Signaling through surface RANKL of activated neutrophils: tyrosine phosphorylation of proteins and regulation of cytokine production. (A) Freshly isolated human neutrophils and (B) LPS-activated neutrophils (20 × 106/mL) were incubated for 30 minutes at 37°C with vehicle alone (C-ve), 1 μg/mL rhRANK polypeptide, 1 μg/mL rhOPG, and 1 μg/mL rhRANK peptide + 1 μg/mL rhOPG. Activated neutrophils were also preincubated for 15 minutes with 10 μM PP2 or PP3, and further incubated for 30 minutes at 37°C with rhRANK peptide + rhOPG. Cells were preincubated with 1 mM di-isopropyl-fluorophosphate for 10 minutes. Equal volume of boiling 2 × modified Laemmli sample buffer was added to incubations. Membranes were immunoblotted with a primary antiphosphotyrosine mAb followed by a secondary HRP-labeled goat anti–mouse IgG antibody. Bottom panels indicate sample loading after reblotting the same membrane with an anti-Lyn mAb. Results are representative of 3 independent experiments. (C) Histograms represent global densitometry (expressed in arbitrary units) of immunorecognized bands. Values are means ± SEM (n = 3). Statistical analysis: one-way ANOVA and Newman-Keuls Multiple comparison posttest (RANK vs RANK + OPG, a for P < .05; OPG vs RANK + OPG, b for P < .01; C-ve vs RANK + OPG, c for P < .05, RANK + OPG + PP2 vs RANK + OPG or RANK + OPG + PP3, d for P < .01). (D) Activated neutrophils (20 × 106/mL) were incubated for 30 minutes at 37°C with vehicle alone (C-ve) and rhRANK peptide + rhOPG. Cells were lysed in nondenaturing lysis buffer containing 0.6% CHAPS, centrifuged at 13 000g at 4°C, and the supernatants were immunoprecipitated (IP) for SHP-1 and Syk proteins using anti–SHP-1 (sc-287) or anti-Syk (MAB88906; Chemicon) antibodies. SHP-1 immunoprecipitates were revealed with antiphosphotyrosine mAb (pY) or anti–SHP-1 mAb (ab660; Abcam) by Western blotting (WB). Syk immunoprecipitates were revealed with antiphosphotyrosine mAb. Results are representative of 3 independent experiments. (E-F) LPS-activated neutrophils (5 × 106/mL) were preincubated with vehicle or with 20 μM SHP-1 inhibitor α-bromo-4-hydroxyacetophenone 4-hydroxyphenacyl Br (Calbiochem) for 15 minutes and then incubated for 24 hours with vehicle or with rhRANK peptide + rhOPG. IL-8 and IL-1β were measured in supernatants by ELISA. Results of IL-8 and IL-1β are expressed in nanograms per milliliter (ng/mL) and picograms per milliliter (pg/mL), respectively (mean ± SEM, n = 5 for IL-8 and n = 4 for IL-1β). Statistics: paired t test, *P < .05 (RANK + OPG vs vehicle).
