Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P05231 (interleukin-6)
 23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.
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PMID:Identification and characterization of a C-terminally extended form of recombinant murine IL-6. 203 66

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.
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PMID:Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. 205 Jan 35

LY303870 [(R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane] is a new, potent and selective nonpeptide neurokinin-1 (NK-1) receptor antagonist. LY303870 bound selectively and with high affinity to human peripheral (Ki = 0.15 nM) and central (Ki = 0.10 nM) NK-1 receptors. LY303870 inhibited [125I]substance P (SP) binding to guinea pig brain homogenates with similar affinity; however, it had approximately 50-fold lesser affinity for rat NK-1 sites. The less active enantiomer, LY306155 [(S)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]-propane], was 1,000- to 15,000-fold less potent in all the species examined. LY303870 antagonized in vitro NK-1 receptor effects as demonstrated by blockade of SP-stimulated phosphoinositide turnover in UC-11 MG human astrocytoma cells (Ki = 1.5 nM) and interleukin-6 secretion from U-373 MG human astrocytoma cells (Ki = 5 nM). In addition, LY303870 inhibited SP-induced rabbit vena cava contractions (pA2 = 9.4) with high (50,000-fold) selectivity vs. NK-2 or NK-3 receptor-mediated responses. In vivo, LY303870 inhibited [Sar9,Met(O2)11]-SP induced guinea pig bronchoconstriction (ED50 = 75 micrograms/kg i.v.) and pulmonary microvascular leakage in the bronchi (ED50 = 12.8 micrograms/kg i.v.) and trachea (ED50 = 18.5 micrograms/kg i.v.). Therefore, LY303870 is a potent and selective NK-1 receptor antagonist in vitro and in vivo. The use of LY303870 will facilitate a better understanding of NK-1 receptors in physiological processes.
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PMID:Pharmacological characterization of LY303870: a novel, potent and selective nonpeptide substance P (neurokinin-1) receptor antagonist. 747 61

Functional NK-1 (substance P) receptors have been demonstrated previously on astrocytes from primary newborn rat brain cultures and human astrocytoma cells lines by specific [125I]-Bolton Hunter substance P (SP) binding and by SP-induced phosphoinositol turnover. In addition, these cells have been shown to release cytokines upon stimulation with interleukin-1 (IL-1) and lipopolysaccharide (LPS). Since SP has also been shown to induce cytokine release from rat glial cells, this neuropeptide may contribute to the pathophysiology of neuronal inflammation in humans by stimulating cytokine production in the brain. We, therefore, explored whether SP could induce U-373 MG human astrocytoma cells, via specific NK-1 receptor activation, to secrete interleukin-6 (IL-6), a cytokine implicated as a key mediator of immune and inflammatory responses. SP stimulated IL-6 production in a concentration-dependent manner with an MC50 (concentration inducing 50% of the maximum response) of 45 nM. IL-6 was detected in the cell culture supernatant fluids 2 h post stimulation and secretion peaked at 12 h. SP induced IL-6 secretion was not mediated by IL-1 since neutralizing anti-IL-1 (alpha and beta) antibody treatment had no effect on the SP response. The selective NK-1 receptor agonist, [Sar9, Met(O2)11]-SP, was comparably effective to SP in stimulating IL-6 secretion; however, selective NK-2 and NK-3 receptor agonists were 250-500-fold less effective. In addition, the non-peptide NK-1 receptor antagonist, (+/-)CP-96,345, inhibited SP (Ki = 4 nM), but not IL-1-induced IL-6 release. These selectivity and specificity studies confirmed the presence of functional NK-1 type receptors linked to IL-6 release. The results of this study support a role for SP as a modulator of immune and/or inflammatory processes in the human CNS.
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PMID:Interleukin-6 secretion from human astrocytoma cells induced by substance P. 751 75

In a human astrocytoma cell line U373 MG, the activation of the neurokinin 1 (NK1) receptor by substance P (SP) increase, in a concentration-related manner (1 nM to 10 microM), the basal release of interleukin-6 (IL-6) as assayed by an ELISA method, in cell supernatants after 18 h of incubation. Septide, a selective NK1 receptor agonist, is equipotent to SP in inducing the IL-6 release showing similar Emax (2644 +/- 285 and 2830 +/- 271 pg/ml) and EC50 (15.6 +/- 3.6 and 13.8 +/- 3.2 nM). However, in binding assays on intact cells, septide was an about 50-fold weaker displacer of the binding of [3H][Sar9,Met(O2)11]SP than SP (Ki's were 0.28 +/- 0.1 nM and 14.2 +/- 5.0 nM for SP and septide, respectively). NK2- and NK3-selective agonists (up to 1 microM) had no binding or functional effect. Highly selective non-peptide (CP96,345) or peptide (GR82,334) NK1 receptor antagonists were more effective in antagonizing septide-(IC50's 0.2 +/- 0.06 nM and 70 +/- 18 nM) than SP-(IC50's 6.7 +/- 1.3 nM and 1.95 +/- 0.4 microM) induced IL-6 secretion. These data support the existence, also in human U373 MG cells, of a septide-sensitive NK1 receptor subtype(s) and/or epitope(s) blocked with high affinity by NK1 antagonist.
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PMID:Different susceptibility to neurokinin 1 receptor antagonists of substance P and septide-induced interleukin-6 release from U373 MG human astrocytoma cell line. 752 49

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.
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PMID:Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. 753 47

