Journal of Orthopaedic Research 8336-3344, Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society

Cathepsin B and Cysteine Protease Inhibitors in Human Osteoarthritis *J. Martel-Pelletier, TJ. M. Cloutier, and *4J. P. Pelletier *Department of Medicine, Universitk de Montrkal and Notre-Dame Hospital Research Center; ?Department of Surgery, Division of Orthopaedic Surgery, Universitk de Montrkal and Hbpital St. Luc; and $Rheumatic Diseases Unit, Universitk de Montrkal and Notre-Dame Hospital Research Center, Montreal, Canada

Summary: The aim of this study was to determine the involvement of cathepsin B and its inhibitors in the proteolytic degradation of human osteoarthritic (OA) tissue. The characteristics of the cathepsin B found in both normal and OA cartilage and synovium were similar to those of the lysosomal cathepsin B. Two inhibitors of cysteine proteases were found with a molecular weight of 67,000 and 16,000 Da. The cartilage cathepsin B level of OA specimens (54.8 f 7.3 units/yg of DNA) was greater than the controls (39.8 t 3.2 units/yg of DNA). Mild-moderate graded samples (78.1 2 12.0 unitdyg of DNA) had significantly higher levels of enzyme activity than the severely graded ones (31.4 f 3.9 unitdyg of DNA, p < 0.001) and controls (p < 0.01). Compared to controls (2.3 2 0.4 unils/rnig of tissue w.w.), cysteine protease inhibitory activity in OA cartilage was decreased in specimens with severe lesions (1.5 t 0.2 units/mg of tissue). This was particularly noted in patients who had not received steroid injections (1.2 2 0.3 units/mg of tissue, p < 0.05). In OA synovia, the cathepsin B level was greater (40.7 f 7.4 units/mg of tissue w.w., p < 0.02) than in the controls (13.6 2 3.7 units/mg of tissue). The cysteine protease inhibitory activity was similar in OA synovium (1.7 ? 0.2 units/mg of tissue w.w.) and in controls (1.5 ? 0.3 units/mg of tissue). This data demonstrated an imbalance between the levels of cathepsin B and cysteine protease inhibitors in OA tissue. A decrease of specific inhibitors could be an important contributing factor, particularly in more severe lesions. Key Words: Cathepsin B-Cysteine protease inhibitors-Cartilage-Synovial membraneOsteoarthritis.

Osteoarthritis (OA) is characterized by a progressive degradation of the major extracellular matrix macromolecules, collagen and proteoglycans, as shown by early ultrastructural and microscopic changes in the cartilage. These changes often occur in the pericellular area around the chondrocytes (22,27), where the matrix pH is in the acid range (10). At first, it was believed that cathepsin D was the prime instigator of degradation in this area (28);

however, in the OA process, this enzyme did not appear to play a role (13). Recent reports indicate that a metalloprotease with maximum activity at acid pH is likely to be involved in the degradation of OA matrix proteoglycans, particularly at the pericellular level (1,23). This metalloenzyme is synthesized as a proform and needs to be activated. Cathepsin B, a lysosomal enzyme, may also play a role in cartilage degradation. An increased level of this enzyme was reported in human degenerative cartilage (5). Cathepsin B belongs to the cysteine protease class, has a maximum activity at pH 6.0, and requires thiol as an activator. In addition to the direct degradative action of the enzyme on both col-

Received February 24, 1989; accepted July 28, 1989. Address correspondenceand reprint requests to Dr. J. MartelPelletier at Rheumatic Diseases Unit, Hapita1 Notre-Dame, 1560 Sherbrooke Street East, Montreal, Quebec, Canada, H2L 4K8.

