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Purification and Characterization of Soluble Human IL-6 Receptor Expressed in CHO Cells
Abstract
An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.
... However, a soluble form of the receptor, the sIL6R that is generated either by the proteolytic shedding of the membrane bound IL6 receptor or by a differently spliced mRNA species is responsible for the signalling of IL6 in many of these cells (Müllberg et al., 1993;Horiuchio et al., 1994). Yasukawa et al. (1990) showed that the sensitivity of cells expressing both IL6R and gp130 to IL6 was further increased in the presence of the soluble IL6 receptor (sIL6R) (Yasukawa et al., 1990). Additionally, a naturally occurring secreted version of the IL6R has been reported in human serum and urine (Novick et al., 1989;Frieling et al., 1994). ...
... However, a soluble form of the receptor, the sIL6R that is generated either by the proteolytic shedding of the membrane bound IL6 receptor or by a differently spliced mRNA species is responsible for the signalling of IL6 in many of these cells (Müllberg et al., 1993;Horiuchio et al., 1994). Yasukawa et al. (1990) showed that the sensitivity of cells expressing both IL6R and gp130 to IL6 was further increased in the presence of the soluble IL6 receptor (sIL6R) (Yasukawa et al., 1990). Additionally, a naturally occurring secreted version of the IL6R has been reported in human serum and urine (Novick et al., 1989;Frieling et al., 1994). ...
Chronic pain affects more than 20% of the UK population. Neurotrophic factors have been identified as therapeutic targets to improve current treatments of chronic pain. This review article focuses on nerve growth factor (NGF) and interleukin-6 (IL-6) as potential therapeutic targets. In this review we highlight the mechanisms of action and the current progress of targeted therapies in clinical trials.
... cDNA encoding both molecules has been cloned and expressed and its biochemical properties were analyzed (3)(4)(5). In particular, a genetically engineered truncated form of the 80-kD IL-6 R, lacking the transmembrane and the intracytoplasmatic domains, has been expressed successfully and has been shown to be able to associate with the 130-kD protein in the presence of IL-6 and to mediate IL-6 functions (6). Moreover, a soluble form ofIL-6 R (sIL-6 R) has been purified as a 50-kD protein from human urine (7) and has been shown to be present in human serum and to bind recombinant human IL-6 with a binding affinity similar to that of the entire recombinant IL-6 R molecule (8). ...
... This second hypothesis is based on the assumption that circulating IL-6/sIL-6 R complexes are present in patients with systemic JRA. Since the serum soluble form of IL-6 R and the genetically engineered truncated form of IL-6 R have been shown to bind IL-6 with a binding affinity similar to the entire 80-kD molecule (6,8) and given the serum concentrations of the sIL-6 R found in this study, it is conceivable to hypothesize that IL-6 circulates bound to its soluble receptor. To verify this hypothesis, we constructed an ELISA based on the use of a monoclonal antibody to IL-6 for capture and a monoclonal antibody to sIL-6 R for revealing the presence ofthe IL-6/sIL-6 R complex. ...
... Purified rHuTPO prepared by the TPO Production Group (Kirin Brewery Co., Ltd.; Takasaki, Japan) and was provided by Dr. Hiroshi Miyazaki (Kirin Brewery; Tokyo, Japan). Recombinant sIL-6R [11] and FP were kind gifts from Tosoh Corp. (Kanagawa, Japan; http://www.tosoh.com). The FP in which Ala (333) of IL-6R residue was linked to Ala (28) of IL-6 was expressed in Pichia pastoris. ...
... Our data also demonstrated that newly generated FP more potently expanded BFU-E and CFU-Mix than the sIL-6R/IL-6 complex. An association constant of IL-6 and sIL-6R was observed to be 5 × 10 -9 mol/l [11]. This indicates that in the solution containing 200 ng/ml (1 × 10 -8 mol/l) of IL-6 (20 kDa) and 500 ng/ml (1 × 10 -8 mol/l) of sIL-6R (50 kDa), 50% of the molecules exist as a monomer. ...
This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34+IL-6R+/– cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.
... IL-6 exerts its biological activities mainly through IL-6R, which exists in two forms: a membrane-bound form (mIL-6R) that exerts its actions through cell membranes and a soluble form (sIL-6R) that is secreted into the extracellular space and in body fluids. The latter mainly binds to IL-6 and exerts its activity in extracellular environments 11,16,17 . Signals mediated by mIL-6R and sIL-6R are termed classical signaling and trans-signaling, respectively. ...
Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).
... IL-6 exerts its biological activities mainly through IL-6R, which exists in two forms, a membrane-bound form (mIL-6R) that exerts its actions through cell membranes, and a soluble form (sIL-6R) that is secreted into the extracellular space and in body uids. The latter mainly binds to IL-6 and exerts its activity in extracellular environments [10,15,16]. Signals mediated by mIL-6R and sIL-6R are termed classical signaling and trans-signaling, respectively. ...
BACKGROUND The biologic and cellular IL-6-related therapies have been used to treat the autoimmune diseases (AID), which prompting us to furtherly explore the IL-6 role in human umbilical cord mesenchymal stem cells (hUCMSCs) therapy.
MATERIALS & METHODS Peripheral blood mononuclear cells (PBMCs) were responder co-cultured with hUCMSCs or exogenous IL-6. PBMC suppression assay was used to analyze the anti-inflammatory effects by using the MTT assay. The IL-6 concentration in supernatant was measured using ELISA. The correlation between anti-inflammation effect of hUCMSCs and IL-6 levels, and the relevant roles of IL-6, IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was furtherly evaluated.
