Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9)

Rita Dreier1, Shona Wallace1, Susanne Fuchs2, Peter Bruckner1 and Susanne Grässel1,*

1 Institut für Physiologische Chemie & Pathobiochemie
2 Klinik und Poliklinik für allgemeine Orthopädie, Westfälische Wilhelms-Universität Münster, Germany

*Author for correspondence (e-mail: graesse{at}uni-muenster.de)

Accepted July 17, 2001


    SUMMARY
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.

Key words: Serum-free coculture, Rheumatoid arthritis, Monocytes, Agarose, MMP-9, Activation


    INTRODUCTION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammatory joint diseases such as rheumatoid arthritis are characterized by a massive invasion of leukocytes into joint capsules. This results in a drastic thickening of the synovial membranes by the formation of a proliferative tissue containing predominantly macrophages and fibroblast-like synoviocytes. As the disease progresses, the joint cartilage surface is covered by a pannus deriving from the inflamed synovial tissue and is infiltrated by macrophages (Hansch et al., 1996; Bresnihan, 1999). This leads to a destruction of the cartilage underlying the pannus. In addition, a shift towards a catabolic state occurs in the metabolism of the cartilage cells themselves, affecting the balance of matrix macromolecules, weakening the structural integrity, and hence causing functional deterioration. In animal models of arthritis there is increased degradation and a reduction of synthesis of matrix macromolecules, including proteoglycans and collagen II, as well as chondrocyte death (Dodge and Poole, 1989; van de Loo et al., 1995). An essential role in inflammatory joint disease is commonly assigned to monocytes and macrophages because the intensity of macrophage infiltration into the synovium correlates with the progression of rheumatoid arthritis (Mulherin et al., 1996; Burmester et al., 1997). The macrophages recruit and activate further peripheral blood monocytes by secretion of chemotactic and inflammatory cytokines (Koch et al., 1991; Chu et al., 1992; Koch et al., 1992; Hahn et al., 1993). There is evidence that cartilage destruction and the maintenance of synovial inflammation are separately controlled (van den Berg and van Lent, 1996).

Cartilage destruction in arthritis is effected, at least in part, by proteolytic enzymes produced by leukocytes and cartilage cells. The proteinases include collagenase 1 (matrix metalloproteinase 1 (MMP-1)), stromelysin-1 (MMP-3) and gelatinase B (MMP-9) (Tetlow et al., 1993; Tetlow et al., 1995), all of which are capable of degrading the molecular components of cartilage matrix suprastructures, the fibrils and the hydrated extrafibrillar matrix. As a result, the load-bearing capacity of joint cartilage is compromised because, in essence, tensile and compressive strengths of the tissue are brought about by the fibrils and the extrafibrillar matrix, respectively.

Matrix metalloproteinases are essential for the remodelling of extracellular matrices under pathological, as well as under normal, physiological conditions. More than 20 mammalian MMP-family members, all of them Zn2+-dependent enzymes, have been identified. Activity of MMPs is regulated at the transcriptional and translational levels. Post-translational events are also crucial, as the enzymes are produced as inactive precursors and are proteolytically processed to their active forms in the extracellular space (Llano et al., 1997; Benbow and Brinckerhoff, 1997; Murphy and Knäuper, 1997). Activation of pro-MMPs to their fully active form proceeds in general in a stepwise manner. Exemplified by MMP-1 and -3, proteinases such as trypsin or plasmin first attack the MMP’s susceptible region located in the propeptide. The removal of a portion of the propeptide results in conformational changes and renders both MMPs to rapid cleavage by MMP-3 to generate stable, fully active enzymes. Both MMPs, in particular MMP-3, are then involved in the activation of pro-gelatinase B by binding to the precursor enzyme/TIMP-1 complex (Nagase et al., 1991; Nagase, 1997). Several MMPs are expressed both in normal and in diseased cartilage (Lark et al., 1997; Smith, 1999) and can be detected at elevated levels in synovial fluids from arthritis patients, suggesting a role in the cartilage destruction associated with joint disease (Fosang et al., 1998). Degradation of cartilage includes breakdown of aggrecan, the major proteoglycan constituent of the extrafibrillar matrix of hyaline cartilage as well as of the collagen-containing fibrillar network by specific members of the MMP-family, but not by most other proteinases (Billinghurst et al., 1997).

However, unlike the ubiquitous occurrence of inflammatory cells in the organism, most of the inflammatory activity associated with arthritis is in the joints, which suggests that cartilage may take an active role in the disease process. We conjectured that an extensive autocrine and paracrine communication between all cell types of inflamed joints results in a mutual control of anabolic and catabolic responses of each cell type. The regulatory contribution of chondrocytes still remains to be established. For these reasons, we have studied the interactions of human articular chondrocytes and human monocytes/macrophages in a well-defined coculture system. We have adapted our serum-free chondrocyte cultures in agarose gels (Tschan et al., 1990) to cocultures of chondrocytes with monocytes or macrophages. Employing this new tool, we have investigated paracrine interactions in vitro between the two cell types that affect metabolism and turnover of cartilage matrix components. Chondrocytes and monocytes or macrophages mutually induce alterations in the expression profiles of degradative enzymes and their inhibitors, accompanied by concurrent changes in the production and/or activation of several MMP-proteins. In the case of MMP-9 we were able to show a direct paracrine interaction of chondrocytes and leukocytes, which is of great interest for degenerative cartilage diseases such as rheumatoid arthritis: the activation of monocyte- and macrophage-produced MMP-9 is dependent on chondrocyte-derived factors.


