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

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

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

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

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.     

A common variant of the interleukin 6 receptor (IL-6r) gene increases IL-6r and IL-6 levels, without other inflammatory effects

Interleukin-6 (IL-6) is a key inflammatory cytokine, signalling to most tissues by binding to a soluble IL-6 receptor (sIL-6r), making a complex with gp130. We used 1273 subjects (mean age 68 years) from the InCHIANTI Italian cohort to study common variation in the IL-6r locus and associations with interleukin 6 receptor (IL-6r), IL-6, gp130 and a battery of inflammatory markers. The rs4537545 single nucleotide polymorphism (SNP) tags the functional non-synonymous Asp358Ala variant (rs8192284) in IL-6r (r(2)=0.89, n=343). Individuals homozygous for the rs4537545 SNP minor allele (frequency 40%) had a doubling of IL-6r levels (132.48 pg/ml, 95% CI 125.13-140.27) compared to the common allele homozygous group (68.31 pg/ml, 95% CI 65.35-71.41): in per allele regression models, the rs4537545 SNP accounted for 20% of the variance in sIL-6r, with P=5.1 x 10(-62). The minor allele of rs4537545 was also associated with higher circulating IL-6 levels (P=1.9 x 10(-4)). There was no association of this variant with serum levels of gp130 or with any of the studied pro- and anti-inflammatory markers. A common variant of the IL-6r gene results in major changes in IL-6r and IL-6 serum levels, but with no apparent effect on gp130 levels or on inflammatory status in the general population.     

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.     

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.     

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.     

Interleukin-6 and the soluble IL-6 receptor are decreased in cerebrospinal fluid of geriatric patients with major depression: no alteration of soluble gp130

Interleukin-6 (IL-6) is hypothesized to play an important role in the interaction between immune mechanisms and the central nervous system. We investigated whether cerebrospinal fluid (CSF) concentrations of interleukin-6 (IL-6), the soluble IL-6 receptor (sIL-6R) and the soluble form of the signal transducing and affinity converting receptor gp130 (sgp130) are altered in geriatric patients with major depression (MD). In 20 geriatric patients with MD and 20 age-matched healthy control subjects CSF concentrations of the three components of the sIL-6R complex were analyzed by enzyme-linked immunosorbent assays (ELISA). All patients except one were treated with psychotropic drugs. We found statistically significant decreased CSF concentrations of IL-6 (P<0.001) and of the sIL-6R (P<0.001) of patients with MD. Levels of sgp130 showed no statistically significant difference between patients and controls.     

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

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

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

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

Interleukin-6 and its receptor: from bench to bedside

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

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.     

Soluble interleukin-6 receptor (sIL-6R) in cerebrospinal fluid of patients with inflammatory and non inflammatory neurological diseases

IL-6 acts on target cells via the ligand-binding protein interleukin-6 receptor (IL-6R) and the affinity-converting and signal-transducing glycoprotein 130 (gp130). Soluble interleukin-6 receptor (sIL-6R) has an agonistic role because the soluble complex (IL-6/sIL-6R) can activate cells that do not express IL-6R and an antagonistic role as it enhances the inhibitory activity of sgp130. Soluble forms of both receptors, sIL-6R and sgp130, regulate the action of IL-6. sIL-6R was measured by a sensitive enzyme-linked immunosorbent assay in paired sera and cerebrospinal fluid (CSF) from 46 patients with inflammatory neurological diseases (IND), 45 patients with relapsing-remitting multiple sclerosis (RR-MS), 13 patients with primary progressive multiple sclerosis (PP-MS), 17 patients with other non inflammatory neurological diseases (NIND) and 13 mentally healthy individuals--healthy controls (HC). Patients with RR-MS had CSF sIL-6R levels comparable to those from patients with IND, but higher than patients with NIND and HC. A positive correlation between the CSF/serum albumin (QAlb) and CSF sIL-6R levels was observed in IND but not in RR-MS patients indicating that CSF sIL-6R levels in IND patients could be influenced by serum sIL-6R and blood brain barrier (BBB) permeability properties. RR-MS patients had higher values of [CSF/serum sIL-6R:CSF/serum albumin] (sIL-6R index) than IND patients suggesting that in multiple sclerosis (MS), the increase in CSF sIL-6R could be due to intrathecal synthesis of sIL-6R. The finding of increased CSF sIL-6R concentrations (>979 pg/ml) with sIL-6R index (>4.66), in correlation with positive oligoclonal bands in RR-MS patients, suggests that values of sIL-6R index > 4.66 indicate intrathecal increase of sIL-6R and might be used as an indicator of neuroimmunoregulatory and inflammatory processes in the central nervous system (CNS).     

