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The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of molecules are intracellular adaptors that regulate cellular signaling through members of the TNFR and Toll-like receptor superfamily. Mammals have seven TRAF molecules numbered sequentially from TRAF1 to TRAF7. Although TRAF5 was identified as a potential regulator of TNFR superfamily members, the in vivo function of TRAF5 has not yet been fully elucidated. We identified an unconventional role of TRAF5 in interleukin-6 (IL-6) receptor signaling involving CD4 + T cells. Moreover, TRAF5 binds to the signal-transducing glycoprotein 130 (gp130) receptor for IL-6 and inhibits the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In addition, Traf5-deficient CD4 + T cells exhibit significantly enhanced IL-6-driven differentiation of T helper 17 (Th17) cells, which exacerbates neuroinflammation in experimental autoimmune encephalomyelitis. Furthermore, TRAF5 demonstrates a similar activity to gp130 for IL-27, another cytokine of the IL-6 family. Additionally, Traf5-deficient CD4 + T cells display significantly increased IL-27-mediated differentiation of Th1 cells, which increases footpad swelling in delayed-type hypersensitivity response. Thus, TRAF5 functions as a negative regulator of gp130 in CD4 + T cells. This review aimed to explain how TRAF5 controls the differentiation of CD4 + T cells and discuss how the expression of TRAF5 in T cells and other cell types can influence the development and progression of autoimmune and inflammatory diseases.

Biochemically, interleukin-6 belongs to the class of four-helical cytokines. The cytokine can be synthesised and secreted by many cells. It acts via a cell surface-expressed interleukin-6 receptor, which is not signalling competent. This receptor, when complexed with interleukin-6, associates with the signalling receptor glycoprotein 130 kDa (gp130), which becomes dimerised and initiates intracellular signalling via the Janus kinase/signal transducer and activator of transcription and rat sarcoma proto oncogene/mitogen-activated protein kinase/phosphoinositide-3 kinase pathways. Physiologically, interleukin-6 is involved in the regulation of haematopoiesis and the coordination of the innate and acquired immune systems. Additionally, interleukin-6 plays an important role in the regulation of metabolism, in neural development and survival, and in the development and maintenance of various cancers. Although interleukin-6 is mostly regarded as a pro-inflammatory cytokine, there are numerous examples of protective and regenerative functions of this cytokine. This review will explain the molecular mechanisms of the, in part opposing, activities of the cytokine interleukin-6., Competing Interests: Competing interests: Stefan Rose-John has acted as a consultant and speaker for AbbVie, Amgen, Janssen, Chugai, Roche, Genentech Roche, Pfizer, Eli Lilly, and Sanofi. He also declares that he is an inventor on patents owned by CONARIS Research Institute, which develops the sgp130Fc protein (olamkicept), and he has stock ownership in CONARIS.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2020 Rose-John S.)

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4 + T cells, prior activation via the T cell antigen receptor limits IL-6's control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4 + T cells. Transcriptomics and chromatin immunoprecipitation-sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4 + T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4 + T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4 + T cells sense and interpret cytokine signals.

Despite remarkable improvements in cancer treatment approaches, breast cancer is still the main cause of cancer-related death in women. Its principal cause is the resistance of the cancer cells against conventional anticancer therapeutics, mainly in advanced disease stages. It has been shown that chronic inflammation in the tumor microenvironment facilitates tumor growth and induces resistance toward chemo- and radiotherapy. Overexpression of interleukin-6 (IL-6) cytokine in the tumor microenvironment has been demonstrated in numerous tumors including breast cancer. Tumor cells and tumor-associated fibroblasts are the major sources of IL-6 secretion in the tumor microenvironment. Several studies have demonstrated the immunopathogenic function of IL-6 and its signaling in the tumor growth, metastasis, and therapeutic resistance in the breast cancer. Therefore, it seems that targeting IL-6 and/or its receptor in combination with other potent anticancer therapies may be a potent therapeutic approach for breast cancer therapy., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

IL-6 is a pleiotropic cytokine involved in many biological functions that affect tissues beyond the immune system and the vasculature. This multifunctional cytokine exerts its actions via the classic signalling pathway when it binds to the transmembrane IL-6 receptor (IL-6R) or via the trans-signalling pathway upon binding to the soluble form of IL-6R (sIL-6R). In general, classic IL-6 signalling is responsible for the anti-inflammatory properties of IL-6, whereas trans-signalling is responsible for the pro-inflammatory actions of IL-6. As a result, dysregulation of the IL-6 axis can lead to the onset or development of several disease states, particularly autoimmune and inflammatory disorders, including RA and GCA. This pathological role of IL-6 means that pharmacologic modulation of the IL-6 axis is a rational therapeutic approach; however, multiple predictable, but often underappreciated, effects on tissues and organs beyond the blood vessels may also occur.

