Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype

Objective. To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) after 1 day and 3 days in culture. Methods. OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring ³⁵S-sulfate incorporation during explant culture over 1–3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1β and TNFα after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1–3 days in culture were examined by agarose–polyacrylamide gel electrophoresis. Results. Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNFα. These differences were no longer detected after 48–72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. Conclusion. Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNFα was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.     

Bone morphogenetic protein 2 stimulates articular cartilage proteoglycan synthesis in vivo but does not counteract interleukin-1α effects on proteoglycan synthesis and content

Objective. To study the effect of bone morphogenetic protein 2 (BMP-2) on articular cartilage proteoglycan (PG) synthesis in vivo and to investigate whether BMP-2 is able to counteract the effects of interleukin-1 (IL-1) on articular cartilage PG synthesis and content. Methods. BMP-2 alone or in combination with IL-1α was injected into murine knee joints. PG synthesis was measured by ³⁵S-sulfate incorporation using an ex vivo method or autoradiography. Cartilage PG content was analyzed by measuring Safranin O staining intensity on histologic sections. Results. BMP-2 appeared to be a potent stimulator of articular cartilage PG synthesis in vivo. However, BMP-2 was not able to counteract the deleterious effects of IL-1α on articular cartilage PG synthesis and content. In addition, intraarticular injections of BMP-2 induced chondrophytes. Conclusion. Although BMP-2 is a very potent stimulator of cartilage PG synthesis in vivo, the therapeutic applications of BMP-2 are limited due to the inability of BMP-2 to counteract the effects of IL-1 and the induction of chondrophytes.     

Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-10 and interleukin-4

Objective. Inflammation-induced articular cartilage degradation is a major problem in rheumatoid arthritis (RA). Type 1 T cell activity (characterized by interferon-γ/interleukin-2 [IL-2] production), and consequently, the production of the proinflammatory cytokines IL-1 and tumor necrosis factor α (TNFα), have been reported to play a major role in cartilage damage. IL-10 and IL-4, both produced by type 2 T cells, are cytokines with the capacity to down-regulate proinflammatory responses. The present study was undertaken to investigate the way in which these cytokines affect activated mononuclear cells (MNC) of RA patients in relation to human articular cartilage degradation in vitro. Methods. MNC from synovial fluid and peripheral blood of RA patients were stimulated with bacterial antigen and treated with IL-10 and/or IL-4. Bacterial antigen is known to activate type 1 T cells and to induce proinflammatory IL-1/TNFα–dependent cartilage damage. Cytokine production and effects of conditioned media, as well as effects of IL-10 and IL-4 on proteoglycan (PG) turnover (as a measure for cartilage damage), were determined. Results. IL-10 and IL-4 inhibited proinflammatory cytokine production of stimulated RA MNC and completely reversed inhibition of cartilage PG synthesis induced by these stimulated RA MNC. IL-10 was more potent than IL-4 in this respect, and the combination of IL-10 and IL-4 had an additive effect. In addition, IL-10 directly stimulated cartilage PG synthesis. Conclusion. IL-10 reverses the cartilage degradation induced by antigen-stimulated MNC, and IL-4 has an additive effect on this process. Furthermore, IL-10 has a direct stimulatory effect on PG synthesis, and IL-4, as a growth factor for type 2 T cells, can reduce the ratio of type 1 to type 2 T cell activity. These results provide evidence in favor of the use of a combination of the two cytokines in the treatment of RA.     

Loss of transforming growth factor counteraction on interleukin 1 mediated effects in cartilage of old mice

OBJECTIVE: To investigate if a difference exists between young and old mice in the response of articular cartilage to interleukin 1 (IL1) and transforming growth factor beta (TGFbeta) alone or in combination. METHODS: The interaction of IL1 and TGFbeta was studied in cartilage of young (three months) and old mice (18 months) both in vivo and in vitro. Therefore, IL1, TGFbeta, or IL1 together with TGFbeta was injected into the knee joints of mice on days 1, 3, and 5 before harvest of the patellae on day 6. Alternatively, isolated patellae were stimulated with IL1, TGFbeta, or IL1 together with TGFbeta in culture for 48 hours. Proteoglycan (PG) synthesis and nitric oxide (NO) production were measured. RESULTS: IL1 inhibited PG synthesis and increased NO production in cartilage of both young and old mice. On the other hand, TGFbeta stimulated PG synthesis and reduced NO production in both age groups. Importantly, TGFbeta was able to counteract IL1 mediated effects on PG synthesis and NO production in young but not in old mice. CONCLUSIONS: Contrary to the findings in young mice, the cartilage of old animals does not antagonise IL1 effects via TGFbeta. This loss of responsiveness to the pivotal cytokine TGFbeta on effects of IL1 can be important in the initiation and progression of osteoarthritis (OA).     

