Toll-like receptors and chondrocytesThe lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 56, No. 6, June 2007, pp 1880–1893 DOI 10.1002/art.22637 © 2007, American College of Rheumatology Toll-like Receptors and Chondrocytes The Lipopolysaccharide-Induced Decrease in Cartilage Matrix Synthesis Is Dependent on the Presence of Toll-like Receptor 4 and Antagonized by Bone Morphogenetic Protein 7 K. Bobacz,1 I. G. Sunk,1 J. G. Hofstaetter,1 L. Amoyo,1 C. D. Toma,2 S. Akira,3 T. Weichhart,1 M. Saemann,1 and J. S. Smolen1 Objective. To assess the presence of Toll-like receptors (TLRs) 1–9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)– induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes. Methods. TLRs 1–9 were assessed in human articular cartilage by reverse transcription–polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4ⴚ/ⴚ mice. Chondrocyte monolayer cultures were incubated with interleukin-1␤ (IL-1␤) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by 35S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1␤ was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis. Results. TLRs 1–9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4ⴚ/ⴚ mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1␤ mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the antianabolic effects of LPS. Blocking of p38, but not ERK1/2, resulted in inhibition of both LPS-mediated IL-1␤ gene expression and the negative effects of LPS on matrix biosynthesis. Conclusion.These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1␤, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases. The immune system functions to eliminate, or at least contain, pathogens threatening an organism. Although the genetically and phenotypically highly sophisticated polymorphic adaptive immune system was long believed to be the most effective component in the host defense system, the role of innate immunity, the socalled first line of defense, has been recognized, in recent years, to be not only a rapidly activated but also a crucial system in the discrimination between self and nonself antigens (1). This discrimination relies on the activities of distinct families of pattern recognition receptors (PRRs) that recognize specific pathogenic signatures (pathogen-associated molecular patterns). The 1 K. Bobacz, MD, I. G. Sunk, MD, J. G. Hofstaetter, MD, L. Amoyo, T. Weichhart, PhD, M. Saemann, MD, J. S. Smolen, MD: Medical University of Vienna, Vienna, Austria; 2C. D. Toma, MD: Medical University of Vienna, Vienna, Austria, and Prince Court Medical Centre, Kuala Lumpur, Malaysia; 3S. Akira, MD: Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. Address correspondence and reprint requests to K. Bobacz, MD, Department of Internal Medicine III, Division of Rheumatology, Allgemeines Krankenhaus, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: firstname.lastname@example.org. Submitted for publication January 22, 2006; accepted in revised form February 20, 2007. 1880 TLRs AND CHONDROCYTES best-characterized signaling PRRs are a family of evolutionarily conserved receptors known as the Toll-like receptors (TLRs). In addition to the role of TLRs in early host defense against invading pathogens (1,2), signaling through TLRs may play an important role in autoimmune and inflammatory diseases (3–5). Many rheumatic diseases, from rheumatoid arthritis (RA) to septic arthritis, are inflammatory in nature. Interestingly, several pathogen-associated structures, such as peptidoglycans and bacterial DNA and RNA, have been found in the joint tissue or synovial fluid of patients with RA and in those with other sterile forms of arthritides (6,7), suggesting that infectious agents may be involved in the initiation and/or perpetuation of these arthritides. A pathogenic role of such agents is supported not only by epidemiologic evidence (8), but also by the capacity of streptococcal cell walls or material contained in Freund’s adjuvant (9,10) to induce severe chronic arthritis. This is further indicated by the frequent occurrence of reactive arthritis (ReA), a subacute to chronic, mostly self-limited, sterile inflammatory joint disease elicited shortly after infection with certain bacterial pathogens, especially Salmonella, Yersinia, and Chlamydia species, in genetically predisposed hosts. Cartilage degradation is highly increased in patients with ReA when compared with patients with other joint diseases, including those with RA and those with psoriatic arthritis (11,12). In addition, direct bacterial invasion of the joint has been shown to lead to rapid joint destruction, especially degradation of cartilage (13). It is currently unclear whether all of these pathogens and pathogenic materials induce arthritis, and especially joint damage, by activation of an adaptive immune response or, primarily, by activation of the innate immune response. Both pathways lead to cytokine secretion and subsequent induction of protease synthesis and other molecules involved in inflammation. Recent findings revealed that synovial inflammation and cytokine production in RA as well as in septic arthritis might be triggered by TLR-2 signaling and subsequent activation of synovial fibroblasts (14–16). Interestingly, in streptococcal cell wall–induced arthritis, TLR-2– dependent inflammation also affected articular cartilage by inducing degradation of matrix and reducing matrix synthesis; TLR-2 gene deficiency ablated these catabolic effects (14). However, it is unknown whether the disturbance in cartilage matrix synthesis was primarily caused by the effects of cytokines originating from synovial cells or by direct activation of the chondrocytes by bacterial products. 