SMART MODE:

NORMAL GENOMIC

• Hiroike T, Higo J, Jingami H, Toh H
• Homology modeling of human leptin/leptin receptor complex.
• Biochem Biophys Res Commun. 2000; 275: 154-8
• Display abstract

• Leptin receptor mediates the weight regulatory signal carried by the adipocyte-secreted peptide hormone, leptin. It is important to understand the atomic interactions between leptin and the receptor for the therapeutic applications. However, the structure of leptin receptor has not yet been determined. Leptin shows structural similarity to G-CSF, while leptin receptor is similar in amino acid sequence to G-CSF receptor. Because of the similarity between leptin/leptin receptor complex and G-CSF/G-CSF receptor complex, we tried to build a model structure of leptin/leptin receptor complex with the crystal structure of the G-CSF/G-CSF receptor complex as the template. The obtained model for the complex was consistent with the results of the amino acid replacement and deletion experiments. The observation suggests that the model is useful to lead the experimental study on the interaction between leptin and the receptor.

• Knezevic-Cuca J et al.
• Neurotrophic role of interleukin-6 and soluble interleukin-6 receptors in N1E-115 neuroblastoma cells.
• J Neuroimmunol. 2000; 102: 8-16
• Display abstract

• Interleukin 6 (IL-6) plays a role in physiological and pathophysiological processes in neuronal cells. We studied whether IL-6 plays a role in neuroblastoma cells in culture. These studies demonstrate that N1E-115 cells constitutively express IL-6 but not IL-6R. Exogenous IL-6 stimulated neuronal proliferation in a dose-dependent manner. Under serum-free conditions soluble IL-6 receptors (sIL-6R) alone or in combination with IL-6 exerted significant proliferative effects, while IL-6 alone failed to promote cell proliferation. Neutralizing anti-IL-6 antibody caused a 30-40% reduction in IL-6 mediated proliferation. Our results suggest the importance of IL-6/sIL-6R for proliferation and survival of N1E-115 adrenergic neuroblastoma cells.

• Pflanz S, Kurth I, Grotzinger J, Heinrich PC, Muller-Newen G
• Two different epitopes of the signal transducer gp130 sequentially cooperate on IL-6-induced receptor activation.
• J Immunol. 2000; 165: 7042-9
• Display abstract

• Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.

• Papanicolaou DA, Vgontzas AN
• Interleukin-6: the endocrine cytokine.
• J Clin Endocrinol Metab. 2000; 85: 1331-3

• Desreumaux P
• Specific targeting of IL-6 signalling pathway: a new way to treat IBD?
• Gut. 2000; 47: 465-6

• Aritomi M, Kunishima N, Morikawa K
• [A new cytokine-receptor recognition scheme revealed by the complex structure of G-CSF and its receptor]
• Tanpakushitsu Kakusan Koso. 2000; 45: 1713-21

• Ogata A, Nishimoto N, Yoshizaki K
• [Advances in interleukin-6 therapy]
• Rinsho Byori. 1999; 47: 321-6
• Display abstract

• Interleukin-6 (IL-6) exhibits multiple biologic activities such as regulation of immunological responses and hematopoiesis, promotion of acute inflammation, and stimulation of some malignant and non-malignant cell growth. The IL-6 receptor system consists of an IL-6 specific binding molecule, IL-6R and a signal transducer, gp130. Following gp130 dimerization, IL-6 activates multiple signaling pathways (Ras dependent MAPk cascade, STAT1-STAT3 heterodimer pathway, and STAT3 homodimer pathway). Several other cytokines including oncostatin M, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotropin-1 (CT-1) use gp130 as a common signal transducing molecule and therefore have similar biological activities. Two major in vivo functions of IL-6 are reported. Firstly, IL-6 acts as a growth factor of some malignant and non-malignant cells such as malignant plasma cells in multiple myeloma, mesangial cells in the kidney, and keratinocytes. Secondly, IL-6 mediates inflammatory and immune responses in rheumatoid arthritis, Castleman disease, psoriasis, cardiac myxoma, cachexia, and other inflammatory conditions. Recently, a humanized anti-IL-6 receptor antibody was developed. Neutralization of IL-6 activity by the humanized anti-IL-6 receptor antibody may be a new therapeutic approach for IL-6 related diseases such as multiple myeloma, Castleman disease and rheumatoid arthritis.

• Genesca J, Marti R, Gonzalez A, Torregrosa M, Segura R
• Soluble interleukin-6 receptor levels in liver cirrhosis.
• Am J Gastroenterol. 1999; 94: 3074-5

• Gaillard JP, Mani JC, Liautard J, Klein B, Brochier J
• Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6.
• Eur Cytokine Netw. 1999; 10: 43-8
• Display abstract

• Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

• Kurth I et al.
• Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes.
• J Immunol. 1999; 162: 1480-7
• Display abstract

• The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.

• Mullberg J, Vollmer P, Althoff K, Marz P, Rose-John S
• Generation and function of the soluble interleukin-6 receptor.
• Biochem Soc Trans. 1999; 27: 211-9

• Croucher PI, Wang F, Hargreaves PG
• Interleukin-6 receptor shedding: a possible role for members of the ADAM family.
• Biochem Soc Trans. 1999; 27: 224-8

• Hirano T
• Interleukin 6 and its receptor: ten years later.
• Int Rev Immunol. 1998; 16: 249-84
• Display abstract

• Ten years have passed since the molecular cloning of interleukin 6 (IL-6) in 1986. IL-6 is a typical cytokine, exhibiting functional pleiotropy and redundancy. IL-6 is involved in the immune response, inflammation, and hematopoiesis. The IL-6 receptor consists of an IL-6 binding alpha chain and a signal transducer, gp130, which is shared among the receptors for the IL-6 related cytokine subfamily. The sharing of a receptor subunit is a general feature of cytokine receptors and provides the molecular basis for the functional redundancy of cytokines. JAK tyrosine kinase is a key molecule that can initiate multiple signal-transduction pathways by inducing the tyrosine-phosphorylation of the cytokine receptor, gp130 in the case of IL-6, on which several signaling molecules are recruited, including STAT, a signal transducer and activator of transcription, and SHP-2, which links to the Ras-MAP kinase pathway. JAK can also directly activate signaling molecules such as STAT and Tec. These multiple signal-transduction pathways intimately regulate the expression of several genes including c-myc, c-myb, junB, IRF1, egr-1, and bcl-2, leading to the induction of cell growth, differentiation, and survival. The deregulated expression of IL-6 and its receptor is involved in a variety of diseases.

• Peters M, Blinn G, Solem F, Fischer M, Meyer zum Buschenfelde KH, Rose-John S
• In vivo and in vitro activities of the gp130-stimulating designer cytokine Hyper-IL-6.
• J Immunol. 1998; 161: 3575-81
• Display abstract

• IL-6 is a multifactorial cytokine mediating acute inflammatory responses in the liver. When IL-6 binds to a specific receptor (IL-6R), the IL-6/IL-6R complex associates with the signal transducer gp130, initiating intracellular signaling. A soluble form of the IL-6R (sIL-6R) renders target cells sensitive to IL-6 that do not express the IL-6R on their surfaces. A designer cytokine, termed Hyper-IL-6, consisting of IL-6 covalently linked to the sIL-6R was fully active on gp130-expressing cells at 100- to 1000-fold lower concentrations than unlinked IL-6 and IL-6R. Mice were injected i.p. with Hyper-IL-6 or IL-6. Upon injection of Hyper-IL-6 into mice, the acute phase response, as measured by haptoglobin mRNA expression in the liver, was markedly increased and lasted significantly longer compared with that in mice injected with a 10-fold higher dose of IL-6 alone. On human hepatoma cells, Hyper-IL-6 caused similar effects, indicating that the longer lasting response to the fusion protein could not only be explained by the longer plasma half-life of the fusion protein. Experiments using iodinated IL-6 and Hyper-IL-6 revealed that Hyper-IL-6 bound with high affinity to gp130 and was less efficiently internalized. This effect might explain the longer lasting activity of this protein on cells. The highly active IL-6/sIL-6R designer protein might be of significant clinical importance for the stimulation of cells that are more responsive to the IL-6/sIL-6R complex than to IL-6 alone. Such cells include hemopoietic progenitor cells and hepatocytes.

• Bonig H et al.
• Interaction between interleukin 10 and interleukin 6 in human B-cell differentiation.
• Immunol Invest. 1998; 27: 267-80
• Display abstract

• Contrary to their opposing action on human T-lymphocytes and monocytes, both Interleukin (IL-)10 and IL-6 are potent differentiation factors of human B-cells. Both are known to induce immunoglobulin (Ig) production. The precise mechanism of this converging effect of IL-6 and IL-10 remains elusive, however. Here we investigated the role of IL-6 in the IL-10 dependent B-cell differentiation into Ig secreting cells. We found that co-stimulation of SAC-stimulated human peripheral B-lymphocytes with IL-10 and IL-6 exhibited no additive effect on Ig production over stimulation with IL-10 alone, and that IL-6 receptor blockade only mildly inhibited IL-10 induced Ig synthesis. In fact, we could show that stimulation of B-cells with IL-10 somewhat suppressed SAC induced autocrine IL-6 production. Despite this suppression IL-6 levels remained sufficiently high to stimulate its receptor, and IL-6 binding to the B-cell surface was not affected. The failure of IL-6 to exert an additional effect on SAC+IL-10 induced Ig production suggests that IL-10 may recruit components of the IL-6 intracellular pathway for Ig induction. In conclusion we could demonstrate that IL-10 acts on B-cell differentiation independently of autocrine IL-6 and that it had a considerably mild effect on B lymphocytic autocrine IL-6 secretion. This still allows an IL-6 effect in the presence of IL-10 which appears adaptive with a view to the converging effects of these two cytokines on human B lymphocytes. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.

• Igaz P, Toth S, Madurka I, Falus A
• [Interleukin-6 acts in different ways via soluble and membrane-bound receptors]
• Orv Hetil. 1998; 139: 1741-4
• Display abstract

• Interleukin-6 is a multifunctional cytokine participating in the regulation of several immunologic and other cell-physiological phenomena. It acts via a receptor consisting of two components, that besides the ligand-specific chain also contains a second component of 130 kD (gp 130). The soluble form of the ligand-specific component of this receptor was shown to occur physiologically in body fluids and -following the binding of interleukin-6-to be capable of associating with the membrane-bound receptor component and inducing signal-transduction. We studied the possible differences between the effects of interleukin-6 exerted via membrane-bound or soluble receptors on HepG2 human hepatoma and primary rat hepatocyte cultures. We used two methods to study the action of interleukin-6: the mRNA expression of the protooncogene junB as an early marker, and the protein production of fibrinogen as a late one. The effect of interleukin-6 on both cell types examined with both methods used was lower via the soluble than the membrane-bound receptor. In addition, the soluble receptors alone (without interleukin-6) could induce the expression of the junB gene. Considering the wide-spread biological and pathological activities of interleukin-6 these phenomena could have some role in the pathogenesis of some diseases.

• Iwasaki T, Hamano T, Fujimoto J, Kakishita E
• Regulation of interleukin-6 and interleukin-6R alpha (gp80) expression by murine immunoglobulin-secreting B-cell hybridomas.
• Immunology. 1998; 93: 498-504
• Display abstract

• We have examined the contribution of endogenous interleukin-6 (IL-6) to the differentiation of murine B-cell hybridomas. AT73 was established by somatic hybridization between BALB/c mice B cells and 2.52M, a hypoxanthine-aminopterine-thymidine (HAT) medium-sensitive B-cell line mutant. It spontaneously secreted IgM, and addition of exogenous IL-6 augmented IgM secretion. Triggering of CD40 led to an augmentation of IL-6 expression and IgM secretion. Blocking the binding of IL-6 to its cellular receptor through the use of inhibitory monoclonal antibodies inhibited CD40-induced IgM secretion, suggesting a possible autocrine role of IL-6 for the differentiation of a CD40-activated B-cell hybridoma. Co-triggering with CD40 and B-cell receptor or activation through CD40 and IL-4 led to a synergistic augmentation of IL-6 expression as well as additive IgM secretion; this was followed by a marked decrease in the expression of B-cell surface markers on the cell membrane. Furthermore, under conditions where IL-6 expression was augmented, gp80 expression was down-regulated, suggesting a negative feedback mechanism in this B-cell hybridoma. These findings provide a role by which T-cell-dependent activation through CD40 regulates an IL-6 autocrine loop, controlling B-cell differentiation.