    MATERIALS AND METHODS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Polyclonal antibodies to human MMP-1, -3, -9, MT1-MMP and TIMP-2 are purchased from Chemicon International Inc., Hofheim/Ts, Germany. Primer pairs (Table 1) used for quantitative PCR were purchased from Biometra, Göttingen. MMP-3 inhibitor II (N-isobutyl-N-(4-methoxy-phenyl-sulfonyl)-glycylhydroxamic acid) was purchased from Calbiochem, Bad Soden, Germany.


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Table 1.
 
Isolation of chondrocytes from human articular tissue
Cartilage was removed under sterile conditions from femoral condyles or from femoral heads of patients aged 40 to 65 years undergoing surgery unrelated to inflammatory joint disease. The tissue fragments were cut into 1 mm slices and were washed four to five times with Krebs-buffer (Dehm and Prockop, 1971) containing 100 units/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml fungizone to reduce the risk of microbial contamination. After a further two rinses with calcium-free Dulbecco’s modified eagles medium (DMEM, Gibco-BRL, Karlsruhe, Germany), slices were cut into small pieces and were incubated for 16-24 hours with 1.5 mg/ml collagenase B (Roche, Mannheim) and 1 mM cysteine in DMEM. The cell suspension was filtered to remove debris and washed three times with calcium-free DMEM. The cells were then suspended in 1 ml 2% low-melting-temperature agarose (FMC, Rockland, ME) in DMEM (resulting in a final concentration of 0.5%) at a density of 0.7-1x106 cells/ml. Cells were seeded onto 35 mm Petri-dishes precoated with 1% agarose autoclaved in water and were allowed to sediment at 37°C to the plane at the interface between the precoating and cell-containing agarose layers. To allow solidification of agarose, the dishes were exposed to 4°C for 15 minutes and then cultured at 37°C, 5% CO2 and 99% humidity (Tschan et al., 1990). Serum-free DMEM was supplemented with 60 µg/ml ß-aminopropionitrile fumarate, 25 µg/ml sodium ascorbate, 1 mM cysteine, 1 mM pyruvate, 100 units/ml penicillin and 100 µg/ml streptomycin. Prior to coculturing with monocytes or macrophages, chondrocytes were kept alone for up to 1 week under above conditions in serum-free agarose.

Preparation of monocytes and macrophages from human buffy coats
Monocytes were isolated from human buffy coats as described previously (Boyum, 1968; Feige et al., 1982). Briefly, buffy coats were layered over Ficoll separating solution (Biochrom, Berlin). After centrifugation for 45 minutes at 540 g, the cells at the serum-Ficoll interphase were collected, washed with PBS, and layered over a preformed Percoll gradient. After centrifugation for 45 minutes at 560 g, the monocyte cell band was aspirated and washed with PBS. As described in detail for chondrocytes (Benya and Shaffer, 1982; Tschan et al., 1990), monocytes were suspended in 0.5% (final concentration) low-melting agarose in DMEM at a density of 2.5-2.7x106 cells/ml. Gels of 0.7 ml were cast into 35 mm Petri dishes.

Monocyte-derived macrophages were generated by the method of Krause and coworkers (Krause et al., 1998). Briefly, human peripheral blood monocytes were cultivated as adherent monolayers for 8 days in RPMI-1640 supplemented with 2% human serum from blood group AB donors.

Coculture system
To set up the chondrocyte-monocyte coculture system, agarose gels containing the monocytes were removed from the culture dishes, inverted and placed on top of similar agarose gels containing chondrocytes. For coculture of macrophages with chondrocytes, adherent macrophages were washed four times with serum-free DMEM and overlayered with agarose gels containing chondrocytes. Cocultures were maintained for up to 10 days; culture medium was changed every 2-3 days. Monocyte viability was assessed by trypan blue exclusion assay. About 400 cells per dish were directly counted with a grid ocular at x100 magnification. For control experiments we added macrophage conditioned medium to chondrocyte cocultures. In some experiments 50 µg/ml plasmin, 50 µg/ml plasminogen, 1,5 µg/ml aprotinin (Sigma-Aldrich, Deisenhofen) and/or 15-225 µM MMP-3 inhibitor II (N-isobutyl-N-(4-methoxy-phenyl-sulfonyl)-glycylhydroxamic acid (Ki 130 nM) was added to the serum-free culture medium up to 24 hours.

Analysis of collagen and aggrecan synthesis
For analysis of proteoglycan metabolism, cocultures and chondrocyte monocultures were metabolically labeled on day 6 with 2 µCi/ml [14C]-L-serine (166 mCi/mmol) in serine-deficient DMEM for 48 hours. Prior to labeling, cells were washed twice with PBS and were incubated for 2-4 hours in 1 ml serine-deficient DMEM. To extract proteoglycans from chondrocyte gels, culture media were stored until use at –20°C and the agarose-gels containing monocytes were discarded. Gel layers containing the chondrocytes were washed twice with PBS and were subsequently extracted for 48 hours at 4°C with 0.05 M sodium acetate buffer, pH 5.2, containing 4 M guanidinium-HCl and 10 mM EDTA. After centrifugation for 15 minutes at 17300 g, the GuHCl-extract was stored until use at –20°C.

