Expression of RANK-L, OPG, and RANK proteins by synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA). (a) Cell-associated materials and supernatants of SF neutrophils (107/ml) from three patients with RA were analyzed by enzyme immunometric assays (EIAs) for RANK-L at day 0 and after 2 and 4 days of incubation in control medium. (b) Surface expression of RANK-L by freshly isolated SF neutrophils from patients with RA. Flow cytometry was performed after incubation of neutrophils with a goat anti-human RANK-L antibody followed by a fluorescein isothiocyanate-conjugated anti-goat F(ab')2 antibody. Control isotype antibody was a normal goat immunoglobulin G (IgG). Results shown are representative of SF neutrophils from three patients with RA. (c) Samples similar to those in (a) were analyzed by EIAs for OPG. (d) Cell-associated materials of SF neutrophils from four patients with RA were solubilized in SDS sample buffer and subjected to SDS-PAGE under reducing conditions (lanes 2 to 5). Protein loading values in lanes 2, 3, 4, and 5 were 123, 163, 135, and 125 μg, respectively. COS-7 cells transfected with a human RANK cDNA were used as a positive control (lane 1). Western blotting was performed with a goat anti-human RANK antibody, a horseradish peroxidase-conjugated anti-goat IgG antibody, and the enhanced chemiluminescence detection system. The position of the molecular weight markers in kilodaltons is indicated on the left. OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-kappa-B; RANK-L, ligand of receptor activator of nuclear factor-kappa-B.