Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

Objective

We reported previously that the orphan nuclear receptor, estrogen receptor–related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA).

Methods

ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short‐term treatment with a variety of OA‐associated factors and signaling pathway agonists and inhibitors.

Results

ERRα expression was lower in OA than in normal articular cartilage. Interleukin‐1β (IL‐1β) markedly up‐regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up‐regulation was dependent on cyclooxygenase 2 (COX‐2; NS398), prostaglandin E₂, cAMP (8‐bromo‐cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up‐regulation of ERRα by IL‐1β, suggesting autoregulation of ERRα in the IL‐1β pathway. Matrix metalloproteinase 13 (MMP‐13) expression was also decreased by treatment with XCT790 plus IL‐1β versus IL‐1β alone, and the down‐regulation of MMP‐13 mRNA and protein observed with XCT790 alone suggests that the up‐regulation of MMP‐13 by IL‐1β is ERRα‐dependent.

Conclusion

We report the first evidence that ERRα expression is regulated by IL‐1β in COX‐2–, cAMP‐, and PKA‐dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP‐13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage.

     

Stress‐induced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models

Objective

The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin‐1β (IL‐1β) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL‐1β is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the “inflammasome,” a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL‐1β and the inflammasome in cartilage breakdown.

Methods

We assessed IL‐1β release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)–, IL‐1α–, and tumor necrosis factor α (TNFα)–induced activity of matrix metalloproteinase 3 (MMP‐3), MMP‐9, and MMP‐13 in NLRP3‐knockout mice and wild‐type mice and the inhibition of caspase 1 with Z‐YVAD‐FMK and the blockade of IL‐1β with IL‐1 receptor antagonist (IL‐1Ra). Cartilage explants from NLRP3‐knockout mice and IL‐1R type I (IL‐1RI)–knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load‐induced glycosaminoglycan (GAG) release and MMP enzymatic activity.

Results

Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL‐1β. LPS, IL‐1α, and TNFα dose‐dependently increased MMP‐3, MMP‐9, and MMP‐13 activity in cultured chondrocytes and in NLRP3^−/− chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL‐1β. The load‐induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL‐1RI in cartilage explants.

Conclusion

OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL‐1β inhibitors observed in previous trials.

     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

Interleukin‐7 stimulates secretion of S100A4 by activating the JAK/STAT signaling pathway in human articular chondrocytes

Objective

S100A4 has been shown to be increased in osteoarthritic (OA) cartilage and to stimulate chondrocytes to produce matrix metalloproteinase 13 (MMP‐13) through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to examine the mechanism of S100A4 secretion by chondrocytes.

Methods

Human articular chondrocytes isolated from ankle cartilage were stimulated with 10 ng/ml of interleukin‐1β (IL‐1β), IL‐6, IL‐7, or IL‐8. Cells were pretreated with either a JAK‐3 inhibitor, brefeldin A, or cycloheximide. Immunoblotting with phospho‐specific antibodies was used to determine the activation of signaling proteins. Secretion of S100A4 was measured in conditioned media by immunoblotting, and MMP‐13 was measured by enzyme‐linked immunosorbent assay.

Results

Chondrocyte secretion of S100A4 was observed after treatment with IL‐6 or IL‐8 but was much greater in cultures treated with equal amounts of IL‐7 and was not observed after treatment with IL‐1β. IL‐7 activated the JAK/STAT pathway, with increased phosphorylation of JAK‐3 and STAT‐3, leading to increased production of S100A4 and MMP‐13. Overexpression of a dominant‐negative RAGE construct inhibited the IL‐7–mediated production of MMP‐13. Pretreatment of chondrocytes with a JAK‐3 inhibitor or with cycloheximide blocked the IL‐7–mediated secretion of S100A4, but pretreatment with brefeldin A did not.

Conclusion

IL‐7 stimulates chondrocyte secretion of S100A4 via activation of JAK/STAT signaling, and then S100A4 acts in an autocrine manner to stimulate MMP‐13 production via RAGE. Since both IL‐7 and S100A4 are up‐regulated in OA cartilage and can stimulate MMP‐13 production by chondrocytes, this signaling pathway could contribute to cartilage destruction during the development of OA.

     

The peroxisome proliferator–activated receptor γ agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: In vivo protective effects mediated through the inhibition of key signaling and catabolic pathways

Objective

Emerging evidence indicates that peroxisome proliferator–activated receptor γ (PPARγ) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARγ agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes.

Methods

OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP‐1), ADAMTS‐5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK‐1/2, p38, JNK, and NF‐κB p65.

Results

Pioglitazone reduced the development of cartilage lesions in a dose‐dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP‐1, ADAMTS‐5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK‐1/2, p38, and NF‐κB.

Conclusion

These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARγ activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.

     

Gene deletion of either interleukin‐1β, interleukin‐1β–converting enzyme,inducible nitric oxide synthase,or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy

Objective

To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin‐1β (IL‐1β), IL‐1β–converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.

Methods

Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild‐type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0–6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti‐VDIPEN, anti‐NITEGE, and Col2‐3/4C_short neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.

Results

All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild‐type mice. ICE‐, iNOS‐, and particularly IL‐1β–knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP‐, aggrecanase‐, and collagenase‐generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.