RESULTS Initially, hUCMSCs did not exert any inhibitory effect on PBMCs, however, a potent inhibitory effect on PBMCs was observed and the IL-6 concentration reached about 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 could inhibit PBMCs inflammations but less than that of hUCMSCs. The hUCMSCs exerts excellent anti-inflammation effect by inducing higher IL-6 level which is different from TCZ IL-6 antagonist
CONCLUSIONS High concentration IL-6 cytokine secretion plays an important role in the anti-inflammation effect of hUCMSCs cell therapy.
... One year later the receptor gene was identified [22] and based on these results, extracellular domains of the receptor of various lengths (soluble receptor) were prepared, and their inhibitory effects on IL-6 were examined. However, even by binding to and capturing IL-6, the function of IL-6 was not inhibited, but instead, induced transmission of IL-6 signals into the cell [23]. ...
The clinical use of monoclonal antibodies is well established in human medicine and has been amongst the most important contributions of basic science to clinical disease. One such antibody, the humanized anti-human IL-6 receptor antibody, is used to treat a variety of autoimmune diseases, particularly rheumatoid arthritis. It is extremely difficult and a laborious process to go from a concept at the research bench, to government approval. Such approval implies not only efficacy but, more importantly, an appropriate safety profile. In this review, the history of anti-human IL-6 receptor antibody is discussed in depth beginning with the author’s experience during a sabbatical visit at the University of California at Davis in 1978. At that time, it was discovered that B cell activation was at least one critical factor in the development of autoimmunity. Approximately six years later, the cDNA encoding for IL-6 was cloned as BSF-2 (B cell stimulatory factor 2) to differentiate B cells to produce antibody. Soon after, it was suggested that this cytokine plays an important role in the development of autoimmune diseases. Based on this evidence, the journey began to search for an IL-6 inhibitor. Although there were numerous obstacles in finding lead compounds, ultimately, basic science developed the methodology for high throughput readouts that would inhibit the biologic function of IL-6. It was finally concluded that a mouse monoclonal antibody against IL-6 receptor would be optimal. In 1991, this antibody was humanized by using CDR-grafting technology in collaboration with the MRC (Medical Research Council). The drug was named tocilizumab and launched as an innovative anti-rheumatic drug in 2008 in Japan. Subsequently, the drug has been used throughout the world and has achieved enormous success in helping patients who suffer from inflammatory arthropathies. The lessons learned in the development of this antibody have application to the study of biologics and their application to other human diseases.
... These actions occur through interactions with specific soluble or membrane bound receptors that form the biologically active IL-6 receptor complex (IL-6RC) [407,466,474]. In addition to the 19.5-26 kDa (depending on glycosylation) cytokine IL-6, two membrane glycoproteins help form the IL-6RC, an 80 kDa protein referred to as the ligand-binding ␣-subunit (gp80, IL-6R, or CD126) and a 130 kDa protein referred to as the nonligand binding, affinity converting, and signal transducing -receptor (gp130 or CD130) [207,628]. All members of the IL-6 cytokine family-IL-6, IL-11, on-costatin M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1)share gp130 as a component critical for signal transduction [207,208,232,260,426,534]. Soluble forms of the two receptors (sIL-6R, sgp130) arise by limited proteolysis (shedding) or differential splicing (sIL-6R with 55 kDa and sgp130 with 100 kDa) [206,378,380,382,448]. ...
... Similar results were reported here showing that CSF levels of IL6r are associated with one IL6r SNP while plasmatic levels are associated with both SNPs (rs7514452 and rs4240872) in ADNI-1 subsample, reflecting a genotype-phenotype effect. The IL6r protein is either a part of the ligand-binding receptor of IL6 or a soluble form (s-IL6r), which binds to IL6 to enhance its activity [61,62]. Deregulation of immune response signaling in AD is evidenced by altered protein expression in the brain [63,64]. ...
Several lines of evidence suggest the involvement of neuroinflammatory changes in Alzheimer’s disease (AD) pathophysiology such as amyloidosis and neurodegeneration. In fact, genome-wide association studies (GWAS) have shown a link between genes involved in neuroinflammation and AD. In order to further investigate whether interactions between candidate genetic variances coding for neuroinflammatory molecules are associated with brain amyloid β (Aβ) fibrillary accumulation, we conducted an epistasis analysis on a pool of genes associated with molecular mediators of inflammation.
[ 18 F]Florbetapir positron emission tomography (PET) imaging was employed to assess brain Aβ levels in 417 participants from ADNI-GO/2 and posteriorly 174 from ADNI-1. IL-1β, IL4, IL6, IL6r, IL10, IL12, IL18, C5, and C9 genes were chosen based on previous studies conducted in AD patients. Using the [ 18 F]florbetapir standardized uptake value ratio (SUVR) as a quantitative measure of fibrillary Aβ, epistasis analyses were performed between two sets of markers of immune-related genes using gender, diagnosis, and apolipoprotein E (APOE) as covariates. Voxel-based analyses were also conducted. The results were corrected for multiple comparison tests. Cerebrospinal fluid (CSF) Aβ 1-42 /phosphorylated tau (p-tau) ratio concentrations were used to confirm such associations.
