Minimal Interleukin 6 (IL-6) Receptor Stalk Composition for IL-6 Receptor Shedding and IL-6 Classic Signaling

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) is coordinated by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The soluble IL-6R is mainly generated by ADAM10- and ADAM17-mediated ectodomain shedding. Little is known about the role of the 52-amino acid-residue-long IL-6R stalk region in shedding and signal transduction. Therefore, we generated and analyzed IL-6R stalk region deletion variants for cleavability and biological activity. Deletion of 10 amino acids of the stalk region surrounding the ADAM17 cleavage site substantially blocked IL-6R proteolysis by ADAM17 but only slightly affected proteolysis by ADAM10. Interestingly, additional deletion of the remaining five juxtamembrane-located amino acids also abrogated ADAM10-mediated IL-6R shedding. Larger deletions within the stalk region, that do not necessarily include the ADAM17 cleavage site, also reduced ADAM10 and ADAM17-mediated IL-6R shedding, questioning the importance of cleavage site recognition. Furthermore, we show that a 22-amino acid-long stalk region is minimally required for IL-6 classic signaling. The gp130 cytokine binding sites are separated from the plasma membrane by ∼96 Å. 22 amino acid residues, however, span maximally 83.6 Å (3.8 Å/amino acid), indicating that the three juxtamembrane fibronectin domains of gp130 are not necessarily elongated but somehow flexed to allow IL-6 classic signaling. Our findings underline a dual role of the IL-6R stalk region in IL-6 signaling. In IL-6 trans-signaling, it regulates proper proteolysis by ADAM10 and ADAM17. In IL-6 classic-signaling, it acts as a spacer to ensure IL-6·IL-6R·gp130 signal complex formation.     

IL-6 Trans-Signaling via the Soluble IL-6 Receptor: Importance for the Pro-Inflammatory Activities of IL-6

Interleukin-6 (IL-6) is a cytokine with many activities. It has functions in the regulation of the immune system and the nervous system. Furthermore, IL-6 is involved in liver regeneration and in the metabolic control of the body. On target cells, IL-6 binds to an 80 kDa IL-6 receptor (IL-6R). The complex of IL-6 and IL-6R associates with a second protein, gp130, which thereupon dimerizes and initiates intracellular signaling. Whereas gp130 is expressed on all cells, IL-6R is only present on few cells in the body including hepatocytes and some leukocytes. Cells, which do not express IL-6R cannot respond to the cytokine, since gp130 alone has no measurable affinity for IL-6. Interestingly, a soluble form of IL-6R (sIL-6R) comprising the extracellular portion of the receptor can bind IL-6 with a similar affinity as the membrane bound IL-6R. The complex of IL-6 and sIL-6R can bind to gp130 on cells, which do not express the IL-6R, and which are unresponsive to IL-6. This process has been called trans-signaling. Here I will review published evidence that IL-6 trans-signaling is pro-inflammatory whereas classic IL-6 signaling via the membrane bound IL-6R is needed for regenerative or anti-inflammatory activities of the cytokine. Furthermore, the detailed knowledge of IL-6 biology has important consequences for therapeutic strategies aimed at the blockade of the cytokine IL-6.     

IL-6 receptor expression and IL-6 effects change during osteoblast differentiation

Studies of the effects of interleukin-6 on osteoblasts have yielded conflicting results. In several earlier in vitro studies it has been stated that IL-6 has no effects on osteoblasts unless soluble IL-6 receptor is added. These results are contradictory to the fact that IL-6 receptors are expressed in osteoblasts in vivo. In this study, MC3T3 preosteoblast cells and rat bone marrow stromal cells were cultured in bone inducing medium containing ascorbic acid, β-glycerophosphate or dexamethasone. We found that IL-6 receptor expression increased in both types of cells during in vitro differentiation. Furthermore in MC3T3 cells IL-6 decreased proliferation and enhanced expression of two osteoblast-specific differentiation markers, Runx2 and osteocalcin, in proper sequential order. Interestingly, in both cell types IL-6-induced apoptosis only in later culture stages. We also found in MC3T3 cells that IL-6 induced STAT3 activation was significantly higher in later culture stages, i.e. when IL-6 receptor expression was high. The present study shows that IL-6 receptor expression increases during in vitro osteoblast differentiation and that IL-6 functions as a differentiation regulator of preosteoblast cells and an apoptosis initiator in more mature cells.     

