The effect of human interleukin 1 on proteoglycan metabolism in human and porcine cartilage explants

Human interleukin 1 (IL-1), up to 100 pg/ml, causes a decrease of the proteoglycan content of human (old and young) as well as porcine cartilage explants, without stimulating the proteoglycan release from the cartilage. The proteoglycan depletion is stronger in young than in old human cartilage and stronger in human than in porcine cartilage. The proteoglycan synthesis is considerably more inhibited by IL-1 in young than in old human cartilage. Our data suggest that an IL-1 induced inhibition of the proteoglycan synthesis, rather than a stimulation of proteoglycan breakdown causes the proteoglycan depletion of the cartilage. The data furthermore suggest a clear difference between young and old human cartilage, with respect to their sensitivity for IL-1. IL-1 in a concentration of 500 pg/ml causes in all 3 kinds of cartilage explants chondrocyte damage that might be relevant in the cartilage destruction during rheumatoid arthritis.     

Influence of rheumatoid synovial fluid and cells on proteoglycans in human cartilage explants. Modulation by piroxicam

Summary We have studied the effects of cell-free rheumatoid synovial fluid (RASF) and the conditioned medium (CM) from these cells on the proteoglycans (PGs) of normal human cartilage and the influence which piroxicam might have on these processes. Both RASF and the CM from RASF cells enhanced the PG release from the cartilage explants. The effects of the above mentioned fluids on the cartilage PG content depended on the metabolic state of the cartilage i.e. correlated inversely with the PG synthesis. Whether this was due to the presence of anabolic and catabolic factors in these fluids is discussed. Piroxicam had no adverse effect on the PGs of human cartilage in vitro. Piroxicam prevented the cartilage PG depletion when it was induced by the CM from RASF cells.     

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).     

Immune Regulation of Collagenase Secretion in Rheumatoid and Osteoarthritic Synovial Cell Cultures

Primary cultures of synovial cells were obtained by proteolytic dispersion of synovial tissue from patients with rheumatoid arthritis (n = 19), psoriatic arthritis (n = 2), osteoarthritis (n = 13) and other joint problems (n = 3). The levels of endogenously secreted collagenase were variable from patient to patient but did not differ significantly between rheumatoid arthritis and osteoarthritis. The endogenous collagenase secretion was likely a consequence of mononuclear cell factor (MCF) release from monocytes/macrophages which have been shown to be present among the heterogeneous primary rheumatoid synovial cell population (Dayer et al. 1976). As also demonstrated by these investigators, medium containing MCF can be generated from peripheral blood mononuclear cells in a T lymphocyte-dependent process by the addition of phytohemagglutinin (PHA). The addition of such medium stimulated collagenase secretion from all our synovial cell cultures regardless of the endogenous level. Protein synthesis but not synovial cell proliferation was required for MCF stimulation of collagenase secretion. The direct addition of PHA to primary synovial cell cultures stimulated collagenase secretion in some but not all cases indicating the presence of T lymphocytes in these positively-responding cultures. In some of these primary synovial cell cultures in which the addition of PHA stimulated collagenase secretion, secretion was also stimulated by the addition of collagen peptides, native collagen, proteoglycan or purified protein derivative of tuberculin. We propose that, in these instances, MCF release is mediated by antigen-sensitized lymphocytes. Antigen-responsive cultures were not restricted to the rheumatoid population. Our data are compatible with the idea that infiltrated lymphocytes in inflamed synovial tissue become sensitized to cartilage and joint capsule components released during tissue degradation and contribute to matrix destruction by mediating MCF release with consequent stimulation of collagenase synthesis and secretion from synovial cells.     

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     

Responses of normal and rheumatic human articular chondrocytes cultured under various experimental conditions in agarose

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.     

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.     

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     

Suppression of human cartilage proteoglycan synthesis by rheumatoid synovial fluid mononuclear cells activated with mycobacterial 60-kd heat-shock protein

Objective. To examine whether T cell reactivity toward heat-shock proteins (HSP) contributes to cartilage destruction in rheumatoid arthritis (RA). Methods. An in vitro system was used, in which human cartilage explants were cocultured with hsp60-activated synovial fluid mononuclear cells (SFMC) from patients with RA, and proteoglycan (PG) synthesis was measured. Results. The hsp60-activated SFMC suppressed cartilage PG synthesis. This effect was dependent on the production of interleukin-1 (IL-1) and tumor necrosis factor α (TNFα). Conclusion. Mycobacterial 60-kd heat-shock protein can activate rheumatoid SFMC to suppress human cartilage PG synthesis. This suppression is mediated by IL-1 and TNFα.     

