Hideo Yawata

Structure-function analysis of human IL-6 receptor : dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130

Here, we report the analysis of the structure‐function relationship of the extracellular region of human interleukin 6 receptor (IL‐6R). Upon binding of IL‐6, IL‐6R becomes associated extracellularly with a non‐IL‐6‐binding but signal transducing molecule, gp130, and the IL‐6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin‐like domain, was responsible both for IL‐6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL‐6R (sIL‐6R) could bind IL‐6 and mediate IL‐6 functions through gp130, amino acid substitutions were introduced into sIL‐6R by site‐directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL‐6 binding have a tendency to be distributed to the hinge region between the two ‘barrel’‐like fibronectin type III modules and to the same side of these two ‘barrels’. Amino acid residues, of which substitutions barely affected the IL‐6‐binding but did abolish the IL‐6 signalling capability of sIL‐6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL‐6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL‐6.

Association of recombinant soluble IL-6-signal transducer, gp130, with a complex of IL 6 and soluble IL-6 receptor, and establishment of an ELISA for soluble gp130

IL-6 mediates its pleiotropic functions through two membrane proteins, a ligand-binding molecule (IL-6 receptor, IL-6R) and a non-ligand-binding signal transducer (gp130). Starting with a previously isolated cDNA clone encoding human gp130, recombinant soluble gp130 (sgp130) lacking the transmembrane and cytoplasmic regions was expressed in COS7 cells or CHO cells. sgp130 could associate with a complex of IL-6 and soluble IL-6R (sIL-6R), also lacking transmembrane and cytoplasmic regions. This indicated that extracellular region of gp130 was responsible for the association with IL-6R which was occupied by IL-6. An enzyme-linked immunosorbent assay (ELISA) for the quantitation of sgp130 was established, which was based on the interaction of sgp130 with the complex of IL-6 and sIL-6R and could detect sgp130 as low as 1 ng/ml.

Preparation of monoclonal antibodies against the IL-6 signal transducer, gp130, that can inhibit IL-6-mediated functions.

mAbs specific to human gp130, a signal transducing component of the IL-6 receptor complex, were prepared by immunizing mice with a previously described recombinant human soluble gp130. Some of the mAbs inhibited the IL-6-induced association of soluble gp130 and soluble IL-6 receptor. Three mAbs (GPX7, GPX22 and GPZ35) were shown to inhibit IL-6-mediated biological responses such as Ig production in a human B cell line and proliferative responses of a human Lennerts lymphoma-derived T cell line, a human myeloma cell line, and a mouse pro-B cell line-derived transfectant expressing human gp130.

A Multifunctional Cytokine (IL-6/BSF-2) and Its Receptor

Interleukin 6 (IL-6)/B cell stimulatory factor 2 is a multifunctional cytokine produced by both lymphoid and nonlymphoid cells. IL-6 regulates immune response, acute phase reaction, and hematopoiesis. It was found that IL-6 production by T cells is dependent on macrophages, and IL-6 is one of essential factors for pokeweed mitogen induced immunoglobulin production. Both high- and low-affinity IL-6 receptors were identified. The molecular cloning of IL-6 receptors demonstrated that this receptor is a member of the immunoglobulin superfamily. The deregulated production of IL-6 is suggested to be involved in the pathogenesis of autoimmune diseases and in the development of multiple myeloma.

NORMAL AND ABNORMAL REGULATION OF HUMAN B CELL DIFFERENTIATION BY A NEW CYTOKINE, BSF2/IL-6

Antibody molecules play an essential role not only in protection against viral or bacterial infections, but also in the induction of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, and immediate-type hypersensitivity. Therefore, the study of the mechanism regulating the activation, proliferation and immunoglobulin secretion of B lymphocytes is essential for the normal and abnormal regulations in the antibody response.

Interleukin 6 and Its Receptor in Immune Regulation

Several factors have been shown to be involved in the regulation of B cell responses into antibody producing cells. The cDNAs for three interleukins, IL-4, IL-5 and IL-6, have been molecularly cloned and functions of these molecules in the activation, proliferation and differentiation of B cells were confirmed utilizing recombinant molecules (Kishimoto and Hirano 1988). However, the studies with recombinant molecules also showed that the functions of these molecules are not restricted to B lineage cells but have a wide variety of biological activities on various tissues and cells. One of the typical examples of multifunctional interleukins is IL-6 (Hirano and Kishimoto in press).

Interleukin 6 and its Receptor: Pathological Role in Autoimmunity and Lymphoid Malignancy

Interleukin 6 (BSF-2/26kD protein/IFN-s 2/HPGF/HSF) is a multifunctional cytokine produced by both lymphoid and non-lymphoid cells. Both high and low affinity IL-6 specific receptors were identified and the cDNA encoding IL-6-R was cloned. The data showed that IL-6 receptor is a member of the immunoglobulin superfamily. IL-6 was demonstrated to be an essential factor for immunoglobulin production by B cells. The results suggested that the deregulation is involved in the pathogenesis of autoimmne diseases and the oncogenesis of multiple myeloma.