Interleukin-6 Synthesis Research Articles

Endogenous microparticles drive the proinflammatory host immune response in severely injured trauma patients.

Severe trauma affects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. A prospective, observational substudy of the ACIT II (Activation of Coagulation and Inflammation in Trauma II) study was performed at our academic level I trauma center. Adult multiple-trauma patients with an injury severity score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole-blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Ten trauma patients and 10 control subjects were included. Trauma patients were significantly injured with a median injury severity score of 19 (range, 17-45). Patients were neither in shock nor bleeding. On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with control subjects, as reflected by decreased production of interleukin 6 (IL-6), IL-10, and tumor necrosis factor α (P < 0.001). In trauma patients, MP-positive plasma was associated with a significantly higher synthesis of IL-6 and tumor necrosis factor α compared with plasma depleted from MPs (P = 0.047 and 0.002, respectively). Compared with control subjects, the number of circulating MPs was significantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in control subjects. On admission, trauma patients have a reduced immune response toward endotoxin challenge, which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an inflammatory host response after injury.

Immune challenge by intraperitoneal administration of lipopolysaccharide directs gene expression in distinct blood-brain barrier cells toward enhanced prostaglandin E(2) signaling.

The cells constituting the blood-brain barrier are critical for the transduction of peripheral immune signals to the brain, but hitherto no comprehensive analysis of the signaling events that occur in these cells in response to a peripheral inflammatory stimulus has been performed. Here, we examined the inflammatory transcriptome in blood-brain barrier cells, including endothelial cells, pericytes, and perivascular macrophages, which were isolated by fluorescent-activated cell sorting, from non-immune-challenged mice and from mice stimulated by bacterial wall lipopolysaccharide. We show that endothelial cells and perivascular macrophages display distinct transcription profiles for inflammatory signaling and respond in distinct and often opposing ways to the immune stimulus. Thus, endothelial cells show induced PGE2 synthesis and transport with attenuation of PGE2 catabolism, increased expression of cytokine receptors and down-stream signaling molecules, and downregulation of adhesion molecules. In contrast, perivascular macrophages show downregulation of the synthesis of prostanoids other than PGE2 and of prostaglandin catabolism, but upregulation of interleukin-6 synthesis. Pericytes were largely unresponsive to the immune stimulation, with the exception of downregulation of proteins involved in pericyte-endothelial cell communication. While the endothelial cells account for most of the immune-induced gene expression changes in the blood-brain barrier, the response of the endothelial cells occurs in a concerted manner with that of the perivascular cells to elevate intracerebral levels of PGE2, hence emphasizing the critical role of PGE2 in immune-induced signal transduction across the blood-brain barrier.

Dexamethasone Controlled Release on TGF-β1 Treated Vocal Fold Fibroblasts.

Corticosteroids may be beneficial in treating vocal fold scarring. Current drug delivery methods do not permit controlled corticosteroid release. Here we investigate the effects of poly-lactic-co-glycolic acid (PLGA) microparticles loaded with the corticosteroid dexamethasone in reducing collagen synthesis and inflammation in vocal fold fibroblasts treated with and without TGF-β1. Experimental, in vitro study. PLGA microparticles of differing molecular weight and terminating moieties were synthesized using a hydrogel template method. The release of dexamethasone was characterized from these microparticles over 4 days. Based on the release studies, ester-terminated low molecular weight PLGA microparticles were loaded with dexamethasone and applied to TGF-β1 treated vocal fold fibroblasts for 4 days. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays (ELISAs) were used to assess the effects of released dexamethasone on collagen synthesis and inflammatory mediators. COL3A1 and COL1A2 were significantly down-regulated after exposure to ester-terminated low molecular weight PLGA microparticles loaded with dexamethasone. The loaded microparticles also reduced interleukin-6 synthesis. These data show promise in using a PLGA microparticle-based delivery system to control dexamethasone release over 4 days. Our findings lay the groundwork for developing more effective treatments for vocal fold scarring.

Biocompatibility of defect-related luminescent nanostructured and microstructured hydroxyapatite.

Three defect-related luminescent hydroxyapatite (HAP) particles, S1, S2, and S3, with different morphologies (the samples S1 and S2 are nanorods with diameters of 25 nm and lengths of 30 and 100 nm, respectively; sample S3 is bur-like microspheres with diameters of 5-6 μm) were synthesized, and their biocompatibility was investigated by MTT, reactive oxygen species (ROS), interleukin-6 (IL-6), comet, and hemolysis assays. The results indicated that all samples were stable in cell culture medium and did not induce the synthesis of proinflammatory cytokine IL-6 or result in hemolysis. It was found that samples S1 and S3 inhibited osteoblast (OB) viability at concentrations of 5, 10, 20, 40, and 80 μg/mL for 24, 48, and 72 h. Sample S2 had no effect on the viability of OB at all tested concentrations for 24 and 48 h, but the viability of OB was increased at concentrations of 20, 40, and 80 μg/mL for 72 h. Samples S1 and S3 could increase the level of cellular ROS; sample S2 had no effect on the level of cellular ROS at a concentration of 20 μg/mL for 48 h. Although samples S1 and S3 induced significant DNA damage, sample S2 could not cause significant DNA damage at a concentration of 20 μg/mL for 72 h. The results suggest that longer nanorod HAP can show excellent biocompatibility and therefore may find potential applications in biomedical fields.

The role of TLR2 and 4-mediated inflammatory pathways in endothelial cells exposed to high glucose.