Radiolabeled materials in media or GuHCl-extracts were precipitated once or twice, respectively, with 9 volumes of ice-cold 96% EtOH overnight at –20°C and were recovered by centrifugation. Pellets were resuspended in 10 mM Tris/HCl pH 8.0 and aliquots were subjected to liquid scintillation counting. Samples were incubated overnight at 37°C with 0.5 units of endo-ß-galactosidase (Roche Diagnostics, Mannheim/Germany) and 1 unit ABC-chondroitinase (Proteus vulgaris, Seikagaku Corp., Tokyo). Fivefold SDS-PAGE loading buffer was added to give final concentrations of 0.125 M Tris/HCl, pH 6.8, 2% SDS, 15% glycerol and 0.1% bromphenol blue. Samples were heated to 95°C and were subjected to SDS-PAGE on 3.5%-12% polyacrylamide gradient gels. The gels were stained with Sypro Red protein gel stain (Molecular Probes, Eugene, OR), dried and subjected to fluoroimaging (Storm, Amersham Pharmacia Biotech, Freiburg, Germany) prior to autoradiography.

For analysis of collagen metabolism cocultures and monocultured chondrocytes were metabollically labeled on day 6 with 100 µCi [35S] Translabel (methionine/cysteine) (1175 Ci/mmol) and 1 µCi [14C] proline (275 Ci/mmol) for 48 hours in methionine/cysteine/proline-free DMEM. Radiolabeled collagens were extracted from cultures after limited digestion with pepsin as described (Böhme et al., 1995). Samples were heated to 95°C in SDS-PAGE loading buffer including 50 mM DTT (dithiothreitol) and subjected to SDS-PAGE on 3.5%-12% polyacrylamid gradient gels which were treated as above.

Extraction of total RNA
After 6 days of coculture with chondrocytes, total RNA was extracted from agarose-embedded cells as described (Szüts et al., 1998). Briefly, agarose-layers containing monocytes and chondrocytes were separately homogenized in 3 M LiCl/6 M urea with a polytron (Kinematica, Lucerne, Switzerland). After partial removal of agarose by centrifugation, the supernatants were kept overnight at 4°C and RNA was pelleted by centrifugation (17300 g; 30 minutes). After digestion with proteinase K, total RNA was purified by phenol/chloroform extraction and precipitated with ethanol at –20°C.

Quantitative ‘real-time’- RT-PCR
Prior to RT-PCR total RNA was treated with 10 units RNase B free DNase (Roche Diagnostics, Mannheim) for 15 minutes at 37°C. For the RT-reaction, 0.5 µg of RNA and 190 ng of random hexamer primers (Roche Diagnostics, Mannheim) were dissolved in 10 µl of water and were denaturated at 70°C for 10 minutes. Four µl of fivefold concentrated RT buffer (Gibco-BRL, Karlsruhe), 2 µl 10 mM dNTPs (Qbiogene, Heidelberg), 1 µl 0.1 M DTT (GibcoBRL, Karlsruhe), 35 units of RNase-inhibitor and 200 units of murine moloney virus reverse transcriptase (200 U/µl, Gibco-BRL, Karlsruhe) were combined with the RNA and were adjusted with H2O to a final volume of 20 µl. The RT-reaction was allowed to continue for 1 hour at 37°C and stopped by heating at 95°C for 5 minutes.

For quantitative PCR, 1 µl cDNA was supplemented with 5 µl 10x SybrGreen PCR-buffer, 6 µl 25 mM MgCl2, 4 µl dNTP blend (2.5 mM of each dCTP, dATP, dGTP and 5 mM of dUTP), 1.5 µl specific primer each (10 pmol/µl), 0.25 µl Ampli-Taq-Gold polymerase and 0.5 units AmpErase (PE Biosystems GmbH, Weiterstadt, Germany) and was diluted with H2O to a final volume of 50 µl. PCR was performed using a 5700 GeneAmp thermocycler (PE Biosystems GmbH). Cycling conditions were 2 minutes at 50°C and 10 minutes at 94°C for the first cycle, and 15 seconds at 94°C and 1 minute at 60°C for 40 subsequent cycles. Relative gene expression levels were assessed with the {Delta}{Delta}CT-method as described by the manufacturer (PE-Biosystems GmbH) using GAPDH as an internal standard. All PCR-products were subcloned and sequenced for identification. Primer sequences for PCR reactions are shown in Table 1.

Gelatin zymography
Contents of total protein in culture media were determined by the bicinchoninic acid protein assay (Smith et al., 1985). Quantities of medium containing 145 µg of protein were mixed with equal volumes of twofold concentrated sample loading buffer (2 mM EDTA, 2% SDS, 20% glycerol, 0.02% bromophenol blue, 20mM Tris/HCl, pH 8.0) and subjected to electrophoresis on a 0.1% gelatin-containing 4.5-15% gradient SDS-polyacrylamide gel. The gels were washed twice for 30 min in 2.5% Triton-X 100, were rinsed in distilled water and were developed for 16 hours at 37°C in 50mM Tris/HCl pH 8.5 containing 5mM CaCl2. Finally, gels were stained with Coomassie brilliant blue R250 (Serva, Heidelberg) to visualize protease activity and were photographed.