Aggregation studies have become a useful criterion for analyzing leukocyte motility and activation in vitro. The T-cell-derived lymphokine human leukocyte inhibitory factor (LIF) is a modulator of many important polymorphonuclear (PMN) functions in addition to aggregation such as chemotaxis, lysosomal degranulation, phagocytosis, bactericidal killing, augmented antibody-dependent cellular cytotoxicity (ADCC), induction of neutrophil Fc-gamma, complement type-1 and FMLP receptors, and production of superoxide and H2O2. Our investigations focused on the ability of LIF to modulate the aggregation of macrophages (MO) induced by calcium ionophore A23187. The ionophore A23187 directly induced potent aggregation of macrophages, which was markedly enhanced when the cells were pretreated with LIF. However, the addition of LIF in the absence of other costimuli did not directly induce MO aggregation. LIF was shown to enhance PMN aggregation induced by N-formyl-Met-Leu-Phe (FMLP), but did not augment the aggregation of FMLP-stimulated macrophages, indicating a cellular specificity of aggregation-inducing costimuli following LIF priming. Additional cytokines examined for possibly inducing MO aggregation were interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), and interleukin-6 (IL-6); all proved to be incapable of inducing aggregation directly, nor did they enhance the effect of A23187 ionophore on macrophage aggregation. Additionally, we found that LIF can directly stimulate MO to activate specific pathways of the arachidonic acid cascade, inducing the synthesis and release of thromboxanes and leukotriene B4. LIF did not augment the potent ability of A23187 to induce increased production of LTB4 or TxA2 by human MO. These new results coupled with our previously published data indicate that LIF can enhance the activation of both MO and PMN leukocytes when exposed to either A23187 or FMLP, respectively. Moreover, these data suggest that LIF can contribute directly to monocyte-macrophage leukocyte activation, in addition to PMN activation, during inflammatory responses, resulting in greater cell aggregation, activation, and specific proinflammatory arachidonic acid product release.
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PMID:Leukocyte inhibitory factor (LIF) potentiates human macrophage aggregation and activation responses to calcium ionophore A23187 and directly induces leukotriene B4 and thromboxane A2 release. 829 72

Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver.
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PMID:Co-expression and regulation of Met and Ron proto-oncogenes in human hepatocellular carcinoma tissues and cell lines. 921 52

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and delta9-tetrahydrocannabinol on the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-gamma, p55 and p75 TNF-alpha soluble receptors by stimulated human peripheral blood mononuclear cells as well as [3H]arachidonic acid release by non-stimulated and N-formyl-Met-Leu-Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3-30 nM) but inhibited the production of TNF-alpha, interferon-gamma, interleukin-4 and p75 TNF-alpha soluble receptors at higher concentrations (0.3-3 microM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-alpha soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-alpha and interferon-gamma production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. delta9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-alpha, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM delta9-tetrahydrocannabinol but stimulated by 3 microM delta9-tetrahydrocannabinol, as was interleukin-8 and interferon-gamma synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-alpha soluble receptors was diminished by 3 microM delta9-tetrahydrocannabinol. [3H]Arachidonate release was stimulated only by high delta9-tetrahydrocannabinol and anandamide concentrations (30 microM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and delta9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.
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PMID:Influence of fatty acid ethanolamides and delta9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells. 925 58

The interleukin-6 (IL-6)/gp-80 and hepatocyte growth factor (HGF)/met ligand/receptor systems have been shown to stimulate biliary epithelial cell (BEC) DNA synthesis in vitro. The mRNA and protein production of these two in vitro mitogens were mapped in vivo during the first week after bile duct ligation (BDL) when peak BEC DNA synthesis is seen. Changes around the biliary tree were compared with those seen in the peripheral liver using a combination of Northern blotting and a unique biliary tree isolation technique, in which the bile ducts and the surrounding portal stroma and inflammatory cells are separated from the hepatocytes by perfusion digestion. Further localization was performed with in situ hybridization and immunohistochemistry. In the normal liver, there is low-level expression of HGF mRNA by periportal stellate cells, and HGF protein localizes to these cells and to neutrophils; extracellular HGF protein is present in the bile. There is no detectable IL-6 mRNA by Northern analysis or IL-6 protein expression in the normal liver, but both met and IL-6 receptor (IL-6R) mRNA are detectable; met mRNA is expressed strongly in the biliary tree, and met protein is expressed weakly on hepatocytes and strongly on BEC. IL-6R mRNA is weakly expressed in the biliary tree, and IL-6R protein is detectable on hepatocytes, with a periportal-to-perivenular gradient, but not on BEC. During the first 3 days after BDL, HGF mRNA expression is increased in both the biliary tree and in the peripheral liver, and production is localized to stellate cells, periductal neutrophils, and stromal cells, which typically accompany the proliferating ductules. IL-6 mRNA and protein were detected only near the biliary tree after BDL, and not in the peripheral liver, and the production was localized to periductal hematolymphoid cells, which had the morphological appearance of macrophages and/or dendritic cells. There is also a distinct up-regulation of met and gp-80 mRNA and protein in the biliary tree, which is stronger than that seen in the peripheral liver. Met protein expression is increased, and IL-6R(gp-80) protein is induced on the proliferating BEC, consistent with the participation of both the HGF/met and IL-6/gp-80 systems in the early phases of type I ductular reactions. These observations show that periductal hematolymphoid and stromal cells are the source of BEC growth factors, and receptors for these factors are up-regulated on BEC during active ductular proliferation. Complex interactions between the inflammatory, stromal, and BEC results in a dysmorphogenic repair response that eventually leads to cirrhosis.
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PMID:Interleukin-6, hepatocyte growth factor, and their receptors in biliary epithelial cells during a type I ductular reaction in mice: interactions between the periductal inflammatory and stromal cells and the biliary epithelium. 979 10


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