Epistasis analysis unveiled two significant single nucleotide polymorphism (SNP)-SNP interactions (false discovery rate (FDR) threshold 0.1), both interactions between C9 gene (rs261752) and IL6r gene (rs4240872, rs7514452). In a combined sample, the interactions were confirmed (p ≤ 10–5) and associated with amyloid accumulation within cognitively normal and AD spectrum groups. Voxel-based analysis corroborated initial findings. CSF biomarker (Aβ 1-42 /p-tau) confirmed the genetic interaction. Additionally, rs4240872 and rs7514452 SNPs were shown to be associated with CSF and plasma concentrations of IL6r protein.
Certain allele combinations involving IL6r and C9 genes are associated with Aβ burden in the brain. Hypothesis-driven search for epistasis is a valuable strategy for investigating imaging endophenotypes in complex neurodegenerative diseases.
... 2).f22] Studies [23,24] have shown that soluble forms of tumour necrosis factor (TNF) receptors may compete with the cell surface receptors, thus may inhibit TNF bioactivity. Conversely, soluble IL-6 receptors may enhance ligand activity. ...
Advances in molecular biology and recombinant DNA technology have led to the development of cytokines as therapeutic agents for a variety of disease states. The pharmacokinetic analysis of cytokines involves the understanding of analytical methods capable of detecting these agents in biological fluids and recognition of several factors which may have an impact on the cytokine concentration-time curves. Enzyme-linked immunosorbent assays (ELISA) have become the most common method of detection and commercial kits are available for a wide variety of cytokines. Monoclonal antibody products are sensitive, have minimal crossreactivity and are relatively inexpensive when compared with high performance liquid chromatography (HPLC). However, the primary limitation of these assays is their inability to measure biologically active protein. Conversely, bioassays do measure a biological event (i.e. proliferation or cytotoxicity) but are generally not used for cytokine analysis because of their high cost, long assay completion time, lack of specificity, poor sensitivity and influence of environmental conditions on the outcome. The pharmacokinetic profile of recombinant cytokines is influenced by a number of variables: endogenous production, circulating soluble receptors and cell-associated receptors, immunocompetence and antibody production against the cytokine all may influence the disposition of the agent. Thus, pharmacokinetic modelling of cytokines may involve complex models capable of characterising these nonlinear processes and resulting effects. An increased understanding of the many factors which can alter the analysis and pharmacokinetics of cytokines is essential to the design of optimal dosage regimens.
... Receptor, Cytokines, and Antibodies. rhlL-6 and slL-6R were prepared as described previously (11). rhSCF was kindly provided by Amgen Biologicals (Thousand Oaks, CA). ...
We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.
... Furthermore, albeit to a much lesser extent, sIL-6R can also be generated by alternative splicing [114]. The soluble receptor is able to bind to gp130, which is ubiquitously expressed by all cell types, where it subsequently activates the IL-6 signaling cascade [115,116]. IL-6 trans-signaling plays a crucial role in chronic inflammatory bowel disease, peritonitis, and rheumatoid arthritis, as well as in colon cancer [117][118][119][120]. Of note, IL-6 trans-signaling rarely occurs under steady-state conditions, where sufficient amounts of inhibitory soluble gp130 are found in the circulation to bind to and block circulating IL-6/sIL-6R complexes [121] (Figure I). ...
Owing to its abundance in inflammatory settings, interleukin IL-6 is frequently viewed as a proinflammatory cytokine, with functions that parallel those of tumor necrosis factor (TNF) and IL-1β in the context of inflammation. However, accumulating evidence points to a broader role for IL-6 in a variety of (patho)physiological conditions, including functions related to the resolution of inflammation. We review recent findings on the complex biological functions governed by IL-6 signaling, focusing on its role in inflammation-associated cancer and metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM). We propose that the anti-inflammatory functions of IL-6 may extend to multiple settings and cell types, and suggest that these dimensions should be incorporated in therapeutic approaches to these diseases. Finally, we outline important areas of inquiry towards understanding this pleiotropic cytokine.
Copyright © 2015. Published by Elsevier Ltd.
... In the case of IL-6, the soluble form of the p80 component of the IL-6 receptor (sIL-6R) binds to IL-6 in solution and is present at a high concentration in serum (17)(18)(19)(20)(21). Therefore, sIL-6R might be a major source of interference in immunoassays of IL-6. ...
... Given that IL-17F provided advantages for both CHO cell growth and transgene expression, we evaluated the benefit of expressing IL-17F during the generation and subsequent upstream processing of a CHO cell line producing a protein of pharmaceutical interest. The model protein studied was the soluble form of the human IL-6 receptor a chain (sIL-6R; Yasukawa et al., 1990). The sIL-6R coding sequence was inserted into a bicistronic IRES expression cassette downstream of the IL-17F cDNA (Fig. 3A, left panel). ...
The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame. Biotechnol. Bioeng. 2013; 110: 1153-1163. © 2012 Wiley Periodicals, Inc.
... Human 1L-6 was expressed in E. coli using the pUC8 vector. Soluble hIL-6 receptor was expressed in Chinese hamster ovary cells and purified as described (Yasukawa et al., 1990). ...
Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183–187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding.
