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β1-Integrin–collagen interaction reduces chondrocyte apoptosis
Matrix Biology 18 Ž1999. 343]355
b 1-Integrin]collagen interaction reduces chondrocyte apoptosis Liu Cao, Vivian Lee, Mark E. Adams1, Chris Kiani, Yaou Zhang, Wendy Hu, Burton B. Yang 2,U Sunnybrook and Women’s College Health Science Centre, and Department of Laboratory Medicine and Pathobiology, Uni¨ ersity of Toronto, Toronto, M4N 3M5 Canada Received 31 July 1998; accepted 24 March 1999
Abstract We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death Žapoptosis.. Moreover, we showed that extracellular matrix ŽECM. molecules in the culture media provide vital chondrocyte]matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin b 1 suggest that the collagen]chondrocyte interactions are mediated through integrin b 1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte]matrix network. Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Collagen; Hyaluronan; Chondrocyte; Adhesion; Apoptosis
1. Introduction The central event in the pathogenesis of joint diseases such as osteoarthritis is the progressive deterioration and destruction of cartilage ŽLohmander et
Abbre¨ iations: HA, hyaluronan; HAse, hyaluronidase; HAR, hyaluronan receptor; LP, link protein; ECM, extracellular matrix; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis U Corresponding author. q1-416-480-5874; fax: q1-416-480-5737. E-mail address: [email protected]. ŽB.B. Yang. 1 On sabbatical from the University of Calgary. 2 Scholar of the Arthritis Society of Canada.
al., 1993; Poole et al., 1994; Lark et al., 1997.. In cartilage, the most abundant macromolecules are the extracellular matrix molecules such as collagen, hyaluronan and aggrecan. It has been observed that expression of collagen and aggrecan increases in the early stages of osteoarthritis ŽMatyas et al., 1995, 1997.. However, the increase in expression of these two molecules is disproportionate. As a result, the concentration of aggrecan is relatively higher than that of collagen in the osteoarthritic cartilage. This concentration disequilibrium may be a contributing factor to this disease pathogenesis. An in vitro model of the three-dimensional culture of chondrocytes is essential to study the metabolism
0945-053Xr99r$ - see front matter Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 9 9 . 0 0 0 2 7 - X
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of the extracellular matrix molecules in detail. Initially, we have found that chondrocytes always adhere to the tissue culture plates and eventually exhibit a fibroblast-like phenotype. Chondrocytes in monolayer express low levels of extracellular matrix molecules and hence their environment is completely different from the in vivo environment. To simulate the in vivo environment more accurately, we grew chondrocytes in an alginate gel so as to obtain a three-dimensional culture. Although the cells expressed increased amounts of matrix molecules, they were immobilized, making it difficult to study their activities. We therefore developed a method to grow chondrocytes as a suspension culture. Cells in such cultures expressed high levels of matrix molecules and thus, at least in this respect, mimic the conditions found in cartilage tissue. Using this model, we observed that complexes including aggrecan, hyaluronan and link protein play an important role in chondrocyte]matrix interaction ŽYang et al., 1998.. Moreover, we also observed that, after the culture media were changed, some chondrocytes looked abnormal and appeared to have undergone programmed cell death, or apoptosis. Chondrocyte apoptosis may be involved in a number of disease states. It has been observed in rheumatoid arthritis synovium ŽFirestein et al., 1995.. Chondrocyte number and DNA concentration are reduced in osteoarthritic cartilage ŽVignon et al., 1983; Matyas et al., 1995., perhaps due to programmed death of chondrocytes. Studies of transgenic mice lacking type II collagen have provided direct evidence that chondrocytes undergo apoptosis in the absence of collagen ŽYang et al., 1997.. In general, in vivo studies of chondrocyte apoptosis are difficult since the degeneration of cartilage is slow, occurring over a long period of time. Cells in culture are a much more convenient model to study apoptosis and a number of studies have been done in this context. For example, chondrocyte apoptosis can be induced by nitric oxide ŽBlanco et al., 1995., FasrFas ligand expression ŽHashimoto et al., 1997. and by terminal differentiation ŽGibson et al., 1997.. These studies have suggested that chondrocyte apoptosis may be involved in cartilage degradation. Our observation that media change resulted in chondrocyte apoptosis may be due to a disruption of the structure of extracellular matrix and the interaction of chondrocyte with matrix. If so, it would provide an appropriate model to investigate the role of extracellular matrix molecules and how they modulate chondrocyte apoptosis, which may be an essential cellular event in osteoarthritic cartilage development. To test this, we investigated the roles of collagen and the aggrecan]hyaluronan-link protein ternary complexes, since they are the major structural molecules in cartilage. We report here that the removal of
collagen induced apoptosis of chondrocytes in suspension cultures; cell death was prevented by re-addition of collagen. Hyaluronidase treatment led to chondrocyte attachment and aggregation, which inhibited chondrocyte apoptosis. An anti-integrin b 1 monoclonal antibody abolished the ability of collagen to mediate chondrocyte aggregation, presumably by hindering essential collagen]integrin interaction.
2. Methods 2.1. Chondrocyte isolation and culture Chondrocytes were isolated from the sterna of 18day chicken embryos. The sterna were removed using sterile scissors and rinsed with PBS. Forty-eight chicken embryos were used each time. The caudal portion Ž1r3. of each sterna was removed with a sterile surgical knife and incubated in dissociation medium Ž0.3% collagenase dissolved in HBSS. at 378C for 30 min. The dissociation medium was changed and incubation took place for an additional 1 h. At this point an equal volume of growth medium was added to stop the enzymatic reaction. The mixture was vortexed and the cells were collected by passing the mixture through a filter and centrifugation at 1100 rev.rmin. The cells were resuspended in growth medium ŽDMEM containing 10% FBS, Gibco.. The sizes and morphology of the cells were uniform. The newly-isolated chondrocytes can be maintained as a suspension culture in tissue culture plates. After several days, some chondrocytes adhered to the tissue culture plates. Gradually, the chondrocytes could be cultured as two pools: an adherent culture and a suspension culture. A small portion of each passage of suspended chondrocytes always attached to the culture plates. Nevertheless, a pool of chondrocytes growing in suspension could be maintained by passing only the suspended chondrocytes to a new culture each time. The adherent chondrocytes exhibited a fibroblast-like morphology and could be maintained for many months. However, chondrocytes growing in suspension could only be maintained for approximately 1 month before they lost the suspension property and adhered to the tissue culture plates. All experiments were performed in the early stages of cultures Ž2 weeks after cell isolation. while cells were actively proliferating and matrix production was sufficient to maintain the cells as suspension cultures. The chondrocytes in these stages were uniform in size and morphology. They were hypertrophic chondrocytes and were matrix-dependent for survival. We also maintained suspension cultures on bacterial petri dishes and agarose-coated plates. These techniques allowed chondrocytes to be maintained
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longer as suspension cultures. As well, to reduce the amount of apoptosis, we changed only half of spent culture medium with fresh medium. In this way, there was always a sufficient amount of extracellular matrix molecules surrounding the cells, and this method has allowed us to maintain the suspension cultures for many weeks. However, the morphology of the cells changed eventually, and the sizes of some cells became much larger. We stopped growing such cultures at this stage. 2.2. Chondrocyte apoptosis analyzed by FACS and DNA fragmentation Apoptosis was analyzed by flow cytometry of propidium iodide ŽPI.-labeled DNA, as described by Nicoletti et al. Ž1991.. Briefly, 5 = 10 5 chondrocytes were grown in 100-mm bacterial petri dishes with appropriate treatment. For the apoptosis assay, the cells were pelleted and resuspended in 1.2 ml of hypotonic PI solution Ž50 mgrml. dissolved in 0.1% sodium citrate plus 0.1% Triton x-100 ŽSigma.. DNA fragmentation of apoptotic chondrocytes was assessed as described by Smith et al. Ž1989.. Chondrocytes were pooled together and resuspended in l ml of lysis solution Ž10 mM Tris]Cl, pH 8.0, 100 mM NaCl, 25 mM EDTA and 1% SDS.. The lysis mixture was incubated at 378C for 1 h with gentle agitation followed by extraction with phenolrchloroform Ž2 = .. The DNA in the upper phase was precipitated with 2.5 volumes of ethanol and resuspended in TE buffer Ž10 mM Tris]Cl, pH 8.0, 1 mM EDTA. at a concentration of 100 mgrml. The DNA samples were treated with RNase-A, subjected to 2% agarose gel electrophoresis, stained with ethidium bromide and photographed. 2.3. Chondrocyte apoptosis and interaction affected by collagen To test the effect of collagen on chondrocyte apoptosis, collagenase ŽSigma, Cat. No. C1913, dissolved in HBSS, 2000 Urmg., was added to the chondrocyte cultures at different concentrations as indicated in the figure legends. The conditions for maintaining the cultures were also provided in detail in each figure. Chondrocyte apoptosis was analyzed by flow cytometry. In collagen-add-back experiments, chondrocyte cultures were digested with collagenase at a concentration of 2 mgrml at 378C for 30 min. Chondrocytes were collected by centrifugation and resuspended in DMEM supplemented with 10% FBS, to which type I collagen Žnamed as type II, Sigma, Cat. No. C 8886, from bovine achilles tendon. was added at a final
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concentration of 50 mgrml. The cultures were maintained at 378C in a tissue culture incubator for 24 h and chondrocyte apoptosis was analyzed. As the type I collagen was difficult to dissolve in water nor in growth medium, we used two methods to make it dissolve. Type I collagen was briefly digested with collagenase Ž0.05%. at 378C for 1 h to reduce viscosity. The collagenase was then heat-inactivated and the collagen solution could be used in this way. Alternatively, we also used a mortar and pestle to ground the collagen to increase solubility and reduce viscosity. After collagen dissolved, the solution was filtered before use. As a control, a different product of type II collagen ŽSigma, Cat. No. C 9301, from chicken sternal cartilage. was used to test its effect on inhibiting chondrocyte apoptosis. To examine the role of collagen in chondrocyte aggregation, adherent chondrocytes were harvested and resuspended in regular culture media with or without exogenous addition of type I collagen at a concentration of 50 mgrml. Chondrocyte aggregation was examined using a light microscope. 2.4. Chondrocyte interaction and apoptosis affected by hyaluronan In cell attachment assay, 24-well tissue culture plates were coated with chicken type II collagen at a concentration of 0.5 mgrml. The following day, the mixture was removed and the plates were dried briefly in a tissue culture incubator. Hyaluronidase or collagenase-treated suspended chondrocytes were seeded on these plates at a concentration of 5 = 10 4 cellsrwell and maintained at 378C for 3 h. Growth medium and unattached chondrocytes were then removed by aspiration and fresh growth medium was added. The attachment of chondrocytes to tissue culture plates was examined using light microscopy. These attached chondrocytes were harvested with a brief incubation with 10 mM EDTA and the cell density was determined with a cytometer. In cell-spreading assay, chondrocyte cultures grown on the collagen-coated plates were also incubated with hyaluronidase ŽSigma, from bovine testes, Cat. No. H 3506. at a final concentration of 200 Urml. The cultures were incubated at 378C in a tissue culture incubator for 48 h. The spreading of chondrocytes to the plates was examined after 48 h of incubation. In a cell aggregation assay, hyaluronidase was also added to the suspended chondrocyte cultures at a concentration of 200 Urml and the cells were incubated at 378C for 3 h. Chondrocyte aggregation was examined using a light microscope.
