Enhanced neutral protease activity in proliferating rheumatoid synovial cells.
код для вставкиСкачать919 ENHANCED NEUTRAL PROTEASE ACTIVITY IN PROLIFERATING RHEUMATOID SYNOVIAL CELLS VICTOR B. HATCHER, JOANNE P. BORG, MINDY A. LEVITT, and CAROL SMITH Proteolytic enzymes associated with rheumatoid synovial cells (RSC) have been implicated in the degradation of articular cartilage. Proteases have been measured in proliferating rheumatoid synovial cells (PRSC), proliferating nonrheumatoid synovial cells (PNSC), and their nonproliferating counterparts (NPRSC and NP-NSC). The P-RSC and P-NSC exhibit enhanced total surface-associated plus secreted neutral protease activity, as compared with NP-RSC and NPNSC. The P-RSC exhibited significantly higher protease activity in this category compared to P-NSC. The RSC also secreted higher levels of secreted proteases alone compared to NSC. The secreted protease activity alone was not related to the proliferative state of the cells. Extractable protease activity was measured in early-passaged and serially-passaged P-RSC, NP-RSC, P-NSC, and NP-NSC. Extractable cellular protease activity measured at pH ranges from 5.0 through 8.0 was not significantly different between P-RSC and NP-RSC or between P-NSC and NP-NSC. The RSC contained elevated extractable cellular protease activity measured at pH ranges from 5.0 through 8.0 compared to extracts from later-passaged cells. The neutral protease activity in the early-passaged RSC was also higher than that From the Departments of Biochemistry and Medicine, Albert Einstein College of Medicine, Montefiore Hospital and Medical Center. Supported in part by the New York Arthritis Foundation, NIH grants AG 01732, HL 16387, and AM 15796, the New York Heart Association, and the Cystic Fibrosis Foundation. Victor B. Hatcher, PhD: Associate Professor, Department of Biochemistry and Medicine (recipient of New York Heart Senior Investigator Award); Joanne P. Borg: student, Tufts University, Boston, (recipient of summer fellowship, New York Arthritis Foundation); Mindy Levitt: Research Assistant, Montefiore Hospital and Medical Center, Bronx, New York; Carol Smith: Associate Professor, Department of Medicine. Address reprint requests to Victor B. Hatcher, PhD, Albert Einstein College of Medicine, Montefiore Hospital and Medical Center, 11 1 East 210th Street, Bronx, NY 10467. Submitted for publication August 25, 1980; accepted in revised form January 28, 1981. Arthritis and Rheumatism, Vol. 24,No. 7 (July 1981) measured at pH 6.0 or 8.0. In synovial cells cellular protease activity was related to the proliferative state and the origin of the cells. The P-RSC exhibited the highest levels of surface associated plus secreted neutral protease activity. The RSC also possessed the highest levels of extractable protease activity compared to NSC. The biological stimulus and the mechanism responsible for the degradation of cartilage in rheumatoid arthritis are not clearly understood. Hypertrophy of the synovial membrane can probably be correlated with articular cartilage destruction. Harris (1) has suggested that the cartilage is destroyed by enzymes released from cells of the advancing pannus. Granda et a1 (2) have reported a correlation between lysosomal cathepsin D in rheumatoid synovia and the amount of damage to articular cartilage. Poo!e et a1 (3) demonstrated the secretion of cathepsin D in synovial tissue removed from rheumatoid and traumatized joints. Collagenase has also been extensively investigated in synovial cells from rheumatoid synovium (48). Dayer et a1 (9) have shown that cells which adhere to tissue culture dishes following proteolytic dispersion of the rheumatoid synovium secrete large amounts of collagenase and prostaglandin E. The major source of collagenase was large stellate cells with many dendritic processes (10,ll). Collagenase and prostaglandin production were stimulated by a soluble