Vascular endothelial growth factor (VEGF) is implicated in the peritoneal membrane remodeling that limits ultrafiltration in patients on peritoneal dialysis (PD). Although the exact mechanism of VEGF induction in PD is unclear, VEGF concentrations in drained dialysate correlate with IL-6 levels, suggesting a link between these cytokines. Human peritoneal mesothelial cells (HPMCs), the main source of IL-6 and VEGF in the peritoneum, do not bear the cognate IL-6 receptor and are thus unable to respond to classic IL-6 receptor signaling. Here, we investigated whether VEGF release by HPMCs is controlled by IL-6 in combination with its soluble receptor (IL-6 trans-signaling). Although treatment with either IL-6 or soluble IL-6 receptor (sIL-6R) alone had no effect on VEGF production, stimulation of HPMCs with IL-6 in combination with sIL-6R promoted VEGF expression and secretion through a transcriptional mechanism involving STAT3 and SP4. Conditioned medium from HPMCs cultured with IL-6 and sIL-6R promoted angiogenic endothelial tube formation, which could be blocked by silencing SP4. In vivo , induction of peritoneal inflammation in wild-type and IL-6-deficient mice showed IL-6 involvement in the control of Sp4 and Vegf expression and new vessel formation, confirming the role of IL-6 trans-signaling in these processes. Taken together, these findings identify a novel mechanism linking IL-6 trans-signaling and angiogenesis in the peritoneal membrane., (Copyright © 2017 by the American Society of Nephrology.)

Interleukin-6 (IL-6) signaling network has been implicated in oncogenic transformations making it attractive target for the discovery of novel cancer therapeutics. In this study, potent antiproliferative and apoptotic effect of diacerein were observed against breast cancer. In vitro apoptosis was induced by this drug in breast cancer cells as verified by increased sub-G1 population, LIVE/DEAD assay, cell cytotoxicity and presence of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, as well as downregulation of antiapoptotic proteins Bcl-2 and Bcl-xL and upregulation of apoptotic protein Bax. In addition, apoptosis induction was found to be caspase dependent. Further molecular investigations indicated that diacerein instigated apoptosis was associated with inhibition of IL-6/IL-6R autocrine signaling axis. Suppression of STAT3, MAPK and Akt pathways were also observed as a consequence of diacerein-mediated upstream inhibition of IL-6/IL-6R. Fluorescence study and western blot analysis revealed cytosolic accumulation of STAT3 in diacerein-treated cells. The docking study showed diacerein/IL-6R interaction that was further validated by competitive binding assay and isothermal titration calorimetry. Most interestingly, it was found that diacerein considerably suppressed tumor growth in MDA-MB-231 xenograft model. The in vivo antitumor effect was correlated with decreased proliferation (Ki-67), increased apoptosis (TUNEL) and inhibition of IL-6/IL-6R-mediated STAT3, MAPK and Akt pathway in tumor remnants. Taken together, diacerein offered a novel blueprint for cancer therapy by hampering IL-6/IL-6R/STAT3/MAPK/Akt network.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

Interleukin (IL)-6-type cytokines are critically involved in health and disease. The duration and strength of IL-6-type cytokine-mediated signaling is tightly regulated to avoid overshooting activities. Here, molecular mechanisms of inter-familiar cytokine cross-talk are reviewed which regulate dynamics and strength of IL-6 signal transduction. Both plasticity and cytokine cross-talk are significantly involved in pro- and anti-inflammatory/regenerative properties of IL-6-type cytokines. Furthermore, we focus on IL-6-type cytokine/cytokine receptor plasticity and cross-talk exemplified by the recently identified composite cytokines IL-30/IL-6R and IL-35, the first inter-familiar IL-6/IL-12 family member. The complete understanding of the intra- and extracellular cytokine networks will aid to develop novel tailor-made therapeutic strategies with reduced side effects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.

Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl₄ induced liver damage. Blockade of IL-6 trans-signaling during CCl₄ induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl₄ was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl₄ treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl₄ induced chemical damage., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Astrocytes have important physiological roles in CNS homeostasis and serve as a bridge between the CNS and immune system. IL-17 and IL-6 are important in many CNS disorders characterized by neuroinflammation. We examined the role of IL-17 on the IL-6 signaling cascade in primary astrocytes. IL-17 functioned in a synergistic manner with IL-6 to induce IL-6 expression in astrocytes. The synergistic effect involved numerous signaling pathways including NF-kappaB, JNK MAPK, and p38 MAPK. The NF-kappaB pathway inhibitor BAY-11, JNK inhibitor JNKi II, and p38 inhibitor SB203580 suppressed the synergistic effect of IL-6 and IL-17 on IL-6 expression. IL-17 synergized with IL-6 to enhance the recruitment of activated NF-kappaB p65, c-Fos, c-Jun, and the histone acetyltransferases CREB-binding protein and p300 to the IL-6 promoter in vivo to induce IL-6 transcription. This was accompanied by enhanced acetylation of histones H3 and H4 on the IL-6 promoter. Moreover, we elucidated an important role for suppressor of cytokine signaling (SOCS) 3 in IL-17 enhancement of IL-6 signaling in astrocytes. SOCS3 small interfering RNA knockdown and SOCS3 deletion in astrocytes augmented the synergistic effect of IL-6 and IL-17 due to an enhancement of activation of the NF-kappaB and MAPK pathways. These results indicate that astrocytes can serve as a target of Th17 cells and IL-17 in the CNS, and SOCS3 participates in IL-17 functions in the CNS as a negative feedback regulator.