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2.5. Detection of integrin b1 by FACS and Western blot The suspended chondrocytes were treated with or without collagenase Ž0.2%. or hyaluronidase Ž200 Urml. for 30 min. The cells were then labeled with anti-integrin b 1 monoclonal antibody ŽSigma, Cat. No. I 8638.. Unlabeled cells were used as a negative control. All samples were then labeled with FITCconjugated goat-anti-mouse IgG and analyzed on a FACScan ŽBecton Dickinson.. Protein samples collected from the same number of cells were subjected to SDS-PAGE ŽLaemmli, 1970.. The buffer system is 1 = TG ŽTris-glycine buffer, Amresco. containing 1% SDS. Proteins separated on SDS-PAGE were transblotted onto a nitrocellulose membrane ŽBio-Rad. in 1 = TG buffer containing 20% methanol. The membrane was blocked in TBST Ž10 mM Tris]Cl pH 8.0, 150 mM NaCl, 0.05% Tween 20. containing 10% non-fat dry milk powder ŽTBSTM. for 1 h at room temperature, and then incubated at 48C overnight with primary antibody in TBSTM. The membranes were washed with TBST Ž3 = 30 min. and then incubated for 1 h with horseradish peroxidase ŽHRP.-conjugated secondary antibody in TBSTM. After washing as above, the bound antibody was visualized with an ECL kit according to the manufacturer’s instructions ŽAmersham..
3. Results 3.1. Loss of collagen induces chondrocyte apoptosis To study the functions of extracellular matrix molecules of cartilage, we maintained chondrocytes as a suspension culture and an adherent culture. We observed that the adherent culture was relatively easy to maintain. It developed a fibroblast-like morphology and we were able to grow this adhesion culture in a way similar to that for fibroblasts. However, the suspension culture was difficult to maintain. This culture tended to lose its ability to grow in suspension, and adhered to the tissue culture plates. To maintain the culture in suspension, it was essential to use only suspended cells at each passage. However, we observed that after changing media, it took 2]3 days for the chondrocytes to recover. Some chondrocytes were broken down and the cell debris could be easily detected using a light microscope ŽFig. 1A.. Initially, the suspension cultures were maintained either in petri dishes or in agarose-coated tissue culture plates. These two culture methods are good for short-term assays. We performed most of our suspension cultures in agarose-coated plates. Those assays performed in petri dishes were indicated in the legend of each figure ŽFig. 1..
We suspected that the chondrocytes underwent apoptosis. To confirm this, chondrocytes were harvested before and after media change for DNA endlabeling to analyze in a FACScan. It showed that 30% of chondrocytes underwent apoptosis after the media were changed as compared to the 11% which were apoptotic prior to the change of media ŽFig. 1b.. This experiment was repeated three times and the results were analyzed statistically. A significant difference was obtained Ždata not shown.. Total DNA was prepared from chondrocytes before and after media change, and analyzed by agarose gel electrophoresis. The smear resulting from DNA fragmentation further confirms severe chondrocyte apoptosis in chondrocytes harvested after media change ŽFig. 1c.. Chondrocyte apoptosis was monitored in both suspension culture and adherent culture after media were changed. Of the two, only the suspension culture underwent significant apoptosis. As the chondrocytes in the suspension cultures recovered and started to grow 1 day after inoculation, the total live chondrocytes increased, and the proportion of apoptotic cells declined each day ŽFig. 1D.. To study the dose-dependency of media removal on chondrocyte apoptosis, different proportions of culture media were exchanged in suspension culture and adherent culture. As seen in Fig. 1E, apoptosis in the suspension culture increased as greater amounts of new media were added, reaching a rate of nearly 30% when all the spent media was replaced with fresh media. The effect on adherent chondrocytes was negligible. We have previously demonstrated that the suspended chondrocytes are surrounded by the extracellular matrix molecules such as hyaluronan, aggrecan and link protein and that expression of the G1 domain of aggrecan induces chondrocyte apoptosis ŽYang et al., 1998; Cao and Yang, 1999.. The most abundant matrix molecule in cartilage and chondrocyte cultures is collagen. When the media is changed, large amounts of collagen would then be removed from the chondrocyte cultures. Loss of collagen may contribute to chondrocyte apoptosis. To test this, chondrocyte cultures were incubated with or without collagenase. After collagenase treatment, cultures exhibited significant apoptosis, as gauged by the amount of fragmented DNA, as compared with the chondrocyte culture without collagenase treatment ŽFig. 2a.. Addition of type I collagen to the collagenase-treated cultures inhibited chondrocyte apoptosis ŽFig. 2a.. These experiments were repeated three times and similar results were obtained each time ŽFig. 2b.. Collagenase was introduced into the suspended chondrocyte cultures and the adherent chondrocyte cultures at different concentrations, and the cultures were maintained at 378C for 24 h. Chondrocyte apoptosis in the suspended culture was promoted by colla-
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genase treatment in a dose-dependent manner ŽFig. 2c.. Collagenase had little effect on the apoptosis of the adherent chondrocytes, further suggesting that degradation of collagen was involved in the apoptosis of the suspended chondrocytes, rather than the effect of any protease contamination in the collagenase preparation. Finally, when exogenous type I collagen was added into collagenase-treated chondrocyte cultures and the cultures were maintained at 378C for 24
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h, collagen inhibited the apoptosis in suspended chondrocytes in a dose-dependent manner, but had little effect on adherent chondrocytes ŽFig. 2d.. 3.2. The roles of HA and collagen in chondrocyte interaction and apoptosis To study the function of collagen in chondrocyte] matrix interactions, adherent chondrocytes were har-
See overleaf for legend to Fig. 1
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vested and incubated with or without exogenous addition of type I collagen and collagenase. Normally, these chondrocytes started to aggregate 3 h after being seeded in petri dishes ŽFig. 3.. Exogenous addition of collagen enhanced the aggregation by forming more and larger conglomerates ŽFig. 3.. Degradation of endogenous collagen by addition of collagenase inhibited the aggregation of chondrocytes Ždata not shown.. To further test the effect of hyaluronidase and collagenase on the interaction of chondrocytes with collagen, the suspended chondrocytes were pre-incubated with or without hyaluronidase or collagenase, and then inoculated in type II collagen-coated plates. Chondrocyte attachment was analyzed. Without treatment, few cells adhered to the collagen-coated plates ŽFig. 4a.. Collagenase treatment caused a small increase in the number of cells adhering, while hyaluronidase ŽHAse. digestion resulted in a large Žmore than 20-fold. increase in the number of adherent cells. Hyaluronidase-digested and untreated chondrocytes were plated on type II collagen-coated plates and maintained in a tissue culture incubator for 2 days. The hyaluronidase-treated chondrocytes exhibited a fibroblast-like morphology and spread on the collagen-coated plates ŽFig. 4b.. The untreated chondrocytes still grew as a suspension culture ŽFig. 4b.. Another major component of cartilage is hyaluronan. To test the effects of this molecule, suspended chondrocytes were pre-incubated with collagenase and then treated with hyaluronidase. The addition of hyaluronidase inhibited chondrocyte apoptosis ŽFig. 5a.. At the concentrations used for suspended chondrocytes, hyaluronidase was also incubated with adherent chondrocytes, and they had little effect on chondrocyte apoptosis Ždata not shown.. This indicated that the effect of any possible protease contami-
nation in the hyaluronidase preparations on chondrocyte apoptosis was negligible. The suspended chondrocytes were pre-digested with collagenase and then the cells were harvested and incubated in type I collagen. Chondrocyte apoptosis was analyzed with FACScan. The results showed that chondrocyte apoptosis induced by collagenase digestion was significantly inhibited by addition of collagen ŽFig. 5b.. To test the effects of collagenase in chondrocyte apoptosis, collagenase was introduced into the suspended chondrocytes. We found that collagenase treatment caused significant apoptosis ŽFig. 5c.. The effects of type I and type II collagen on chondrocyte apoptosis were then tested by addition of collagen solution to suspended chondrocytes that had been treated with collagenase for 30 min. The cultures were maintained in an incubator for 24 h followed by analysis of cell apoptosis. Chondrocytes that had been treated with collagenase but were not incubated with collagen were used as controls. As expected, the effect of type II collagen was similar to type I collagen on inhibiting apoptosis of chondrocytes ŽFig. 5d.. 3.3. HA moderated the effect of collagen and integrin on aggregation and apoptosis Integrin b 1 , a cell surface molecule expressed by chondrocytes, binds to collagen ŽEnomoto et al., 1993; Loeser et al., 1995.. Thus, we reasoned that integrin b 1 may be involved in collagen’s mediation of chondrocyte aggregation. To test this, suspended chondrocytes were incubated with hyaluronidase, with hyaluronidase and collagenase, or hyaluronidase and anti-integrin b 1 antibody. Chondrocyte aggregation was examined with a light microscope. Hyaluronidase digestion induced aggregation of the suspended chondrocytes as compared with the untreated chondro-
Fig. 1. The effect of media-change on chondrocyte apoptosis. Ža. The suspended chicken chondrocytes were inoculated in 100-mm bacterial petri dishes at a concentration of 1 = 10 6 cellsrml Žsubconfluence ., the cultures were divided into two groups. In one group, culture media was removed by aspiration and replaced with fresh media Žmedia changed.. The other group was maintained in the same conditions Žcontrol., without the media change. The cultures were incubated at 378C in a tissue culture incubator for 24 h. The cultures were examined under a light microscope. A large amount of chondrocyte debris was detected in the cultures which were maintained in the fresh media, indicating increased cell death. Žb. Apoptosis was tested in chondrocytes cultured in petri dishes with or without media changed. Chondrocytes were collected from the culture by centrifugation, stained with PI and analyzed in a FACScan. The media-changed chondrocytes exhibited a twofold increase in apoptosis as compared with the controls Ž30% vs. 10.8% apoptosis.. Žc . Chondrocytes cultured in petri dishes were harvested 24 h after culture media was changed. Cultures not subjected to media change were used as a control. Total DNA isolated from each sample was subjected to 2% agarose gel electrophoresis and the gel was stained with ethidium bromide to visualize the DNA. DNA from cells collected after media change was fragmented Žbracket area., a characteristic property of apoptosis. Žd. Suspended chondrocytes were maintained in petri dishes, and adherent chondrocytes were cultured in tissue culture plates. At day 0, all media in both types of treatments were carefully removed, and fresh media were introduced into each dish. The cultures were maintained at 378C in an incubator. Chondrocyte apoptosis was analyzed after media change in suspended and adherent chondrocyte cultures. In suspended cultures, apoptosis increased significantly within 24 h and then declined. The adherent chondrocytes showed little apoptosis. Že. The culture media were changed at different proportions in suspended cultures Žin petri dishes. and adherent cultures Žin tissue culture plates.. The cultures were maintained at 378C for 1 day and chondrocyte apoptosis was analyzed by FACScan. Apoptosis in suspended chondrocytes was dose-dependent. The change of culture media in adherent chondrocytes had no effect on apoptosis.