A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
Les anticorps monoclonaux anti-cytokine ont constitué une révolution dans le traitement des maladies inflammatoires chroniques, mais leur utilisation présente des inconvénients (non réponse, résistance, effets secondaires, coûts élevés).Notre équipe développe une stratégie alternative originale, l’immunisation active à base de peptides de cytokines. Elle a pour but de faire synthétiser, par l’organisme même du patient, des anticorps neutralisant les effets pathogènes dus à l’excès de cytokines.Durant ma thèse, j’ai montré que l’immunisation active contre un peptide dérivé de l’IL-6 murine est protectrice dans un modèle murin de sclérodermie systémique. L’immunisation de singes avec l’équivalent humain entraîne une réduction significative des réactions inflammatoires locales suite à l’induction d’une réaction d’HSR. De plus, l’immunisation active contre deux peptides dérivés de l’IL-1β et de l’IL-23 conduit à la réduction de la sévérité de l’EAE.Ces résultats confortent l’intérêt de cibler les cytokines par l’approche d’immunisation active à base de peptides, qui pourra permettre de diversifier l’offre thérapeutique actuellement disponible.
The interleukin-6 (IL-6) signal is transduced through membrane-anchored gp130, which is associated with IL-6 receptor (IL-6R) in the presence of IL-6. Soluble forms of gp130 (sgp130) with molecular weights of 90 and 110 Kd were found in human serum. In the presence of recombinant IL- 6 (rIL-6), serum sgp130 were capable of associating with serum sIL-6R. By the sandwich enzyme-linked immunosorbent assay, healthy human sera was shown to contain 390 +/- 72 ng/mL of sgp130. A mouse pro-B-cell line-derived transfectant, BAF-130, expressing human gp130 was used to examine the function of serum sgp130. When supplemented with rIL-6, human serum induced DNA synthesis in BAF-130 cells, whereas the serum deprived of sIL-6R did not. In contrast, the DNA synthesis induced in BAF-130 cells by rIL-6-supplemented serum was increased when the serum was deprived of sgp130. These results indicated that serum sgp130 could negatively regulate the IL-6 signal. Recently, gp130 has been shown to be involved in the signaling processes of oncostatin M, leukemia inhibitory factor, and ciliary neurotropic factor, in addition to those of IL-6. Recombinant sgp130 showed inhibitory effect on the biologic function of such cytokines. This work implies physiologic roles of naturally produced serum sgp130 in modulating signals through gp130.
We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.
We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.
Liver pathologies (fibrosis, cirrhosis, alcoholic, non-alcoholic diseases and hepatocellular carcinoma) represent one of the most common causes of death worldwide. A number of genetic and environmental factors contribute to the development of liver diseases. Interleukin-6 (IL-6) is a pleiotropic cytokine, exerting variety of effects on inflammation, liver regeneration, and defence against infections by regulating adaptive immunity. Due to its high abundance in inflammatory settings, IL-6 is often viewed as a detrimental cytokine. However, accumulating evidence supports the view that IL-6 has a beneficial impact in numerous liver pathologies, due to its roles in liver regeneration and in promoting an anti-inflammatory response in certain conditions. IL-6 promotes proliferation, angiogenesis and metabolism, and downregulates apoptosis and oxidative stress; together these functions are critical for mediating hepatoprotection. IL-6 is also an important regulator of adaptive immunity where it induces T cell differentiation and regulates autoimmunity. It can augment antiviral adaptive immune responses and mitigate exhaustion of T cells during chronic infection. This review focuses on studies that present IL-6 as a key factor in regulating liver regeneration and in supporting effector immune functions and suggests that these functions of IL-6 can be exploited in treatment strategies for liver pathologies.
This chapter discusses the reconstitution in vitro of the interleukin-6/ interleukin-6 receptor interaction. In a study described in the chapter, the recombinant IL-6 samples were purified from insoluble inclusion bodies. Extraneous proteins were extracted from the inclusion bodies by selective urea washing. The washed inclusion bodies were solubilized in 8 M GuHCl, 50 mM Tris–HCl, and pH 7.5, and the IL-6 variants were purified to homogeneity using a two-step procedure of size-exclusion chromatography, and 6 M GuHCl, 50 mM Tris–HCl, pH 7.5, and RP-HPLC. The yield was ∼50 mg of pure mIL-6 from 5L cultures of Echerichia coli. No refolding step was required because correct disulfide bond formation occurred during the GuHCl solubilization step. Purified mIL-6, hIL-6, pMC5, and pMC5H chromatographed as single symmetrical peaks on RP-HPLC. pMC5H had a distinctly longer retention time relative to mIL-6, which reflects the more hydrophobic nature of the C-terminal residues in hIL-6. Molecular masses of hIL-6, pMC5H, pMC5, and mIL-6, determined by electrospray mass spectrometry were 21.854, 21.281, 20.680, and 21.253 kDa, respectively.
Growth hormone (GH) acts on a variety of terminally differentiated cells, including adipocytes, myocytes, hepatocytes, and pancreatic beta cells, to regulate energy balance (1). In adipocytes GH produces an immediate insulin-like response that is most readily demonstrated by accelerated glucose metabolism in cells that have been deprived of GH for at least 3 h (2) and a delayed anti-insulin-like response typified by increased lipolysis (3). Because these responses are independently affected by chemical modifications of GH (4, 5) or the GH receptor (6), it was suggested that different responses may require different hormone receptor interactions (5, 6). However, consistent findings of linear Scatchard isotherms argue for a single class of binding sites (6–9). In the absence of any definitive mechanism for how GH might produce any of its biological actions, the question of whether GH interacts with a single receptor or a variety of receptor complexes cannot yet be answered.
