Inhibition of interleukin 1 beta induced rat and human cartilage degradation in vitro by the metalloproteinase inhibitor U27391.

Interleukin 1 induced proteoglycan loss from cartilage in vitro was prevented by a biochemical inhibitor of metalloproteinase activity. The inhibitor also partially relieved the inhibition of proteoglycan synthesis caused by interleukin 1. The loss of glycosaminoglycan by rat and human femoral head cartilage in response to human recombinant interleukin 1 beta (rhIL-1 beta) was established, and the modulation of this loss by the metalloproteinase inhibitor U27391 was investigated. Rat femoral head cartilage consistently lost glycosaminoglycan in response to rhIL-1 beta whereas only a proportion (30%) of normal human femoral head cartilage did so. Concentrations of 10-100 mumol/l U27391 inhibited the action of rhIL-1 beta on rat femoral head cartilage, reversing both the loss of glycosaminoglycan and the inhibition of glycosaminoglycan synthesis. U27391 also prevented the reduction in glycosaminoglycan content of those human femoral head cartilage explants responsive to rhIL-1 beta. Metalloproteinase inhibition therefore prevents rhIL-1 beta induced glycosaminoglycan loss by rat and human femoral head cartilage, suggesting that inhibitors of such enzymes may prove to be of therapeutic benefit in erosive diseases in humans.     

Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture.

Explants of articular cartilage from young pigs were maintained in organ culture for 10--16 days, and degradation of matrix was induced by retinol or complement-sufficient antiserum. The percentage breakdown of proteoglycan and collagen (as hydroxyproline release) was measured. The response of the cartilage depended on whether or not the explants were cut so as to include some of the invading marrow ('invasion zone'). In media containing retinol, cartilage lost up to three-quarters of its proteoglycan whether the invasion zone was present or not, but very little of its collagen unless this region was included. In the presence of complement-sufficient anti-serum, however, cartilage without the invasion zone was virtually unaffected, but both proteoglycan and hydroxyproline were released when invasion zone was included; here proteoglycan release began almost immediately, but there was a time-lag of 6--8 days before a substantial amount of hydroxyproline appeared in the medium. Histological examination of sample explants from the experiments supported the biochemical findings. The possible significance of the results in relation to rheumatoid arthritis is discussed.     

Carrageenin-induced arthritis. II. Effect of intraarticular injection of carrageenin on the synthesis of proteoglycan in articular cartilage

An inflammation of the periarticular tissue induced by the intraarticular injection of carrageenin caused a 40–75% decrease in the rate of proteoglycan synthesis in the articular cartilage. This decrease was determined both in vitro and in vivo. Provided that the collagen network of the cartilage was not disrupted, the chondrocytes were able to recover this synthetic ability and exhibited rates of synthesis above those of the controls. This ability resulted in a net replacement of the proteoglycan lost during the initial period of inflammation.     

Carrageenin-induced arthritis: V. A morphologic study of the development of inflammation in acute arthritis

Following a single injection of the polysaccharide carrageenin into the rabbit knee joint, a rapid inflammatory process occurs in the joint space and synovial membrane, followed by changes in the articular cartilage. Initially there is an influx of cells, mainly PMNs, into the synovial fluid, accompanied by proliferation of the synovial lining cells and infiltration of the synovial membrane. The numbers of synovial fluid cells decline gradually after 24 hr. The reaction in the synovial membrane is greatest at day 7, and inflammation is still evident at day 21. Initially, the infiltrate consists mainly of PMNs, but by day 7 it is predominantly mononuclear, with small clusters of lymphocytes.The articular cartilage shows loss of metachromasia with toluidine blue at 3–14 days after injection, but stains normally after day 21. Electron microscopy shows damage to the chondrocytes at days 1 and 7, with complete destruction of cells in the surface layer. At day 7 cells in the deeper layers have lost the apparatus required for proteoglycan synthesis, but at day 21 the cells appear virtually normal.There was no evidence for a direct inhibitory effect of carrageenin on proteoglycan biosynthesis. Most labeled carrageenin was rapidly cleared from the joint space, but about 10% was retained in the synovial membrane and 0.6% in articular cartilage at 48 hr after injection.Since the increase and decline in PMN numbers respectively precede the cartilage damage and recovery, it is suggested that there may be a correlation between the clinical activity of arthritis and the number of PMNs in the synovial fluid.     