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Fig. 2. The effect of collagen on chondrocyte apoptosis. Ža. Suspended chondrocytes cultured in agarose-coated tissue culture plates were incubated with or without collagenase Ž500 mgrml. at 378C for 2 h. Untreated chondrocytes were maintained as a control. Chondrocyte apoptosis was enhanced significantly by collagenase digestion Ž45% apoptosis., but exogenous addition of type I collagen Ž50 mgrml, 13% apoptosis. abolished collagenase’s effect on chondrocyte apoptosis as compared with untreated cells Ž11% apoptosis.. Žb. The experiment was repeated three times and data were analyzed statistically. Chondrocyte apoptosis caused by collagenase digestion was completely reversed by exogenous addition of type I collagen after collagenase digestion Ž n s 3, P- 0.01.. Žc . The suspended and adherent chondrocyte cultures were incubated with different concentrations of collagenase in a tissue culture incubator at 378C for 24 h. Collagenase treatment promoted apoptosis of suspended chondrocytes in a concentration-dependent manner. Žd. Suspended and adherent chondrocyte cultures were incubated with collagenase Ž2 mgrml. for 30 min at 378C. Collagenase was removed and type I collagen was added as shown. The exogenous addition of type I collagen protected suspended chondrocytes from apoptosis in a dose-dependent manner.
cytes ŽFig. 6a.. A combination of hyaluronidase and collagenase Ž collagenase first followed by hyaluronidase. reduced the effect of hyaluronidase on chondrocyte aggregation significantly. Addition of the
anti-integrin b 1 antibody at the presence of hyaluronidase also inhibited chondrocyte aggregation indicating that the antibody can block the effect of hyaluronidase.
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Fig. 3. The role of collagen in chondrocyte aggregation. Adherent chicken chondrocytes were harvested by treatment with 10 mM EDTA, collected by centrifugation and the cells were resuspended in DMEM containing 10% FBS. Type I collagen was added to the cultures at a final concentration of 50 mgrml. Untreated chondrocytes were used as controls. The cultures were maintained at 378C for 3 h. Chondrocyte aggregation was examined under a light microscope and photographed. Exogenous addition of collagen induced chondrocyte aggregation.
To test the effect of integrin]antibody interaction on chondrocyte apoptosis, suspended chondrocytes
were pre-incubated with collagenase and then inoculated in bacterial petri dishes in DMEM supplemented with 10% FBS. Type I collagen andror anti-b 1 antibody were added and the cultures were maintained in a tissue culture incubator for 24 h prior to analysis. Chondrocyte apoptosis induced by collagenase treatment was ameliorated by the addition of collagen but not by the addition of anti-integrin b 1 antibody ŽFig. 6b.. Nevertheless, addition of anti-integrin b 1 antibody partially inhibited collagen’s effects when both were added simultaneously. To test if the interaction of collagen and integrin b 1-mediated chondrocyte aggregation, suspended chondrocytes were incubated with or without collagenase and the resultant chondrocytes were analyzed for the presence of integrin b 1 by labeling the chondrocytes with a monoclonal antibody against integrin b 1. The labeled chondrocytes were analyzed in a FACScan. Treatment with collagenase resulted in enhanced staining for integrin b 1 on the cell surface ŽFig. 7a.. There are two mechanisms which may account for this result: digestion of chondrocytes with collagenase could make more integrin b 1 accessible for staining; or treatment with collagenase might upregulate the expression of integrin b 1. Since either
Fig. 4. The role of hyaluronidase in cell]matrix interactions. Ža. The suspended chondrocytes were pre-incubated with or without hyaluronidase Ž200 Urml. or collagenase Ž2 mgrml. at 378C for 30 min and then plated on type II collagen-coated plates. Untreated chondrocytes were used as controls. The cultures were maintained at 378C for 3 h. Cell attachment was determined by counting. Žb. The suspended chondrocytes were incubated with or without hyaluronidase Ž200 Urml. at 378C for 30 min, and the cells were then plated on type II collagen-coated tissue culture plates. The cultures were maintained at 378C in a tissue culture incubator for 48 h. Cell spreading was examined under a light microscope.
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Fig. 5. The effect of hyaluronidase and collagenase on apoptosis. Ža. Suspended chondrocytes were treated with collagenase Ž2 mgrml. for 30 min, and then incubated in culture media with or without hyaluronidase Ž200 Urml. at 378C in a tissue incubator for 24 h. Chondrocyte apoptosis was analyzed by FACS. Without the addition of hyaluronidase, chondrocytes underwent significant apoptosis due to collagenase digestion. Addition of hyaluronidase reduced the apoptosis. Žb. Suspended chondrocytes were pre-treated with collagenase Ž2 mgrml. at 378C for 30 min and then the treated chondrocytes were harvested by centrifugation and incubated with type I collagen Ž50 mgrml.. The cultures were maintained at 378C for 24 h and chondrocyte apoptosis was analyzed by FACS. Apoptosis induced by collagenase treatment was reduced significantly by exogenous addition of type I collagen. Žc. Suspended chondrocytes were treated with collagenase Ž2 mgrml. at 378C for 24 h. Untreated chondrocytes were used as controls. Chondrocyte apoptosis was determined. Treatment with collagenase induced chondrocyte apoptosis significantly. Žd. To compare the protecting effects of type I and type II collagen on chondrocyte apoptosis, suspension chondrocytes were pre-treated with collagenase and cells harvested as described above. Type I and type II collagen were added to the chondrocytes at a concentration of 50 mgrml, respectively. Collagenase-treated chondrocytes without addition of collagen were used as controls. Apoptosis of chondrocytes was analyzed as above. Addition of type II collagen, similar to type I collagen, inhibited chondrocyte apoptosis as compared with the controls.
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in cultures treated with or without collagenase ŽFig. 7b. and this result was confirmed by repeating the experiment, it seems unlikely that increased b 1 expression is responsible for the increased b 1 staining observed in collagenase-treated cultures. Under normal conditions, collagen probably binds to integrin b 1 in a way that obstructs the interaction of anti-integrin b 1 antibody with b 1. Collagenase treatment would thus remove this obstruction and result in enhanced b 1 staining. Another major component surrounding chondrocytes in cartilage is hyaluronan. To test the possibility that hyaluronan may also block the availability of integrin b 1 , the suspended chondrocytes were incubated with or without hyaluronidase and the resultant chondrocytes were stained for accessible integrin b 1 as described above. The result showed that hyaluronidase digestion also enhanced the availability of integrin b 1 stained by the anti-integrin b 1 antibody ŽFig. 7c.. However, hyaluronidase treatment did not enhance the expression of integrin b 1 , as analyzed by Western blot ŽFig. 7d.. This experiment was repeated three times and similar result was obtained. To assure that collagenase and hyaluronidase had no effect on integrin b 1 expression, adherent chondrocytes were incubated with collagenase and hyaluronidase for 3 days. Expression of integrin b 1 did not change in the adherent chondrocytes analyzed with Western blot assays Ždata not shown.. The results described above suggest that the interaction of collagen and integrin can modulate chondrocyte aggregation and apoptosis. Hyaluronan inhibits chondrocyte aggregation. We present a model to explain such relationships ŽFig. 8.. In this model, hyaluronan binds to aggrecan and link protein to form stable ternary complexes. These complexes mediate chondrocyte]matrix interactions by binding to chondrocyte surface through hyaluronan-binding receptors and inhibit chondrocyte aggregation. Degradation of hyaluronan will destroy the complexes and induce chondrocyte aggregation. As our experiments using the anti-integrin b 1 antibody suggest, aggregation seems to be mediated by an interaction between integrin, an integral membrane protein found on the surface of the plasma membrane, and collagen, a ubiquitous extracellular matrix molecule.
4. Discussion one could result in chondrocyte aggregation, it was necessary to test which mechanism was responsible. Cell lysate was prepared from the collagenase-treated chondrocytes and the untreated chondrocytes, and analyzed for integrin b 1 expression in Western blot stained with the same antibody as above. Since little change in the level of b 1 expression could be detected
Our studies stemmed from our observation that chondrocytes maintained as a suspension culture suffered from significant levels of cell death every time culture media were changed. Chondrocytes secrete large amounts of extracellular matrix molecules, and these spent culture media contained collagen,
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Fig. 6. Interaction of collagen and integrin b1 modulated chondrocyte aggregation and apoptosis. Ža. Suspended chondrocytes were incubated with or without hyaluronidase ŽHAse, 200 Urml., collagenase Žcols, 0.2 mgrml., and monoclonal antibody against integrin b1 Ž20 mgrml., as shown in the figure, at 378C for 3 h. Chondrocyte aggregation was examined under a light microscope and photographed. Žb. Suspended chondrocytes were pre-incubated with collagenase Ž2 mgrml. at 378C for 30 min. The chondrocytes were then inoculated in bacterial petri dishes in DMEM supplemented with 10% FBS. Reagents were added to the cultures at a concentration of 50 mgrml Žtype I collagen. or 10 mgrml Žanti-b 1 antibody.. The cultures were maintained in a tissue culture incubator for 24 h and then analyzed for chondrocyte apoptosis. Type I collagen prevented apoptosis to a great degree, but anti-b 1 antibody interfered with this effect. Antibody to b1 alone had no effect.
