Human -defensin 3 mediates tissue remodeling processes in articular cartilage by increasing levels of metalloproteinases and reducing levels of their endogenous inhibitors.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 6, June 2005, pp 1736–1745 DOI 10.1002/art.21090 © 2005, American College of Rheumatology Human ␤-Defensin 3 Mediates Tissue Remodeling Processes in Articular Cartilage by Increasing Levels of Metalloproteinases and Reducing Levels of Their Endogenous Inhibitors Deike Varoga,1 Thomas Pufe,2 Jürgen Harder,3 Jens-Michael Schröder,3 Rolf Mentlein,2 Ulf Meyer-Hoffert,3 Mary B. Goldring,4 Bernhard Tillmann,2 Joachim Hassenpflug,3 and Friedrich Paulsen5 Objective. Beta-defensins are broad-spectrum antimicrobial peptides (APs) that are components of innate immunity. Recent investigations showed the induction of ␤ -defensins in synovial membranes of osteoarthritic (OA) joints and suggested that they have functions other than the ability to kill microbes. As a result of these findings, we undertook this study to investigate the production of human ␤-defensin 3 (HBD-3) in OA cartilage and to determine its influence on chondrocyte function. Methods. Healthy and OA cartilage were assessed for HBD-3 expression by reverse transcriptase– polymerase chain reaction (RT-PCR) and immunohistochemistry. HBD-3 expression in C28/I2 chondrocytes after administration of tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1) was determined by real-time RT-PCR and immunodot blot. Enzyme-linked immunosorbent assay experiments were used to study the effects of HBD-3 in cultured articular chondrocytes and in healthy and OA cartilage discs. Immunohistochemical analyses were performed to study the expression of mouse ␤-defensins (MBDs) in OA cartilage of STR/Ort mice. Results. HBD-3 was induced in OA cartilage without bacterial challenge. Cytokines involved in the pathogenesis of OA, namely, TNF␣ and IL-1, were strong inducers of HBD-3 in cultured chondrocytes. Application of the recombinant HBD-3 protein to cultured chondrocytes and cartilage discs resulted in increased production of cartilage-degrading matrix metalloproteinases and in down-regulation of their endogenous regulators, tissue inhibitors of metalloproteinases 1 and 2. Furthermore, STR/Ort mice, which are genetically predisposed to develop OA-like lesions in the knee joint, demonstrated an increased expression of MBDs 3 and 4 in cartilage compared with that in healthy animals. Conclusion. These findings widen our knowledge of the functional spectrum of APs and demonstrate that HBD-3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissueremodeling processes in articular cartilage. Moreover, our data suggest that HBD-3 is an additional factor in the pathogenesis of OA. Drs. Varoga and Pufe’s work was supported in part by a grant from the Hensel-Stiftung of the University of Kiel. Drs. Harder and Schröder’s work was supported by the SFB 617. Dr. Goldring’s work was supported by the NIH (grants R01-AR-45378 and R01-AG22021). 1 Deike Varoga, MD: University Hospital Schleswig-Holstein, Campus Kiel, and Christian Albrechts University of Kiel, Kiel, Ger2 many; Thomas Pufe, PhD, Rolf Mentlein, PhD, Bernhard Tillmann, PhD: Christian Albrechts University of Kiel, Kiel, Germany; 3Jürgen Harder, PhD, Jens-Michael Schröder, PhD, Ulf Meyer-Hoffert, MD, Joachim Hassenpflug, PhD: University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 4Mary B. Goldring, PhD: Beth Israel Deaconess Medical Center, New England Baptist Bone and Joint Institute, and Harvard Institutes of Medicine, Boston, Massachusetts; 5 Friedrich Paulsen, PhD: Christian Albrechts University of Kiel, Kiel, and University of Halle, Wittenberg, Germany. Address correspondence and reprint requests to Deike Varoga, MD, Department of Orthopaedic Surgery, University of Kiel, Michaelisstrasse 1, 24105 Kiel, Germany. E-mail: d.varoga@ orthop.uni-kiel.de. Submitted for publication August 25, 2004; accepted in revised form March 2, 2005. Antimicrobial peptides (APs) are effector molecules of the innate immune system. They act as antibiotics by directly killing microorganisms and are primarily