IL-6 transsignaling: the in vivo consequences.

Cytokine receptors exist in membrane-bound and soluble forms. They bind their ligands with comparable affinity. Although most soluble receptors are antagonists because they compete with their membrane counterparts for their ligands, some soluble receptors are agonists. In this case, on target cells, the complex of cytokine and soluble cytokine receptor binds to a second receptor subunit and initiates intracellular signal transduction. The soluble receptors of the interleukin-6 (IL-6) family of cytokines--soluble IL-6 receptor (sIL-6R), sIL-11R, and soluble ciliary neurotrophic factor receptor (sCNTFR)--are agonists. In vivo, the IL-6/sIL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, as they do not express the membrane- bound IL-6R. This process has been named transsignaling. We have shown recently that in several chronic inflammatory diseases, such as chronic inflammatory bowl disease, peritonitis, and rheumatoid arthritis, as well as in colon cancer, transsignaling via the sIL-6R complexed to IL-6 is a crucial point in the maintenance of the disease. The mechanism by which the IL-6/sIL-6R complex regulates the inflammatory or neoplastic state is discussed.     

Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer

Cytokine receptors, which exist in membrane-bound and soluble forms, bind their ligands with comparable affinity. Although most soluble receptors are antagonists and compete with their membrane-associated counterparts for the ligands, certain soluble receptors are agonists. In these cases, complexes of ligand and soluble receptor bind on target cells to second receptor subunits and initiate intracellular signaling. The soluble receptors of the interleukin (IL)-6 family of cytokines (sIL-6R, sIL-11R, soluble ciliary neurotrophic factor receptor) are agonists capable of transmitting signals through interaction with the universal signal-transducing receptor for all IL-6 family cytokines, gp130. In vivo, the IL-6/sIL-6R complex stimulates several types of cells, which are unresponsive to IL-6 alone, as they do not express the membrane IL-6R. We have named this process trans-signaling. The generation of soluble cytokine receptors occurs via two distinct mechanisms-limited proteolysis and translation-from differentially spliced mRNA. We have demonstrated that a soluble form of the IL-6 family signaling receptor subunit gp130, which is generated by differential splicing, is the natural inhibitor of IL-6 trans-signaling responses. We have shown that in many chronic inflammatory diseases, including chronic inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma, as well as colon cancer, IL-6 trans-signaling is critically involved in the maintenance of a disease state, by promoting transition from acute to chronic inflammation. Moreover, in all these models, the course of the disease can be disrupted by specifically interfering with IL-6 trans-signaling using the soluble gp130 protein. The pathophysiological mechanisms by which the IL-6/sIL-6R complex regulates the inflammatory state are discussed.     

Local delivery of soluble interleukin-6 receptors to improve the outcome of alpha-toxin producing Staphylococcus aureus infection in mice

Staphylococcal alpha-toxin enhances interleukin (IL)-6 secretion in mice infected with Staphylococcus aureus. The role of alpha-toxin-induced IL-6 secretion in host defense has not been sufficiently clarified. In the present study, IL-6 signaling was transiently regulated using soluble IL-6 receptors (sIL-6R) to investigate the role of IL-6 in the early stage of abdominal S. aureus infection. In mice challenged with bacteria producing high alpha-toxin levels, the local delivery of sIL-6R was effective in improving the survival rate, the resolution of neutrophilia and the bacteria clearance. Mice that had received sIL-6R and survived showed high levels of IL-6, monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha. In contrast, mice that died in spite of the delivery of sIL-6R showed high levels of interferon (IFN)-gamma and IL-1alpha and low TNF-alpha level. When the effect of soluble gp130, a sIL-6R antagonist, was examined, the number of neutrophils increased significantly and the MCP-1 level decreased significantly, compared to the group that received sIL-6R alone; the number of viable bacteria also tended to increase as a result of the inhibition of IL-6 signaling. The cellular phosphotyrosine level in alpha-toxin-treated macrophages was reduced in cultures supplemented with recombinant IL-6 in vitro. These results suggest that IL-6 enhances bactericidal activity and reduces the number of immune cells that are activated abnormally through the regulation of inflammatory cytokines during the early stage of infection in alpha-toxin producers.     

