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CALCIUM-DEPENDENT CYSTEINE PROTEINASE (CALPAIN) IN HUMAN ARTHRITIC SYNOVIAL JOINTS SHINGO YAMAMOTO, KATSUJI SHIMIZU, KAZUYA SHIMIZU, KIICHI SUZUKI, YASUAKI NAKAGAWA, and TAKAO YAMAMURO Objective. To study the roles of calpains in the synovial joint in rheumatoid arthritis (RA) and osteoarthritis (OA) and to verify the hypothesis that calpains present in the synovial fluid come from the synovium. Methods. We performed immunohistochemical, biochemical, and immunoblotting analyses for calpains in synovial tissues, synovial cell cultures, and synovial fluids. Results. Immunohistochemical staining of RA synovium demonstrated specific cytoplasmic staining of cells in the synovial lining layer, storomal fibroblasts, and endothelial cells. OA synovium showed almost the same intensity and distribution of calpain staining. DEAE-cellulose chromatography of RA and OA synovial extracts and synovial fluids showed a peak of caseinolytic activity attributable to calpain, as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anticalpain antibody from the calpain peak of RA and OA synovium and synovial fluid showed identity with the heavy subunit of calpain (80 kd), Similarly, calpain existed in the same form (80 kd) in conditioned media (supernatant) From the Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan. Shingo Yamamoto, MD: Graduate Student, Department of Orthopaedic Surgery; Katsuji Shimizu, MD: Lecturer, Department of Orthopaedic Surgery; Kazuya Shimizu, MD: Assistant Professor, Department of Orthopaedic Surgery; Kiichi Suzuki, MD: Research Fellow, Department of Orthopaedic Surgery; Yasuaki Nakagawa, MD: Graduate Student, Department of Orthopaedic Surgery; Takao Yamamuro, MD: Professor and Director, Department of Orthopaedic Surgery. Address reprint requests to Katsuji Shimizu, MD, Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, 54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606, Japan. Submitted for publication March 2, 1992; accepted in revised form June 2, 1992. Arthritis and Rheumatism, Vol. 35, No, 11 (November 1992)

obtained from synovial cell cultures, as well as in the synoviocytes. The total specific activity of the 2 calpains in the synovial fluid of RA patients was higher than that of calpastatin. Conclusion. The findings suggest that the extracellular appearance of calpains could be due to the secretion of these proteins from the synovial cells and that calpains may playa role in cartilage damage of RA and OA that occurs in synovial joints. The pathologic changes in both rheumatoid arthritis (RA) and osteoarthritis (OA) are associated with degradation of components of the extracellular matrix (I). Much study has been devoted to proteolytic enzymes, such as collagenase (2-4), stromelysin (5-7), plasminogen activator (8), and cathepsin B (9). These proteases are capable of degrading components of the connective tissue matrix and are thought to play an important role in cartilage destruction in RA (10). Calpain, also called calcium-activated neutral proteinase (CANP), is a calcium ion-dependent neutral cysteine proteinase. Two forms of calpain are now known to exist, and they differ in their Ca2+ requirements for activation: JL-calpain, or calpain I, requires low concentrations (micromolar) and m-calpain, or calpain II, requires high concentrations (millimolar) (11,12). Calpastatin is the natural specific inhibitor of calpains I and II (13). Calpains and calpastatin are present in many cells and tissues such as the brain, muscle, and submandibular gland (13,14). Originally thought to be an intracellular proteinase (13), recent studies have demonstrated the calpain-calpastatin system extracellularly, in the calcifying zone of growth cartilage (15) and in fracture callus of rats (16), as well as in OA and RA synovial fluid in humans (17,18).

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Our previous studies have shown that calpain degrades cartilage proteoglycan in vitro (15,19). It is possible that calpain functions as a matrix proteoglycanase, facilitating articular cartilage degradation in RA and OA, since its action is highly regulated by calcium ion concentration and it has enzymatic activity at neutral pH. In the present study, we sought to investigate the source of calpains in synovial fluid (extracellular component). Through immunohistochemical and biochemical analyses, calpains were demonstrated in the synovial joint and in cultured synovial cells.