factor released by the mononuclear cells (12,13). Monocyte-macrophages were primarily responsible for the production of the factor (14). Recently, Dayer et a1 (15) observed that adherent rheumatoid synovial cells which possess decreased collagenase activity following serial passage of the celIs could be stimulated by a soluble factor released in vitro from human blood monocyte-macrophages. Increased levels of the factor were produced by monocyte-macrophages stimulated by aggregated immunoglobulin, Fc fragment of the immunoglobulin, or concanavalin A. HATCHER ET AL 920 Neutral proteases have been investigated in synovial lining cells. Hams and Krane (16) isolated an endopeptidase from RSC which degraded gelatin to small fragments. Werb and Reynolds (17) have reported that endocytosis causes increased secretion of neutral proteinase from rabbit synovial fibroblasts. Werb et a1 (18) have also shown that synovial cell cultures produced plasminogen activator which can generate plasmin from plasminogen. Latent collagenase produced by rheumatoid synovium can be activated by plasmin. In this study, surface associated neutral protease plus secreted neutral protease, secreted neutral protease alone, and extractable cellular proteases assayed at pH values from 5.0 through 8.0 have been compared in proliferating versus nonproliferating and early-passage versus later-passage rheumatoid (RSC)and non-rheumatoid synovial cell (NCS)cultures. Several observations have now been made concerning each of these cell types. MATERIALS AND METHODS Synovial cell culture. Synovial cell cultures were prepared by using a procedure modified from Dayer et a1 (9). Rheumatoid cell cultures were isolated from samples of synovium obtained at synovectomy from patients with clinically defined rheumatoid arthritis. Nonrheumatoid cell cultures were obtained from samples of synovium from patients with osteoarthritic joint disease who required reconstructive procedures. Following collection of the specimens in Dulbecco modified Eagle's medium (DMEM), the lining cell layer was stripped off and minced into 1-2 mm pieces and placed in sterile flasks. The minced tissue was incubated for 3 4 hours on a Bellco rocker platform at 37OC in 50-100 ml of DMEM containing 1 mg/ml of collagenase (Sigma Type I, Saint Louis, MO, 125-250 units/ml) and 3-5 mg/100 ml of testicular hyaluronidase (Wyeth, personal supply) supplemented with 210 units/ml of penicillin and 80 pg/ml of streptomycin (Pfizer Laboratories, New York, NY). The Bask was rapidly agitated by hand for 5-10 minutes to break up any remaining pieces of tissue. The cells were centrifuged for 10 minutes at 500g, and the cell pellet was resuspended in DMEM containing 1Wo newborn calf serum (Gibco) and placed in flasks or in plastic Petri dishes (Falcon Plastics, Oxnard, CA). After overnight incubation, the floating cells were removed and the adherent cells were washed several times with phosphate buffered saline (PBS) and treated with 1 ml of 0.125% trypsin (weight/volume) (Gibco) containing 0.004M EDTA for several minutes in order to temove lymphocytes. The cells were then placed in DMEM containing 20% newborn calf serum. Primary cultures which were not passaged for longer than 2 weeks were placed in DMEM containing 5% newborn calf serum. Cultures were carried in DMEM containing 10% newborn calf serum and subcultured at a 1 :2 split ratio using 0.125% trypsin (Gibco), containing 0.004M EDTA. Cell counts were done in a hemocytometer after trypsinization. In experiments utilizing nonproliferating cells, cultures between passage 0-5 were used in which no increase in the cell counts was observed for at least 3 days following confluence. In experiments using proliferating cells, synovial cells between passages 0-5 were used in the rapid growth phase, usually 48 to 72 hours following subculture. Surface plus secreted protease activity. The surface protease determination was