TNF-α secretion and macrophage mortality induced by cobalt and chromium ions in vitro-Qualitative analysis of apoptosis

Metal ion toxicity is a major cause for concern in metal–metal hip replacements. A previous study in our laboratory demonstrated that Co2+ and Cr3+ induce macrophage apoptosis in vitro at 24 h, with the implication of a caspase-3 pathway. The aim of the present study was to look at the effect of a prolonged incubation time on macrophage response with regards to TNF-α secretion and macrophage mortality, more specifically apoptosis. J774 macrophages were exposed for up to 48 h to 0–10 ppm Co2+ and 0–500 ppm Cr3+. ELISA results demonstrated that Co2+ and Cr3+ induced a concentration- and time-dependent increase of TNF-α secretion, but a decrease at the highest concentrations of Cr3+ (350–500 ppm). This decrease was most likely due to a high toxicity of Cr3+ at such concentrations. Higher levels of TNF-α were observed with Co2+ than Cr3+, demonstrating a higher stimulatory effect of this ion. Trypan blue and flow cytometry results demonstrated that both Co2+ and Cr3+ ions induce macrophage mortality in a dose- and time-dependent manner. The number of cells decreased when ion concentrations increased, especially at 48 h. In parallel with the TNF-α results, Co2+ was more toxic than Cr3+ since the maximal effects were reached with lower concentrations (8–10 ppm vs. 350–500 ppm, respectively). DNA analysis demonstrated that both Co2+ and Cr3+ ions induce macrophage apoptosis, with a stronger signal at 24 h than at 48 h, suggesting the presence of more necrosis after 48 h. PARP cleavage, another marker of apoptosis, was observed at both 24 and 48 h, with a maximum intensity at 48 h and with the highest concentrations of ions. In conclusion, this study demonstrates that both Co2+ and Cr3+ ions can induce the release of TNF-α and macrophage mortality in a dose- and time-dependent manner. More specifically, Co2+ and Cr3+ ions induced apoptosis after both 24 and 48 h incubation, although DNA analysis suggested the presence of necrosis at 48 h. The relative importance of apoptosis and necrosis in the induction of macrophage mortality by these metal ions remains to be investigated.