Receptor complexes for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) utilize membrane glycoprotein gp130 as a common signal-transducing component. In order to investigate detailed physiological roles of gp130 and to determine pathological consequences of complete lack or abnormal activation of gp130, mice deficient for gp130 protein or expressing continuously activated gp130 protein have been made. A gp130 null mutation is lethal, and embryos deficient for gp130 protein progressively die between 12.5 days post coitum (dpc) and term. They show, at 16.5 dpc, hypoplastic development of the ventricular myocardium. They have greatly reduced numbers of pluripotential and committed hematopoietic progenitors in the liver, as measured on 13.5 dpc. gp130-/- placentas on and after 14.5 dpc are smaller than controls and exhibit impaired maternofetal transport. Continuous activation of gp130 in vivo by overexpressing both IL-6 and IL-6R leads to hypertrophy of ventricular myocardium and thickened ventricular walls of the heart in adulthood. These results indicate crucial roles of gp 130 in cardiomyocyte regulation, hematopoiesis, and placental development.
Multiple myeloma (MM) is a malignant, still incurable, neoplastic disease of unclear etiology and pathogenesis, which is characterised by a slow proliferation of monoclonal plasmocytes or plasmoblasts in the bone marrow. About 15 000 new cases of MM occur in the USA annually. Multiple myeloma incidence in the USA is 3 in 100 000, majority in men (60%), while 98% of patients are over 40 years of age. Primary in vitro animal studies suggest that interaction of neoplastic cells and interstitium in regulation of their growth migration and resistance to chemotherapy. Neoplastic cells in bone marrow microenvironment grow and survive notwithstanding genetic instability and chromosomal aberrations and migrate due to cytokines. According to most recent studies the greatest significance in MM occurrence and development are interleukin 6 (IL-6), vascular endothelium growth factor (VEGF), tumor necrosis factor-α (TNF-α), hepatocyte growth factor (HGF). There is a view that drug resistance of MM is to a large axtent associated with a pathological constellation of cytokines promoting uninhibited growth of neoplastic cells and their migration.
The interleukin-6 (IL-6) is a cytokine associated with auto-immune thyroid diseases and could be also involved in pathological cardiac hypertrophy. In FRTL-5 thyroid cells and in adult rat cardiomyocytes, we show that both IL-6 secretion and mRNA expression, stimulated by interleukin-1 (IL-1), are enhanced by cAMP/PKA pathway in a synergistic manner. In FRTL-5 cells, this synergistic effect occurs also at a post-transcriptional level by increasing IL-6 promoter activation. This synergic effect involves AP-1 and CRE binding sites of IL-6 promoter and also c-Fos and Fra2 transcription factors. IL-6 and its membrane-bound receptor stimulate STAT3 and ERK-5 signaling pathways in FRTL-5 cells. In cardiomyocytes, STAT3 is activated by IL-6 only in the presence of its soluble receptor. We show that this mechanism conduces to increase protein synthesis and expression of markers associated to the pathological hypertrophy.
Functional pleiotropy and redundancy are characteristic features of cytokines, including interleukins, interferons, colony-stimulating factors and growth factors. These molecules exert their biological functions through specific receptors expressed on the surface of target cells. Originally it was thought that each cytokine exerts a specific effect on its particular target cell, but in fact, most cytokines exhibit a wide range of biological effects on various tissues and cells. One receptor may interfere with different signal transduction systems and multiple receptors can couple to the same signaling pathwayviacommon effectors. Therefore signaling pathways within a cell form a network with multiple interactions between different cytokine pathways. Interleukin-6 (IL-6) is an example of such a multifunctional cytokine [1] and exerts multiple effects on the immune, hematopoietic and nervous system in a finely regulated interaction with its two receptors: interleukin-6 receptor (IL-6R) and glycoprotein 130 (gp130), the component critical for signal transduction.
Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.
In this study, we have developed a multiscale systems model of interleukin (IL)‐6–mediated immune regulation in Crohn's disease, by integrating intracellular signaling with organ‐level dynamics of pharmacological markers underlying the disease. This model was linked to a general pharmacokinetic model for therapeutic monoclonal antibodies and used to comparatively study various biotherapeutic strategies targeting IL‐6–mediated signaling in Crohn's disease. Our work illustrates techniques to develop mechanistic models of disease biology to study drug–system interaction. Despite a sparse training data set, predictions of the model were qualitatively validated by clinical biomarker data from a pilot trial with tocilizumab. Model‐based analysis suggests that strategies targeting IL‐6, IL‐6Rα, or the IL‐6/sIL‐6Rα complex are less effective at suppressing pharmacological markers of Crohn's than dual targeting the IL‐6/sIL‐6Rα complex in addition to IL‐6 or IL‐6Rα. The potential value of multiscale system pharmacology modeling in drug discovery and development is also discussed.
CPT Pharmacometrics Syst. Pharmacol . (2014) 3, e89; doi: 10.1038/psp.2013.64 ; advance online publication 8 January 2014
Individuals with chronic fatigue syndrome (CFS) have significantly increased proportions of activated CD8+T cells, decreased natural killer (NK) cell cytotoxic and lymphoproliferative activities, elevated serum levels of tumor necrosis factor (TNF)-α and detectable TNF-β, interleukin (IL)-lβ, and IL-6 mRNA in peripheral blood mononuclear cells (PBMC). We report here that CFS patients as a group also have significantly higher levels, as compared to controls, of soluble TNF receptor type I (sTNF-RI or sCDl20a), sIL-6R (sCD126) and β2-microglobulin (β2-m), but not of IL-1 receptor antagonist (IL-1Ra). Correlative and population distribution studies that included lymphoid phenotypic distributions and function as well as soluble immune mediator expression levels revealed the existence of at least two mainly nonoverlapping immunological categories among CFS patients with either: (1) dysregulaled TNF-α/β expression in association with changes in the serum levels of IL-lα, IL-4, sIL-2R and IL-lRa, PBMC-associated expression of IL-1β, IL-6 and TNF-β mRNA, and T cell activation; or, (2) interrelated and dysregulated expression of sTNF-RI, sIL-6R, and β2-m and significantly decreased lymphoproliferative and NK cell cytotoxic activities. This preliminary systematization is of usefulness in the diagnosis, follow-up, and characterization of possible etiological agents for CFS.