1881 On the basis of this information, we hypothesized that the activation of TLRs by bacterial constituents may directly affect the biosynthetic activity of chondrocytes and consequently lead to a decrease in matrix production. Given the previously described catabolic effects of lipopolysaccharide (LPS) on cartilage matrix (17–19), we particularly focused on the LPS-induced activation of TLR-4, the receptor for LPS, in human and murine articular chondrocytes. Our results consistently showed the expression of TLR-4 in human articular chondrocytes on both the messenger RNA (mRNA) and protein level. Furthermore, our findings revealed that LPSinduced activation of TLR-4 in articular chondrocytes decreases matrix biosynthesis, and therefore may compromise cartilage tissue replenishment in various joint diseases. PATIENTS AND METHODS Cell culture. Human articular cartilage samples from the femoral heads and knee joints of 35 patients (ages 18–87 years, mean age 60.3 years) with or without macroscopic and histologic cartilage damage were obtained at the time of endoprosthetic replacement for acute transcervical fractures (n ⫽ 3) or endoprosthetic knee replacement due to osteoarthritis (OA) (n ⫽ 23) or osteosarcoma (n ⫽ 9). Articular cartilage was also obtained from the knee joints of female C57BL/6 mice (Harlan Winkelmann, Borchen, Germany) and TLR-4⫺/⫺ mice with a C57BL/6 background. Murine samples were obtained from mice ages 6–8 weeks. Removal of all cartilage specimens was in accordance with our ethics committee policies. Sections of human and murine articular cartilage were carefully, aseptically dissected from the joint surface and finely minced. Chondrocytes were released by overnight digestion of the cartilage in 0.2% collagenase B (Boehringer Mannheim, Mannheim, Germany), followed by filtration through a cell strainer (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) to remove debris and undissociated cell clusters. The cell filtrate was then centrifuged at 500g for 10 minutes. Pellets were resuspended in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (25 mM HEPES plus 4,500 mg/liter glucose plus pyridoxine, without sodium pyruvate; Life Technologies, Gaithersburg, MD) and Ham’s F-12 (Ham’s F-12 plus Lglutamine; Life Technologies) containing 10% fetal bovine serum (PAA Laboratories, Linz, Austria) and antibiotics/ antimycotics (100 units/ml penicillin G, 100 mg/ml streptomycin, and 0.25 g/ml amphotericin B; Life Technologies). Evaluation of the chondrocyte number was done after trypan blue staining in a Buerker-Tuerk chamber. The isolated human cells were grown in quadruplicate as monolayer cultures in 24-well plates (Costar, Cambridge, MA) at a density of 1 ⫻ 105 cells/cm2, for 35S-sulfate incorporation and extraction of proteins for Western immunoblotting. For extraction of RNA, 1 ⫻ 106 cells were seeded in 6-well culture plates (Costar). Murine chondrocytes were cultured in triplicate in 24-well plates at a density of 5 ⫻ 104 cells, for 1882 BOBACZ ET AL Table 1. Primer sequences of oligonucleotides used in the study* Primer sequence Oligonucleotide (ref.) Forward Reverse Aggrecan (77) Type II collagen (78) Interleukin-1␤ (79) ␤-actin TLR-1 (80) TLR-2 (81) TLR-3 (81) TLR-4 (25) TLR-5 (81) TLR-6 (80) TLR-7 (80) TLR-8 (80) TLR-9 (81) 5⬘-CGCTACGACGCCATCTGCTAC-3⬘ 5⬘-CCCTGAGTGGAAGAGTGGAG-3⬘ 5⬘-AGATGATAAGCCCACTCTACA-3⬘ 5⬘-TGTGATGGTGGGAATGGGTCAG-3⬘ 5⬘-AGTTGTCAGCGATGTGTTCGG-3⬘ 5⬘-GCCAAAGTCTTGATTGATTGG-3⬘ 5⬘-CCATTCCAGCCTCTTCGTAA-3⬘ 5⬘-TACAAAATCCCCGACAACCTCCCCT-3⬘ 5⬘-GGAACCAGCTCCTAGCTCCT-3⬘ 5⬘-CCTGGGAGGTAAACATCTGA-3⬘ 5⬘-GATAACAATGTCACAGCCGTCC-3⬘ 5⬘-GTGTCACCCAAACTGCCAAGCTCC-3⬘ 5⬘-CGTGACAATTACCTGGCCTT-3⬘ 5⬘-GCCTGCTGTGCCTCCTCAAA-3⬘ 5⬘-GAGGCGTGAGGTCTTCTGTG-3⬘ 5⬘-ACATTCAGCACAGGACTCTC-3⬘ 5⬘-TTTGATGTCACGCACGATTTCC-3⬘ 5⬘-GATCAAGTACCTTGATCCTGGG-3⬘ 5⬘-TTGAAGTTCTCCAGCTCCTG-3⬘ 5⬘-GGATGTTGGTATGGGTCTCG-3⬘ 5⬘-AACGATGGACTTCTAAACCAGCCAGA-3⬘ 5⬘-GATGGCATCCTGGATATTGG-3⬘ 5⬘-CCCTCAACCACATAGAAACGA-3⬘ 5⬘-GTTCCTGGAGTTTGTTGATGTTC-3⬘ 5⬘-GATCCAGCACCTTCAGATGAGGC-3⬘ 5⬘-GTCCTGTGCAAAGATGCTGA-3⬘ * TLR-1 ⫽ Toll-like receptor 1. 35 S-sulfate incorporation and reverse transcription–polymerase chain reaction (RT-PCR). When the chondrocyte cultures reached 90% confluence, serum-containing medium was changed to a recently described, chemically defined serum-free basal medium (20). For evaluation of sulfated glycosaminoglycan (sGAG) synthesis and RT-PCR, the cells were subsequently cultured with either interleukin-1␤ (IL-1␤) (10 ng/ml), LPS (1 g/ml) (Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO), recombinant bone morphogenetic protein 7 (BMP-7) (100 ng/ml) (R&D Systems, Minneapolis, MN), or LPS plus BMP-7 for 5 days. Some cultures were incubated with recombinant IL-1 receptor antagonist (IL-1Ra) at 10 g/ml (Amgen, Thousand Oaks, CA) prior to stimulation with IL-1␤ or LPS. Chondrocyte cultures that were incubated with serum-free basal medium alone served as negative controls. The medium, IL-1␤, LPS, BMP-7, and IL-1Ra were replaced every other day. For Western blot analyses, subconfluent cultures