hyaluronan and aggrecan. To investigate if these molecules play a role in chondrocyte growth, we tested the roles of collagen and hyaluronan in chondrocyte survival, since collagen is the most abundant molecule in the extracellular matrix of cartilage ŽHollander et al., 1995; Matyas et al., 1997., and hyaluronan is the central molecule in the aggrecan]hyaluronan-link protein ternary complexes. Our findings suggest that collagen is very important in chondrocyte viability, and hyaluronan, through its effects on chondrocyte aggregation, interfered with collagen’s effects on apoptosis. Degradation of collagen and reduced chondrocyte number are associated with a number of joint diseases. For example, in the late stages of osteoarthritis,
collagen is severely degraded due to proteolytic activity. Severe arthritis is also characterized by irregular chondrocyte distribution: a large proportion of chondrocytes disappear from the cartilage while the small groups of chondrocytes are easily detected. However, the relationships among the collagen degradation, chondrocyte disappearance and chondrocyte conglomeration are not known. Our results here indicate that collagen degradation induces chondrocyte apoptosis while the conglomeration of small groups of chondrocytes protects those chondrocytes from apoptosis. This interpretation is supported by the recent report that chondrocytes obtained from collagen knock-out mice undergo apoptosis ŽYang et al., 1997.. The role of integrin has been studied extensively. It
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Fig. 7. Collagen and hyaluronan hindered detection of integrin b1 . Ža. Suspended chondrocytes were incubated with or without collagenase Ž2 mgrml. at 378C for 30 min. The cells were labeled with monoclonal antibody against integrin b1 and analyzed on FACScan. Unlabeled chondrocytes were used as controls Žleft peak.. Digestion with collagenase enhanced the staining of integrin b1 significantly, resulting in an increase in fluorescence intensity Žmiddle peak, without collagenase; right peak, with collagenase.. Žb. Cell lysate was prepared from chondrocytes treated with or without collagenase as described in Ža. and analyzed in a Western blot stained with the monoclonal antibody against integrin b1 . Untreated cells served as controls. The density of immunostaining was similar for both treatments. Žc. Suspended chondrocytes were incubated with or without hyaluronidase Ž200 Urml. at 378C for 30 min. The cells were labeled with antibody to integrin b1 and analyzed on FACScan. Unlabeled chondrocytes were used as controls Žleft peak.. Hyaluronidase digestion resulted in enhanced staining of integrin b1 Žmiddle peak, without hyaluronidase; right peak, with hyaluronidase.. Žd. Cell lysate was prepared from chondrocytes treated with or without hyaluronidase as described in Žc. and analyzed in a Western blot stained with the antibody against integrin b1. Untreated cells served as controls. The band density was similar in both samples.
binds to fibronectin and modulates signal transduction. Insufficient interaction of integrin and fibronectin induces apoptosis of endothelial cells ŽMeredith et al., 1993; Bates et al., 1994; Frisch and Francis, 1994; Ruoslahti and Reed, 1994; Chen et al., 1997; McGill et al., 1997.. It is known that integrin also binds to type II collagen ŽEnomoto et al., 1993.. The effect of their interaction is not clear, but it may be critical. We show here that collagenase digestion and treatment with anti-integrin b 1 antibody produce a similar effect, an inhibition of chondrocyte aggregation. Furthermore, we demonstrated using FACS analysis that collagenase digestion seems to enhance the accessibility of integrin b 1 on the chondrocyte surface. This observation cannot be explained by increased expression of integrin b 1 , as similar levels of expression were observed in treated and untreated cells on Western blot. Taken together, these suggest that the interaction of collagen and integrin b 1 is responsible for chondrocyte aggregation. Degradation of collagen induced chondrocyte apoptosis which was not reversed
by addition of the anti-integrin b 1 antibody. In fact, simultaneous addition of collagen with the antibody to collagenase-treated chondrocytes resulted in more severe apoptosis as compared to the addition of collagen alone. The interaction of collagen and integrin b 1 may be important to chondrocyte survival and thus addition of collagen to collagenase-treated chondrocytes brought the apoptosis to a basic level Ž; 12%, Fig. 6b.. Recently, it has been demonstrated that collagen degradation in osteoarthritis is the result of collagenase activity ŽHollander et al., 1995.. The collagenases are synthesized and secreted by chondrocytes, and the pericellular collagen would be the most likely substrate for these enzymes. As a result of collagenase activity, collagen]integrin interaction would be disrupted and the chondrocytes, without any collagen to bind, would undergo apoptosis. In addition to collagen, other important molecules in cartilage are aggrecan, link protein and hyaluronan, which form ternary complexes and help to maintain the stable three-dimensional cartilage networks, necessary for joint function ŽPerkins et al., 1989;
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Fig. 8. A model for inhibition of collagen]chondrocyte interaction by hyaluronan. Hyaluronan ŽHA. binds to aggrecan and link protein ŽLP. forming stable ternary complexes. The complexes bind to chondrocyte surface through hyaluronan-binding receptor ŽHAR. on the cell surface. These complexes are important in chondrocyte]matrix interaction and they prevent the aggregation of chondrocytes. Degradation of hyaluronan destroys the complexes. As a result, chondrocytes conglomerate through the interaction of collagen and integrin.
Barry et al., 1992; Li et al., 1993; Neame and Barry, 1993; Margolis and Margolis, 1994.. We have demonstrated that the aggrecan]hyaluronan-link protein ternary complexes are important in mediating chondrocyte]matrix interaction ŽYang et al., 1998.. However, the role of these ternary complexes in chondrocyte aggregation and apoptosis is complicated in the studies presented here. Degradation of hyaluronan with hyaluronidase induces chondrocyte aggregation, and the aggregation inhibits apoptosis. Hyaluronidase also degraded chondroitin sulfate, a major component of chondrocyte products, and this also enhanced chondrocyte aggregation. The aggregation also reduced the level of chondrocyte apoptosis. It is conceivable that, even in the presence of hyaluronidase, newly synthesized collagen could mediate chondrocyte aggregation and, therefore, inhibit apoptosis. It should be noted that degradation of hyaluronan inducing cell aggregation might involve other mechanisms. For example, removal of hyaluronan will allow other adhesion molecules on the cell surface to interact with each other. In the chondrocyte]substratum interaction assay, we sought to directly test the role of hyaluronan in chondrocyte]collagen interaction by introducing hyaluronidase into the suspended chondrocyte cultures to degrade hyaluronan. Chondroitin sulfate might also be degraded and this might result in better interactions of chondrocytes with collagen. Indeed, chondrocytes treated with hyaluronidase bound to the collagen-coated plates strongly. Hyaluronan interfered with the interaction of chondrocytes with colla-
gen. The result of FACScan analysis showed that hyaluronidase digestion enhanced immunostaining of integrin b 1 in the chondrocyte surface. We also observed that collagenase digestion enhanced the interaction of chondrocytes with the collagen-coated plates. Removal of collagen from the chondrocytes increased the accessibility of endogenous integrin b 1 and enhanced chondrocyte binding to collagen-coated plates. The result of FACScan analysis also demonstrated that collagen inhibited the immunostaining of integrin b 1. In normal chondrocyte cultures, addition of hyaluronan had no effect on chondrocyte apoptosis, because collagen]chondrocyte interaction plays a dominant function in protecting chondrocytes from apoptosis. In collagenase-treated chondrocyte cultures, however, apoptosis is high. Addition of hyaluronidase also inhibited chondrocyte apoptosis and this might be due to interactions between chondrocytes and newly synthesized collagen, in the absence of hyaluronan. The newly synthesized collagen-mediated chondrocyte aggregation and small conglomerates could be seen. Our results here demonstrate that hyaluronan, which binds to aggrecan and link protein to form stable ternary complexes, plays an important function in inhibiting chondrocyte aggregation, and collagen plays an important role in protecting chondrocytes from apoptosis. Perhaps, degradation and insufficient production of the hyaluronan]aggrecan-link protein ternary complexes cause chondrocyte aggregation. Degradation of collagen by proteolytic activities induces chondrocyte apoptosis. Since these phenomena are associated with many kinds of joint diseases, further studies are required to uncover the detailed mechanisms by which diseases develop and to find ways to block such processes.
Acknowledgements We thank Dr R.S. Kerbel for helpful comments. This work was supported by Grant MT-13730 from the Medical Research Council of Canada to BBY References Barry, F.P., Gaw, J.U., Young, C.N., Neame, P.J., 1992. Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate. Biochem. J. 286, 761]769. Bates, R.C., Buret, A., van Helden, D.F., Horton, M.A., Burns, G.F., 1994. Apoptosis induced by inhibition of intercellular contact. J. Cell Biol. 125, 403]415. Blanco, F.J., Ochs, R.L., Schwarz, H., Lotz, M., 1995. Chondrocyte apoptosis induced by nitric oxide. Am. J. Pathol. 146, 75]85. Cao, L., Yang, B.B., 1999. Chondrocyte apoptosis induced by aggre-
L. Cao et al. r Matrix Biology 18 (1999) 343]355 can G1 domain as a result of decreased cell adhesion. Exp. Cell Res. 246, 527]537. Chen, C.S., Marksich, M., Huang, S., Whitesides, G.M., Ingber, D.E., 1997. Geometric control of cell life and death. Science 276, 1425]1428. Enomoto, M., Leboy, P.S., Menko, A.S., Boettiger, D., 1993. ß1 Integrins mediate chondrocyte interaction with type I collagen, type II collagen, and fibronectin. Exp. Cell Res. 205, 276]285. Firestein, G.S., Yeo, M., Zvaifler, N.J., 1995. Apoptosis in rheumatoid Aarthritis synovium. J. Clin. Invest. 96, 1631]1638. Frisch, S.M., Francis, H., 1994. Disruption of epithelial cell]matrix interactions induces apoptosis. J. Cell Biol. 124, 619]626. Gibson, G., Lin, D.-L., Roque, M., 1997. Apoptosis of terminally differentiated chondrocytes in culture. Exp. Cell Res. 233, 372]382. Hashimoto, S., Setareh, M., Ochs, R.L., Lotz, M., 1997. FASrFAS ligand expression and induction of apoptosis in chondrocytes. Arthritis Rheum. 40, 1749]1755. Hollander, A.P., Pidoux, I., Reiner, A., Rorabeck, C., Bourne, R., Poole, A.R., 1995. Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration. J. Clin. Invest. 96, 2859]2869. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680]685. Lark, M.W., Bayne, E.K., Flanagan, J., 1997. Aggrecan degradation in human cartilage: Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J. Clin. Invest. 100, 93]106. Li, H., Schwartz, N.B., Vertel, B.M., 1993. cDNA cloning of chick cartilage chondroitin sulfate Žaggrecan. core protein and identification of a stop codon in the aggrecan gene associated with the chondrodystrophy, nanomelia. J. Biol. Chem. 268, 23504]23511. Loeser, R.F., Carlson, C.S., McGee, M.P., 1995. Expression of b1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage. Exp. Cell Res. 217, 248]257. Lohmander, L.S., Neame, P., Sandy, J.D., 1993. The structure of aggrecan fragments in human synovial fluid: Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury and osteoarthritis. Arthritis Rheum. 36, 1214]1222. Margolis, R.U., Margolis, R.K., 1994. Aggrecan-versican-neurocan family proteoglycans. Meth. Enz. 245, 105]126.
