Expression of RANKL by human blood neutrophils. (A) Purity of neutrophils isolated from healthy human subjects. Flow cytometry was performed after incubation of neutrophils with FITC-conjugated mouse anti–human CD66b (blue line), and FITC-conjugated mouse anti–human CD3 (red dotted line). Isotype controls (black dotted line) were performed with equal concentrations of corresponding mouse IgG1. Results shown are representative of neutrophils from 10 healthy subjects. (B) Flow cytometry analysis of RANKL expression by neutrophils incubated with the TLR4 agonist LPS (100 ng/mL), the TLR2 agonist Pam3Cys-Ser-(Lys)4 hydrochloride (1 μg/mL), the TLR5 agonist flagellin (500 ng/mL), and the TLR7 and TLR 8 agonist gardiquimod (at 0.1 μg/mL and 1 μg/mL, respectively) for 48 hours. After incubation, neutrophils were treated with an anti–human RANK-L mAb followed by a FITC-conjugated anti–mouse F(ab′)2 antibody. Isotype controls (dotted line) were performed for each condition. Results are representative of 3 independent experiments. Histograms represent means (± SEM) of the percentage of cells expressing RANKL; control cells (Ctl) are freshly isolated neutrophils (n = 3). Statistical analysis: unpaired t test, *P < .05 (TLR vs Ctl). (C) Expression of RANKL mRNA by human blood neutrophils incubated with 100 ng/mL LPS for 24, 48, and 72 hours. RNA extraction and semiquantitative reverse-transcription–PCR were performed for freshly isolated neutrophils (0) and LPS-stimulated neutrophils; β-actin gene served as an internal control. Histograms represent means (± SEM) of ratios of densitometric values for RANKL/β-actin (n = 3). Student paired t test: *P < .05 (LPS-stimulated vs freshly isolated neutrophils). (D) RANKL is detected in membranes of LPS-activated neutrophils by Western blotting with a primary mouse anti–human RANKL IgG Ab and a secondary HRP-conjugated anti–mouse Ab. Membranes correspond to 500 000 freshly isolated or LPS-activated neutrophils. CD32 serves to verify the integrity of neutrophil membranes.