of human articular chondrocytes were stimulated with LPS for 15–30 minutes. Moreover, some cell cultures were incubated for 1 hour with specific inhibitors of p38 MAPK, p44/p42 MAPK (ERK-1/2), and NF-B; these inhibitors were SB203580 (20 M), U0126 (50 M) (both from Calbiochem, San Diego, CA), and pyrrolidine dithiocarbamate (PDTC) (50 M) (Sigma-Aldrich), respectively. The cultures were then stimulated with LPS (1 g/ml) for another 30 minutes. Cultures that were incubated with LPS plus SB203580, U0126, or PDTC were also subjected to 35S-sulfate incorporation assays to determine the sGAG synthesis rate. Cultures were maintained at 37°C in an atmosphere of humidified air and 5% CO2. Histology and immunohistochemistry. Thirteen specimens obtained from the knee joints of patients were assessed by histology and immunohistochemistry. Punch biopsy specimens of joint tissue from each sample (5 mm in diameter), including cartilage and subchondral bone, were obtained from 2 different locations of the tibial plateaus and fixed in 4.0% formalin overnight. Thereafter, samples were decalcified in 14% EDTA (Sigma-Aldrich) at 4°C, pH adjusted to 7.2 by the addition of ammonium hydroxide (Sigma-Aldrich), until the bones were pliable. Specimens were embedded in paraffin and sectioned at 4 m. After deparaffination, the sections were stained with Safranin O according to standard protocols. The histopathologic changes in the cartilage sections were then evaluated by 2 independent assessors using the modified Mankin score (scale 0–14) (21). For immunohistochemistry, deparaffined, ethanoldehydrated tissue sections were blocked for 60 minutes in phosphate buffered saline (PBS) containing 1.5% goat serum, followed by overnight incubation with a mouse anti-human TLR-4 monoclonal antibody (R&D Systems) diluted 1:50. After rinsing, the sections were incubated with a species-specific anti-IgG secondary antibody (Vector, Burlingame, CA). The bound antibodies were visualized using the appropriate Vectastain ABC kit (Vector). Cultures in which the primary antibody was omitted served as negative controls. Biosynthesis of matrix macromolecules. To study the stimulatory effects of LPS on sGAG synthesis, assays of 35 S-sulfate incorporation into sGAG were performed as previously described (22). Briefly, cell cultures were washed twice with PBS and labeled with 20 Ci/ml of 35S-sulfate (carrierfree; Amersham, Buckinghamshire, UK) in a sulfate-free/ serum-free medium for 3 hours at 37°C. Chondrocytes were extracted in guanidine-HCl buffer (4M guanidine-HCl, 50 mM sodium acetate buffered at pH 7.2, in the presence of protease inhibitors). Unincorporated isotope was removed using Sephadex G-25 (PD-10 columns; Pharmacia Biotech, Piscataway, NJ) gel chromatography. Results were calculated by liquid scintillation counting (1410 liquid scintillation counter; Wallac Oy, Turku, Finland) of aliquots from void volume fractions, with values normalized to DNA content. To determine the DNA content, the dye bisbenzimide (Hoechst 33258; Sigma) was used (23). RNA isolation and RT-PCR. Total RNA was extracted using a commercially available kit (RNeasy kit; Qiagen, Valencia, CA). For RT-PCR, 1 g total RNA from each sample was copied into complementary DNA (cDNA) in a 20-l reaction using the First-Strand cDNA synthesis kit (Pharmacia, Uppsala, Sweden). One-microliter aliquots were amplified in a previously described reaction mixture (24). The following reaction profile was used for all experiments: an initial dena- TLRs AND CHONDROCYTES Figure 1. A, Endogenous expression of Toll-like receptors (TLRs) in human articular cartilage, by age groups of ⬍35 years and ⱖ35 years. Total RNA was extracted from monolayers of unstimulated chondrocytes cultured in serum-free basal medium for 5 days. Complementary DNA, prepared as described in Patients and Methods, was subjected to polymerase chain reaction (PCR) amplification using primer sequences for TLRs 1–9. The PCR mixture was separated on a 1.5% agarose gel and visualized on a FluorImager system. Equal loading of the samples was confirmed against ␤-actin expression. Peripheral blood mononuclear cells (PBMCs) served as a positive control. B, Detection of TLR-4 in human articular cartilage tissue extracts (n ⫽ 5) by Western immunoblotting using specific polyclonal antibodies. Cell extracts from PBMCs served as a positive control. turation step at 94°C for 1 minute, followed by 30 cycles at 94°C for 1 minute, 52°C for 1 minute, and 72°C for 40 minutes, and an additional extension step at 72°C for 10 minutes after the last cycle. Amplification reactions were performed in an air thermal cycler (Idaho Technology, Idaho Falls, ID). The amplified DNA fragments were visualized with a FluorImager (Bio-Rad, Hercules, CA). Positive and negative control experiments were run in parallel. The primer sequences used are listed in Table 1. Western blot analysis. For the analysis of TLR-4 expression, cartilage slices of 5 freshly isolated articular cartilage specimens (0.5–1 gm wet weight) were washed with PBS, finely minced, and extracted in guanidine-HCl buffer (1M guanidine-HCl, 50 mM sodium acetate buffered at pH 7.2, in the presence of protease inhibitors) for 3 hours at 4°C and subsequently dialyzed (Slide-A-Lyzer Dialysis Cassette; Pierce, 1883 Rockford, IL) against water for 48 hours at 4°C. Extracts were then lyophilized