IL-6 responses are classically orchestrated via a membrane-bound IL-6R (CD126) alpha subunit (classical IL-6R signaling) or through a soluble form of this cognate receptor (IL-6 trans signaling). Appraisal of IL-6R expression on human and mouse T cells emphasized that IL-6R expression is closely linked with that of CCR7 and CD62L. In this regard, infiltrating effector T cells from clinical and experimental peritonitis episodes lose IL-6R expression, and anti-CD3/CD28 Ab costimulation of peripheral T cells in vitro leads to a downregulation in IL-6R expression. Consequently, IL-6 signaling through membrane-bound IL-6R seems to be limited to naive or central memory T cell populations. Loss of IL-6R expression by activated T cells further suggests that these effector cells might still retain IL-6 responsiveness via IL-6 trans signaling. Using IL-6R-deficient mice and recombinant tools that modulate the capacity of IL-6 to signal via its soluble receptor, we report that local control of IL-6 trans signaling regulates the effector characteristics of the T cell infiltrate and promotes the maintenance of IL-17A-secreting CD4(+) T cells. Therefore, we concluded that classical IL-6R signaling in naive or central memory CD4(+) T cells is required to steer their effector characteristics, whereas local regulation of soluble IL-6R activity might serve to maintain the cytokine profile of the Th cell infiltrate. Therefore, the activation status of a T cell population is linked with an alteration in IL-6 responsiveness.

Activation of the IL-6/Stat3 via IL-6 trans-signaling plays an important role in the pathogenesis of inflammatory bowel disease. Colitis-associated cancer (CAC) is a large bowel cancer and occurs with long-standing inflammatory bowel disease. The role of the IL-6/Stat3 in the development of CAC has not been fully understood. We investigate whether IL-6 trans-signaling contributes to the development of CAC using a mouse colitis-associated premalignant cancer (CApC) model. Chronic colitis (CC) was induced in BALB/c mice using dextran sodium sulfate. CApC was induced by dextran sodium sulfate treatment to CC-affected mice. IL-6 expression was determined by quantitative RT-PCR and immunofluorescence staining in colon. Phospho-Stat3 expression was examined by Western blotting and immunofluorescence analysis. The expression of IL-6 receptors (i.e., the IL-6R alpha-chain and gp130) and tumor necrosis factor-alpha converting enzyme in the colon was examined by laser-capture microdissection and immunofluorescence staining. Soluble IL-6R alpha (sIL-6R alpha) was examined by Western blotting of epithelial cell-depleted colonic tissues. We also investigated whether a soluble gp130-Fc fusion protein could prevent CApC. IL-6 expression was increased in the colon of CC- and CApC-affected mice and was restricted to lamina propria-macrophages. The expression of IL-6R alpha and tumor necrosis factor-alpha converting enzyme was increased in the lamina propria CD11b-macrophages of CC-affected mice. sIL-6R alpha expression was also increased in these tissues. Reduced levels of IL-6R alpha generation were observed in the colonic epithelial cells of CC- and CApC-affected mice and were associated with the increased expression of gp130 and phospho-Stat3. Treatment with soluble gp130Fc significantly reduced the CApC. IL-6 trans-signaling in epithelial cells induced by macrophage-derived IL-6/sIL-6R alpha plays a crucial role in the development of CAC.

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.

Interleukin 6 (IL-6) belongs to a large group of secreted proteins called cytokines functioning to mediate and regulate immunity, inflammation, and hematopoiesis with direct effects on cell proliferation, apoptosis, and differentiation. Along with the IL-6 protein, two of its splice variants, IL-6delta2 and IL-6delta4, were reported to be transcribed or expressed in vivo in human, and the mRNAs of IL-6delta3 and IL-6delta5 had been observed in mouse. While the existence of different splice variants of IL-6 has been shown, very little is known on how the structural modifications of IL-6 resulting from the formation of the different splice variants may alter cytokine functions. We have analyzed the potential effects splicing would have on interactions with the cell surface receptor complex. We (1) constructed three-dimensional structures of the IL-6 splice variants, IL-6delta2, IL-6delta3, and IL-6delta4, with the assumption that an interleukin splice variant as a folded protein should retain a functional hydrophobic core; (2) reconstructed the ternary structural complexes consisting of the modeled IL-6 splice variants, the IL-6 receptor molecule (IL-6R) and the dimeric signal-transducing protein, gp130, and (3) analyzed all complexes and made comparisons with the X-ray structure of the wild-type IL-6 complex. We identified three separate sites on IL-6 where interactions are made with IL-6R and with each of the two copies of gp130. The structural consequences of losing an exon lead to a unique pattern of lost interaction with different components of the receptor complex. Thus, in IL-6 and its splice variants, the exons appear to have compartmentalized roles contributing to the combined function of the cytokine. The modeled interactions suggest that splice variants could act as antagonists, and that IL-6delta2, missing the signal peptide, would be a cytoplasmic protein and be released and interact with nearby cell-surface receptors when cells are damaged. We argue that in the case of IL-6, helix E may act as a "silent secondary structure," which only has an active role when it substitutes for a part of the hydrophobic core, for example, replacing helix A in IL-6delta2.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) occurring in the gut of genetically susceptible individuals independent of a specific pathogen. The interaction between antigen-presenting cells and the local bacterial flora contributes to an uncontrolled activation of mucosal CD4+ T lymphocytes with the consecutive release of proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, IL-23, IL-27, and also IL-17, which is attributed to a specific, differentiated CD4+ lineage called Th17 (TH-IL17, THi). Recent data suggest that IL-6 contributes to Th17 differentiation. However, to clarify the importance of Th17 cells in IBD further data are needed. So far, CD has been attributed to a Th1-mediated disease, whereas UC exhibits a modified Th2 cytokine response. In both diseases CD4+ T cells at the site of inflammation are critically dependent on antiapoptotic IL-6 signaling. Thereby, IL-6 induces the transcription factor STAT-3 via transsignaling (activation of a cell lacking membrane-bound IL-6 receptor via soluble IL-6 receptor). STAT-3 itself induces the antiapoptotic factors bcl-2 and bcl-xL, thus resulting in T-cell resistance against apoptosis. This vicious circle of T-cell accumulation, mediated by apoptosis resistance, finally leading to chronic inflammation, can be blocked by anti-IL-6 receptor antibodies. This review highlights the role of IL-6 in IBD immunopathogenesis and its clinical relevance in IBD therapy and diagnostics.