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Matyas, J.R., Adams, M.E., Huang, D., Sandell, L.J., 1995. Discoordinate gene expression of aggrecan and Type II collagen in experimental osteoarthritis. Arthritis Rheum. 38, 420]425. Matyas, J.R., Adams, M.E., Huang, D., Sandell, L.J., 1997. Major role of collagen IIB in the elevation of total Type II procollagen messenger RNA in the hypertrophic phase of experimental osteoarthritis. Arthritis Rheum. 40, 1046]1049. McGill, G., Shimamura, A., Bates, R.C., Savage, R.E., Fisher, D.E., 1997. Loss of matrix adhesion triggers rapid transformationselective apoptosis in fibroblasts. J. Cell Biol. 138, 901]911. Meredith Jr, J.E., Fazeli, B., Schwartz, M.A, 1993. The extracellular matrix as a cell survival factor. Mol. Biol. Cell. 4, 953]961. Neame, P.J., Barry, F.P., 1993. The link proteins. Experientia 49, 393]402. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., Riccardi, C., 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271]279. Perkins, S.J., Nealis, A.S., Dudhia, J., Hardingham, T.E., 1989. Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein. J. Mol. Biol. 206, 737]748. Poole, A.R., Ionescu, M., Swan, A., Dieppe, P.A., 1994. Changes in cartilage metabolism in arthritis are reflected by altered serum and synovial fluid levels of the cartilage proteoglycan aggrecan. J. Clin. Invest. 94, 25]33. Ruoslahti, E., Reed, J.C., 1994. Anchorage dependence, integrins, and apoptosis. Cell 77, 477]478. Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J., Owen, J.J.T., 1989. Antibodies to CD3rT-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337, 181]184. Vignon, E., Arlot, M., Hartmann, D.J., Moyen, G., Ville, G., 1983. Hypertrophic repair of articular cartilage in experimental osteoarthrosis. Ann. Rheum. Dis. 42, 82]88. Yang, C., Li, S.-W., Helminen, H.J., Khillan, J.S., Bao, Y., Prockop, D.J., 1997. Apoptosis of chondrocytes in transgenic mice lacking collagen II. Exp. Cell Res. 235, 370]373. Yang, B., Zhang, Y., Cao, L., Yang, B.L., 1998. Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen. Matrix Biol. 16, 541]561.
b 1-Integrin]collagen interaction reduces chondrocyte apoptosis Liu Cao, Vivian Lee, Mark E. Adams1, Chris Kiani, Yaou Zhang, Wendy Hu, Burton B. Yang 2,U Sunnybrook and Women’s College Health Science Centre, and Department of Laboratory Medicine and Pathobiology, Uni¨ ersity of Toronto, Toronto, M4N 3M5 Canada Received 31 July 1998; accepted 24 March 1999
Abstract We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death Žapoptosis.. Moreover, we showed that extracellular matrix ŽECM. molecules in the culture media provide vital chondrocyte]matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin b 1 suggest that the collagen]chondrocyte interactions are mediated through integrin b 1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte]matrix network. Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Collagen; Hyaluronan; Chondrocyte; Adhesion; Apoptosis
1. Introduction The central event in the pathogenesis of joint diseases such as osteoarthritis is the progressive deterioration and destruction of cartilage ŽLohmander et
Abbre¨ iations: HA, hyaluronan; HAse, hyaluronidase; HAR, hyaluronan receptor; LP, link protein; ECM, extracellular matrix; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis U Corresponding author. q1-416-480-5874; fax: q1-416-480-5737. E-mail address: [email protected]. ŽB.B. Yang. 1 On sabbatical from the University of Calgary. 2 Scholar of the Arthritis Society of Canada.
al., 1993; Poole et al., 1994; Lark et al., 1997.. In cartilage, the most abundant macromolecules are the extracellular matrix molecules such as collagen, hyaluronan and aggrecan. It has been observed that expression of collagen and aggrecan increases in the early stages of osteoarthritis ŽMatyas et al., 1995, 1997.. However, the increase in expression of these two molecules is disproportionate. As a result, the concentration of aggrecan is relatively higher than that of collagen in the osteoarthritic cartilage. This concentration disequilibrium may be a contributing factor to this disease pathogenesis. An in vitro model of the three-dimensional culture of chondrocytes is essential to study the metabolism
0945-053Xr99r$ - see front matter Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 9 9 . 0 0 0 2 7 - X
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of the extracellular matrix molecules in detail. Initially, we have found that chondrocytes always adhere to the tissue culture plates and eventually exhibit a fibroblast-like phenotype. Chondrocytes in monolayer express low levels of extracellular matrix molecules and hence their environment is completely different from the in vivo environment. To simulate the in vivo environment more accurately, we grew chondrocytes in an alginate gel so as to obtain a three-dimensional culture. Although the cells expressed increased amounts of matrix molecules, they were immobilized, making it difficult to study their activities. We therefore developed a method to grow chondrocytes as a suspension culture. Cells in such cultures expressed high levels of matrix molecules and thus, at least in this respect, mimic the conditions found in cartilage tissue. Using this model, we observed that complexes including aggrecan, hyaluronan and link protein play an important role in chondrocyte]matrix interaction ŽYang et al., 1998.. Moreover, we also observed that, after the culture media were changed, some chondrocytes looked abnormal and appeared to have undergone programmed cell death, or apoptosis. Chondrocyte apoptosis may be involved in a number of disease states. It has been observed in rheumatoid arthritis synovium ŽFirestein et al., 1995.. Chondrocyte number and DNA concentration are reduced in osteoarthritic cartilage ŽVignon et al., 1983; Matyas et al., 1995., perhaps due to programmed death of chondrocytes. Studies of transgenic mice lacking type II collagen have provided direct evidence that chondrocytes undergo apoptosis in the absence of collagen ŽYang et al., 1997.. In general, in vivo studies of chondrocyte apoptosis are difficult since the degeneration of cartilage is slow, occurring over a long period of time. Cells in culture are a much more convenient model to study apoptosis and a number of studies have been done in this context. For example, chondrocyte apoptosis can be induced by nitric oxide ŽBlanco et al., 1995., FasrFas ligand expression ŽHashimoto et al., 1997. and by terminal differentiation ŽGibson et al., 1997.. These studies have suggested that chondrocyte apoptosis may be involved in cartilage degradation. Our observation that media change resulted in chondrocyte apoptosis may be due to a disruption of the structure of extracellular matrix and the interaction of chondrocyte with matrix. If so, it would provide an appropriate model to investigate the role of extracellular matrix molecules and how they modulate chondrocyte apoptosis, which may be an essential cellular event in osteoarthritic cartilage development. To test this, we investigated the roles of collagen and the aggrecan]hyaluronan-link protein ternary complexes, since they are the major structural molecules in cartilage. We report here that the removal of
collagen induced apoptosis of chondrocytes in suspension cultures; cell death was prevented by re-addition of collagen. Hyaluronidase treatment led to chondrocyte attachment and aggregation, which inhibited chondrocyte apoptosis. An anti-integrin b 1 monoclonal antibody abolished the ability of collagen to mediate chondrocyte aggregation, presumably by hindering essential collagen]integrin interaction.