and resuspended in sample buffer (NuPAGE lithium dodecyl sulfate sample buffer) to which 8M urea was added, and stored at ⫺80°C. For Western blot analysis of cell lysates, chondrocyte cultures were washed 3 times with PBS. Thereafter, 50 l of sample buffer (4⫻ NuPAGE LDS sample buffer; Invitrogen, Carlsbad, CA) per well was added. Cells were disrupted by ultrasound, and samples were stored at ⫺80°C. Aliquots of 1 l of samples per lane were analyzed under reducing conditions (2% ␤-mercaptoethanol). Samples were boiled for 5 minutes in a water bath at 95°C and loaded onto a 10% Bis-Tris gel (NuPAGE 10% Bis-Tris Gel; Invitrogen). Electrophoresis was carried out in a commercially available running buffer (20⫻ NuPAGE MOPS sodium dodecyl sulfate [SDS] running buffer; Invitrogen) at 200V for 60 minutes. Proteins were transferred from the gel onto a nitrocellulose membrane for 120 minutes at 30V in a buffer containing 0.4M glycine, 0.5M Tris base, and 0.01M SDS. Nonspecific binding sites were blocked with 4% bovine serum albumin for 60 minutes. To determine the presence of TLR-4, blots were probed with a polyclonal antibody against TLR-4 (Santa Cruz Biotechnology, Santa Cruz, CA). Cell extracts from peripheral blood mononuclear cells (PBMCs) were used as a positive control. LPS-mediated TLR signaling was evaluated in cell lysates using the PhosphoPlus p38 MAPK antibody kit, PhosphoPlus p44/42 MAPK antibody kit, or PhosphoPlus IB␣ (inhibitor of NF-B) antibody kit (all from New England Biolabs, Beverly, MA) according to the manufacturer’s instructions. For enhancement of the detected signal, a chemiluminescent substrate (SuperSignal West Pico; Pierce) was applied. The blots were visualized on Kodak X-Omat Blue XB-1 imaging film (Eastman Kodak, Rochester, NY). Statistical analysis. Results are expressed as the mean ⫾ SEM. Comparisons of treated and untreated samples were performed using Student’s t-test. P values less than 0.05 were considered significant. RESULTS Expression of TLRs 1–9 in human articular chondrocytes. The expression of some members of the TLR family has been examined in previous studies, although the existing data are contradictory (25–27). To determine the expression profiles of TLRs 1–9 in postnatal articular chondrocytes, RT-PCR analysis of total RNA in chondrocytes obtained from human articular cartilage was performed; total RNA derived from PBMCs served as a positive control. This analysis yielded amplification products of fragments specific for mRNA encoding all TLRs, including the intracellular TLRs of the TLR-9 subfamily (Figure 1A). However, in some of the cells, RT-PCR analysis of TLR-7, an intracellular TLR, displayed only a very faint signal. Interestingly, chondrocytes from patients younger 1884 BOBACZ ET AL Figure 2. Immunohistochemical detection of Toll-like receptor 4 in articular chondrocytes from human knee cartilage. Results from immunohistochemical analyses of 6 specimens are presented, arranged according to their respective modified Mankin score (scale 0–14) of cartilage damage (scores of 2–7, respectively) (original magnification ⫻ 100). Insets, Higher-magnification general view of all cartilage zones (original magnification ⫻ 50). Articular chondrocytes were obtained from patients ages 65 years (A), 74 years (B), 59 years (C), 61 years (D), 63 years (E), and 71 years (F). A negative control experiment in which the primary antibody was omitted was run in parallel. than age 35 years expressed TLR-7, whereas TLR-7 expression was undetectable in chondrocytes of patients age 35 years or older (Figure 1A), suggesting that aging cartilage may lose the capacity to express this molecule. Detailed analysis of TLR-4 expression in human articular chondrocytes. Since this study was primarily focused on LPS-mediated effects, we extended our RT-PCR analyses to an investigation of TLR-4 expression on the protein level. Protein from 5 human articular cartilage tissue specimens was extracted ex vivo and evaluated by immunoblotting; cell extracts from PBMCs served as a positive control. Consistent with the data on mRNA expression, we demonstrated the presence of TLR-4 on the protein level, with individual variations in basal protein expression (Figure 1B). To further elucidate the expression of TLR-4, human cartilage tissue specimens obtained from 13 knees were evaluated by immunohistochemistry. TLR-4 was entirely and predominantly expressed in the superficial and transitional zones of the cartilage, with only a few positively stained cells in the deeper zones (Figure 2). Interestingly, the expression of TLR-4 appeared to be up-regulated in parallel with increasing Mankin scores of histopathologic features and cartilage damage (Figure 2); the most intense staining was found in areas of severe surface disruption (Figure 2D). In very advanced OA, however, only a few of the remaining chondrocytes expressed TLR-4 (Figure 2F). Suppressive effects of LPS with dependence on the presence of TLR-4. It is generally accepted that LPS from gram-negative bacteria stimulates an inflammation response (28,29). Moreover, LPS was previously shown to induce matrix breakdown in articular cartilage explants (17–19) and is widely used as a catabolic molecule in cell culture. Likewise, the presence of IL-1␤ leads to reductions in biosynthetic activity. In the present study, murine chondrocyte