• Mackiewicz A et al.
• Immunogene therapy of human melanoma. Phase I/II clinical trial.
• Adv Exp Med Biol. 1998; 451: 557-60

• Peters M, Muller AM, Rose-John S
• Interleukin-6 and soluble interleukin-6 receptor: direct stimulation of gp130 and hematopoiesis.
• Blood. 1998; 92: 3495-504

• Fischer M et al.
• I. A bioactive designer cytokine for human hematopoietic progenitor cell expansion.
• Nat Biotechnol. 1997; 15: 142-5
• Display abstract

• Efficient expansion of hematopoietic progenitor cells requires, at least, the simultaneous stimulation of the receptors c-kit and gp130. While c-kit is activated by SCF; gp130, in cells which do not express sufficient amounts of IL-6R, can be activated by the complex of soluble IL-6R (sIL-6R) and IL-6. The therapeutic use of IL-6/sIL-6R, however, has been hampered by the high concentrations of the sIL-6R protein required. We have designed a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain, that was expressed to high levels. On gp130 expressing cells the fusion protein turned out to be fully active at 100 to 1,000-fold lower concentration than the combination of unlinked IL-6 and IL-6R. The fusion protein was used to effectively expand human hematopoietic progenitor cells ex vivo in a dose dependent fashion.

• Taga T
• The signal transducer gp130 is shared by interleukin-6 family of haematopoietic and neurotrophic cytokines.
• Ann Med. 1997; 29: 63-72
• Display abstract

• Receptors for many of the cytokines functioning in the haematopoietic system belong to the class I cytokine receptor family. In most cases these receptors share common signal transducing receptor components in the same family, which explains the functional redundancy of haematopoietic cytokines. Interleukin-6 and related cytokines, interleukin-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1, are all pleiotrophic, from the haematopoietic to the nervous system, and exhibit overlapping biological activities. Receptors for these cytokines fall into the class I cytokine receptor family. Functional receptor complexes for the interleukin-6 family of cytokines share a membrane glycoprotein 130 (gp130) as a critical component for signal transduction. In these receptor complexes, gp130 and ligand-specific chains possess no intrinsic tyrosine kinase domain but are associated with cytoplasmic tyrosine kinases. Ligand stimulation triggers homo- or heterodimerization of gp130, leading to activation of the associated cytoplasmic tyrosine kinases and subsequent modification of transcription factors. This paper reviews the recent progress in the study of gp130 and the background information from biomedical and biochemical viewpoints.

• Xu GY et al.
• Solution structure of recombinant human interleukin-6.
• J Mol Biol. 1997; 268: 468-81
• Display abstract

• Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

• Fukutoku M, Shimizu S
• [gp-130 related cytokines in mechanisms of myeloma cell growth]
• Rinsho Ketsueki. 1997; 38: 262-5

• Simpson RJ, Hammacher A, Smith DK, Matthews JM, Ward LD
• Interleukin-6: structure-function relationships.
• Protein Sci. 1997; 6: 929-55
• Display abstract

• Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor, and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affinity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.

• Hirano T, Nakajima K, Hibi M
• Signaling mechanisms through gp130: a model of the cytokine system.
• Cytokine Growth Factor Rev. 1997; 8: 241-52
• Display abstract

• The interleukin-6 cytokine family plays roles in a wide variety of tissues and organs, including the immune hematopoietic and nervous systems. Gp130 is a signal-transducing subunit shared by the receptors for the IL-6 family of cytokines. The binding of a ligand to its receptor induces the dimerization of gp130, leading to the activation of JAK tyrosine kinase and tyrosine phosphorylation of gp130. These events lead to the activation of multiple signal-transduction pathways, such as the STAT, Ras-MAPK and PI-3 kinase pathways whose activation is controlled by distinct regions of gp130. We propose a model showing that the outcome of the signal transduction depends on the balance or interplay among the contradictory signal transduction pathways that are simultaneously generated through a cytokine receptor in a given target cell.

• Franchimont N, Rydziel S, Delany AM, Canalis E
• Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoblast cultures.
• J Biol Chem. 1997; 272: 12144-50
• Display abstract

• Interleukin-6 (IL-6), a cytokine produced by skeletal cells, increases bone resorption, but its effects on collagenase expression are unknown. We tested the effects of IL-6 and its soluble receptor on collagenase 3 expression in osteoblast-enriched cells from fetal rat calvariae (Ob cells). IL-6 caused a small increase in collagenase mRNA levels, but in the presence of IL-6-soluble receptor (IL-6sR), IL-6 caused a marked increase in collagenase transcripts after 2-24 h. In addition, IL-6sR increased collagenase mRNA when tested alone. IL-6 and IL-6sR increased immunoreactive collagenase levels. Cycloheximide and indomethacin did not prevent the effect of IL-6 and IL-6sR on collagenase mRNA levels. IL-6 and IL-6sR did not alter the decay of collagenase mRNA in transcriptionally arrested Ob cells and increased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. IL-6 and IL-6sR increased collagenase 3 mRNA in MC3T3 cells but only modestly in skin fibroblasts. IL-6 and IL-6sR enhanced the expression of tissue inhibitor of metalloproteinases 1. In conclusion, IL-6, in the presence of IL-6sR, increases collagenase 3 synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the action of IL-6 on bone matrix degradation and bone resorption.

• Grotzinger J, Kurapkat G, Wollmer A, Kalai M, Rose-John S
• The family of the IL-6-type cytokines: specificity and promiscuity of the receptor complexes.
• Proteins. 1997; 27: 96-109
• Display abstract

• The cytokines IL-6, LIF, CNTF, OSM, IL-11, and CT-1 have been grouped into the family of IL-6-type cytokines, since they all require gp130 for signal transduction. Interestingly, gp130 binds directly to OSM, whereas complex formation with the other cytokines depends on additional receptor subunits. Only limited structural information on these cytokines and their receptors is available. X-ray structures have been solved for the cytokines LIF and CNTF, whose up-up-down-down four-helix bundle is common to all of these cytokines, and for the receptors of hGH and prolactin, which contain two domains with a fibronectin III-like fold. Since cocrystallization and x-ray analysis of the up to four different proteins forming the receptor complexes of the IL-6-type cytokines is unlikely to be achieved in the near future, model building based on the existing structural information is the only approach for the time being. Here we present model structures of the complexes of human and murine IL-6 with their receptors. Their validity can be deduced from the fact that published mutagenesis data and the different receptor specificity of human and murine IL-6 can be understood. It is now possible to predict the relative positions and contacts for all molecules in their respective complexes. Such information can be used for the rational design of cytokine and receptor antagonists, which may have a valuable therapeutic perspective.

• Bank U, Reinhold D, Kunz D, Ansorge S
• Selective proteolytical cleavage of the ligand-binding chains of the IL-2-receptor and IL-6-receptor by neutrophil-derived proteases.
• Adv Exp Med Biol. 1997; 421: 231-42

• Young DC, Zhan H, Cheng QL, Hou J, Matthews DJ
• Characterization of the receptor binding determinants of granulocyte colony stimulating factor.
• Protein Sci. 1997; 6: 1228-36
• Display abstract

• We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.

• Shizuya K, Komori T, Fujiwara R, Miyahara S, Ohmori M, Nomura J
• The influence of restraint stress on the expression of mRNAs for IL-6 and the IL-6 receptor in the hypothalamus and midbrain of the rat.
• Life Sci. 1997; 61: 13540-13540
• Display abstract

• Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated the influence of restraint stress on the expression of the mRNA for interleukin-6 (IL-6) and the mRNA for the IL-6 receptor (IL-6R) in the rat brain. After rats had been restrained for 4 hours, the hypothalamus and midbrain were removed at fixed intervals up to 24 hours, and levels of IL-6 mRNA and of IL-6R mRNA in these regions were determined by RT-PCR. Restraint stress significantly enhanced the expression of IL-6 mRNA and reduced that of IL-6R mRNA in the midbrain, whereas the stress caused the reduced expression of IL-6R mRNA without any change in the level of IL-6 mRNA in the hypothalamus. After the stress, the expression of mRNAs for IL-6 and IL-6R continued to diminish in both regions. These findings indicate that the levels of mRNAs for both of IL-6 and IL-6R in the rat brain can be influenced by restraint stress.

• Nishimoto N, Shima Y, Sasai M, Danno N, Yoshizaki K
• [Clinical application of interleukin-6 receptor antibody]
• Nihon Rinsho Meneki Gakkai Kaishi. 1997; 20: 87-94
• Display abstract

• Interleukin-6 (IL-6) is a pleiotropic cytokine which shows multiple biological functions. Pathological significance of IL-6 has been elucidated in various diseases including multiple myeloma, Castleman's disease, and rheumatoid arthritis. Thus the blockade of IL-6 signal transduction may be therapeutically effective for these diseases. For this purpose, humanized anti-IL-6 receptor antibody was prepared, and its therapeutic efficacy has been examined. Immediately after administration of humanized anti-IL-y receptor antibody to the patients with multiple myeloma, fever and systemic edema disappeared followed by the stability of M-protein which had been rapidly increased before the treatment. Humaniged anti-IL-6 receptor antibody also improved not only the chronic inflammatory symptoms but also laboratory findings such a hemoglobin, C-reactive protein, erythrocyte sedimentation rate observed both in Castleman's disease and in rheumatoid arthritis. The data suggest that the blockade of IL-6 signal transduction can be a new therapeutic approach based on the pathological significance of IL-6 in these diseases.

• Korolenko TA, Heinrich PK, Hemmann U, Weiergraber O, Dittrich E, Graeve L
• [Cell endocytosis of the complex interleukin-6-soluble interleukin-6 receptor and its intralysosomal degradation]
• Biull Eksp Biol Med. 1997; 124: 527-9

• Hisano S, Sakamoto K, Ishiko T, Kamohara H, Ogawa M
• IL-6 and soluble IL-6 receptor levels change differently after surgery both in the blood and in the operative field.
• Cytokine. 1997; 9: 447-52
• Display abstract

• To investigate alterations in post-operative levels of IL-6 and soluble IL-6 receptor (sIL-6R), we examined their levels in serum and samples of drainage fluids from 26 patients who underwent thoracoabdominal surgery. Serum IL-6 levels reached the maximum within the first post-operative day and decreased thereafter. The IL-6 levels in the drainage fluid were much higher than in the serum (458 +/- 101-fold; mean +/- SEM) in the early post-operative phase. A large quantity of sIL-6R levels was present in blood samples. The time course of serum sIL-6R levels in 26 patients showed no significant change. sIL-6R concentrations in the drainage fluid were significantly lower than in serum (4.5 +/- 1.1-fold; mean +/- SEM) in the early post-operative phase. We propose that IL-6 is produced in the operative field and enters the peripheral blood stream to induce elevation of serum IL-6. On the other hand, sIL-6R levels in the operative field are lower than in the serum, and the serum sIL-6R levels are not influenced by surgical trauma. These data suggest that sIL-6R is being constantly produced in areas other than the operative field, while sIL-6R level is reduced by consumption in the operative field. Mechanisms to cope with surgical stress, involving sIL-6R together with its ligand IL-6 may thus exist.

• Ryan JJ
• Interleukin-4 and its receptor: essential mediators of the allergic response.
• J Allergy Clin Immunol. 1997; 99: 1-5

• Kalai M et al.
• Analysis of the human interleukin-6/human interleukin-6 receptor binding interface at the amino acid level: proposed mechanism of interaction.
• Blood. 1997; 89: 1319-33
• Display abstract

• The interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6-inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their alpha-subunit receptors suggests that the structural location of certain binding sites are conserved.

