX-2, chondrocytes were seeded (500,000/well) in 6-well culture plates and treated with peptidoglycan or LPS for 6 hours. Cellular protein was extracted in lysis buffer containing 50 mM sodium acetate (pH 5.8), 10% volume/ volume SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin at 4°C, and protein concentrations were measured using the bicinchoninic acid method with bovine serum albumin as standard. Electrophoresis and blotting procedures were the same as those for MMPs. SAPK/JNK assay. SAPK/JNK activity was measured using a solid-phase kinase assay method. Briefly, 100 g of cell lysate was incubated with 2 g of glutathione S-transferase–cJun beads (Cell Signaling Technology, Beverly, MA) at 4°C overnight with gentle rocking. The beads were then pelleted, washed, and resuspended in kinase buffer (25 mM Tris, pH 7.5, KIM ET AL 5 mM ␤-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2) containing a final concentration of 100 M ATP. After incubation at room temperature for 30 minutes, the reaction was terminated by adding SDS sample buffer. The proteins were separated by 12% SDS– polyacrylamide gel electrophoresis and blotted with anti– phospho–c-Jun antibody (Cell Signaling Technology). Nitrate/nitrite quantification. Chondrocyte culture medium was harvested after a 24-hour incubation with peptidoglycan or LPS and analyzed with a nitrate/nitrite colorimetric assay kit as recommended by the manufacturer. Briefly, nitrate was converted to nitrite utilizing nitrate reductase, and then Griess reagents were added to convert nitrite to a deep-purple azo compound. The absorbance of the azo chromophore was measured to determine the nitrite concentration at 540 nm using the plate reader. The detection limit of the assay was 1 M nitrite. ELISA. Culture supernatants from chondrocytes were harvested after a 24-hour incubation with peptidoglycan or LPS and stored frozen at ⫺70°C. MMP-1, MMP-3, and MMP-13 proteins and PGE2 were quantitated in cell supernatants by ELISA using the Quantikine human proMMP-1, MMP-3, proMMP-13, and PGE2 immunoassay kits according to the instructions of the manufacturer (R&D Systems). The detection limits were 0.021, 0.009, and 0.0077 ng/ml for MMP-1, MMP-3, and MMP-13, respectively, and 13.4 pg/ml for PGE2. Electrophoretic mobility shift assay. Nuclear extracts from chondrocytes were prepared from 2 ⫻ 106 cells as described previously, with minor modifications (19). Protein content was measured, and 5-g portions of extracts were used for the binding reaction. A consensus double-stranded NF-B probe was obtained from Promega (Madison, WI) and endlabeled by using ␥32P–adenosine-5-triphosphate. Nuclear extracts were incubated in gel binding buffer (Promega) in a volume of 9 l. Afterward, the end-labeled probe was added (100,000 counts per minute/sample). Samples were then incubated for 20 minutes and loaded onto a 4% nondenaturing polyacrylamide gel. Electrophoresis was run for 3 hours in a cold room. Protein complexes were identified by autoradiography. Reverse transcription–polymerase chain reaction (RTPCR). Chondrocytes cultured as described above were serum starved and treated with 1 ng/ml IL-1␤, 10 ng/ml TNF␣, 0.2 g/ml peptidoglycan, or 0.2 g/ml LPS for 4 hours. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA). Two hundred nanograms of total RNA was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Gaithersburg, MD) with oligo(dT)20 primers. PCR amplification of complementary DNA (cDNA) aliquots was performed by adding 2.5 mM dNTPs, 2.5 units Taq DNA polymerase, and 0.25 M sense and antisense primers. The reaction was done in PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris HCl, pH 8.3) in a total volume of 25 l. The following sense and antisense primers for each molecule were used: for TLR-2, 5⬘-GCC-AAA-GTC-TTGATT-GAT-TGG-3⬘ (sense) and 5⬘-TTG-AAG-TTC-TCCAGC-TCC-TG-3⬘ (antisense); for TLR-4, 5⬘-TGG-ATACGT-TTC-CTT-ATA-AG-3⬘ (sense) and 5⬘-TGG-ATA-CGTTTG-CTT-ATA-AG-3⬘ (antisense); and, for GAPDH, 5⬘- TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES 2155 Figure 1. Expression and regulation of Toll-like receptors (TLRs) 2 and 4. A, Expression and regulation of TLR-2 and TLR-4 mRNA in monolayercultured articular chondrocytes as detected by reverse transcription–polymerase chain reaction (RT-PCR). Top, Chondrocytes were obtained from patients with knee osteoarthritis (OA), cultured in monolayer, and treated with 1 ng/ml interleukin-1␤ (IL-1␤), 10 ng/ml tumor necrosis factor ␣ (TNF␣), 0.2 g/ml peptidoglycan (PGN), or 0.2 g/ml lipopolysaccharide (LPS) for 4 hours. Total RNA was isolated, and RT-PCR was performed using TLR-2, TLR-4, and GAPDH primer sets. The PCR products were separated on an agarose gel and stained with ethidium bromide. Results are representative of samples obtained from 4 different donors. Bottom, The band densities were quantified, the percentage GAPDH density was calculated for TLRs, and the value for control culture was set at 1. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control. B, Expression of TLR-2 and TLR-4 in articular cartilage as detected by immunohistochemistry. Top, Cartilage obtained from OA patients at the time of joint replacement surgery was cut from both lesional and nonlesional areas. Macroscopically and microscopically normal cartilage obtained from the femoral heads of patients with fractures served as non-OA controls. Compared with non-OA and OA nonlesional cartilage, the expression of both TLR-2 and TLR-4 was significantly up-regulated in OA lesional cartilage (original magnification ⫻ 200). Results are representative of samples obtained from 5 different OA and 5 different non-OA donors. Bottom, The percentage of chondrocytes positive for TLR-2 and TLR-4 was calculated for OA lesional cartilage and for non-OA and OA nonlesional control cartilage. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control cartilage. CGA-TGC-TGG-GCG-TGA-GTA-C-3⬘ (sense) and 5⬘-CGTTCA-GCT-CAG-GGA-TGA-CC-3⬘ (antisense). Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Wellesley, MA) through 25 cycles of 30 seconds of denaturation at 94°C and 1 minute of annealing at 55°C for GAPDH and 30 cycles of 45 seconds of denaturation at 95°C and 30 seconds of annealing at 55°C for TLR-2 and TLR-4, followed by 1 minute of elongation at 72°C. For MMPs and inducible NO synthase (iNOS) RTPCR, chondrocytes were treated with 0.2 g/ml peptidoglycan or 0.2 g/ml LPS for 4 hours. The procedures for RNA extraction, cDNA synthesis, and RT-PCR were similar to those for TLRs, except for annealing at 60°C for 35 cycles for MMP-1 and annealing at 64°C for 24 cycles for MMP-13. The following sense and antisense primers were used: for MMP-1, 5⬘-CTG-TTC-AGG-GAC-AGA-ATG-TGC-T-3⬘ (sense) and 5⬘-TTG-GAC-TCA-CAC-CAT-GTG-TT-3⬘ (antisense); for MMP-3, 5⬘-TGC-GTG-GCA-GTT-TGC-TCA-GCC-3⬘ (sense) and 5⬘-GAA-TGT-GAG-TGG-AGT-CAC-CTC-3⬘ (antisense); for MMP-13, 5⬘-GGC-TCC-GAG-AAA-TGC-AGT-CTT-TCTT-3⬘ (sense) and 5⬘-ATC-AAA-TGG-GTA-GAA-GTC-GCCATG-C-3⬘ (antisense); and, for iNOS, 5⬘-GTG-AGG-ATCAAA-AAC-TGG-GG-3⬘ (sense) and 5⬘-ACC-TGC-AGGTTG-GAC-CAC-3⬘ (antisense). PCR conditions were chosen to be nonsaturating in all cases. PCR products were run on a 1.5% agarose gel, stained with ethidium bromide, and visual- 2156 KIM ET AL Figure 2. Activation of MAPKs and NF-B by TLR ligands in chondrocytes. A, Monolayer-cultured human articular chondrocytes were treated with 0.2 g/ml peptidoglycan or 0.2 g/ml LPS for the indicated periods. Protein was extracted from chondrocytes, and 20 g of each protein sample was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Phosphorylation of ERK-1/2, p38, and SAPK/JNK was analyzed by Western blotting or solid-phase kinase assay. Blots were stripped and reprobed with non–phospho-specific antibodies. Stimulation with IL-1␤ served as a positive control. Results are representative of samples obtained from 3 different donors. B, Human articular chondrocytes were treated with 0.2 g/ml peptidoglycan or 0.2 g/ml LPS for 30 minutes, and activation of NF-B was analyzed by electrophoretic mobility shift assay. The arrow indicates activated NF-B bands. Results are representative of samples obtained from 3 different donors. See Figure 1 for definitions. ized using a UVP transilluminator (Ultraviolet Products, Upland, CA). The band densities were quantified using image analysis software (NIH Image, National Institutes of Health, Bethesda, MD; online at: http://rsb.info.nih.gov/nih-image/). Proteoglycan and type II collagen degradation product measurement. Cartilage explants were incubated in the media containing peptidoglycan or LPS, and the media were collected on days 3, 6, 9, 12, 15, and 18 for measurement of proteoglycan and type II collagen degradation products. The amount of sulfated glycosaminoglycans (GAGs), reflecting