PATIENTS AND METHODS Tissue specimens. Samples of synovium were obtained during therapeutic joint surgery (knee or hip) from patients with clinically and biologically defined RA or OA joint disease. The criteria used for the diagnosis of RA were those proposed by the American College of Rheumatology (formerly, the American Rheumatism Association) (20). Multiple pieces of synovium were obtained from each joint studied. Samples were selected randomly. No attempt was made to select only inflamed, erythematous tissue for analysis, nor was any attempt made to group RA or OA patients according to duration of disease, disease activity, or therapeutic regimen. Immunohistochemistry. Calpains I and II were purified from human red blood cells and porcine kidney, respectively, as previously described (21,22). Antisera were raised by repeated injections of rabbits with the respective heavy subunits of calpains I and II in Freund's complete adjuvant. Preparation of these antibodies has been described in detail elsewhere, and lack of immunologic cross-reactivity of the 2 antibodies has been demonstrated by immunoblotting (23). Tissue specimens were preserved in 10% formalin (FormaIde-Fresh; Fisher, Fairlawn, NJ). Specimens were embedded in paraffin and sectioned onto gelatin-coated microscope slides at a thickness of 6 ILm. Sections were stained by the peroxidase-anti-peroxidase (PAP) method (24). Details of the immunohistochemical studies are described elsewhere (15). Control specimens were obtained by incubating sections with normal rabbit serum or with the same anti-calpain I or anti-calpain II antibody after preincubation with purified human erythrocyte calpain I or porcine kidney calpain II instead of the first antibody. Positive control slides were cut and prepared in the same way from a rat submandibular gland, known to contain calpains I and II. Tissue preparation for biochemical study. Synovial lining tissue was carefully dissected away from surrounding fat, capsule, and subsynovium. All the following procedures were the same as previously reported (15). Briefly, the sample was added to a 3-fold volume of 20 mM Tris HCI buffer, pH 7.5, containing I mM EGTA, I mM EDTA, 5 mM 2-mercaptoethanol, and 0.25M sucrose. Samples were homogenized using a Polytron homogenizer (Kriens, Lucerne, Switzerland). The synovial homogenate was centrifuged at 105,000g for 60 minutes in a Hitachi 70P-72 ultracentrifuge

using a Hitachi SRP-70AT rotor (Hitachi, Ibaragi, Japan), and the supernatant was collected (crude synovial extract). The extract was dialyzed overnight against 20 mM Tris HCI buffer, pH 7.5, containing calpain inhibitors and 50 mM NaCl. Preparation of synovial fluid samples. Synovial fluid samples from 23 RA and 14 OA patients were aspirated with an 18-gauge needle. Only transparent synovial fluid was used. The sample volume ranged from 8.0 ml to 20.2 ml. Before biochemical analysis, all the samples were diluted 2-fold with concentrated Tris buffer, pH 7.5. The final buffer consisted of 50 mM Tris HCI, pH 7.5, with 50 mM NaCI, I mM EDT A, I mM EGT A, and 5 mM 2-mercaptoethanol (buffer A). Samples were then centrifuged at 2,000 revolutions per minute for 10 minutes, filtered through disc membranes (pore size 5IL diameter), and centrifuged again at 2,000 rpm for 10 minutes to remove tissue debris and cellular components. Only 0-2 cells/mrrr' remained in the samples (17,18). DEAE-cellulose chromatography. Diluted synovial fluid and dialyzed crude synovial extract was applied to a DEAE-cellulose column that had been equilibrated with buffer A and washed several times with the same buffer. The protein was then eluted with a linear gradient of 50-500 mM NaCI. The concentration of NaCI was monitored by measuring the conductivity of the eluted fractions. Assay for calpain. Calpain activity was determined as previously described (25,26), using casein as the substrate. The increase in trichloroacetic acid-soluble products was measured colorimetrically, by absorption at 750 nm (27). The caseinase reaction was performed on each chromatographic fraction (0.5-ml volume) in buffer A, pH 7.5, in the presence of 5 mM cysteine, 10 mM CaCl z , and 0.4% casein, in a total volume of 1.0 ml. One unit of cal pain was defined as the amount of enzyme that increased the absorbance by 1.0 unit after incubation at 30°C for 30 minutes. Calpastatin levels were assayed in each fraction by measuring the reduction of caseinase activity, using a fixed amount (usually 0.4 units) of purified human erythrocyte calpain I (21). One unit of calpastatin was defined as the amount that inhibited I unit of human erythrocyte calpain I in the caseinase assay. Samples were heated to 100°C for 5 minutes before assay to assure the heat stability of inhibitor activity, which is characteristic of calpastatin (13). Immunoblotting. Polyacrylamide slab gel electrophoresis (PAGE) in the presence of 0.1% sodium dodecyl sulfate (SDS) was performed with 10% resolving gels and 3% stacking gels. After gel electrophoresis, sample proteins were transferred to a nitrocellulose membrane, according to the method of Towbin et al (28). Nitrocellulose membranes were cut into strips and some of them were stained with 0.1% amide black in 40% methanolllO% acetic acid. The other strips were first incubated with anticalpain antibodies and then with a peroxidase-conjugated second antibody directed against the first antibody. Antigens were visualized by peroxidase staining according to the method of Hawkes et al (29) using DAB (3,3' -diaminobenzidine tetrahydrochloride) as substrate. Cell culture. Within 3 hours after surgery, synovial tissues were aseptically disaggregated and cultured according to the method of Hamerman et al (30). Briefly, the tissue