Bacterial arthritis. A staphylococcal proteoglycan-releasing factor

We report the effect of staphylococci on induction of proteoglycan release from cartilage in organ culture and document the presence of a staphylococcal proteoglycan-releasing factor in the bacterial growth medium. Staphylococci cocultured with cartilage increased release of proteoglycan 3-4-fold within 48 hours. Viable cartilage was essential for maximum loss of proteoglycan. Killed bacteria had no effect on proteoglycan release. Four Staphylococcus aureus strains yielded growth media preparations active in proteoglycan release. Analysis of the proteoglycan subunits released from cartilage showed only limited degradation, and glycosaminoglycan chain length was not significantly altered.     

Role of TNF alpha in the induction of antigen induced arthritis in the rabbit and the anti-arthritic effect of species specific TNF alpha neutralising monoclonal antibodies.

OBJECTIVE--To investigate the role of tumour necrosis factor alpha (TNF alpha) in the development of antigen induced arthritis (AIA) in rabbits. METHODS--Monoclonal antibodies to rabbit TNF alpha were developed in rats and were used to detect TNF alpha in synovial fluid by enzyme linked immunosorbent assay and to localise it in tissue sections of synovium and cartilage from rabbits up to 21 days after induction of AIA. An antibody which neutralised TNF alpha activity in vitro was injected into rabbits to block TNF alpha action in vivo in AIA. Joint swelling, leucocyte infiltration into synovium and proteoglycan loss from cartilage were measured and compared with a control group, which were injected with sterile saline. RESULTS--Monoclonal antibodies to purified rabbit TNF alpha were prepared in rats and two were selected which were able to neutralise rabbit TNF alpha in a cytotoxicity bioassay. TNF alpha was detected in significant concentrations (21.7 (SE 0.5) pg/ml) in the arthritic joint fluid of rabbits with AIA only at one day after induction and it was then also sparsely localised in cells of the synovium, but from day 3 onwards it was localised more strongly in the deep zone of articular cartilage. Injection of anti-TNF monoclonal antibody R6 over three days into rabbits with AIA reduced joint swelling and leucocyte infiltration into joint fluid and decreased the expression of CD11b and CD18 on cells in the joint fluid. However, there was no significant reduction in the loss of proteoglycan from articular cartilage, although the joint fluid at three days contained a lower glycosaminoglycan content. The antibody R6 gave most effect at a dose of 0.6 mg/kg and there was no increase in its effectiveness at a fivefold greater dose (3.0 mg/kg). Treatment over 10 days gave a more complete suppression of joint swelling, but did not result in any less proteoglycan loss from cartilage. Treatment for five days with a 16 day follow up gave a significant reduction in swelling for several days beyond the treatment, but the swelling then slowly returned, until by day 21 there was no significant difference in joint swelling and there was also no recovery of cartilage proteoglycan content. A rabbit anti-rat immunoglobulin response was detected at 21 days, which may have limited the long term effectiveness of the antibody. CONCLUSIONS--In AIA in rabbits, TNF alpha was only detected in synovial fluid at one day after induction and there was only limited cellular localisation of TNF alpha in synovium and cartilage from three days. However, neutralising TNF alpha with a monoclonal antibody was effective in suppressing inflammatory changes in the joint during the acute onset of AIA, but it had little effect on the loss of proteoglycan from cartilage. The results suggest that blocking inflammation and synovitis with anti-TNF alpha may be more easily achieved than preventing damage to articular cartilage.     

Effects of murine recombinant interleukin 1 on synovial joints in mice: measurement of patellar cartilage metabolism and joint inflammation.

Murine recombinant interleukin 1 was injected intra-articularly into mice. It induced a clear effect on patellar cartilage within 24 hours. A low dose of interleukin 1 (1 ng) elicited a significant reduction in [35S]sulphate incorporation (50%) into proteoglycans and an accelerated breakdown (twofold) of 35S prelabelled proteoglycan. Proteoglycan breakdown returned to normal rates (approximately 10%/day) 48 hours after a single interleukin 1 injection. Recovery of proteoglycan synthesis was delayed by up to 72 hours, however, which implies that repair of the depleted cartilage matrix is retarded. Interleukin 1 induced only minor joint inflammation, too slight to be held responsible for the strong suppression of proteoglycan synthesis. Vehement joint inflammation was found after repeated interleukin 1 injections. The plasma extravasation and massive infiltration and exudation of leucocytes, predominantly polymorphonuclear leucocytes, were not a mere summation of single interleukin 1 effects, but point to interleukin 1 induced local hypersensitivity. The cartilage matrices of patella and femur were heavily depleted. Measurement of the extent of loss of 35S prelabelled proteoglycan and the prolonged inhibition of [35S]sulphate incorporation indicate that both inhibition of proteoglycan synthesis and enhanced loss of proteoglycan contributed substantially to this depletion.     