based on the measurement of trichloroacetic acid (TCA) soluble radioactive peptides released from 'H-labeled casein (19,20). The 'H-acetyl-casein assay is capable of detecting proteolysis produced by 50-100 pg of trypsin (2 1,22). The 'H-acetyl-casein, which was prepared by acetylating casein with 'H-acetic anhydride, had a specific activity of 3,840 cpm/pmol, assuming a molecular mass of 121,800 daltons. In order to measure protease activity bound to the cell surface, plus ptotease secreted into the medium, the cell cultures grown in 60 mm Falcon petri dishes were washed 6 times with DMEM without serum. The wash was removed and 300 11 'H-labeled casein (60 pg casein. 2.0 X lo6 cpm) which was previously dialyzed against serum-free medium was added to the plates together with fresh serum-free medium (600 1.11).The plates were then incubated for 180 minutes at 38°C under 7% CO, in room air. The reaction was terminated by the addition of unlabeled 3% casein (100 pl) dissolved in 1M KCl followed by chilled 6% TCA (200 pl). The mixture was allowed to remain overnight in an ice slurry after which the samples were centrifuged at 27,ooOg for 40 minutes, 200 pl of supernatant was added to 10 ml of Riafluor (New England Nuclear), and then the radioactivity was determined. In the measurement of surface plus secreted protease activity, plates without cells were used as negative controls. Control plates without cells were incubated for 25 hours in DMEM containing 10%newborn calf serum. The plates were washed 6 times with DMEM without serum. The medium was removed, and 300 pl 'H-labeled casein (6 pg casein, 2.0 X lo5 cpm) was added to the plates together with serum-free medium (600 pl). The plates were incubated, and the reaction was terminated as described for the plates containing cells. The TCA soluble counts measured on plates not containing cells were substrated from the TCA soluble counts in the cell cultures grown in Falcon petri dishes. Approximately 2.0% of the total radioactivity in the 'H-acetyl casein was TCA soluble following TCA precipitation on the control plates after incubation for 180 minutes. In the plates containing cells from 2.0-6.0% of the total 'H-acetyl casein, radioactivity was TCA soluble after incubation for 180 minutes. Following each experiment, the cells were trypsinized and counted with a hemocytometer. The viability of the cells was examined by eosin-y exclusion at the end of the experiment (21). Viable cells were observed as evaluated by eosin-y exclusion. In some experiments, the cells were trypsinized, washed 3 times, centrifuged, lysed, and the cell button counted in Riafluor. Less than 0.02% of the total amount of 'H-labeled casein was measured in the cell button. Secreted protease activity. In order to determine if neutral proteolytic activity was secreted during the assay, replicate plates containing serum-free medium (900 pl) were incubated for 180 minutes at 37OC under 7% CO, in room air. The medium was removed, centrifuged at 21,OOOg for 5 minutes, and an aliquot (100 pl) incubated for 120 minutes at 37°C with 'H-labeled casein (40 pl). The reaction was termi- NEUTRAL PROTEASE ACTIVITY nated with unlabeled 3%casein (100 pl) and 6% TCA (200 pl) under the conditions described above. An aliquot (100 p l ) was counted for radioactivity. Extractable cellular protease activity. NSC and RSC cultures were extracted by freezing and thawing (3 times) in I ml of 0.05Msodium phosphate buffer, pH 7.5, containing 1M KCI and 0.1% Triton X-100. After centrifugation at 26,OOOg for 60 minutes, the precipitate was removed and the supernatant was used for the determination of proteolytic activity. In the enzyme blank supernatant boiled for 1 minute was used. A 20-pl aliquot in triplicate of the supernatant was added to 20 p1 of buffer (1Mphosphate buffer, p H 5.0, 6.0, 7 . 