the amount of proteoglycans released in culture medium, was determined using a commercial kit (Biocolor, Belfast, UK) according to the manufacturer’s recommendations. Briefly, collected media were mixed with dye reagent containing 1,9-dimethylene blue for 30 minutes, and then the GAG–dye complex was separated by centrifugation. The dye pellet was dissociated with propan1-ol solution, and the absorbance was read at 656 nm with a plate reader. For type II collagen degradation, the Urine CartiLaps ELISA kit measuring C-telopeptide of type II collagen (CTX-II; Nordic Bioscience Diagnostics, Herlev, Denmark) was used according to the manufacturer’s recommendations. Statistical analysis. Data are expressed as the mean ⫾ SD. Differences between OA and non-OA cartilage and between treatment groups were tested by using the MannWhitney U test (GraphPad Prism, version 3; GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant. RESULTS Expression and regulation of TLR-2 and TLR-4 in articular chondrocytes. In monolayer-cultured chondrocytes obtained from the knees of OA patients, we detected TLR-2 mRNA in all samples and TLR-4 mRNA in 50% of the samples (in 4 of 4 samples for TLR-2 and in 2 of 4 samples for TLR-4). Basal expression of TLR-2 and TLR-4 varied from patient to patient. Treatment of chondrocytes with IL-1␤ or TNF␣ significantly up-regulated the mRNA expression of TLR-2 TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES 2157 Figure 3. Production of matrix metalloproteinases (MMPs) in cultured chondrocytes by TLR ligands. A, Monolayers of human chondrocytes were treated with varying concentrations of peptidoglycan or LPS for 24 hours, and production of MMPs 1, 3, and 13 was analyzed by Western blotting of conditioned media. Stimulation with IL-1␤ served as a positive control. Results are representative of samples obtained from 3 different donors. B, Explants obtained from patients with knee OA were cultured with varying concentrations of peptidoglycan or LPS for 11 days, and production of MMPs 1, 3, and 13 was analyzed by Western blotting of conditioned media. Stimulation with IL-1␤ served as a positive control. Results are representative of samples obtained from 5 different donors. C, Up-regulation of MMP mRNA by TLR ligands was verified by RT-PCR. Top, Chondrocytes were treated with 0.2 g/ml peptidoglycan or 0.2 g/ml LPS for 4 hours. Total RNA was isolated, and RT-PCR was performed using MMP-1, MMP-3, MMP-13, and GAPDH primer sets. The PCR products were separated on an agarose gel and stained with ethidium bromide. Results are representative of samples obtained from 4 different donors. Bottom, The band densities were quantified, the percentage GAPDH density was calculated for MMPs, and the value for the control culture was set at 1. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure 1 for other definitions. (Figure 1A). In addition, treatment of chondrocytes with peptidoglycan or LPS significantly up-regulated the mRNA expression of TLR-2, suggesting a positive regulatory loop. TLR-4 mRNA tended to increase in response to the above stimuli. To demonstrate TLR expression at the protein level, immunohistochemistry was performed on human OA knee cartilage and non-OA hip cartilage. Compared with non-OA and OA nonlesional cartilage, expression of both TLR-2 and TLR-4 was significantly up-regulated in OA lesional cartilage (Figure 1B). The expression of TLR-2 and TLR-4 was rare in non-OA and OA nonlesional cartilage and did not differ significantly between the 2 types of cartilage. Activation of MAPKs and NF-B in cultured chondrocytes by TLR ligands. Because activation of MAPKs has been reported in OA cartilage, and because signaling downstream leading to NF-B activation could contribute to cartilage degradation through upregulation of MMP expression (20), we were interested in defining the activation of MAPKs and NF-B in TLR-mediated signaling of articular chondrocytes. When chondrocytes were treated with peptidoglycan or LPS, phosphorylation of all 3 MAPKs (p38, ERK, and JNK) and activation of NF-B were observed (Figure 2). Activation of p38 by peptidoglycan or LPS was prolonged and was observed until 60 minutes of stimulation, while JNK activation was brief, with its peak at 30 2158 KIM ET AL Figure 4. Inhibition of matrix metalloproteinase (MMP) up-regulation induced with TLR ligands by blocking anti-TLR antibodies (␣-TLR), MAPK