expressed in epithelial tissues, where they help to limit infections in the first hours after microbial colonization. The human innate defense system includes several different subfamilies of APs, such as defensins, RNase 7, cationic antimicrobial protein (CAP-37), and the cathe1736 HBD-3 IN OSTEOARTHRITIC CARTILAGE licidin LL-37 (1). Expression of some APs is constitutive and contributes to the noninflammatory antimicrobial barrier of epithelial surfaces, whereas other APs are induced after appropriate stimulation (2–5). Defensins represent an important peptide family among APs. These small (3–5 kd), cysteine-rich, cationic peptides are divided into ␣- and ␤-defensins based on the location and the connectivity of 6 conserved cysteine residues. Human ␤-defensins 2 and 3 (HBDs 2 and 3) have been isolated from human lesional psoriatic scales (3,4), and various studies demonstrated an up-regulation in response to stimulation by tumor necrosis factor ␣ (TNF␣) (3), interleukin-1 (IL-1) (6), or bacteria (7). Moreover, ␤-defensins serve as a link between innate and adaptive immune responses by acting as chemotactic factors for immature dendritic cells and T cells (8). Recent data revealed the induction of HBD-3 in keratinocytes by insulin-like growth factor 1 (9). The upregulation by growth factors suggests the existence of functions in addition to antimicrobial functions and might reflect a potential influence of HBD-3 in remodeling processes after tissue degradation. Recently, Paulsen et al (10,11) demonstrated that “inner surfaces” such as synovial membranes of articular joints are also protected from microbial invasion through the endogenous production of APs. In the case of osteoarthritis (OA), the expression pattern of APs in the synovial membranes changes. HBD-3 and LL-37 messenger RNA (mRNA), which are not expressed in healthy synovial membranes, are up-regulated without bacterial challenge in OA. OA in general is characterized by a breakdown of extracellular matrix (ECM) of articular cartilage in the affected joints. The pathogenesis involves multiple etiologies including mechanical, genetic, and biochemical factors. Various in vitro and in vivo studies indicate that IL-1 and TNF␣ are involved in the initiation and progression of articular cartilage destruction (12,13). Matrix metalloproteinases (MMPs) are thought to play a central role in cartilage degradation (14). The collagenases (MMPs 1, 8, and 13) are distinguished from other MMPs by their unique ability to cleave type II collagen, the major component of articular cartilage ECM (15). MMPs may be induced in synoviocytes or chondrocytes after induction of gene expression by numerous cytokines such as IL-1␤, TNF␣ (16,17), or vascular endothelial growth factor (18). MMP activity is controlled in part by tissue inhibitors of metalloproteinases (TIMPs), which form inhibitory complexes in a 1:1 stoichiometry (19). The imbalance between proteinases and inhibitors ultimately leads to an altered net proteolysis of cartilage 1737 components. Once damaged, articular cartilage has a poor intrinsic repair capacity. STR/Ort mice, which are genetically predisposed to develop OA-like lesions in the knee before the age of 6 months (20,21), have been used to study the pathogenesis of OA in vivo. Comparable with the situation in humans, the development of OA in mice correlates with the up-regulation of proinflammatory cytokines such as TNF␣ and IL-1 (22). In order to investigate the role of ␤-defensins in vivo, genes for homologous peptides in laboratory animal models should be identified. To date, more than 10 different mouse ␤-defensins (MBDs) have been found (23). Similar to the human homologs, they are involved in innate, antibacterial defense mechanisms, although only MBD-2 (24) and MBD-3 (25,26) have been verified to be inducible. Controversies exist regarding the murine homolog of HBD-3, since the similarity of the amino acid sequence to that of the human counterpart is low (23). The induction of HBD-3 in synovial membranes of OA joints suggests that it has functions other than the ability to kill microbes. This encouraged us to investigate its