Effect of Apo2L/TRAIL on osteoclast differentiation and bone resorption. (A) Human peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from buffy coats acquired from the Australian Red Cross Blood Service. The cells were diluted in Hanks balanced salt solution (HBSS) and separated by gradient centrifugation with Lymphoprep (Nycomed Pharma, Oslo, Norway). Isolated cells (2.5 × 105 cells/well) were then plated in minimal essential medium (MEM), supplemented with 10% fetal calf serum, l-glutamine (2 mM), Hepes (20 mM), recombinant human M-CSF (25 ng/mL; Genetics Institute, Cambridge, MA), 1α,25(OH)2vitamin D3 (10 nM; Wako Pure Chemicals, Osaka, Japan) and dexamethasone (10 nM; Fauldings, Adelaide, Australia) into 96-well plates containing slices of sperm whale dentine, for the bone resorption assay, or directly into wells for tartrate resistant acid phosphatase (TRAP) staining. The following day, media were removed and replaced with media as above, additionally supplemented with recombinant human RANKL (50 ng/mL; Roche, Indiannapolis, IN), in the presence or absence of 100 ng/mL of Apo2L/TRAIL (Genentech, South San Francisco, CA). Media and treatments were replaced every 3 days. Cells were fixed on day 9 and stained histochemically for TRAP (Sigma-Aldrich, St Louis, MO), and TRAP-positive cells were visualized by light microscopy. To assess bone resorption, dentine slices were washed in 0.1% Extran detergent, rinsed with distilled water, washed in 70% ethanol, and air-dried overnight. The dentine slices were then mounted on stubs, carbon-gold coated, and examined on a Philips XL-20 scanning electron microscope. Images were then analyzed and resorption pits counted using ImageQuant software (GE Healthcare, Little Chalfont, United Kingdom; quadruplicate dentine slices for each treatment). Results shown are average numbers of pits (± SEM) and the significant differences between treatments determined using Student t tests (2-tailed, unpaired). (B) RAW264.7 cells were left untreated or cultured in the presence of increasing concentrations of Apo2L/TRAIL with or without RANKL for 5 days. Shown are representative fields of the cell cultures treated as indicated after TRAP staining. The number of TRAP-positive multinucleated cells (containing 3 or more nuclei) was scored. The viability of identically-treated cells was assessed by crystal violet staining. Data represent the means (± SD) of 3 independent experiments. Osteoclasts generated after 5 days of RANKL-stimulation were treated with Apo2L/TRAIL for a further 24 hours as indicated to assess the effect of Apo2L/TRAIL on the survival of the mature osteoclasts. Apo2L/TRAIL had no effect on the survival of mature osteoclasts. (C) To determine the effect of Apo2L/TRAIL on bone resorption by mature osteoclasts, we used cells isolated from human primary giant cell tumors of bone (GCT) specimens known to contain abundant numbers of osteoclast-like cells, as we have described previously. GCT cells were plated onto dentine slices in 96-well plates at a density of 105/well and treated for 5 days with 100 ng/mL of Apo2L/TRAIL or with 25 μM of the osteoclast-ablating bisphosphonate zoledronic acid (ZOL). Medium and treatments were replaced on day 3 and pit formation was determined, as described above, after 5 days. Whereas treatment with ZOL completely inhibited bone resorption, Apo2L/TRAIL treatment was without effect. (D) RAW264.7 cells were cultured in the presence of RANKL (100 ng/mL) with or without recombinant Fc-OPG (100 ng/mL) or conditioned media from cultured K-HOS human osteosarcoma cells in the presence or absence of 100 ng/mL of Apo2L/TRAIL. The number of TRAP+ osteoclasts were assessed as described in panel A. The data represent the means (± SD) of 3 different experiments.