2. Methods 2.1. Chondrocyte isolation and culture Chondrocytes were isolated from the sterna of 18day chicken embryos. The sterna were removed using sterile scissors and rinsed with PBS. Forty-eight chicken embryos were used each time. The caudal portion Ž1r3. of each sterna was removed with a sterile surgical knife and incubated in dissociation medium Ž0.3% collagenase dissolved in HBSS. at 378C for 30 min. The dissociation medium was changed and incubation took place for an additional 1 h. At this point an equal volume of growth medium was added to stop the enzymatic reaction. The mixture was vortexed and the cells were collected by passing the mixture through a filter and centrifugation at 1100 rev.rmin. The cells were resuspended in growth medium ŽDMEM containing 10% FBS, Gibco.. The sizes and morphology of the cells were uniform. The newly-isolated chondrocytes can be maintained as a suspension culture in tissue culture plates. After several days, some chondrocytes adhered to the tissue culture plates. Gradually, the chondrocytes could be cultured as two pools: an adherent culture and a suspension culture. A small portion of each passage of suspended chondrocytes always attached to the culture plates. Nevertheless, a pool of chondrocytes growing in suspension could be maintained by passing only the suspended chondrocytes to a new culture each time. The adherent chondrocytes exhibited a fibroblast-like morphology and could be maintained for many months. However, chondrocytes growing in suspension could only be maintained for approximately 1 month before they lost the suspension property and adhered to the tissue culture plates. All experiments were performed in the early stages of cultures Ž2 weeks after cell isolation. while cells were actively proliferating and matrix production was sufficient to maintain the cells as suspension cultures. The chondrocytes in these stages were uniform in size and morphology. They were hypertrophic chondrocytes and were matrix-dependent for survival. We also maintained suspension cultures on bacterial petri dishes and agarose-coated plates. These techniques allowed chondrocytes to be maintained
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longer as suspension cultures. As well, to reduce the amount of apoptosis, we changed only half of spent culture medium with fresh medium. In this way, there was always a sufficient amount of extracellular matrix molecules surrounding the cells, and this method has allowed us to maintain the suspension cultures for many weeks. However, the morphology of the cells changed eventually, and the sizes of some cells became much larger. We stopped growing such cultures at this stage. 2.2. Chondrocyte apoptosis analyzed by FACS and DNA fragmentation Apoptosis was analyzed by flow cytometry of propidium iodide ŽPI.-labeled DNA, as described by Nicoletti et al. Ž1991.. Briefly, 5 = 10 5 chondrocytes were grown in 100-mm bacterial petri dishes with appropriate treatment. For the apoptosis assay, the cells were pelleted and resuspended in 1.2 ml of hypotonic PI solution Ž50 mgrml. dissolved in 0.1% sodium citrate plus 0.1% Triton x-100 ŽSigma.. DNA fragmentation of apoptotic chondrocytes was assessed as described by Smith et al. Ž1989.. Chondrocytes were pooled together and resuspended in l ml of lysis solution Ž10 mM Tris]Cl, pH 8.0, 100 mM NaCl, 25 mM EDTA and 1% SDS.. The lysis mixture was incubated at 378C for 1 h with gentle agitation followed by extraction with phenolrchloroform Ž2 = .. The DNA in the upper phase was precipitated with 2.5 volumes of ethanol and resuspended in TE buffer Ž10 mM Tris]Cl, pH 8.0, 1 mM EDTA. at a concentration of 100 mgrml. The DNA samples were treated with RNase-A, subjected to 2% agarose gel electrophoresis, stained with ethidium bromide and photographed. 2.3. Chondrocyte apoptosis and interaction affected by collagen To test the effect of collagen on chondrocyte apoptosis, collagenase ŽSigma, Cat. No. C1913, dissolved in HBSS, 2000 Urmg., was added to the chondrocyte cultures at different concentrations as indicated in the figure legends. The conditions for maintaining the cultures were also provided in detail in each figure. Chondrocyte apoptosis was analyzed by flow cytometry. In collagen-add-back experiments, chondrocyte cultures were digested with collagenase at a concentration of 2 mgrml at 378C for 30 min. Chondrocytes were collected by centrifugation and resuspended in DMEM supplemented with 10% FBS, to which type I collagen Žnamed as type II, Sigma, Cat. No. C 8886, from bovine achilles tendon. was added at a final
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concentration of 50 mgrml. The cultures were maintained at 378C in a tissue culture incubator for 24 h and chondrocyte apoptosis was analyzed. As the type I collagen was difficult to dissolve in water nor in growth medium, we used two methods to make it dissolve. Type I collagen was briefly digested with collagenase Ž0.05%. at 378C for 1 h to reduce viscosity. The collagenase was then heat-inactivated and the collagen solution could be used in this way. Alternatively, we also used a mortar and pestle to ground the collagen to increase solubility and reduce viscosity. After collagen dissolved, the solution was filtered before use. As a control, a different product of type II collagen ŽSigma, Cat. No. C 9301, from chicken sternal cartilage. was used to test its effect on inhibiting chondrocyte apoptosis. To examine the role of collagen in chondrocyte aggregation, adherent chondrocytes were harvested and resuspended in regular culture media with or without exogenous addition of type I collagen at a concentration of 50 mgrml. Chondrocyte aggregation was examined using a light microscope. 2.4. Chondrocyte interaction and apoptosis affected by hyaluronan In cell attachment assay, 24-well tissue culture plates were coated with chicken type II collagen at a concentration of 0.5 mgrml. The following day, the mixture was removed and the plates were dried briefly in a tissue culture incubator. Hyaluronidase or collagenase-treated suspended chondrocytes were seeded on these plates at a concentration of 5 = 10 4 cellsrwell and maintained at 378C for 3 h. Growth medium and unattached chondrocytes were then removed by aspiration and fresh growth medium was added. The attachment of chondrocytes to tissue culture plates was examined using light microscopy. These attached chondrocytes were harvested with a brief incubation with 10 mM EDTA and the cell density was determined with a cytometer. In cell-spreading assay, chondrocyte cultures grown on the collagen-coated plates were also incubated with hyaluronidase ŽSigma, from bovine testes, Cat. No. H 3506. at a final concentration of 200 Urml. The cultures were incubated at 378C in a tissue culture incubator for 48 h. The spreading of chondrocytes to the plates was examined after 48 h of incubation. In a cell aggregation assay, hyaluronidase was also added to the suspended chondrocyte cultures at a concentration of 200 Urml and the cells were incubated at 378C for 3 h. Chondrocyte aggregation was examined using a light microscope.
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2.5. Detection of integrin b1 by FACS and Western blot The suspended chondrocytes were treated with or without collagenase Ž0.2%. or hyaluronidase Ž200 Urml. for 30 min. The cells were then labeled with anti-integrin b 1 monoclonal antibody ŽSigma, Cat. No. I 8638.. Unlabeled cells were used as a negative control. All samples were then labeled with FITCconjugated goat-anti-mouse IgG and analyzed on a FACScan ŽBecton Dickinson.. Protein samples collected from the same number of cells were subjected to SDS-PAGE ŽLaemmli, 1970.. The buffer system is 1 = TG ŽTris-glycine buffer, Amresco. containing 1% SDS. Proteins separated on SDS-PAGE were transblotted onto a nitrocellulose membrane ŽBio-Rad. in 1 = TG buffer containing 20% methanol. The membrane was blocked in TBST Ž10 mM Tris]Cl pH 8.0, 150 mM NaCl, 0.05% Tween 20. containing 10% non-fat dry milk powder ŽTBSTM. for 1 h at room temperature, and then incubated at 48C overnight with primary antibody in TBSTM. The membranes were washed with TBST Ž3 = 30 min. and then incubated for 1 h with horseradish peroxidase ŽHRP.-conjugated secondary antibody in TBSTM. After washing as above, the bound antibody was visualized with an ECL kit according to the manufacturer’s instructions ŽAmersham..
3. Results 3.1. Loss of collagen induces chondrocyte apoptosis To study the functions of extracellular matrix molecules of cartilage, we maintained chondrocytes as a suspension culture and an adherent culture. We observed that the adherent culture was relatively easy to maintain. It developed a fibroblast-like morphology and we were able to grow this adhesion culture in a way similar to that for fibroblasts. However, the suspension culture was difficult to maintain. This culture tended to lose its ability to grow in suspension, and adhered to the tissue culture plates. To maintain the culture in suspension, it was essential to use only suspended cells at each passage. However, we observed that after changing media, it took 2]3 days for the chondrocytes to recover. Some chondrocytes were broken down and the cell debris could be easily detected using a light microscope ŽFig. 1A.. Initially, the suspension cultures were maintained either in petri dishes or in agarose-coated tissue culture plates. These two culture methods are good for short-term assays. We performed most of our suspension cultures in agarose-coated plates. Those assays performed in petri dishes were indicated in the legend of each figure ŽFig. 1..
We suspected that the chondrocytes underwent apoptosis. To confirm this, chondrocytes were harvested before and after media change for DNA endlabeling to analyze in a FACScan. It showed that 30% of chondrocytes underwent apoptosis after the media were changed as compared to the 11% which were apoptotic prior to the change of media ŽFig. 1b.. This experiment was repeated three times and the results were analyzed statistically. A significant difference was obtained Ždata not shown.. Total DNA was prepared from chondrocytes before and after media change, and analyzed by agarose gel electrophoresis. The smear resulting from DNA fragmentation further confirms severe chondrocyte apoptosis in chondrocytes harvested after media change ŽFig. 1c.. Chondrocyte apoptosis was monitored in both suspension culture and adherent culture after media were changed. Of the two, only the suspension culture underwent significant apoptosis. As the chondrocytes in the suspension cultures recovered and started to grow 1 day after inoculation, the total live chondrocytes increased, and the proportion of apoptotic cells declined each day ŽFig. 1D.. To study the dose-dependency of media removal on chondrocyte apoptosis, different proportions of culture media were exchanged in suspension culture and adherent culture. As seen in Fig. 1E, apoptosis in the suspension culture increased as greater amounts of new media were added, reaching a rate of nearly 30% when all the spent media was replaced with fresh media. The effect on adherent chondrocytes was negligible. We have previously demonstrated that the suspended chondrocytes are surrounded by the extracellular matrix molecules such as hyaluronan, aggrecan and link protein and that expression of the G1 domain of aggrecan induces chondrocyte apoptosis ŽYang et al., 1998; Cao and Yang, 1999.. The most abundant matrix molecule in cartilage and chondrocyte cultures is collagen. When the media is changed, large amounts of collagen would then be removed from the chondrocyte cultures. Loss of collagen may contribute to chondrocyte apoptosis. To test this, chondrocyte cultures were incubated with or without collagenase. After collagenase treatment, cultures exhibited significant apoptosis, as gauged by the amount of fragmented DNA, as compared with the chondrocyte culture without collagenase treatment ŽFig. 2a.. Addition of type I collagen to the collagenase-treated cultures inhibited chondrocyte apoptosis ŽFig. 2a.. These experiments were repeated three times and similar results were obtained each time ŽFig. 2b.. Collagenase was introduced into the suspended chondrocyte cultures and the adherent chondrocyte cultures at different concentrations, and the cultures were maintained at 378C for 24 h. Chondrocyte apoptosis in the suspended culture was promoted by colla-
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genase treatment in a dose-dependent manner ŽFig. 2c.. Collagenase had little effect on the apoptosis of the adherent chondrocytes, further suggesting that degradation of collagen was involved in the apoptosis of the suspended chondrocytes, rather than the effect of any protease contamination in the collagenase preparation. Finally, when exogenous type I collagen was added into collagenase-treated chondrocyte cultures and the cultures were maintained at 378C for 24
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h, collagen inhibited the apoptosis in suspended chondrocytes in a dose-dependent manner, but had little effect on adherent chondrocytes ŽFig. 2d.. 3.2. The roles of HA and collagen in chondrocyte interaction and apoptosis To study the function of collagen in chondrocyte] matrix interactions, adherent chondrocytes were har-
See overleaf for legend to Fig. 1