production in OA cartilage and to determine its influence on chondrocyte function. MATERIALS AND METHODS Tissues. OA cartilage (n ⫽ 10 samples) was obtained, with approval of the Institutional Review Board, from patients (ages 38–78 years) who underwent knee joint replacement at the Department of Orthopaedic Surgery, University of Kiel. Tissue samples were graded according to the Mankin scale (27), and only samples with moderate-to-severe OA (Mankin score 6–14) were included. Healthy cartilage (n ⫽ 5 samples) was obtained from patients (ages 21–52 years) who underwent resection arthroplasty because of an extraarticular tumor. Cell culture. The human chondrocyte cell line C28/I2 was used to examine the regulation of ␤-defensin expression in vitro. These cells, immortalized with SV40 large T antigen, express proteins such as aggrecan, type II collagen, and other markers that are typical of the differentiated phenotype (28) and have been used to study the regulation of gene expression and signaling in response to cytokines and other factors (29,30). For experiments, 1 ⫻ 106 cells were seeded in 25-cm2 flasks and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS). When they reached 80% confluency, the medium was changed to serum-free DMEM containing 0.05% bovine serum albumin. IL-1␤ (10 ng/ml; Tebu, Offenbach, Germany) or TNF␣ (10 ng/ml; Tebu) was added 3 hours later, and the incubation was continued for 6 hours. In addition, OA and healthy cartilage discs (3-mm diameter ⫻ 1-mm thickness) were cultured under standard conditions as described by Kurz et al (31). After 24 hours of 1738 incubation with different amounts of HBD-3 peptide, conditioned medium was withdrawn and aliquots were assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits. Analysis of HBD-3 mRNA in human cartilage by reverse transcriptase–polymerase chain reaction (RT-PCR). Frozen tissue samples (20 mg) of healthy and OA cartilage were crushed in an achate mortar under liquid nitrogen. The RNA from the cultured chondrocytes and cartilage was extracted and prepared for PCR as recently described (32). For the PCR, 4 l of complementary DNA (cDNA) was incubated with 30.5 l of water, 4 l of 25 mM MgCl2, 1 l of dNTP, 5 l of 10⫻ PCR buffer, and 0.5 l (2.5 units) of Platinum Taq DNA polymerase (Gibco, Karlsruhe, Germany) and an intron-spanning primer pair for HBD-3 (forward 5⬘AGCCTAGCAGCTATGAGGATC-3⬘, reverse 5⬘-CTTCGGCAGCATTTTGCGCCA-3⬘), which yielded a 206-bp amplified product at an annealing temperature of 60°C. A GAPDH-specific intron-spanning primer pair (forward 5⬘-CCAGCCGAGCCACATCGCTC-3⬘, reverse 5⬘-ATGAGCCCCAGCCTTCTCCAT3⬘), which yielded a 360-bp amplified product, served as the internal control. Thirty-five cycles were performed with each primer pair. All primers were synthesized by MWG Biotech (Ebersberg, Germany). The positive control cDNA included samples from human tonsils. For the negative control reaction, the cDNA was replaced with water. Analysis of HBD-3 mRNA levels in cultured chondrocytes by real-time RT-PCR. For real-time RT-PCR, RNA was isolated from the cultured human chondrocytes with the RNeasy-Total RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Real-time RT-PCR was carried out using a one-step system (QuantiTect SYBR Green RT-PCR; Qiagen). For this purpose, 100 ng of total RNA was added. Real-time RT-PCR was used to monitor gene expression using an iCycler (Bio-Rad, Munich, Germany) according to the standard procedure. PCR was performed using Hot Star Taq DNA polymerase, which is activated by an initial heating step while Omniscript reverse transcriptase is deactivated. The temperature profile included an initial denaturation at 95°C for 15 minutes, followed by 37 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, elongation at 72°C (the elongation time depended on the size of the fragment; the number of basepairs divided by 25 yielded the time in seconds), and fluorescence monitoring at 72°C. Bio-Rad iCycler Data Analysis software was used for PCR data analysis. The specificity of the amplification reaction was determined by performing a melting curve analysis. Relative quantification was performed by normalizing the signals of the different genes against those of ␤-actin (forward primer 5⬘-CTCCTTAATGTCACGCAGGATTTC-3⬘, reverse primer 5⬘-GTGGGGCGCCCCAGGCACCA-3⬘). The data were assessed from 3 independent experiments performed in triplicate. Immunohistochemistry. After fixation of the human cartilage in 4% paraformaldehyde, the tissue was embedded in paraffin, sectioned, and dewaxed. Endogenous peroxidases in tissue sections were blocked with 3% H2O2, and tissue sections were subsequently incubated with normal serum (1:5 in Tris buffered saline) from the species in which the primary antibody was raised. Immunohistochemical staining was performed on 6-m paraffin sections, using polyclonal primary VAROGA ET AL antibody against HBD-3 (diluted 1:50; Santa Cruz Biotechnology, Santa Cruz, CA). Prior to the incubation, the slides were pretreated either by microwave heating or by trypsinization. This was followed by incubation with the biotinylated secondary antibody and by staining with a technique using peroxidaselabeled streptavidin–biotin (Dako, Glostrup, Denmark). After counterstaining with hemalum, the sections were finally mounted with Aquatex (Boehringer, Mannheim, Germany). Negative control studies were carried out by absorption of the primary antibody by recombinant protein (1:500 dilution). HBD-3 immunodot blot. An immunodot blot assay was performed for HBD-3 protein detection. Standard curves were generated with a recombinant HBD-3 peptide (4). The peptide was diluted serially from 100 ng/ml to 10 g/ml. After 24-hour stimulation of 1 ⫻ 106 C28/I2 chondrocytes with IL-1 (10 ng/ml) or TNF␣ (10 ng/ml), conditioned medium was withdrawn, and 3 l of each sample was spotted in triplicate on nitrocellulose membrane. The membrane was blocked with 10% nonfat dry milk and Tris buffered saline–0.1% Tween 20 (TBST) at room temperature for 1 hour, then incubated at 4°C overnight with anti–HBD-3 antibody (diluted 1:50). The membranes were then incubated at room temperature for 1 hour with rabbit anti-goat antibody (diluted 1:100,000 in blocking solution). After washing in TBST, the membranes were developed and visualized using the ECL chemiluminescence system (Amersham Biosciences, Freiburg, Germany) followed by apposition with autoradiographic X-Omat AR film (Eastman Kodak, Rochester, NY). For quantification of HBD-3, the signal intensities of aliquots were compared directly with those of simultaneously prepared standards consisting of known amounts of HBD-3 peptide, using PC BAS (LAS-1000; Fujifilm Medical Systems, Stamford, CT). ELISAs. For ELISAs, aliquots of supernatant of cartilage discs and cultured chondrocytes were analyzed. C28/I2 cells (1 ⫻ 106) were seeded into fresh dishes and cultivated for 72 hours in DMEM/Ham’s F-12 medium (1/1 [volume/ volume]) plus 10% FCS. The cultures were then changed to serum-free medium, and after 3 hours of equilibration, the cells were treated for 24 hours with recombinant HBD-3 (4) at 0.5, 1, 5, and 10 g/ml. For ELISAs, conditioned medium was withdrawn and aliquots were assayed using the following commercial kits from Amersham Biosciences: RPN 2610 (for activated MMP-1), RPN 2613 (for MMP-3), RPN 2614 (for MMP-9), RPN 2621 (for MMP-13), RPN 2611 (for TIMP-1), and RPN 2618 (for TIMP-2). Signals were detected by chemiluminescence reaction (ECL-Plus; Amersham Biosciences). The data were assessed from 3 independent experiments performed in triplicate. For ELISA experiments, 3 wells were analyzed for each sample. Animals. STR/Ort mice are genetically predisposed to develop an OA-like lesion of the medial tibial cartilage. More than 85% of male STR/Ort mice show signs of degenerative joint disease in the tibia by age 6 months (20,21). After the mice (ages 22–45 weeks) were killed, their knee joints were removed and demineralized by a procedure explained in detail in an earlier report (33). To study the in vivo expression of MBDs in OA cartilage, we immunostained 8-m serial histologic sections of joints with different stages of OA (grades 0–IV) with anti–MBD-3 and anti–MBD-4 antibodies (diluted 1:100; Santa Cruz Biotechnology). The OA-like lesions were classified in accordance with a recent report (34). Sections of HBD-3 IN OSTEOARTHRITIC CARTILAGE 1739 Figure 1. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis and immunohistochemistry showing human ␤-defensin 3 (HBD-3) in osteoarthritic (OA) cartilage. A, HBD-3 mRNA was detected by RT-PCR in 2 different samples of OA cartilage (lanes 1 and 2). In contrast, healthy articular cartilage did not express HBD-3 transcripts (lanes 3 and 4). GAPDH mRNA was assayed as the internal control for equal amounts of cDNA. B, By immunohistochemistry, there was no staining for HBD-3 protein in samples of healthy articular cartilage. C, Expression of HBD-3 was confirmed in OA cartilage. Immunoreactivity (arrows) was found in the cytoplasm and pericellular matrix of clustered chondrocytes in all layers of articular cartilage. The polyclonal antibody against HBD-3 was diluted 1:50. Negative control studies were carried out by absorption of the primary antibody by recombinant protein (1:500 dilution). Bars ⫽ 10 m. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org. mouse skin were used as positive controls. Age- and sexmatched BALB/c mice, which do not develop OA, were used as control animals. The animal study was approved by the Institutional Review Board. Statistical analysis. Data are expressed as the mean ⫾ SD of tested samples. Statistical significance was evaluated by t-test. RESULTS Induction of HBD-3 in OA cartilage. To investigate the expression pattern of HBD-3 in human OA cartilage, tissue samples were analyzed by RT-PCR and immunohistochemistry. Examination of OA cartilage showed an induction of HBD-3 mRNA and protein by RT-PCR (lanes 1 and 2 in Figure 1A) and immunohistochemistry (Figure 1C), respectively, in the majority (8 of 10) of the tissue samples. HBD-3 was absent in healthy articular cartilage by RT-PCR (lanes 3 and 4 in Figure 1A) and immunohistochemistry (Figure 1B). Immunohistochemical labeling was found in the cytoplasm of clustered chondrocytes and in the pericellular matrix of all layers of articular cartilage. Induction of HBD-3 expression in cultured articular chondrocytes by IL-1 and TNF␣. We next investigated whether IL-1 and TNF␣, cytokines involved in the pathogenesis of OA, influence HBD-3 gene expression in the C28/I2 chondrocyte culture model. Real-time RT-PCR revealed induction of HBD-3 mRNA after stimulation with 10 ng/ml of IL-1 or TNF␣ at physiologically relevant concentrations. Six hours after exposure to TNF␣, basal HBD-3 mRNA levels increased more than 4-fold compared with the unstimulated samples, and addition of IL-1 nearly doubled the amount of 1740 VAROGA ET AL ELISAs. Exposure to different amounts (0.5, 1, 5, and 10 g/ml) of HBD-3 protein resulted in a clear upregulation of MMPs 1 and 13. Compared with basal expression levels, treatment with HBD-3 for 24 hours Figure 2. Induction of human ␤-defensin 3 (HBD-3) in C28/I2 chondrocytes by proinflammatory cytokines. A, Interleukin-1 (IL-1; 10 ng/ml) increased HBD-3 transcription levels nearly 2-fold compared with those in untreated samples after 6 hours of stimulation. Tumor necrosis factor ␣ (TNF␣; 10 ng/ml) increased HBD-3 mRNA levels more than 4-fold. B, For immunodot blot, 1 ⫻ 106 cells were incubated with TNF␣ or IL-1 for 24 hours. Immunodot blot revealed upregulation of HBD-3 protein to 3,500 ng/ml supernatant in response to stimulation. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls. HBD-3 transcripts (Figure 2A). Immunodot blot assaying was done to estimate the quantities of HBD-3 protein in chondrocytes after challenge with IL-1 and TNF␣. Stimulation of cultured chondrocytes for 24 hours revealed an increase in HBD-3 production (to 3.5 g/ml) upon treatment with TNF␣. IL-1 increased the amount of chondrocyte-derived HBD-3 protein to 3 g/ml (Figure 2B). HBD-3 stimulates the expression of MMPs in cultured chondrocytes and cartilage discs and reduces the production of their endogenous regulators, TIMPs 1 and 2. To understand why HBD-3 is up-regulated in OA cartilage without bacterial challenge, we investigated the influence of recombinant human HBD-3 on metalloproteinases that are involved in remodeling the