Immunoblotting
TCA-precipitated medium samples from mono- and cocultures were subjected to electrophoresis on 10% or 4.5-15% SDS-polyacrylamide gels according to their protein content (Smith et al., 1985). Prestained protein standards within the range of 207 kDa to 7.5 kDa were used as molecular weight markers (BIO-RAD, München). Proteins were electrotransferred at a constant current of 495 mA to nitrocellulose filters for 4 hours at 4°C. The filters were blocked with Tris buffered saline 20 mM Tris/HCl, 137 mM sodium chloride, 0.1% Tween-20 (TBS-T), containing 5% dried milk at 4°C for 4-5 hours and incubated with polyclonal antibodies to matrix metalloproteinases (diluted as recommended by the manufacturer) in TBS-T/2% dried skim milk at 4°C overnight. Fifteen minute washes were performed twice with TBS-T, once with TBS plus 0.5% Tween-20, once with TBS-T and once with TBS. The filters were incubated for 1-1.5 hours with secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (Kirkegard, Gaithersburg, MD), diluted 1:5000 in TBS-T/2% dried milk at room temperature. After washing (as above) the filters were developed with ECLplus-chemiluminescence substrate (Amersham Pharmacia Biotech, Freiburg) and detection was performed by short exposures to blue-light sensitive autoradiography film.


    RESULTS
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 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coculture of chondrocytes with monocytes or macrophages
Human articular chondrocytes were cultured without serum for up to 2 weeks in 0.5% agarose at a density of 0.75-1x106 cells/ml (Tschan et al., 1990). The differentiated cartilage phenotype is stably expressed under these rather than other, more commonly used, culture conditions. Agarose gels containing 2.3-2.5x106 monocytes/ml or macrophages adhering to culture dishes and covered by agarose gels were cocultured with articular chondrocytes as schematically represented in Fig. 1. This culture arrangement effectively prevented direct contact between the two cell types but allowed communication by soluble mediators. The consequences of this paracrine interaction could be interpreted satisfactorily because serum was strictly omitted from the culture media. The development of individual cells could be separately monitored by revising the same light microscopic fields during the entire culture period. Under these conditions, migration of neither chondrocytes nor leukocytes was observed microscopically. In control experiments, macrophage conditioned medium was added to chondrocyte monocultures to assess whether observed pro-MMP-9 activating effects were due to direct cell-cell contacts of chondrocytes and macrophages. No differences between chondrocytes cocultured with macrophages and those supplied with macrophage conditioned medium could be detected.



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Fig. 1. Schematic representation of the serum-free coculture systems. (A) Monocyte/chondrocyte coculture system. Both chondrocytes (panel A, left phase contrast micrograph) and monocytes (panel A, right phase contrast micrograph) were embedded into agarose gels, stacked and cultured up to 10 days in 35 mm dishes in serum-free DMEM containing 60 µg/ml ß-aminopropionitrile fumarate, 25 µg/ml sodium ascorbate, 1 mM cysteine and 1 mM pyruvate (complete medium). (B) Macrophage/chondrocyte coculture system. Monocyte-derived macrophages (panel B, right phase contrast micrograph) were generated as adherent monolayer in a 35 mm dish and overlayered with a chondrocyte containing agarose layer (panel B, left phase contrast micrograph) as described in Materials and Methods. The cells were cultured up to 10 days in complete medium (see panel A).

 
For their survival, most if not all types of cells require a conducive environment established by the cells themselves or by other parts of the organism (Jacobson et al., 1997). When cultured without serum, monocytes (Fig. 2) and macrophages survived substantially longer in cocultures than in monocultures. Thus, unlike chondrocytes (Bruckner et al., 1989), monocytes and macrophages did not establish their own survival conditions by secreting autocrine mediators. However, chondrocyte-derived factors not investigated further had a life-supporting effect on monocytes and macrophages in cocultures.



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Fig. 2. Monocyte viability in percent of living cells for 2 to 10 days. At each time point, monocytes were stained with trypan blue and cells were revised throughout the cultures in representative preselected micrographic fields at x100 magnification. More than 400 cells per field were counted using a grid ocular. Diamonds, monocytes cultured alone; squares, coculture of monocytes with chondrocytes; n=3.

 
By contrast, neither monocytes nor macrophages markedly altered the numbers of viable chondrocytes in coculture for at least two weeks. Chondrocytes rarely divided to form occasional cell pairs and also did not undergo extensive cell death. As judged by phase contrast microscopy, coculture with monocytes or macrophages also did not noticeably affect their morphology.