• Liu H, Duan J, Wang J, Peng S, Zou M
• Residues K128-Q175 of human interleukin-6 are essential for its biological activity.
• Biochem Mol Biol Int. 1997; 42: 1045-50
• Display abstract

• Internal deletion K128-Q175 of the human interleukin-6 (hIL-6) has been generated at the cDNA level. With pBV220 as expressing vector, the recombinant pBV*-DIL-6 encoding the deletion mutant (12 kD) of hIL-6 has been constructed. The resulted recombinant plasmids were then used to transform E. coli strain HB101, and the expression in the PLPR promoter system, which is temperature-regulatable, was achieved. After purification and renaturation, the biological activity of the expressed product, designated as DM120, was measured by MTT method in an IL-6-dependent cell line 7TD1. The results show that the amino acid residues of IL-6 128 to 175 are crucial for IL-6 activity. Receptor binding assay in vitro indicates that the entire region is not involved in forming the receptor binding surface.

• Horsten U et al.
• Molecular modeling-guided mutagenesis of the extracellular part of gp130 leads to the identification of contact sites in the interleukin-6 (IL-6).IL-6 receptor.gp130 complex.
• J Biol Chem. 1997; 272: 23748-57
• Display abstract

• The transmembrane protein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal-transducing subunit. In the case of interleukin-6 (IL-6), the signal-transducing complex is composed of the ligand IL-6, the IL-6 receptor (IL-6R, gp80, CD126), and at least two gp130 (CD130) molecules. The extracellular part of the signal transducer gp130 consists of six fibronectin type III-like domains. It has recently been shown that the three membrane distal domains bind to the IL-6. IL-6R complex. A structural model of the IL-6.IL-6R.gp130 complex enabled us to propose amino acid residues in these domains of gp130 interacting with IL-6 bound to its receptor. The proposed amino acid residues located in the B'C' loop (Val252) and in the F'G' loop (Gly306, Lys307) of domain 3 and in the hinge region (Tyr218) connecting domains 2 and 3 of gp130 were mutated to disturb ternary complex formation. Binding of wild type and mutants of the extracellular region of gp130 was studied by use of a co-precipitation assay and Scatchard analysis. All mutants showed decreased binding to the IL-6.IL-6R complex. Biological function of the membrane-bound gp130 mutants was studied by STAT (signal transducer and activator of transcription) activation in COS-7 cells and by proliferation of stably transfected Ba/F3 cells. Reduced binding of the mutants was accompanied by decreased biological activity. The combined approach of molecular modeling and site-directed mutagenesis has led to the identification of amino acid residues in gp130 required for complex formation with IL-6 and its receptor.

• Duan J, Wang J, Cai X, Wang L, Liu H, Zou M
• Characterization of a soluble IL-6 receptor alpha mutant C277D/H280I expressed in E.coli.
• Biochem Mol Biol Int. 1997; 41: 1101-8
• Display abstract

• The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal transducer gp130. To construct the antagonist of IL-6, a DNA segment encoding the soluble human IL-6R alpha (shIL-6R alpha) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold. DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells. DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.

• Rettig MB
• Interleukin-6: an antagonizing problem becomes a solution.
• Nat Biotechnol. 1997; 15: 952-3

• Hooper WC, Phillips DJ, Evatt BL
• Endothelial cell protein S synthesis is upregulated by the complex of IL-6 and soluble IL-6 receptor.
• Thromb Haemost. 1997; 77: 1014-9
• Display abstract

• We have recently demonstrated that the proinflammatory cytokine, interleukin-6 (IL-6), could upregulate the production of protein S in the human hepatoma cell line, HepG-2, but not in endothelial cells. In this study, we have demonstrated that the combination of exogenous IL-6 and soluble IL-6 receptor (sIL-6R) could significantly upregulate protein S production in both primary human umbilical vein endothelial cells (HUVEC) and in the immortalized human microvascular endothelial cell line, HMEC-1. The IL-6/sIL-6R complex was also able to rapidly induce tyrosine phosphorylation of the IL-6 transducer, gp130. Neutralizing antibodies directed against either IL-6 or gp130 blocked protein S upregulation by the IL-6/sIL-6R complex. It was also observed that exogenous sIL-6R could also upregulate protein S by forming a complex with IL-6 constitutively produced by the endothelial cell. Two other cytokines which also utilize the gp130 receptor, oncostatin M (OSM) and leukemia inhibitory factor (LIF), were also able to upregulate endothelial cell protein S. This study demonstrates a mechanism that allows endothelial cells to respond to IL-6 and also illustrates the potential importance of circulating soluble receptors in the regulation of the anticoagulation pathway.

• Peters M et al.
• Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.
• J Immunol. 1997; 159: 1474-81
• Display abstract

• There is a growing number of soluble agonistic (IL-6, ciliary neurotropic factor, IL-11, and glia-derived neurotropic factor receptors) and antagonistic (IL-1 and TNF receptors) receptor proteins, modulating the biological functions of their cognate ligands. The physiologic role of these receptor molecules in vivo is unclear. In particular, it is not known how the specificity of function of soluble receptors after release into the blood stream is maintained. We addressed this question by studying the function of the soluble IL-6R (sIL-6R) at the cellular level in the liver. We have generated double transgenic mice coexpressing human sIL-6R and human IL-6 in the liver and have analyzed the expression patterns by in situ hybridization. The expression of the human sIL-6R, driven by the phosphoenolpyruvate carboxykinase promoter, is located mainly in periportal areas, whereas human IL-6 under the control of the metallothionein promoter is uniformly expressed throughout the liver. We show here by in situ hybridization that acute phase protein gene expression induced by human IL-6 and human sIL-6R correlated with the periportal expression of sIL-6R, indicating that sIL-6R acts mainly in an area where it is generated. We conclude that in a concentration-dependent manner, at low concentrations of sIL-6R, there is a predominantly paracrine action at the site of its generation, whereas at higher concentrations of the sIL-6R there are both local and systemic effects.

• Fujii I, Nagahara Y, Yamasaki M, Yokoo Y, Itoh S, Hirayama N
• Structure of KW-2228, a tailored human granulocyte colony-stimulating factor with enhanced biological activity and stability.
• FEBS Lett. 1997; 410: 131-5
• Display abstract

• KW-2228 is a tailored human granulocyte colony-stimulating factor (hG-CSF) which has more potent granulopoietic activity and is more stable than wild-type hG-CSF. Analysis of the 2.3 A resolution crystal structure of KW-2228 unambiguously revealed a four-alpha-helix bundle motif with up-up-down-down connectivity. The structures of long overhand loops connecting the helices and the N-terminus have been definitively determined. The present analysis has clearly revealed that substituted residues play important roles in fastening a long overhand loop to the N- and C-termini to fix the conformation. This conformation should be responsible for a substantial enhancement of the biological activity and stability.

• Taga T, Kishimoto T
• Gp130 and the interleukin-6 family of cytokines.
• Annu Rev Immunol. 1997; 15: 797-819
• Display abstract

• Receptors for most interleukins and cytokines that regulate immune and hematopoietic systems belong to the class I cytokine receptor family. These molecules form multichain receptor complexes in order to exhibit high-affinity binding to, and mediate biological functions of, their respective cytokines. In most cases, these functional receptor complexes share common signal transducing receptor components that are also in the class I cytokine receptor family, i.e. gp130, common beta, and common gamma molecules. Interleukin-6 and related cytokines, interleukin-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1 are all pleiotropic and exhibit overlapping biological functions. Functional receptor complexes for this interleukin-6 family of cytokines share gp130 as a component critical for signal transduction. Unlike cytokines sharing common beta and common gamma chains that mainly function in hematopoietic and lymphoid cell systems, the interleukin-6 family of cytokines function extensively outside these systems as well, e.g. from the cardiovascular to the nervous system, owing to ubiquitously expressed gp130. Stimulation of cells with the interleukin-6 family of cytokines triggers homo- or hetero-dimerization of gp130. Although gp130 and its dimer partners possess no intrinsic tyrosine kinase domain, the dimerization of gp130 leads to activation of associated cytoplasmic tyrosine kinases and subsequent modification of transcription factors. This paper reviews recent progress in the study of the interleukin-6 family of cytokines and gp130.

• Marz P, Herget T, Lang E, Otten U, Rose-John S
• Activation of gp130 by IL-6/soluble IL-6 receptor induces neuronal differentiation.
• Eur J Neurosci. 1997; 9: 2765-73
• Display abstract

• Interleukin-6 (IL-6) on target cells binds to the specific IL-6 receptor (IL-6R) and subsequently induces homodimerization of the signal-transducing protein gp130. Cells which express gp130 but no IL-6R and which therefore do not respond to IL-6 can be stimulated by the complex of IL-6 and soluble IL-6R (slL-6R). Here we show that on rat pheochromocytoma cells (PC12), the combination of IL-6 and slL-6R but not IL-6 alone induces expression of c-fos, GAP-43 and neuron-specific enolase followed by neuron-specific differentiation and formation of a neuronal network. The differentiation was dose-and time-dependent and followed the same kinetics as nerve-growth factor (NGF)-induced differentiation. The responses of PC12 cells to IL-6/sIL-6R and NGF were additive, suggesting independent signaling pathways. We demonstrate that activation of gp130 generates a neuronal differentiation signal that is equivalent to and independent of trk/NGF receptor tyrosine kinase. Interestingly, the failure of IL-6 to induce differentiation of PC12 cells is not due to lack of surface expression of IL-6R as IL-6 alone triggered expression of GAP-43 mRNA and protein. We hypothesize that PC12 cells express more gp130 than IL-6R and that the extent of activated gp130 molecules determines the quality of the response.

• Zwadlo-Klarwasser G, Hamann W, Gatsios P, Graeve L, Schmutzler W
• Expression of interleukin-6 receptor subunits in human macrophage subsets.
• Int Arch Allergy Immunol. 1997; 113: 300-1

• Romano M et al.
• Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment.
• Immunity. 1997; 6: 315-25
• Display abstract

• IL-6-/- mice showed impaired leukocyte accumulation in subcutaneous air pouches. Defective leukocyte accumulation was not due to a reduced migratory capacity of IL-6-/- leukocytes and was associated with a reduced in situ production of chemokines. These observations led to a reexamination of the interaction of IL-6 with endothelial cells (EC). EC express only the gp130 signal transducing chain and not the subunit-specific IL-6R and are therefore unresponsive to IL-6. However, EC are responsive to a combination of IL-6 and soluble IL-6R as measured by the activation of STAT3, chemokine expression, and augmentation of ICAM-1. Activation by IL-6-IL-6R complexes was inhibited by an IL-6 receptor antagonist and potentiated by a superagonist. Hence, in vivo and in vitro evidence supports the concept that the IL-6 system plays an unexpected positive role in local inflammatory reactions by amplifying leukocyte recruitment.

• Kalai M et al.
• Analysis of the mechanism of action of anti-human interleukin-6 and anti-human interleukin-6 receptor-neutralising monoclonal antibodies.
• Eur J Biochem. 1997; 249: 690-700
• Display abstract

• Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.

• Takizawa H, Ohtoshi T, Yamashita N, Oka T, Ito K
• Interleukin 6-receptor expression on human bronchial epithelial cells: regulation by IL-1 and IL-6.
• Am J Physiol. 1996; 270: 34652-34652
• Display abstract

• Airway epithelial cells have a potential to participate in regulation of local homeostasis by releasing active compounds including cytokines and growth factors. Several factors such as transforming growth factor-beta and endothelin have been shown to regulate airway epithelial cell functions through an autocrine mechanism. We studied the expression of the specific receptor for a multifunctional cytokine interleukin 6 (IL-6), which is expressed and released by airway epithelial cells. Specific binding assay demonstrated a single set of binding sites on human primary and transformed bronchial epithelial cells. Human interleukin-1alpha (IL-1alpha) increased maximal binding sites to IL-6. Northern blot analysis demonstrated that airway epithelial cells constitutively expressed mRNA for IL-6 receptor (IL-6R), and IL-1alpha and IL-6 itself upregulated IL-6R gene expression. Moreover, exogenously added human recombinant IL-6 had a stimulatory effect on IL-8 release from human bronchial epithelial cells. These results indicated that human bronchial epithelial cells expressed IL-6R, and IL-6 might be involved in the regulation of the epithelial functions via an autocrine as well as a paracrine mechanism.