Proteoglycan metabolism in tissue cultured human articular cartilage. Influence of piroxicam

Proteoglycan metabolism was investigated in longterm tissue cultured human cartilage. Visually intact cartilage from adult donors showed improving accumulation rates for 35sulfate labelled proteoglycans over a 6-week period. The loss of newly synthesized molecules in the nutrient culture media was low and constant. Fibrillated cartilage from a 17-year-old male showed higher basal 35S incorporation rates and the proportions of 35S proteoglycan aggregates were higher than in normal tissue. These observations may reflect the immature status of the tissue or an attempt at repair. However samples lost increasing amounts of 35S proteoglycans in the incubation media. This material appeared to be monomeric proteoglycan. The amount of 35S activity retained in the fibrillated tissue matrix fell during culture as did the proportion of proteoglycan aggregates. Thus catabolic events were postulated in these fibrillated cartilage samples. When piroxicam was added to the incubation media more newly synthesized proteoglycans were retained in the intercellular matrix of the fibrillated samples. Increased accumulation of 35S activity was seen in some of the batches of visually intact cartilage.     

Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin b and an inhibitor of matrix metalloproteinases. evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation

Objective. To investigate mechanisms of cartilage matrix destruction by a study of the effects of a specific inactivator of cathepsin B and an inhibitor of several matrix metalloproteinases (MMP) on cartilage proteoglycan release. Methods. Cartilage explants were treated with either recombinant human interleukin-1α (rHuIL-1α) or retinoic acid in the presence or absence of the inhibitors, and proteoglycan release was quantitated. Tests for nonspecific effects of the inhibitors included reversibility, rates of protein synthesis and glycolysis, and effects on other rHuIL-1α–mediated events. Results. The cathepsin B inactivator inhibited rHuIL-1α–stimulated proteoglycan release at nanomolar concentrations, but failed to significantly inhibit retinoic acid–stimulated proteoglycan release. An inhibitor of MMP was inhibitory to both rHuIL-1α–stimulated release and retinoic acid–stimulated release. Conclusion. Cathepsin B is implicated in rHuIL-1α–stimulated loss of cartilage proteoglycan. Its lack of involvement in retinoic acid–stimulated proteoglycan release suggests the existence of at least 2 pathways of cartilage proteoglycan breakdown, which may converge at the activation of a matrix prometalloproteinase.     

Differential responses of human articular cartilage to retinol.

An in-vitro study has been made of the response of aged human articular cartilage to the catabolic agent retinol. Weight bearing cartilage from the femoral condyle degrades and releases proteoglycan with an associated reduction of sulphate incorporation. Similar cartilage from the femoral head responds to the retinol with an inhibition of sulphate incorporation but no degradation or loss of proteoglycan. Extraction of the proteoglycan from the femoral head samples failed to demonstrate any evidence of breakdown.     

Development and reversal of a proteoglycan aggregation defect in normal canine knee cartilage after immobilization.

Healthy adult dogs were studied for a defect in proteoglycan aggregation by immobilizing one limb for varying periods of time. Immobilization for 6 days resulted in a 41% reduction in proteoglycan synthesis by articular cartilage from the restrained knee compared with the contralateral control knee. After 3 weeks of immobilization, proteoglycan aggregation was no longer demonstrable in cartilage from the constrained limb. The aggregation defect was rapidly reversible and aggregates were again normal size 2 weeks after removal of a cast that had been worn for 6 weeks.     

Protection against cartilage proteoglycan synthesis inhibition by antiinterleukin 1 antibodies in experimental arthritis.