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vested and incubated with or without exogenous addition of type I collagen and collagenase. Normally, these chondrocytes started to aggregate 3 h after being seeded in petri dishes ŽFig. 3.. Exogenous addition of collagen enhanced the aggregation by forming more and larger conglomerates ŽFig. 3.. Degradation of endogenous collagen by addition of collagenase inhibited the aggregation of chondrocytes Ždata not shown.. To further test the effect of hyaluronidase and collagenase on the interaction of chondrocytes with collagen, the suspended chondrocytes were pre-incubated with or without hyaluronidase or collagenase, and then inoculated in type II collagen-coated plates. Chondrocyte attachment was analyzed. Without treatment, few cells adhered to the collagen-coated plates ŽFig. 4a.. Collagenase treatment caused a small increase in the number of cells adhering, while hyaluronidase ŽHAse. digestion resulted in a large Žmore than 20-fold. increase in the number of adherent cells. Hyaluronidase-digested and untreated chondrocytes were plated on type II collagen-coated plates and maintained in a tissue culture incubator for 2 days. The hyaluronidase-treated chondrocytes exhibited a fibroblast-like morphology and spread on the collagen-coated plates ŽFig. 4b.. The untreated chondrocytes still grew as a suspension culture ŽFig. 4b.. Another major component of cartilage is hyaluronan. To test the effects of this molecule, suspended chondrocytes were pre-incubated with collagenase and then treated with hyaluronidase. The addition of hyaluronidase inhibited chondrocyte apoptosis ŽFig. 5a.. At the concentrations used for suspended chondrocytes, hyaluronidase was also incubated with adherent chondrocytes, and they had little effect on chondrocyte apoptosis Ždata not shown.. This indicated that the effect of any possible protease contami-
nation in the hyaluronidase preparations on chondrocyte apoptosis was negligible. The suspended chondrocytes were pre-digested with collagenase and then the cells were harvested and incubated in type I collagen. Chondrocyte apoptosis was analyzed with FACScan. The results showed that chondrocyte apoptosis induced by collagenase digestion was significantly inhibited by addition of collagen ŽFig. 5b.. To test the effects of collagenase in chondrocyte apoptosis, collagenase was introduced into the suspended chondrocytes. We found that collagenase treatment caused significant apoptosis ŽFig. 5c.. The effects of type I and type II collagen on chondrocyte apoptosis were then tested by addition of collagen solution to suspended chondrocytes that had been treated with collagenase for 30 min. The cultures were maintained in an incubator for 24 h followed by analysis of cell apoptosis. Chondrocytes that had been treated with collagenase but were not incubated with collagen were used as controls. As expected, the effect of type II collagen was similar to type I collagen on inhibiting apoptosis of chondrocytes ŽFig. 5d.. 3.3. HA moderated the effect of collagen and integrin on aggregation and apoptosis Integrin b 1 , a cell surface molecule expressed by chondrocytes, binds to collagen ŽEnomoto et al., 1993; Loeser et al., 1995.. Thus, we reasoned that integrin b 1 may be involved in collagen’s mediation of chondrocyte aggregation. To test this, suspended chondrocytes were incubated with hyaluronidase, with hyaluronidase and collagenase, or hyaluronidase and anti-integrin b 1 antibody. Chondrocyte aggregation was examined with a light microscope. Hyaluronidase digestion induced aggregation of the suspended chondrocytes as compared with the untreated chondro-
Fig. 1. The effect of media-change on chondrocyte apoptosis. Ža. The suspended chicken chondrocytes were inoculated in 100-mm bacterial petri dishes at a concentration of 1 = 10 6 cellsrml Žsubconfluence ., the cultures were divided into two groups. In one group, culture media was removed by aspiration and replaced with fresh media Žmedia changed.. The other group was maintained in the same conditions Žcontrol., without the media change. The cultures were incubated at 378C in a tissue culture incubator for 24 h. The cultures were examined under a light microscope. A large amount of chondrocyte debris was detected in the cultures which were maintained in the fresh media, indicating increased cell death. Žb. Apoptosis was tested in chondrocytes cultured in petri dishes with or without media changed. Chondrocytes were collected from the culture by centrifugation, stained with PI and analyzed in a FACScan. The media-changed chondrocytes exhibited a twofold increase in apoptosis as compared with the controls Ž30% vs. 10.8% apoptosis.. Žc . Chondrocytes cultured in petri dishes were harvested 24 h after culture media was changed. Cultures not subjected to media change were used as a control. Total DNA isolated from each sample was subjected to 2% agarose gel electrophoresis and the gel was stained with ethidium bromide to visualize the DNA. DNA from cells collected after media change was fragmented Žbracket area., a characteristic property of apoptosis. Žd. Suspended chondrocytes were maintained in petri dishes, and adherent chondrocytes were cultured in tissue culture plates. At day 0, all media in both types of treatments were carefully removed, and fresh media were introduced into each dish. The cultures were maintained at 378C in an incubator. Chondrocyte apoptosis was analyzed after media change in suspended and adherent chondrocyte cultures. In suspended cultures, apoptosis increased significantly within 24 h and then declined. The adherent chondrocytes showed little apoptosis. Že. The culture media were changed at different proportions in suspended cultures Žin petri dishes. and adherent cultures Žin tissue culture plates.. The cultures were maintained at 378C for 1 day and chondrocyte apoptosis was analyzed by FACScan. Apoptosis in suspended chondrocytes was dose-dependent. The change of culture media in adherent chondrocytes had no effect on apoptosis.
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Fig. 2. The effect of collagen on chondrocyte apoptosis. Ža. Suspended chondrocytes cultured in agarose-coated tissue culture plates were incubated with or without collagenase Ž500 mgrml. at 378C for 2 h. Untreated chondrocytes were maintained as a control. Chondrocyte apoptosis was enhanced significantly by collagenase digestion Ž45% apoptosis., but exogenous addition of type I collagen Ž50 mgrml, 13% apoptosis. abolished collagenase’s effect on chondrocyte apoptosis as compared with untreated cells Ž11% apoptosis.. Žb. The experiment was repeated three times and data were analyzed statistically. Chondrocyte apoptosis caused by collagenase digestion was completely reversed by exogenous addition of type I collagen after collagenase digestion Ž n s 3, P- 0.01.. Žc . The suspended and adherent chondrocyte cultures were incubated with different concentrations of collagenase in a tissue culture incubator at 378C for 24 h. Collagenase treatment promoted apoptosis of suspended chondrocytes in a concentration-dependent manner. Žd. Suspended and adherent chondrocyte cultures were incubated with collagenase Ž2 mgrml. for 30 min at 378C. Collagenase was removed and type I collagen was added as shown. The exogenous addition of type I collagen protected suspended chondrocytes from apoptosis in a dose-dependent manner.
cytes ŽFig. 6a.. A combination of hyaluronidase and collagenase Ž collagenase first followed by hyaluronidase. reduced the effect of hyaluronidase on chondrocyte aggregation significantly. Addition of the
anti-integrin b 1 antibody at the presence of hyaluronidase also inhibited chondrocyte aggregation indicating that the antibody can block the effect of hyaluronidase.
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Fig. 3. The role of collagen in chondrocyte aggregation. Adherent chicken chondrocytes were harvested by treatment with 10 mM EDTA, collected by centrifugation and the cells were resuspended in DMEM containing 10% FBS. Type I collagen was added to the cultures at a final concentration of 50 mgrml. Untreated chondrocytes were used as controls. The cultures were maintained at 378C for 3 h. Chondrocyte aggregation was examined under a light microscope and photographed. Exogenous addition of collagen induced chondrocyte aggregation.
To test the effect of integrin]antibody interaction on chondrocyte apoptosis, suspended chondrocytes
were pre-incubated with collagenase and then inoculated in bacterial petri dishes in DMEM supplemented with 10% FBS. Type I collagen andror anti-b 1 antibody were added and the cultures were maintained in a tissue culture incubator for 24 h prior to analysis. Chondrocyte apoptosis induced by collagenase treatment was ameliorated by the addition of collagen but not by the addition of anti-integrin b 1 antibody ŽFig. 6b.. Nevertheless, addition of anti-integrin b 1 antibody partially inhibited collagen’s effects when both were added simultaneously. To test if the interaction of collagen and integrin b 1-mediated chondrocyte aggregation, suspended chondrocytes were incubated with or without collagenase and the resultant chondrocytes were analyzed for the presence of integrin b 1 by labeling the chondrocytes with a monoclonal antibody against integrin b 1. The labeled chondrocytes were analyzed in a FACScan. Treatment with collagenase resulted in enhanced staining for integrin b 1 on the cell surface ŽFig. 7a.. There are two mechanisms which may account for this result: digestion of chondrocytes with collagenase could make more integrin b 1 accessible for staining; or treatment with collagenase might upregulate the expression of integrin b 1. Since either
Fig. 4. The role of hyaluronidase in cell]matrix interactions. Ža. The suspended chondrocytes were pre-incubated with or without hyaluronidase Ž200 Urml. or collagenase Ž2 mgrml. at 378C for 30 min and then plated on type II collagen-coated plates. Untreated chondrocytes were used as controls. The cultures were maintained at 378C for 3 h. Cell attachment was determined by counting. Žb. The suspended chondrocytes were incubated with or without hyaluronidase Ž200 Urml. at 378C for 30 min, and the cells were then plated on type II collagen-coated tissue culture plates. The cultures were maintained at 378C in a tissue culture incubator for 48 h. Cell spreading was examined under a light microscope.
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Fig. 5. The effect of hyaluronidase and collagenase on apoptosis. Ža. Suspended chondrocytes were treated with collagenase Ž2 mgrml. for 30 min, and then incubated in culture media with or without hyaluronidase Ž200 Urml. at 378C in a tissue incubator for 24 h. Chondrocyte apoptosis was analyzed by FACS. Without the addition of hyaluronidase, chondrocytes underwent significant apoptosis due to collagenase digestion. Addition of hyaluronidase reduced the apoptosis. Žb. Suspended chondrocytes were pre-treated with collagenase Ž2 mgrml. at 378C for 30 min and then the treated chondrocytes were harvested by centrifugation and incubated with type I collagen Ž50 mgrml.. The cultures were maintained at 378C for 24 h and chondrocyte apoptosis was analyzed by FACS. Apoptosis induced by collagenase treatment was reduced significantly by exogenous addition of type I collagen. Žc. Suspended chondrocytes were treated with collagenase Ž2 mgrml. at 378C for 24 h. Untreated chondrocytes were used as controls. Chondrocyte apoptosis was determined. Treatment with collagenase induced chondrocyte apoptosis significantly. Žd. To compare the protecting effects of type I and type II collagen on chondrocyte apoptosis, suspension chondrocytes were pre-treated with collagenase and cells harvested as described above. Type I and type II collagen were added to the chondrocytes at a concentration of 50 mgrml, respectively. Collagenase-treated chondrocytes without addition of collagen were used as controls. Apoptosis of chondrocytes was analyzed as above. Addition of type II collagen, similar to type I collagen, inhibited chondrocyte apoptosis as compared with the controls.
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in cultures treated with or without collagenase ŽFig. 7b. and this result was confirmed by repeating the experiment, it seems unlikely that increased b 1 expression is responsible for the increased b 1 staining observed in collagenase-treated cultures. Under normal conditions, collagen probably binds to integrin b 1 in a way that obstructs the interaction of anti-integrin b 1 antibody with b 1. Collagenase treatment would thus remove this obstruction and result in enhanced b 1 staining. Another major component surrounding chondrocytes in cartilage is hyaluronan. To test the possibility that hyaluronan may also block the availability of integrin b 1 , the suspended chondrocytes were incubated with or without hyaluronidase and the resultant chondrocytes were stained for accessible integrin b 1 as described above. The result showed that hyaluronidase digestion also enhanced the availability of integrin b 1 stained by the anti-integrin b 1 antibody ŽFig. 7c.. However, hyaluronidase treatment did not enhance the expression of integrin b 1 , as analyzed by Western blot ŽFig. 7d.. This experiment was repeated three times and similar result was obtained. To assure that collagenase and hyaluronidase had no effect on integrin b 1 expression, adherent chondrocytes were incubated with collagenase and hyaluronidase for 3 days. Expression of integrin b 1 did not change in the adherent chondrocytes analyzed with Western blot assays Ždata not shown.. The results described above suggest that the interaction of collagen and integrin can modulate chondrocyte aggregation and apoptosis. Hyaluronan inhibits chondrocyte aggregation. We present a model to explain such relationships ŽFig. 8.. In this model, hyaluronan binds to aggrecan and link protein to form stable ternary complexes. These complexes mediate chondrocyte]matrix interactions by binding to chondrocyte surface through hyaluronan-binding receptors and inhibit chondrocyte aggregation. Degradation of hyaluronan will destroy the complexes and induce chondrocyte aggregation. As our experiments using the anti-integrin b 1 antibody suggest, aggregation seems to be mediated by an interaction between integrin, an integral membrane protein found on the surface of the plasma membrane, and collagen, a ubiquitous extracellular matrix molecule.