0 or 8.0) in microcentrifuge tubes. The ’H-acetyl-casein (6 pg, 2 x 1CP cpm) was added in a volume of 20 pl, and the incubation was performed for 60 minutes at 4OOC. At the end of the incubation, 5 0 pl of the unlabeled cold casein (3% w/v in 5 0 m M sodium phosphate buffer, pH 7.0, containing 2M KCI) and 100 p1 of ice cold 6% TCA (w/v) were added. The contents of the tube were agitated vigorously on a Vortex mixer and incubated overnight in an ice slurry. Following centrifugation at 26.000g for 45 minutes, the top of each tube was sliced off and 100 pl of the supernatant was removed, added to 10 ml of Riafluor, and the radioactivity was determined. The standard Student’s t-test was used for comparison of means between the different groups (23). 921 Table 1. Surface plus secreted neutral protease activity in rheumatoid and nonrheumatoid synovial cells __ Synovial cells. ~ ~~~ Passage number Protease activity, units/l05 cellst 4 0 0.4 1.1 2.0 0.8 ~. ~ Nonrheumatoid Nonproliferating N3 N5 N6 N6 N8 N8 N 12 N 13 0 0 0 Mean f SD 0.5 f 0.7 Proliferating N3 N7 N8 N9 N 13 Mean SD Total Mean f SD * I 2 4 2 3 I .6 3.6 2.6 2.4 3.1 2.7 f 0.8$ 1.4 2 1.3 Rheumatoid Nonproliferating RA 4 0.1 RA4 1.8 I .o RA 5 RA 5 RESULTS RA6 RA 1 1 Surface plus secreted neutral protease activity. Proteolytic activity due to proteases associated with the cell surface, plus that secreted into the medium, were measured on proliferating rheumatoid synovial cells (PRSC), nonproliferating rheumatoid synovial cells (NPRSC), proliferating nonrheumatoid synovial cells (PNSC), and nonproliferating nonrheumatoid synovial cells (NP-NSC). The results are presented in Table 1. Significant differences were observed between proliferating and nonproliferating cultures and between the RSC and NSC derived cells. In both cell types, the proliferative state was associated with higher levels (P< 0.001, P < 0.01) of surface plus secreted proteases. This observation has been previously reported with other cell types (20,27). The NP-RSC contained significantly higher surface plus secreted protease activity compared t o NPNSC (P< 0.02). Higher levels of surface plus secreted protease activity were also observed on the P-RSC compared to the P-NSC (P < 0.05). The proliferating plus nonproliferating rheumatoid synovial cells exhlbited significantly higher surface plus secreted protease activity compared to the proliferating plus nonproliferating nonrheumatoid synovial cells (Pc 0.05). The proliferating cells (RSC plus NSC) also exhibited significantly higher surface plus secreted protease activity compared to the nonproliferating cells -~ RA 13 Mean f SD Prolijeraring RA4 KA5 RA 5 RA 6 RA 1 1 RA 13 * Mean SD Total Mean f SD 2.6 1.6 1.2 2.4 1.6 f 0.78 4.1 4.5 5.5 3.8 7.9 3.1 4.8 f 1.71 3.1 f 2,1# -. *N = nonrheumatoid adherent synovial cells; RA = rheumatoid ad- herent synovial cell. t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,ooO cpm of TCA soluble 3H-acetyl peptides after incubation for 180 minutes at 37°C. Each value represents mean value of protease activity from duplicate plates. $ Proliferating nonrheumatoid synovial cells significantly higher than nonproliferating nonrheumatoid synovial cells, P < 0.001. 5 Rheumatoid nonproliferating synovial cells significantly higher protease activity compared to nonproliferating nonrheumatoid synovial cells (P < 0.02). 7 Proliferating rheumatoid synovial cells significantly higher than nonproliferating rheumatoid synovial cells, P < 0.01. Proliferating rheumatoid synovial cells significantly higher than proliferating nonrheumatoid synovial cells, P < 0.05. tt Rheumatoid synovial cells significantly higher than nonrheumatoid synovial cells, P < 0.05. Proliferating rheumatoid synovial cells plus proliferating nonrheumatoid synovial cells exhibit significantly higher surface plus secreted protease activity compared to nonproliferating rheumatoid synovial cells plus nonproliferating nonrheumatoid synovial cells, P < 0.001. HATCHER ET AL 922 (RSC plus NSC) (P < 