Effects of IL-6 and Soluble IL-6 Receptor on the Expression of Cartilage Matrix Proteins in Human Chondrocytes

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Rα) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Rα on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Rα, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Rα, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Rα. These results suggest that IL-6 and sIL-6Rα suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.     

Soluble IL-6 receptor induces calcium flux and selectively modulates chemokine expression in human dermal fibroblasts.

Truncated forms of cytokine receptors have been regarded as modulators of the activity of their cognate ligands. In addition to inhibiting effects of their respective ligands, soluble receptors can also facilitate ligand-mediated signaling. Several studies have demonstrated that exogenous IL-6 in association with the soluble IL-6 receptor alpha (sIL-6Ralpha) can activate cells expressing the gp130 signal transducer lacking the specific, membrane-bound IL-6Ralpha. Since cell cultures of human dermal fibroblasts express high amounts of IL-6, we examined whether the addition of sIL-6Ralpha in association with endogenous IL-6 would be sufficient to stimulate these cells via gp130. As an early rapid signal we analyzed changes in intracellular free calcium concentrations ([Ca2+]i). Addition of sIL-6Ralpha induced an acute and transient increase in cytosolic free calcium concentrations in a dose-dependent fashion. This Ca2+-signal was abolished when cells were pretreated with anti-IL-6 or anti-gp130 antibodies. Using flow cytometric analysis we could demonstrate membrane-associated IL-6 and gp130, but not IL-6Ralpha on fibroblasts. We also analyzed MCP-1 and IL-8 expression as a response involved in the more recently recognized chemoattractant functions of fibroblasts, and found MCP-1 to be up-regulated, but not IL-8. These data suggest that sIL-6Ralpha binds to cell-associated, endogenous IL-6 produced by fibroblasts and this complex then activates the cells via gp130. This pathway of fibroblast activation by sIL-6Ralpha adds another dimension to the role of fibroblasts in the cytokine network.     

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

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

Characterization of anti-mouse interleukin-6 receptor antibody

Hybridoma that produces rat anti-mouse interleukin 6 receptor (IL-6R) antibody, MR16-1, was established by the fusion of mouse P3U1 myeloma cells and spleen cells from mouse soluble IL-6R (sIL-6R)-immunized Wistar rat. In the present study, we examined the characteristics of MR16-1 in vitro and in vivo. MR16-1 bound to mouse sIL-6R dose-dependently. MR16-1 suppressed IL-6-induced proliferation of 7TD1 cells in a dose-dependent manner and this inhibitory effect was reversed by the addition of a higher concentration of IL-6. Cross-reactivity study using T cells from mouse, rat, and human revealed that MR16-1 did not cross-react with human and rat IL-6R. Binding region analysis using several human-mouse chimeric IL-6Rs showed that half of the fibronectin domain II of mouse IL-6R (amino acids 214-285) was required for MR16-1 binding. Furthermore, MR16-1 completely suppressed IL-6-induced antibody production in DNP-KLH immunized mice. These lines of evidence demonstrate that MR16-1 is useful to investigate the physiological and pathological roles of IL-6 and sIL-6R in mice.     

Release of the Soluble Interleukin-6 Receptor from Human T-Cells

The soluble Interleukin-6 receptor (sIL-6R) is capable of confering the Interleukin-6 (IL-6) signal onto cells lacking the gp80 ligand binding protein. Here we investigate the release of sIL-6R from T-cells. After 2 h stimulation with PMA, a release of sIL-6R from peripheral human T-cells was observed which was insensitive to the protein synthesis inhibitor cycloheximide. This release was accompanied by a decrease of membrane-bound (mb) IL-6R. After 24 h, however, the observed sIL6-R release did prove to be sensitive to cycloheximide. These results suggest that both shedding and de-novo-synthesis may be responsible for the PMA-induced sIL-6R release. In contrast to PMA, neither anti-CD3, a positive, nor IL-10, a negative regulator of IL-6 release from T-cells affected the production of the sIL-6R. The differential regulation of sIL-6R and IL-6 production by T-cells might be relevant for the immunomodulatory potential of the sIL-6R with respect to the interaction of T- and non-T-cells.     

