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

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

View larger version (43K): [in a new window]   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).

View larger version (11K): [in a new window]   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.

View larger version (38K): [in a new window]   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).

View larger version (29K): [in a new window]   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.

View larger version (51K): [in a new window]   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.

View larger version (9K): [in a new window]   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.