Binding of soluble interleukin-18 receptor α complex to human natural killer cells expressing interleukin-18 receptor α. (A) The reaction of the human interleukin (IL)-18 receptor α (hIL-18Rα) complex with the human natural killer (NK) cell line NK0 is shown. NK0 cells were stained with control biotin-labeled mouse immunoglobulin G1 (mIgG1-Bio), biotin-labeled anti-IL-18Rα monoclonal antibody clone 44 (H44-Bio) or biotin-labeled hIL-18Rα complex protein at 4°C for 30 minutes. For blocking assays (bottom), excess nonlabeled IL-18Rα complex proteins (80 μg) were preincubated with the cells at room temperature for 30 minutes. The cells were then stained with biotin hIL-18Rα complex at 4°C for 30 minutes. Streptavidin-PE was used for the second-step staining. FACS analysis was performed as described in Materials and methods. Percentages of immunopositive cells are shown in each graph. sIL-18Rα-Bio, biotin-labeled soluble interleukin-18 receptor α complex. (B) The neutralizing activity of hIL-18Rα complex protein against recombinant hIL-18 (rhIL-18) protein is shown. NK0 cells (5 × 105 cells/mL) were suspended in RPMI 1640 medium with 10% fetal calf serum and then pretreated with hIL-18Rα complex protein (0.003 to 10 μg/mL) for 15 minutes at room temperature. The cells were then stimulated with rhIL-2 (rhIL-2) (50 IU/mL) plus rhIL-18 (50 ng/mL) for 18 hours. Human interferon-γ in the supernatants was then analyzed using an enzyme-linked immunosorbent assay kit (R&D Systems, Inc.).