Activation of Protein Kinase C Induces De Novo Synthesis of the Soluble Interleukin-6 Receptor in Human B Cells

The mechanism of protein kinase C (PKC) induced release of the soluble interleukin-6 receptor (sIL-6R) from human B cells was investigated. Phorbot myristat acetat (PMA)-induced activation of PKC significantly enhanced the release of sIL-6R from the human B-cell line SKW 6.4. The PMA effect was completely blocked by cycloheximide, whereas different inhibitors of proteases had no effect. In contrast to the effect on sIL-6R release, FACS analysis did not reveal any effect of PMA on the expression of IL-6R on the surface of SKW 6.4 cells. After 6h of stimulation with PMA, analysis of mRNA expression using a polymerase chain reaction-(PCR)-assisled mRNA amplification assay, showed increased expression of a spliced mRNA encoding for a soluble form of IL-6R. Comparable to the results in SKW 6.4 cells, activation of purified human B cells with PMA induced a significant augmentation of sIL-6R release which was also sensitive to cycloheximide. In conclusion, a novel mechanism of sIL-6R release is reported involving de novo synthesis. Thus, sIL-6R release from human B cells is completely different compared with that described in hepatocytes, which involved rapid, proteolytic cleavage of the membrane-bound receptor but not de novo synthesis. The results of this study may help to understand the molecular control of sIL-6R release from human B cells.     

Release of the Soluble Interleukin-6 Receptor from Human T-Cells

The soluble Interleukin-6 receptor (sIL-6R) is capable of confering the Interleukin-6 (IL-6) signal onto cells lacking the gp80 ligand binding protein. Here we investigate the release of sIL-6R from T-cells. After 2 h stimulation with PMA, a release of sIL-6R from peripheral human T-cells was observed which was insensitive to the protein synthesis inhibitor cycloheximide. This release was accompanied by a decrease of membrane-bound (mb) IL-6R. After 24 h, however, the observed sIL6-R release did prove to be sensitive to cycloheximide. These results suggest that both shedding and de-novo-synthesis may be responsible for the PMA-induced sIL-6R release. In contrast to PMA, neither anti-CD3, a positive, nor IL-10, a negative regulator of IL-6 release from T-cells affected the production of the sIL-6R. The differential regulation of sIL-6R and IL-6 production by T-cells might be relevant for the immunomodulatory potential of the sIL-6R with respect to the interaction of T- and non-T-cells.     

Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.     

Novel pathogenic mechanism of microbial metalloproteinases: liberation of membrane-anchored molecules in biologically active form exemplified by studies with the human interleukin-6 receptor.

Certain membrane-anchored proteins, including several cytokines and cytokine receptors, can be released into cell supernatants through the action of endogenous membrane-bound metalloproteinases. The shed molecules are then able to fulfill various biological functions; for example, soluble interleukin-6 receptor (sIL-6R) can bind to bystander cells, rendering these cells sensitive to the action of IL-6. Using IL-6R as a model substrate, we report that the metalloproteinase from Serratia marcescens mimics the action of the endogenous shedding proteinase. Treatment of human monocytes with the bacterial protease led to a rapid release of sIL-6R into the supernatant. This effect was inhibitable with TAPI [N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl) L-3-(2' naphthyl)-alanyl-L-alanine, 2-aminoethyl amide], a specific inhibitor of the membrane-bound intrinsic metalloproteinase, but not with other conventional proteinase inhibitors. sIL-6R-liberating activity was also detected in culture supernatants of Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes, organisms that are known to produce metalloproteinases. sIL-6R released through the action of S. marcescens metalloproteinase retained biological activity and rendered IL-6-unresponsive human hepatoma cells sensitive to stimulation with IL-6. This was shown by Northern (RNA) blot detection of haptoglobin mRNA and by quantitative measurements of de novo-synthesized haptoglobin in cell supernatants. Analysis of immunoprecipitated, radiolabeled sIL-6R revealed that the bacterial protease cleaved IL-6R at a site distinct from that utilized by the endogenous protease. These studies show that membrane-anchored proteins can be released in active form through cleavage at multiple sites, and they uncover a novel mechanism via which microbial proteases possibly provoke long-range biological effects in the host organism.     

Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.     