• Oppmann B, Stoyan T, Fischer M, Voltz N, Marz P, Rose-John S
• Alternative assay procedures for cytokines and soluble receptors of the IL-6 family.
• J Immunol Methods. 1996; 195: 153-9
• Display abstract

• Human hepatoma cells (HepG2 cells) were transfected with expression vectors for human IL-6 (hIL-6) and rat IL-6R (rIL-6-R). The cell lines were used for testing the biological activity of different IL-6 species, soluble hIL-6R (shIL-6R) and some members of the IL-6 cytokine family by means of an ELISA procedure. The assay is based on induction of the gene expression of the acute phase protein haptoglobin in hepatoma cells and provides an alternative bioassay taking advantage of the hepatocyte stimulatory activity of IL-6 (as opposed to the B9 proliferative assay). A dose-response experiment with IL-6 showed that half-maximal stimulation was achieved with approx. 5 ng/ml of hIL-6 in HepG2 cells and with 5-10 ng/ml muIL-6 in HepG2-rIL-6R cells after 24 h. The same response was achieved with 10 ng/ml shIL-6R in HepG2-IL6 cells. In conclusion, the assay is fast and reliable and might be adopted for other cytokines and receptors with hepatocyte stimulating activity.

• Hofbauer LC, Heufelder AE
• Intercellular chatter: osteoblasts, osteoclasts and interleukin 6.
• Eur J Endocrinol. 1996; 134: 425-6

• Kalai M et al.
• Participation of two Ser-Ser-Phe-Tyr repeats in interleukin-6 (IL-6)-binding sites of the human IL-6 receptor.
• Eur J Biochem. 1996; 238: 714-23
• Display abstract

• The alpha-subunit of interleukin-6 (IL-6) receptor is a member of the hematopoietin receptor family. The alignment of its amino acid sequence with those of other members of this family (human somatotropin receptor/murine IL-3 receptor beta and human IL-2 receptor beta) has suggested that amino acids included in two SSFY repeats found in each of its hematopoietin receptor domains, contribute to the binding of the ligand. The involvement of these amino acids in IL-6 binding and signal transduction was studied by site-directed mutagenesis and molecular modelling. We present a computer-derived three-dimensional model of the IL-6/IL-6 receptor complex based on the structure of the human somatotropin/human somatotropin receptor complex. This model allowed the location of distinct regions important for IL-6 and gp130 binding. We show that some of the residues included in the SSFY repeats located in our IL-6 receptor model in the loops between beta-strands E and F of domain-I and B' and C', of domain-II, participate in the formation of a major IL-6-binding site. These residues are necessary for IL-6 and gp130 binding and for signal transduction. Using our IL-6 receptor mutants we mapped the epitopes of our anti-(IL-6 receptor) neutralising monoclonal antibodies to these residues. Our results demonstrate that a generic hematopoietin receptor family structural module can be used for the study of both alpha and beta receptor subunits belonging to this family.

• Mundy GR
• An OAF by any other name.
• Endocrinology. 1996; 137: 1149-50

• Bencosme A, Warner A, Healy D, Verme C
• Prognostic potential of cytokines, nitrates, and APACHE II score in sepsis.
• Ann Clin Lab Sci. 1996; 26: 426-32
• Display abstract

• The prognostic potentials of physiological ([Acute Physiologic and Chronic Health Evaluation] APACHE II score) and biochemical (Interleukin 6 [IL-6], Interleukin 6 soluble receptor [IL-6sR], and Interleukin 2 receptor [IL-2R], nitrates) measures were evaluated in sepsis. The APACHE II scores were calculated, and concentrations of the biochemical markers were measured, based upon information and samples obtained from 68 septic patients at time of diagnosis. Outcome (survival/non-survival) was determined at the end of the hospital stay associated with the septic episode. Statistically significant differences between survivors (S) and non-survivors (non-S) were found for APACHE II (p < 0.0001), IL-6 (p < 0.005), IL-6sR (p < 0.05), IL-2R (p < 0.02). No significant differences were found for nitrates. None of the markers could serve individually as an effective prognostic indicator. However, those markers demonstrating a significant difference between survivors and non-survivors may be able to contribute to a multi-parameter prognostic model.

• Reyes TM, Coe CL
• Interleukin-1 beta differentially affects interleukin-6 and soluble interleukin-6 receptor in the blood and central nervous system of the monkey.
• J Neuroimmunol. 1996; 66: 135-41
• Display abstract

• Two studies were conducted to investigate whether behavioral and physiological responses induced by administration of interleukin-1 beta (IL-1 beta) were also associated with changes in interleukin-6 (IL-6) and soluble IL-6 receptor levels (sIL-6R). Following intravenous injection of rhIL-1 beta, blood and cerebrospinal fluid (CSF) samples were collected from juvenile rhesus monkeys. Marked increases in IL-6 levels were evident at 1 h in both blood and intrathecal compartments. IL-1 beta also induced significant elevations in the release of ACTH and cortisol into the blood stream, and following high doses, the monkeys evinced signs of sickness behavior. The second study characterized the IL-beta dose-response relationship showing that these physiological changes were most evident at doses between 0.5 microgram and 1.0 microgram IL-1/kg body weight. Soluble IL-6 receptor concentration was also increased, but only in plasma. There was no detectable sIL-6R in CSF. The large release of IL-6 into CSF suggests that some behavioral symptoms may be due to intrinsic changes in central nervous system activity concomitant with the alterations in peripheral physiology.

• Teraoka H, Mikoshiba M, Takase K, Yamamoto K, Tsukada K
• Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines: dimethyl sulfoxide inhibits IL-6-induced differentiation of SKW6-CL4 into IgM-secreting plasma cells.
• Exp Cell Res. 1996; 222: 218-24
• Display abstract

• We have previously found that dimethyl sulfoxide (DMSO), a known inducer of differentiation in several kinds of myeloid cells, arrests proliferation of human lymphoid cells including Raji and Akata Burkitt's lymphoma cells at the G1 phase. We investigated whether DMSO affects cell proliferation and differentiation of the lymphoid cell line SKW6-CL4, which is capable of differentiating terminally into IgM-producing cells. As in the case of Raji, Akata, and Molt-4, the proliferation of SKW6-CL4 was reversibly arrested at the G1 phase by treatment with 2% DMSO for 5 days even in the presence of interleukin-6 (IL-6). DMSO inhibited spontaneous IgM secretion as well as IL-6-induced IgM production in SKW6-CL4 at a concentration lower than that affecting cell proliferation. Of the cell-surface differentiation markers CD10, CD20, CD21, and CD23, the expression of CD20 was suppressed by DMSO treatment, and partial restoration of the expression was observed 24 to 48 h after release from DMSO. The level of IL-6 receptor protein was not affected by DMSO treatment. These results indicate that DMSO not only arrests the cell cycle of a human lymphoid cell line SKW6-CL4 at the G1 phase but also inhibits the differentiation into IgM-secreting cells at a concentration lower than that affecting cell proliferation and that DMSO overcomes the effect of IL-6 on terminal differentiation of SKW6-CL4. As a whole, proliferation of human lymphoblastoid cell lines was revealed to be reversibly arrested at the G1 phase by DMSO, which is known to induce differentiation in several myeloid cells, without inducing cell differentiation.

• Ishiyama T, Koike M, Nakamura S, Kakimoto T, Akimoto Y, Tsuruoka N
• Interleukin-6 receptor expression in the peripheral B cells of patients with multicentric Castleman's disease.
• Ann Hematol. 1996; 73: 179-82
• Display abstract

• Interleukin-6 (IL-6) is an important regulator of terminal B-cell differentiation. Inappropriate oversynthesis of IL-6 may play primary role in the pathogenesis of multicentric Castleman's disease (MCD). We investigated the expression of the IL-6 receptor (IL-6R) in peripheral B cells from three patients with MCD, as well as the responsiveness of these cells to IL-6. Flow-cytometric analysis showed that IL-6R was significantly expressed on the peripheral B cells of two of three patients. The B cells expressing IL-6R spontaneously produced increased levels of immunoglobulin G (IgG). IL-6R-expressing B cells from one patient showed hyper-responsiveness to IL-6.

• Ehlers M, Grotzinger J, Fischer M, Bos HK, Brakenhoff JP, Rose-John S
• Identification of single amino acid residues of human IL-6 involved in receptor binding and signal initiation.
• J Interferon Cytokine Res. 1996; 16: 569-76
• Display abstract

• The pleiotropic cytokine interleukin-6 (IL-6) has been predicted to be a protein with four antiparallel alpha-helices. On target cells, IL-6 interacts with a specific ligand binding receptor subunit (IL-6R), and this complex associates with the signal-transducing subunit gp130. Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of chimeric human/murine IL-6 proteins has allowed us to define a region (residues 77-95, region 2c) within the human IL-6 protein that is important for IL-6R binding and a region (residues 50-55, region 2a2) that is important for IL-6R dependent gp130 interaction. Guided by sequence alignment and molecular modeling, we have constructed several IL-6 variants with point mutations in these regions and have tested them for receptor binding and signal initiation. Within region 2c, phenylalanine 78 was involved in receptor binding, whereas lysine 54 within region 2a2 participated in gp130 activation. Furthermore, some IL-6 variants with lysine 54 replacements could be used to construct muteins that retained receptor binding but failed to activate gp130. Such IL-6 muteins were efficient IL-6 receptor antagonists.

• Gaillard JP, Liautard J, Mani JC, Fernandez Suarez JM, Klein B, Brochier J
• Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain.
• Immunology. 1996; 89: 135-41
• Display abstract

• The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6 IL-6R to the gp130 chain, we showed that M182 was directed against a structure directly involved in the IL-6R gp130 interaction. M182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling.

• Kelly RA, Smith TW
• de modulatione cordis.
• Circulation. 1996; 94: 2361-3

• Smith SR, Morgan L
• Clinical significance of elevated soluble interleukin 6 receptor levels in patients with plasma cell disorders.
• Br J Haematol. 1996; 92: 767-8

• Paquet P, Pierard GE
• Interleukin-6 and the skin.
• Int Arch Allergy Immunol. 1996; 109: 308-17
• Display abstract

• Interleukin-6 is a pleiotropic cytokine with numerous biological activities. It is produced by normal constituents of the skin, including epidermal cells, fibroblasts and dermal endothelial cells. It is also synthesized by inflammatory cells infiltrating the skin in various pathological conditions. We reviewed the presumed activities of interleukin-6 in normal skin and in some diseases with cutaneous involvement. The action of some drugs used in dermatology was also considered with regard to the modulation of the cytokine release. It is concluded that several important cellular lineages of the skin release interleukin-6. The cytokine appears to play a crucial role in the pathogenesis of both local and systemic inflammation, tumor development and autoimmune diseases.

• Hibi M, Nakajima K, Hirano T
• IL-6 cytokine family and signal transduction: a model of the cytokine system.
• J Mol Med. 1996; 74: 1-12
• Display abstract

• The interleukin 6 (IL-6) cytokine family, which includes IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-11 and cardiotrophin-1 (CT-1), exhibits pleiotropy and redundancy in biological activities. The IL-6 family cytokines exhibit a helical structure. Their receptors belong to the type 1 cytokine receptor family. The receptors of the IL-6 family cytokines share a receptor subunit, which explains one of the mechanisms of functional redundancy. In this review, we describe the general features of the IL-6 cytokine family and its signal transduction mechanisms. Many functional properties of the IL-6 family of cytokines and their receptors are general features of the cytokine system.

