Rheumatn!OUY

Rheumatol Int (1992) 12: 141-!46

Clinical and Experimental Investigations

9 Springer-Verlag t992

Originals Damage of cultured chondrocytes by hydrogen peroxide derived from polymorphonuclear leukocytes: a possible mechanism of cartilage degradation R. Saura 1, I". Matsubara a, K. Hirohata

1, and H.

Itoh 2

x Department of Orthopaedic Surgery, School of Medicine, Kobe University, Hyogo 650, Japan 2 Department of Pathology, School of Medicine, Kobe University, Hyogo 650, Japan Received September 27, 1991/Accepted May 18, 1992

Summary. To study the mechanisms o f c h o n d r o c y t e damage, chondrocyte cytotoxicity as shown by chromium-51 release induced by polymorphonuclear leukocytes (PMNLs) was examined. P M N L s significantly enhanced chondrocyte cytotoxicity in the presence of phorbol dibutyrate. This chondrocyte damage was abolished by the addition of catalase, whereas superoxide dismutase and scavengers of hydroxyl radicals and protease inhibitors failed to reverse it. When cartilage matrix components such as hyaluronic acid and various proteoglycans were added to the P M N L - c h o n d r o c y t e cultures, these components failed to affect the chromium-51 release. These results suggest that the increase in chondrocyte cytotoxicity is due to hydrogen peroxide generated by the P M N L s , and that cartilage matrix components do not prevent it. H y d r o g e n peroxide from P M N L s m a y therefore play an important role in cartilage degradation through direct damage of chondrocytes during inflammatory process.

Key words: Chondrocyte - H y d r o g e n peroxide - Polymorphonuclear leukocytes - Cell damage - Inflammation

Introduction Cartilage is composed histologically of chondrocytes (cellular components) and extracellular matrix, which consists of proteoglycans (PGs), link protein, hyaluronic acid (HA), and various collagens. Cartilage matrix in inflammatory sites is degraded by a variety of proteases derived from inflammatory cells such as p o l y m o r p h o n u clear leukocytes (PMNLs) and macrophages [1 -3]. It has also been reported that activated P M N L s produce superoxide anion ( 0 2 ) during respiratory burst [4, 5], and that hydrogen peroxide (H202) and hydroxyl radicals (OH.) converted from 0 2 can alter the functional properties of cartilage matrix [6-8], which leads to matrix degrada-

Correspondence to: T. Matsubara

tion. Although m a n y reports have suggested mechanisms of cartilage matrix degradation in the inflammatory process [2, 9-12], the mechanisms of cellular damage in inflammatory cartilage is not well understood. To study t h e mechanisms of chondrocyte damage in inflammation we studied the chondrocyte cytotoxicity in P M N L - c h o n d r o cyte interaction. The possible effects of various cartilage matrix glycoproteins on P M N L - i n d u c e d chondrocyte damage were also investigated.

Materials and methods

Preparation of chondrocytes Chondrocytes were obtained from costal cartilage of 3-week-old rats or adult porcine hind legs as described previously [13] with some modification. The cartilage was diced with a surgical blade. Small pieces of the obtained cartilage were pretreated with 0.05% hyaluronidase (Sigma Chemical, St. Louis, Mo, USA) in Dulbecco's phosphate-buffered solution (D-PBS) for 3 min at 37 ~ The pellet was then resuspended in 5 ml 0.2% collagenase (Washington Biochemical, Freehold, N J, USA) and incubated for 30 rain at 37 ~ Supernate was aspirated and placed into a 50-ml plastic tube with adequate Hank's balanced salt solution (HBBS) containing 40% heat-inactivated fetal calf serum (FCS; M. A. Bioproducts, Walkersville, Mass., USA) to stop the collagenase digestion. This procedure was repeated five times. The solution containing residual tissue fragments was filtered through platinum mesh. The filtered solution was centrifuged at 735 g for 5 min, and the pellet was washed with HBBS three times. After washing the pellet was resuspended in the complete medium, Ham F-12 (Flow Laboratories, Irvine, UK) containing 10% heat-inactivated FCS and antibiotics (20 U/ml penicillin and 100 gg/ml streptomycin). Chondrocytes were cultured overnight in 75 cm2 tissue culture flasks (Corning Glass Works, Corning, NY, USA) at 37 ~ in 5% CO 2, 95% air). The culture flasks were rinsed vigorously to remove nonadherent cells, and fresh complete medium was added. When the primary culture reached confluency, the chondrocytes were trypsinized, resuspended in complete medium, and seeded into flasks coated with gelatin (DIFCO Laboratories, Detroit, Mich., USA) for further passage. The chondrocytes were then used in the second or third passage for the experiments described below. The identity of the chondrocytes was established by their ability to synthesize glycosaminoglycans (GAGs) as measured by the up-

