Human primary osteoblasts express Fn14 and induce RANTES and RANKL upon TWEAK stimulation. (a) Cell surface expression of Fn14 on human osteoblasts. Human osteoblasts were stained with anti-Fn14 monoclonal antibody (open histogram) or control Ig (filled histogram) followed by phycoerythrin-labeled rabbit anti-mouse Ig antibody and were analyzed by flow cytometry. (b) Human osteoblasts were stimulated with 100 ng/ml TWEAK for 48 hours in the absence or presence of 1 μg/ml Fn14-Fc chimera (Fn14-Fc). The culture supernatants were then collected, and the RANTES concentrations were measured by enzyme-linked immunosorbent assay. Values represent the mean ± standard deviation. *p < 0.05 compared with corresponding control. (c) Human osteoblasts were grown on slide chambers, stimulated with 100 ng/ml TWEAK for 48 hours, and then stained for RANKL as described in Materials and methods. RANKL-positive cells are shown in green. Goat IgG antibody was used as a negative control for the immunofluorescence staining, with negative results (data not shown). BMP, bone morphogenetic protein; Fn14, fibroblast growth factor-inducible 14; Ig, immunoglobulin; RANKL, receptor activation of nuclear factor-κB ligand; RANTES, regulated upon activation, healthy T cell expressed and secreted; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.