Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro.

Bisphosphonates (BPs) inhibits bone resorption by reducing osteoclastic activity; they induce osteoclast apoptosis. Pathophysiology of prostheses loosening is complex and implies an inflammatory reaction secondary to the phagocytosis of wear debris by macrophages with a secondary increased bone resorption by osteoclasts. BPs inhibit proliferation and cause cell death in macrophages by induction of apoptosis. We have used mouse macrophage-like J774.1 cells to evaluate the effects of five BPs. J774A.1 cells were cultured in a standard culture medium for 2-days. BPs (alendronate, pamidronate, etidronate, risedronate, zoledronic acid) were added in the medium at concentration of 10(-6) to 10(-4)M during 3 days. Cells were studied by fluorescence microscopy after staining with the fluorescent dye Hoescht H33342 and the percentage of apoptotic cells was determined on 300 nuclei. Cells were analyzed by flow cytofluorometry after staining with annexin V-FITC (for counting apoptotic cells) and propidium iodide (for necrotic/late-apoptotic cells) on 2000 cells. Etidronate did not cause significant apoptosis or necrosis, at any concentration. Alendronate and pamidronate caused apoptosis and death only at very high concentration [10(-4)M]. On the contrary, apoptotic and necrotic cells were evidenced with risedronate or zoledronic acid at lower concentrations. These effects were dose-dependant and occurred when concentration reached [10(-5)M]. The number of apoptotic cells was higher with zoledronic acid and then with risedronate. Cytofluorometry appeared superior to cytologic analysis in the investigation of macrophage apoptosis, since necrotic cells loose contact with the glass slides and are not identifiable in cytological counts. Some amino-BPs appear to induce apoptosis in macrophages.