We have used neutralizing antibodies raised against murine recombinant interleukin 1 (IL-1) to demonstrate a role for IL-1 in the cartilage destruction and inflammation of antigen induced arthritis. Ex vivo production of IL-1 was demonstrated in tissue cultures of joint cross sections shortly after arthritis induction. Neutralizing antimurine IL-1 antibodies identified the activity to be about 80% IL-1 alpha 24 h after onset of arthritis. In animals receiving a single injection of anti-IL-1 antisera at Day -3, cartilage proteoglycan synthesis suppression during the first 2 days of arthritis was prevented. Normal proteoglycan synthesis was maintained until Day 4 when anti-IL-1 antisera was given at Days -2, 0, and 2 or arthritis. Dose response experiments showed that the reduction in inflammation was insufficient to account for the clearcut reduction in cartilage proteoglycan synthesis inhibition. Our results demonstrate that IL-1 plays a role in cartilage pathology in murine antigen induced arthritis.     

Spontaneous secretion of a proteoglycan releasing factor by mononuclear cells in juvenile arthritis

Patients with juvenile arthritis (JA) spontaneously produced a substance which accelerated proteoglycan loss from cultured articular cartilage. Peripheral blood mononuclear cells (PBMC) from 15 patients with JA were cultured for varying days, and cell-free PBMC conditioned media were added to articular cartilage cultures. Release of proteoglycan and collagen from cartilage was quantified by analysis of chondroitin sulfate and hydroxyproline content, respectively, after 4 days of culture. Conditioned media from PBMC of patients with systemic onset JA (3/3) and 5/7 patients with polyarticular JA increased release of proteoglycan when added to cartilage cultures. Mitogen stimulation of the PBMC was unnecessary for activity and addition of mitogen did not alter proteoglycan release. The PBMC conditioned media from the other patients (2/7) with polyarticular JA, from patients (3/3) with systemic onset JA which had progressed to polyarticular JA, and from patients with pauciarticular JA, did not enhance proteoglycan release without mitogen stimulation. PBMC of normal children produced media which enhanced proteoglycan release after mitogen stimulation. No conditioned medium accelerated proteoglycan release if cartilage was freeze killed before culture and none tested reduced cartilage collagen content.     

A staphylococcal proteoglycan-releasing factor

We report the effect of staphylococci on induction of proteoglycan release from cartilage in organ culture and document the presence of a staphylococcal proteoglycan-releasing factor in the bacterial growth medium. Staphylococci cocultured with cartilage increased release of proteoglycan 3-4-fold within 48 hours. Viable cartilage was essential for maximum loss of proteoglycan. Killed bacteria had no effect on proteoglycan release. Four Staphylococcus aureus strains yielded growth media preparations active in proteoglycan release. Analysis of the proteoglycan subunits released from cartilage showed only limited degradation, and glycosaminoglycan chain length was not significantly altered.     

Significance of connective tissue proliferation in the breakdown of cartilage: a novel in vivo model.

The implantation of homologous femoral head cartilage in subcutaneous tissues of random bred Wistar rats results in both subchondral and articular surfaces becoming overlaid by an adherent granulation tissue comprising predominantly fibroblast-like cells. The response of the tissue to cartilage encapsulated with cotton fibres was also similar but erosions, mainly subchondral, were more evident and proteoglycan loss markedly greater. The connective tissue response to cotton was the progressive formation of a foreign body granuloma comprising mononuclear cells, multinucleated giant cells, and fibroblasts with very few polymorphonuclear leucocytes.     

The Effect of Synovial Tissue on the Breakdown of Articular Cartilage in Organ Culture

Living (pig) cartilage in contact with synovium lost both proteoglycan and collagen and sometimes became reduced to a mass of fibroblast-like chondrocytes without matrix; dead cartilage lost proteoglycan but less collagen. Similar changes appeared in living cartilage grown at a distance from the synovium but in the same dish; dead cartilage was unaffected. Conclusion: the synovium has a) a direct, presumably enzymatic action on cartilage matrix and b) an indirect effect mediated through the chondrocytes.     

