Proteomic analysis by two‐dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin‐1β

Objective

To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) and to ascertain differences in the protein expression patterns of these 2 cytokines.

Methods

Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL‐1β (5 ng/ml) or with TNFα (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2‐dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix‐assisted laser desorption ionization−time‐of‐flight/time‐of‐flight technology.

Results

We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL‐1β–stimulated chondrocytes, IL‐1β was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up‐regulated. Proteins involved in cellular metabolism and energy (23%) that were up‐regulated or induced by IL‐1β included nicotinamide phosphoribosyltransferase, long‐chain fatty acid–coenzyme A ligase 4, δ‐aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde‐3‐phosphate dehydrogenase. In TNFα‐stimulated chondrocytes, TNFα was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3–like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up‐regulated. Eighteen proteins were differently modulated by IL‐1β and TNFα. Of these, 45% were related to metabolism.

Conclusion

IL‐1β and TNFα induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

     

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.

     

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.

     

The impact of anterior cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor necrosis factor α on chondroprotection in an animal model

Objective

To examine the effects of anterior cruciate ligament transection (ACLT) in a rat model on lubricin metabolism and its relationship to markers of inflammation and cartilage damage, and to determine whether blocking the metabolic effects of tumor necrosis factor α (TNFα) by etanercept increases the chondroprotection provided by lubricin.

Methods

Unilateral ACLT was performed in Lewis rats. Levels of lubricin, TNFα, interleukin‐1β (IL‐1β), and sulfated glycosaminoglycans (sGAG) in synovial fluid (SF) lavage specimens and synovial tissue lubricin gene expression were evaluated at 1 week and 4 weeks following ACLT. Histologic evaluation of articular cartilage included staining with lubricin‐specific monoclonal antibody 9G3 and Safranin O. The percentage of lubricin staining on the surface of articular cartilage in weight‐bearing areas was estimated by digital imaging. Blocking of TNFα was performed using etanercept, which was administered subcutaneously at a dose of 0.5 mg/kg around the ACL‐transected joints, using different dosing strategies. The ACL‐transected and contralateral joints of these rats were harvested 4 weeks following surgery.

Results

Four weeks following ACLT, SF lubricin concentrations and the percentage of cartilage surface lubricin staining were significantly lower in the injured joints compared with the contralateral joints. A significant decrease in synovial tissue lubricin gene expression was associated with elevated TNFα and IL‐1β concentrations in SF lavage samples. With all of the etanercept treatment strategies, blocking of TNFα significantly increased the amount of lubricin bound to cartilage, coupled with a significant decrease in sGAG release. However, changes in the concentrations of lubricin in SF were variable.

Conclusion

Blocking TNFα resulted in a chondroprotective effect, exemplified by increased lubricin deposition on articular cartilage and a decrease in sGAG release from articular cartilage in an animal model of posttraumatic arthritis.

     

A sodium dodecyl sulfate–polyacrylamide gel electrophoresis–liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor α and interleukin‐1β

Objective

To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα), by characterizing proteins lost to the medium from cartilage explant culture.

Methods

Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL‐1β or 100 ng/ml of TNFα or were subjected to uniaxial, radially‐unconfined injurious compression (50% strain; 100%/second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel‐based separation (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis.

Results

More than 250 proteins were detected, including extracellular matrix (ECM) structural proteins, pericellular matrix proteins important in cell–cell interactions, and novel cartilage proteins CD109, platelet‐derived growth factor receptor–like, angiopoietin‐like 7, and adipocyte enhancer binding protein 1. IL‐1β and TNFα caused increased release of chitinase 3–like protein 1 (CHI3L1), CHI3L2, complement factor B, matrix metalloproteinase 3, ECM‐1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins, including Grp58, Grp78, α4‐actinin, pyruvate kinase, and vimentin. Injurious compression also caused increased release and evidence of proteolysis of type VI collagen subunits, cartilage oligomeric matrix protein, and fibronectin.

Conclusion

Overload compression injury caused a loss of cartilage integrity, including matrix damage and cell membrane disruption, which likely occurred through strain‐induced mechanical disruption of cells and matrix. IL‐1β and TNFα caused the release of proteins associated with an innate immune and stress response by the chondrocytes, which may play a role in host defense against pathogens or may protect cells against stress‐induced damage.

     

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.

     

ADAMTS‐9 is synergistically induced by interleukin‐1β and tumor necrosis factor α in OUMS‐27 chondrosarcoma cells and in human chondrocytes

Objective

To compare induction of the aggrecanases (ADAMTS‐1, ADAMTS‐4, ADAMTS‐5, ADAMTS‐8, ADAMTS‐9, and ADAMTS‐15) by interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) in chondrocyte‐like OUMS‐27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.