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.
Starting with a previously established hybridoma producing a monoclonal antibody (F1α75) against a synthetic glycolipid, in which the carbohydrate moiety was artificially designed to be putatively cancer-specific, we cloned complementary DNA (cDNA) for active variable regions of both heavy and light chains of the antibody. We established a Chinese hamster ovary (CHO) cell line expressing a recombinant F1α75 of human IgG class. The recombinant F1α75 retained the binding ability to its antigen and was easily purified. The result suggests that the conversion into the IgG class by genetic engineering is useful for antibodies of the IgM class which are difficult to be purified due to aggregation. Sequence analysis of variable regions of F1α75 revealed no strong homology with an antibody against galactose, the terminal residue of the synthetic glycolipid, suggesting the clinical usefulness of the recombinant F1α75.
FP6, a novel recombinant fusion protein of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), was prepared in the methylotrophic yeast Pichia pastoris. This protein was a potent activator of a cell surface transducing glycoprotein, gp130 and is a potential therapeutical reagent in the hemopoietic field. A linker is generally thought to be required for two fused molecules to retain their proper structures although it should preferably be removed to reduce possible antigenicity. It was found that the C-terminal residue of IL-6R could be directly linked to the N-terminal residue of IL-6 without decreasing the ability of IL-6 to bind gp130 and send the IL-6 signal. It was also found that the peptide bond between Lys-37 and Asp-38 of IL-6 was prone to proteolytic cleavage and that the immunoglobulin (Ig)-like region of IL-6R underwent extensive and heterogeneous glycosylation when expressed in P. pastoris. Based on these findings, we designed FP6 without the Ig-like region, in which the C-terminal residue of Ala-333 of IL-6R was directly linked to Asp-38 of IL-6 by a peptide bond. Purified FP6 had both an in vitro effect on hemopoietic progenitors to generate various colonies and an in vivo effect on megakaryocyte progenitors to increase platelet counts. Four purified FP6s were obtained, which had the same molecular mass and different isoelectric points without any detectable modification in the course of purification. The difference in isoelectric points was shown to be due to microheterogeneity of the carbohydrate chains. Each FP6 had the same specific activity in the cell growth assay with or without endoglycosidase digestion. Homogeneous FP6 with respect to isoelectric point as well as molecular mass merits more detailed characterization and evaluation for possible clinical application.
New type of plasmids named pEdHCG1 and pEdHC?.were constructed. A combination of two plasmids containing the amplified gene product for the variable region of the antibody (Ab) heavy or light chains by the polymerase chain reaction (PCR) was co-transfected into COS cells to transiently express and in Chinese hamster ovary (CHO) cells to constitutively express chimeric Ab composed of the mouse-derived variable region and human-derived constant region.
It has been reported that soluble interleukin (IL)-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and human immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates IL-6 action, its physiological role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant mouse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6R) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In contrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrate-resistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation on dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding monoclonal anti-mouse IL-6R antibody (MR16-1). It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gp130, even though they may have no or a very small number of IL-6Rs. Factors such as IL-11, oncostatin M, and leukemia inhibitory factor, which are known to exert their functions through gp130 (the signal-transducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced sIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanism involving gp130. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption.
Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal
antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6
was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.
Interleukin-5 (IL-5) mediates pleiotropic functions in various types of cells through its specific receptor (IL-5R) which is composed of two distinct subunits, α and β. In mice, the α subunit (IL-5Rα) specifically binds IL-5 with low affinity. The β subunit (IL-5Rβ) does not bind IL-5 by itself, but constructs the high affinity receptor with IL-5Rα. We have isolated cDNA encoding the soluble form of IL-5Rα (sIL-5Rα). To elucidate the biochemical and functional properties of sIL-5Rα, we developed an expression system for sIL-5Rα cDNA in insect cell line Sf21 using baculovirus expression vector and obtained conditioned medium containing large quantities of mouse sIL-5Rα. Mouse sIL-5Rα was purified from the conditioned medium by using anti-IL-5Rα mAb-coupled beads. Immunoaffinity-purified sIL-5Rα with an approximate molecular mass of 42 kDa inhibited the binding of 125I-labeled IL-5 to IL-5R. By using purified sIL-5Rα, we prepared rabbit anti-sIL-5Rα antibody and developed a sandwich ELISA for detection of sIL-5Rα. Significant amounts of sIL-5Rα were detected in sera and ascitic fluids of mice bearing tumors (BCL1 and MOPC104E) that responded to IL-5 for DNA synthesis, but not in sera of normal mice. Interestingly, elevated levels of serum sIL-5Rα were observed in NZB and NZW mice. The sIL-5Rα may, therefore, have an immunoregulatory role in vivo.
