Identification of a critical Ig-like domain in IL-18 receptor α and characterization of a functional IL-18 receptor complex

Abstract

Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R α-chain (IL-18Rα) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [125I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [125I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (Kd) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent Kd of 146 pM. Each domain of extracellular IL-18Rα was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-γ production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Rα prevented the formation of IL-18R ternary complex with IL-18R β-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Rα for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-γ in NK cells.