monolayer cultures that were treated with IL-1␤ showed a significant decrease in the synthesis of sGAG, as measured by 35S-sulfate incorporation, in both wild-type (WT) and TLR-4⫺/⫺ mouse strains (Figure 3). In particular, IL-1␤ reduced matrix production by almost 30% in TLR-4⫺/⫺ mice (mean ⫾ SEM 29.3 ⫾ 2.5 counts per minute/g DNA in unstimulated control cultures versus 20.7 ⫾ 2.4 cpm/g DNA in IL-1␤–treated cultures; P ⬍ 0.003) and by ⬃27% in WT mice (31.6 ⫾ 1.3 cpm/g DNA in unstimulated control cultures versus 23.2 ⫾ 1.6 TLRs AND CHONDROCYTES 1885 Figure 3. Suppressive effects of lipopolysaccharide (LPS) on articular cartilage in relation to the presence of Toll-like receptor 4 (TLR-4). Murine articular chondrocytes were incubated in serum-free basal medium (BM) alone as the unstimulated control or in BM with the addition of interleukin-1␤ (IL-1␤) or LPS. On day 5 of the stimulation period, cell cultures were labeled with 35S-sulfate for 3 hours. In wild-type mice (n ⫽ 3), stimulation with LPS led to a significant decrease in matrix macromolecule synthesis (A), whereas in TLR-4⫺/⫺ mice (n ⫽ 3), no such suppressive effect was observed (C). Results are the mean and SEM rate of incorporated radiolabel in the newly synthesized matrix macromolecules on the cell layer, normalized to DNA content. Results from polymerase chain reaction analysis of aggrecan and IL-1␤ gene expression (B and D) support the findings from the isotope incorporation experiments (A and C). * ⫽ P ⬍ 0.009; ** ⫽ P ⬍ 0.0005; *** ⫽ P ⬍ 0.003, versus BM. cpm/g DNA in IL-1␤–treated cultures; P ⬍ 0.009) (Figures 3A and C). When chondrocytes from WT mice were subjected to LPS, a marked reduction in isotope uptake was observed, with a decrease of ⬃50% compared with controls (31.6 ⫾ 1.3 cpm/g DNA in unstimulated control cultures versus 15.7 ⫾ 1.7 cpm/g DNA in LPS-stimulated cultures; P ⬍ 0.0005) (Figure 3A). In contrast, when chondrocytes derived from TLR4–deficient mice were stimulated with LPS, there was no appreciable decrease in sGAG synthesis (29.3 ⫾ 2.5 cpm/g DNA in unstimulated control cultures versus 28.7 ⫾ 2.7 cpm/g DNA in LPS-stimulated cultures; P ⫽ 0.89) (Figure 3C). Furthermore, in contrast to the results obtained in WT mice and to the effects of IL-1␤ described above, but consistent with the abrogation of LPS-induced effects in the absence of TLR-4, analysis of gene expression profiles in chondrocytes from TLR-4⫺/⫺ mice dem- 1886 BOBACZ ET AL Figure 4. A, Expression of mRNA for aggrecan, type II collagen, interleukin-1␤ (IL-1␤), and Toll-like receptor 4 (TLR-4) was evaluated by immunoblotting in human articular cartilage specimens after incubation with basal medium (BM) as the unstimulated control or with recombinant bone morphogenetic protein 7 (BMP-7), IL-1␤, lipopolysaccharide (LPS), or LPS plus BMP-7. Equal loading of the samples was confirmed against ␤-actin expression. Results are representative of 3 experiments. B, Isotope incorporation assays were performed in articular chondrocytes obtained from human cartilage specimens (n ⫽ 5). Cells were cultured as monolayers, incubated with BMP-7, IL-1␤, LPS, or LPS plus BMP-7 for 5 days, and subsequently labeled with 35S-sulfate for 3 hours. * ⫽ P ⬍ 0.00005; ** ⫽ P ⬍ 0.001; *** ⫽ P ⬍ 0.003, versus BM. C, An additional 7 cartilage specimens were studied with isotope incorporation assays after stimulation with IL-1␤ or LPS or costimulation with IL-1␤ plus IL-1 receptor antagonist (IL-1Ra) or LPS plus IL-1Ra. * ⫽ P ⬍ 0.03 versus IL-1␤; ** ⫽ P ⬍ 0.005 versus LPS. In B and C, results are the mean and SEM rate of incorporated radiolabel in the newly synthesized matrix macromolecules, normalized to DNA content. onstrated that there was no up-regulation of IL-1␤ after LPS stimulation (Figure 3D), and, conversely, there was only minor down-regulation of aggrecan after LPS stimulation, as compared with the findings in WT controls (Figure 3B). Effects of BMP-7 and IL-1Ra and role of IL-1␤ in LPS-stimulated human articular cartilage. In human articular chondrocytes, stimulation with LPS led to a significant decrease in cartilage sGAG biosynthesis as compared with that in unstimulated control cultures (1,168.1 ⫾ 225.7 cpm/g DNA in controls versus 517.9 ⫾ 57.2 cpm/g DNA in LPS-stimulated cultures; P ⬍ 0.003) (Figure 4B). This decrease was comparable with the effects observed in IL-1␤–incubated human chondrocyte cultures (347.1 ⫾ 31.5 cpm/g DNA; P ⬍ 0.001 versus controls) (Figure 4B). The results from isotope incorporation assays were supported by the observed reduction in aggrecan and type II collagen mRNA expression levels in LPS- and IL-1␤–treated chondrocyte cultures (Figure 4A). Moreover, with re- TLRs AND CHONDROCYTES 1887 Figure 5. Evaluation of lipopolysaccharide (LPS)–induced activation of A, p38 MAPK, B, p44/p42 MAPK, or C, IB␣ after 15–30 minutes. Chondrocyte cultures were incubated with specific inhibiting agents (SB203580, U0126, and pyrrolidine dithiocarbamate [PDTC]) for 1 hour, and LPS was then added to the cultures for another 30 minutes. The phosphorylation/activation of p38 MAPK, p44/p42 MAPK, or IB␣ was measured by Western immunoblotting. A marked increase in the phosphorylation of p38 MAPK was evident after stimulation with LPS. BM ⫽ basal medium (as negative control). gard to mRNA expression of IL-1␤, we found a significant up-regulation of this cytokine after stimulation