cartilage ECM, as well as on the major endogenous regulators of their activity, TIMPs 1 and 2. MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2 protein could all be detected in C28/I2 chondrocyte supernatants by specific Figure 3. Human ␤-defensin 3 (HBD-3) stimulates production of matrix metalloproteinases (MMPs) and reduces levels of their endogenous inhibitors, tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in cultured chondrocytes. C28/I2 cells were exposed to recombinant HBD-3 (at 0.5, 1, 5, and 10 g/ml) for 24 hours. A, Clear up-regulation of MMP-1, up to 8-fold compared with untreated control (c), was revealed by specific enzyme-linked immunosorbent assay (ELISA). MMP-3 levels were much lower under standard conditions (2.1 ng/ml), but increased to a maximum level of 2.9 ng/ml upon addition of HBD-3 protein. B, Exposure to 1 g/ml HBD-3 resulted in a 30% down-regulation of the gelatinase MMP-9, whereas higher concentrations had no additional effect. Expression of the collagenase MMP-13 increased nearly 3-fold after 24 hours of incubation with HBD-3. C, To examine the effects of HBD-3 on the endogenous regulators of MMP activity, TIMPs 1 and 2, C28/I2 chondrocytes were treated with HBD-3 for 24 hours. ELISA revealed down-regulation of both proteins, with maximum down-regulation at 5 g/ml HBD-3, which reduced the level of TIMP-2 to one-sixth of that at baseline. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls. HBD-3 IN OSTEOARTHRITIC CARTILAGE 1741 Figure 4. HBD-3 induces expression of MMPs in cartilage explants and reduces production of their regulators, TIMPs 1 and 2. To evaluate the effects of recombinant HBD-3 on cartilage explants with an extracellular matrix structure, different amounts of this protein (0.5, 1, and 5 g/ml) were incubated for 24 hours. Shown are expression of MMP-1 (A), MMP-3 (B), MMP-9 (C), MMP-13 (D), TIMP-1 (E), and TIMP-2 (F). Similar to the results in chondrocyte monolayer culture, HBD-3 stimulated the expression of MMPs 1 and 13 while reducing that of TIMPs 1 and 2. Interestingly, healthy cartilage explants showed higher levels of MMP-1 and MMP-13 expression than did osteoarthritic (OA) explants following treatment with HBD-3. Levels of MMPs 3 and 9 were nearly unchanged following treatment with HBD-3. Down-regulation of TIMPs following treatment with HBD-3 was much more pronounced in OA explants. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls. See Figure 3 for other definitions. resulted in up to an 8-fold increase of MMP-1 (Figure 3A) and in a nearly 3-fold induction of MMP-13 (Figure 3B). The stromelysin MMP-3 was expressed at much lower levels (2.1 ng/ml). After addition of 5 g HBD-3 into the culture medium, specific ELISA revealed a maximum peak of 2.9 ng/ml (Figure 3A). In contrast, MMP-9 levels in the culture medium were unchanged after exposure to 5 and 10 g/ml HBD-3, but a 30% down-regulation was observed after treatment with 1 g/ml HBD-3 (Figure 3B). Specific ELISAs for TIMPs 1 and 2 revealed down-regulation following treatment with HBD-3 (0.5, 1, and 5 g/ml) (Figure 3C). In general, TIMP-2 was suppressed to a greater degree than was TIMP-1. To confirm the results obtained in the C28/I2 monolayer culture using a cartilage organ culture model with an ECM structure, discs of healthy and OA cartilage were incubated with 0.5, 1, and 5 g/ml HBD-3 protein for 24 hours. Similar to the results in the cell culture, MMPs 1 and 13 were strongly induced in healthy articular cartilage discs with a normal matrix composition (Figures 4A and D). In contrast, incubated OA cartilage discs were much more resistant to HBD-3 protein. Compared with a 300% increase in healthy 1742 VAROGA ET AL Figure 5. Induction of mouse ␤-defensins (MBDs) 3 and 4 in osteoarthritic (OA) cartilage of STR/Ort mice. To investigate the in vivo expression pattern of MBDs in cartilage, knee joints of BALB/c and STR/Ort mice were examined by immunohistochemistry. In contrast to BALB/c mice (A), STR/Ort mice with low-grade OA (grades I and II) demonstrated induction of MBD-3 (arrows in B) and MBD-4 (arrows in C) in articular cartilage. Surprisingly, mice with late-stage OA (grade