Soluble interleukin-6 receptor (sIL-6R) was found to be spontaneously released from human myeloma cell line U266 cells into culture supernatant, and was quantitatively measured with a fluorescence sandwich enzyme-linked immunosorbent assay employing antibodies specific to IL-6R. The supernatant IL-6R was generated only from IL-6R-positive cell lines; myeloma cell lines RPMI8226 and RPMI1788, and myelomonocytic cell lines U937, THP-1, and HL-60. In contrast, it was not released from the IL-6R-negative cells; T cell line MoIt-4 and Burkitt lympboma cell line Raji. SDS-PAGE analysis of the soluble IL-6R from U266 cells suggested a molecular weight of approximately 50-55 kDa, 25–30 kDa smaller than the mature cell surface receptor. These results suggest that the generation of soluble IL-6R may be a maker of myeloma cells and myelomonocytic cells.
Amino acid substitutions of human interleukin-6 (IL-6) were performed. Single substitution Met162 → Ala and double substitutions Leu159, 166 → Val resulted in a significant decrease of IL-6 activity in the production of immunoglobulin (lg) from B-cells. Single substitution Leu166→Val or Leu159→Val gave a slight or no significant decrease in the Ig-induction activity, respectively. The receptor-binding activity of each IL-6 mutant was also examined. It was observed that the decrease of the receptor-binding activity was generally in parallel with that of the Ig-induction activity. We therefore suggest that hydrophobic side-chains existing in Met162, Leu159, and Leu164 are significantly involved in the receptor-binding of IL-6.
The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp 130. In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp 130. The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing. We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris. We obtained, however, no detectable expression and secretion of the recombinant protein. When we used only the coding sequence of the cytokine receptor domain of the sIL-6R for the construction of an expression plasmid, this truncated version of the sIL-6R accumulated in the supernatant to 1-5 mg/l. The protein was purified by a single affinity chromatography step using a monoclonal antibody directed against the human IL-6R. Following the same approach, we expressed a truncated splice variant of the sIL-6R. Both, the secreted truncated sIL-6R and the splice variant showed full agonistic biological activity on human hepatoma cells. The described expression strategy will be useful for large scale production of biologically active sIL-6R and might be adapted for the expression of other members of the hematopoietic cytokine receptor family.
The characteristics of soluble interleukin-6 receptor (sIL-6R) in murine sera were examined. To investigate a relationship between serum sIL-6R level and autoimmune diseases, quantitative analysis of serum sIL-6R in MRL/lpr mice was performed by an enzyme-linked immunosorbent assay. The serum sIL-6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 ± 9.3 ng/ml in female and 31 ± 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age-associated increase in serum sIL-6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL-6R level at the age of 30 weeks was observed in female and male (NZB × NZW)F1 mice (32 ± 10 ng/ml and 17 ± 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 ± 18 ng/ml), suggesting that elevated serum sIL-6R in aged mice is one of the characteristics of autoimmune-prone mice. Quantitative analysis of serum IL-6 in MRL/lpr revealed that the serum sIL-6R level correlated well with the serum IL-6 level. We also showed that sIL-6R in the sera from MRL/lpr mice could mediate the IL-6 functions through the IL-6 signal-transducing receptor component gpl30, suggesting that elevated production of sIL-6R may partly contribute to development of autoimmune disease in MRL/lpr mice.
Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.
Cytokines (IL-1, IL-6, IL-8, IL-11, TNF, IFN-γ, and TGF-β) and growth factors (EGF, bFGF, aFGF, and KGF) play an important role in modulation of hormone secretion by directly influencing specific enzyme steps of steroidogenesis in various endocrine cell types. For this tabular data collection, the following enzyme steps were considered: steroidogenic acute regulatory protein (StAR), side chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase, 17-alpha-hydroxylase/17,20-lyase (P450c17), 17-beta-hydroxysteroid-dehydrogenase, aromatase complex, 5-alpha-reductase, P450c21, DHEAS sulfatase, and DHEA sulfotransferase. This collection summarizes the current information on how the mentioned cytokines and growth factors influence particular enzyme steps.
Soluble human interleukin‐6 receptor (sIL‐6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be sIL‐6R, because of its binding capacity to IL‐6 and its molecular mass of 55 kDa.
All sera of healthy individuals contained sIL‐6R (mean value: 89 ng/ml, range 17‐300 ng/ml). Serum sIL‐6R levels were increased by 51% in patients with MGUS (mean value: 135 ng/ml, p <0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p <0.001) and by 116 % in patients with overt MM (mean value: 193 ng/ml, p <0.001). In patients with MM, a complete lack of correlation ( p >0.7) was found between serum sIL‐6R levels and other previously recognized prognostic factors in this disease, particularly serum IL‐6 levels and those factors related to tumor cell mass. The independence of serum sIL‐6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL‐6R were found to increase by tenfold the sensitivity of human myeloma cell lines to IL‐6. These observations suggest a high control of the sIL‐6R level in vivo , and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.