CALPAINS IN ARTHRITIC SYNOVIAL JOINTS

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Figure 1. Immunohistochemical localization of calpains I and II in human synovial tissues. Representative sections of formalin-fixed synovial tissue from patients with rheumatoid arthritis (A-F), and osteoarthritis (G and H) were stained with anti-calpain II (A and C), with anti-calpain I (E and G), or with non immunized rabbit serum (controls) (B, F, and H) and absorbed immune serum (control) (D), as described in Patients and Methods. Calpain-containing cells were found mainly in the synovial cell layer (A, E, and G), in perivascular distribution with endothelial and smooth muscle cells (C, E, and G). Intense brown cytoplasmic staining was absent from the control slides stained with nonimmunized rabbit serum (B, F, and H) and absorbed immune serum (D). Healthy, nonarthritic synovial membranes showed no calpain staining (I). (Aqueous hematoxylin counterstained, original magnification x 400.)

was cut into 2-mm pieces or smaller and incubated in 10-20 ml of serum-free Dulbecco's modified Eagle's medium (DMEM) containing 4 mg/ml of collagenase and 3 mg/IO ml of testicular hyaluronidase (Sigma, St. Louis, MO), 100units of penicillin and 100 J,Lg of streptomycin per ml, at 37°C, with rocking. After 3-4 hours, the suspension was centrifuged at 1,500 rpm for 5 minutes, and the cells were washed 3 times with medium containing 10% fetal bovine serum (FBS) and antibiotics, and then resuspended in the same medium for culture. After 24-48 hours, the nonadherent cells (red cells, lymphocytes) were removed; the adherent cells were then treated as primary cultures. Ten milliliters of the suspension was placed in a l00-mm tissue culture dish (Corning, Corning, NY) and incubated at 37°C under an atmosphere of 5% CO 2 , The medium was changed every 3-7 days until the cells

reached confluence (2-3 weeks). Cells at confluence were passaged using trypsin. For experiments, cells from the second to the eighth passage were plated in lOO-mm tissue culture dishes at a density of 4 x 105 cells in 10 ml of DMEM containing 10% FBS and antibiotics. The medium was changed daily until day 14. The supernatant was centrifuged at 1,500 rpm for 5 minutes, and 1.5 ml of the supernatant was used for lactate dehydrogenase (LDH) measurement and the rest (8.5 ml) was used for immunoblotting after application to a DEAEcellulose column and concentration using Centricon 10 devices (Amicon, Lexington, MA). For immunoblotting, synovial cell layers in culture were collected, suspended in SDS-PAGE sample buffer, and sonicated on ice. Measurement of LDH. Release of LDH into the culture medium has been shown to correlate with the rate of

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cellular breakdown during culture (31). Enzyme activity in the culture medium was determined at the end of each 24-hour period for 14 days, using the ultraviolet method described by Wroblewski et al (32). Statistical analysis. Data were compared using Student's r-test. P values less than 0.05 were considered significant.