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lagen and proteoglycans (9,26), cathepsin B may act as an activator of metalloproteases (1 1,29). The degradative action of cathepsin B on connective tissue is likely to occur both intra- and extracellularly. At neutral pH, the chondrocytes and synovial membrane cathepsin B can survive in their native form for a limited time (2,20), therefore exerting their proteolytic action before being inactivated. The proteolytic action of cathepsin B in human articular joints appears to be regulated by cysteine protease inhibitors (12,14). As such, the balance between the level of the enzyme and inhibitors could be of utmost importance in the OA degradative process. Therefore, the aim of this study was to determine the involvement of cathepsin B and cysteine protease inhibitors in the human OA process. First, the enzyme and inhibitors were purified from articular cartilage and synovial membrane and the activities of both were determined in each tissue. The data were in turn correlated to the severity of OA lesions. MATERIALS AND METHODS Materials

Iodoacetic acid amide was obtained from Eastman Kodak Co. (Rochester, NY, U.S.A.), CbzArg-Arg-4-O-MenA from Enzyme Systems Products (CA, U.S.A.), and DNA calf thymus type 1, collagenase, trypsin and fast garnet GBC base (4-amino-2',3-dimethylazobenzene)from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Mersalyl acid (2-{[3-(hydroxymercuri)-2-methoxypropyl] carbomoyl} phenoxyacetic acid) and sodium nitrite were acquired from Aldrich Chemicals (Milwaukee, WS, U.S.A.). Aquacid I1 was obtained from Behring Diagnostics (La Jolla, CA, U.S.A.); Sephadex G75 superfhe from Pharmacia (Uppsala, Sweden); pronase and papain from Bcehringer-Mannheim (Dorval, Quebec, Canada); and Dulbecco modified Eagle's medium (DMEM) and fetal calf serum (FCS) from Gibco (Grand Island, NY, U.S.A.). Other chemicals used were either certified by the American Chemical Society or were the best commercial grade available. Methods Specimen Selection

Articular cartilage and synovial membrane specimens were obtained from patients with primary osteoarthritis who underwent total knee replacement surgery at St. Luc and Notre-Dame Hospitals in

Montreal. The diagnostic criteria were based on clinical and radiological evaluations. At the time of hospitalization, all patients were taking nonsteroidal anti-inflammatory drugs. Cartilage and synovial membrane specimens from 19 OA patients (5 men and 14 women; mean age of 67 ? 7 years) and 8 controls (2 men and 6 women; mean age of 50 2 4 years) were analyzed to determine cathepsin B and cysteine protease inhibitor activities. Ten of the OA patients had received at least one intra-articular injection within 1 year of surgery. The specimens were washed in a cold physiological saline solution and immediately placed on ice for dissection. At the time of resection, cartilage from tibia1 plateaus and femoral condyles were tested for carbon black retention (19) on a scale of 0-4.Cartilage samples with no surface disruptions were graded 0, and those with the most severe lesions were graded 4. For each OA patient, blocks of cartilage were excised from the mildmoderate lesion, grades 1-2, and from the severe lesions, grades 3 4 . Samples of synovium from the peripatellar area were selected and the synovial lining was carefully dissected from underlying fibrous and fatty tissues under a microscope (Stereozoom; Bausch & Lomb, Rochester, NY, U.S.A.). A portion of each synovial membrane specimen was processed for histological grading as previously described (18). Specimens were fixed in 10% formalin, embedded in paraffin, and sections (6 pm) were cut and stained with hematoxylin and eosin. The severity of synovitis was graded on a scale of 0-10 (18). Briefly, two specimens from each knee were graded and an average of the two scores was taken. Three histological criteria were used: (a) synovial lining cell hyperplasia, graded 0 to 2 + ; (b) villous hyperplasia, graded 0 to 3 + ; and (c) mononuclear cell infiltration, graded 0 to 4 + and polymorphonuclear leukocytes, graded 0 (absent) to 1 + (present). The remaining tissue was dissected and used for biochemical studies. Control cartilage and synovium of age-matched patients with no history of joint disease were obtained during autopsy, within 12 h of death. Before the dissection, a macroscopic evaluation of each joint was performed (19). Only joints with macroscopically normal cartilage (grade 0) and synovial membrane were used for this study. Cathepsin B and Cysteine Protease Inhibitor Purifications Cell culture: Chondrocytes and synoviocytes were obtained from human OA and normal articular J Orthop Res, Vol. 8, No. 3 , 1990