0.001). Surface plus secreted protease activity was not related to the passage number of the proliferating or nonproliferating cells, but was related only to the proliferative state and probably to the rheumatoid origin of the cells. That is, the cultures of PRSC contained the highest levels of surface plus secreted protease activity. Secreted protease activity. Secreted protease activity was examined in the RSC and NSC. Synovial cells were permitted to secrete proteases into serum-free culture medium for 180 minutes. The medium was removed, centrifuged, and assayed for secreted activity. In this assay, protease activity associated with the cell surface was not measured. No difference in the secreted protease activity was observed in proliferating and nonproliferating synovial cells in the limited number of cultures evaluated (Table 2). Secreted protease activity was observed in only one NSC line. In the rheumatoid cell lines, higher levels of secreted protease activity (P < 0.01) were observed compared to the 4 NSC lines. In comparing surface plus secreted protease activity (Table 1) with secreted protease activity alone (Table 2) in proliferating nonrheumatoid and rheumatoid cell lines, higher levels of protease activity were observed in the Table 2. Secreted neutral protease activity in rheumatoid and nonrheumatoid synovial cells ~ Passage number Synovial cells* Nonrheumatoid Proliferating Nonproliferating Proliferating Nonproliferating Proliferating Nonproliferating Mean f SD N6 N6 N8 N 12 N 13 N 13 Rheumatoid Proliferating Nonproliferating Proliferating Nonproliferating Nonproliferating Nonproliferating Nonproliferating Nonproliferating Nonproliferating Mean -+ SD RA 4 RA 4 RA 5 RA 5 RA 6 RA 10 RA I 1 RA 12 RA 13 ~ ~ _ Protease activity, units/105 cellst 0 0 I .8 0 0 0 0.3 k 0.7 2.8 2.3 3.2 I .7 2.9 1.9 I .4 0.8 1.5 2.1 f 0.84~~ * N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells. t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides after incubation for 120 minutes at 37°C. Protease activity represents mean values from duplicate plates. 4- Rheumatoid synovial cells exhibit significantly higher secreted protease activity compared to nonrheumatoid synovial cells, P < 0.01. Table 3. Extractable acidic protease activity in rheumatoid and nonrheumatoid synovial cells ~ Cell lines* Passage number ~ ~~ Protease activity, units/lO5 cellst - - - - _ _ _ ~ pH 5.0 pH 6.0 Nonrheumatoid N3 N5 N6 N8 Mean rt SD 36.5 43.2 15.9 18.5 28.5 f 13.4 26.6 20.4 11.4 14.4 18.2 f 6.7 N3 N5 N6 N8 Mean f SD Total Mean f SD 20.5 25.0 27.2 26.2 24.7 f 3.0 26.6 f 8.6 7.9 15.9 15.6 14.0 13.4 f 3.7 17.7 f 7.8 Rheumatoid RA 5 RA 5 RA 6 RA 10 RA 11 RA 13 Mean f SD 53.0 55.4 35.9 46.5 155.0 56.3 67.0 f 43.8 27.1 40.5 30.5 33.3 93.8 60.8 47.7 f 25.6 49.8 66.7 31.1 49.2 f 17.8 62.0 f 34.84- 27. I 47.1 22.4 32.2 13.1 48.5 f 28.68 RA 5 RA 5 RA 6 Mean f. SD Total Mean f SD 3 4 3 * ~ ~~~~ * N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells. _ _ t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 3H-acetyl peptides after incubation for 60 minutes at 40°C. Protease activity represents mean values from duplicate plates. 4- Rheumatoid synovial cells significantly higher than nonrheumatoid synovial cells, P < 0.05. § Rheumatoid synovial cells significantly higher than nonrheumatoid synovial cells, P < 0.02. experiments in which a combination of surface associated plus secreted protease activity was measured. Extractable protease activity. Proteases were also extracted from proliferating and nonproliferating RSC and NSC. No significant difference was observed in the amount of extractable protease activity measured at pH 5.0, 6.0, 7.0, and 8.0 between proliferating and nonproliferating cells of both types. Extractable proteolytic enzyme activity measured at pH 5.0 and pH 6.0 is shown in Table 3. No difference was observed in the proteolytic activity in the extracts at different passages