RESULTS Immunohistochemical demonstration of calpain. Synovial membranes from RA patients contained cells positive for cytoplasmic staining with anti-calpain I and II. Calpains containing cells were found mainly in the synovial lining layer (which consists of fibroblastlike and monocytic elements) (Figures lA and E) and blood vessels, including the endothelial and smooth muscle cells (Figures IC and E). Some calpainpositive cells were also detected in the deeper interstitium, not only in cell aggregates, but also in interaggregate areas. Controls using nonimmunized rabbit serum and absorbed immune serum showed no staining (Figures lB, D, and F). Similarly, OA synovial membrane sections showed calpain-containing cells in the same distribution as in RA specimens (Figure IG). Controls using nonimmunized rabbit serum showed no staining (Figure IH). Most of the staining was observed in the areas of greatest cellularity, such as the lining cell layer. However, the extent and intensity of immunostaining were greater in RA than in OA samples. As a control, we used synovial tissues obtained from otherwise healthy individuals undergoing surgery for patellar subluxation, ligamental injuries, and meniscal tears. No cells in these healthy synovial membranes showed staining with the antibodies against calpains (Figure II). Immunolocalization of calpains in primary cultures of synovial cells from patients with RA and OA. Since the synthesis of calpains had not been demonstrated in fibroblast-like cells dissociated from the synovium of patients with RA and OA, we evaluated the intracellular localization of calpains in these dissociated synovial cells. Cultured, nonstimulated cells (passages 2-8) were fixed in paraformaldehyde, extracted with acetone to render the cell membrane permeable to antibodies, and subsequently incubated with anticalpain antibodies in the same manner as for the synovial tissues. Calpains were present in discrete areas of the cytoplasm; however, the location, size, and number of these areas varied considerably from cell to cell. In many cells, the cytoplasmic region

showed intense staining with the anticalpain antibodies (Figure 2). Calpain activity in synovial fluid. As the previous study showed (17), 2 positive caseinolytic peaks were seen at NaCl concentrations of 180 mM and 300 mM, as described for calpains I and II in other tissues. A negative peak was seen at an NaCI concentration of 120 mM; this corresponds to the elution pattern of calpastatin in other tissues. When the data for RA and OA synovial fluid were compared statistically, the following results were obtained. 1) In RA synovial fluid, calpain I activity was about 1.5 times greater than that in OA synovial fluid (P < 0.05). 2) RA synovial fluid contained about 3 times more calpain I than calpain II (P < 0.001). 3) The specific activity of total calpains exceeded that of calpastatin in RA (P < 0.05). 4) There was no significant difference in the calculated mean calpain 11- and calpastatin-specific activity between RA and OA synovial fluid (Table 1). Calpain activity in synovial tissue. Levels of calpains I and II and calpastatin were measured (expressed as units/gm tissue wet weight). In RA and OA synovial tissue, calpains I and II activities were lower, but calpastatin was higher, in RA tissue than in OA tissue. However, calpain levels exceeded calpastatin levels in both RA and OA tissues. When the data for RA and OA synovial tissue were compared statistically, there was no significant difference in the specific activity of the calpains and calpastatin between the 2 groups (Table 2). Immunoblotting of calpains from synovium and synovial fluid. The dialyzed crude synovial tissue sample and the synovial fluid eluates from the DEAEcellulose column (washed with 200 mM NaCI, was eluted stepwise with 400 mM NaCl, and concentrated in Centricon 10 devices) were subjected to SDSPAGE. The immunoblots with the anti-calpain II antibody revealed a single band of 80 kd, both in the dialyzed crude synovial tissue sample and in the synovial fluid concentrated eluate (Figure 3, lanes 7 and 8). This finding was consistent both in RA and OA samples. Although the dialyzed crude synovial sample showed a single band of calpain I at 80 kd, there was an obscure band just below 80 kd in the synovial fluid eluate (data not shown). Lactate dehydrogenase levels. High concentrations of LDH (mean ± SEM 76 ± 2 IV/liter, n = 3) were present in the culture medium after 24 hours. Thereafter, the rate of enzyme release declined and remained between 60 and 68 IV/liter between 2 and 10 days of culture. LDH levels in the medium of cultures

CALPAINS IN ARTHRITIC SYNOVIAL JOINTS

Figure 2. Calpains were present in rheumato id arthritis synovial cells in discrete areas of cytopl asm ; however, the location , size, and numbe r of these areas varied considerably among cells. In many cells, the cytoplasmic region showed inten se staining with the antic alpain antibodies (A); the staining was less intense with the normal rabbit serum (B). (Methyl green countersta ined , original magnification x 400.)

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Table 1.