Cocultured monocytes reduce aggrecan and collagen II protein expression and increase aggrecan degradation
It is well established that synthesis and degradation of cartilage matrix are both altered in inflammatory joint disease. Activated monocytes and macrophages produce matrix-degrading proteinases. At the same time, they secrete paracrine signals instructing chondrocytes to reduce synthesis and to increase degradation of matrix components, including aggrecan and collagen II. To ascertain the validity of our in vitro model, the metabolism of these matrix macromolecules was studied in serum-free cocultures. After 6 days of culture, the core proteins of newly synthesized proteoglycans were metabolically labeled for 48 hours with [14C]-serine. Labelled macromolecules were extracted from gels containing chondrocytes, cultured either alone or with monocytes, and digested overnight with chondroitinase ABC and ß-endo-galactosidase. Monocytes cultured alone and all culture media were treated likewise. The proteins were subjected to SDS-PAGE on 3.5%-12% gradient gels, followed by autoradiography. Aggrecan gave prominent signals because it is an abundant chondrocyte product and because it is rich in serine residues. Its intact core-protein migrated with an apparent molecular mass of about 490 kDa (Fig. 3, panel A), and was identified by comparison with purified and chondroitinase/endo-ß-galacosidase-treated bovine aggrecan stained with Sypro Red protein stain (Fig. 3A, lane 1). Additional bands, presumably corresponding to discrete degradation products of aggrecan, were also visible. These included a doublet of 230 and 240 kDa, as well as a band corresponding to 58 kDa. The agarose layers of monocultured chondrocytes (Fig. 3A, lane 3) and chondrocytes cocultured with monocytes (lane 4) contained comparable amounts of intact radiolabelled aggrecan core protein, and the culture media of chondrocyte monocultures also gave a strong signal (lane 6). By contrast, the relative amounts of intact aggrecan were substantially reduced in the media of cocultures (lane 7). The doublet produced by degraded aggrecan appeared in all culture media (lanes 6, 7), albeit at a lower level in those of chondrocyte monocultures (lane 6). The agarose layers of the mono- and coculture (lanes 3, 4) also contained these degradation products, although the latter contained a significantly higher amount. Further degradation of aggrecan producing the 58 kDa fragment in the culture media was only detected in cocultures (lane 7). Therefore, chondrocytes in serum-free culture can rapidly degrade their newly synthesized aggrecan and this effect is greatly enhanced in cocultures with monocytes.



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Fig. 3. Protein expression and degradation of aggrecan and collagen II in mono- and cocultures. (A) [14C]-serine-labeled proteoglycans extracted from cells (lanes 2-4) and culture media (lanes 5-7) were digested with ABC-chondroitinase and ß-endo-galactosidase before being subjected to 3.5-12% gradient SDS-PAGE under nonreducing conditions. Lane 1: 30 µg purified bovine aggrecan stained with Sypro Red protein stain (standard); lanes 2, 5: monocyte monoculture; lanes 3, 6: chondrocyte monoculture; lanes 4, 7: coculture of chondrocytes with monocytes. Stars indicate degradation products. (B) [35S]-methionine/cysteine and [14C]-proline-labelled collagens were extracted from cells plus culture media and subjected to 3.5-12% gradient SDS-PAGE under reducing conditions. Lane 1: chondrocyte monoculture; lane 2: coculture of chondrocytes with monocytes; lane 3: 15 µg purified bovine collagen II stained with Sypro Red protein stain (standard).

 
Radiolabeled collagen II protein was present at progressively reduced levels in cocultures with monocytes cultured up to 30 days, indicating that the metabolism of collagen II was also affected under these circumstances. However, after 6 days of culturing, differences in protein levels between mono- and cocultures were still minor (Fig. 3B, lanes 1, 2).

Monocytes also downregulate chondrocyte expression of aggrecan and collagen II at the transcriptional level. Total RNA was extracted from chondrocytes kept in mono- or cocultures for 6 days (Szüts et al., 1998), and steady-state levels of mRNA for aggrecan and {alpha}1(II)-chains of collagen II were determined by real-time PCR and evaluation by the {Delta}{Delta}CT-method normalized by GAPDH-expression (see Materials and Methods). Steady-state expression of aggrecan-mRNA in cocultured chondrocytes was reduced to 10% of the monoculture controls. The corresponding value for ({alpha}1) collagen II mRNA-levels was about 50% of the controls.

Taken together, the metabolic parameters observed in our cocultures are consistent with well-known effects of monocytes/macrophages on chondrocytes in inflammatory arthritis and, hence, warrant further studies on paracrine interactions between chondrocytes and monocytes or macrophages in our culture models.