Background: Vascular access failure is the main cause of morbidity in hemodialysis patients. Stenosis of the arteriovenous fistula (AVF) is similar histologically to atherosclerosis. Recent studies showed that interleukin 6 (IL-6) has a key role in the pathogenesis of atherosclerosis by binding 2 specific receptors, gp80 and gp130. When activated, gp130 interacts with a tyrosine kinase, Janus kinase (JAK2), which then activates a transcription factor, signal transducers and activators of transcription (STAT3), directly turning on several proinflammatory genes. The aim of this study is to evaluate gp130 expression and JAK2/STAT3 activation within stenotic AVFs., Methods: 44 patients undergoing surgery for AVF creation were enrolled; 10 of them had AVF failure with histologically proven AVF stenosis (wall-lumen ratio > 1). A venous fragment of the AVFs was collected during creation and revision of the vascular access. gp130 and gp80 expression, as well as JAK/STAT activation, were evaluated by means of confocal microscopy. Peripheral-blood mononuclear cells were isolated at the time of AVF creation and revision., Results: gp130 protein expression, barely detectable in native AVFs, was strikingly increased within the venous branch of stenotic AVFs. The signaling subunit of the IL-6 receptor broadly colocalized with gp80, the IL-6-binding subunit. gp130-expressing cells were mainly CD34(+), suggesting that this receptor is expressed primarily by neovasculature endothelial cells. At the same time, a significant increase in phosphorylation of JAK2/STAT3 was observed in endothelial cells of stenotic AVFs. Interestingly, peripheral-blood mononuclear cells isolated at the time of AVF failure presented strikingly greater IL-6 expression compared with dialysis age-matched controls., Conclusion: IL-6 receptor activation may have a role in the pathogenesis of AVF failure in hemodialysis patients and may represent a potential therapeutic target in this setting.

Cytokines are pleiotropic substances that are known to participate in inflammatory and immune responses as well as cell differentiation and proliferation. Interleukin-6 (IL-6) is a key cytokine with pro-inflammatory function. Wound healing is a complex cascade of physiologic events comprising inflammation, proliferation and remodeling, and proceeds with the integrated actions of different cells, cytokines, and the extracellular matrix. Aberrant wound healing results in keloid formation which causes disfigured appearance, discomfort, psychological stress, and patient frustration. In this review, the role of IL-6 signaling pathway in the pathogenesis of keloid is assessed and its potential as a therapeutic target is addressed. The existing data suggest that IL-6 mediated inflammation is a key player and may be considered as a common causative factor for development of keloid. Furthermore, in a recent comprehensive study, we confirmed the functional role of IL-6 signaling in keloid pathogenesis. Accordingly, inhibitory strategies of IL-6 signaling pathway by targeting the IL-6 receptors, its downstream effecters, or other molecules influencing this pathway appear to have considerable potential as new therapeutic or preventive challenges for keloid. Hopefully, several IL-6 blocking agents including a humanized antibody to IL-6 receptor have been developed and successfully used in clinical trials of inflammatory diseases. It is likely that these agents may prove worthy in the treatment or prevention of keloid as well. Future in-depth exploration of such challenges will shed light on their efficacy and safety for clinical application in keloid.

The molecular mechanism(s) behind keloid pathogenesis remains unclear. Previously by global gene expression analysis of keloid fibroblasts (KFs), we implicated the IL-6 signaling pathway in keloid pathogenesis. Here, we determine a functional role of IL-6 signaling in keloid scars. Primary cultures of KFs and surrounding nonlesional fibroblasts (NFs) were subjected to induction or inhibition of IL-6 or its specific receptor IL-6 receptor alpha (IL-6R alpha) and detection of their effects on extracellular matrix gene expression. The levels of gp130 and several downstream targets in IL-6 signaling were also examined. IL-6 secretion was significantly higher in KFs than NFs. Addition of IL-6 peptide to NFs culture or inhibition of IL-6 or its receptor IL-6R alpha by their corresponding antibodies in KFs culture revealed a dose-dependent increase or decrease in collagen type I alpha 2 and fibronectin 1 mRNAs, respectively. Induction of IL-6 by IL-1beta peptide and stimulation by IL-6 peptide in NFs, or inhibition of IL-6 or IL-6R alpha in KFs cultures demonstrated a dose-dependent increase or decrease in procollagen I synthesis, respectively. The mRNA and protein expressions of gp130 and several downstream targets in IL-6 signaling (JAK1, STAT3, RAF1, and ELK1) were upregulated in KFs versus NFs. Our results indicate that IL-6 signaling may play an integral role in keloid pathogenesis and provide clues for development of IL-6 receptor blocking strategies for therapy or prophylaxis of keloid scars.