Interleukin‐6 plays an essential role in hypoxia‐inducible factor 2α–induced experimental osteoarthritic cartilage destruction in mice

Objective

Hypoxia‐inducible factor 2α (HIF‐2α) (encoded by Epas1) causes osteoarthritic (OA) cartilage destruction by regulating the expression of catabolic factor genes. We undertook this study to explore the role of interleukin‐6 (IL‐6) in HIF‐2α–mediated OA cartilage destruction in mice.

Methods

The expression of HIF‐2α, IL‐6, and catabolic factors was determined at the messenger RNA and protein levels in primary culture mouse chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild‐type, HIF‐2α–knockdown (Epas1^+/−), and Il6^–/– mice was caused by intraarticular injection of Epas1 adenovirus or destabilization of the medial meniscus. The role of IL‐6 was determined by treating with recombinant IL‐6 protein or by injecting HIF‐2α adenovirus (AdEpas1) intraarticularly in mice with or without IL‐6–neutralizing antibody.

Results

We found that Il6 is a direct target gene of HIF‐2α in articular chondrocytes. Both Epas1 and Il6 were up‐regulated in human and mouse OA cartilage, whereas HIF‐2α knockdown in mice led to inhibition of both Il6 expression and cartilage destruction. Treatment with IL‐6 enhanced Mmp3 and Mmp13 expression; conversely, Il6 knockdown inhibited HIF‐2α–induced up‐regulation of Mmp3 and Mmp13. Injection of IL‐6 protein into mouse knee joints triggered OA cartilage destruction, whereas IL‐6 neutralization led to blocking of HIF‐2α–induced cartilage destruction with concomitant modulation of Mmp3 and Mmp13 expression. Moreover, Il6 knockout resulted in inhibition of AdEpas1‐induced and destabilization of the medial meniscus–induced cartilage destruction as well as inhibition of Mmp3 and Mmp13 expression.

Conclusion

Our findings indicate that IL‐6 acts as a crucial mediator of HIF‐2α–induced experimental OA cartilage destruction in mice via regulation of Mmp3 and Mmp13 levels.

     

Role of interleukin-1, tumor necrosis factor α, and interleukin-6 in cartilage proteoglycan metabolism and destruction effect of in situ blocking in murine antigen- and zymosan-induced arthritis

Objective. To determine the involvement of interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 in the cartilage pathology of murine antigen-induced arthritis (AIA) and zymosan-induced arthritis (ZIA). Methods. Arthritis was induced by intraarticular injection of zymosan in naive mice or by subcutaneous injection of methylated bovine serum albumin in sensitized animals. Mini-osmotic pumps releasing human recombinant IL-1 receptor antagonist (IL-1ra) protein were implanted intraperitoneally 2 days before arthritis induction, and neutralizing antibodies directed against murine IL-1α, IL-1β, TNFα, or IL-6 were administered 1 day before. Proteoglycan (PG) synthesis and degradation were assessed in patellar cartilage. Results. Murine IL-1α and IL-1β injected intraarticularly at doses of 0.1–100 ng suppressed chondrocyte PG synthesis. The highest dose of TNF tested (100 ng) decreased PG synthesis marginally. In contrast, the maximum dose of IL-6 (1 μg) stimulated PG synthesis 2 days after injection. Treatment of AIA with neutralizing monoclonal antibodies against either TNFα or IL-6 did not reduce either the PG degradation or the suppression of its synthesis. However, treatment with anti-IL-1 (α + β) polyclonal antibodies totally prevented PG suppression, although the initial breakdown of PG was unaffected. This effect was confirmed when IL-1ra was administered in high doses. Moreover, treatment of ZIA with anti-IL-1 (α + β), but not with anti-TNF, resulted in normal PG synthesis, confirming the key role played by IL-1 in the inhibition of PG synthesis. Treatment of AIA with anti-IL-1 did not affect inflammation during the acute phase, but a significant reduction of ongoing inflammation was noted at day 7, and there was a marked reduction in the loss of cartilage PG. Conclusion. The suppression of PG synthesis in both ZIA and AIA in mice is due to the combined local action of IL-1 (α + β), and neither IL-6 nor TNF is involved. Moreover, the normalization of PG synthesis brought about by blocking of IL-1 ameliorates the cartilage damage associated with AIA.     

