Quantitative analysis of macrophage apoptosis vs. necrosis induced by cobalt and chromium ions in vitro

The potential toxicity of metal ions in tissues surrounding metal–metal hip replacements is a cause for concern. Previous studies conducted in our laboratory demonstrated that Co2+ and Cr3+ induce TNF-α secretion in macrophages, as well as cell mortality. However, the degree of apoptosis and necrosis remained to be investigated. The aim of the present study was to quantify the rate of macrophage mortality by apoptosis vs. necrosis induced by Co2+ and Cr3+. J774 mouse macrophages were incubated in growth medium containing 0–10 ppm Co2+ and 0–500 ppm Cr3+ for 24 and 48 h under conventional cell culture conditions. Transmission electron microscopy, flow cytometry (Annexin-V fluorescein isothiocyanate/propidium iodide assay) and a specific cell death detection ELISA were used to illustrate cell death and differentiate between apoptotic and necrotic cells. Cell culture exposed to low concentrations of Co2+ (0–6 ppm) revealed a low degree of mortality. In contrast, at the highest concentrations (8–10 ppm), late apoptosis occurred within 24 h. After 48 h, however, there was a clear evidence for an increase in the rate of necrosis while apoptosis occurred at much lower rate. Macrophages exposed to Cr3+ demonstrated a predominance of apoptosis after 24 h. At concentrations lower than 250 ppm, early and late apoptosis occurred at the same rate. At higher concentrations (250–500 ppm), the number of early apoptotic cells decreased in favor of late apoptosis. After 48 h, lower concentrations of Cr3+ (⩽150 ppm) induced a higher degree of early apoptosis than after 24 h, and some necrosis. At higher concentrations, the percentage of early apoptotic cells decreased, while necrosis became predominant over late apoptosis. In conclusion, this study demonstrates that macrophage mortality induced by metal ions depends on the type and concentration of metal ions as well as the duration of their exposure. Overall, apoptosis was predominant after 24 h with both Co2+ and Cr3+ ions, but high concentrations induced mainly necrosis at 48 h. These results point to the potential for these ions of inducing tissue damage by necrosis if present in large concentrations in vivo.