Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP

Abstract

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6R␣ and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFP-IL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6R␣, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6⅐IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.Cytokines are important mediators in the regulation of immune responses and inflammation. Dysregulated cytokine signaling leads to chronic inflammation and cancer. Therefore, pro-inflammatory cytokines, such as tumor necrosis factor (TNF) 2 and interleukin-1 and -6, have been identified as promising therapeutic targets. First approaches to specifically block the action of pro-inflammatory cytokines have focused on the use of neutralizing antibodies against a specific cytokine or its receptor. Only recently, the value of soluble cytokine receptors as cytokine antagonists for the treatment of inflammatory diseases has been fully recognized. Pro-inflammatory cytokines signal through receptor proteins consisting of an extracellular part, a single transmembrane region and a cytoplasmic domain. Ligand binding to the extracellular part of the receptor results in the activation of signal transduction cascades by the cytoplasmic domain. Soluble receptors consisting only of the extracellular part are potent inhibitors of cytokine activity. They bind the cytokine with the same specificity and affinity as the membranebound receptors without eliciting an intracellular signal. A dimeric form of the soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1).Most cytokines signal through heteromeric receptor complexes consisting of two or more different r...

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