Functional pleiotropy and redundancy are characteristic features of cytokines. Interleukin 6 (IL-6) is a typical example: IL-6 induces cellular differentiation or expression of tissue-specific genes; it is involved in processes such as antibody production in B cells, acute-phase protein synthesis in hepatocytes, megakaryocyte maturation, cytotoxic T cell differentiation, and neural differentiation of PC12 (pheochromocytoma) cells. It promotes growth of myeloma/plasmacytoma cells, T cells, keratinocytes and renal mesangial cells, and it inhibits growth of myeloid leukaemic cell lines and certain carcinoma cell lines.The IL-6 receptor consists of two polypeptide chains, a ligand-binding chain (ILdR) and a non-ligand-binding, signal-transducing chain (gp130). Interaction of IL-6 with IL-6R triggers the association of gp130 and ILdR, and the signal can be transduced through gp130. Association of gp130 with IL-6R is involved in the formation of high affinity binding sites. This two-chain model has been shown to be applicable to receptor systems for several other cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-5 and nerve growth factor (NGF). The pleiotropy and redundancy of cytokines may be explained on the basis of this unique receptor system.
The chapter describes interleukin-5 (IL-5) that is a cytokine with important biological activity in inflammation. The gene for IL-5 clusters with the genes for IL-3, IL-4, and granulocyte macrophage colony stimulating factor (GM-CSF), and their similar exon-intron organization suggests an evolutionary relationship. The three-dimensional structure of IL-5 demonstrates a four alpha helical bundle motif that is conserved among several hematopoietic cytokines. However, IL-5 is unusual as two monomers interdigitate to form a pair of these helical bundle motifs. The major cellular sources of IL-5 are mast cells, eosinophils, and lymphocytes. Although IL-5 has pleiotrophic effects on a variety of leukocytes, it is unique among cytokines in its ability to specifically promote the terminal maturation of eosinophils. In humans, the biologic effects of IL-5 are best characterized for eosinophils. In addition to inducing terminal maturation of eosinophils, IL-5 prolongs eosinophil survival by delaying apoptotic death, possesses eosinophil chemotactic activity, increases eosinophil adhesion to endothelial cells and enhances eosinophil effector function. Physiologically, IL-5 and its associated eosinophil activation serve a protective role against helminth infections and facilitate the immunologic destruction of tumors.
Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.
We investigated the possible influence of recombinant (r) sIL-6R on the growth of three IL-6 non-responsive or weakly IL-6 responsive long-term myeloma cell lines. The three cell lines chosen for the study (U266, L363 and Fravel) all expressed gp130 but differed in their expression of IL-6R and IL-6. mRNA analysis by northern blot and reverse transcriptase polymerase reaction showed that the cell line U266 was the only one that expressed IL-6 mRNA. Only U266 and L363 expressed IL-6R mRNA. 125I-rIL-6 binding studies and FACS analysis, using biotinylated IL-6 and antibodies directed against the IL-6R and gp130, showed corresponding results on the protein level. Addition of rsIL-6R resulted in induction of IL-6 responsiveness in L363 cells, whereas the 3H-thymidine incorporation of the cell lines U266 and Fravel was unaffected by rsIL-6R addition. In conclusion, the IL-6 unresponsive growth of several long-term myeloma cell lines in vitro can in some, but not all cases, be due to a deficiency in exogenous sIL-6R.
Contradictory results have been reported on the effects and role of IL-6 on proteoglycan (PG) synthesis. Having shown recently that in vitro IL-6 depends on the presence of soluble IL-6 receptor α (sIL-6Rα) to fully exert its effects on chondrocytes, we conducted the present study to analyse the effects of IL-6 on PG synthesis by human articular chondrocytes in the presence of sIL-6Rα. PG synthesis was quantified by specific ELISA using a monoclonal antibody (MAB) raised against the keratan sulphate region of PG as a capture antibody, and a MAB to the acid binding region as a detector. It proved specific for PG from primary (differentiated) chondrocytes. In the absence of sIL-6Rα, IL-6 had a slight inhibitory effect on PG synthesis by articular chondrocytes. sIL-6Rα alone also had slight but consistent inhibitory effects. When adding sIL-6Rα at concentrations of 50 ng/ml corresponding to levels found in synovial fluid, the effects of IL-6 increased consistently. However, even at optimal concentrations (30–100 ng/ml of IL-6sR per 100 ng/ml of IL-6), maximal inhibition (48%) did not equal the degree of inhibition achieved by IL-1 at 1 ng/ml (66%). Similar effects, although slightly weaker, were observed on osteoarthritic cells. Dexamethasone, over a wide range of concentrations, markedly enhanced proteoglycan synthesis and completely reversed the downregulatory effects of IL-1 and IL-6+sIL-6Rα. The effects of IL-1 were partially inhibited by an anti-IL-6 antibody. Finally, unlike IL-1, IL-6+sIL-6Rα only weakly stimulated nitric oxide (NO) synthesis. In conclusion, sIL-6Rα potentiates the inhibitory effect of IL-6 on PG synthesis by articular chondrocytes, but the overall effect of IL-6+IL-6sR is moderate compared to the effects of IL-1.
The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 10⁶ cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium⁻¹ day⁻¹. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10⁻¹⁰ and 2.81 × 10⁻¹⁰ M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10⁻⁸ M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in β-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.
Serum levels of interleukin-6 (IL-6) and interleukin-6 soluble receptor (IL-6sR) were measured in 41 patients (23 female and 18 male, mean age 72.5 years) with Alzheimer's disease (AD) and in 32 controls (14 women and 18 men, mean age 69.2 years) using enzyme-linked immunosorbent assays (ELISA). Proportions of individuals with detectable serum IL-6 concentrations did not differ significantly between patients and controls. There was however, a significant decrease in IL-6sR levels in Alzheimer's patients when compared with controls. Our results suggest that there is a dysregulation of IL-6 and its soluble receptor in AD.
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