with LPS and IL-1␤ as compared with that in unstimulated control cultures (Figure 4A). Interestingly, the effects of LPS on human articular chondrocytes were antagonized by BMP-7, a potent anabolic factor in cartilage homeostasis (30). In fact, incubation of chondrocytes with BMP-7 induced the expected marked increase in sGAG production (3,358.8 ⫾ 528.8 cpm/g DNA in BMP-7–treated cultures; P ⬍ 0.00005 versus controls). Of note, when BMP-7 was present in chondrocyte cultures subjected to LPS, a 3-fold increase in isotope uptake, as compared with incubation with LPS alone, was found (1,626.8 ⫾ 348.3 cpm/g DNA with LPS plus BMP-7; P ⬍ 0.003 versus LPS alone); this effect more than counteracted the LPS-induced degradative effects (Figure 4B). In accordance with previous results demonstrating that 1888 BOBACZ ET AL Figure 6. A, Effects of inhibition of p38 MAPK and p44/p42 MAPK by SB203580 or U0126 in chondrocyte cultures in relation to involvement of kinases in the lipopolysaccharide (LPS)/Toll-like receptor 4 signaling cascade in human articular chondrocytes. A, Results from reverse transcription–polymerase chain reaction analysis of interleukin-1␤ (IL-1␤) mRNA regulation. Results are representative of 3 independent experiments. Peripheral blood mononuclear cells (PBMCs) stimulated with LPS served as the positive control, while cultures in basal medium (BM) served as the negative control. B, Human articular chondrocytes (n ⫽ 7) were cultured as monolayers. Stimulation with LPS, LPS plus SB203580, or LPS plus U0126 was performed. Cultures incubated in BM served as controls. The cultures were labeled with 35S-sulfate for 3 hours, and the mean and SEM rate of incorporated isotope was measured. Values were normalized to DNA content of the cells. * ⫽ P ⬍ 0.007 versus LPS alone. BMP-7 counteracts the inhibitory effects of IL-1␤ on cartilage matrix synthesis (31), the present results suggest a major involvement of IL-1␤ in the LPS-mediated effects on chondrocytes. These findings were further supported by the determination of the endogenous expression of aggrecan and IL-1␤. The LPS-induced increase in IL-1␤ expression was antagonized by the concomitant presence of BMP-7. Conversely, the LPS-induced reduction in aggrecan mRNA expression was restored to basal levels in the presence of BMP-7 (Figure 4A). Nevertheless, BMP-7 did not increase the expression of type II collagen mRNA, a phenomenon that has been observed previously in studies with BMP-7 (22) and other BMPs (32). Moreover, BMP-7 did not counteract the LPSdependent reduction in type II collagen expression levels (Figure 4A), indicating that the effects of LPS on type II collagen may be mediated by pathways other than IL-1␤–dependent pathways. We also assessed the influence of chondrocyte stimulation on the mRNA expression of TLR-4. Our results revealed that TLR-4 mRNA expression in human articular chondrocytes did not differ among the cultures, irrespective of the various stimuli used (Figure 4A). To further evaluate whether the effects of LPS were mediated by IL-1␤, recombinant IL-1Ra was added to the culture medium prior to stimulation and isotope incorporation. When we compared the rate of 35Ssulfate uptake in cultures stimulated with LPS in the presence of IL-1Ra with that in cultures incubated with LPS alone, we observed an ⬃30% increase in the sGAG synthesis rate (719 ⫾ 46.9 cpm/g DNA with LPS alone versus 942.3 ⫾ 57.3 cpm/g DNA with LPS plus IL-1Ra; P ⬍ 0.005) (Figure 4C), bolstering the notion that the LPS-mediated effects are, at least in part, mediated by IL-1␤. In fact, IL-1Ra antagonized the IL-1␤–induced impairment of sGAG synthesis to an extent similar to that seen in cultures with LPS (776.9 ⫾ 67.7 cpm/g DNA with IL-1␤ alone versus 1,026.1 ⫾ 81.5 cpm/g DNA with IL-1␤ plus IL-1Ra; P ⬍ 0.03). The introduction of IL-1Ra, however, could not restore the isotope uptake levels to basal values prior to stimulation (1,237.9 ⫾ 119.2 cpm/g DNA) (Figure 4C). This finding suggests that IL-1Ra may not fully abrogate the IL-1␤–mediated effects, which could be attributable to the fact that the avidity of IL-1Ra to the IL-1 receptors is not higher than that of IL-1 (33). LPS-mediated TLR-4 signaling through p38 MAPK in articular chondrocytes. To characterize the TLR-4 signaling pathways mediated by LPS in articular chondrocytes, the phosphorylation of p38 MAPK, ERK1/2, and IB␣, important factors in the LPS/TLR-4 signaling cascade, was investigated by immunoblotting. TLRs AND CHONDROCYTES Human articular chondrocytes were incubated with LPS for either 15 minutes or 30 minutes. Unstimulated cells served as controls. Moreover, specific inhibitors of p38 MAPK, ERK-1/2, and NF-B, SB203580, U0126, and PDTC, respectively, were added to some cultures for 1 hour prior to stimulation with LPS for another 30 minutes. Although the various kinases analyzed were already preactivated in the cultured chondrocytes, which is consistent with previous observations (34), stimulation with LPS caused an increase in phosphorylation of p38 MAPK in human articular chondrocytes after both 15 minutes and 30 minutes, whereas the phosphorylation of ERK-1/2 and IB␣ remained constant when compared with that in unstimulated controls (Figures 5A–C). SB203580, a selective inhibitor of p38, decreased the LPS-induced phosphorylation of p38 (Figure 5A). Moreover, p38 phosphorylation