in the limited number of cells examined. Rheumatoid cell extracts contained significantly higher protease activity at pH 5.0 (P< 0.05) and at pH 6.0 (P< 0.02). In comparing protease activity from early passage NSC ex- NEUTRAL PROTEASE ACTIVITY tracts at pH 5.0 and pH 6.0, no significant differences were observed. Extracts from NSC at passage 3,4, and 5 exhibited higher protease activity at pH 5.0 (P< 0.01) compared to activity at pH 6.0. Extractable neutral proteolytic activity in rheumatoid and nonrheumatoid synovial cells is shown in Table 4. At neutral pH, significant differences were observed between the passage number and the amount of protease activity. In the extracts from NSC, significantly (P< 0.05) higher amounts of neutral protease activity were observed at passage 0 and 1 compared with extracts from cells at passage 3, 4, and 5. In the RSC lines, significantly higher amounts (P < 0.02) of neutral protease activity were also present in the cell lines at passage 0 and 1 compared with extracts obtained from cells at passage number 3 and 4. The early passaged RSC extracts also contained higher levels of neutral protease activity compared with the earlypassaged NSC (P < 0.02). In comparing the neutral protease activity in the rheumatoid extracts from all cell lines with the extracts from all nonrheumatoid cell lines, the extracts from rheumatoid cell lines had significantly higher levels of neutral protease activity (P < 0.02). Synovial cells at passage 0 and 1 (RSC plus NSC) exhibited higher neutral protease activity compared to synovial cells at passage 3, 4, and 5 (RSC plus NSC) (P < 0.01). Nonrheumatoid early passage synovial cells contained higher protease activity at pH 7.0 (Table 4) compared to activity at pH 6.0 (P< 0.02) (Table 3) and at pH 8.0 (P c 0.02) (Table 5). Early passaged RSC had higher neutral protease activity (Table 4) compared to activity at pH 6.0 (P < 0.05) (Table 3). Protease activity was compared in extracts from RSC and NSC at pH 8.0 (Table 5). No significant difference was observed between the passage number and the protease activity measured at pH 8.0 in RSC and NSC. The RSC contained elevated cellular protease activity measured at pH 8.0 compared with the extracts obtained from the NSC (P< 0.02). The RSC at passage 0 and 1 contained higher protease activity at pH 8.0 compared to NSC at passage 0 and 1 (P < 0.05). The early passaged RSC exhibited significantly more extractable protease activity at pH 7.0 (Table 4) compared to early passaged RSC measured at pH 8.0 (P< 0.02) (Table 5). DISCUSSION The present investigation demonstrates that cultured adherent RSC possess higher levels of protease 923 Table 4. Extractable neutral protease activity in rheumatoid and nonrheumatoid synovial cells -_ -. Cell lines* Passage number Protease activity, units/ lo5 cells, pH 7.0t Nonrheumatoid N3 N5 N6 N8 Mean f SD 31.2 37.0 28.1 31.1 3 1.9 & 3.7$ N3 N5 N7 N8 Mean f SD Total Mean f SD 27.1 14.7 26.0 16.5 21.1 f 6.4 26.5 7.5 Rheumatoid RA 4 RA 5 RA 5 RA 6 RA 7 RA 8 RA 10 RA 1 1 RA 13 Mean f SD RA 4 RA 5 RA 5 RA 6 Mean f SD Total Mean 2 SD * 53.6 39. I 76.7 77.2 75.4 83.9 44.1 120.3 47.0 68.6 f 25.69 26.0 39.1 24.1 26.0 28.8 f 6.9 56.3 -c 28.61 * N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells. t One unit of protease activity is defined as the amount of protease activity which soluhilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides after incubation for 60 minutes at 40°C. Mean values from duplicate plates. $Nonrheumatoid synovial cells at passage 0 and 1 contain significantly higher neutral protease compared to nonrheumatoid synovial cells at passage 3, 4, and 5, P < 0.05. 5 Rheumatoid synovial cells at passage 0 and I contain significantly higher neutral protease activity compared to nonrheumatoid synovial cells at passage 0 and 1, P < 0.02. Rheumatoid synovial cells at passage 0 and 1 contain significantly higher neutral protease compared to rheumatoid synovial cells at passage 3 and 4, P < 0.02. 