Objective: To determine whether interleukin-6 (IL-6) trans-signaling directs the expression of pre-B cell colony-enhancing factor (PBEF) in vitro and in vivo., Methods: Complementary DNA from rheumatoid arthritis (RA) synovial fibroblasts treated with IL-6 and soluble IL-6 receptor (sIL-6R) was used to probe a cytokine microarray. PBEF regulation by the IL-6-related cytokines, IL-6, sIL-6R, oncostatin M (OSM), IL-11, and leukemia inhibitory factor (LIF) was determined by reverse transcription-polymerase chain reaction analysis. IL-6-mediated STAT-3 regulation of PBEF was determined using a cell-permeable STAT-3 inhibitor peptide. Antigen-induced arthritis (AIA) was induced in wild-type (IL-6(+/+)) and IL-6-deficient (IL-6(-/-)) mice. PBEF and STAT were detected by immunohistochemistry, immunoblotting, and electrophoretic mobility shift assay. Synovial levels of PBEF were quantified by enzyme immunoassay., Results: IL-6 trans-signaling regulated PBEF in a STAT-3-dependent manner. In addition, PBEF was regulated by the IL-6-related cytokine OSM, but not IL-11 or LIF. Flow cytometric analysis of the IL-6-related cognate receptors suggested that OSM regulates PBEF via its OSM receptor beta and not its LIF receptor. The involvement of PBEF in arthritis progression was confirmed in vivo, where induction of AIA resulted in a 4-fold increase in the synovial expression of PBEF. In contrast, little or no change was observed in IL-6(-/-) mice, in which the inflammatory infiltrate was markedly reduced and synovial STAT-1/3 activity was also impaired. Analysis of human RA synovial tissue confirmed that PBEF immunolocalized in apical synovial membrane cells, endothelial cells, adipocytes, and lymphoid aggregates. Synovial fluid levels of PBEF were significantly higher in RA patients than in osteoarthritis patients., Conclusion: Experiments presented herein demonstrate that PBEF is regulated via IL-6 trans-signaling and the IL-6-related cytokine OSM. PBEF is also actively expressed during arthritis. Although these data confirm an involvement of PBEF in disease progression, the consequence of its action remains to be determined.

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.

Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.

A fundamental mechanism of immune privilege in the eye is the induction of T lymphocyte apoptosis. Intraocular inflammation in uveitis implies compromise of immune privilege. This study sought to determine whether apoptosis of T cells is actively inhibited in patients with uveitis and by what pathways this may occur. Apoptotic lymphocytes were found to be absent from aqueous humor (AqH) of virtually all patients with recent-onset uveitis. However, T cells removed from the eye were highly susceptible to both spontaneous and Fas ligand-induced apoptosis in vitro. AqH from patients with uveitis had no modulatory effect on Fas ligand-induced apoptosis, but strongly suppressed survival factor deprivation-induced apoptosis. In contrast, noninflammatory AqH from patients undergoing cataract surgery had no modulatory effects on apoptosis at all. These data suggest that triggering of the Fas pathway is diminished in uveitis, and also that homeostatic resolution through survival factor deprivation-induced apoptosis is inhibited by factors present in AqH. The most widely recognized pathways, common gamma-chain cytokines and type I IFNs, did not contribute to AqH-mediated T cell survival. High levels of both IL-6 and soluble IL-6R were found in AqH. IL-6 alone did not induce T cell survival, because IL-6R expression on T cells in AqH was too low to facilitate signaling. However, combinations of IL-6 and soluble IL-6R were highly effective inhibitors of T cell apoptosis, suggesting that the trans-signaling pathway is likely to be a key mediator of T cell apoptosis inhibition mediated by uveitis AqH.

The acute phase response is an evolutionarily conserved response of the liver to inflammatory stimuli, which aids the body in host defense and homeostasis. We have previously reported that CCAAT enhancer-binding protein alpha (C/EBPalpha) is required for the induction of acute phase protein (APP) genes in newborn mice in response to lipopolysaccharide. In this paper, we describe a mechanism by which C/EBPalpha knock-out mice are unable to induce APP gene expression in response to inflammatory stimuli. We demonstrate that the lack of acute phase response in C/EBPalpha knock-out mice is because of a hepatocyte autonomous defect. C/EBPalpha knock-out hepatocytes do not activate STAT3 in response to recombinant interleukin (IL)-6, indicating a defect in the IL-6 pathway. C/EBPalpha knock-out hepatocytes also do not show activation of other IL-6 receptor (IL-6R)-mediated Janus kinase substrates, gp130, SHP-2, and Tyk2. Further examination of the IL-6 pathway demonstrated that C/EBPalpha knock-out hepatocytes have decreased IL-6Ralpha protein levels caused, in part, by reduced protein stability. However, other components of the IL-6 pathway are intact, as demonstrated by rescue of STAT3 activation and APP gene induction with recombinant-soluble IL-6R linked to IL-6 cytokine (Hyper-IL-6) or with another gp130 signaling cytokine, Oncostatin M. In conclusion, C/EBPalpha is required for the proper regulation of IL-6Ralpha protein in hepatocytes resulting in a lack of acute phase protein gene induction in newborn C/EBPalpha null mice in response to lipopolysaccharide or cytokines.

Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the shared human cytokine signaling receptor gp130 without the requirement for pre-complexation to a specific alpha-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in addition to the "alpha-receptor-independent" tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human alpha-receptor (IL-6Ralpha) to form a hexameric vIL-6/IL-6Ralpha/gp130 complex with enhanced signaling potency. In contrast to the assembly sequence of the hIL-6 hexamer, the preformed vIL-6/gp130 tetramer can be decorated with IL-6Ralpha, post facto, in a "vIL-6-dependent" fashion. A detailed comparison of the viral and human cytokine/gp130 interfaces indicates that vIL-6 has evolved a unique molecular strategy to interact with gp130, as revealed by an almost entirely divergent structural makeup of its receptor binding sites. Viral IL-6 appears to utilize an elegant combination of both convergent, and unexpectedly divergent, molecular strategies to oligomerize gp130 and activate similar downstream signaling cascades as its human counterpart.

The physiological benefit of the febrile response is poorly understood. Here we show that fever-range thermal stress enhances the function of the L-selectin lymphocyte homing receptor through an interleukin-6 (IL-6)-dependent signaling mechanism. Thermal stimulation of L-selectin adhesion in vitro and in vivo is mediated by engagement of the gp130 signal-transducing chain by IL-6 and a soluble form of the IL-6 receptor-alpha (sIL-6Ralpha) binding subunit. Thermal control of adhesion is maintained in IL-6-deficient mice through a gp130-dependent compensatory mechanism mediated by IL-6-related cytokines (i.e., oncostatin M [OSM], leukemia inhibitory factor [LIF], and IL-11). Combined biochemical and pharmacological inhibitor (PD98059, U0126, SB203580, SP600125) approaches positioned MEK1/ERK1-2, but not p38 MAPK or JNK, in the IL-6/sIL-6Ralpha signaling pathway upstream of activation of L-selectin/cytoskeletal interactions and L-selectin avidity/affinity. These results highlight a role for gp130-linked IL-6/sIL-6Ralpha transsignaling in amplifying lymphocyte trafficking during febrile inflammatory responses.

The molecular consequence of the fever response has been illuminated by a recent study showing that a temperature shift to 40 degrees C resulted in increased leukocyte adhesion to tissue sections, which was mediated by L-selectin activation in lymphocytes. This L-selectin activation during heat responses was dependent on IL-6 trans-signaling via the soluble IL-6R.

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed.

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.

Interleukin (IL)-6 is a pleiotropic cytokine that not only affects the immune system, but also acts in other biological systems and many physiological events in various organs. In a target cell, IL-6 can simultaneously generate functionally distinct or sometimes contradictory signals through its receptor complex, IL-6Ralpha and gp130. One good illustration is derived from the in vitro observations that IL-6 promotes the growth arrest and differentiation of M1 cells through gp130-mediated STAT3 activation, whereas the Y759/SHP-2-mediated cascade by gp130 stimulation has growth-enhancing effects. The final physiological output can be thought of as a consequence of the orchestration of the diverse signaling pathways generated by a given ligand. This concept, the signal orchestration model, may explain how IL-6 can elicit proinflammatory or anti-inflammatory effects, depending on the in vivo environmental circumstances. Elucidation of the molecular mechanisms underlying this issue is a challenging subject for future research. Intriguingly, recent in vivo studies indicated that the SHP-2-binding site- and YXXQ-mediated pathways through gp130 are not mutually exclusive but affect each other: a mutation at the SHP-2-binding site prolongs STAT3 activation, and a loss of STAT activation by gp130 truncation leads to sustained SHP-2/ERK MAPK phosphorylation. Although IL-6/gp130 signaling is a promising target for drug discovery for many human diseases, the interdependence of each signaling pathway may be an obstacle to the development of a nonpeptide orally active small molecule to inhibit one of these IL-6 signaling cascades, because it would disturb the signal orchestration. In mice, a consequence of the imbalanced signals causes unexpected results such as gastrointestinal disorders, autoimmune diseases, and/or chronic inflammatory proliferative diseases. However, lessons learned from IL-6 KO mice indicate that IL-6 is not essential for vital biological processes, but a significant impact on disease progression in many experimental models for human disorders. Thus, IL-6/gp130 signaling will become a more attractive therapeutic target for human inflammatory diseases when a better understanding of IL-6 signaling, including the identification of the conductor for gp130 signal transduction, is achieved.