was also decreased by blocking of ERK-1/2. In contrast, PDTC markedly increased the phosphorylation of p38 MAPK (Figure 5A). The LPS-induced phosphorylation of ERK-1/2 was completely blocked by the MEK inhibitor U0126, whereas SB203580 and PDTC further increased the phosphorylation of both ERK-1 and ERK-2 kinases (Figure 5B). IB␣ phosphorylation was not significantly affected by SB203580, U0126, or PDTC (Figure 5C). We thus evaluated the role of p38 MAPK and ERK-1/2 in matrix biosynthesis in LPS-activated chondrocytes. Our results revealed that blocking of p38 MAPK resulted in an increase in sGAG synthesis compared with that in cultures with LPS alone (719 ⫾ 46.9 cpm/g DNA with LPS alone versus 958.6 ⫾ 46.2 cpm/g DNA with LPS plus SB203580; P ⬍ 0.007). The use of ERK-1/2 inhibitor U0126 had no effect on the LPS-induced reduction in isotope incorporation (703.6 ⫾ 68.1 cpm/g DNA with LPS plus U0126) (Figure 6B). We also examined the mRNA expression of IL-1␤ after blocking distinct signaling pathways. The results showed that the endogenous expression of IL-1␤ was noticeably down-regulated by inhibition of p38 MAPK, but not by inhibition of ERK-1/2 (Figure 6A). DISCUSSION In the present study we have shown that human articular chondrocytes express TLRs 1–9, suggesting that articular chondrocytes can be directly activated by the respective ligands of each TLR. We further demonstrated that, in the presence of TLR-4, LPS directly activates articular chondrocytes to induce up-regulation of IL-1␤ and inhibition of biosynthetic activity. The 1889 LPS-induced suppression of matrix synthesis of articular cartilage observed in the present study was, at least in part, governed by up-regulation of IL-1␤ and could be antagonized by BMP-7 and IL-1Ra. TLRs have only recently been identified as important molecules in the mammalian innate immune response. These receptors are expressed primarily in macrophages and dendritic cells (35) and act as an effective early host defense system. However, TLRs not only are expressed by cells involved in activation of the immune system, but also have been found, among other molecules, in human aortic endothelial cells, lung epithelial cells, hepatocytes, and synoviocytes (36–39). The observations reported herein, that TLRs are also expressed in articular chondrocytes, expand these findings and are consistent with the notion that these cells display functional properties in common with monocytes/macrophages (40–45). In contrast to hitherto-inconsistent observations regarding the presence of some of the TLRs in articular chondrocytes (25–27), we found clear evidence of the expression of TLRs 1–9 in human articular chondrocytes on the mRNA level. Interestingly, TLR-7 was only demonstrable in chondrocytes derived from patients younger than age 35 years, suggesting that a patient’s age may influence TLR expression, which is consistent with previous observations of the effects of aging in cartilage biology, particularly with regard to matrix synthesis and cell numbers, each of which shows a dramatic decrease in patients around age 30 years (24,46–50). However, in our culture settings, the presence of TLRs 1–6 as well as TLR-8 and TLR-9 in human articular chondrocytes was independent of age. The presence of TLR-4, the main focus of this study, was shown not only on the mRNA level, but also on the protein level in ex vivo tissue extracts from human articular cartilage, as well as by immunohistochemistry. We made the striking observation that the expression of TLR-4 tends to be up-regulated in parallel with greater severity of histopathologic changes as measured by increasing modified Mankin scores, but its expression is reduced in severely damaged cartilage. The reason that a receptor may be up-regulated in chondrocytes, with its activation leading to matrix catabolism, remains the subject of speculation; the mechanism of action might be attributable to mechanical stimuli or to binding of tissue breakdown products. The presence of TLR-4, however, appears to contribute to the onset of inflammation, as demonstrated in a murine model of immune complex– mediated arthritis (51). Given the presence of TLR-4 in human articular 1890 chondrocytes, we investigated the effects of TLR-4 activation via LPS on cartilage matrix synthesis. Recently, Joosten and coworkers demonstrated that cartilage breakdown as well as inhibition of cartilage proteoglycan synthesis in a mouse model of arthritis was initiated by intraarticular triggering of either TLR-2 or TLR-4 (14). These results provided a first hint of the TLR-dependent impairment of cartilage matrix synthesis. Nevertheless, when addressing the mechanisms of cartilage breakdown after intraarticular application of various agents, investigators should take into account the activation of synoviocytes via TLR-mediated or other mechanisms, and consider the secondary effects on cartilage (16). Using the sGAG production rate as a surrogate marker, we consistently showed a marked decrease in biosynthetic activity in primary human articular chondrocyte cultures after incubation with LPS, which was comparable with that induced by IL-1␤. Consistent with these results, expression of aggrecan mRNA and type II collagen mRNA was also reduced. In contrast, IL-1␤, a cytokine known to be involved in the pathogenesis of RA and OA (52–54), was up-regulated in