Astrocytic alterations in interleukin-6/Soluble interleukin-6 receptor alpha double-transgenic mice

Interleukin-6 (IL-6), a major cytokine with diverse effects on cells mainly of the immune and hematopoietic systems, has been linked to several neurological disorders such as acquired immune deficiency syndrome dementia, multiple sclerosis, and Alzheimer's disease. Central nervous system (CNS)-specific expression of IL-6 caused neurodegeneration, massive gliosis, and vascular proliferation in transgenic mice. However, the effects of systemically circulating IL-6 and its receptor IL-6Ralpha on the CNS are unknown. IL-6Ralpha is the specific component of the IL-6 receptor system and hence an important co-factor of IL-6. IL-6Ralpha is bioactive in a membrane-bound and in a soluble (s) form. We investigated the effects of systemically elevated levels of either human IL-6 or human sIL-6Ralpha or both on the CNS of transgenic mice. Although IL-6 and sIL-6Ralpha single transgenic mice were free of neurological disease, IL-6/sIL-6Ralpha double-transgenic mice showed neurological signs, such as tremor, gait abnormalities, and paresis. However, these mice also frequently showed prominent general weakness probably because of the systemic effects of IL-6/IL-6Ralpha such as liver damage and plasmacytomas. IL-6/sIL-6Ralpha transgenic mice exhibited massive reactive gliosis. Lack of signs of neuronal breakdown versus ample astrogliosis suggested that astrocytes were selectively affected in these mice. There was neither vascular proliferation nor inflammatory infiltration. Ultrastructural analysis revealed blood-brain barrier (BBB) changes manifested by hydropic astrocytic end-feet. However, albumin immunohistochemistry did not reveal major BBB leakage. Our results indicate that increased and constitutive systemic expression of IL-6 together with its soluble receptor sIL-6Ralpha is less harmful to the brain than to other organs. The BBB remains primarily intact. IL-6/IL-6Ralpha, however, might be directly responsible for the selective activation of astrocytes.     

Role of neutrophils in release of some cytokines and their soluble receptors

Available data suggest that cytokines and soluble cytokine receptors, which are being produced by immunological cells, can modulate the immune response of the host. Although the production of mediators such as TNF- and IL-6 as well as that of their soluble receptors has been extensively studied in tissue cells and mononuclear cells, it has not been fully investigated in neutrophils (PMN). In the present study we examined the ability of PMN to simultaneously release TNF-, IL-6 and their soluble receptors-sTNFRp55, sTNFRp75 and sIL-6R. Concentrations of soluble receptors were compared with expression of membrane-bound TNF and IL-6 receptors. For comparative purposes, similar examinations with autologous peripheral blood mononuclear cells (PBMC) were performed. We found that PMN and PBMC have the same ability to release IL-6 and sTNFRp75. In contrast, there were significant differences in the release of TNF-, sTNFRp55 and sIL-6R between these cells. Reduction in membrane TNF receptor expression, observed in this study, was associated with increase secretion of soluble TNF receptors by PMN and PBMC. The results suggest that PMN can play an essential role in modulating the inflammatory response by affecting the balance between pro-inflammatory mediators, such as TNF-, and anti-inflammatory mediators, such as soluble TNF receptors.     

Role of neutrophils in release of some cytokines and their soluble receptors

Available data suggest that cytokines and soluble cytokine receptors, which are being produced by immunological cells, can modulate the immune response of the host. Although the production of mediators such as TNF-alpha and IL-6 as well as that of their soluble receptors has been extensively studied in tissue cells and mononuclear cells, it has not been fully investigated in neutrophils (PMN). In the present study we examined the ability of PMN to simultaneously release TNF-alpha, IL-6 and their soluble receptors-sTNFRp55, sTNFRp75 and sIL-6R. Concentrations of soluble receptors were compared with expression of membrane-bound TNF and IL-6 receptors. For comparative purposes, similar examinations with autologous peripheral blood mononuclear cells (PBMC) were performed. We found that PMN and PBMC have the same ability to release IL-6 and sTNFRp75. In contrast, there were significant differences in the release of TNF-alpha, sTNFRp55 and sIL-6R between these cells. Reduction in membrane TNF receptor expression, observed in this study, was associated with increase secretion of soluble TNF receptors by PMN and PBMC. The results suggest that PMN can play an essential role in modulating the inflammatory response by affecting the balance between pro-inflammatory mediators, such as TNF-alpha, and anti-inflammatory mediators, such as soluble TNF receptors.     

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

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