4. Discussion one could result in chondrocyte aggregation, it was necessary to test which mechanism was responsible. Cell lysate was prepared from the collagenase-treated chondrocytes and the untreated chondrocytes, and analyzed for integrin b 1 expression in Western blot stained with the same antibody as above. Since little change in the level of b 1 expression could be detected
Our studies stemmed from our observation that chondrocytes maintained as a suspension culture suffered from significant levels of cell death every time culture media were changed. Chondrocytes secrete large amounts of extracellular matrix molecules, and these spent culture media contained collagen,
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Fig. 6. Interaction of collagen and integrin b1 modulated chondrocyte aggregation and apoptosis. Ža. Suspended chondrocytes were incubated with or without hyaluronidase ŽHAse, 200 Urml., collagenase Žcols, 0.2 mgrml., and monoclonal antibody against integrin b1 Ž20 mgrml., as shown in the figure, at 378C for 3 h. Chondrocyte aggregation was examined under a light microscope and photographed. Žb. Suspended chondrocytes were pre-incubated with collagenase Ž2 mgrml. at 378C for 30 min. The chondrocytes were then inoculated in bacterial petri dishes in DMEM supplemented with 10% FBS. Reagents were added to the cultures at a concentration of 50 mgrml Žtype I collagen. or 10 mgrml Žanti-b 1 antibody.. The cultures were maintained in a tissue culture incubator for 24 h and then analyzed for chondrocyte apoptosis. Type I collagen prevented apoptosis to a great degree, but anti-b 1 antibody interfered with this effect. Antibody to b1 alone had no effect.
hyaluronan and aggrecan. To investigate if these molecules play a role in chondrocyte growth, we tested the roles of collagen and hyaluronan in chondrocyte survival, since collagen is the most abundant molecule in the extracellular matrix of cartilage ŽHollander et al., 1995; Matyas et al., 1997., and hyaluronan is the central molecule in the aggrecan]hyaluronan-link protein ternary complexes. Our findings suggest that collagen is very important in chondrocyte viability, and hyaluronan, through its effects on chondrocyte aggregation, interfered with collagen’s effects on apoptosis. Degradation of collagen and reduced chondrocyte number are associated with a number of joint diseases. For example, in the late stages of osteoarthritis,
collagen is severely degraded due to proteolytic activity. Severe arthritis is also characterized by irregular chondrocyte distribution: a large proportion of chondrocytes disappear from the cartilage while the small groups of chondrocytes are easily detected. However, the relationships among the collagen degradation, chondrocyte disappearance and chondrocyte conglomeration are not known. Our results here indicate that collagen degradation induces chondrocyte apoptosis while the conglomeration of small groups of chondrocytes protects those chondrocytes from apoptosis. This interpretation is supported by the recent report that chondrocytes obtained from collagen knock-out mice undergo apoptosis ŽYang et al., 1997.. The role of integrin has been studied extensively. It
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Fig. 7. Collagen and hyaluronan hindered detection of integrin b1 . Ža. Suspended chondrocytes were incubated with or without collagenase Ž2 mgrml. at 378C for 30 min. The cells were labeled with monoclonal antibody against integrin b1 and analyzed on FACScan. Unlabeled chondrocytes were used as controls Žleft peak.. Digestion with collagenase enhanced the staining of integrin b1 significantly, resulting in an increase in fluorescence intensity Žmiddle peak, without collagenase; right peak, with collagenase.. Žb. Cell lysate was prepared from chondrocytes treated with or without collagenase as described in Ža. and analyzed in a Western blot stained with the monoclonal antibody against integrin b1 . Untreated cells served as controls. The density of immunostaining was similar for both treatments. Žc. Suspended chondrocytes were incubated with or without hyaluronidase Ž200 Urml. at 378C for 30 min. The cells were labeled with antibody to integrin b1 and analyzed on FACScan. Unlabeled chondrocytes were used as controls Žleft peak.. Hyaluronidase digestion resulted in enhanced staining of integrin b1 Žmiddle peak, without hyaluronidase; right peak, with hyaluronidase.. Žd. Cell lysate was prepared from chondrocytes treated with or without hyaluronidase as described in Žc. and analyzed in a Western blot stained with the antibody against integrin b1. Untreated cells served as controls. The band density was similar in both samples.
binds to fibronectin and modulates signal transduction. Insufficient interaction of integrin and fibronectin induces apoptosis of endothelial cells ŽMeredith et al., 1993; Bates et al., 1994; Frisch and Francis, 1994; Ruoslahti and Reed, 1994; Chen et al., 1997; McGill et al., 1997.. It is known that integrin also binds to type II collagen ŽEnomoto et al., 1993.. The effect of their interaction is not clear, but it may be critical. We show here that collagenase digestion and treatment with anti-integrin b 1 antibody produce a similar effect, an inhibition of chondrocyte aggregation. Furthermore, we demonstrated using FACS analysis that collagenase digestion seems to enhance the accessibility of integrin b 1 on the chondrocyte surface. This observation cannot be explained by increased expression of integrin b 1 , as similar levels of expression were observed in treated and untreated cells on Western blot. Taken together, these suggest that the interaction of collagen and integrin b 1 is responsible for chondrocyte aggregation. Degradation of collagen induced chondrocyte apoptosis which was not reversed
by addition of the anti-integrin b 1 antibody. In fact, simultaneous addition of collagen with the antibody to collagenase-treated chondrocytes resulted in more severe apoptosis as compared to the addition of collagen alone. The interaction of collagen and integrin b 1 may be important to chondrocyte survival and thus addition of collagen to collagenase-treated chondrocytes brought the apoptosis to a basic level Ž; 12%, Fig. 6b.. Recently, it has been demonstrated that collagen degradation in osteoarthritis is the result of collagenase activity ŽHollander et al., 1995.. The collagenases are synthesized and secreted by chondrocytes, and the pericellular collagen would be the most likely substrate for these enzymes. As a result of collagenase activity, collagen]integrin interaction would be disrupted and the chondrocytes, without any collagen to bind, would undergo apoptosis. In addition to collagen, other important molecules in cartilage are aggrecan, link protein and hyaluronan, which form ternary complexes and help to maintain the stable three-dimensional cartilage networks, necessary for joint function ŽPerkins et al., 1989;
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Fig. 8. A model for inhibition of collagen]chondrocyte interaction by hyaluronan. Hyaluronan ŽHA. binds to aggrecan and link protein ŽLP. forming stable ternary complexes. The complexes bind to chondrocyte surface through hyaluronan-binding receptor ŽHAR. on the cell surface. These complexes are important in chondrocyte]matrix interaction and they prevent the aggregation of chondrocytes. Degradation of hyaluronan destroys the complexes. As a result, chondrocytes conglomerate through the interaction of collagen and integrin.
Barry et al., 1992; Li et al., 1993; Neame and Barry, 1993; Margolis and Margolis, 1994.. We have demonstrated that the aggrecan]hyaluronan-link protein ternary complexes are important in mediating chondrocyte]matrix interaction ŽYang et al., 1998.. However, the role of these ternary complexes in chondrocyte aggregation and apoptosis is complicated in the studies presented here. Degradation of hyaluronan with hyaluronidase induces chondrocyte aggregation, and the aggregation inhibits apoptosis. Hyaluronidase also degraded chondroitin sulfate, a major component of chondrocyte products, and this also enhanced chondrocyte aggregation. The aggregation also reduced the level of chondrocyte apoptosis. It is conceivable that, even in the presence of hyaluronidase, newly synthesized collagen could mediate chondrocyte aggregation and, therefore, inhibit apoptosis. It should be noted that degradation of hyaluronan inducing cell aggregation might involve other mechanisms. For example, removal of hyaluronan will allow other adhesion molecules on the cell surface to interact with each other. In the chondrocyte]substratum interaction assay, we sought to directly test the role of hyaluronan in chondrocyte]collagen interaction by introducing hyaluronidase into the suspended chondrocyte cultures to degrade hyaluronan. Chondroitin sulfate might also be degraded and this might result in better interactions of chondrocytes with collagen. Indeed, chondrocytes treated with hyaluronidase bound to the collagen-coated plates strongly. Hyaluronan interfered with the interaction of chondrocytes with colla-
gen. The result of FACScan analysis showed that hyaluronidase digestion enhanced immunostaining of integrin b 1 in the chondrocyte surface. We also observed that collagenase digestion enhanced the interaction of chondrocytes with the collagen-coated plates. Removal of collagen from the chondrocytes increased the accessibility of endogenous integrin b 1 and enhanced chondrocyte binding to collagen-coated plates. The result of FACScan analysis also demonstrated that collagen inhibited the immunostaining of integrin b 1. In normal chondrocyte cultures, addition of hyaluronan had no effect on chondrocyte apoptosis, because collagen]chondrocyte interaction plays a dominant function in protecting chondrocytes from apoptosis. In collagenase-treated chondrocyte cultures, however, apoptosis is high. Addition of hyaluronidase also inhibited chondrocyte apoptosis and this might be due to interactions between chondrocytes and newly synthesized collagen, in the absence of hyaluronan. The newly synthesized collagen-mediated chondrocyte aggregation and small conglomerates could be seen. Our results here demonstrate that hyaluronan, which binds to aggrecan and link protein to form stable ternary complexes, plays an important function in inhibiting chondrocyte aggregation, and collagen plays an important role in protecting chondrocytes from apoptosis. Perhaps, degradation and insufficient production of the hyaluronan]aggrecan-link protein ternary complexes cause chondrocyte aggregation. Degradation of collagen by proteolytic activities induces chondrocyte apoptosis. Since these phenomena are associated with many kinds of joint diseases, further studies are required to uncover the detailed mechanisms by which diseases develop and to find ways to block such processes.
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