inhibitors, and NF-B inhibitors. A, Blocking anti–TLR-2 or anti–TLR-4 antibodies (Ab) were added to the chondrocytes at 20 g/ml 2 hours before stimulation with peptidoglycan or LPS, respectively. MMP production in conditioned media was measured 24 hours after peptidoglycan or LPS treatment, by enzyme-linked immunosorbent assay (ELISA). Results are the mean and SD percentage of MMP production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 4 donors. ⴱ ⫽ P ⬍ 0.05 versus control. B, MAPK inhibitors SB202190 (SB; 0.5–2 M), PD98059 (PD; 5–25 M), or SP600125 (SP; 5–10 M) were added to the chondrocytes 1 hour before stimulation with peptidoglycan or LPS. For NF-B suppression, hypoestoxide (50 M) or SN50 (20 M) was added to the chondrocytes 1 hour before stimulation with peptidoglycan or LPS. MMP production in the conditioned media was measured after 24 hours, by ELISA. Results are the mean and SD percentage of MMP production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 4 donors. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure 1 for other definitions. minutes. The pattern of ERK activation was brief (with its peak at 30 minutes) for peptidoglycan, but it was more prolonged in LPS-treated chondrocytes. The degree of activation of MAPKs by TLR ligands was similar to that induced by IL-1␤. Increase in MMP production in cultured chondrocytes by TLR ligands. We next determined whether TLR-mediated signaling stimulates the production of MMPs. Treatment with peptidoglycan or LPS resulted in a marked up-regulation of MMP-1, MMP-3, and MMP-13 revealed by Western blotting analysis of monolayer chondrocytes (Figure 3A). Up-regulation of MMPs by TLR ligands was also verified in explant-cultured chondrocytes (Figure 3B). Both peptidoglycan and LPS were potent in their stimulation of MMPs, with significant up-regulation observed at 0.05 g/ml. Upregulation of MMPs in cultured chondrocytes was also verified at the mRNA level by RT-PCR (Figure 3C). MMP production by peptidoglycan and LPS was significantly down-regulated by blocking anti–TLR-2 and anti–TLR-4 antibodies, respectively, consistent with TLR stimulation by peptidoglycan and LPS (Figure 4A). In order to delineate the role of MAPKs and NF-B signaling in the up-regulation of MMPs mediated by TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES 2159 Figure 5. Increase in nitric oxide (NO) and prostaglandin E2 (PGE2) production in cultured chondrocytes by TLR ligands. Monolayers of human chondrocytes were treated with varying concentrations of peptidoglycan or LPS for 24 hours, and the culture supernatants were used for analysis. A, Production of NO was analyzed by the Griess reaction. Stimulation with IL-1␤ served as a positive control. Results are the mean and SD M NO released into the culture medium (representative of duplicate experiments with chondrocytes obtained from 4 donors). ⴱ ⫽ P ⬍ 0.05 versus TLR ligand treatment alone. B, Production of PGE2 was analyzed by enzyme-linked immunosorbent assay (ELISA). Stimulation with IL-1␤ served as a positive control. Results are the mean and SD pg/ml PGE2 released into the culture medium (representative of duplicate experiments with chondrocytes obtained from 4 donors). ⴱ ⫽ P ⬍ 0.05 versus TLR ligand treatment alone. C, Up-regulation of inducible NO synthase (iNOS) mRNA and cyclooxygenase 2 (COX-2) protein by TLR ligands. For iNOS, chondrocytes were treated with 0.2 g/ml peptidoglycan or 0.2 g/ml LPS for 4 hours. Total RNA was isolated, and RT-PCR was performed using iNOS and GAPDH primer sets. Results are representative of samples obtained from 3 different donors. For COX-2, chondrocytes were treated for 6 hours and protein expression was analyzed by Western blotting. Results are representative of samples obtained from 3 different donors. D, Inhibition of NO and PGE2 production by MAPK inhibitors (SB202190 [SB], PD98059 [PD], or SP600125 [SP]) and NF-B inhibitors (hypoestoxide or SN50). MAPK inhibitors or NF-B inhibitors were added to the chondrocytes 1 hour before stimulation with peptidoglycan or LPS. Top, NO production in conditioned media was measured after 24 hours by the Griess reaction. Results are the mean and SD percentage of NO production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 3 donors. ⴱ ⫽ P ⬍ 0.05 versus control. Bottom, PGE2 production in conditioned media was measured after 24 hours by ELISA. Results are the mean and SD percentage of PGE2 production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 3 different donors. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure 1 for other definitions. TLR ligands, inhibition experiments were performed (Figure 4B). Blocking of NF-B was first attempted by transduction of chondrocytes with adenovirus expressing IB super-repressor (Ad-IB SR). However, Ad-IB SR at multiplicities of infection as low as 1:10 was found to stimulate MMP and NO production in chondrocytes, and this precluded its use for blocking experiments (data not shown). Therefore, we used 2 inhibitors that inhibit NF-B activation through different mechanisms. Pretreatment either with hypoestoxide (a direct inhibitor of IB kinase) or with SN50 peptide (an NF-B translocation inhibitor) led to significant inhibition of MMP-1, MMP-3, and MMP-13 up-regulation in peptidoglycan- or LPS-stimulated chondrocytes. These 2160 observations suggest a crucial role of NF-B signaling in the mediation of MMP production induced by TLR ligands. Among MAPK inhibitors, the JNK inhibitor SP600125 significantly inhibited up-regulation of MMP-1 and MMP-3 in peptidoglycan- or LPSstimulated chondrocytes, while the p38 inhibitor SB202190 and the MEK-1/2 inhibitor PD98059 did not. MMP-13 production by TLR ligands was inhibited by the JNK inhibitor and by the highest concentration of the p38 inhibitor employed (2 M). Thus, it is postulated that the TLR ligand–induced MMP up-regulation is mediated mainly by the JNK and NF-B pathways. Increase in NO production in cultured chondrocytes by TLR ligands. TLR-mediated NO production was next investigated. Treatment with peptidoglycan or LPS resulted in significant up-regulation of NO after 24 hours of stimulation in monolayer articular chondrocytes and in explant cultured chondrocytes (Figure 5A and data not shown). NO production by peptidoglycan or LPS was significantly down-regulated by blocking anti–TLR-2 and anti–TLR-4 antibodies, respectively (Figure 5A). Treatment with TLR ligands induced significant up-regulation of iNOS mRNA in cultured chondrocytes after 4 hours of stimulation (Figure 5C). In inhibition experiments using MAPK and NF-B inhibitors, pretreatment with NF-B inhibitors led to significant inhibition of NO as in MMPs. SP600125 significantly inhibited up-regulation of NO in both peptidoglycan- and LPS-stimulated chondrocytes, and the highest concentration of p38 inhibitor (2 M) inhibited LPS-induced NO up-regulation (Figure 5D). Up-regulation of PGE2 in cultured chondrocytes by TLR ligands. TLR-mediated PGE2 production was investigated. Treatment with peptidoglycan or LPS also resulted in significant up-regulation of PGE2 after 24 hours of stimulation in monolayer articular chondrocytes and in explant cultured chondrocytes with significant inhibition by blocking anti–TLR-2 and anti–TLR-4 antibodies, respectively (Figure 5B and data not shown). Treatment with TLR ligands induced significant upregulation of COX-2 protein in cultured chondrocytes after 6 hours of stimulation (Figure 5C). All 3 MAPK inhibitors and NF-B inhibitors led to significant inhibition of PGE2 in peptidoglycan- or LPS-stimulated chondrocytes (Figure 5D). Increased release of proteoglycan and type II collagen degradation product by treatment with peptidoglycan or LPS. To confirm the influence of peptidoglycan or LPS on cartilage matrix catabolism, we cultured cartilage explants with peptidoglycan or LPS for prolonged periods of time and observed the release KIM ET AL Figure 6. Increased release of proteoglycan and type II collagen degradation product by treatment with peptidoglycan or LPS. Cartilage slices were cultured in explants and incubated in media containing peptidoglycan or LPS for 3–18 days, and media were collected. A, The amount of sulfated glycosaminoglycans (GAGs), reflecting the amount of proteoglycans released into the culture medium, was determined with a commercial kit using 1,9-dimethylene blue. Results are the mean and SD g of GAG released into the culture medium per mg cartilage and are representative of duplicate experiments with samples obtained from 3 different donors. ⴱ ⫽ P ⬍ 0.05 versus background levels (BG) at the same time point. B, For type II collagen degradation, the Urine CartiLaps enzyme-linked immunosorbent assay kit measuring C-telopeptide of type II collagen (CTX-II) was used. Results are the mean and SD ng of CTX-II released into the culture medium per mg of cartilage and are representative of duplicate experiments with samples obtained from 3 different donors. