Neutrophils from LPS-injected mouse air pouch express RANKL and mediate functional osteoclastogenesis from murine monocyte-macrophage RAW264.7 cells. Cells obtained from (A) PBS-injected and (B) LPS-injected air pouches were labeled with a FITC-conjugated anti–mouse neutrophil antibody (clone 7/4), and PE-conjugated anti-mRANKL antibody simultaneously. Nonspecific sites were blocked by murine Fc block for 1 hour. Graphs represent flow cytometric analysis of pooled neutrophils from 5 mice/group and are representative of 3 such groups. Percentages in each quadrant correspond to numbers of cells present in each quadrant relative to 10 000 cells counted. RAW264.7 mononuclear cells were incubated (C) with medium (DMEM + 10% FBS) alone, (D) with medium + 20 ng/mL recombinant murine RANKL, (E) with medium + murine neutrophils (2.5 × 105/well) from LPS-injected air pouches, and (F) with medium + murine neutrophils (2.5 × 105/well) from LPS-injected air pouches and preincubated with 1 μg/mL recombinant OPG for 1 hour at 37°C. After 5 days of incubation, TRAP-positive multinucleated cells were analyzed by light microscopy (magnification, ×200). Results shown are representative of 3 independent experiments. (G) RAW264.7 cells (2 × 104/well) deposited on Osteologic discs were incubated with murine neutrophils (2.5 × 105/well) from LPS-injected air pouches. Cells were removed after 10 days of culture and resorption lacunae were observed under light microscopy (magnification, ×100). (H) Histograms represent total resorption area by RAW264.7 cells incubated in the absence of exogenous RANKL and neutrophils (C-ve), and in the presence of murine neutrophils (2.5 × 105/well) from PBS- or LPS-injected air pouches with or without 1 μg/mL recombinant OPG. Results are expressed in square millimeter (mm2) of resorption area (mean ± SEM, pool of 5 mice/group representative of 4 such groups). Statistical analysis: unpaired t test, *P < .05 (LPS vs PBS; LPS vs + OPG).