1Neutral protease activity from rheumatoid synovial cells significantly higher compared to neutral protease from nonrheumatoid synovial cells, P < 0.02. Neutral protease activity from nonrheumatoid synovial cells plus rheumatoid synovial cells at passage 0 and 1 significantly higher than neutral protease activity from nonrheumatoid synovial cells at passage 3 and 4 plus rheumatoid synovial cells at passage 3 , 4 , and 5, P < 0.01. activity compared to NSC. P-RSC have elevated surface associated plus secreted protease activity compared to NP-RSC and P-NSC. Several investigators have reported a relationship between cell proliferation and cellular protease activity. Chou et a1 (24) reported that the HATCHER ET AL 924 Table 5. Protease activity a t pR 8 . 0 i n rheumatoid and nonrheumatoid synovial cells Cell lines. Passage number Protease activity, units/105 cells, pH 8.0t Nonrhcumatoid N3 N5 N6 N8 N9 Mean f SD 15.2 12.6 25.8 28.7 22.5 21.0 f 6.9 N3 N5 N9 N 10 N I1 Mean f SD Total Mean +- SD 19.1 11.6 26.3 19.7 22.5 20.0 f 5.4 20.5 f 5.8 Rheumatoid RA 4 RA 5 RA 5 RA 6 RA I RA 9 RA 10 RA 13 Mean SD * RA 4 RA 5 RA 5 RA 6 Mean f SD Total Mean f SD 0 0 1 0 0 1 I I 4 3 4 3 21.5 39.1 34.5 69.9 37.1 41.9 36.1 25. I 38.3 f 14.6* 19.7 36.1 28.1 21.1 26.4 f 7.8 34.3 f 13.68 -- N = Nonrheumatoid synovial cells; RA = rheumatoid synovial Cells. t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides after incubation for 60 minutes at 40°C. Mean values from duplicate plates. $ Rheumatoid synovial cells at passage 0 and I contain higher protease activity compared to nonrheumatoid synovial cells at passage 0 and 1, P < 0.05. 8 Rheumatoid synovial cells contain higher protease activity compared to nonrheumatoid synovial cells, P < 0.02. maximal secretion of plasminogen activator occurs when Swiss 3T3 cells were proliferating. Rohrlick and Rifkin (25) reported that cultured low passaged embryo fibroblasts elaborate increasing plasminogen activator activity before they reach confluence. Bauer (26) observed the maximal secreted collagenase activity shortly after the skin fibroblasts reached confluence. The secreted collagenase activity decreased in post-confluent cells. Surface associated neutral protease activity has also been observed in a large number of proliferating cells (20). The maximal surface protease activity was observed just before, during, or just after mitosis. Nonproliferating cells have decreased surface associated neutral protease activity (27,28). Cartilage derived factors and heparin which inhibit cell proliferation also inhibit surface associated protease activity (29). In the present study, it was not possible to differentiate surface associated protease activity from secreted protease activity, since RSC and some NSC secrete protease into the medium under experimental conditions in which surface protease activity has been determined in previous studies (20,27). Although RSC secrete higher amounts of proteases into the medium than do NSC, no differences in secreted protease activity alone were observed between proliferating and nonproliferating cells (Table 2). significant differences in protease activity were observed between proliferating and nonproliferating cells in experiments in which a combination of surface associated plus secreted protease activity was measured (Table 1). T h e results from previous experiments (20,27,29), together with the results presented on Table 1 and Table 2, suggest that proteases associated with the cell may be responsible for part of the protease activity in P-RSC and P-NSC. The functional role of proteases associated with the membrane is not known. One could speculate that during proliferation, membrane proteases are responsible for the degradation of specific proteins in the extracellular matrix. During proliferation, specific membrane proteins could also be