IL-4 inhibits the surface expression of RANK in osteoclast precursors. (A) Osteoclast precursors from wild-type mice were cultured for 72 hours in the presence of rmM-CSF (20 ng/mL), rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) or rmM-CSF (20 ng/mL), RANKL (150 ng/mL), and 10 ng/mL rmIL-4, respectively. Cells were stained using FITC-RANKL and were analyzed by flow cytometry for RANK expression. These data reflect RANK expression on the cell surface in one representative experiment. RANK-positive cells were divided into low- and high-expressing cells based on fluorescence intensity; the numbers in each column indicate the percentages of cells expressing low and high levels of RANK. (B) Osteoclast precursors from wild-type mice were cultured in the presence or absence of rmM-CSF (20 ng/mL), RANKL (150 ng/mL), and rmIL-4 (10 ng/mL) for 48 hours. For one group, media were removed. After several washes with complete media, new media were added to the cells containing rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) for another 24 hours, as indicated. After that, cells were harvested and analyzed. The left graph represents the percentage of RANK-expressing cells analyzed by flow cytometry using FITC-RANKL at 48 and 72 hours. Mean percentages from 2 independent experiments are shown. The right graph represents the ratio values in arbitrary fluorescent units for RANK versus actin mRNA using Light-cycler PCR, with the same methodology as used in Figure 6. ▦ indicates rmM-CSF; □, rmM-CSF + RANKL; ▪, rmM-CSF + RANKL + IL-4; and ▧, IL-4 washout at 48 hours.