ROLE OF TNF{alpha}, IN RELATION TO IL-1 AND IL-6 IN THE PROTEOGLYCAN TURNOVER OF HUMAN ARTICULAR CARTILAGE

In both young and old human articular cartilage explants, TNFinduced a concentration-dependent, reversible suppression ofthe proteoglycan (PG) synthesis. Young cartilage was more sensitiveto TNF than old cartilage: 50% suppression of PG synthesis wasreached at a TNF concentration of 5 U/ml for young and 30 U/mlfor old cartilage, whereas at 10³ U/ml the PG synthesis of youngcartilage was blocked and that of old cartilage suppressed by80%. These inhibition levels of PG synthesis resulted in 25%PG depletion of the explants after 8 days of culture. The releaseof cartilage PG not enhanced. TNF induced no detectable amountsof IL-1 (<0.01 U) in young or old cartilage but did induceIL-6 production. The induced amounts of IL-6 were higher inyoung than in old cartilage but no dose-dependency was evident.Antibodies to neither IL-1 nor IL-6 had any influence on theTNF-induced suppression of PG synthesis. The combination ofTNF and IL-1 led to an additive inhibition of PG synthesis whichhad no relationship to induced IL-6. TNF was about 100-foldless active than IL-1. KEY WORDS: Explant culture, Cytokines, Bioassay     

A fibronectin fragment induces type II collagen degradation by collagenase through an interleukin‐1—mediated pathway

Objective

To examine the effects of a fibronectin (FN) fragment containing the COOH‐terminal heparin‐binding domain (HBFN‐f) on chondrocyte‐mediated type II collagen (CII) cleavage by collagenase and proteoglycan (PG) degradation in articular cartilage in explant culture.

Methods

Intact FN or HBFN‐f was added to explant cultures of mature bovine articular cartilage. We investigated collagenase‐mediated cleavage of CII caused by HBFN‐f in explant cultures using a new immunoassay for detection and measurement of the primary collagenase cleavage site of CII. CII denaturation in cartilage was also measured using a specific enzyme‐linked immunosorbent assay. Degradation of PG (principally aggrecan) was analyzed by a dye‐binding assay. APMA and/or a matrix metalloproteinase 13 (MMP‐13) preferential inhibitor or interleukin‐1 receptor antagonist (IL‐1Ra) were added to some cultures to examine the presence of latent procollagenase or the involvement of MMP‐13 or IL‐1, respectively, in cartilage breakdown induced by HBFN‐f. Secretion of MMP‐3 and MMP‐13 into media was detected by immunoblotting.

Results

In contrast to intact FN, HBFN‐f was shown to stimulate CII cleavage by collagenase in a dose‐dependent manner following PG degradation, similar to cartilage breakdown induced by IL‐1. Treatment with HBFN‐f also resulted in elevated denaturation of CII. Immunoblotting demonstrated that HBFN‐f enhanced pro–matrix metalloproteinase 13 (proMMP‐13) production as well as that of proMMP‐3. APMA, which activates latent proMMPs, enhanced the HBFN‐f–mediated cleavage of CII by collagenase. An MMP‐13 preferential inhibitor or IL‐1Ra suppressed HBFN‐f–induced collagen cleavage to control levels.

Conclusion

Our data demonstrate that HBFN‐f can induce early PG degradation and subsequent CII cleavage. The latter is probably mediated by early proMMP‐13 induction involving an IL‐1–dependent pathway. Activation of latent collagenase is delayed. This new information, together with existing data on other FN fragments, reveals that increased levels of these fragments, found in diseased joints such as in osteoarthritis and rheumatoid arthritis, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the present study.

