To analyze human articular chondrocyte cell-associated matrix aggrecan, hyaluronan (HA) and type II collagen metabolism using flow cytometry, and to compare the results obtained for aggrecan with classic(35)Sulfate incorporation methods and an enzyme linked immunosorbent assay (ELISA).
Human articular chondrocytes obtained from five donors were cultured in gelled agarose and tested for their response to different concentrations of interleukin-1beta (IL-1beta). Synthesis and distribution of aggrecan in the cell-associated matrix (CAM), in the interterritorial matrix and in the nutrient medium of the chondrocytes in culture were analyzed using(35)Sulfate incorporation. The results were expressed as pg SO(4)incorporated in aggrecan per 1 x 10(6)cells/h. Flow cytometry with FITC-conjugated monoclonal antibodies against aggrecan and type II collagen, and with the biotinylated hyaluronic acid binding protein (b-HABP), was used to investigate the synthesis and accumulation of aggrecan, type II collagen and HA in the CAM of the cultured cells. The packing of these macromolecules in the CAM of the chondrocytes was assessed by measuring the mean fluorescence intensity (MFI) of the cell sample due to the binding of the specific monoclonal antibodies or b-HABP used. ELISA was used in parallel to quantify CAM aggrecans after these macromolecules were brought into solution with guanidinium chloride. Detection of aggrecan by flow cytometry was compared with(35)S-incorporation in chondrocytes from two subjects and with ELISA in a further two donors.
IL-1beta suppressed aggrecan synthesis by chondrocytes in agarose. An IL-1beta dose-dependent suppression of(35)S-aggrecan in the CAM reflected the changes in the interterritorial matrix. IL-1beta-induced aggrecan breakdown was followed by a rise in(35)S-aggrecan metabolites in the incubation media of the cells in culture. Flow cytometry and ELISA confirmed this decreased accumulation of aggrecan in the CAM of the chondrocytes. The results obtained with flow cytometry were closely related to those obtained with ELISA.(35)S-incorporation, on the other hand, indirectly measures the glycosaminoglycan content of the aggrecan and does not necessarily reflect the absolute amount of aggrecan molecules. Therefore, the effects of IL-1beta on cell-associated aggrecan, where assessed with(35)S-incorporation, did not correlate with the results of the flow cytometric assays. Flow cytometry enabled the detection of an impaired synthesis and accumulation of HA and of type II collagen in the CAM of the cultured chondrocytes. IL-1beta-induced changes in CAM aggrecan and hyaluronan closely agreed.
Flow cytometry offers an efficient tool to study the metabolism of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM.