Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus.

Neutral proteases and cathepsin D in human articular cartilage.

Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.

Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

The action of cathepsin D in human articular cartilage on proteoglycans.

In recent years the lysosomal cathepsins have been implicated as important agents in the physiological degradation of various cartilages. In the present study, the nature of cathepsin present in human articular cartilage was investigated by microtechniques and a possible role for cathepsins in the cartilage degradation observed in osteoarthritis was sought. The results of this study indicated that the hemoglobin and proteoglycan-digesting activity in the human cartilage observed is predominantly that of a cathepsin D-type enzyme. This cathepsin D-type enzyme activity was present in two to three times greater amounts in yellowish or ulcerated articular cartilage from patients with primary osteoarthritis than in control "normal" human cartilages. The human cathepsin D-type enzyme, as well as a highly purified cathepsin D from bovine uterus degraded proteoglycan subunit (PGS) maximally at pH 5. Both enzyme preparations were inactive on hemoglobin at pH 6-8, but degraded PGS considerably at neutral pH. The activity of the human cathepsin extract was not affected by reagents which inhibit or activate cathepsins A and B. Neutral proteases which are active on hemoglobin or are inhibited by diisopropylfluorophosphate (DFP) were not detected in these preparations, but contamination by another type of neutral protease cannot be excluded. Chloroquine inhibited the degradation of PGS at neutral pH by the human cartilage enzyme extract.

Neutral proteinases from articular chondrocytes in culture. 2. Metal-dependent latent neutral proteoglycanase, and inhibitory activity.

Monolayer and spinner cultured rabbit articular chondrocytes released into the medium latent metal-dependent enzyme with activity against bovine proteoglycan. Pretreatment of medium with p-aminophenylmercuric acetate or trypsin followed by soybean trypsin inhibitor significantly increased enzyme activity. The monolayer-cultured chondrocytes released more of this activity than spinner cultures. The neutral proteoglycanase activity increased with medium concentration and incubation time. Like the human cartilage proteoglycanase, its pH optimum on proteoglycan subunit was 7.25. Gel filtration on BioGel P-30 indicated that the proteoglycanase occurred in two molecular weight forms: 20 000--30 000 and 13 000. The latent enzyme was about 30 000--40 000. The metal-chelators, o-phenanthroline (5 mM) and EDTA (10 mM) inhibited the activated proteoglycanase almost completely, but trypsin and chymotrypsin inhibitors had little effect. The cultured chondrocytes also released into the media a heat-labile inhibitor against the proteoglycanase. The inhibitory activity was present in the nonactivated media and eluted on Sephadex G-100 chiefly at a position corresponding to molecular weights of 10 000--13 000.

Neutral proteinases from articular chondrocytes in culture. I. A latent collagenase that degrades human cartilage type II collagen.

Culture media collected from secondary monolayer and spinner cultures of rabbit articular chondrocytes showed evidence of collagenolytic activity by the following criteria: (1) Amicon PM-10 concentrates of culture medium released [14C] glycine from reconstituted rabbit skin collagen fibrils at 37 degrees C; (2) medium concentrated by lyophilization decreased the relative viscosity of human cartilage collagen in solution. The loss in viscosity was partially inhibited if medium was preincubated with o-phenanthroline, and (3) degradation of human cartilage collagen after 60 h incubation at 24 degrees C was characterized primarily by the appearance of 75 000 dalton (TCA) and 25 000 dalton ((TCB) products. The majority of the collagenase (EC 3.4.24.3) from cultured chondrocytes was secreted in latent form, since preincubation with either trypsin or p-aminophenylmercuric acetate significantly increased activity against human cartilage collagen. Chondrocyte collagenase may be important in mediating the normal slow turnover of cartilage collagen and may be particularly active in collagen destruction associated with early stages of synovial joint arthritides, before attack by non-cartilage cells or extra-articular soft tissues.

'Gelatinase-like' activity from articular chondrocytes in monolayer culture.

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TCA and TCB fragments of human Type II collagen into smaller peptides at 37 degrees C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1.10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.

Further characterization of a neutral metalloprotease isolated from human articular cartilage.

The neutral metalloprotease extracted from 1,200 gm of human articular cartilage was purified 1,400- to 2,400-fold by diethylaminoethyl- and carboxymethyl-Sephadex chromatography. Disc electrophoresis and an isoelectric focusing method resolved the neutral enzyme activity into 4 bands. All bands had a similar amino acid analysis and a similar molecular weight by sodium dodecylsulfate electrophoresis and gel filtration: 24,000-27,000 daltons. The enzyme degraded proteoglycan subunit and proteoglycan aggregate to products with a sedimentation coefficient of 3S, but at low dilutions the enzyme produced 19.3S fragments. It is postulated that this protease may contribute to the development of osteoarthritis from within the cartilage matrix.

Metal-dependent neutral proteoglycanase activity from monolayer-cultured lapine articular chondrocytes.

Neutral proteoglycanase and other protease activity from cellular and CM fractions of monolayer-cultured rabbit articular chondrocytes were studied. The cellular fraction comprising soluble cytoplasmic enzymes possessed concentration-dependent elastase-like esterase activity and activity against trypsin and chymotrypsin synthetic substrates but had little caseinase activity. The 20% ammonium sulfate precipitate of CM possessed more neutral caseinase activity than the 60% ammonium sulfate precipitate and the bulk of activity against the synthetic substrates. Activity against bovine nasal septum PG was present in these fractions. Both the 20% and 60% ammonium sulfate fractions reduced the viscosity and the S of the PG substrate. This activity was incompletely inhibited by preincubation with either 5 mM o-phenanthroline or 10 mM EDTA, indicating that it was paritally metal-dependent. The activity in the cellular fraction was also partially inhibited by o-phenanthroline but more so by EDTA. These data indicate that chondrocytes synthesize and secrete into the culture medium neutral proteoglycanase(s) capable of initiating degradation of PG derived from the neutral pH cartilage matrix. The inhibitory profiles, together with recent evidence of enzymes with similar activity extracted from cartilage suggested that the proteoglycanase enzyme(s) may occur in multiple forms.