Coculturing of chondrocytes with monocytes or macrophages leads to new protease activities in media
Gelatinolytic activities in coculture media were compared with monoculture controls by gelatin zymography sensitive to picogram levels of gelatinases. Aliquots of media from mono- and cocultured chondrocytes, normalized to their total protein content, produced a band corresponding to a protein with an apparent molecular mass below 66 kDa (Fig. 4A, lanes 5-9). The enzyme causing this band is presumably pro-MMP-2, because it comigrated with a gelatinolytic protein in media of melanoma cells kept in collagen gels known to content latent and active MMP-2 (C. Mauch, personal communication). Two bands with proteolytic activity occurred slightly above the 45 kDa standard and were attributed to the 53 and 51 kDa forms of pro-MMP-1 by immunoblotting (Fig. 5A, lanes 2, 3). A further double band with a molecular mass of around 116 kDa was not identified, but may correspond to pro-MMP-2 complexed with isoforms of TIMPs. Media conditioned by macrophages for 24 hours produced a single prominent band with an electrophoretic mobility slightly less than that of a 97 kDa standard protein (Fig. 4A, lane 1). This protein was identified as pro-MMP-9 by immunoblotting (Fig. 5C, lane 1). Strikingly, however, coculturing of monocytes or macrophages with chondrocytes led to the production of two additional gelatinolytic molecules with an electrophoretic mobility slightly less than that of pro-MMP-9 (Fig. 4A, lane 5). The two polypeptides reacted with antibodies to MMP-9 in immunoblotting (Fig. 5C, lane 3) and could thus be attributed to the 86 kDa intermediate and the 82 kDa active forms of MMP-9. These polypeptides did not occur in media of monocytes or macrophages cultured alone (Fig. 4A, lane 1) and, in addition, were not produced by chondrocytes (Fig. 4A, lane 8). To examine the activation mechanism of pro-MMP-9, culture media were supplemented with 50 µg/ml plasmin. In monocultured macrophages, addition of plasmin caused only incomplete activation of MMP-9 (Fig. 4A, lane 3), which was abolished by further addition of aprotinin, a plasmin inhibitor (Fig. 4A, lane 4). Activation of pro-MMP-9 in cocultures was not inhibited by aprotinin (Fig. 4A, lane 6). However, addition of plasmin to cocultures led to a complete activation of pro-MMP-9 to the active 82 kDa form of MMP-9 (Fig. 4A, lane 7). Under these conditions, proteolytic activation also occurred in pro-MMP-1 and led to active 42 and 44 kDa enzymes (Fig. 4A, lane 7) identified by immunoblotting with antibodies to MMP-1 recognizing all forms. In contrast to plasmin (Fig. 4B, lane 2) plasminogen (Fig. 4B, lane 3) had no pro-MMP-9 activating effect in coculture. MMP-3 inhibitor II, however, prevented pro-MMP-9 activation in a dose-dependent manner (Fig. 4C, lanes 2-5).



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Fig. 4. Gelatin zymographic analysis of culture medium. Activators and inhibitors were added directly at the beginning of culture. Medium aliqouts were collected after a culture period of 20 hours and then subjected to SDS-PAGE under nonreducing conditions on a 4.5-15% gradient gel containing 1% gelatin. The zymograph was developed and stained as described in Materials and Methods. (A) Lanes 1-4: medium of monocultured macrophages; lanes 5-7: medium of cocultures with chondrocytes and macrophages; lanes 8-9: medium of monocultured chondrocytes. Plasmin and/or aprotinin was added as indicated. (B) Lanes 1-3: coculture medium. Plasmin or plasminogen was added as indicated. (C) Lanes 1-5: coculture medium. MMP-3 inhibitor II was added as indicated.

 


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Fig. 5. Immunoblot analysis of culture medium from cocultures and monocultures. Medium aliquots were subjected to SDS-PAGE on 4.5-15% gradient gels under reducing conditions. Proteins were transferred to nitrocellulose and analyzed with specific antibodies recognizing pro- and active forms of: (A) MMP-1, (B) MMP-3, (C) MMP-9 and (D) TIMP-2. Lane 1: monocyte monoculture, lane 2: chondrocyte monoculture, lane 3: coculture of chondrocytes with monocytes. Stars indicate the active enzyme forms.

 
Analysis of the MMPs in coculture medium by immunoblotting
Metalloproteinases and their inhibitors were also investigated by immunoblotting of proteins in conditioned media after different culture regimens. Antibodies to MMP-1, MMP-3, MMP-9 and TIMP-2 were used to detect specific protein bands (Fig. 5). The antibody to MMP-1 (collagenase-1) recognized two bands at 51 and 53 kDa, which represented the unglycosylated and the glycosylated precursor form of the enzyme, respectively (Fig. 5A). After 2 days of culture, both forms were secreted by chondrocytes alone (Fig. 5A, lane 2), but not by monocytes alone (Fig. 5A, lane 1), and increasing amounts of the enzyme occurred in coculture media (Fig. 5A, compare lanes 2 and 3). MMP-1 was activated under coculture conditions to enzyme forms with apparent molecular masses of 42 and 44 kDa. In addition, two bands appeared with molecular masses of 28 and 33 kDa (Fig. 5A, lane 3) possibly constituting the hemopexin-like domain of MMP-1.

MMP-3-proteins, in precursor as well as in activated forms, were present in media of chondrocytes alone and, in increased amounts, in those of cocultures (Fig. 5B, compare lanes 2 and 3). Monocytes cultured in agarose without chondrocytes did not secrete immunodetectable MMP-3 (Fig. 5B, lane 1). By contrast, MMP-9 (gelatinase B) was found in media of cocultures after 2 days as the 92 kDa enzyme precursor and as active 82 and 86 kDa enzymes (Fig. 5C, lane 3). Monocytes cultured in agarose without chondrocytes secreted only the 92 kDa precursor form (Fig. 5C, lane 1). Chondrocytes cultured alone did not synthesize MMP-9 precursor regardless of culture period (Fig. 5C, lane 2).

The secretion of TIMP-2, a 21 kDa protein detected as early as after 7 days in cultures containing chondrocytes, was essentially unaffected by the presence of monocytes (Fig. 5D, lanes 2 and 3) and media of monocytes cultivated alone lacked TIMP-2 protein (Fig. 5D, lane 1).