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus background levels at the same time point. See Figure 1 for other definitions. of proteoglycan or type II collagen degradation product into the culture media. Treatment with either peptidoglycan or LPS led to a significantly increased release of matrix degradation products (Figure 6). Increase in proteoglycan release was observed from day 3 in both peptidoglycan- and LPS-treated explants, and proteoglycan release continued to increase until day 15 of culture in LPS-treated explants. However, the increase in CTX-II was slower and reached significance on days 6 and 9 with peptidoglycan and on days 15 and 18 with LPS. DISCUSSION There is a continuing challenge in defining the catabolic triggers of articular chondrocytes in the patho- TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES genesis of cartilage degradation in various arthritides. Findings from this study suggest that TLR ligands play a pivotal role as potent catabolic stimuli in articular chondrocytes. The role of TLR ligands in the pathogenesis of inflammatory arthritis is not well understood. However, the presence of bacterial peptidoglycan and functional TLR-4 ligand in RA joints raises the possibility that TLRs may be involved in the joint degradation of RA as well as in septic arthritis (21,22). TLRs 2 and 4 are expressed in the synovial tissue of patients with clinically active RA and are associated with the levels of both IL-12 and IL-18 (13,23). In addition, TLRs 3 and 7 are highly expressed in RA synovium, and combined stimulation of TLR-4 and TLR-7/8 or TLR-3 and TLR-7/8 results in a synergy of the production of mediators of inflammation (24). In our study, both TLR-2 and TLR-4 mRNA were detected in cultured OA chondrocytes. Our findings differ from those in 2 previous studies that showed TLR-2 mRNA in both normal and OA chondrocytes but did not find TLR-4 mRNA (17,18). This discrepancy may result from variations in the donors and in the RT-PCR conditions employed. We believe that functional TLR-4 exists in chondrocytes because our results, as well as the results of many others, demonstrate that chondrocytes respond consistently to LPS, a traditional TLR-4 ligand, and this response is effectively blocked by anti–TLR-4 antibody. Immunohistochemistry revealed that OA lesional cartilage expressed abundant TLR-2 and TLR-4 proteins compared with non-OA and nonlesional cartilage. TLR-2 expression increased in response to TLR ligand itself as well as in response to proinflammatory cytokines, suggesting a positive amplification loop contributing to the inflammatory response in articular chondrocytes. This hypothesis is also supported by the finding that the fibronectin fragment, a well-known catabolic mediator in chondrocytes, also up-regulates TLR-2 expression in articular chondrocytes (17). Recent studies show that IL-1␤ up-regulates TLR-2 mRNA in hepatocytes and epithelial cells via the NF-B–dependent pathway (25,26). Both peptidoglycan and LPS were found to stimulate phosphorylation of all 3 MAPKs as well as NF-B nuclear translocation in chondrocytes. In addition, both ligands stimulated MMP, NO, and PGE2 release in chondrocytes and increased the release of proteoglycan and collagen degradation product into the explant culture media. Increases in MMPs, NO, and PGE2 were completely inhibited by the NF-B inhibitor, which is consistent with previous studies showing a link between 2161 the cytoplasmic Toll/IL-1R domain and NF-B activation in terms of iNOS and MMP up-regulation in a variety of cells (27,28). Our results show that in addition to NF-B, the JNK pathway mediates the induction of mediators of inflammation in response to TLR ligands in chondrocytes. The MAPKs p38 and ERK primarily regulated the TLR-mediated PGE2 response. Interaction between TLR signaling and MAPK in the proinflammatory response is not fully understood, and different patterns seem to exist in different cells. TLR-mediated signaling depends upon adaptor molecules such as MyD88 and TRIF and is often classified as an MyD88- or TRIFdependent pathway (29). TLR interaction with MyD88 leads to recruitment of IL-1R–associated kinase 1 (IRAK-1), activation of IRAK-1, its association with