• Vos JP, Gard AL, Pfeiffer SE
• Regulation of oligodendrocyte cell survival and differentiation by ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6.
• Perspect Dev Neurobiol. 1996; 4: 39-52
• Display abstract

• The regulation and maintenance of developmental lineages by trophic factors, both cell-mediated and soluble, is a key aspect of cellular differentiation in the nervous system. In this review we focus on oligodendrocytes and their progenitors and how differentiation and survival are regulated by four neuropoietic cytokines: ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6 (IL-6). We discuss how these cytokines act as "broad spectrum" factors. That is, how, even within a specific cell lineage, a given cytokine may have different effects on the target cells at various stages of differentiation.

• Callard RE, Matthews DJ, Hibbert L
• IL-4 and IL-13 receptors: are they one and the same?
• Immunol Today. 1996; 17: 108-10
• Display abstract

• Interleukin 4 (IL-4) and IL-13 share several biological properties, suggesting that they also share a common receptor or receptor component. Indeed, as discussed here by Robin Callard and colleagues, the IL-13 receptor appears to be a functional receptor for IL-4.

• Akira S, Kishimoto T
• Role of interleukin-6 in macrophage function.
• Curr Opin Hematol. 1996; 3: 87-93
• Display abstract

• Interleukin-6 is a multifunctional cytokine important for host defense. Macrophages are potent producers of interleukin-6. Conversely, interleukin-6 acts on monocytes to induce their differentiation to macrophages. This paper reviews the in vivo roles of interleukin-6 and nuclear factor for interleukin-6 expression that have been revealed by gene targeting as well as recent progress in understanding the interleukin-6 gene regulation and signaling pathway in macrophages.

• Biffl WL, Moore EE, Moore FA, Peterson VM
• Interleukin-6 in the injured patient. Marker of injury or mediator of inflammation?
• Ann Surg. 1996; 224: 647-64
• Display abstract

• OBJECTIVE: The effects of interleukin (IL)-6 in the injured patient are examined in an attempt to clarify the potential pathophysiologic role of IL-6 in the response to injury. SUMMARY BACKGROUND DATA: Interleukin-6 is an integral cytokine mediator of the acute phase response to injury and infection. However, prolonged and excessive elevations of circulating IL-6 levels in patients after trauma, burns, and elective surgery have been associated with complications and mortality. The mechanistic role of IL-6 in mediating these effects is unclear. METHODS: A review of current literature is performed to summarize the origins, mechanisms of action, and biologic effects of IL-6 and to characterize the IL-6 response to injury. RESULTS: Interleukin-6 is a multifunctional cytokine expressed by a variety of cells after a multitude of stimuli, under complex regulatory control mechanisms. The IL-6 response to injury is uniquely consistent and related to the magnitude of the insult. Moreover, the early postinjury IL-6 response correlates with complications as well as mortality. CONCLUSIONS: Interleukin-6 appears to play an active role in the postinjury immune response, making it an attractive therapeutic target in attempts to control hyperinflammatory provoked organ injury.

• Waelti ER, Inaebnit SP, Weismann U, Limat A, Hunziker T
• Interleukin 6 receptors on human outer root sheath cells and interfollicular epidermal keratinocytes in vitro: density-induced down regulation (DIDR) of receptors.
• In Vitro Cell Dev Biol Anim. 1996; 32: 255-8

• Barton BE
• The biological effects of interleukin 6.
• Med Res Rev. 1996; 16: 87-109

• Graeve L, Korolenko TA, Hemmann U, Weiergraber O, Dittrich E, Heinrich PC
• A complex of the soluble interleukin-6 receptor and interleukin-6 is internalized via the signal transducer gp130.
• FEBS Lett. 1996; 399: 131-4
• Display abstract

• In human body fluids a soluble form of the interleukin-6 receptor (sIL-6R) has been found which together with interleukin-6 (IL-6) acts agonistically on cells expressing the signal transducer gp130. The means by which the sIL-6R is removed from the circulation is unknown. Here, we show that a complex of 125I-labelled recombinant sIL-6R and IL-6 is internalized by MDCK cells stably transfected with gp130 and by human hepatoma cells HepG2 that endogenously express the IL-6R and gp130. We further show that most of the internalized sIL-6R is degraded within lysosomes. Our studies suggest that cells expressing gp130 are capable of endocytosing an IL-6/sIL-6R complex, thereby removing both from the circulation.

• Crichton MB, Nichols JE, Zhao Y, Bulun SE, Simpson ER
• Expression of transcripts of interleukin-6 and related cytokines by human breast tumors, breast cancer cells, and adipose stromal cells.
• Mol Cell Endocrinol. 1996; 118: 215-20
• Display abstract

• The expression of transcripts of cytokines of the interleukin-6 (IL-6) family has been examined in human breast tumors, breast cancer cell lines, and adipose stromal cells, by means of reverse transcription polymerase chain reaction amplification. Of the six breast tumor samples examined, all expressed transcripts encoding IL-6 and Leukemia Inhibitory Factor (LIF). Four of the samples also expressed transcripts for oncostatin M (OSM) and IL-11, and three expressed the IL-6 receptor. Adipose stromal cells expressed IL-6, IL-11 and LIF, but not the IL-6 receptor, consistent with previous conclusions that IL-6 activity in these cells required addition of IL-6 soluble receptor. In the case of T47D cells, expression of IL-11 protein was confirmed by immunotitration. Moreover, in these cells, expression of IL-11 transcripts was induced 3-fold by addition of estradiol to the culture medium. These results add credence to our previous proposal that breast cancer development is regulated in part by local autocrine and paracrine mechanisms via epithelial/mesenchymal interactions, in which estrogen produced by stromal cells surrounding the tumor acts to stimulate the production of growth factors and cytokines by the tumor cells. Some of these may act to stimulate further the growth and development of the tumor, while these or other factors may act on the surrounding mesenchymal cells in a paracrine fashion to stimulate aromatase expression in the presence of glucocorticoids. Thus, a positive feedback loop is established which leads to the development and growth of the tumor.

• Demartis A, Bernassola F, Savino R, Melino G, Ciliberto G
• Interleukin 6 receptor superantagonists are potent inducers of human multiple myeloma cell death.
• Cancer Res. 1996; 56: 4213-8
• Display abstract

• Interleukin-6 (IL-6) plays a central role in the pathogenesis of multiple myeloma, acting as both a growth and a survival factor for myeloma cells. A series of IL-6 receptor antagonists that are IL-6 variants has been recently obtained, the affinity of which for the ligand-specific receptor chain IL-6R alpha has been maintained or even increased, but the signaling of which is impaired by not being able to bind and/or dimerize the signaling chain gp130. Although IL-6 antagonists have been shown to inhibit the growth of IL-6-dependent myeloma, no information has been gathered on their ability to induce myeloma cell death. We show here that IL-6 receptor antagonists are pro-apoptotic factors for the IL-6-dependent human myeloma cell line XG-1. Their capacity to induce cell death is in direct relation to their affinity for IL-6R alpha, degree of gp130 binding impairment, and efficiency to inhibit intracellular signaling events. Interestingly, the most potent pro-apoptotic molecule, Sant7, counteracts the protective autocrine effect exercised by the limited amounts of IL-6 produced by XG-1 cells and is thus able to induce cell death at higher rate than just IL-6 deprivation. These findings are particularly relevant for the therapy of multiple myeloma.

• Maeba T et al.
• Increase in portal blood interleukin-6 soon after the commencement of digestive surgery.
• Surg Today. 1996; 26: 890-4
• Display abstract

• To determine whether cytokines produced in the operative field during digestive surgery selectively spill over into the portal blood, the changes in interleukin-6 (IL-6) levels in portal and peripheral venous blood were assayed at several points in time from the commencement of surgery until 14 days later, in 11 patients. Similar changes in the IL-6 levels were observed in the portal and peripheral blood samples; however, the IL-6 levels in the portal blood reached a maximum 6-12h after the commencement of surgery, being earlier than in the peripheral venous blood. In fact, between 3 and 12h after the commencement of surgery, the IL-6 levels were higher in the portal blood by 33-81 pg/ml. By 24h or more after the commencement of surgery, the IL-6 levels did not differ significantly in the two types of blood samples. Moreover, the C-reactive protein levels 2 days after surgery were even more closely correlated to the maximum IL-6 levels in the portal blood than to those in the peripheral venous blood. These results suggest that IL-6 produced during intra-abdominal digestive surgery initially enters the portal blood, being trapped by IL-6 receptors in the liver, where it may regulate the synthesis of acute-phase proteins as a hepatocyte-stimulating factor.

• Cichy J, Rose-John S, Pryjma J, Travis J
• Effect of soluble interleukin-6 receptor on interleukin-6 synthesis in human skin fibroblasts.
• Biochem Biophys Res Commun. 1996; 227: 318-21
• Display abstract

• In this study the ability of soluble interleukin-6 receptor (sIL-6R) to stimulate interleukin-6 (IL-6) synthesis in human fibroblasts is described. It was found that sIL-6R, in combination with endogenous or exogenous IL-6, markedly upregulated IL-6 synthesis. These data suggest that increased IL-6 production after stimulation by either interleukin-1 or tumor necrosis factor-alpha would result in complex formation with sIL-6R, rapid uptake, and further synthesis of this cytokine. Furthermore, it would explain the decrease in sIL-6R plasma levels observed in patients suffering from sepsis.

• Weiergraber O et al.
• Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor.
• FEBS Lett. 1996; 379: 122-6
• Display abstract

• Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding. Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified.

• Kong B, Isozaki T, Sasaki S
• IL-6 antisense-mediated growth inhibition of a choriocarcinoma cell line: an intracellular autocrine growth mechanism.
• Gynecol Oncol. 1996; 63: 78-84
• Display abstract

• The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of choriocarcinoma and explore a new therapeutic approach for the carcinoma, we examined the role of IL-6 in the growth of a human choriocarcinoma cell line (JEG-3). IL-6 was identified in the supernatant of the cell culture medium by enzyme-linked immunosorbent assay, indicating that this cell secreted IL-6. The mRNAs of IL-6, IL-6 receptor and gP130, the IL-6 signal transducer, in this cell were shown to be present by reverse transcriptase polymerase chain reaction assay and confirmed by Southern blot hybridization and direct sequencing. The addition of hrIL-6 to the cell culture failed to stimulate cell growth. Monoclonal antibodies against IL-6, IL-6 receptor, and gP130 were also unable to inhibit the proliferation of this cell line. The antisense oligonucleotides targeting IL-6 mRNA, however, inhibited both cell growth and IL-6 production. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of the JEG-3 cell line, and it exerts its action by an intracellular autocrine growth mechanism. The results also suggest that the therapeutic trials with monoclonal antibodies designed to neutralize IL-6 or block its receptor will likely fail, whereas the antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of choriocarcinoma and other cancers with intracellular autocrine growth fashion mediated by IL-6.

• Chung U, Tanaka Y, Fujita T
• Association of interleukin-6 and hypoaldosteronism in patients with cancer.
• N Engl J Med. 1996; 334: 473-473

• Peters M, Meyer zum Buschenfelde KH, Rose-John S
• The function of the soluble IL-6 receptor in vivo.
• Immunol Lett. 1996; 54: 177-84
• Display abstract

• Interleukin-6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover, IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B-cells, T-cells, keratinocytes, neuronal cells, osteoclasts and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific IL-6 receptor (IL-6R) and a signal-transducing subunit (gp130). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as AIDS, rheumatoid arthritis and others. The function of the soluble IL-6R in vivo is unknown. To discriminate between the biologic function of hIL-6 alone and that of the hIL-6/hsIL-6R complex, mice transgenic for human IL-6, for the human soluble IL-6R and for both, human IL-6 and the human soluble IL-6R were analyzed and compared with nontransgenic littermates. While IL-6 transgenic mice exhibit elevated acute phase protein levels and develop plasmacytomas, hsIL-6R single transgenic mice are hypersensitized towards human IL-6, mounting an acute phase protein gene induction at significantly lower IL-6 dosages compared to control animals. Furthermore, in hsIL-6R transgenic mice, the acute phase response persists for a longer period of time and the IL-6 plasma half life was markedly prolonged. IL-6/sI1-6R mice, however, develop massive hepatosplenomegaly caused by extramedullary hematopoisis in these organs. In IL-6- and IL-6R-single transgenic mice, no such effects were observed. Our study discloses a novel biologic effect of the hIL-6/hsIL-6R complex, which is clearly distinct from that of hIL-6 alone. We provide evidence that the activation of the gp130 signal transducer represents a major stimulation of growth and differentiation of hematopoietic progenitor cells.