Degradation of type II collagen,but not proteoglycan,correlates with matrix metalloproteinase activity in cartilage explant cultures

Objective. To determine the contribution of certain matrix metalloproteinases (MMPs) to the degradation of proteoglycan and type II collagen in cartilage. Methods. Bovine nasal and articular cartilage explants were cultured with recombinant human interleukin-1α (IL-1α) for up to 4 weeks. Release of proteoglycan and type II collagen into the medium was determined by colorimetric assay and immunoassay, respectively. The activity of MMPs in the medium was assayed using a quenched fluorescent substrate, as well as with a collagen fibril assay, by zymography, and in Western immunoblots. In some experiments, the effects of specific MMP inhibitors on type II collagen degradation were studied. Results. In cultures of nasal cartilage with IL-1α, almost all the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collagen occurred in the third or fourth week. MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of culture, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gelatinolytic activity (by zymography) during the third week of culture. Articular cartilage cultured with IL-1α lost proteoglycan progressively during the 4-week period; however, there was no loss of type II collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal cartilage stimulated with IL-1α was inhibited by BB87, an inhibitor of both collagenases and gelatinases, and by BB3003, a selective inhibitor of gelatinase A. In Western immunoblots, procollagenase and active interstitial collagenase could be readily detected in nasal cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be present. Conclusion. MMP activity correlates with degradation of type II collagen, but not proteoglycan, in cartilage cultures. Interstitial collagenase, collagenase 3, and gelatinases are all likely to contribute to cleavage and removal of collagen from the cartilage matrix. The proteinase(s) responsible for aggrecan breakdown remains unclear.     

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.     

In vivo model of cartilage degradation--effects of a matrix metalloproteinase inhibitor.

OBJECTIVES--To develop a model of cartilage degradation that (i) enables the testing of synthetic, small molecular weight matrix metalloproteinase (MMP) inhibitors as agents to prevent cartilage erosion, (ii) permits the direct assay of the principal constituents of the extracellular matrix (collagen and proteoglycan) in both the non-calcified articular cartilage and the calcified cartilage compartments, and (iii) is mediated by a chronic, granulomatous tissue that closely apposes intact articular cartilage, and in this respect resembles the pannus-cartilage junction of rheumatoid arthritis. METHODS--Femoral head cartilage was obtained from donor rats, wrapped in cotton and implanted subcutaneously into recipient animals. After a two stage papain digestion procedure, the proteoglycan and collagen contents were measured by assaying for glycosaminoglycans and hydroxyproline, respectively, in both the non-calcified cartilage that comprises the articular surface layer and the calcified cartilage compartment. The incorporation in vitro of [35S]-sulphate into glycosaminoglycans was assayed as a measure of proteoglycan biosynthesis. An osmotic minipump was cannulated to the implanted femoral head cartilage and synthetic MMP inhibitors (MI-1 and MI-2) were infused continuously over a 14 day period. RESULTS--The implanted, cotton wrapped femoral head cartilages provoked a granulomatous response that resulted in the removal of collagen and proteoglycan from the cartilage matrix. The removal of proteoglycan and collagen was exclusively from the non-calcified articular cartilage, whereas the proteoglycan and collagen content of the calcified compartment increased during the experiments. MI-1 reproducibly reduced the degradation of proteoglycan and collagen in implanted femoral head cartilage. CONCLUSIONS--We have described an in vivo model of cartilage degradation that permits the measurement of proteoglycan and collagen in both non-calcified articular cartilage and calcified cartilage compartments. The model can be used to test the effects of agents of unknown systemic bioavailability and pharmacokinetic profile by infusing them directly to the site of cartilage degradation. The removal of cartilage extracellular matrix by granulomatous tissue was inhibited by an MMP inhibitor, thus proving the involvement of this family of proteinases in cartilage catabolism in this model.     

Impaired activity of protease inhibitors towards neutrophil elastase bound to human articular cartilage.

OBJECTIVE: To investigate the effects of protease inhibitors on the ability of free and cartilage bound neutrophil elastase to degrade cartilage proteoglycan in vitro. METHODS: Cryostat sections of human articular cartilage were used as substrate, and proteoglycan loss induced by free or cartilage bound elastase was quantified by alcian blue staining, followed by scanning and integrating microdensitometry. RESULTS: High molecular mass protease inhibitors (alpha 1 protease inhibitor, alpha 2 macroglobulin, and soya bean trypsin inhibitor) and synovial fluid from patients with rheumatoid arthritis were effective in blocking proteoglycan loss from sections treated with free elastase, but their activity towards cartilage bound elastase was much reduced. In contrast, low molecular mass elastase inhibitors (N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone and ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulphonylamino]benzoyl] amino-acetic acid) were effective against free and cartilage bound elastase. CONCLUSION: The binding of elastase to cartilage appears to be a mechanism whereby the enzyme can remain active in the presence of high molecular mass protease inhibitors.