The activation of cells that do not express the membrane bound interleukin-6 6 receptor (IL-6R) by IL-6 and the soluble IL-6 receptor (sIL-6R) is termed transsignalling. Transsignalling may be an pathogenetic factor in human diseases as diverse as multiple myeloma (MM), Castleman's disease, prostate carcinoma, Crohn's disease, systemic sclerosis, Still's disease, osteoporosis and cardiovascular diseases. IL-6 and sIL-6R may directly or indirectly enhance their own production on endothelial or bone marrow stromal cells. Positive feedback autocrine loops thus created in affected organs may either cause or maintain disease progression. In autoimmune or vasculitic disease, the ability of the IL-6/sIL-6R complex to inhibit apoptosis of autoreactive T-cells may be central to the development of tissue specific autoimmunity. The anti-apoptotic effect of the IL-6/sIL-6R complex may be involved in tumour genesis and resistance to chemotherapy. Only in rare cases, where counterregulation has failed, there is a notable systemic effect of IL-6/sIL-6R. Appropriate animal models are necessary to establish the pathogenetic role of the IL-6/sIL-6R complex. A specific treatment option for diseases influenced by the sIL-6R could be based on gp130-Fc, a soluble gp130 (sgp130) linked to the Fc-fragment of IgG1. gp130-Fc has shown efficacy in vivo in animal models of Crohn's disease.

The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.

Recently, constitutive activation of JAK kinases (JAKs) and/or signal transducers and activators of transcription (STATs) has been reported in growing numbers of human cancer cells as well as oncogene-transformed cells. JAB/SOCS-1 has been shown to be an intrinsic JAK tyrosine kinase inhibitor and to suppress the cytokine-dependent JAK-STAT pathway. In this report, we investigated the effect of ectopic expression of JAB on v-Src-induced JAK-STAT activation. Forced expression of JAB in v-Src-transformed NIH3T3 cells neither suppressed phosphorylation of STAT3 and JAK1/JAK2 nor blocked STAT3-reporter gene activation. Colony forming assay also showed that JAB did not suppress v-Src-induced transformation of NIH3T3 cells, while dominant negative STAT3 suppressed it. In contrast, JAB could downregulate phosphorylation of STAT1 and STAT3 induced by interferon gamma (IFNgamma) and interleukin-6 (IL-6) plus soluble IL6 receptor (sIL-6R), respectively. Furthermore, in vitro kinase assay indicated that JAB suppressed hyperactivation of JAK1/JAK2 and JAK1 induced by IFNgamma and IL-6 plus sIL-6R respectively, but not v-Src-induced basal JAK1/JAK2 activity. Nevertheless, both JAK1/JAK2 activated by v-Src and that activated by IL-6 plus sIL-6R could similarly bind JAB. These results clearly demonstrate that JAB distinguishes cytokine-induced JAK-STAT signaling from v-Src-induced one and can not suppress the transformation with v-Src.

The effect of IL-6 on the growth of mouse biliary epithelial cells (BEC), in vitro, was tested by comparing BEC obtained IL-6-deficient mice (IL-6(-/-)) to wild-type littermate controls (IL-6(+/+)), in two different media: simple serum-free media (S-SFM), and complete serum-free media (C-SFM) containing forskolin, which stimulates BEC IL-6 production. In S-SFM, neither IL-6(+/+)nor IL-6(-/-)BEC constitutively produced IL-6 mRNA or protein, and there was no difference between IL-6(+/+)and IL-6(-/-)BEC growth. In contrast, when the BEC were maintained in C-SFM, over 48 h, the growth of IL-6(+/+)BEC was 40% greater than IL-6(-/-)BEC (P<0.006). Enhanced IL-6(+/+)BEC growth in C-SFM was associated with induced expression of IL-6 mRNA and IL-6 protein secretion into the medium, upregulation of the IL-6Ralpha (gp80) and phosphorylation of the signal transducing molecule gp130. In C-SFM, anti-IL-6 neutralizing antibodies blocked enhanced IL-6(+/+)BEC growth, whereas exogenous rhIL-6 stimulated retarded growth of IL-6(-/-)BEC. Thus, under conditions that mimic an inflammatory or stressful microenvironment in vivo, BEC produce, secrete and respond to IL-6, via upregulation and activation of the IL-6Ralpha (gp80)/gp130 signaling system in an autocrine/paracrine manner., (Copyright 2000 Academic Press.)

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.

The physiological function of interleukin-6 (IL-6) within the central nervous system (CNS) is complex; IL-6 exerts neurotrophic and neuroprotective effects, and yet can also function as a mediator of inflammation, demyelination, and astrogliosis, depending on the cellular context. In the normal brain, IL-6 levels remain low. However, elevated expression occurs in injury, infection, stroke, and inflammation. Given the diverse biological functions of IL-6 and its expression in numerous CNS conditions, it is critical to understand its regulation in the brain in order to control its expression and ultimately its effects. Accumulating data demonstrate that the predominant CNS source of IL-6 is the activated astrocyte. Furthermore, a wide range of factors have been demonstrated to be involved in IL-6 regulation by astrocytes. In this review, we summarize information concerning IL-6 regulation in astrocytes, focusing on the role of proinflammatory factors, neurotransmitters, and second messengers.