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cartilage and synovial membrane. The cells were released within 1 h of enzymatic digestion with pronase (1 mg/ml) for cartilage or trypsin (2.5 mg/ml) for synovium, followed by 6 h with collagenase (4 mg/ml) at 37°C in DMEM containing L-glutamine and penicillin and streptomycin (50 U/ml, 50 pg/ml). The resulting solution was centrifuged (400 g for 10 min) and the cells in the pellet were seeded in tissue culture flasks (Gibco # 156502) containing DMEM supplemented with 10% FCS and antibiotics. Cultures were performed at 37°C in an atmosphere of 5% C0,/95% air. At confluency (8 days), the cells were trypsinized (trypsin-EDTA; 0.05%, 0.53 mM) in a sterile phosphate buffer solution. Isolated cells were seeded (medium cell density) in tissue culture flasks (Gibco #165250) containing DMEM and FCS and incubated for 5 days. The milieu was then replaced by DMEM without FCS and incubated for another 5 days. At the end of the incubation period, the latter milieu was concentrated with Amicon membrane YM5, and dialyzed against MES [2-(N-morpholino)ethanesulphonate]buffer (50 mM MES, 1M NaC1, 0.05% Brij-35 (polyethylene lauryl ether) at pH 6.0. The cells were trypsinized and homogenized in the MES buffer. Cathepsin B and cysteine protease inhibitors were purified using gel chromatography and electrophoresis techniques described below. Tissue extracts: Cathepsin B and cysteine protease inhibitors were extracted (2 g for each set of experiments) from both normal and OA cartilage and synovium. The tissue was first homogenized in 10 volumes of MES buffer (50 mM MES, 150 mM NaC1, 0.05% Brij-35) at pH 6.0, containing guanidine hydrochloride (2M for cartilage, 1M for synovial membrane). The suspension was extracted overnight at 4°C then centrifuged (30,OOOg for 20 min). The supernatant was ultrafiltered using an Amicon membrane XM100, concentrated on a YM5 membrane and dialyzed against MES buffer pH 6.0 containing 1M NaCl and 0.05% Brij-35. Chromatography: The concentrates were chromatographed on a Sephadex G-75 column (Pharmacia, 1.5 cm x 95 cm) and eluted in the MES buffer. Fractions were collected and assayed for cysteine inhibitory activity using cathepsin B and papain. The fractions containing the enzyme or the inhibitors were pooled separately, concentrated with Aquacid 11, and rechromatographed on Sephadex G-75 or Sephadex G-100 superfine columns (1.5 cm x 95 cm) in a 50 mM MES buffer, pH 6.0 containing 1M NaCl and 0.05% Brij-35. Fractions showing ac-

J Orthop Res, Vol. 8, No. 3, 1990

tivity were pooled and exhaustively dialyzed against the MES buffer before use. The Sephadex columns were calibrated with a mixture of Dextran blue 2,000 (void volume, Vo),bovine serum albumin (Mr 67 ,OOO), ovalbumin (Mr 43 ,OOO), chymotrypsinogen (Mr 25,000), ribonuclease A (Mr 13,700), and potassium ferricyanide (total volume, VJ .