Methods

OUMS‐27 cells were stimulated for different periods of time and with various concentrations of IL‐1β and/or TNFα. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL‐1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real‐time polymerase chain reaction and Northern blotting. ADAMTS‐9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL‐1β–stimulated OUMS‐27 cells was investigated.

Results

IL‐1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL‐1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL‐1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS‐9 protein was also induced in IL‐1β–stimulated OUMS‐27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up‐regulation in OUMS‐27 cells.

Conclusion

ADAMTS9 is an IL‐1β– and TNFα‐inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS‐9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS‐9 may have a pathologic role in arthritis.

     

Tumor necrosis factor α and interleukin‐1β modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes

Objective

Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β) modulate the osteocyte response to mechanical loading.

Methods

MLO‐Y4 osteocytes were incubated with TNFα (0.5–30 ng/ml) or IL‐1β (0.1–10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean ± amplitude 0.7 ± 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca²⁺]_i) using Fluo‐4/AM. Focal adhesions and filamentous actin (F‐actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell‐generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry.

Results

Pulsatile fluid flow increased [Ca²⁺]_i within seconds (in 13% of cells) and NO production within 5 minutes (4.7‐fold). TNFα and IL‐1β inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow–induced NO production. TNFα and IL‐1β affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFα and IL‐1β decreased the amount of F‐actin. Incubation with IL‐1β for 24 hours stimulated osteocyte apoptosis.

Conclusion

Our results suggest that TNFα and IL‐1β inhibit mechanical loading–induced NO production by osteocytes via abrogation of pulsatile fluid flow–stimulated [Ca²⁺]_i, and that IL‐1β stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

     

Tumor necrosis factor α activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro‐resorptive factors

Objective

Tumor necrosis factor α (TNFα) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNFα have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNFα on chondrocytes.

Methods

We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNFα or cartilage destruction, in murine articular chondrocytes isolated from wild‐type mice and TNFα receptor–null (p55/p75^−/−) mice.

Results

TNFα induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosis‐induced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2‐fold to >10‐fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony‐stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNFα was confirmed by comparison with the effects of TNFα on chondrocytes isolated from receptor‐null mice.

Conclusion

These findings demonstrate that TNFα alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNFα in human cartilage cells.

     

Interleukin‐1α induction of tensile weakening associated with collagen degradation in bovine articular cartilage

Objective

To determine whether interleukin‐1α (IL‐1α) induces tensile weakening of articular cartilage that is concomitant with the loss of glycosaminoglycans (GAGs) or the subsequent degradation of the collagen network.

Methods

Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured with or without IL‐1α for 1 week or 3 weeks. Then, portions of the explants were analyzed for their tensile properties (ramp modulus, strength, and failure strain); other portions of explants and spent culture medium were analyzed for the amount of GAG and the amount of cleaved, denatured, and total collagen.

Results

The effect of IL‐1α treatment on cartilage tensile properties and content was dependent on the duration of culture and the depth of the explant from the articular surface. The tensile strength and failure strain of IL‐1α–treated samples from the superficial and middle layers were lower after 3 weeks of culture, but not after 1 week of culture. However, by 1 week of culture, IL‐1α had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3 weeks of culture, IL‐1α induced significant collagen degradation, as indicated by the amount of total, cleaved, or denatured collagen in the medium or in explants from the superficial and middle layers.

Conclusion

IL‐1α–induced degradation of cartilage results in tensile weakening that occurs subsequent to the depletion of GAG and concomitant with the degradation of the collagen network.

     

Oncostatin M in combination with tumor necrosis factor α induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

Objective

To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.

Methods

The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.

Results

The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.

Conclusion

This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

     

Interleukin‐6 receptor shedding is enhanced by interleukin‐1β and tumor necrosis factor α and is partially mediated by tumor necrosis factor α–converting enzyme in osteoblast‐like cells

Objective

Interleukin‐6 (IL‐6) and soluble IL‐6 receptor (sIL‐6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL‐6R levels in response to the inflammatory cytokines IL‐1β and tumor necrosis factor α (TNFα) and to determine the role of TNFα‐converting enzyme (TACE) in this process.

Methods

Levels of sIL‐6R in the culture media of MG63 and SAOS‐2 osteoblast‐like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL‐6R and TACE to determine the potential involvement of TACE in IL‐6R shedding.

Results

IL‐1β and TNFα increased the levels of sIL‐6R in the culture media of MG63 osteoblast‐like cells. This effect was not influenced by cycloheximide or 5,6‐dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL‐1β and TNFα had no influence on the alternatively spliced form of IL‐6R RNA. Levels of sIL‐6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS‐2 cells with expression plasmids for IL‐6R and TACE produced a dose‐dependent increase in sIL‐6R levels.

Conclusion

IL‐1β‐ and TNFα‐mediated induction of IL‐6R shedding in osteoblast‐like cells is at least partly dependent on TACE activation.

     

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.