YAMAMOTO ET AL

Levels of ca lpains I and II and calpastatin in synovia l fluid fro m patients with rheumatoi d arthriti s (RA) and osteoa rthritis (OA)' Calpain I

RA (n = 23) OA (n = 14)

0. 190 O. 120

~ ~

Calpains I and II

Calpain II

0.021t 0.024

0.068 ~ 0.019 0.081 ~ 0.021

0.258 0.201

~ ~

Calpastatin

0.03 ):j: 0.035

0.165 0.182

~ ~

0.020 0.029

• Cent rifuged , filtrated synov ial fluid (&-20.2 mil was applied to a 1.9 x 6.0-cm DEAE-cellul ose co lumn. Inh ibition and enzy me activities were calc ulate d by summing the ac tivity of eac h frac tion. Inhib ition activity was assayed against human er yth rocyte ca lpain I. Values are the mean ~ SEM specific ac tivity , in units/mI. t p < 0.05 versus OA ca lpain I, by Stude nt's r-test ; P < 0.001 versus RA cal pain II , by Stude nt's paired r-test. :j: P < 0.05 vers us RA ca lpas tatin, by Student ' s paired r-test,

without cells (containing 10% FBS ) remained between 62 and 65 IV/liter. This result showed that cells were viable until day 10. Immunoblotting of calpains from synovial cell cultures. Synovial cell samples and synovial cell cultu re supernatants eluted and concentrated as described above were subjected to SDS-PAGE. Both the synovia l cell samples and cell culture supernatants from RA and OA patients showe d single bands of 80 kd on immunoblotting with ant i-calpain II ant ibody until day 10 (Figure 3, lane s 2-5 ). When the immunoblotting was carried out using anti-calpain I antibody. a similar , but les s distinct , band below 80 kd was ob served in the blot s with cell culture supernatants (da ta not shown).

patients with OA. The patterns of staining of ca lpa ins I and II were similar, but was slightly darker, in the RA tissues than the OA tissue s. There was no positive staining of synovial tissues obtained from otherwise healthy individuals undergoing surgery for patellar subluxation , ligamental inj uries, and meniscal tears. The se results demonstrate the pre sence of calpains in synov ial lining cells from pat ients with inflamm atory synovitis, and the ir existe nce so clo se to the joint space may be responsible for their presence in sy novial fluid. We also showed the presence of calpain s in the endothelial cell s of some, but not all , blood vessels

1 2

1

DISCUSSION Calp ain s have generally been referred to as intrac ellular proteinase ; the y cat alyze selective and limited proteolytic modification of proteins (14). Our pre viou s study (17) demonstrated that synovial fluid from osteoarthritis patients contains extracellular calp ain s and that these calpains exert enzyme activity . To study the source of calpains in the synovial fluid of the hum an arthritic joint, we used anticalpain antibodies to identify calpains. Calpain s were found in the synovial lining cells both in pat ients with RA and in Table 2, Levels of ca lpains I and II and calpas tatin in synovial tissues fro m patient s with rheum atoid art hritis (RA) and osteoarthritis (OA)' Calpain I RA (n = 6) OA (n = 4)

Calpain II

1.18 ~ 0.48 0.60 1.40 ± 0.69 0.84

~ ~

0.27 0.43

Calpain s I and II

Calpastatin

1.78 ~ 0.72 1.25 ~ 0.60 2.24 ± 1.08 0.87 ± 0.51

• Dialyzed crude extract fro m 3-5 gm wet weight of sy novial tissue s was applied to a 1.9 x 3.G-cm DEA E-cellulose column, and the result s were de termined as described in Table I. Values are the mean ± SE M specific activity, in units/gm wet weight.

... • kd ... 1 kd ...

kd

... 0 kd

Figure 3. Immunoelect roph oretic blotting of ca lpain II fro m the synovial joi nt of a patient with rheumatoi d arthritis (RA) by anticalpain II antibody. Lane I, Control por cine kidne y calpain II (80 kd); lane 2, concentrated ca lpain II fraction fro m RA synovial ce ll culture (80 kd); lane 3, co nce ntra ted calpain II fraction from the medium (superna tant) of RA synov ial ce ll cultures (day 0--1) (80 kd); lane 4, medium (superna tant) fro m RA synovial cell cultures (da y 3-4) (80 kd); lane 5, medi um (superna tant) from RA synovial cell cultures (day 6-7) (80 kd). (The med ium was cha nged daily.) Lane 6, Concentrated cal pain II frac tion of med ium alone (co nta ining 10% fetal bovine seru m; no ca lpain II band) ; lane 7, ca lpain II fraction from RA synovial tissu es (80 kd); lane 8, concentra ted ca lpain II fraction from RA synovia l fluid (80 kd) .

CALPAINS IN ARTHRITIC SYNOVIAL JOINTS

in RA and OA synovial membranes. Immunohistochemical studies have localized calpains in rabbit organs and epithelial tissues, and calpains are thought to be involved in directional transport of substances in epithelial tissues through basal lamina (33). Most of the epithelial cells receive many stimuli from tissue fluid and blood, and respond to them. It is thus reasonable to assume that calpains are abundant in these cells. Conditioned medium from cultured synovial cells, as well as the synoviocytes themselves, obtained from RA and OA tissues showed a large subunit of calpain II (80 kd) by Western blotting until day 10 in culture. However, when anti-calpain I antibody was used, an obscure band just below 80 kd was identified in eluates from synovial cell cultures of RA and OA samples (data not shown). Although this calpain I band was obscure, calpains nevertheless existed. Release of LDH into the synovial cell culture medium was the same as in the control medium (containing 10% FBS) until day 10. This indicates that synovial cells are viable and that the calpains which appeared in the culture medium were not derived from destroyed cells, but from viable synovial cells. Media containing 1% FBS obtained from viable cultured synovial cells showed a faint single band of calpain II (80 kd), and media devoid of FBS obtained from these cells showed no calpain band. Thus, it may indicate that such substances as cytokines or growth factors are necessary for calpain to appear in the extracellular space. There are matrix proteinases such as collagenase that are secreted extracellularly from the Golgi apparatus and from lysosomes such as cathepsin D (34). It is well known that most secretory proteins have signal peptides that are a typical feature of secreted proteins that have been transported from an intracellular to an extracellular environment. Calpain is not a lysosomal enzyme, and there is not a signal peptide in calpain and calpastatin (35-40). Similar to interleukin-l, however, some proteins act extracellularly without signal peptides. How the interleukin-l protein is transported from an intracellular to an extracellular environment without a characteristic signal peptide is puzzling, but it is reported that interleukin-l simultaneously undergoes sequential enzymatic cleavage into progressively smaller peptides while it is secreted extracellularly (41). Our study showed that in the synovial cell culture, calpain exists extracellularly and intracellularly in the same form (80 kd). These data suggest that calpains may be released from syno-

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vial cells by a different mechanism than proteinases with signal peptides and interleukin-l. Human blood cells are known to contain calpain and calpastatin (42), and in inflammatory arthritis, these blood cells, lymphocytes and neutrophils in particular, appear in the synovial fluid. In the present study, by a low-rpm centrifugation and filtration step, contaminating cells in the synovial fluid samples were removed; the samples were examined microscopically and were determined to be free of cells (17,18). We therefore believe there was no possibility that the source of calpain and calpastatin in the synovial fluid samples might be contaminating cells. The specific activity of calpain I in RA synovial fluid was higher than that in OA synovial fluid, but both contained almost the same level of calpain II and calpastatin. However, the total specific activity of the calpains was higher than that of calpastatin in both RA and OA synovial fluid. In synovium, the specific activity of the calpains was lower, and that of calpastatin was higher, in RA than in OA. But the total specific activity of calpains exceeded that of calpastatin in both RA and OA. Although wide variation of the calpain-calpastatin system in various tissues has been well documented (12,43,44), this shows that in human arthritic joints, the calpains are more abundant than calpastatin. We demonstrated that by means ofimmunoblot analysis, the 80-kd heavy subunit of calpain II was found in synovial tissues and synovial fluid in both RA and OA. There was an obscure broad band (below 80 kd) of calpain I in both RA and OA synovial fluid. Although the smaller molecular mass of the calpain II heavy subunit has been reported in synovial fluid (17,18), this can be attributed to limited cleavage by some unidentified proteinases in the synovial fluid in vivo and/or in vitro during the preparation of the samples. In the synovial fluid, the Ca2+ concentration is in the millimolar range, and the pH is neutral (45), and the requirements for activation of the calpains are fully met. The active proteinase form of calpains (smaller molecular form) in the synovial fluid may be induced by some unidentified proteinases. Our preliminary immunohistochemical data concerning the presence of calpain in human arthritic articular cartilage showed variable results. Calpain activity in arthritic cartilage was very low compared with that in synovium (data not shown). These data suggest that the synovium is the major source of calpains in the synovial fluid (extracellular). In conclusion, the findings reported herein in-

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dicate that secretion from synovial cells may be the extracellular source of calpains, and calpains are more abundant than calpastatin in human arthritic synovial joints. Since the active form of calpains have a smaller molecular mass, calpains may playa role in cartilage damage with other proteinases in the synovial joint.