LPS-treated cultures. This increase in IL-1␤ expression may be the linchpin in LPS-induced TLR-4 signaling, ultimately leading to suppression of cartilage matrix production, which would be in accordance with previous studies showing the importance of IL-1␤ in cartilage destruction (55,56). Indeed, in the presence of IL-1Ra, the LPSmediated effects on the biosynthetic activity of chondrocytes were significantly inhibited. We further validated our hypothesis by investigating the stimulatory effects of LPS as well as IL-1␤ on articular chondrocytes derived from TLR-4⫺/⫺ mice, with the results showing that in a TLR-4–deficient state, LPS had no effect on sGAG neosynthesis or on IL-1␤ expression, unlike the effects observed in TLR-4– competent WT mice. Interestingly, despite the absence of TLR-4, aggrecan mRNA expression was slightly decreased after LPS stimulation compared with that in unstimulated control cultures; this phenomenon could be due to internalization of LPS and binding to its intracellular receptor, Nod1, that potentially activates a TLR-4–independent signaling cascade (57,58). Furthermore, we assessed whether the catabolic events triggered by LPS could be counteracted by BMP-7, a major growth factor in cartilage biology (30,31). Our findings revealed that BMP-7 interfered with the inhibitory effects of LPS on chondrocyte sGAG synthesis and restored aggrecan and IL-1␤ mRNA expression to basal levels. This anabolic/anti-catabolic BOBACZ ET AL effect could be exerted either indirectly by activating the BMP-specific pathways via BMP receptors and receptorassociated Smads or directly by activating MAPKs (p38 MAPK) via transforming growth factor ␤–activated kinase (Tak1) (59,60). Alternatively, since BMP and TLR signaling pathways are linked by the protein known as evolutionarily conserved signaling intermediate in Toll pathways, a direct crosstalk between BMP and the TLRs might modulate the signaling competence of the TLRs, as has been observed in Drosophila during dorsoventral patterning (61). However, when we investigated the phosphorylation of p38 MAPK in BMP-7– and LPS/ BMP-7–costimulated chondrocytes, no difference was evident (results not shown), which supports the presumption that BMP-7 signaling is affected via the Smad pathway only, as proposed recently (62). The signaling cascade participating in LPSinduced TLR activation has been extensively characterized in macrophages (63–68). Generally, MAPKs and transcription factors such as NF-B become activated upon binding of IL-1R–associated kinase 1 to tumor necrosis factor receptor–associated factor 6, and concomitantly induce the synthesis and release of cytokines (64,66,69,70). An important event in LPS/TLR-4 signaling is the phosphorylation of p38 MAPK, as has been previously demonstrated in macrophages (71). In the present study of human articular chondrocytes, LPS stimulation led to an increased phosphorylation of p38 MAPK, and blocking of p38 MAPK with SB203580 resulted in a significant increase in sGAG synthesis as well as a decrease in IL-1␤ mRNA expression. The ERK-1/2 pathway, however, seemed to play no role in our experimental settings, since blocking of its activity did not affect sGAG synthesis or IL-1␤ expression. Our inability to detect changes in the phosphorylation status of IB␣, however, might have been due to its preactivation in the cultured chondrocytes, which could have rendered alterations in phosphorylation beyond the limits of detection. According to these findings, the LPS-mediated TLR-4 signaling involved in decreased chondrocyte biosynthesis is dependent on the activation of p38 MAPK, a finding that is fully consistent with results reported in the literature. On the one hand, up-regulation of IL-1␤ is governed by p38 MAPK (72), while, on the other hand, the activation of p38 MAPK has been implicated in the catabolic and anti-anabolic actions of IL-1␤ in articular chondrocytes (73–75). This study was able to show the presence of TLRs 1–9 in postnatal human articular chondrocytes. It further showed that the suppressive effects of LPS on articular TLRs AND CHONDROCYTES cartilage biosynthetic activity are dependent on the presence of TLR-4 and are exerted, at least in part, by up-regulation of IL-1␤, which could be antagonized by BMP-7 or IL-1Ra. Furthermore, this in vitro study characterized the involvement of p38 MAPK, but not ERK-1/2, in LPS-mediated TLR-4 signaling in human articular chondrocytes. These results suggest that therapeutic targeting of TLRs, which has been proposed as a potential approach for treating RA (76), may be a viable way to specifically address and ameliorate the degradation of cartilage in this disease as well as in ReA or septic arthritis. 1891 12. 13. 14. 15. 16. ACKNOWLEDGMENT The opportunity to carry out this investigation was provided under the auspices of the Joint and Bone Center (Schwerpunkt muskuloskelettale Erkrankungen) of the Medical University of Vienna. AUTHOR CONTRIBUTIONS Dr. Bobacz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Bobacz, Saemann, Smolen. Acquisition of data. Bobacz, Sunk, Hofstaetter, Amoyo, Toma, Akira, Weichhart, Saemann. Analysis and interpretation of data. Bobacz, Sunk, Smolen. Manuscript preparation. Bobacz, Sunk, Smolen. Statistical analysis. Bobacz. 17. 18. 19. 20. 21. 22. REFERENCES 1. 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