Do arachidonic acid and its metabolites, secreted by rheumatoid and osteoarthritic synovial tissue, account for the strong inhibition of DNA synthesis in cultured human articular chondrocytes? A novel approach to the mechanism of tissue damage.

AIMS: To further characterize factors secreted in vitro by osteoarthritic and rheumatoid arthritis synovial membranes that inhibit DNA synthesis by cultured human articular chondrocytes, and extend these findings to synovial fluid. MATERIAL AND METHODS: Synovial tissue, synovial fluid and articular cartilage were obtained at surgery from two patients suffering rheumatoid arthritis and two other patients suffering from osteoarthritis. Synovial tissue was incubated in DMEM, then condition media and synovial fluids were extracted with methanol. Methanol extracts and extracted residues (hyaluronic acid, proteins) were assayed for their capacity to inhibit DNA synthesis in articular chondrocytes. Methanol extracts were also fractionated by thin layer chromatography on silica-coated plates and recovered fractions similarly tested. RESULTS: All extracts exhibited strong and concentration-dependent inhibition of [3H]-thymidine incorporation. The most potent inhibition was obtained with the extracts from rheumatoid joints and the least potent inhibition was with synovial fluids. The removal of active substances with methanol leaves an inactive residue. Methanol extraction does not alter the mitogenic activity of five exogenous growth factors and two cytokines, thus suggesting that such activity is entirely due to lipids. The bulk of anti-mitotic factors extracted by methanol co-migrate when fractionated by thin layer chromatography on silica-coated plates with arachidonic acid and its lipo-oxygenase metabolites. IN CONCLUSION: Inflamed synovium produces and releases lipids, most probably arachidonic acid metabolites that inhibit cell proliferation thus limiting inflammation and pannus formation in arthritis.

Method for selecting populations of rat articular chondrocytes that exhibit distinct growth and metabolic characteristics, and their responses to growth factors, PMA and vitamin D3.

Chondrocytes were released from articular cartilage fragments of 6-week-old Wistar rats by a 2-hour treatment with bacterial collagenase. The cells from one animal were seeded in a 25-cm2 culture flask at a density of 10(5) cells/cm2. After 1 h, the flask was gently shaken and the medium, containing nonadherent cells, was transferred to a new flask. The attached cells were incubated with 5 ml of fresh medium. This procedure was repeated after 3, 24, 48 and 96 h. Resulting cell populations were then analyzed. The earlier cells attached, the more rapidly they proliferated, and the less collagen and proteoglycan (PG) they produced. The cells that attached after 24 h grew much more slowly, piled up in many areas, exhibited strong alkaline phosphatase activity and calcified extracellular matrix (ECM). Differences in deoxyribonucleic acid (DNA) and protein/PG synthesis were also observed when these cell populations were challenged with growth factors and 12-myristate 13-acetate (PMA). Pretreatment of cells for 2 h with PMA strongly enhanced DNA and PG synthesis only in cultures containing insulin-like growth factor-1. Nonselected rat articular chondrocytes (AC) subcultured at least four times as monolayers still expressed mRNA specific for aggrecan and type II collagen, suggesting conservation of the chondrogenic phenotype. In conclusion, AC of young individuals seem to be heterogeneous with respect to their capacity to proliferate and synthesize ECM. By selecting and expanding in vitro the appropriate cell population, this method could be potentially useful for studies aimed at repairing damaged cartilage.