ACKNOWLEDGMENTS The authors wish to thank Michiko Veda for excellent technical assistance, and Greg Cossu, director of Bridges English Academy, for proofreading assistance.

II.

12.

13. 14. 15.

REFERENCES I. Krane SM, Conca W, Stephenson ML, Amento EP,

2.

3.

4.

5.

6.

7.

8.

9.

10.

Goldring MB: Mechanisms of matrix degradation in rheumatoid arthritis. Ann N Y Acad Sci 580:340-354, 1990 Evanson JM, Jeffrey JJ, Krane SM: Human collagenase: identification and characterization of an enzyme from rheumatoid synovium in culture. Science 158:499-502, 1967 Harris ED Jr, Cohen GL, Krane SM: Synovial collagenase: its presence in culture from joint disease of diverse etiology. Arthritis Rheum 12:92-102, 1969 Brinckerhoff CE, Auble DT: Regulation of collagenase gene expression in synovial fibroblasts. Ann N Y Acad Sci 580:355-374, 1990 Okada Y, Nagase H, Harris ED Jr: A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components: purification and characterization. J BioI Chern 261: 14245-14255, 1986 Wilhelm SM, Collier IE, Kronberger A, Eisen AZ, Marmer BL, Grant GA, Bauer EA, Goldberg GI: Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells. Proc Natl Acad Sci V S A 84:6725-6729, 1987 Case JP, Lafyatis R, Remmers EF, Kumkumian GK, Wilder RL: Transin/stromelysin expression in rheumatoid synovium: a transformation-associated metalloproteinase secreted by phenotypically invasive synoviocytes. Am J Pathol 135:1055-1064, 1989 Pelletier J-P, Mineau F, Faure M-P, Martel-Pelletier J: Imbalance between the mechanisms of activation and inhibition of metalloproteases in the early lesions of experimental osteoarthritis. Arthritis Rheum 33:14661476, 1990 Trabandt A, Gay RE, Fassbender H-G, Gay S: Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis. Arthritis Rheum 34: 1444-1451 , 1991 Harris ED Jr: Rheumatoid arthritis: pathophysiology

16.

17.

18.

19.

20.

21.

22.

23.

24.

and implications for therapy. N Engl J Med 322: 12771289, 1990 Mellgren RL: Canine cardiac calcium-dependent proteases: resolution of two forms with different requirements for calcium. FEBS Lett 109:129-133, 1980 Murachi T, Tanaka K, Hatanaka M, Murakami T: Intracellular Ca 2 + -dependent protease (calpain) and its high-molecular-weight endogenous inhibitor (calpastatin). Adv Enzyme Regul 19:407-424, 1981 Murachi T: Calpain and calpastatin. Trends Biochem Sci 8:167-169,1983 Pontremoli S, Melloni E: Extralysosomal protein degradation. Ann Rev Biochem 55:455-481, 1986 Shimizu K, Hamamoto T, Hamakubo T, Lee WJ, Suzuki K, Nakagawa Y, Murachi T, Yamamuro T: Immunohistochemical and biochemical demonstration of calciumdependent cysteine proteinase (calpain) in calcifying cartilage of rats. J Orthop Res 9:26-36, 1991 Nakagawa Y, Shimzu K, Hamamoto T, Suzuki K, Veda M, Yamamuro T: Calcium-dependent neutral proteinase (calpain) in fracture healing of rats. Submitted for publication Suzuki K, Shimizu K, Hamamoto T, Nakagawa Y, Hamakubo T, Yamamuro T: Biochemical demonstration of calpains and calpastatin in osteoarthritic synovial fluid. Arthritis Rheum 33:728-732, 1990 Fukui I, Tanaka K, Murachi T: Extracellular appearance of calpain and calpastatin in the synovial fluid of the knee joint. Biochem Biophys Res Commun 162:559566, 1989 Suzuki K, Shimizu K, Hamamoto T, Nakagawa Y, Hamakubo T, Yamamuro T: Characterization of prot eoglycan degradation by calpains. Biochem J (in press) Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp IT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988 Hatanaka M, Kikuchi T, Murachi T: Calpain I, a low Ca2+-requiring protease, from human erythrocytes: purification and subunit structure. Biomed Res 4:381-388, 1983 Kitahara A, Sasaki T, Kikuchi T, Yumoto N, Yoshimura N, Hatanaka M, Murachi T: Large-scale purification of porcine calpain I and calpain II and comparison of proteolytic fragments of their subunits. J Biochem (Tokyo) 95:1759-1766,1984 Yoshimura N, Ohtuki H, Hamakubo T, Kitahara A, Kannagi R, Murachi T: Distribution of calpain in various organs of the rat: an immunohistochemical study. Biomed Res 5:419-424, 1984 Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG: The unlabeled antibody enzyme method of immunohis-