Electrophoresis: Polyacrylamide gels (12%) were cast according to the Laemmli method (15) in slab gels (1 mm thick). Each sample was denatured at 100°C for 5 min in 10 mM Tris buffer containing 1 mM EDTA and 2.5% sodium dodecyl sulfate, 5% P-mercaptoethanol, and 12.5% glycerol. A trace of bromophenol blue was added before layering the samples on top of the gel. Electrophoresis was performed in a Tridglycine buffer, pH 8.3, containing 0.1% sodium dodecyl sulfate. Electrophoresis was run at 20 mA for 5 h. The gels were silver stained and calibrations were done using the Bio-Rad (BioRad Laboratories, Mississauga, Ontario, Canada) low molecular weight kit. Cathepsin B and Cysteine Protease Inhibitor Quantifications Enzyme assay: Cathepsin B activity was measured by the colorimetric method described by Barrett (4), using a synthetic substrate (Cbz-ArgArg4-0-MenA). The samples of cartilage and synovial membranes were cut and homogenized in MES buffer (50 mM MES, 150 mM NaCl, 0.05% Brij-35) at pH 6.0, containing 2 mM cysteine and 1 mM EDTA. Cbz-Arg-Arg4-0-MenA was used in a final concentration of 0.5 mM and was added as 1/40 of the assay volume in 5% dimethylsulfoxide. Aliquots of homogenate were distributed in test tubes along with 0.5 mM of the synthetic substrate. The blank tubes were constituted with the addition of 5 mM iodoacetamide. Each assay was performed in duplicate. After 3 h of incubation at 37"C, the test tubes were centrifuged (12,OOOg for 15 min). A color reagent, as described by Barrett (4), was added to the supernatant. This color solution was prepared at 4°C by mixing a fast garnet solution (4-amino2',3-dimethylazobenzene) in ethanol with sodium nitrite and a mersalyl (2-{[3-(hydroxymercuri)2-methoxypropyl] carbomoyl} phenoxyacetic acid) Brij-35 reagent. The colored coupling product was then allowed to develop for 10 min and read at 520 nm. The enzyme activity was expressed as units/pg of DNA for the cartilage and units/mg of tissue wet

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weight (w.w.) for the synovial membrane. One unit of cathepsin B hydrolyzed 1 p,g of substrate/h at 37°C. Cysteine protease inhibitors assay: The inhibitory activity in the tissue homogenates was determined after the destruction by heat of the endogenous cathepsin B. This method is based on the principle that the enzyme is heat labile (9), while, on the other hand, the inhibitors are stable at a temperature that destroys the enzyme (16,17). This was verified in preliminary experiments done with both purified cathepsin B and cysteine protease inhibitors from synovial membrane and cartilage. Curve responses were carried out by varying either the temperature or time, using different concentrations of purified enzymes and inhibitors. These experiments were also repeated using tissue homogenates. For convenience, an incubation period at 60°C for 20 min was chosen for the routine inhibitory assay. Each specimen, after being sliced and homogenized in the MES buffer as described above, was first heated (60°C for 20 min) to denature the endogenous cathepsin B, and then preincubated with purified synovial cathepsin B (35 units) (37°C for 30 min). Thirty-five units of cathepsin B is the amount needed to hydrolyze 70% of the substrate under the present experimental conditions. A blank tube with no cathepsin B was used for each experiment using the same procedure. After preincubation, 0.5 mM Cbz-Arg-Arg-4-0-MenA was added to each test tube and the mixture was incubated for 3 h at 37°C. This was followed by centrifugation (12,OOOg for 15 min) at 4°C. The resulting supernatant was used to determine enzyme activity, as described for the cathepsin B assay. For each experiment, appropriate controls for cathepsin B and the substrate were used. One unit of cathepsin B inhibitor was defined as the amount needed to block 1 unit of cathepsin B activity. Since the inhibitors were found extracellularly (see the Results section), their activity was expressed as units/mg of tissue wet weight. The activity of cysteine protease inhibitors during and after purification was tested for both cathepsin B (35 units) and papain (25 nglml). This was done following an incubation period of 3 h at 37°C for cathepsin B and 60°C for papain. D N A Assay

Tissue samples (15 mg wet weight) were homogenized in the MES buffer, and the DNA content

was determined by colorimetric estimation of deoxyribonucleic acid using Burton’s method (6). Statistical Analysis

The mean values and the standard errors of the mean for the resulting data were calculated and compared using the Student’s two-tailed t test. p values