TNF receptor–associated factor 6 (TRAF6), and activation of MAPKs and NF-B (30). A recent study showed that MSU and CPPD crystals induce NO production in articular chondrocytes, which is dependent on IKK-2– mediated NF-B activation (18). Upstream of NF-B, parallel to the canonical MyD88, IRAK, and TRAF6 signaling, the Rac1/phosphatidylinositol 3-kinase pathway has been found to mediate TLR-2–mediated NO production by MSU and CPPD crystals (18). Despite numerous reports on the stimulatory function of LPS in articular chondrocytes, a paucity of data exists on the pertinent signaling mechanism. Delineation of a catabolic signaling pathway other than MAPK and NF-B mediated by peptidoglycan and LPS will be the subject of future investigations in our laboratory. Because the IL-1 receptor and TLRs share a common signaling pathway, we examined the induction of IL-1 in peptidoglycan- or LPS-treated chondrocytes. Peptidoglycan and LPS at a concentration up to 1 g/ml failed to induce significant IL-1 in chondrocytes (data not shown). In addition, the catabolic response mediated by peptidoglycan or LPS was not inhibited by the IL-1 receptor antagonists (data not shown). In a study investigating the in vivo role of TLR-mediated signaling in an SCW-induced arthritis model using TLR and MyD88knockout mice (16), it was found that signaling via the TLR/MyD88 pathway is responsible for early cartilage damage independent of IL-1 and IL-18 before induction of the inflammatory response. In our study, chondrocytes were obtained from OA cartilage samples both for monolayer culture and for explant culture. It would have been more relevant to study the effect of TLR ligands in chondrocytes obtained from patients with RA or septic arthritis, because the catabolic signaling of TLR would be more pronounced 2162 KIM ET AL in these diseases compared with that in OA. For culture, we excluded severely affected areas, and the chondrocytes mostly came from the nonlesional area, which is similar to non-OA cartilage in terms of the low level of TLR expression. However, we cannot exclude the possibility that OA pathology itself might have influenced the chondrocyte responses to TLR ligands. Our results thus may not be extrapolated to the situation in RA or normal cartilage. In summary, we have shown that TLR is present in articular cartilage and cultured OA chondrocytes and is increased in the presence of OA lesions. TLR-2 expression is increased by catabolic cytokines and by the TLR ligand itself. TLR-2 and TLR-4 ligands strongly induce catabolic responses in chondrocytes. Recently, TNF␣ blockade was reported to down-regulate the systemic and local expression of TLR-2 and TLR-4 in spondylarthropathy, raising the possibility that modulation of TLR might contribute both to therapeutic aspects and to side effects (31). TLR ligands include components of the extracellular matrix, and TLRmediated catabolic responses may further trigger chronic inflammation, leading to self-perpetuating loops of cell activation in arthritis, with a minimal requirement for adaptive immune mechanisms (32). Modulation of TLR-mediated signaling as a potential therapeutic strategy for arthritis requires detailed elucidation of the signaling pathways and endogenous ligands involved. REFERENCES 1. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front Biosci 2006;11:529–43. 2. Murrell GA, Jang D, Williams RJ. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 1995;206:15–21. 3. Sasaki K, Hattori T, Fujisawa T, Takahashi K, Inoue H, Takigawa M. Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes. J Biochem (Tokyo) 1998; 123:431–9. 4. Hauselmann HJ, Oppliger L, Michel BA, Stefanovic-Racic M, Evans CH. Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture. FEBS Lett 1994;352: 361–4. 5. Cao M, Westerhausen-Larson A, Niyibizi C, Kavalkovich K, Georgescu HI, Rizzo CF, et al. Nitric oxide inhibits the synthesis of type-II collagen without altering Col2A1 mRNA abundance: prolyl hydroxylase as a possible target. Biochem J 1997;324: 305–10. 6. Blanco FJ, Ochs RL, Schwarz H, Lotz M. Chondrocyte apoptosis induced by nitric oxide. Am J Pathol 1995;146:75–85. 7. Ahmed S, Rahman A, Hasnain A, Lalonde M, Goldberg VM, Haqqi TM. Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 ␤-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes. Free Radic Biol Med 2002;33:1097–105. 