     

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.

     

Effect of ebselen,a scavenger of reactive oxygen species,on chondrocyte metabolism

Abstract

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a synthetic heterocyclic seleno-organic compound, has been shown to act as a scavenger of reactive oxygen species (ROS). We have previously reported that interleukin-1 (IL-1) inhibited proteoglycan (PG) synthesis and induced the production of ROS in cartilage explants and isolated chondrocyte cultures. In this study, we report the protective effect of ebselen against IL-1-mediated inhibition of PG synthesis and ROS induction in cultured cartilage explants and chondrocytes. Ebselen also reversed the inhibition of PG synthesis in mechanically stressed cultured chondrocytes. These data suggest that the use of the antioxidant ebselen may be a useful tool for studying the mechanisms of cartilage degradation.     

Inhibition of toll‐like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis

Objective

Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll‐like receptor 4 (TLR‐4). The objective of this study was to investigate the involvement of TLR‐4 activation in the development and progression of autoimmune destructive arthritis.

Methods

A naturally occurring TLR‐4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen‐induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin‐1 receptor antagonist (IL‐1Ra) gene deficiency were treated with TLR‐4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL‐1 in joints were evaluated after treatment.

Results

The TLR‐4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR‐4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR‐4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti–type II collagen immunity crucial for this model. Treatment with TLR‐4 antagonist strongly reduced IL‐1β expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL‐1–mediated autoimmune arthritis in IL‐1Ra^−/− mice and protected the mice against cartilage and bone pathology.

Conclusion

In the present study, we demonstrate for the first time that inhibition of TLR‐4 suppresses the severity of experimental arthritis and results in lower IL‐1 expression in arthritic joints. Our data suggest that TLR‐4 might be a novel target in the treatment of rheumatoid arthritis.

     

Impaired activation‐induced cell death promotes spontaneous arthritis in antigen (cartilage proteoglycan)–specific T cell receptor–transgenic mice

Objective

To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis.

Methods

We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAY QDK peptide of human cartilage PG. PG‐specific TCR–transgenic (PG‐TCR–Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG‐TCR–Tg mice.

Results

Spontaneous arthritis developed as early as 5–6 months of age, and the incidence increased to 40–50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4+ T cells, enhanced interferon‐γ and interleukin‐17 (IL‐17) production, and elevated levels of serum autoantibodies. PG‐TCR–Tg mice lacking IL‐4 developed arthritis earlier and at a higher incidence than IL‐4–sufficient mice. Antigen‐specific activation–induced cell death was diminished in vitro in CD4+ T cells of PG‐TCR–Tg mice with spontaneous arthritis, especially in those lacking IL‐4.

Conclusion

The presence of CD4+ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL‐4 appears to be a negative regulator of this disease, through attenuation of activation‐induced cell death.

     

Cyclic compression loaded on cartilage explants enhances the production of reactive oxygen species

OBJECTIVE: Although mechanical forces are an essential factor in the regulation of cartilage metabolism, the precise mechanisms involved have not yet been determined. We previously demonstrated that mechanical forces on chondrocytes inhibited proteoglycan (PG) synthesis. We also demonstrated the induction of reactive oxygen species (ROS) is loaded on the chondrocytes. Our purpose was to determine the ROS induction with mechanical compression and its involvement in PG synthesis of cartilage slices. METHODS: Bovine articular cartilage slices were subjected to cyclic compression loading. Synthesis of PG and ROS was measured using Na2[35S]-SO4 and a chemiluminescent probe, respectively. The induction of nitrotyrosine was determined using immunohistochemistry. RESULTS: The synthesis of PG was significantly inhibited with 2.0 MPa of compression stress; 1 h of compression was sufficient to inhibit PG synthesis. The ROS inhibitor ebselen reversed the compression-inhibited synthesis of PG. Compression on the cartilage induced synthesis of ROS and the expression of nitrotyrosine. CONCLUSION: Mechanical compression at 2.0 MPa inhibited PG synthesis by cartilage explants. ROS were involved in this action.     