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tochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333, 1970 25. Murachi T, Hatanaka M, Yasumoto Y, Nakayama N, Tanaka K: A quantitative distribution study on calpain and calpastatin in rat tissues and cells. Biochem Int 2:651-656, 1981 26. Murakami T, Hatanaka M, Murachi T: The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin). J Biochem 90:1809-1816, 1981 27. Ross E, Schatz G: Assay of protein in the presence of high concentrations of sulfhydryl compounds. Anal Biochern 54:304-306, 1973 28. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat! Acad Sci USA 76:4350-4354, 1979 29. Hawkes R, Niday E, Gordon J: A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochern 119:142-147, 1982 30. Hamerman D, Smith C, Keiser HD, Craig R: Glycosaminoglycans produced by human synovial cell cultures. Coil Relat Res 2:313-329, 1982 31. Rae T: Tolerance of mouse macrophages in vitro to barium sulfate used in orthopedic bone cement. J Biomed Mater Res 11:839-846, 1977 32. Wroblewski F, LaDue JS: Lactic dehydrogenase activity in blood. Proc Soc Exp BioI Med 90:210-213, 1955 33. Hayashi M, Kasai Y, Kawashima S: Preferential localization of calcium-activated neutral protease in epithelial tissues. Biochem Biophys Res Commun 148:567-574, 1987 34. Werb Z: Proteinases and matrix degradation, Textbook of Rheumatology. Third edition. Edited by WN Kelley, ED Harris Jr, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1989 35. Emori Y, Kawasaki H, Sugihara H, Imajoh S, Kawashima S, Suzuki K: Isolation and sequence analyses of

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cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease. J BioI Chern 261: 9465-9471, 1986 36. Emori Y, Kawasaki H, Imajoh S, Kawashima S, Suzuki K: Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease. J BioI Chern 261:9472-9476, 1986 37. Ohno S, Emori Y, Irnajoh S, Kawasaki H, Kisaragi M, Suzuki K: Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312:566-570, 1984 38. Sakihama T, Kakidani H, Zenita K, Yumoto N, Kikuchi T, Sasaki T, Kannagi R, Nakanishi S, Ohmori M, Takio K, Titani K, Murachi T: A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis. Proc Nat! Acad Sci USA 82:6075-6079, 1985 39. Suzuki K, Hayashi H, Hayashi T, Iwai K: Amino acid sequence around the active site cysteine residue of calcium-activated neutral protease (CANP). FEBS Lett 152:67-70, 1983 40. Suzuki K, Ohno S, Imajoh S, Emori Y, Kawasaki H: Identification and distribution of mRNA for calciumactivated neutral protease (CANP). Biomed Res 6:323327, 1985 41. Oppenheim JJ, Kovacs EJ, Matsushima K, Durum SK: There is more than one interleukin 1. Immunol Today 7:45-56, 1986 42. Takano E, Park YH, Kitahara A, Yamagata Y, Kannagi R, Murachi T: Distribution of calpains and calpastatin in human blood cells. Biochem Int 16:391-395, 1988 43. Murachi T, Hatanaka M, Hamakubo T: Calpains and neuropeptide metabolism, Neuropeptides and Their Peptidases. Edited by AJ Turner. Chichester, Ellis Horwood, 1987 44. Murachi T: Intracellular regulatory system involving calpain and calpastatin. Biochem Int 18:263-294, 1989 45. Ward TT, Steigbigel RT: Acidosis of synovial fluid correlates with synovial fluid leukocytosis. Am J Med 64:933-936, 1978