Relative gene expression levels of MMPs and TIMPs
To quantitate MMP expression revealed in zymograms and immunoblot analysis, 500 ng of total RNA from either chondrocytes or monocytes, each after 6 days of coculture, were subjected to cDNA synthesis and subsequent quantitative PCR. Specific primers for TIMP-1, TIMP-2, MMP-1, MMP-3, MMP-9 and MT1-MMP were used (Table 1). To account for variations in initial total RNA quantities, gene expression levels were normalized with steady-state expression levels of GAPDH-mRNA. To visualize changes induced by the coculture regimen, GAPDH-normalized steady-state levels of TIMP- or MMP-mRNA in cocultures were then related to the corresponding levels in chondrocytes and monocytes cultured alone (Fig. 6). Monocytes upregulated MMP-1 gene expression in chondrocytes about twofold, whereas MMP-3-mRNA expression appeared to be slightly downregulated. MT1-MMP gene expression levels are doubled after coculturing with monocytes. Messages for both tissue inhibitors of MMPs, TIMP-1 and TIMP-2, are found in chondrocytes cultured in agarose. TIMP-2 is expressed less in chondrocytes alone but is upregulated in the presence of monocytes by about twofold, whereas TIMP-1 gene expression levels in chondrocytes are not affected by monocytes.



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Fig. 6. Relative gene expression levels of MMPs and TIMPs. Filled bars: relative gene expression levels of TIMP-1 (n=5), TIMP-2 (n=5), MMP-1 (n=5), MMP-3 (n=4) and MT1-MMP (n=5) in cocultured chondrocytes normalized to monocultured chondrocytes. Open bar: relative gene-expression level of MMP-9 (n=4) in cocultured monocytes normalized to monocultured monocytes. RNA was extracted separately from chondrocyte and monocyte layers after coculturing. Expression levels of gene sequences of interest, normalized to an internal standard (GAPDH) were calculated relative to a calibrator sample. These calibrator samples constitute either chondrocytes cultured without monocytes (TIMP-1, TIMP-2, MMP-1, MMP-3, MT1-MMP) or monocytes cultured without chondrocytes (MMP-9) set at 1.0, represented by the x-graph lines. Standard deviation was calculated with Microsoft Excel from mean values of total number of experiments, each done in triplicate.

 
Interestingly, mRNA for MMP-9 (gelatinase B) is not expressed in cultured chondrocytes and also fails to be induced by coculture with monocytes. However, monocultured monocytes express MMP-9-mRNA regardless of culture time. Notably, coculture with chondrocytes results in a markedly reduced expression of the monocyte MMP-9-gene compared to monocultured monocytes.


    DISCUSSION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Articular cartilage contains neither blood nor lymph vessels and is not innervated. Because chondrocytes occupy only a small volume fraction of the tissue and are surrounded by abundant extracellular matrix, not only nutrients and gases but also instructive metabolic signals are distributed to the cells by diffusion over large distances and, thus, are available at low concentrations. Consequently, many aspects of cartilage metabolism do not depend on systemic instructions from other tissues. However, it has now become clear that, in spite of this metabolic autonomy, there exists an extensive paracrine communication between different regions of cartilage or between cartilage and tissues of the immediate environment. For example, such paracrine interactions can control chondrocyte differentiation during endochondral ossification (Böhme et al., 1995; Vortkamp et al., 1996; Pathi et al., 1999).

Furthermore, there is a close interaction between cartilage and blood vessels of adjacent tissues. For example, progression through subsequent stages of differentiation in growth plate cartilage depends on stimulatory signals originating from blood vessels of subchondral bone (Trueta and Morgan, 1960). Soluble plasma factors, including thyroid hormones, are essential for chondrocyte maturation (Burch and Lebovitz, 1982; Quarto et al., 1992; Böhme et al., 1992), and chronic thyroid hormone deficiency causes skeletal abnormalities in cretinism (Raiti and Newns, 1971). However, cells of the vessel walls also produce cross-talk signals in cartilage metabolism. We have recently shown that diffusing proteinases derived from endothelial cells can abrogate autocrine barriers in chondrocytes against their own terminal differentation (Bittner et al., 1998).

Circulating blood cells have a profound influence on cartilage metabolism, not only in scenarios such as tissue remodelling during endochondral bone formation but also in inflammatory joint diseases. To address latter issue, we employed a coculture system of chondrocytes and monocytes/macrophages to explore the paracrine cross-talk that may take place between these cells. An important objective of the experimental design was to provide an opportunity to show the distinction between chondrocyte signals addressed to monocytes and macrophages and signals operating vice versa.

Chondrocyte responses to monocyte/macrophage signals are well known. They lead to a preponderance of catabolic activities of cartilage cells and, thus, contribute to the establishment of chronic inflammatory joint disease (Dayer and Burger, 1994; Mulherin et al., 1996; Burmester et al., 1997). This has been confirmed and extended here. After exposure to monocytes/macrophages, chondrocytes increase their secretion of catabolic pro-enzyme activities (e.g. MMP-1) and their activated forms. In parallel, chondrocytes reduce synthesis of major matrix components, aggrecan and collagen II.