142 take of 35804 (New England Nuclear, Boston, Mass., USA) into the synthesized GAGs, characteristic morphology under phase-contrast microscope [t 4], and indirect immunostaining with rabbit antiS-100 antibody, which is reported to be specific for chondrocytes or nerve cells (Wako Pure Chemical, Osaka, Japan) by peroxidase-antiperoxidase method, with over 90% of the cells positively stained. Chondrocytes were detached from confluent monolayer by tripsinization; cell viability was 96%-98% as determined by trypan blue exclusion. Further cultures were carried out in triplicate in sterile, gelatin-coated, flat-bottomed microtiter wells (6.4 mm in diameter; Coming). Each well contained 2 x 104 responding cells in 0.2 ml complete culture medium, consisting of Ham F-t2 containing 10% heat-inactivated FCS and antibiotics. This medium was used in all chondrocyte cytotoxicity assays unless otherwise stated. The microtiter wells were than placed in a CO 2 incubator overnight to obtain chondrocyte monolayers.

Preparation of PMNLs PMNLs were obtained from heparinized venous blood from healthy human donors. PMNLs were isolated using standard techniques of sequential Ficoll-Hypaque density centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes as previously described [15]. The preparation comprised over 95% PMNLs as determined by Wright's stain, and over 98% were determined viable by trypan blue exclusion. Purified PMNLs were suspended in RPMI 1640 (Flow Laboratories, Irvine, UK) containing 10% heatinactivated FCS and antibiotics for further studies.

Assay for chondrocyte cytotoxicity The PMNL-induced cytotoxicity of chondrocytes was determined in a manner similar to that of fibroblasts [16] and endothelial cells [17, 18]. The chondrocytes for SlCr labeling were grown to confluence in the flat-bottomed microtiter wells (6.4 mm in diameter) and then incubated in 0.2 ml complete culture medium containing 4 gCi Na 2 [51Cr] 0 4 (New England Nuclear) for 24 h. The plates were then washed with D-PBS and used immediately for the cytotoxicity studies. S*Cr-labeled chondrocytes (20000) were cultured with various numbers of PMNLs in RPMI 1640 containing 10% heat-inactivated FCS in the presence or absence of phorbol dibutyrate (PDB; Sigma), concanavalin A (con A; Sigma) or heat-aggregated IgG (HAG) made from human IgG (Sigma) by heat treatment (63 ~ 30 rain). Assays were performed in triplicate, and the final volume of the reaction mixture was 0.2 ml. Experiments without PMNLs were performed in an identical manner, in which the supernatant of the PMNLs preincubated with PDB for 30 rain was used in place of the activated ceils. In the inhibitor experiments bovine catalase (25 000 U/mg), various concentrations of mannitol (Man), 2-mercaptoethanol (2-ME), cq-antitrypsin (cqAT), or ~2-macroglobulin (~2-MG; Sigma) was added to the PMNL-chondrocyte cultures simultaneously with PDB. To study the effects of cartilage matrix components on PMNLinduced chondrocyte cytotoxicity, various GAGs such as HA with various molecular weights, PGs (Seikagaku, Tokyo, Japan), keratan sulfate (Sigma), chondroitin sulfate (Sigma) were used in place of the above inhibitors. After incubation in a humidified CO 2 incubator (5% CO 2, 95% air) at 37 ~ for 24 h supernatant was removed to determine the release of SlCr, which was measured in a gamma-counter (Model 1260 Multigamma II, Pharmacia, LKB Biotechnology, Uppsala, Sweden). Cytotoxicity (specific release of 51Cr) was expressed as the percentage of 51Cr release calculated by the following formula, as described previously [18]: specific release of 51Cr = (A -- B)/(C-- B) x 100 (%), where A is the experimental release, determined by the measurement of 51Cr released into the supernatant of activated PMNLs and chondrocytes; B is the spontaneous release, the mean cpm in the supernate of the samples containing only chondrocytes; and C is the total release, determined by measurement of 51Cr