IL-6 and soluble IL-6 receptors (sIL-6R and sgp130) in human pleural effusions: massive IL-6 production independently of underlying diseases

IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) levels were measured in sera and pleural effusions from 42 patients with metastatic carcinoma, non-Hodgkin's lymphoma, tuberculosis, cardiac failure and miscellaneous diseases. Pleural IL-6 levels measured by ELISA were very high in all patient groups (mean 34.8 ± 15.3 ng/ml) without significant difference according to diseases. IL-6 was shown to be biologically active in a proliferative assay. Serum IL-6 levels were low (0.049 ± 0.014 ng/ml) and did not correlate with pleural fluid levels. Pleural IL-6 levels correlated with the number of polymorphonuclear cells in pleural fluid (P< 0.03). Pleural sIL-6R levels (76 ± 8 ng/ml) were always lower than serum levels (196 ± 12 ng/ml; P< 0.0001) but correlated with them (P< 0.01). Pleural sIL-6R and albumin levels correlated (P< 0.01), suggesting a transudation of sIL-6R from the serum. Pleural sgp130 levels (10.9 ± 1.0 ng/ml) were lower than serum levels (24.6 ± 2.8 ng/ml; P< 0.002). After gel filtration of pleural fluid, the bulk of IL-6 (>90%) was recovered in a 15 000–30 000 fraction, corresponding to the expected mol. wt of free IL-6. These results suggest a production and a sequestration of IL-6 in the pleural cavity in all studied conditions.     

Expression of the interleukin 6 receptor in primary renal cell carcinoma.

AIMS: Interleukin 6 (IL-6) is expressed in the majority of renal cell carcinomas and has an important role in the proliferation of some renal cell carcinoma cell lines. This action is mediated by two membrane proteins, gp80 (the IL-6 receptor; IL-6R), which binds IL-6, and gp130, which transduces the signal. The soluble form of gp80 (sIL-6R) is able to activate gp130 when complexed to the IL-6 molecule. These considerations prompted an investigation of IL-6R expression in this malignancy. IL-6, C reactive protein (CRP), and sIL-6R were also measured in serum and correlated to clinical and pathological features. METHODS: Immunostaining was performed on cryostat sections from renal cell carcinoma tumours with M91, an anti-IL-6R monoclonal antibody, using the alkaline phosphatase antialkaline phosphatase technique. The proliferation index was measured using the KI-67 monoclonal antibody. CRP, IL-6, and sIL-6R were measured in serum before nephrectomy, using an immunoenzymatic or immunoradiometric assay. RESULTS: There were significant differences in survival in patients with tumours larger than 8 cm, metastasis at diagnosis, high nuclear grade tumours, detectable serum concentrations of IL-6 (correlated to CRP serum concentration), more than 4% proliferating cells, and the presence of the IL-6R in situ. Furthermore, the serum IL-6 concentration correlated with tumour size and stage. The mean serum sIL-6R concentration was not significantly different from that observed in 40 normal subjects. Tumour IL-6R expression was present in 10 samples. There was a significant association between the presence of the IL-6 receptor in tumours and tumour stage, nuclear grade, proliferation index, and serum IL-6. CONCLUSIONS: This study revealed the importance of IL-6/CRP and IL-6R expression in situ as potential new prognostic factors and opens the way to new therapeutic strategies in renal cell carcinoma.     

抽动秽语综合征患者血清自身抗体与可溶性白介素-6受体表达增高

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

Central role of IL-6 receptor signal-transducing chain gp130 in activation of L-selectin adhesion by fever-range thermal stress

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.