Preparation of soluble murine IL-6 receptor and anti-murine IL-6 receptor antibodies

Starting with a previously isolated cDNA clone encoding murine IL-6R, a stable transformed Chinese hamster ovary cell line constitutively expressing soluble murine IL-6R (smIL-6R) has been established. The smIL-6R was purified to homogeneity by sequential filtration and chromatography of culture medium. The smIL-6R augmented the sensitivity of M1 cells to IL-6 in their growth inhibition in a dose-response manner. Rat hybridomas producing mAb specific to murine IL-6R were also established. One of the clones, RS13, produced IgG2a isotype that was capable of inhibiting IL-6 activity. ELISA for the quantitation of smIL-6R was established, which could detect smIL-6R in a quantity as low as 1 ng/ml.     

IL-6 receptor independent stimulation of human gp130 by viral IL-6

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.     

IL-1 stimulates IL-6 production in endothelial cells

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of IL-6 as a communication signal between vascular and immunocompetent cells. IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant IL-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to 20 h of incubation. Human and murine rIL-1 alpha stimulated HGF production in HEC. Anti-IL-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. IL-1-treated HEC expressed high levels of IL-6 mRNA as detected by Northern blot analysis. Inasmuch as IL-1 elicits a complex series of changes in HEC, it was important to assess whether IL-6, produced after exposure to IL-1, modified HEC function. Natural or rIL-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce IL-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.     

应用椭偏光学显微成像对IL-6与其受体的相互应用的初步观察

白细胞介素6(IL-6)是一种具有复杂生物功能的细胞因子,可由多种淋巴类和非淋巴类细胞产生.它对机体多种组织及细胞均有不同程度的作用[1-3].近年来发现,临床上免疫异常性疾病,如发热、淋巴结肿大、血沉增快、急性期蛋白增高、高γ球蛋白血症、自身抗体阳性等症状都与IL-6的异常表达密切相关.IL-6的生物活性是通过细胞膜表面特异性受体介导的[4].研究IL6与其受体的相互作用对于揭示某些疾病的发病机制,监测疾病进程以及指导临床治疗等均具有重要意义.     

应用椭偏光学显微成像对IL-6与其受体的相互应用的初步观察

白细胞介素 6 (ＩＬ 6 )是一种具有复杂生物功能的细胞因子 ,可由多种淋巴类和非淋巴类细胞产生。它对机体多种组织及细胞均有不同程度的作用[1～ 3 ] 。近年来发现 ,临床上免疫异常性疾病 ,如发热、淋巴结肿大、血沉增快、急性期蛋白增高、高γ球蛋白血症、自身抗体阳性等症状都与ＩＬ 6的异常表达密切相关。ＩＬ 6的生物活性是通过细胞膜表面特异性受体介导的[4] 。研究ＩＬ 6与其受体的相互作用对于揭示某些疾病的发病机制 ,监测疾病进程以及指导临床治疗等均具有重要意义。用于研究ＩＬ 6与其受体相互作用的方法主要有ＩＬ 6依赖株细胞…     

IL-2 induces IL-6 production in human monocytes.

IL-2 is a potent activator of effector and secretory activities of human monocytes. Since monocytes are an important source of IL-6, we investigated whether IL-2 can induce IL-6 production and whether regulatory circuits can modulate this process. We found that stimulation of monocytes with IL-2 induced expression of IL-6 mRNA and bioactivity in a dose-dependent manner. Production of IL-6 in monocytes can be induced by other cytokines such as IL-1 beta. By using mAb alpha-IL-1 beta we showed that IL-2-induced IL-6 production is not mediated by the autocrine stimulation of IL-1 beta elicited by IL-2. IL-6 induction by monocytes is not a common response to activating signals because IFN-gamma did not induce IL-6 expression under conditions in which it elicits tumoricidal activity. In contrast, IFN-gamma could completely abrogate the induction of IL-6 expression by IL-1 beta but did not affect the levels of mRNA and the secretion of IL-2-elicited IL-6. We have previously reported that transforming growth factor-beta inhibits IL-6 production in response to IL-1 beta. Studies on the inhibitory activity of transforming growth factor-beta demonstrated that this cytokine differs from IFN-gamma because it inhibited both IL-1- and IL-2-induced IL-6 expression. These data demonstrate that, in human monocytes, both IL-1 and IL-2 stimulate IL-6 expression by independent mechanisms that can be dissociated by the susceptibility to the inhibitory effect of IFN-gamma. IL-6 production is also down-regulated by TGF-beta, whose inhibitory activity is stimulus-unrelated.     

Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms

Skeletal muscle cells have been established as significant producers of IL-6 during exercise. This IL-6 production is discussed as one possible mediator of the beneficial effects of physical activity on glucose and fatty acid metabolism. IL-6 itself could be the exercise-related factor that upregulates and maintains its own production. We investigated this hypothesis and the underlying molecular mechanism in cultured C₂C₁₂ cells. IL-6 led to a rapid and prolonged increase in IL-6 mRNA, which was also found in human myotubes. Because IL-6 has been shown to activate AMP-activated kinase (AMPK), we studied whether, in turn, activated AMPK induces IL-6 expression. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside upregulated IL-6 mRNA expression, which was blocked by knockdown of AMPK ₁ and ₂ using small, interfering RNA (siRNA) oligonucleotides. However, the effect of IL-6 was shown to be independent of AMPK, since the siRNA approach silencing the AMPK -subunits did not reduce the upregulation of IL-6 induced by IL-6 stimulation. The self-stimulatory effect of IL-6 partly involves a Ca²⁺-dependent pathway: IL-6 increased intracellular Ca²⁺, and intracellular blockade of Ca²⁺ with a Ca²⁺ chelator reduced the IL-6-mediated increase in IL-6 mRNA levels. Moreover, inhibition of Ca²⁺/calmodulin-dependent kinase kinase with STO-609 or the siRNA approach decreased IL-6 mRNA levels of control and IL-6-stimulated cells. A major, STO-609-independent mechanism is the IL-6-mediated stabilization of its mRNA. The data suggest that IL-6 could act as autocrine factor upregulating its mRNA levels, thereby supporting its function as an exercise-activated factor in skeletal muscle cells. 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside; AMP-activated kinase; STO-609; calcium/calmodulin-dependent kinase kinase; C₂C₁₂ cells     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice

Transgenic mice carrying human IL-6 cDNA fused with a murine major histocompatibility class-I promoter (H-2L(d)) were serially administered with anti-interleukin-6 receptor (IL-6R) monoclonal antibody (mAb), MR16-1, from the age of 4 weeks to estimate its efficacy on a variety of disorders developed in these mice, most of which are similar to the disorders associated with Castleman's disease. In the control mice treated with isotype-matched mAb, a massive and multiple IgG1 plasmacytosis, mesangial proliferative glomerulonephritis, leukocytosis, thrombocytosis, anemia and abnormalities of blood chemical parameters have developed in accordance with the elevation of serum IL-6, and 50% of mice have died of renal failure by 18 weeks of age. In contrast, the treatment with MR16-1 prevented all these symptoms and prolonged the lifetime of the majority of the mice. Thus, the constitutive overexpression of IL-6 caused various disorders, and the treatment with anti-IL-6R mAb completely prevented from these symptoms. These results clearly confirm that IL-6 indeed plays an essential role in the pathogenesis of a variety of disorders. Furthermore, anti-IL-6R mAb could provide novel therapy for Castleman's disease and MR16-1 should be a useful tool to estimate therapeutic potential of IL-6 antagonists in a variety of murine models for human disease.     

An IL-6/IL-6 soluble receptor (IL-6R) hybrid protein (H-IL-6) induces EPO-independent erythroid differentiation in human CD34(+) cells

H-IL-6 is a hybrid protein constructed to contain IL-6 and its soluble receptor linked by a flexible peptide chain. Here we show that H-IL-6 strongly enhances proliferation of human CD34(+)cells in serum-free liquid culture, and that the majority of the cells generated belong to the erythroid lineage, being positive for the marker Glycophorin A. Conversely, H-IL-6 does not increase the number of myeloid, CD13-positive cells. Comparable effects are observed on progenitors from cord blood and adult peripheral blood. Therefore, H-IL-6 triggers an erythroid-inducing signal in haematopoietic progenitor cells, independently from erythropoietin (EPO).     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

IL-1，IL-6，IL-8 与胃癌的关系

胃癌是常见的恶性肿瘤之一,在我国其发病率居各类肿瘤前列,导致的死亡人数占所有肿瘤的四分之一,而且每年有近40万新增的胃癌病人,但是其早期诊断率低于20%,胃癌已经成为危害人民健康的最严重的疾病之一。白细胞介素(interleukin,IL)作为在白细胞或免疫细胞间相互作用的淋巴因子,不仅在介导T、B细胞活化、增殖与分化以及炎症反应中起着重要作用,近年来,越来越多的学者发现其与肿瘤的发生及发展也有着密不可分的联系。目前为止,已经发现了29种白细胞介素,分别被命名为IL-1~IL-29,它们各自承担着相应的使命。国内外大量实验及文献表明,IL-1,IL-6,IL-8和胃癌有着密切的关系,这对于胃癌的早期诊断及治疗提出了新的思路,进而提高胃癌的早期诊断率,改善胃癌的治疗状况。本文对IL-1,IL-6,IL-8的来源、分子结构及受体方面进行简要概述,同时阐述了其生物学特性及与胃癌的关系。