Impact of interleukin-6 classic- and trans-signaling on liver damage and regeneration

Interleukin-6 (IL-6) has been suggested to play a pivotal role in liver regeneration. IL-6 on target cells activates a receptor complex consisting of the IL-6 receptor (IL-6R) and the signal transducing receptor subunit gp130. Not all cells in the body express the IL-6R on the cell surface. IL-6 can signal via two different pathways: classical signaling via the membrane bound IL-6R and IL-6 trans-signaling via a naturally occurring soluble IL-6R (sIL-6R). This second pathway widens the scope of IL-6 signaling since also cells expressing no membrane bound IL-6R can be stimulated by the trans-signal pathway. Mimicking IL-6 trans-signaling via a designer molecule, Hyper-IL-6 has been shown to accelerate liver regeneration. Another designer molecule, sgp130Fc, specifically blocks IL-6 trans-signaling. Using these proteins we investigated the contribution of IL-6 classic- and trans-signaling in the liver. Here we review the role of IL-6 signaling in response to liver damage and during liver regeneration.     

Therapeutic targeting of interleukin-6 trans-signaling does not affect the outcome of experimental tuberculosis

Treatment of autoreactive inflammatory diseases such as rheumatoid arthritis with anti-inflammatory drugs is associated with an increased rate of reactivation tuberculosis (TB). Interleukin-6 (IL-6) plays a pivotal role in inflammation and protection against various infectious diseases. IL-6 signals by two mechanisms via the ubiquitous transmembrane protein gp130: 'classic' signaling using the membrane-bound IL-6 receptor (IL-6R), which is expressed mainly on hepatocytes and some leukocytes, and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling by the IL-6/sIL-6R complex is selectively inhibited by natural soluble gp130 (sgp130) and by sgp130 designer proteins. As specific blockade of IL-6 trans-signaling represents a promising approach for the therapy of inflammatory diseases, we evaluated the potential risk of interfering with this alternative pathway and analyzed the outcome of experimental TB after treatment with an IgG1-Fc fusion protein of soluble gp130 (sgp130Fc) and in sgp130Fc-overexpressing transgenic (sgp130Fc(tg)) mice. In contrast to treatment with anti-tumor necrosis factor (TNF) antibodies, administration of sgp130Fc did not interfere with protective immune responses after infection with Mycobacterium tuberculosis (Mtb). Moreover, Mtb-infected sgp130Fc(tg) mice were capable of controlling mycobacterial growth. Our finding that IL-6 trans-signaling plays no role for protective immune responses against Mtb supports the superior safety of therapeutic targeting of IL-6 trans-signaling compared to anti-TNF treatment.     

Teleost IL-6 promotes antibody production through STAT3 signaling via IL-6R and gp130

Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM(+) B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM(+) B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.     

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.     

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.     

The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.

The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.     

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.     

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.     

Pathological role of IL-6 in the experimental allergic bronchial asthma in mice

Although allergic asthma was described to be associated with the presence of mucosal T-helper (Th)2 cells, it is not entirely clear which factors are responsible for priming of T cells to differentiate into Th2 effector cells in this disease. Interleukin (IL)-6 has been recognized as important because it is secreted by cells of the innate immunity and induces the expansion of the Th2 effector cells, which are major players of the adaptive immune responses. Additionally, IL-6 released by dendritic cells (DCs) inhibits the suppressive function of CD4⁺CD25⁺ T-regulatory cells, thus inhibiting the peripheral tolerance. The signal transduction of IL-6 has recently taught us how this cytokine influences different aspects of the immune response, especially under pathological conditions. IL-6 can bind to the soluble IL-6R, increased after allergen challenge in asthmatic patients, and, through a mechanism called trans-signaling, induces proliferation of cells expressing the cognate receptor gp130. This mechanism appears to be used for proliferation by developed Th2 cells in the airways. In contrast, through the membrane-bound IL-6R, IL-6 controls CD4⁺CD25⁺ survival, as well as the initial stages of the Th2 cells development in the lung. These findings impact the establishment of new therapies for allergic diseases; indeed, blockade of the soluble IL-6R through the fusion protein gp130Fc reduces Th2 cells in the lung, and by blocking the membrane-bound Il-6R, anti-IL-6R antibody treatment induces the number of T-regulatory cells in the lung, thereby reducing the local number of CD4⁺ T-effector cells in experimental asthma.     

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.     

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.     

Regulated expression of gp130 and IL-6 receptor alpha chain in T cell maturation and activation

The functional receptor for the inflammatory cytokine IL-6 is composed of the ligand binding IL-6 receptor alpha chain (IL-6R alpha) and the signal transducing chain gp130, which is a shared component of multiple cytokine receptors. We analyzed the surface expression of gp130 and IL- 6R alpha in thymocytes and peripheral T cells. While all thymocytes expressed gp130 throughout thymic maturation, they gained expression of IL-6R alpha at the CD4 or CD8 single-positive stage. Approximately 10- 30% of the CD4-CD8+ and 40-50% of the CD4+CD8- thymocytes expressed IL- 6R alpha. Within the CD4+CD8- population, the IL-6R alpha- subpopulation was cortisone sensitive, appeared immature according to the cell surface markers expressed and failed to proliferate after TCR cross-linking. Peripheral T cells were predominantly gp130+ and IL-6R alpha+, but down-regulated gp130 and IL-6R alpha expression upon TCR engagement in vitro and in vivo. Peripheral gp130low/-IL-6R alphalow/- T cells expressed surface markers characteristic of memory T cells. We show that gp130 and IL-6R alpha are expressed in a regulated manner in T cells, depending on the developmental and functional stage.      

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.     

Hyper-IL-6融合基因的构建及在真核细胞中的表达

目的： 构建融合基因Hyper-IL-6并在真核细胞中进行表达.方法： 采用基因拼接（GeneSOEing）法将人类可溶性白细胞介素6受体和白细胞介素6的编码基因用一富含甘氨酸序列的接头经PCR扩增融合, 并定向克隆至pIRES2-EGFP, 构建与绿色荧光蛋白（GFP）共表达的融合重组质粒, 转染哺乳动物细胞, 通过绿色荧光蛋白、 RT-PCR和免疫组化检测Hyper-IL-6蛋白的表达.结果： PCR扩增出1 224 bp的Hyper-IL-6编码序列并成功构建重组质粒pIRES-HIL-6, 重组质粒转染真核细胞后经检测证实Hyper-IL-6能在真核细胞中表达.结论： 人类Hyper-IL-6重组表达质粒的成功构建与真核表达为以后对其进行活性分析和生物学功能的研究提供了基础.