Activated human neutrophils mediate functional osteoclastogenesis from human monocyte precursors. (A) TRAP expression by osteoclast precursors in culture with activated neutrophils: human monocytes were incubated with (i) M-CSF (25 ng/mL), (ii) M-CSF + exogenous rhRANKL (40 ng/mL), and (iii) M-CSF + LPS-activated neutrophils (106/well). After 10 days, TRAP-positive multinucleated cells were observed with light microscope. Results were reproduced in 3 independent experiments. (i-iii) Magnification, × 200. (Inset, iii): Enlarged view of a TRAP-positive multinucleated cell in coincubation with activated neutrophils (magnification, ×400). (B) Formation of resorption pits in neutrophil-osteoclast cocultures: mature OCs deprived of exogenous RANKL were incubated on Osteologic discs for 10 days in the presence of activated neutrophils and were then stained by toluidine blue vital coloration. Osteoclasts (multinucleated giant cells) are visualized in the interior and in the periphery of the resorption lacunae. Histograms represent total resorption area by mature human OCs incubated on Osteologic discs in the absence of exogenous RANKL and activated neutrophils (C-ve), and in the presence of activated neutrophils (106/well) with or without 2 μg/mL recombinant OPG. Results are expressed in square millimeter (mm2) of resorption area (mean ± SEM, n = 3 independent experiments). Statistical analysis: unpaired t test, *P < .002, **P < .003 (activated neutrophils vs C-ve; activated neutrophils vs activated neutrophils + OPG). (C) TRAP expression by osteoclast precursors in culture with neutrophil membranes: monocytes were incubated with (i) M-CSF (25 ng/mL), (ii) M-CSF + exogenous rhRANKL (40 ng/mL), and (iii) M-CSF + membranes of LPS-activated neutrophils added at a cellular equivalent of 500 000 cells/well. After 10 days of cultures, TRAP-positive cells were observed with light microscope (i-iii: magnification, ×200). Results were reproduced in 3 independent experiments. (D) Formation of resorption pits in cultures of osteoclasts with neutrophil membranes: mature OCs without exogenous RANKL were incubated on Osteologic discs with (i) membranes of activated neutrophils added at a cellular equivalent of 500 000 cells/well, and (ii) conditioned medium from activated neutrophils (200 μL/well). Formation of resorption pits was evaluated after an additional 10 days of coculture. Histograms represent total resorption area by mature human OCs incubated on Osteologic discs in the absence of exogenous RANKL and neutrophil membranes (C-ve), and in the presence of membranes from activated neutrophils (cellular equivalent of 500 000 cells/well) with or without 2 μg/mL recombinant OPG. Results are expressed in square millimeter (mm2) of resorption area (mean ± SEM, n = 3 independent experiments). Statistical analysis: Student unpaired t test, *P < .02, +P < .02 (neutrophil membranes vs C-ve; neutrophil membranes vs neutrophil membranes + OPG).