The superficial layer of human articular cartilage is more susceptible to interleukin-1–induced damage than the deeper layers

Objective. To compare the responses of chondrocytes from superficial and deep layers of normal human articular cartilage to interleukin-1 (IL-1) and IL-1 receptor antagonist protein (IRAP), and to evaluate the binding sites for IL-1 on these cells. Methods. Cartilage and chondrocytes from superficial and deeper layers of human femoral condyles were cultured with and without IL-1 in the presence and absence of IRAP. The effect of these agents on ³⁵S-proteoglycan synthesis and catabolism and production of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by biochemical and immunologic assays. Receptor binding was evaluated using ¹²⁵I-labeled IL-1. Results. IL-1 induced more severe inhibition of proteoglycan synthesis and a lower ratio of secreted TIMP-1:stromelysin in chondrocytes from superficial cartilage than those from deeper cartilage. IRAP blocked responses to IL-1 more effectively in chondrocytes from deep cartilage than those from superficial cartilage. Chondrocytes from the articular surface showed approximately twice the number of high-affinity binding sites for IL-1 as did cells from deep cartilage. Conclusion. Chondrocytes from the surface of articular cartilage show a greater vulnerability to the harmful effects of IL-1 and are less responsive to the potential therapeutic effects of IRAP than cells in the deeper layers of the tissue.     

Induction of cartilage damage by overexpression of T cell interleukin‐17A in experimental arthritis in mice deficient in interleukin‐1

Objective

To examine the capacity of T cell interleukin‐17A (IL‐17A; referred to hereinafter as IL‐17) to induce cartilage damage during experimental arthritis in the absence of IL‐1.

Methods

Local IL‐17 gene transfer was performed in the knee joint of IL‐1–deficient mice and wild‐type controls during streptococcal cell wall (SCW)–induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS‐4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP‐mediated cartilage damage. In addition, systemic anti–IL‐1α/β antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL‐17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion.

Results

During SCW‐induced arthritis, local T cell IL‐17 gene transfer turned this acute, macrophage‐driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL‐1 dependency of cartilage PG depletion was fully abrogated when IL‐17 was locally overexpressed in the joint. Moreover, local IL‐17 gene transfer increased MMP expression without the need for IL‐1, although IL‐1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL‐17 was overexpressed in the knee joints of mice with collagen‐induced arthritis, anti–IL‐1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion.

Conclusion

These data show the capacity of IL‐17 to replace the catabolic function of IL‐1 in cartilage damage during experimental arthritis.

     

Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis

The objective of this study was to quantify insulin-like growth factor (IGF) binding proteins (IGFBPs) in the synovial fluid (SF) and plasma of patients with rheumatic diseases and to study the role of these proteins in the regulation of cartilage proteoglycan (PG) synthesis. Immunological determination of IGFBP-2, IGFBP-3, IGF-I, IGF-II, interleukin-1β (IL-1β) and tumour necrosis fac-tor α (TNFα) was undertaken in the SF and plasma of 115 patients with rheumatoid arthritis (RA; n = 53), osteoarthritis (OA; n = 44) and other rheumatic disorders. We also determined the effects of SF on bovine cartilage PG synthesis in culture. IGFBP-2 and IGFBP-3 were elevated in the plasma (by 38% and 28%, respectively) and SF (by 56% and 59%, respectively) of patients with RA compared to age- and sex-matched OA controls (determined by RIA and confirmed by Western ligand blot). IGF-I and IGF-II did not differ significantly between the two groups. OA SF, and, to a lesser extent, RA SF stimulated cartilage PG synthesis in culture, and more than 60% of this activity was neutralised by a specific monoclonal anti-IGF-I antibody. Human IGFBP-3 dose-dependently inhibited the stimulation of cartilage PG synthesis effected by SF or human IGF-I. In RA patients, the SF concentration of IGFBP-3 was positively correlated with SF levels of IL-1β and TNFα, with the serum level of C-reactive protein and with the erythrocyte sedimentation rate. We concluded that IGF-I is, under the conditions studied, the most important anabolic factor in human SF with respect to articular cartilage PG synthesis. The bioactivity of IGF-I in joints is modulated by IGFBP-3, which is elevated in RA SF compared to OA SF. Elevated IGFBP-3 in RA SF may reduce the availability of IGF-I to articular chondrocytes, thus interfering with cartilage PG synthesis in RA. Received: 30 November 1996 / Accepted: 19 February 1997