IV) revealed no MBD expression in articular cartilage (D). Negative control studies were carried out by absorption of the primary antibody by recombinant protein (1:500 dilution). fc ⫽ femoral cartilage; m ⫽ meniscus; tc ⫽ tibial cartilage; sb ⫽ subchondral bone. Bars ⫽ 10 m. tissue samples, levels of MMP-1 increased only 50% after 24 hours of stimulation with different amounts of HBD-3 in OA tissue samples (Figure 4A). MMP-13 was not inducible at all in the OA cartilage discs (Figure 4D). Further, levels of MMP-3 (Figure 4B) and MMP-9 (Figure 4C) were nearly unchanged in the collected supernatants of HBD-3–incubated cartilage discs. Moreover, ELISAs revealed a decrease in levels HBD-3 IN OSTEOARTHRITIC CARTILAGE of TIMP-1 (Figure 4E) and TIMP-2 (Figure 4F) following treatment with HBD-3 in the cartilage culture. Similar to the results in the monolayer culture, TIMP-2 was suppressed more strongly than TIMP-1. The observed effects of HBD-3 were much more intense in OA cartilage explants, suggesting an influence of the ECM composition on the induction process. Taken together, these results suggest that HBD-3 causes a disruption of the balance between destructive enzymes and their inhibitors. Increased expression of MBDs 3 and 4 in cartilage of STR/Ort mice. To investigate the in vivo expression pattern of MBDs in cartilage with OA-like pathology, the knee joints of STR/Ort mice (ages 22–45 weeks) were examined by immunohistochemistry. In contrast to the findings in age- and sex-matched BALB/c animals (Figure 5A), immunohistochemical staining of knee joints of STR/Ort mice revealed increased expression of MBD-3 (Figure 5B) and MBD-4 (Figure 5C) in cartilage with low-grade OA (grades I and II). Immunoreactivity was found in the cytoplasm and pericellular matrix of chondrocytes in all layers of articular cartilage. Moreover, cross-sections of articular cartilage from mice with late-stage OA (grade IV) demonstrated no immunoreactivity for MBDs 3 and 4 (Figure 5D). DISCUSSION OA is characterized by an imbalance between biosynthesis and degradation of matrix components in articular cartilage, ultimately leading to progressive destruction of the tissue. In the present report, we demonstrate the expression of HBD-3 in mesenchymal OA cartilage. Similar to the findings of Paulsen and coworkers (11) in synovial membranes, our results show that HBD-3 is present in OA cartilage in the absence of bacterial challenge. These results suggest another function of these antibacterial molecules, and they encouraged us to examine the role of HBD-3 in OA. First, we tested the capacity of the proinflammatory cytokines IL-1 and TNF␣, which have roles in the initiation and progression of OA (35), to induce HBD-3 in cultured chondrocytes. After exposure to these stimulators, real-time RT-PCR and immunodot blot revealed a clear induction of HBD-3 mRNA and protein in cultured chondrocytes. Our results showing upregulation of HBD-3 expression after stimulation with proinflammatory cytokines are consistent with findings on defensin expression and regulation in major epithelia such as skin (3,36), respiratory tract (37,38), urogenital tract (39), and gut (40); however, all of those investigations focused on the antibacterial properties of de- 1743 fensins. Recent data revealed more than just the catabolic functions of TNF␣ and IL-1. Aside from their destructive abilities, they are able to stimulate growth factors such as bone morphogenetic protein 2 (BMP-2), osteogenic protein 1 (BMP-7), and transforming growth factor ␤, which results in an increased synthesis of aggrecan and type II collagen in articular cartilage (41,42). Regardless of their ultimately deleterious effects on articular cartilage, TNF␣ and IL-1 could initiate the repair response displayed by injured cartilage in early stages of OA through their ability to enhance anabolic pathways in chondrocytes. To elucidate the possible involvement of HBD-3 in cartilage destruction or repair, we assessed the effects of recombinant proteins in vitro. We first analyzed the influence of HBD-3 on the major ECM-degrading metalloproteinases and their endogenous inhibitors (TIMPs). ECM of articular cartilage consists mainly of type II collagen, aggrecan and other proteoglycans, minor collagens (types V, VI, IX, X, and XI), and other noncollagenous matrix proteins. MMP-1 and MMP-13 (collagenases 1 and 3, respectively) are able to cleave the triple-helical domain of collagens, including types II and X collagen (15), and they therefore play a decisive role in cartilage degradation. MMP-3 (stromelysin) cleaves the telopeptide regions or noncollagen domains of types IX and XI collagen. As a consequence, incubation of cartilage explants with MMP-3 results in the breakdown of the collagen network, a very early feature of OA (43). MMPs are known to be induced by IL-1 and TNF␣ in chondrocytes (16) and inactivated by their negative regulators, the TIMPs (15). Up-regulation of the metalloproteinases after HBD-3 stimulation of chondrocytes suggests that ␤-defensins may be involved in catabolic pathways in articular cartilage monolayer culture. To evaluate the effects of cationic HBD-3 in cartilage discs, where chondrocytes were surrounded by negatively charged proteoglycans, organ culture experiments were performed. These experiments demonstrated that the difference in the charges of the ECM and the effector protein HBD-3 does not neutralize the influence of HBD-3 on ECM-degrading pathways in articular cartilage. Interestingly, the observed effects of HBD-3 were much more pronounced in the presence of a normal matrix composition. It is reasonable to propose that the grade of cartilage degradation might influence the susceptibility of articular cartilage to HBD-3. Moreover, TIMPs, the regulators of MMP activity, were clearly down-regulated even in the presence of a normal ECM structure. Taken together, our results indicate that HBD-3 contributes to ECM-degrading processes in ar- 1744 VAROGA ET AL ticular cartilage by activating MMPs 1 and 13 and simultaneously reducing the expression of TIMPs 1 and 2. Recent studies revealed the involvement of the chemokine receptor CCR6 in the immunomodulating processes of HBD-3 (44), but the putative receptor on chondrocytes remains to be determined in future investigations. The in vivo induction of MBDs in the cartilage of STR/Ort mice supports the present in vitro results. Comparable with the situation in humans, the development of OA-like pathology is connected with the upregulation of proinflammatory cytokines such as TNF␣ and IL-1 (45). Recent investigations revealed the induction of MMPs and TIMP-2 in OA cartilage of STR/Ort mice (46). These observations demonstrate the similarities with the induction of OA in humans and confirm the usefulness of this model for studying OA in vivo. To our knowledge, there are no in vivo data concerning the induction of ␤-defensins in sealed-off compartments without contamination due to microbial colonization. The available reports are of investigations that have focused on the expression of APs in epithelial surfaces due to bacterial invasion (25) or in models of wounding with an increased risk of infection (47,48). It is interesting to speculate why ␤-defensins are up-regulated in OA cartilage without microbial threat. Supported by the in vitro results, we hypothesize that ␤-defensins augment catabolic pathways in articular cartilage, ultimately leading to a timely breakdown of the ECM. Until now, the expression of ␤-defensins was connected solely with antibacterial tasks involving direct killing of microorganisms and chemotactic activity. This report shows a novel function of HBD-3. Exposure of articular chondrocytes and cartilage to HBD-3 results in increased synthesis of ECM-degrading metalloproteinases and reductions in TIMPs 1 and 2. Taken together, our findings demonstrate that HBD-3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissue remodeling processes in articular cartilage. The role of HBD-3 in OA requires further elucidation. ACKNOWLEDGMENTS We thank F. Lichte, M. Lorenzen, I. Kronenbitter, R. Worm, and S. Seiter for their excellent technical assistance, and B. Muller-Hilke (Berlin, Germany) for providing the STR/Ort mice. REFERENCES 1. 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