Characterization of a potent human interleukin-11 agonist.

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand-receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) alpha subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor beta subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Ralpha subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Ralpha with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60-400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.