Figure 4

Induction of the expression of RANK-L, OPG, and RANK by normal human blood neutrophils incubated in the presence of synovial fluid (SF) from patients with rheumatoid arthritis (RA) or patients with osteoarthritis (OA). (a) Surface expression of RANK-L by normal blood neutrophils incubated in SF from patients with RA (RA-SF) or OA (OA-SF) for 2 days. Flow cytometry was performed after incubation of neutrophils with a goat anti-human RANK-L antibody followed by a fluorescein isothiocyanate (FITC)-conjugated anti-goat F(ab')₂ antibody. Control isotype antibody was a normal goat immunoglobulin G (IgG). Results shown are representative of three RA-SF and nine OA-SF. (b) Expression of mRNA for OPG and RANK by normal blood neutrophils incubated for 2 days in SF from four patients with RA and two patients with OA. Total RNA was isolated from freshly isolated normal blood neutrophils (D0) and from neutrophils of the same healthy donors after 2 days of incubation in SF (RA-1 to -4, OA-1, -2). RNA was then analyzed by reverse transcriptase-polymerase chain reaction. Results shown are representative of two different healthy donors. (c) Surface expression of RANK by normal blood neutrophils incubated in SF from patients with RA (RA-SF) or OA (OA-SF) for 3 days. Flow cytometry was performed after cellular fixation, permeabilization, and staining with a mouse monoclonal anti-human RANK antibody followed by a FITC-conjugated anti-mouse F(ab')₂ antibody. Control isotype antibody was a non-specific mouse IgG. Results shown are representative of neutrophils from two different healthy subjects incubated in three different RA-SF and two OA-SF. OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-kappa-B; RANK-L, ligand of receptor activator of nuclear factor-kappa-B.