Cytokines, tumor necrosis factor-α and interleukin-1β, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes

ObjectiveThis study addresses the effects of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on cell death in human chondrocytes.MethodsOsteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively.ResultsTNF-α and IL-1β differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-α induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1β. The time sequence of caspase-3 and -7 inductions by TNF-α differs from that induced by IL-1β. Cell viability was not modified by TNF-α or IL-1β in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-α and ActD (TNF-α/ActD) increased cell death induced by ActD (23%). Treatment with IL-1β and ActD (IL-1β/ActD) did not modulate ActD-induced cell death. Similarly, IL-1β/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-α/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1β/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-α/ActD-induced cell death (58%).ConclusionTNF-α and IL-1β differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.