degraded by membrane proteases. Linsley et a1 (30) have reported that epidermal growth factor (EGF) receptor complex binding undergoes proteolytic modifications in murine 3T3 cells and human foreskin fibroblasts. Two distinct proteases, one of unknown specificity and one similar to trypsin, seem to be involved in the cleavages of EFG-receptor complexes (31). Recently, Bach et a1 (32) suggested that plasma membrane proteases control the binding of IgE to specific receptors in rat peritoneal mast cells. It is not understood why P-RSC possess higher surface plus secreted protease activity than do P-NSC. Adherent RSC dispersed by proteolysis of rheumatoid synovium consist of at least two cell types (9-12). The majority of the population are large stellate cells with many dendritic processes lacking macrophage markers. Small adherent monocyte macrophages with receptors for the Fc portion of the immunoglobulin constitute the second type of adherent cell in primary cultures. Serial passaged synovial cells have less than 2% of the small adherent cells. Primary cultures of NSC have fewer small adher- 925 NEUTRAL PROTEASE ACTIVITY ent cells compared to primary adherent RSC (unpublished results). No significant difference was observed in surface plus secreted protease activity between the primary cultures containing both types of cells and the serial passaged cells consisting of mainly large stellate cells. The enhanced levels of surface plus secreted protease activity in the proliferating rheumatoid and nonrheumatoid cells were not related to the presence of the small adherent cells. It was not possible to determine if different populations of stellate cells exist between the rheumatoid and nonrheumatoid cells. Proteolytic enzymes were extracted from proliferating and nonproliferating synovial cells. No significant difference was observed between the extracted protease activity in proliferating or nonproliferating adherent synovial cells. Rheumatoid synovial cell extracts contained elevated protease activity measured at pH 5.0, 6.0, 7.0, and 8.0. Lysosomal enzymes have been proposed in the destruction of cartilage matrix (33,34). The protease activity measured at pH 5.0 and 6.0 are probably due to lysosomal enzymes (33) since lysosomal enzymes are increased in rheumatoid synovia (34). Neutral proteases have also been implicated in the degradative process of articular cartilage (16- 18). Werb et a1 (35) isolated a neutral metalloprotein protease from rabbit synovial fibroblasts which degrade bovine nasal proteoglycan and azo-casein. In the present study, human synovial adherent cells contain large amounts of extractable neutral protease when assayed with 'H-acetyl casein. The amount of neutral protease was related to the passage number of the cells. Nonrheumatoid cells at passage 0 and 1 contain higher neutral protease activity compared to nonrheumatoid cells at passage 3, 4, and 5. Rheumatoid early passaged cells also exhibited enhanced neutral protease activity. The differences in extractable protease activity in the early passaged cells compared to serially passaged cells are probably due to the presence of different types of cells. Primary cells contain both large stellate cells and small monocyte macrophages, while the serially passaged cells contain mainly large stellate cells (9-12). In nonrheumatoid cells, difference in extractable protease activity between pH 5.0 and 6.0 was only observed in serially passaged cells. Enhanced protease activity at pH 7.0 compared to pH 6.0 and 8.0 was observed in primary and first passaged cells. The differences in protease activity measured at neutral pH were not seen in synovial cells at passage 3, 4, and 5. The results suggest that the monocyte-macrophages contribute either directly or indirectly to the enhanced neutral protease levels in extracts of rheumatoid and nonrheumatoid cells. 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