Celecoxib has a positive effect on the overall metabolism of hyaluronan and proteoglycans in human osteoarthritic cartilage

OBJECTIVE: To assess the effects of celecoxib, a cyclooxygenase (COX-2) selective inhibitor, on the metabolism of hyaluronan (HA) and proteoglycans (PG) in human cartilage explants with midrange severity of osteoarthritis (OA). Results were compared with those of diclofenac, a non-selective COX inhibitor. METHODS: Cartilage specimens (OA grade 4-8 on Mankin's scale) were pulsed with 3H -glucosamine and chased in the absence or presence of 1-10 micro g/ml of celecoxib or diclofenac. After papain digestion, the labeled chondroitin sulfate and HA molecules were purified by anion-exchange chromatography. RESULTS: Diclofenac did not affect the metabolic balance of PG and HA whereas, in a relatively dose-dependent manner, celecoxib increased the synthesis of HA and PG; celecoxib also reduced the net loss of labeled HA and PG molecules from cartilage explants. CONCLUSION: In short term in vitro cultures, celecoxib has a favorable effect on the overall metabolism of PG and HA. It is therefore unlikely that this drug would have a detrimental effect on articular cartilage during longterm administration. Further, celecoxib might help counteract the depletion of HA seen in OA cartilage.     

Modulation of synovial cell products by a factor from a human cell line: T lymphocyte induction of a mononuclear cell factor.

A human cell line (U937) can be stimulated to produce a soluble factor (MCF) by either lectin-activated T lymphocytes or their soluble products. In prior studies, we showed that MCF produced by peripheral blood mononuclear cells can increase production of collagenase and prostaglandin E2 by adherent synovial cells obtained from enzymatically dispersed rheumatoid synovium. We show here that peripheral blood T lymphocytes or cloned human T lymphocyte lines are capable of inducing MCF production by the monocyte-like U937 cells. MCF can be demonstrated in the supernatant fluid from cocultures of U937 cells and T lymphocytes that have been stimulated with phytohemagglutinin or concanavalin A for 24-48 hr. In addition, the supernatant fluid from 24-hr lectin-stimulated T lymphocytes can be transferred onto the U937 cells and subsequently, MCF activity can be recovered from the U937 culture medium. The activity of the soluble T cell product on the U937 cells is both time- and dose-dependent. A human cell line capable of MCF production in continuous culture has not been previously available. The use of a monocyte-like cell line (U937) and cloned T lymphocytes now makes it possible to demonstrate the role of discrete cell populations in the production of MCF and other mediators.     

In vivo effects of interleukin-1 on articular cartilage. Prolongation of proteoglycan metabolic disturbances in old mice

We investigated the effects of intraarticular injections of interleukin-1 alpha (IL-1 alpha) into the knee joints of young (3-month-old) and old (18-month-old) C57Bl/10 mice. In this in vivo study, 35S-sulfate incorporation and release were used to compare the effects of IL-1 on patellar cartilage proteoglycan metabolism. IL-1-induced stimulation of proteoglycan degradation was confined to the first 24 hours after injection in both young and old animals, and was only slightly lower in old cartilage than in young. In old patellar cartilage, IL-1-induced suppression of proteoglycan synthesis appeared to be more prolonged. Also, the amount of time needed to restore the cartilage matrix, characterized by proteoglycan synthesis above normal levels, was longer in old animals. Histologic analysis confirmed the retarded recovery in the cartilage of old mice. Autoradiography showed that the chondrocytes of the medial side of the femorotibial area were most vulnerable to IL-1-induced suppression of proteoglycan synthesis, especially the medial tibial plateau. As with the patellar cartilage, IL-1-induced suppression of proteoglycan synthesis in this area of articular cartilage was more prolonged in old animals. Our data indicated that the impact of IL-1 on articular cartilage is higher in old mice and that, consistent with certain loci found to be at risk in experimental osteoarthritis, there are sites at which IL-1 is more likely to act.     

Inhibition of cartilage breakdown by hydrocortisone in a tissue culture model of rheumatoid arthritis.

