G-CSF enhances osteoclastogenesis in vitro. (A) RT-PCR was performed on mRNA from primary murine macrophages (Day 0), primary murine OCs (Day 5), a murine OB cell line, ST-2, and primary calvarial OBs. Macrophages and OCs express G-csf-r, while OBs do not. Spleen cDNA was a positive control. (B) Primary murine BMMs were grown in the presence of M-CSF and RANKL to promote differentiation of macrophages into OCs. Cultures were supplemented with 50 ng/mL G-CSF or PBS. Day-3 OCs were stained with TRAP; multinucleated TRAP-positive cells were counted. The addition of G-CSF led to a significant enhancement in osteoclastogenesis (P = .03). (C) A TRAP solution assay was performed to obtain relative levels of TRAP. The addition of G-CSF resulted in significant increases in TRAP activity at all days examined (P = .005, Day 2; P = .01, Day 3; and P < .005, Day 4). Representative images of the cells are shown. (D) To assess a dose response to G-CSF, a TRAP solution assay was performed to obtain relative levels of TRAP activity 4 days after plating BMMs in M-CSF and RANKL with 0, 10, 30, or 50 ng/mL G-CSF. Increasing doses of G-CSF resulted in increasing TRAP activity (P < .05 for each dose of G-CSF compared with vehicle). (E) Primary murine BMMs were grown in the presence of M-CSF and RANKL on osteologic calcium-phosphate–coated slides for 4 days. Cultures were supplemented with either 50 ng/mL G-CSF or PBS. Cultures supplemented with G-CSF demonstrated increased areas of calcium-phosphate resorption (P = .01). All error bars represent standard error of the mean. Asterisks indicate statistical significance.