Less is known, however, about the paracrine dialogue in the opposite direction, i.e. reactions of monocytes or macrophages to input from chondrocytes. Here, we provide evidence for this exchange. First, we found that soluble chondrocyte signals support the viability of monocytes in serum-free agarose cultures. Chick embryo sternal chondrocytes produce factors supporting their own viability (Bruckner et al., 1989), some of which are low-molecular-weight compounds eliminating pro-apoptotic effects of oxygen metabolites (Tschan et al., 1990). It is not unlikely that the same chondrocyte products also support monocyte or macrophage survival. However, media of chondrocyte cultures contain additional autocrine survival factors. Unfortunately, their molecular identity remains elusive, probably because they are extremely unstable. Nevertheless, anti-apoptotic activity has been assigned tentatively to insulin-like growth factor 1 (IGF-1) (Ishizaki et al., 1994). Our own observations indicate, however, that IGF-1 is a chondrocyte mitogen if viability factors are present in adequate concentrations (Böhme et al., 1992). Otherwise, IGF-1 acts as a death signal (G. Schürmann and P.B., unpublished).

Second, diffusing paracrine signals derived from chondrocytes can influence metabolic activities of monocytes and macrophages. By coculturing these cell types, we have determined that chondrocytes direct the expression and activation of pro-MMP-9 (gelatinase B) in monocytes/macrophages. A limited population of cells at the junction between hypertrophic cartilage and the vascular invasion front in growing bone expresses MMP-9-mRNA (Vu et al., 1998), but articular chondrocytes do not. In freshly isolated monocytes, very low expression levels of pro-MMP-9 are found and, hence, both cell types do not constitutively produce the enzyme. By contrast, monocytes cultured in agarose or monocyte derived macrophages express MMP-9-mRNA and secrete high levels of pro-MMP-9, but do not activate the proenzyme. The functionally important contribution of articular chondrocytes in our coculture system is to trigger proteolytic activation of monocyte- or macrophage-derived pro-MMP-9. The nature of the activation suggests that chondrocytes supply factors and/or proteinases necessary for this conversion.

Whenever activated MMP-9 occurred in coculture media we also detected active MMP-1. A candidate proteinase for activating procollagenases is MMP-3 (Suzuki et al., 1990), which is also a proven activator of pro-MMP-9 (Ogata et al., 1992) and is synthesized by chondrocytes in our cocultures at constitutive levels. Addition of plasmin as a partial activator of pro-MMP-3 (Nagase et al., 1990) and pro-MMP-1 (Nagase, 1997) to our coculture system enhances the activation process of pro-MMP-9. Conversion of pro-MMP-9 into its activated forms possibly occurs in a stepwise manner and requires proteolysis by MMP-3 and/or MMP-1. This conclusion derives from the fact that inhibitors of MMP-3 dose-dependently prevented activation of pro-MMP-9 in our cocultures. Also, plasmin added to macrophages alone only partially activates pro-MMP-9 since macrophages do not synthesize MMP-1 and MMP-3. In cocultures, however, chondrocyte-derived pro-MMP-1 and pro-MMP-3 are readily available and will be activated by plasmin. Thus, pro-MMP-9 conversion is easily accomplished in cocultures treated with plasmin. This is consistent with a proteolytic activation mechanism of MMP-9 starting with systemically derived plasmin activating the chondrocyte-product pro-MMP-3 which, in turn, acts on monocyte or macrophage-generated pro-MMP-9. However, in other experimental systems, activation of pro-MMP-9 may be effected by alternative routes. Generation of active MMP-9 mediated by plasminogen activators has been reported (Murphy et al., 1992) whereas others, more consistently with our observations made here, reported inefficient (Okada et al., 1992; Goldberg et al., 1992) or indirect (Ramos-De Simone et al., 1999) activation of MMP-9 by plasmin.

A third consequence of the paracrine cross-talk between chondrocytes and monocytes was the downregulation of pro-MMP-9 expression in cocultures. This may be an important chondrocyte activity, not only to prevent direct damage to cartilage matrix by MMP-9, but also as a part of a general strategy to prevent overt inflammation in normal joints. Under pathological conditions, however, chondrocytes may abolish this paracrine restriction of catabolic monocyte activities. Interestingly, a positive correlation between the level of MMP-9 in the synovial fluid and the severity of the disease has been shown in patients with rheumatoid arthritis (Ahrens et al., 1996). This may lead to a shift in the turnover balance towards increased degradation of cartilage. Additionally, diffusion of plasmin from the synovium to the cartilage in vivo could be an important contributing factor in the enzymatic cascade responsible for the degradation of the extracellular matrix.


    ACKNOWLEDGMENTS
 
Human articular cartilage samples were kindly provided by Halm, Steinbeck, Lindner and Rödl (Department of Orthopedics) and from Brinkschmidt and Bürger (Department of Pathology, University of Münster). We also thank Marianne Ahler and Heidi Bracht for their excellent technical assistence. This work was supported by the Deutsche Forschungsgemeinschaft (Br 1497/1-2,3 and Sonderforschungsbereich 293, A9) and by a grant to P.B. by UCB-Pharma S.A., Brussels, Belgium.


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 SUMMARY
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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