Bovine nasal cartilage discs cocultured with human rheumatoid synovial membrane or synovial-membrane-conditioned media release proteoglycan largely as a result of cartilage breakdown. We assessed the effects of hydrocortisone on proteoglycan distribution between cartilage and culture medium, and on cartilage breakdown expressed as the release of either proteoglycan or 35S-products from prelabelled discs. The presence of synovial membrane inhibited the capacity for net proteoglycan synthesis, preventing its accumulation in cartilage; this was little affected by hydrocortisone. The major response to pharmacological concentrations of hydrocortisone was suppression of both spontaneous and synovial-membrane-induced cartilage breakdown. The autolysis of synovial protein that normally occurred during culture was similarly prevented by comparable doses of corticosteroid. Changes in chromatographic distribution of the 35S-labelled degradation products released from cartilage conformed with a corticosteroid-induced inhibition of endogenous lysosomal or related proteinase activity. Additionally, inhibition of the early events in synovial membrane that are responsible for chondrocyte-mediated breakdown of cartilage may contribute significantly to the overall corticosteroid effect.     

The role of plasminogen in interleukin-1 mediated cartilage degradation

Explants of 35SO4 labelled rabbit articular cartilage, cultured for 3 days with either 5 X 10(4) rabbit peritoneal cells (PEC) or 1:10 macrophage conditioned medium (MCM), released 30-40% of labelled proteoglycans into the medium while controls released 8-12%. The addition of 1 mM 4 aminophenylmercuric acetate (APMA) or 0.2 U/ml plasminogen increased proteoglycan release to 85%. Similar results were obtained when recombinant human interleukin-1 (IL-1) was used instead of MCM. Further, supernatant from MCM stimulated chondrocytes, incubated with dead cartilage explants for 3 days, did not significantly increase proteoglycan release above the background level of cartilage alone (7-10%), nor did the addition of 5 X 10(4) PEC to cultures of dead cartilage explants plus supernatant from MCM stimulated chondrocytes make any significant difference, indicating that supernatant from MCM stimulated chondrocytes and PEC alone had negligible cartilage proteoglycan degrading activity in these experiments. The inclusion of 0.1 mM APMA or 0.2 U/ml plasminogen in cultures of dead cartilage explants plus supernatant from MCM stimulated chondrocytes, however, increased proteoglycan release to 80-93%, with or without PEC. Our results suggest that plasminogen, activated by a product from IL-1 stimulated chondrocytes, greatly enhanced IL-1 mediated cartilage degradation by activating latent metalloproteinases.     

Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage

Cartilage from normal controls, patients with osteoarthritis, and patients with rheumatoid arthritis produced no interleukin-6 (IL-6) in culture. However, IL-1 induced massive production of IL-6 (up to 135 ng/ml) in cartilage from all 3 sources, in a dose-dependent manner (in some cases, a peak value was reached). The levels of induced IL-6 were similar to those found in rheumatoid arthritis synovial fluid. At IL-1 concentrations that induced almost complete inhibition of proteoglycan (PG) synthesis, IL-6 production could still be increased considerably. Exogenous IL-6 inhibited PG synthesis by up to 25%. IL-1-induced inhibition of PG synthesis was reversed by antibodies against recombinant human IL-6. These results suggest that IL-6 is required for the IL-1-induced inhibition of PG synthesis.     

Degradation of human articular cartilage by neutrophils in synovial fluid

Objective. To determine whether surface-adherent immunoglobulins are capable of mediating synovial fluid (SF) neutrophil degradation of proteoglycan and collagen in intact, normal human articular cartilage, and to define the respective roles of neutrophil serine proteases and metalloproteases in degrading these cartilage constituents. Methods. Pellet explants of normal human articular cartilage pretreated with bovine serum albumin (BSA) or IgG were incubated with polymorphonuclear cells suspended in SF (PMN-SF), or with supernatants derived from neutrophils stimulated with surface-associated IgG. Proteoglycan degradation was measured by assaying release of ³⁵S-proteoglycan fragments from cartilage explants prelabeled with ³⁵S-sulfate. Collagen degradation was measured by assaying hydroxyproline content in the PMN-SF preparations or neutrophil supernatants following their incubation with unlabeled explants. Results. Significant release of both ³⁵S fragments and hydroxyproline was noted following incubation of PMN-SF with IgG-treated pellets, compared with pellets treated with BSA. IgG preparations derived from pooled normal serum or rheumatoid arthritis SF were equally efficacious in mediating PMN degradation of cartilage collagens. Explant release of ³⁵S fragments during incubation with PMN supernatant was completely inhibited when serine proteases were inactivated by diisopropyl fluorophosphate (DFP); however, release of ³⁵S fragments was enhanced when metalloprotease activity was present in the supernatant. Release of hydroxyproline during incubation of explants with PMN supernatant was comparable in the presence of DFP or EDTA, but was markedly enhanced when both serine and metalloprotease activity were present in the supernatant. Conclusion. Neutrophils in SF are capable of degrading both proteoglycans and collagens in intact human articular cartilage. Degradation of these cartilage constituents is facilitated by immunoglobulins adherent to the cartilage surface and by the synergistic action of PMN serine and metalloproteases released during activation of neutrophils with surface-associated immunoglobulin.     

