High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.     

Immunoadhesins of interleukin-6 and the IL-6/soluble IL-6R fusion protein hyper-IL-6

Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-1000 times more effective than unlinked IL-6 and soluble IL-6R. Hyper-IL-6 acts on cells only expressing gp130 and is a potent stimulator of in vitro expansion of early hematopoietic precursors. Here we show that a Fc fusion protein of H-IL-6 (H-IL-6Fc) has the same biological activity on BAF/gp130 cells as H-IL-6. Furthermore, both H-IL-6 forms have a similar ability to induce the synthesis of acute phase proteins in human hepatoma cells HepG2 and in mice in vivo. The introduction of a thrombin cleavage site between H-IL-6 and the Fc portion of H-IL-6Fc made it possible to specifically recover biologically active monomeric H-IL-6 by limited proteolysis of the fusion protein. A more general use of cleavable immunoadhesins expressed in mammalian cells is discussed.     

IL-10-induced gp130 expression in mouse mast cells permits IL-6 trans-signaling

It is reported that human and mouse mast cells express the IL-27R, which consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Although it has been proposed that IL-27 may negatively regulate mast cell-dependent, immediate hypersensitivity responses directly, this has yet to be examined specifically. We found that mouse BMMC and primary peritoneal mast cells are unresponsive to IL-27. Consistent with this, gp130 protein in resting BMMC was not on the cell surface to a measurable degree but was found intracellularly, and data are consistent with incompletely processed N-linked glycosylation. Furthermore, BMMC constitutively expressed SOCS3, a major negative regulator of gp130 signaling. However, BMMC stimulation with IL-10 and consequential STAT3 activation increased gp130 expression, which resulted in a functional gp130 receptor on the BMMC cell surface. IL-10 has not been previously shown to regulate gp130 expression, which on the BMMC surface, permitted IL-6 trans-signaling, found to increase survival under limiting conditions and enhance IL-13 and TNF-α secretion. This study identifies factors that regulate mouse mast cell gp130 expression and signaling and makes conspicuous the limitations of using cultured mouse mast cells to study the effects of the IL-6/IL-12 cytokine family on mast cell biology.     

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

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

Preeclampsia Status Controls Interleukin-6 and Soluble IL-6 Receptor Release from Neutrophils and Endothelial Cells: Relevance to Increased Inflammatory Responses

Increased neutrophil–endothelial binding and inflammatory responses are significant pathophysiological events in the maternal vascular system in preeclampsia, a hypertensive disorder in human pregnancy. Interleukin 6 (IL-6) and its soluble receptors (soluble IL-6R (sIL-6R) and soluble gp130 (sgp130)) are critical inflammatory mediators. During pregnancy, maternal IL-6 and sgp130 levels were increased, but sIL-6R levels were decreased, in women with preeclampsia compared to normotensive pregnant women. However, little is known about differences in IL-6, sIL-6R, and sgp130 production by neutrophils and endothelial cells between normal pregnancy and preeclampsia. To study this, we isolated neutrophils and cultured human umbilical vein endothelial cells (HUVECs) from normal and preeclamptic pregnancies. Production of IL-6, sIL-6R, and sgp130 was measured. The role of placental factor(s)-mediated neutrophil production of IL-6, sIL-6R, and sgp130 was also determined by pretreating neutrophils with placental conditioned medium generated from placental villous cultures. We found that IL-6 and sgp130 were mainly produced by endothelial cells, while sIL-6R was mainly produced by neutrophils. Endothelial cells from preeclampsia produced significantly more IL-6 and sgp130, and neutrophils from preeclampsia produced significantly less sIL-6R than normal pregnancy cells. Interestingly, production of IL-6, sIL-6R, and sgp130 were time-dependently increased when neutrophils and endothelial cells were co-cultured. We also found that neutrophils from normal pregnancies produced more IL-6, but less sIL-6R, after being primed by preeclamptic-placental conditioned medium. These results demonstrated that neutrophils and endothelial cells have different capacities in producing IL-6, sIL-6R, and sgp130 between normal pregnancy and preeclampsia. These results also provide evidence that the placenta plays a role in inducing neutrophil activation in preeclampsia.     

Advances in interleukin-6 therapy]

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.     

Development of a monoclonal antibody-based enzyme-linked immunoabsorbent assay for the binding of gp130 to the IL-6/IL-6R complex and its competitive inhibition

The proinflammatory cytokine IL-6 binds to the membrane bound or soluble IL-6 receptor (IL-6R) and activates an intracellular signaling cascade after complex formation with two gp130 molecules. These mediate general homeostasis and orchestrates the immune response during disease. Trans-signalling via the soluble IL-6R has importance for the development and maintenance of human diseases like Crohn's disease, peritonitis and rheumatoid arthritis. We have developed an enzyme-linked immunoabsorbent assay (ELISA) that detects the binding of gp130 to the IL-6/sIL-6R complex. Competitive binding of sgp130-Fc, viral IL-6, and the inhibitory drug Suramin to gp130 has been demonstrated. The assay can be used for high-throughput screening of peptide and chemical compound libraries for the identification of new agonists and antagonists of the binding between gp130 and IL-6/sIL-6R.     