• Path G, Bornstein SR, Spath-Schwalbe E, Scherbaum WA
• Direct effects of interleukin-6 on human adrenal cells.
• Endocr Res. 1996; 22: 867-73
• Display abstract

• Interleukin-6 (IL-6), which is expressed in the human adrenal gland, was found to be a very potent activator of the human HPA axis. So far nothing is known about a local paracrine or autocrine influence of IL-6 within the human adrenal. In this study, the expression of IL-6 and the IL-6 receptor by human adrenal cells in vitro could be demonstrated by immunohistochemistry. Possible effects of IL-6 on steroid release were tested by incubating human adrenal cells in vitro with IL-6 [10(-8) M]. Adrenal steroids were stimulated by IL-6: aldosterone 184 +/- 23, cortisol 198 +/- 19, DHEA 140 +/- 8 and androstenedione 136 +/- 5 (results are means +/- s.e.m. in %). In conclusion, IL-6 can act directly on human adrenal cells and appears to be an important paracrine or autocrine factor.

• Sehgal PB
• Interleukin-6-type cytokines in vivo: regulated bioavailability.
• Proc Soc Exp Biol Med. 1996; 213: 238-47
• Display abstract

• Investigators have traditionally thought of the class of inflammation- and injury-associated cytokines in large part as "free" entities in the peripheral circulation. In the case of interleukin-6 (IL-6), the cytokine can be found in blood in complexes of molecular mass 400-500, 150-200, and 25-35 kDa in association with binding proteins that can include soluble IL-6 receptor (sIL-6R), anti-IL-6, and anti-sIL-6R IgG, and others. Sustained high levels of different particular IL-6 complexes are observed in the human circulation in cancer patients subjected to particular active anticancer immunotherapy regimens. In the "chaperoned" state, circulating IL-6 complexes display differential immunoreactivity in different ELISAs and possess differential biological activity as assayed ex vivo. The discovery of "chaperoned" circulating IL-6 in humans points to a new level of modulation of cytokine function, that of regulated bioavailability of IL-6 in vivo.

• Hammacher A, Ward LD, Weinstock J, Simpson RJ
• Structural and biological characterization of murine-human interleukin-6 chimeras.
• Ann N Y Acad Sci. 1995; 762: 422-3

• Kishimoto T, Akira S, Narazaki M, Taga T
• Interleukin-6 family of cytokines and gp130.
• Blood. 1995; 86: 1243-54

• de Hon FD et al.
• Leucine-58 in the putative 5th helical region of human interleukin (IL)-6 is important for activation of the IL-6 signal transducer, gp130.
• FEBS Lett. 1995; 369: 187-91
• Display abstract

• A model of the tertiary structure of human IL-6, derived from the crystal-structure of granulocyte-colony stimulating factor, reveals a 5th helical region in the loop between the first and second alpha-helix. To investigate the importance of this region for biological activity of IL-6, residues Glu-52, Ser-53, Ser-54, Lys-55, Glu-56, Leu-58, and Glu-60 were individually replaced by alanine. IL-6.Leu-58Ala displayed a 5-fold reduced biological activity on the IL-6 responsive human cell lines XG-1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL-6 receptor-alpha-chain-dependent binding to the IL-6 signal transducer, gp130.

• Heinrich PC et al.
• Membrane-bound and soluble interleukin-6 receptor: studies on structure, regulation of expression, and signal transduction.
• Ann N Y Acad Sci. 1995; 762: 222-36

• Kishimoto T, Tanaka T, Yoshida K, Akira S, Taga T
• Cytokine signal transduction through a homo- or heterodimer of gp130.
• Ann N Y Acad Sci. 1995; 766: 224-34

• Sayinalp N, Haznedaroglu IC, Ozdemir O, Ozcebe OI, Dundar S, Kirazli S
• Interleukin-1 beta and interleukin-6 in clonal versus reactive thrombocytosis.
• Eur J Haematol. 1995; 55: 339-40

• Zeni F, Tardy B, Vindimian M, Pain P, Gery P, Bertrand JC
• Soluble interleukin-6 receptor in patients with severe sepsis.
• J Infect Dis. 1995; 172: 607-8

• May LT, Ndubuisi MI, Patel K, Garcia D
• Interleukin-6 chaperones in blood.
• Ann N Y Acad Sci. 1995; 762: 120-8
• Display abstract

• There is increasing evidence that many, perhaps all, cytokines have a soluble form of their receptor in the systemic circulation at all times. There is also evidence that endogenous antibodies to some cytokines, including IL-6, are also found in blood. Initially these findings were evaluated in vitro, and associated with inhibiting the respective effects of those cytokines. However, it is now becoming clear that the in vivo effects are paradoxically the reverse of what is seen in vitro. As we have explained here for IL-6 it is evident that many or all of these molecules that bind and/or associate with IL-6 maintain this molecule in the systemic circulation and constitute a reservoir of masked, but potentially active IL-6. The mode of regulation of the biological activity of these IL-6-associated complexes remains unknown, but needs to be uncovered in order to pharmacologically exploit many of the potentially beneficial effects or to prevent any potential pathological effects.

• Lahm A et al.
• The molecular design of human IL-6 receptor antagonists.
• Ann N Y Acad Sci. 1995; 762: 136-50

• Rose-John S, Ehlers M, Grotzinger J, Mullberg J
• The soluble interleukin-6 receptor.
• Ann N Y Acad Sci. 1995; 762: 207-20

• Ehlers M et al.
• Residues 77-95 of the human interleuken-6 protein are responsible for receptor binding and residues 41-56 for signal transduction.
• Ann N Y Acad Sci. 1995; 762: 400-2

• Gadient RA, Otten U
• Interleukin-6 and interleukin-6 receptor mRNA expression in rat central nervous system.
• Ann N Y Acad Sci. 1995; 762: 403-6

• Ward LD, Howlett GJ, Hammacher A, Moritz RL, Simpson RJ
• Stoichiometry of the interleukin-6 high affinity receptor complex.
• Ann N Y Acad Sci. 1995; 762: 471-3

• Wulf P, Piekorz RP, Hocke GM
• Functional reconstitution of IL-6 signaling in a myeloid leukemic cell line.
• Ann N Y Acad Sci. 1995; 762: 485-7

• Breton J et al.
• Structure, stability and biological properties of a N-terminally truncated form of recombinant human interleukin-6 containing a single disulfide bond.
• Eur J Biochem. 1995; 227: 573-81
• Display abstract

• A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys-->Ser substitution at positions 45 and 51 was produced in Escherichia coli. The 163-residue protein des-(A1-S22)-[C45S, C51S]interleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18,695 +/- 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukin-6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 +/- 0.4 kJ.mol-1 for the thermodynamic stability of the protein at 25 degrees C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent.

• Hansen MB, Svenson M, Diamant M, Abell K, Bendtzen K
• Interleukin-6 autoantibodies: possible biological and clinical significance.
• Leukemia. 1995; 9: 1113-5
• Display abstract

• The pleiotropic cytokines, interleukin (IL)-1 alpha, type I interferons and IL-6 also act on cells involved in antibody production. Somehow the immunologic tolerance to these cytokines is often spontaneously broken--even in healthy individuals. Thus, relatively high concentrations of high affinity IgG antibodies against IL-1 alpha and IL-6 frequently occur in the circulation of healthy adults. The autoantibodies specifically antagonize the respective cytokines in vitro. Thermodynamic estimations strongly suggest that autoimmunity can play a significant role in the regulation of certain cytokines. In the light of IL-6 autoantibodies the possible biological and clinical significance of cytokine autoimmunity is discussed.

• Chen-Kiang S
• Regulation of terminal differentiation of human B-cells by IL-6.
• Curr Top Microbiol Immunol. 1995; 194: 189-98

• Tani Y, Nishimoto N, Ogata A, Shima Y, Yoshizaki K, Kishimoto T
• Gp130 in human myeloma/plasmacytoma.
• Curr Top Microbiol Immunol. 1995; 194: 229-33

• Lehrnbecher T, Schrod L, Kraus D, Roos T, Martius J, von Stockhausen HB
• Interleukin-6 and soluble interleukin-6 receptor in cord blood in the diagnosis of early onset sepsis in neonates.
• Acta Paediatr. 1995; 84: 806-8

• Markman M
• 86th annual meeting of the American Association for Cancer Research. Toronto, Canada, March 18-22, 1995.
• J Cancer Res Clin Oncol. 1995; 121: 439-40

• Duan J et al.
• Cloning, expression and purification of the ligand-binding region of human IL-6R in E. coli and its preliminary functional identification.
• Sci China B. 1995; 38: 1321-31
• Display abstract

• The ligand-binding region of human IL-6R is taken as the target gene fragment to be cloned and expressed. With pET-3b as expressing vector, two recombinants pET-6R(B) and pET-6R(B)4 have been constructed encoding the ligand-binding region (28 kD) of hIL-6R and its dimmer (53 kD), respectively. After induction with IPTG, they produced two proteins rIL6R-28 of 28 kD and rIL6R-53 of 53 kD amounting to 50% and 30% of total bacteria proteins, respectively. The expressed products were mainly recovered as inclusion bodies. After purification and renaturation, both of them were capable of augmenting the growth-stimulating effect of IL-6 on 7TD1 cells, an IL-6 dependent cell line. The result of ELISA also revealed that both rIL6R-28 and rIL6R-53 had the obvious ligand-binding activity.

• Mackiewicz A et al.
• Gene therapy of human melanoma. Immunization of patients with autologous tumor cells admixed with allogeneic melanoma cells secreting interleukin 6 and soluble interleukin 6 receptor.
• Hum Gene Ther. 1995; 6: 805-11

• Jones TH
• Interleukin-6 an endocrine cytokine.
• Clin Endocrinol (Oxf). 1994; 40: 703-13

• Borden EC, Chin P
• Interleukin-6: a cytokine with potential diagnostic and therapeutic roles.
• J Lab Clin Med. 1994; 123: 824-9
• Display abstract

• Interleukin-6 is a pleiotropic cytokine and may be a pivotal mediator in the pathogenesis of shock and sepsis, in modulating megakaryocytopoiesis, and in inhibition of tumor growth. Among characteristics of interleukin-6 are regulation of expression of other cytokines, induction of differentiation and proliferation of normal and malignant cells, and inhibition of tumor growth in vivo under experimental conditions. As a major inducer of the acute phase response, interleukin-6 is produced and sets off a chain of events as it acts on effector targets. Preclinical anti-tumor studies with interleukin-6 have provided rationale for probing its role in the therapy of malignancy. The probability is that in the near future interleukin-6 will have established clinical roles as a protein of diagnostic and therapeutic import.

• Brailly H, Montero-Julian FA
• [Cytokine binding proteins: a model of interleukin 6]
• Arch Inst Pasteur Tunis. 1994; 71: 535-48

• Fontaine V, Ooms J, Content J
• Mutagenesis of the human interleukin-6 fourth predicted alpha-helix: involvement of the Arg168 in the binding site.
• Eur J Immunol. 1994; 24: 1041-5
• Display abstract

• Random substitutions of amino acid 161-184 of human interleukin-6 (hIL-6) have been generated at the cDNA level using oligonucleotide-directed mutagenesis. Among the majority of the mutant proteins showing a reduced biological activity on murine hybridoma cells, only those having a substitution of Met161, Arg168, Arg179 or Met184, retained a tertiary structure similar to the IL-6 folding. These residues are thus probably involved in the interaction with the IL-6 receptor. However, the contacts established by Arg168 and Arg179 seem far more important for the biological activity. According to Bazan's model of cytokine folding and the receptor binding site on the fourth alpha-helix, based on growth hormone similarity, we propose that Arg168 and Arg179 are located on the exposed surface of this presumed helix.