Somatomedin activity in synovial fluid from patients with joint diseases.

The somatomedin activity in synovial fluids from 50 patients with a variety of joint diseases has been studied and compared with the activity in each of the patient's own serum and a standard reference serum (SRS). The porcine costal cartilage bioassay of Van den Brande and Du Caju (1974a) has been used with the isotopes 3H-thymidine and 35S-sulphate. Synovial fluids from most patients with post-traumatic and post-operative effusions, osteoarthritis and arthritis associated with psoriasis, Reiter's disease, and ankylosing spondylitis stimulated the synthesis of DNA and proteoglycans in cartilage. Synovial fluids from patients with rheumatoid arthritis either had impaired capacity to stimulate DNA synthesis, or they inhibited it; a similar, but less evident pattern was observed for proteoglycan synthesis. Some synovial fluids from patients with miscellaneous synovitides stimulated, while others inhibited cartilage metabolism. It is concluded that the synovial fluid from patients with rheumatoid arthritis and from some patients with miscellaneous synovitides contained an inhibitor(s) to DNA and possibly proteoglycan synthesis. The sera from nearly all the patients stimulated both DNA and proteoglycan synthesis, but the somatomedin potency ratios for serum in terms of SRS were generally less than 1.0. There was a significant inverse correlation between the serum somatomedin potency ratio and the age of the patient.     

Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis.

OBJECTIVE: To investigate whether apocynin, 1-(4-hydroxy-3-methoxyphenyl)ethanone, is able to diminish inflammation-induced cartilage destruction in rheumatoid arthritis (RA), studied in a human in vitro model. METHODS: Apocynin was added to cultures of RA peripheral blood mononuclear cells (PBMNC). Cartilage-destructive activity was determined by addition of culture supernatant to tissue samples of human articular cartilage. In addition, the proliferation of PBMNC, their production of tumour necrosis factor alpha (TN-Falpha), interleukin (IL)-1 and IL-10, and T-cell production of interferon gamma (IFN-gamma) and IL-4, as measures for T1 and T2 cell activity, were determined. RESULTS: Apocynin was able to counteract RA PBMNC-induced inhibition of cartilage matrix proteoglycan synthesis, while no effect on inflammation-enhanced proteoglycan release was found. The effect was accompanied by a decrease in IL-1 and TNF-alpha production by the MNC. No effect on T-cell proliferation was found, but the production of IFN-gamma, IL-4 and T-cell-derived IL-10 was strongly diminished. Most important, apocynin did not show any direct adverse effects on chondrocyte metabolism; on the contrary, it diminished the release of proteoglycans from the cartilage matrix. CONCLUSION: Apocynin in vitro inhibits inflammation-mediated cartilage destruction without having adverse effects on cartilage. The latter may be an advantage of apocynin over many other non-steroidal anti-inflammatory drugs. Therefore, apocynin might have an added beneficial effect in protecting RA patients from joint destruction.     

Chondrocyte nonresponsiveness to insulin-like growth factor 1 in experimental arthritis

Inhibition of chondrocyte proteoglycan (PG) synthesis is one of the mechanisms leading to cartilage destruction in joint inflammation. Using murine cartilage from normal and arthritic knee joints, we examined this process. We found that for normal, anatomically intact murine articular cartilage, insulin-like growth factor 1 (IGF-1) is a potent anabolic factor. Recombinant IGF-1 at physiologic concentrations in a completely synthetic medium sustained PG synthesis, at the in vivo rate, of patellar cartilage in organ culture. Using an experimental arthritis model, we found that cartilage from an arthritic joint could not be stimulated in vitro with IGF-1. This nonresponsiveness was not caused by a generalized metabolic inhibition of the chondrocytes, because PG synthesis in arthritic cartilage could still be stimulated by forskolin, an activator of adenylate cyclase. Our data suggest that during joint inflammation, inhibition of chondrocyte PG synthesis is, at least partially, caused by a defect in the IGF-1 responsiveness of the chondrocyte. We propose this finding as a possible pathogenetic mechanism for cartilage destruction in joint diseases.     

Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.