8. Martel-Pelletier J, Mineau F, Fahmi H, Laufer S, Reboul P, Boileau C, et al. Regulation of the expression of 5-lipoxygenaseactivating protein/5-lipoxygenase and the synthesis of leukotriene B4 in osteoarthritic chondrocytes: role of transforming growth factor ␤ and eicosanoids. Arthritis Rheum 2004;50:3925–33. 9. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001;1:135–45. 10. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197–216. 11. O’Neill LA. TLRs: Professor Mechnikov, sit on your hat. Trends Immunol 2004;25:687–93. 12. Bowie A, O’Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol 2000;67:508–14. 13. Seibl R, Birchler T, Loeliger S, Hossle JP, Gay RE, Saurenmann T, et al. Expression and regulation of Toll-like receptor 2 in rheumatoid arthritis synovium. Am J Pathol 2003;162:1221–7. 14. Kyburz D, Rethage J, Seibl R, Lauener R, Gay RE, Carson DA, et al. Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling. Arthritis Rheum 2003;48:642–50. 15. Pierer M, Rethage J, Seibl R, Lauener R, Brentano F, Wagner U, et al. Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands. J Immunol 2004;172:1256–65. 16. Joosten LA, Koenders MI, Smeets RL, Heuvelmans-Jacobs M, Helsen MM, Takeda K, et al. Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88. J Immunol 2003;171:6145–53. 17. Su SL, Tsai CD, Lee CH, Salter DM, Lee HS. Expression and regulation of Toll-like receptor 2 by IL-1␤ and fibronectin fragments in human articular chondrocytes. Osteoarthritis Cartilage 2005;13:879–86. 18. Liu-Bryan R, Pritzker K, Firestein GS, Terkeltaub R. TLR2 signaling in chondrocytes drives calcium pyrophosphate dihydrate and monosodium urate crystal-induced nitric oxide generation. J Immunol 2005;174:5016–23. 19. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’ prepared from a small number of cells. Nucleic Acids Res 1989;17:6419. 20. Pelletier JP, Fernandes JC, Brunet J, Moldovan F, Schrier D, Flory C, et al. In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes. Arthritis Rheum 2003;48:1582–93. 21. Van der Heijden IM, Wilbrink B, Tchetverikov I, Schrijver IA, Schouls LM, Hazenberg MP, et al. Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides. Arthritis Rheum 2000;43:593–8. 22. Martin CA, Carsons SE, Kowalewski R, Bernstein D, Valentino M, Santiago-Schwarz F. Aberrant extracellular and dendritic cell (DC) surface expression of heat shock protein (hsp)70 in the rheumatoid joint: possible mechanisms of hsp/DC-mediated crosspriming. J Immunol 2003;171:5736–42. 23. Radstake TR, Roelofs MF, Jenniskens YM, Oppers-Walgreen B, van Riel PL, Barrera P, et al. Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-␥. Arthritis Rheum 2004;50:3856–65. 24. Roelofs MF, Joosten LA, Abdollahi-Roodsaz S, van Lieshout AW, Sprong T, van den Hoogen FH, et al. The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arthritis Rheum 2005;52:2313–22. 25. Matsumura T, Degawa T, Takii T, Hayashi H, Okamoto T, Inoue J, et al. TRAF6-NF-B pathway is essential for interleukin-1- TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES induced TLR2 expression and its functional response to TLR2 ligand in murine hepatocytes. Immunology 2003;109:127–36. 26. Sakai A, Han J, Cato AC, Akira S, Li JD. Glucocorticoids synergize with IL-1␤ to induce TLR2 expression via MAP Kinase Phosphatase-1-dependent dual inhibition of MAPK JNK and p38 in epithelial cells. BMC Mol Biol 2004;5:2. 27. Fieber C, Baumann P, Vallon R, Termeer C, Simon JC, Hofmann M, et al. Hyaluronan-oligosaccharide-induced transcription of metalloproteases. J Cell Sci 2004;117:359–67. 28. Vazquez de Lara LG, Umstead TM, Davis SE, Phelps DS. Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells. Am J Physiol Lung Cell Mol Physiol 2003; 285:L899–906. 2163 29. Shinohara H, Inoue A, Toyama-Sorimachi N, Nagai Y, Yasuda T, Suzuki H, et al. Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling. J Exp Med 2005;201: 333–9. 30. Nakayama K, Okugawa S, Yanagimoto S, Kitazawa T, Tsukada K, Kawada M, et al. Involvement of IRAK-M in peptidoglycaninduced tolerance in macrophages. J Biol Chem 2004;279:6629–34. 31. De Rycke L, Vandooren B, Kruithof E, De Keyser F, Veys EM, Baeten D. Tumor necrosis factor ␣ blockade treatment downmodulates the increased systemic and local expression of Toll-like receptor 2 and Toll-like receptor 4 in spondylarthropathy. Arthritis Rheum 2005;52:2146–58. 32. Cor M. The Tolls of arthritis. Arthritis Rheum 2005;52:2233–6.