One of the major actions of interleukin-6 (IL-6) is the transcriptional activation of acute-phase plasma proteins (APP) genes in liver cells. Signaling by the IL-6 receptor is mediated through the signal transducing subunit gp130 and involves the activation of Janus-associated kinases (JAKs), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein (MAP) kinase. Functional analysis of gp130 in rat hepatoma cells by using transduced chimeric G-CSFR-gp130 receptor constructs demonstrates that SHP-2, the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase, acts as a negative regulator of the JAK/STAT signaling in part by downregulating JAK activity, thereby indirectly moderating the induction of STAT3-dependent APP genes. This study shows that in hepatoma cells, the recruitment and tyrosine phosphorylation of SHP-2, but not SHC, is the primary signaling event associated with the activation of MAP kinases (ERK1/2) by gp130. Overexpression of truncated SHP-2 that lacks Grb2-interacting sites, but not the full-length catalytically inactive SHP-2, reduces ERK activation by IL-6, confirming the signal-mediating role of SHP-2. Activation of ERK1/2 is correlated with induction of the immediate-early response genes. Stimulation of the c-fos, c-jun, and egr-1 genes is essentially absent in cells expressing gp130 with a Y759F mutation, which is unable to recruit SHP-2. Interestingly, both JAK/STAT and SHP-2 pathways regulate the induction of the junB gene. Moreover, disengagement of SHP-2 from gp130 signaling not only enhances APP gene induction but also further reduces cell proliferation, in part correlated with the attenuated expression of immediate-early response genes. These results suggest that IL-6 regulation of APP genes is affected by SHP-2 in two ways: SHP-2 acts as a phosphatase on the JAK/STAT pathway and serves as linker to the MAP kinase pathway, which in turn moderates APP production.

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

AIDS-associated Kaposi's sarcoma (KS) cell, a key element for development of KS lesions, proliferates in response to external cytokines, such as oncostatin M, the soluble IL-6R-IL-6 complex, TNF-alpha, and IL-1beta. In addition, the KS cell-produced basic fibroblast growth factor (bFGF) was reported to function as an autocrine growth factor. However, little is known of the exact roles of these external growth factors and endogenous bFGF on proliferation of KS cells, and underlying intracellular events have remained to be defined. We obtained evidence that anti-bFGF Ab abolished growth of KS cells by preventing S phase entry of the cell cycle, even in the presence of the external growth factors. Blockade of the FGF action profoundly inhibited cyclin E expression and cyclin-dependent kinase-2 (CDK2) activity, but not D-type cyclin expression and CDK4 activity. Exogenously added acidic FGF (aFGF), which generated a rapid tyrosine phosphorylation of FGFR1 and FGFR2 on KS cells, reversed the inhibitory effects of anti-bFGF Ab. Thus, FGF actions are essential for cyclin E-CDK2 activity and S phase entry. We also observed that the presence of external growth factors markedly induced cyclin E-CDK2 activity and S phase entrance, while the addition of aFGF or bFGF alone was insufficient to induce these responses. All this evidence shows that integration of the activities of external growth factors and endogenous bFGF is required for full activation of cyclin E-CDK2 activity and KS cell proliferation.

We assessed the biologic role of signaling through gp130, a signal-transducing receptor (R) component, in human hematopoiesis in vitro. Although peripheral blood-derived CD34(+) cells ubiquitously expressed gp130 and interleukin-3 receptor alpha (IL-3Ralpha), IL-6Ralpha was only detected on 80% of these CD34(+) cells. We sorted CD34(+)IL-6R+ or CD34(+)IL-6R- cells and studied the effect on hematopoietic colony formation of signaling through gp130 activated by IL-6 or a combination of IL-6 and recombinant soluble human IL-6R (sIL-6R) in the presence or absence of stem cell factor (SCF ) and/or IL-3. Signals activated by SCF, IL-6, or IL-6/sIL-6R complex alone did not induce significant colony formation. However, a combination of IL-3, SCF, and IL-6/sIL-6R complex dramatically induced many neutrophil (colony-forming unit-granulocyte [CFU-G]), erythroid burst (burst-forming unit-erythrocyte [BFU-E]), erythrocyte-containing mixed (CFU-Mix), and megakaryocyte (CFU-Meg) colony formations when CD34(+)IL-6R- cells were used as the target. CFU-G colony formation induced by the three signals was more evident when CD34(+)IL-6R+ cells were used as the target. This distinct synergistic effect of the three different signals was confirmed by single-cell clone-sorting experiments. Moreover, colony formation (including CFU-G, BFU-E, CFU-Mix, and CFU-Meg) was observed even in the presence of neutralizing antibodies for granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin (c-Mpl), whereas neutralizing antibodies for gp130, IL-6R, IL-3, and SCF partially or completely blocked the synergistic effect. The maturation of neutrophilic, erythroid, and megakaryocytic cells supported by the three signals in serum-free cultures was confirmed by immunostaining using anti-CD66b, antiglycophorin A, antihemoglobin alpha, and anti-CD41 monoclonal antibodies, respectively. In contrast, any two of the three signals were insufficient for effective blood cell production in the absence of maturation factors. These results suggest that simultaneous activation of the three signals through gp130, c-kit, and IL-3R can induce in vitro proliferation and differentiation of trilineage hematopoietic progenitors in the absence of terminally acting lineage-specific factors.

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.