In vitro effects of tiaprofenic acid, sodium salicylate and hydrocortisone on the proteoglycan metabolism of human osteoarthritic cartilage

We examined the in vitro effects of therapeutic doses of tiaprofenic acid (26 micrograms/ml; 2.6 micrograms/ml), sodium salicylate (160 micrograms/ml), and hydrocortisone (7.25 micrograms/ml; 0.725 micrograms/ml) on the proteoglycan metabolism (catabolism and synthesis) and chondrocyte ultrastructure of organ explant cultures of human osteoarthritic (OA) articular cartilage. The effect of these drugs on chondrocyte neutral metalloprotease synthesis was also examined. Tiaprofenic acid and the higher concentration of hydrocortisone had a similar suppressive effect on proteoglycan catabolism (38%). The effect of the lower concentration of hydrocortisone was less marked (29%), while sodium salicylate had the least effect (21%). The proteoglycan released in each treated group was significantly lower (p less than 0.05) than that of the untreated one. The suppression of proteoglycan catabolism by tiaprofenic acid was reversible in all but one specimen. This reversal was only seen in 4 of the 8 patient specimens treated with sodium salicylate and in 2 of the 8 patient specimens treated with the higher dose of hydrocortisone. The decrease in proteoglycan catabolism induced by these drugs correlated with their potential to reduce synthesis of neutral metalloprotease. The proteoglycan synthesis in cartilage organ explant cultures was reduced by sodium salicylate and hydrocortisone, but not by tiaprofenic acid. These findings were corroborated through an electron microscopic study, showing extensive vesicular dilatation of chondrocytic endoplasmic reticulum seen only in explants treated with hydrocortisone or sodium salicylate. Our data suggests that some nonsteroidal antiinflammatory drugs (NSAID) are able to decrease OA cartilage catabolism. However, caution should be taken since certain NSAID, like salicylate, may also possibly jeopardize the cartilage repair process by inhibiting the proteoglycan synthesis.     

Rheumatoid synovial fluid contains bioactive leukemia inhibitory factor with cartilage degrading activity--another target for chondroprotective intervention

OBJECTIVE: To determine if the procatabolic activity of inflammatory synovial fluids (SF) from patients with rheumatoid arthritis (RA) could be attenuated by the cytokine antagonists murine leukemia inhibitory factor (LIF) binding protein (mLBP) and interleukin 1 receptor antagonist (IL-1ra). METHODS: Pig articular cartilage explants were cultured in the presence of either 20% v/v rheumatoid (RA) or osteoarthritic (OA) SF and varying concentrations of either mLBP and/or IL-1ra. The catabolic activity of the SF and the relative effects of mLBP and/or IL-1ra were assessed by determining the percentage release of sulfated glycosaminoglycans from cartilage explants. LIF concentrations were measured by ELISA. RESULTS: RA SF but not OA SF stimulated release of proteoglycans from pig cartilage explants in vitro (47.3 +/- 2.2% vs 24.6 +/- 2.0%; p < 0.0001). Murine LBP at 100 ng/ml and recombinant human (rh) IL-1ra at 5000 ng/ml produced a dose dependent inhibition of this proteoglycan release (p < 0.0067 and p < 0.0111, respectively). The RA SF stimulated proteoglycan release was attenuated by mLBP and rhIL-1ra independently. No additive effect of this attenuation was observed when maximal inhibitory doses were used in combination. The decrease in proteoglycan release produced by mLBP correlated significantly with LIF concentrations in RA SF. CONCLUSION: These findings are consistent with the concept that IL-1 stimulates cartilage proteoglycan resorption in RA. They also support the hypothesis that LIF, too, contributes to cartilage proteoglycan resorption in RA. The residual stimulation not accounted for by IL-1 or LIF suggests other cytokines may contribute. The role of LIF and related or unrelated cytokines may need to be taken into account to optimize chondroprotection in RA and other rheumatic diseases.     

Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

OBJECTIVE: To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). METHODS: Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum-free medium with or without IGF-1 (100 ng/ml), OP-1 (100 ng/ml), or bFGF (0-100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants. RESULTS: Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF-1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF-1 and OP-1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF-1 or OP-1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF-1 and OP-1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures. CONCLUSION: The anabolic activity of IGF-1 and OP-1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.     

Interferon-gamma increases human neutrophil-mediated cartilage proteoglycan degradation.

Interferon gamma (IFN-gamma) is known to activate neutrophils and is produced by the synoviocytes, macrophages, T cells and other inflammatory cells in rheumatoid arthritis. Neutrophils participate in articular cartilage destruction and predominate in the synovial fluid of inflamed joints during the early stages and acute exacerbations of the disease. In this study we investigated the effect of recombinant human IFN-gamma on human neutrophil damage to bovine articular cartilage explants which had been coated with heat-aggregated IgG (HAGG). Cartilage proteoglycan degradation by neutrophils was augmented by IFN-gamma at 10(2)-10(4) U/ml. IFN-gamma also increased neutrophil-mediated proteoglycan degradation when the cells were preincubated with IFN-gamma and washed before addition to cartilage. This activity of IFN-gamma was abolished by boiling and was unaffected by polymixin B, indicating that the effects of IFN-gamma were not due to endotoxin contamination. In the absence of neutrophils IFN-gamma had no effect on proteoglycan metabolism, and the ability of IFN-gamma to increase neutrophil-mediated proteoglycan degradation did not require living chondrocytes. Adherence of neutrophils to HAGG-coated and uncoated cartilage explants was enhanced by IFN-gamma. Opsonisation of the cartilage with HAGG also increased the adhesion of neutrophils. In contrast to the enhancement of neutrophil-mediated proteoglycan degradation, IFN-gamma had no effect on the inhibition of proteoglycan synthesis by neutrophils.     

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.     

The influence of hydrocortisone on aggrecan metabolism in human articular chondrocyte cultures: comparison of two different matrices

OBJECTIVE: Numerous reports of negative effects as well as protective effects of glucocorticoids on articular cartilage convinced us to study the influence of hydrocortisone on aggrecan synthesis of isolated phenotypically stable human articular chondrocytes cultured in two different matrices. METHODS: Macroscopically normal human articular cartilage was obtained from femoral condyles within 24 hours postmortem. Chondrocytes were isolated and cultured in gelled agarose or in alginate. After 14 days in culture, hydrocortisone was added for 5 days at concentrations ranging from 0.005 microgram to 1 mg/ml for the agarose cultures and from 0.005 microgram to 1 microgram/ml for the alginate culture system. Aggrecan synthesis was measured by the incorporation of 35Sulphate, and the proportion of neosynthesized aggrecan that bound to hyaluronan to form aggrecan aggregates was analyzed by gel chromatography. RESULTS: At concentrations from 0.005 to 1 microgram/ml, hydrocortisone was found to produce a similar dose-dependent stimulation of aggrecan synthesis in both matrices. The synthesis of aggrecans remained at the same level for concentrations of 1 microgram/ml up to 100 micrograms/ml of hydrocortisone. When supraphysiological concentrations of hydrocortisone were added the aggrecan synthesis rate plateau declined. Simultaneously with the increase in aggrecan synthesis, the proportion of low-molecular weight 35S-proteoglycans decreased in favour of 35S-aggrecan aggregates and monomers in the agarose system. The chondrocytes cultured in alginate showed this increase of aggrecan aggregates and monomeric aggrecans in both the cell-associated and the inter-territorial matrix. CONCLUSION: Hydrocortisone is a stimulator of aggrecan synthesis by normal human articular chondrocytes cultured in vitro. The two culture systems (agarose and alginate) tested in this experiment showed a comparable aggrecan synthesis rate, increasing under the influence of hydrocortisone at concentrations up to 1 microgram/ml. The proportions of 35Sulphate incorporated in aggrecan aggregates and monomeric aggrecan were also higher under the influence of hydrocortisone.     

