Figure 1.

IL-4 blocks RANKL-mediated osteoclastogenesis by bone marrow macrophages. Osteoclast precursor cells were isolated from the bone marrow of 4- to 6-week-old mice as described in Methods. Pure (∼90%) marrow macrophages were plated in 48-well plates at 1 × 10⁶ cells/ml using α-MEM supplemented with 10% heat-inactivated (HI) FCS and 10 ng/ml M-CSF. Cells were treated with PBS, 10 ng/ml mIL-4 for 30–60 minutes, 20 ng/ml soluble RANKL for 4 days, or a combination of IL-4 and RANKL (as shown). Cultures were placed at 37°C in a 5% CO₂ incubator and supplemented with an additional dose of M-CSF and RANKL on the third day of culture. Osteoclasts developed on days 4–5, after which they were washed, fixed, and stained for TRAP activity following manufacturers’ directions. TRAP-positive (purple) mono- and multinucleated large cells are osteoclasts and their committed precursors. The average number of osteoclasts in RANKL-treated cultures was 182 ± 22/cm² compared with no osteoclasts in all other conditions. Results represent average number of quadruplicate wells from three independent experiments. ×20 taken by light microscope.