Conclusion

We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.

     

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n‐3 fatty acids

Objective

To determine if n‐3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n‐6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage.

Methods

The metabolic profile of human OA cartilage was determined at the time of harvest and after 24‐hour exposure to n‐3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin‐1 (IL‐1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]).

Results

Supplementation with n‐3 PUFA (but not other fatty acids) reduced, in a dose‐dependent manner, the endogenous and IL‐1–induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS‐4, MMP‐13, and MMP‐3 (but not TIMP‐1, ‐2, or ‐3) was also specifically abolished with n‐3 PUFA supplementation. In addition, n‐3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5‐lipoxygenase, 5‐lipoxygenase–activating protein, tumor necrosis factor α, IL‐1α, and IL‐1β) without affecting the expression of message for several other proteins involved in normal tissue homeostasis.

Conclusion

These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n‐3 PUFA, but not to other classes of fatty acids.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin‐1β and with chondrocyte resistance to insulin‐like growth factor 1

Objective

To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).

Methods

The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.

Results

The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.

Conclusion

Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.

     

Cyclooxygenase 2‐dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants

Objective

To examine cyclooxygenase‐2 (COX‐2) enzyme expression, its regulation by interleukin‐1β (IL‐1β), and the role of prostaglandin E₂ (PGE₂) in proteoglycan degradation in human osteoarthritic (OA) cartilage.

Methods

Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL‐1β and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE₂. Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium.

Results

IL‐1β induced robust expression of COX‐2 and PGE₂ in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL‐1β also induced cartilage proteoglycan degradation in OA synovial membrane‐cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX‐2 protein expression in, and PGE₂ production from, the synovial membrane. Dexamethasone, neutralizing IL‐1β antibody, or the selective COX‐2 inhibitor, SC‐236, attenuated both the IL‐1β‐induced PGE₂ production and cartilage proteoglycan degradation in these cocultures. The addition of PGE₂ reversed the inhibition of proteoglycan degradation caused by SC‐236.

Conclusion

IL‐1β‐induced production of COX‐2 protein and PGE₂ was low in OA articular cartilage compared with that in the other OA tissues examined. IL‐1β‐mediated degradation of cartilage proteoglycans in OA synovial membrane‐cartilage cocultures was blocked by the selective COX‐2 inhibitor, SC‐236, and the effect of SC‐236 was reversed by the addition of exogenous PGE₂. Our data suggest that induction of synovial COX‐2‐produced PGE₂ is one mechanism by which IL‐1β modulates cartilage proteoglycan degradation in OA.

     

Freshly isolated osteoarthritic chondrocytes are catabolically more active than normal chondrocytes,but less responsive to catabolic stimulation with interleukin‐1β

Objective

Interleukin‐1β (IL‐1β) is one potentially important cytokine during cartilage destruction. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL‐1β on the expression level of anabolic genes (type II collagen, aggrecan), catabolic genes (matrix metalloproteinase 1 [MMP‐1], MMP‐2, MMP‐3, MMP‐13, and ADAMTS‐4), and cytokines (IL‐1β, IL‐6, and leukemia inhibitory factor [LIF]) by articular chondrocytes (normal and osteoarthritic). Determination of whether there was a difference in reactivity between normal and osteoarthritic chondrocytes was also a goal of this study.

Methods

Gene expression levels were detected by real‐time polymerase chain reaction from isolated (nonpassaged) chondrocytes (normal [n = 6]; osteoarthritic [n = 7]) after stimulation with 0.01 ng, 0.1 ng, 1 ng, and 10 ng/ml IL‐1β.

Results

In normal adult articular chondrocytes the expression of both aggrecan and type II collagen genes was significantly down‐regulated, whereas matrix‐degrading proteases (except MMP‐2), as well as the investigated cytokines, were induced by IL‐1β in a dose‐dependent manner. The strongest regulation was found for IL‐6 and LIF. Osteoarthritic chondrocytes showed strongly increased levels of catabolic enzymes and mediators, but were less responsive to further stimulation with IL‐1β.

Conclusion

Our study confirms that IL‐1β activity is critically dependent on both the applied concentration and the reactivity of the cells stimulated. The responsiveness appears to be significantly reduced in late‐stage osteoarthritic chondrocytes. However, these cells show high basic expression levels of catabolic enzymes and mediators. Thus, it remains open whether our data indicate that osteoarthritic chondrocytes are per se not responsive to IL‐1β or are already so strongly stimulated (e.g., by IL‐1) during the disease process that they are refractory to further stimulation.

     

Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3

Objective

To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP‐3) in the pathology of early and late OA.

Methods

The role of synovial macrophages in MMP‐mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase‐induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP‐3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results

On day 14 following induction of OA, MMP‐mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean ± SD positively stained surface area 20 ± 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP‐induced neoepitopes was largely prevented (mean ± SD positively stained surface area 5 ± 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP‐mediated cartilage damage. We observed a strong decrease in MMP‐3 and MMP‐9 expression in synovial but not cartilage tissue in macrophage‐depleted joints. Among 2‐year‐old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP‐3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP‐3–knockout mice. In addition, MMP‐mediated VDIPEN expression was significantly decreased, indicating reduced MMP‐mediated cartilage breakdown.

Conclusion

The results of this study prove that MMP‐3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.