• Ershler WB, Sun WH, Binkley N
• The role of interleukin-6 in certain age-related diseases.
• Drugs Aging. 1994; 5: 358-65
• Display abstract

• Interleukin-6 (IL-6) is a pro-inflammatory cytokine with a wide range of functions. Perhaps the most important physiologically is its role as a mediator of the acute phase inflammatory response. Normally, there is little measurable IL-6 in the circulation, but levels increase abruptly to nanogram amounts during an inflammatory process. During aging, it has been proposed that the tight regulation of IL-6 gene expression becomes less effective and levels are measurable even when there is no evidence for inflammation. Several investigators have identified this cytokine as being involved in the pathogenesis of various disease processes and we have suggested that certain age-associated diseases are directly related. Among these are late-life lymphoma and myeloma, osteoporosis and possibly Alzheimer's disease.

• Sato N, Miyajima A
• Multimeric cytokine receptors: common versus specific functions.
• Curr Opin Cell Biol. 1994; 6: 174-9
• Display abstract

• The class I cytokine receptors consist of multiple subunits without any intrinsic enzymatic activities. Receptors for a subset of cytokines with overlapping biological activities often share a common receptor subunit with a signaling function. Each receptor regulates its specific signaling pathways, as well as common pathways depending on the target cell type.

• Yamamori T, Sarai A
• Evolution of the IL-6/class IB cytokine receptor family in the immune and nervous systems.
• J Physiol Paris. 1994; 88: 165-71
• Display abstract

• It has been suggested that the cytokine receptor has a structure similar to immunoglobulin and this structural similarity has raised the possibility that they have evolved from a common ancestral molecule. In the early 1970s, it was discovered that developing sympathetic neurons could switch from an adrenergic to cholinergic phenotype. The search for a diffusible factor responsible for this eventually led to the identification of leukemia inhibitory factor (LIF). Cholinergic differentiation factor (CDF)/LIF has turned out to belong to the IL-6/class IB cytokine family. In this article we further speculate on a plausible molecular pathway for the IL6/class IB receptor family in the immune and nervous systems. We think that the evolution of the IL-6/class IB receptor family may have occurred in at least two major steps. Firstly, binding subunits of an IL-6 receptor and for a CDF/LIF receptor evolved and secondly, a third binding subunit of a CNTF receptor evolved. Our evolutional consideration predicts that the binding subunits generally determine the specificity of the receptors and it is possible that novel members of the cytokine family and their receptors exist in the nervous system.

• Okada K, Shimizu Y, Nambu S, Higuchi K, Watanabe A
• Interleukin-6 functions as an autocrine growth factor in a cholangiocarcinoma cell line.
• J Gastroenterol Hepatol. 1994; 9: 462-7
• Display abstract

• The tumour cells of a human cholangiocarcinoma cell line, HuCC-T1, were found to express mRNA of interleukin-6 (IL-6) and to secrete a large amount of biologically active IL-6 in the culture medium at the concentration of 22.6 ng/mL. Interleukin-6 was demonstrated in the cytoplasm of the cells by immunohistochemical staining. Furthermore, these cells showed the presence of receptors for IL-6 on the surface, and DNA synthesis of the cells was stimulated by the exogenous addition of recombinant human IL-6 into the culture medium. The cell growth was significantly inhibited in the presence of anti-human IL-6 antibody in the culture medium. These findings indicate that IL-6 is one of the autocrine growth factors of this cell line in vitro.

• Hoffmann R, Henninger HP, Schulze-Specking A, Decker K
• Regulation of interleukin-6 receptor expression in rat Kupffer cells: modulation by cytokines, dexamethasone and prostaglandin E2.
• J Hepatol. 1994; 21: 543-50
• Display abstract

• Interleukin-6 has a variety of biological effects, mainly on the immune system. The regulation of this signal at both the site of production and the site of action is necessary to maintain the organism's homeostasis. In the microenvironment of the hepatic sinusoids, Kupffer cells as resident macrophages are the most potent source of interleukin-6 during inflammation. This cytokine is an important signal to hepatocytes during the early stages of the acute-phase response, leading to the expression of several major plasma proteins. Kupffer cells were found to express interleukin-6 receptor constitutively. Interleukin-6 decreased the level of interleukin-6 receptor mRNA, indicating an autocrine pathway by which Kupffer cells regulate their responsiveness to interleukin-6. Furthermore, lipopolysaccharide, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 beta and phorbol ester induced interleukin-6 production and, at the same time, suppressed the level of interleukin-6 receptor mRNA. The existence of an autocrine loop in rat Kupffer cells may be physiologically relevant, as it would contribute to a regulated interleukin-6 signal chain in the liver. The anti-inflammatory mediators dexamethasone or PGE2 and its second messenger, cyclic AMP, increased interleukin-6 receptor mRNA, whereas prostaglandin D2 or the Ca2+ ionophore, A 23187, were without effect. The changes in interleukin-6 mRNA were paralleled by the number of interleukin-6 receptors present on Kupffer cells as detected by binding of 125I-interleukin-6. These results suggest the existence of control mechanisms involving several soluble mediators that help balance the level of interleukin-6-R mRNA in rat liver macrophages.

• Hirano T, Matsuda T, Nakajima K
• Signal transduction through gp130 that is shared among the receptors for the interleukin 6 related cytokine subfamily.
• Stem Cells. 1994; 12: 262-77
• Display abstract

• Interleukin 6 (IL-6) and related cytokines, such as leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and IL-11 exhibit multiple functions and redundancy in biological activities and play important roles in the immune response, hematopoiesis, the nervous system and acute phase reactions. These IL-6 family cytokines exhibit a similar helical structure, and their receptors are structurally similar and constitute a cytokine receptor super family. In addition, a receptor subunit is shared among these IL-6 related cytokine subfamily receptors, contributing to one of the mechanisms of functional redundancy of cytokine activities and suggesting the presence of a common signal transduction pathway among these receptors. In this review, we describe the structure of the receptors for IL-6 and its related cytokine subfamily members. Furthermore, we propose a novel mechanism for the generation of cytokine diversity, i.e. the complex of a cytokine and one of its receptor subunits act as a novel cytokine on the cells that express the other receptor subunit(s) capable of acting as a receptor for the complex. Finally, we describe a Ras-independent novel signal transduction pathway that utilizes Jak tyrosine kinase family, Stat protein family and yet unidentified H-7-sensitive pathway. This signal transduction pathway is commonly generated through the receptors for a wide range of cytokines and growth factors.

• Zhang JG, Reid GE, Moritz RL, Ward LD, Simpson RJ
• Specific covalent modification of the tryptophan residues in murine interleukin-6. Effect on biological activity and conformational stability.
• Eur J Biochem. 1993; 217: 53-9
• Display abstract

• Modification of recombinant murine interleukin-6 (mIL-6) with the tryptophan-specific reagent 2-nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2-nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp-N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL-6 caused a 50% reduction in both the biological activity in the murine-hybridoma-growth-factor assay using 7TD1 cells and receptor-binding affinity to mIL-6 receptors. Sulfenylation of mIL-6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti-mIL-6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [delta delta G(H2O) = 3.98 kJ/mol] than unmodified mIL-6 as measured by urea-gradient gel electrophoresis.

• Jean LF, Waters CA, Keemy D, Murphy JR
• Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6.
• Protein Eng. 1993; 6: 305-11
• Display abstract

• We have used site-directed and in-frame deletion mutational analysis in order to explore the structural features of the IL-6 portion of the diphtheria toxin-related interleukin-6 (IL-6) fusion toxin DAB389-IL-6 that are essential for receptor-binding and subsequent inhibition of protein synthesis in target cells. Deletion of the first 14 amino acids of the IL-6 component of the fusion toxin did not alter either receptor binding affinity or cytotoxic potency. In contrast, both receptor binding and cytotoxic activity were abolished when the C-terminal 30 amino acids of the fusion toxin were deleted. In addition, we explored the relative role of the disulfide bridges within the IL-6 portion of DAB389-IL-6 in the stabilization of structure required for receptor-binding. The analysis of mutants in which the substitution of either Cys440, Cys446, Cys469 or Cys479 to Ser respectively, demonstrates that only the disulfide bridge between Cys469 and Cys479 is required to maintain a functional receptor binding domain. In addition, the internal in-frame deletion of residues 435-451, which includes Cys440 and Cys446, was found to reduce, but not abolish receptor binding affinity. These results further demonstrate that the disulfide bridge between Cys440 and Cys446 is not essential for receptor-binding. However, the reduced cytotoxic potency of DAB389-IL6(delta 435-451) suggests that the conformation and/or receptor binding sites associated with this region of the fusion toxin is/are important for maintaining the wild type receptor binding affinity and cytotoxic potency.

• Lotz M
• Interleukin-6.
• Cancer Invest. 1993; 11: 732-42

• Moutabarrik A et al.
• Effect of FK 506 and cyclosporine on the expression of IL-6 and its receptor on stimulated monocytes.
• Transplant Proc. 1993; 25: 2320-1

• van Dam M et al.
• Structure-function analysis of interleukin-6 utilizing human/murine chimeric molecules. Involvement of two separate domains in receptor binding.
• J Biol Chem. 1993; 268: 15285-90
• Display abstract

• As an approach to understanding the interaction of interleukin-6 (IL-6) and its 80-kDa receptor (gp80), we have constructed chimeric human/murine IL-6-molecules, which were expressed in Escherichia coli and analyzed for biological activity and receptor binding. This experimental strategy was based on the observation that human IL-6 acts on human and murine cells, whereas murine IL-6 stimulates only murine cells. The regions to be exchanged were chosen according to the four antiparallel helix model of the hematopoietic cytokine family. All 14 chimeras constructed showed biological activity on murine cells. From the differential biological activities on human cells we deduced that three out of four domains of IL-6 are involved in species specificity, whereas only two domains are necessary for specific recognition by the gp80 IL-6-receptor protein.

• Junker U, vd Heyden-Rynsch B, Diener C, Vogelsang H, Jager L
• In patients with common variable immunodeficiency, interleukin-6 and expression of its receptor on B-cells are normal.
• Cell Biol Int. 1993; 17: 609-14
• Display abstract

• In a group of patients suffering from common variable immunodeficiency (CVID), levels of interleukin-6 (IL6) were measured and found to be in the normal range or even increased, for single patients. IL6 production of peripheral blood mononuclear cells was shown to be in the normal range when corrected for monocyte number. To exclude the possibility that the Interleukin-6 receptor (IL6R) on B-cells is missing, its expression was measured by assessing the binding of a Phycoerythrin-derivative of IL6 by flow cytometry and correlated with various markers for B-cells and T-cells. As compared with normal controls, no statistically significant deviation of the group as a whole nor for individual patients could be shown. It was concluded that lack of B cell differentiation in the presence of normal to high IL6 as shown for these patients is not due to inability of the B cells to detect IL6 in the serum.

• Kishimoto T
• [Diseases associated with cytokine dysregulation]
• Nippon Sanka Fujinka Gakkai Zasshi. 1993; 45: 724-34
• Display abstract

• Communication between cells is essential for a wide variety of biological functions. One way cells interact in immune and hemopoietic systems is through soluble mediators called interleukins or cytokines. Many cytokines and their receptors have been identified and characterized at the molecular level. These studies have observed that most cytokines function in a pleiotropic and redundant manner. Receptor studies have shown that many cytokine receptors consist of two polypeptide chains, a ligand-binding receptor, and a nonbinding signal transducer. This arrangement may explain the pleiotropic and redundant effects of cytokines. For example, leukemia inhibitory factor (LIF) and IL-6 share many biological activities including platelet production, and the receptors of these cytokines utilize gp130 as a common signal transducer. IL-6 is multifunctional and produces both favorable and unfavorable effects on human health. Dysregulation of IL-6 expression is linked to the occurrence of cancer and autoimmune diseases, such as multiple myeloma, Castleman's disease, mesangial proliferative glomerulonephritis, and rheumatoid arthritis. Studies in transgenic mice in which the IL-6 gene was overexpressed have confirmed these pathogenic actions of IL-6. The pathogenesis of these diseases and therapies to treat them are discussed here based on insights derived from cytokine research.

