PLB-985 neutrophil–like cells express RANKL and mediate monocyte maturation into OCs. Flow cytometric analysis of RANKL expression by undifferentiated PLB-985 cells (A) and by PLB-985 cells differentiated for 48 hours with dbcAMP (B). Cells were incubated with an anti–human RANKL mAb followed by a FITC-conjugated anti–mouse F(ab′)2 antibody. Isotype controls are dotted tracings. Results are representative of 5 independent experiments. Monocytes isolated from peripheral blood of healthy human donors were incubated (C) with M-CSF (25 ng/mL) alone, (D) with M-CSF + rhRANKL (40 ng/mL), and (E) with M-CSF + PLB-985 cells (106/well) differentiated for 48 hours with dbcAMP and subsequently fixed with 2% PFA. After 10 days of incubation, cells were stained for TRAP to evaluate TRAP-positive multinucleated cells by light microscopy. (C-E) Magnification, ×200. Mature OCs (100 000/well) on Osteologic discs were incubated (F) with M-CSF (25 ng/mL) alone, (G) with addition of PFA-fixed PLB-985 neutrophil-like cells, and (H) with addition of rhRANKL (40 ng/mL). Cells were removed after 10 days of incubation and bone resorption was evaluated by light microscopy and Imagepro software. (I) Mature OCs deprived of exogenous RANKL were incubated on dentine discs with PFA-fixed PLB-985 neutrophil-like cells. After 10 days, dentine discs were stained with toluidine blue (0.5%). Results were reproduced in 3 independent experiments. (F-I) Magnification, × 100.