released into the supernatant of chondrocytes treated with 0.2% Triton X-100 (Wako Pure Chemical Industries).

Results

PMNL-mediated chondrocyte cytotoxieity C h o n d r o c y t e s f r o m r a t costal c a r t i l a g e were c u l t u r e d w i t h P M N L s in the presence o r a b s e n c e o f 10 n g / m l P D B . A u g m e n t a t i o n o f the specific release o f 51Cr was o b s e r v e d in the increasing n u m b e r o f P D B - a c t i v a t e d P M N L s . A significant increase in specific release o v e r the c o n t r o l level ( P < 0 . 0 1 ) was o b t a i n e d at the r a t i o o f 1:4 (chondrocytes:PMNLs). Human dermal fibroblasts (Det r o i t 548, I C N M e d i c a l s , M c L e a n , Va) were also d a m a g e d b y P M N L s in the presence o f P D B . P M N L s in the a b s e n c e o f P D B d i d n o t significantly increase the release o f 51Cr f r o m either cell type (Fig. 1). W h e n the l a b e l e d c h o n d r o c y t e s were c u l t u r e d w i t h P M N L s in the presence o f P D B at the r a t i o o f 1:4 ( c h o n d r o c y t e s : P M N L s ) , the specific release o f 5~Cr i n c r e a s e d linearly u p to 18 h o f i n c u b a t i o n ( 1 4 . 3 + 1 . 7 0 % ) a n d leveled off. A c t i v a t e d P M N L s also e n h a n c e d s ~Cr release f r o m the p o r c i n e articular c h o n d r o c y t e s with 17 % o f the specific ~ 1Cr release at the r a t i o o f 1:4 ( P < 0.05), w h e r e a s c o s t a l c h o n d r o c y t e s yielded 19.9_+ 3.30% o f the specific release. N e i t h e r costal n o r a r t i c u l a r c h o n d r o c y t e s were affected by P M N L s in the a b s e n c e o f P D B . W h e n freshly o b t a i n e d a r t i c u l a r c h o n d r o c y t e s were c o c u l t u r e d with P M N L , these cells were also d a m a g e d b y P M N L in the presence o f P D B (17.1 -t- 0.870% at the cell r a t i o o f 1:4). It is therefore suggested t h a t P M N L - i n d u c e d c h o n d r o c y t e d a m a g e is n o t significantly different b e t w e e n the different s u b p a s -

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Cell ratio (PMNLs / Chondrocytes or Fibroblasts)

Fig. 1. Effect of PDB-activated PMNLs on both the costal chondrocytes and fibroblasts, s 1Cr-labeled costal chondrocytes or fibroblasts were cultured with PMNLs in the presence (e .... e, 9 .... 9 or absence (o .... o, A----A) of 10 ng/ml PDB for 24 h, and the specific release of SlCr was measured. Data represent means __+SEof five separate experiments, each performed in duplicate. The data with or without PDB at the same cell ratio were analyzed statistically by paired Student's t-test (** P

Damage of cultured chondrocytes by hydrogen peroxide derived from polymorphonuclear leukocytes: a possible mechanism of cartilage degradation.

To study the mechanisms of chondrocyte damage, chondrocyte cytotoxicity as shown by chromium-51 release induced by polymorphonuclear leukocytes (PMNLs...
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