Proteoglycan metabolism in isolated chondrocytes from human cartilage and in short-term tissue-cultured human articular cartilage

The effect of piroxicam on proteoglycan metabolism of human cartilage cells was investigated in two in vitro models. Cells or tissue samples were obtained from six different donors. Piroxicam levels used in the test systems ranged from 2 to 6 micrograms r/ml and were comparable with serum concentrations in humans after oral intake. Piroxicam increased the synthesis rates of proteoglycan in some batches of isolated and monolayer-cultured chondrocytes and in tissue-cultured articular cartilage. The fact that this increase in the synthesis of proteoglycan was restricted to some of the donors whereas isolated cells or tissue samples from other individuals remained unaffected illustrates the heterogeneity of different human donors. Depression of proteoglycan synthesis in the presence of the drug was not observed.     

Pentosan polysulfate (Cartrophen) prevents the hydrocortisone induced loss of hyaluronic acid and proteoglycans from cartilage of rabbit joints as well as normalizes the keratan sulfate levels in their serum

The intraarticular injection of a sterile solution of 25 mg hydrocortisone succinate into rabbit knee joints once a week for 8 weeks reduced the levels of proteoglycans and hyaluronic acid (HA) in articular cartilage. In contrast, the keratan sulfate (KS) peptide levels present in sera of these animals were elevated relative to a saline treated control group. By injecting 25 mg hydrocortisone succinate combined with pentosan polysulfate (Cartrophen) (5 mg) into rabbit joints over the 8 week period, the loss of proteoglycans and HA from articular cartilage was abrogated and serum KS-peptides were restored, with time, to within control levels. These findings suggest that intraarticular administration of high dose hydrocortisone succinate to rabbits has a deleterious effect on articular cartilage HA and proteoglycan metabolism but this effect can be attenuated by co-intraarticular administration of Cartrophen.     

Basic fibroblast growth factor inhibits the anabolic activity of insulin‐like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

Objective

To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin‐like growth factor 1 (IGF‐1) and osteogenic protein 1 (OP‐1).

Methods

Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum‐free medium with or without IGF‐1 (100 ng/ml), OP‐1 (100 ng/ml), or bFGF (0–100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants.

Results

Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF‐1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF‐1 and OP‐1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF‐1 or OP‐1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF‐1 and OP‐1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures.

Conclusion

The anabolic activity of IGF‐1 and OP‐1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.

     

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.     

Thyroid hormone enhances aggrecanase-2/ADAM-TS5 expression and proteoglycan degradation in growth plate cartilage

Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T(4) reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T(3) also induced breakdown of proteoglycan. T(3) increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T(3) treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T(3)-exposed explants underwent cleavage at Glu(373)-Ala(374), the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T(3) was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T(3)-induced aggrecan degradation and found that T(3) enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.     

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.     

Effects of murine recombinant interleukin 1 on intact homologous articular cartilage: a quantitative and autoradiographic study.

Murine recombinant interleukin 1 (IL1) was tested for its ability to affect intact murine articular cartilage. IL1 caused enhanced proteoglycan degradation and severe inhibition of chondrocyte synthetic function at a concentration of 3 U/ml (100 pg/ml). Inhibition of proteoglycan synthesis appeared to be delayed in onset but occurred consistently after 24 hours. Pulse chase experiments made it clear that proteoglycan degradation and inhibition of proteoglycan synthesis are two distinct actions of IL1. No indications were obtained for selective degradation of either newly synthesised or processed proteoglycan. Moreover, chondrocyte synthetic activity appeared to be inhibited uniformly throughout the cartilage matrix, i.e., no evidence was found for selective suppression of cells in certain regions. IL1 uptake measurement in the cartilage, using [125I]IL1, yielded a partition coefficient far below 1, and autoradiography demonstrated a faint but even distribution within the cartilage matrix. The coordinated induction of enhanced breakdown of proteoglycan and inhibition of proteoglycan synthesis, with such low concentrations of IL1 reaching the chondrocytes, underlines the impressive destructive potential of IL1.     

Effect of homologous synovial membrane on adult human articular cartilage in organ culture, and failure to influence it with D-penicillamine.