Interleukin-6 and its receptor: from bench to bedside

Interleukin-6 (IL-6) is an inflammatory cytokine with a well-documented role in inflammation and cancer. The cytokine binds to a membrane bound IL-6 receptor (IL-6R) and this complex associates with two molecules of the signal transducing protein gp130 thereby initiating intracellular signaling. While gp130 is present on most if not all cells of the body, the IL-6R is only present on some cells, mainly hepatocytes and several leukocytes. Cells, which only express gp130 and no IL-6R are refractory to IL-6 signals. We have shown earlier that the IL-6R can exist as a soluble protein generated by limited proteolysis of the membrane bound receptor or by translation from an alternatively spliced mRNA. This soluble IL-6R (sIL-6R) can bind the ligand IL-6 and the soluble complex of sIL-6R and IL-6 can bind to gp130 on cells which lack the membrane bound IL-6R and trigger gp130 signaling. We have named this process ‘trans-signaling’. We will review data, which clearly show that IL-6 uses classical signaling via the membrane bound receptor and trans-signaling via the soluble receptor in various physiological and pathophysiological situations. Furthermore, we have developed designer cytokines, which can specifically enhance or inhibit IL-6 trans-signaling. These designer cytokines have been shown to be extremely useful to in therapeutic applications ranging from the long-term culture of stem cells and enhancing liver regeneration up to the blockade of chronic inflammation and cancer.There are inclusions in this text from a number of different articles, which are cited in the reference section.     

抽动秽语综合征患者血清自身抗体与可溶性白介素-6受体表达增高

目的探讨抽动秽语综合征(TS)病程中自身免疫损伤相关机制。方法用ELISA法检测TS患者血清中抗脑抗体(ABAb)、抗核抗体(ANAb)、可溶性白介素-6受体(sIL-6R)及可溶性gp130(sgp130)的含量,用胶乳增强免疫比浊法检测抗链球菌溶血素O抗体(ASO)水平。结果TS组血清sIL-6R和sgp130含量显著高于对照组[(44.1±15.8)ng/mLvs(30.3±9.0)ng/mL和(69.0±24.6)ng/mLvs(47.3±14.1)ng/mL,P<0.01];血清ABAb和ANAb检出率显著高于对照组(66%vs4%和53%vs25%,P<0.01),并且ASO增高的比例高于对照组(P<0.01);TS组ABAb水平与sgp130呈负相关(r=0.375,P<0.05)。结论TS患者依赖IL-6信号传导的生理功能上调和反馈抑制的网络机制启动;自身免疫相关的损伤机制均可能参与了疾病发生发展的病理生理过程。     

Interleukin-6 trans-signalling in chronic inflammation and cancer

Interleukin-6 (IL-6) is a cytokine, which plays an important role in many chronic inflammatory diseases. IL-6 belongs to a family of 10 cytokines, which all act via receptor complexes containing the cytokine receptor subunit gp130. On cells, IL-6 first binds to a specific membrane-bound IL-6R and the complex of IL-6 and IL-6R interacts with gp130 leading to signal initiation. Whereas gp130 is widely expressed throughout the body, the IL-6R is only found on some cells including hepatocytes and some leucocytes. A soluble form of the IL-6R is an agonist capable of transmitting signals through interaction with the gp130 protein. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells, which are unresponsive to IL-6 alone, as they do not express the membrane-bound IL-6R. We have named this process trans-signalling. We provided evidence that a soluble form of the IL-6 family signalling receptor subunit gp130 is the natural inhibitor of IL-6 trans-signalling responses. We showed that in chronic inflammatory diseases such as inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma as well as in colon cancer, IL-6 trans-signalling is critically involved in the maintenance of the disease state. Moreover, in all these animal models, the progression of the disease can be interrupted by specifically interfering with IL-6 trans-signalling using recombinant-soluble gp130Fc protein. The pathophysiologic mechanisms by which the IL-6/soluble IL-6R complex perpetuates the inflammatory state are discussed.     

A role for IL-27p28 as an antagonist of gp130-mediated signaling

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.     

Interleukin-6: From basic biology to selective blockade of pro-inflammatory activities

Cytokines receptors exist in membrane bound and soluble form. A soluble form of the human IL-6R is generated by limited proteolysis and alternative splicing. The complex of IL-6 and soluble IL-6R stimulates target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. We have named this process trans-signaling. Soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses. Recombinant soluble gp130 protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. Neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein for selective blockade of IL-6 trans-signaling have been used in several animal models of human diseases. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate in models of inflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis pancreatitis, colon cancer, ovarian cancer and pancreatic cancer, that IL-6 trans-signaling via the soluble IL-6R is the crucial step in the development and the progression of the disease. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer and it undergoes phase I clinical trials as an anti-inflammatory drug since June 2013.     

Soluble interleukin-6 receptor enhanced by oncostatin M induces major changes in gene expression profile of human hepatoma cells

Interleukin-6 (IL-6) binds to a receptor complex consisting of an 80 kDa binding unit (IL-6R) and gp130 responsible for signal transduction. Due to alternative splicing and/or proteolytic digestion IL-6R occurs in soluble form (sIL-6R), as well. Soluble IL-6R is able to bind to gp130 expressing on nucleated cells, thus sIL-6R makes most cells responsive to IL-6. In this study we found that oncostatin M (OSM), an other gp130 dependent cytokine with proliferation inhibitory potential, increases the expression of both membrane-bound IL-6R and sIL-6R generated by alternative splicing in hepatic and mammary carcinoma cell lines. Furthermore, we studied the functional relevance of the presence and binding of soluble IL-6R to HepG2 cells. Using a cDNA expression array, mRNA levels of about 580 human genes were tested by differential display analysis. Our findings suggest, that elevation of surface density of IL-6R by attachment of sIL-6R induces major modulation in gene expression profile of the hepatoma cells. Soluble IL-6R alone has minor effect, it rather decreases expression of some genes, while incubation with IL-6 and sIL-6R together induces major changes in the mRNA pattern of HepG2 cells. These data strongly suggest that presence and binding of soluble cytokine receptors are important elements of inter-cytokine cross talk and affects actual gene expression profile of responding cells.