Soluble Human Interleukin‐6 Receptor

The extracellular domain of the human interleukin‐6 (IL‐6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus‐infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL‐6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 × 10 4 l · mol −1 · cm −1 was calculated from the ultraviolet spectrum of the soluble IL‐6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N ‐glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41–44 kDa. As expected, the soluble human IL‐6 receptor bound human 125 I‐labeled IL‐6 with low affinity ( K d = 500 pM). Furthermore, the binding of soluble human IL‐6 receptor to immobilized IL‐6 was studied using real‐time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL‐6 (HepG2‐IL‐6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL‐6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1–67 and 68–143 of the soluble IL‐6 receptor, respectively. The plasma clearance of soluble 125 I‐labeled IL‐6 receptor in the absence and presence of IL‐6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL‐6 receptor/IL‐6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125 I‐labeled IL‐6 receptor, as well as complexes with IL‐6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys, Subsequently, the radioactivity appeared in the gut content.