Adult human articular cartilage has been maintained in organ culture for 8 days, and the culture medium, which was changed on alternate days, was pooled. Normal and rheumatoid cartilage was obtained from patients and 4 types of culture were prepared: (1) cartilage alone; (2) cartilage + D-penicillamine; (3) cartilage + homologous synovium; (4) cartilage, synovium, and D-penicillamine. The hexosamines and hexuronic acid were measured in the cartilage explants and in the medium. The quantity released was divided by the amount measured in the original cartilage explant and the different culture variables were compared. D-penicillamine did not alter the release of cartilage proteoglycan, but the addition of synovium did. The rheumatoid cartilage released significantly more proteoglycan than normal cartilage whether or not homologous synovium was present.     

Influence of oxygen tension on interleukin 1-induced peroxynitrite formation and matrix turnover in articular cartilage

OBJECTIVE: Osteoarthritis is characterized by the degradation of articular cartilage. The catabolic activity of chondrocytes is partly regulated by nitric oxide (NO), which with superoxide (O2-) leads to the formation of peroxynitrite (OONO-), a potentially damaging reactive species. Cartilage is avascular and functions at reduced oxygen tension. We investigated whether oxygen tension influences the effects of interleukin 1 (IL-1) on peroxynitrite formation and cartilage matrix metabolism. METHODS: Porcine cartilage explants were incubated at either 1% O2 or 20% O2 with either 1 ng/ml IL-1alpha, 25 microM MnTE-2-PyP5+ [Mn porphyrin-based catalytic antioxidant, Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin], or 1 ng/ml IL-1 + 25 microM MnTE-2-PyP5+ to decrease peroxynitrite formation. Nitrotyrosine, formed by nitration of tyrosine by peroxynitrite, was measured by immunoblot. Proteoglycan and collagen synthesis and proteoglycan degradation were also determined. RESULTS: IL-1-induced peroxynitrite formation was decreased in 1% O2 as compared to 20% O2. MnTE-2-PyP5+ inhibited IL-1-induced peroxynitrite formation in either 1% O2 or 20% O2. In 1% O2 (but not in 20% O2), Mn porphyrin significantly inhibited IL-1-induced proteoglycan degradation. IL-1 decreased both proteoglycan and collagen II synthesis in cartilage explants in 1% O2 or 20% O2, but MnTE-2-PyP5+ did not prevent these anti-anabolic effects. MnTE-2-PyP5+ alone caused a significant decrease in collagen synthesis at 20% O2 but not at 1% O2. CONCLUSION: Our findings show that oxygen tension alters IL-1-induced peroxynitrite formation, which can influence proteoglycan degradation. Oxygen tension may influence the effects of reactive oxygen and nitrogen species on matrix homeostasis.     

Mycobacterial antigens stimulate rheumatoid mononuclear cells to cartilage proteoglycan depletion

In a coculture with porcine articular cartilage explants unstimulated blood mononuclear cells (BMC) from patients with rheumatoid arthritis (RA), but not from healthy controls, induced proteoglycan depletion of dead cartilage. Specific stimulation of the RA BMC with Mycobacterium tuberculosis (MT), in comparison with concanavalin A (Con-A), strongly enhanced the proteoglycan depletion of living cartilage; this was not found with the BMC of healthy controls. However, the MT induced proliferative responses of the same BMC were similar in healthy controls and patients with RA. Neither the proliferative response nor the proteoglycan depletion was influenced by the presence of HLA-DR4 in the donor, whether patients with RA or healthy control. The proliferative responses of the RA BMC seemed to correlate inversely with the proteoglycan depletion. We conclude that stimulation of RA BMC with mycobacterial antigens may elicit effector pathways that induce proteoglycan depletion, independent of T cell proliferation.     

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.     

Biologic effects of an interleukin-1 receptor antagonist protein on interleukin-1-stimulated cartilage erosion and chondrocyte responsiveness

Recombinant human interleukin-1 alpha (IL-1 alpha) and recombinant human IL-1 beta stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-1 alpha and IL-1 beta in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-1 alpha to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-1-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.     

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase

OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.