RA-SF neutrophils and blood neutrophils preactivated by cell-free RA-SF mediate functional osteoclastogenesis from human monocyte precursors via RANKL. (A) Purity of neutrophils from RA-SF. Flow cytometry was performed after incubation of neutrophils with FITC-conjugated mouse anti–human CD66b (solid line). Isotype controls (dashed line) were performed with equal concentrations of corresponding mouse IgG1. Inset represents forward and side light scatter. Results are representative of neutrophils from SF of 3 RA patients with flare-up. (B) Surface expression of RANKL by neutrophils from RA-SF. Flow cytometry was performed after incubation of neutrophils with an anti–human RANKL mAb followed by a FITC-conjugated anti–mouse F(ab′)2 antibody. Results are representative of neutrophils from SF of 3 RA patients with flare-up. (C) RA-SF neutrophils were fixed with 2% PFA, repeatedly washed, and added (106/well) to cultures of human peripheral blood monocytes in 96-well plates without addition of exogenous RANKL. After 10 days of coculture, TRAP-positive cells were observed with light microscope (magnification, ×200). Results are representative of neutrophils from 3 RA-SF. (D) Mature OCs without exogenous RANKL were incubated on Osteologic discs with fixed RA-SF neutrophils in the presence or absence of 2 μg/mL rhOPG. Resorption pits were evaluated after an additional 10 days of coculture. Histograms represent resorption area expressed in square millimeter (mm2; mean ± SEM). Results are representative of 3 independent experiments performed in duplicates. Statistical analysis: unpaired t test, *P < .02. (E) Freshly isolated neutrophils preincubated in cell-free RA-SF or cell-free OA-SF for 24 hours were fixed with 2% PFA, repeatedly washed, and added (106/well) to mature OCs on Osteologic discs without addition of exogenous RANKL, and with or without pretreatment with 2 μg/mL rhOPG. Cells were removed after 10 days of culture and resorption lacunae were analyzed under light microscope. Histograms represent resorption area expressed in square millimeter (mm2; mean ± SEM). Results shown are representative of 3 independent experiments performed in duplicates. Statistical analysis: unpaired t test, *P < .02 (RA-SF vs RA-SF + OPG); +P < .02 (RA-SF vs OA-SF).