• Rossi G, Schmitt M, Owsianowski M, Thiele B, Gollnick H
• IL-6 and its high affinity receptor during differentiation of monocytes into Langerhans cells.
• Adv Exp Med Biol. 1993; 329: 191-8

• Ward LD et al.
• Role of the C-terminus in the activity, conformation, and stability of interleukin-6.
• Protein Sci. 1993; 2: 1472-81
• Display abstract

• Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

• Savino R, Lahm A, Giorgio M, Cabibbo A, Tramontano A, Ciliberto G
• Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.
• Proc Natl Acad Sci U S A. 1993; 90: 4067-71
• Display abstract

• Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.

• Brouet JC, Levy Y
• [Interleukin 6 and lymphoplasmocytic proliferation]
• Nouv Rev Fr Hematol. 1993; 35: 225-6

• Elias JA
• Interleukin-6: on target for disease and approaching the bedside.
• J Lab Clin Med. 1992; 120: 672-4

• Gauldie J, Richards C, Baumann H
• IL6 and the acute phase reaction.
• Res Immunol. 1992; 143: 755-9

• Leebeek FW, Fowlkes DM
• Construction and functional analysis of hybrid interleukin-6 variants. Characterization of the role of the C-terminus for species specificity.
• FEBS Lett. 1992; 306: 262-4
• Display abstract

• We have constructed several hybrid human interleukin-6 (IL-6) variants in which the carboxyl-terminus, which includes a receptor binding site of IL-6 has been replaced with the C-terminus of various proteins homologous to human IL-6. IL-6 hybrids with the C-terminus of human growth hormone and human granulocyte-colony stimulating factor maintain part of the biological activity of human IL-6. Replacing the C-terminus of human IL-6 with the C-terminus of mouse and rat IL-6 resulted in a normal or increased activity on a mouse cell line; however, this gave a low (to 200-fold less) activity on a human cell line compared to wild-type human IL-6. We therefore conclude that the C-terminus of IL-6 plays an important role in the species specificity of IL-6.

• Cai YP, Zhu LP
• [Human interleukin-6 receptors]
• Sheng Li Ke Xue Jin Zhan. 1992; 23: 357-60

• Roodman GD
• Interleukin-6: an osteotropic factor?
• J Bone Miner Res. 1992; 7: 475-8

• Hirano T
• The biology of interleukin-6.
• Chem Immunol. 1992; 51: 153-80

• Revel M
• Growth regulatory functions of IL6 and antitumour effects.
• Res Immunol. 1992; 143: 769-73

• Fiorillo MT, Cabibbo A, Iacopetti P, Fattori E, Ciliberto G
• Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding.
• Eur J Immunol. 1992; 22: 2609-15
• Display abstract

• The multifunctional cytokine interleukin-6 (IL-6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse. Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL-6 shows species specificity. Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL-6 molecule are involved in human receptor binding. In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding. Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL-6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.

• Akira S, Kishimoto T
• The evidence for interleukin-6 as an autocrine growth factor in malignancy.
• Semin Cancer Biol. 1992; 3: 17-26
• Display abstract

• Interleukin-6, IL-6, is a pleiotropic cytokine which plays a central role in defense mechanisms, including the immune response, acute phase reaction and hematopoiesis. Abnormal expression of the IL-6 gene has been suggested to be involved in the pathogenesis of a variety of diseases, especially rheumatoid arthritis, Castleman's disease, mesangial proliferative glomerulonephritis, multiple myeloma and Kaposi's sarcoma. In the case of multiple myeloma and Kaposi's sarcoma, the existence of an IL-6-IL-6 receptor autocrine loop has been implicated in the oncogenesis process. On the other hand, IL-6 has a potent anti-tumor activity against certain types of tumors. This anti-tumor effect is mediated by in vivo induction of tumor specific cytotoxic T cells and in part by a growth inhibitory activity of IL-6.

• Kishimoto T, Akira S, Taga T
• Interleukin-6 and its receptor: a paradigm for cytokines.
• Science. 1992; 258: 593-7
• Display abstract

• Many cytokines and cytokine receptors involved in the regulation of hematopoiesis, immune responses, and inflammation have been identified and characterized at the molecular level. Several characteristic features of cytokines, such as pleiotropy and redundancy, are now more clearly understood on the basis of their molecular structures. Accumulating evidence has demonstrated an intimate link between cytokines and various diseases such as allergy, autoimmune diseases, and cancer. The pathogenesis of these diseases and therapies to treat them will be discussed based on insights derived from cytokine research.

• Leebeek FW, Kariya K, Schwabe M, Fowlkes DM
• Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.
• J Biol Chem. 1992; 267: 14832-8
• Display abstract

• To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.

• Yoshizaki K, Kuritani T, Kishimoto T
• Interleukin-6 in autoimmune disorders.
• Semin Immunol. 1992; 4: 155-66
• Display abstract

• Interleukin-6, a pleiotropic cytokine, appears to play a key role as a physiologically functioning molecule in host defense mechanisms. Previous reports have suggested that dysregulated interleukin-6 production may be involved in lymph node hyperplasia, plasmacytosis, immunoglobulin hyperproduction, thrombocytosis, mesangial cell proliferation and acute phase response, all of which are frequently observed in autoimmune disorders. In this report, we discuss the possible involvement of interleukin-6 in the pathogenesis of a variety of autoimmune diseases and the regulatory mechanism of expression of the interleukin-6 gene.

• Williams N
• Is thrombopoietin interleukin 6?
• Exp Hematol. 1991; 19: 714-8
• Display abstract

• Recent data concerning to ability of interleukin 6 (IL-6) to stimulate platelet production have raised the possibility that platelet production is not specifically regulated by a unique feedback mechanism, but is part of a network encompassing several hemopoietic growth factors. Hypotheses are presented about the nature of thrombopoietin, its relationship to known growth factors, especially IL-6, and the specificity of a thrombopoietic response following change in the circulating platelet mass.

• Bataille R, Klein B
• The bone-resorbing activity of interleukin-6.
• J Bone Miner Res. 1991; 6: 1143-6

• Hopkins SJ, Rothwell NJ
• Further functions of IL-6.
• Immunol Today. 1991; 12: 170-170

• Ling ZD, Gillis S, Hart LJ, Matheson DS
• Particle concentration fluorescence immunoassay for measuring interleukin-6 receptor numbers.
• Cytokine. 1991; 3: 17-20
• Display abstract

• Analysis of the number of receptors per cell and the affinity of the ligand/receptor interaction has provided considerable insight into the functioning of numerous cytokines. Interleukin-6 (IL-6) is a multifunctional cytokine which may have considerable clinical relevance in inflammatory or immunodeficiency diseases. Using particle concentration fluorescence immunoassay (PCFIA) technology, an assay is described which calculates the receptor number and affinity on small numbers of human cells. Resting B cells are shown to lack IL-6 receptors but activation of B cells induces up to 1,300 receptors per cell, with Kd of 1 x 10(-11) to 2 x 10(-11) M. Other recombinant mediators do not alter the binding of labeled IL-6 to the cells. PCFIA avoids the use of radioactivity and requires very small numbers of cells (2 x 10(4) per well). Potential application to the study of regulatory mechanisms and to clinical situations where small samples of blood are available is feasible.

• Paul W
• IL-6: a multifunctional regulator of immunity and inflammation.
• Jpn J Cancer Res. 1991; 82: 1458-9

• Matsuda T, Hirano T
• [Interleukin-6 and its receptor]
• Tanpakushitsu Kakusan Koso. 1991; 36: 1184-94

• Hirano T
• [Cytokines and diseases. Abnormal expression of interleukin 6 and diseases]
• Nippon Naika Gakkai Zasshi. 1991; 80: 1401-8

• Nishimura C et al.
• Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6.
• Eur J Biochem. 1991; 196: 377-84
• Display abstract

• Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.

• Van Snick J
• Interleukin-6: an overview.
• Annu Rev Immunol. 1990; 8: 253-78

• Miki Y, Miki S
• Change of genetic expression by physical compression forces.
• Cell Biol Int Rep. 1990; 14: 473-7
• Display abstract

• Despite intense investigation of transmembrane signalling mediated by lymphocyte surface molecules, the subcellular mechanisms responsible for these phenomena remain poorly understood (Kishimoto, T. et al., 1983). In the present study, an answer for the mechanism is demonstrated by using human B blastoid cell line (CESS) which has IgG and Interleukin 6 (IL-6) receptors on the cell surface and produces IgG in response to IL-6 without any requirement for proliferation (Muraguchi, A. et al., 1981).

• Hirano T, Akira S, Taga T, Kishimoto T
• Biological and clinical aspects of interleukin 6.
• Immunol Today. 1990; 11: 443-9
• Display abstract

• Interleukin 6 (IL-6) is a multi-functional cytokine that is produced by a range of cells and plays a central role in host defense mechanisms. Abnormal production of IL-6 has been suggested to be involved in glomerulonephritis, plasmacytomagenesis and in the pathogenesis of autoimmune diseases. In this review, Toshio Hirano and colleagues discuss the possible involvement of IL-6 in a variety of diseases, the regulatory mechanism(s) of expression of the IL-6 gene and the structure and function of the IL-6 receptor.

• Matsuda T, Hirano T
• Interleukin 6 (IL-6).
• Biotherapy. 1990; 2: 363-73

• Aderka D, Levo Y
• [Interleukin-6: biological characteristics and clinical importance]
• Harefuah. 1990; 118: 402-4

• Sehgal PB
• Interleukin 6 in infection and cancer.
• Proc Soc Exp Biol Med. 1990; 195: 183-91

• Murakami M, Hirano T
• [IL-6 and IL-6 receptor]
• Seikagaku. 1990; 62: 32-5

• Snyers L, Fontaine V, Content J
• Modulation of interleukin-6 receptors in human cells.
• Ann N Y Acad Sci. 1989; 557: 388-93

• Matsuda T, Yamasaki K, Taga T, Hirano T, Kishimoto T
• Current concepts of B cell modulation.
• Int Rev Immunol. 1989; 5: 97-109
• Display abstract

• Three interleukins with distinct functions, IL-4, IL-5 and IL-6, are involved in the regulation of B cell response into antibody producing cells. The studies with recombinant interleukins, however, demonstrated that the activities of these interleukins were not restricted to B lineage cells but showed a wide variety of biological functions on various tissues and cells. One of the most typical example of multifunctional interleukins is IL-6. It acts not only on B cells as B cell differentiation factor but also on T cells, hematopoietic stem cells, hepatocytes, nerve cells and myeloma cells. Deregulation of the expression of these interleukins was shown to be responsible for various diseases, such as i) IL-4 vs. immediate type hypersensitivity and ii) IL-6 vs. autoimmunity and multiple myelomas.

• Bauer J
• Interleukin-6 and its receptor during homeostasis, inflammation, and tumor growth.
• Klin Wochenschr. 1989; 67: 697-706
• Display abstract

• This review focuses on describing the specific role of interleukin-6 within the network of inflammatory mediators in man. Sites of interleukin-6 synthesis, regulation of its expression, and the biological functions of this molecule are here outlined. The potential role of interleukin-6 as a diagnostic monitor is discussed. Particular attention is paid to experimental evidence that interleukin-6 and its receptor may be involved in the pathogenesis of autocrine tumor growth. A recently proposed therapeutical use of cytotoxic interleukin-6 fusion proteins in order to selectively, destroy certain interleukin-6 receptor bearing tumor cells is discussed in the light of the finding, that not only hepatocytes, but also normal peripheral blood monocytes express the interleukin-6 receptor.

• Andus T, Heinrich PC, Castell JC, Gerok W
• [Interleukin-6: a key hormone of the acute phase reaction]
• Dtsch Med Wochenschr. 1989; 114: 1710-6

© 2021 EMBL | Privacy Policy