Postprandial hyperglycemia induces inflammation and endothelial dysfunction resulting in vascular complications in patients with diabetes. Toll-like receptors (TLRs) are central to the regulation of inflammatory responses through activation of nuclear factor-kappa B (NF-ĸB). This study examined the role of TLR2 and 4 in regulating inflammation and endothelial dysfunction when exposed to fluctuating glucose concentrations. HMEC-1 cells (a human microvascular endothelial cell line) were exposed to control (5 mM), 30 mM (high), fluctuating (5/30 mM) and 11.2 mM glucose (approximate glycaemic criteria for the diagnosis of diabetes mellitus) for 72 h. Cells were assessed for TLR2, 4, high mobility group box -1 (HMGB1), NF-ĸB, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Fluctuating glucose concentrations maximally upregulated TLR4 but not TLR2 expression with increased NF-ĸB activation, IL-8 and ICAM-1 expression. HMGB1 was increased in the supernatants of cells exposed to 30 mM and 11.2 mM glucose compared to control. The addition of recombinant HMGB1 induced NF-ĸB activation and synthesis of proinflammatory cytokines and chemokines, which were prevented by TLR2 or 4 signalling inhibition. An additive effect when both TLR2 and 4 signalling pathways were inhibited was observed. However, only inhibition of TLR4 signalling suppressed the synthesis of MCP-1, IL-8 and ICAM-1. In vivo, streptozotocin-induced diabetic mice exhibited an increase in glomerular ICAM-1 which was not evident in TLR2-/- or TLR4-/- diabetic mice. Collectively, our results suggest that targeting the signalling pathway of TLR2 and 4 may be of therapeutic benefit in attenuating vascular inflammation in diabetic microangiopathy.

Abstract 2254: Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer

Abstract Introduction and Objectives: Bicalutamide induces a rapid and sustained hypoxia in growing LNCaP prostatic tumors. This treatment-induced hypoxia (&amp;gt;14 days) increases synthesis of the CXC-chemokine Interleukin-8(CXCL8) co-incident with increasing oxygen tension and the restoration of tumor vascularisation1. Consequently, our current hypothesis is that hypoxia induced CXCL8 signalling underpins resistance to bicalutamide-treated tumors and facilitates the adoption of more aggressive cancer cells. Results: Culturing of LNCaP (PTEN null) and 22Rv1 (PTEN wt) cells under hypoxic conditions (mimicking a bicalutamide-treated tumor microenvironment) increased transcriptional activity of the androgen receptor (AR), Hypoxia-Inducible Factor (HIF-1) and NF-kB, inducing expression of downstream target genes associated with survival, metabolic adaptation and angiogenesis. The magnitude and duration of these responses was greater in PTEN-null LNCaP cells. Experiments were conducted to determine how bicalutamide-mediated inhibition of AR signalling would affect hypoxia-induced potentiation of AR, HIF and NF-kB transcription. Bicalutamide or an AR-targeted siRNA failed to reduce expression of pro-angiogenic, pro-survival and metabolism-associated genes. However, concurrent knockdown of both HIF-1 and NF-kB activity using gene-targeted siRNA's repressed expression of these hypoxia-induced disease-progressing genes. Amongst the genes up-regulated in hypoxic and PTEN-deficient LNCaP cells was expression of CXCL8 and its receptors, CXCR1 and CXCR2, which we have previously shown to be regulated by HIF-1 and NF-kB activity. Potentiation of CXCL8 signalling under hypoxia was found to sustain AR, HIF and NF-kB transcription. Combination of bicalutamide with the proteasome inhibitor bortezomib attenuated hypoxia-induced AR expression and transcriptional activity and decreased HIF-1 activity but failed to reduce NF-kB activity. In contrast, only direct inhibition of CXCL8 signalling was shown to repress activity of AR, HIF-1 and NF-kB in hypoxic LNCaP cells and increased LNCaP cells sensitivity to bicalutamide. Conclusion: Our data suggests that targeting CXCL8 signalling may therefore be a relevant strategy to enhance tumor response to anti-androgen therapies. Inhibiting signalling of this chemokine attenuates key transcriptional approaches induced in the hypoxic microenvironment of bicalutamide-treated tumors. Moreover, inhibition of CXCL8 signalling may prevent the re-vascularization previously observed in hypoxic LNCaP tumors. Citation Format: Melanie McKechnie, Pamela J. Maxwell, David J. J. Waugh. Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2254. doi:10.1158/1538-7445.AM2014-2254

IL-6 in inflammation, immunity, and disease.

Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Although its expression is strictly controlled by transcriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6 plays a pathological effect on chronic inflammation and autoimmunity. For this reason, tocilizumab, a humanized anti-IL-6 receptor antibody was developed. Various clinical trials have since shown the exceptional efficacy of tocilizumab, which resulted in its approval for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Moreover, tocilizumab is expected to be effective for other intractable immune-mediated diseases. In this context, the mechanism for the continual synthesis of IL-6 needs to be elucidated to facilitate the development of more specific therapeutic approaches and analysis of the pathogenesis of specific diseases.

Defining a Role for Acid Sphingomyelinase in the p38/Interleukin-6 Pathway

Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid β-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4β-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid β-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.

Dexmedetomidine suppresses interleukin-1β-induced interleukin-6 synthesis in rat glial cells.

Dexmedetomidine, an α2-adrenoceptor agonist, is used as a sedative medication for criticalyl ill patients and is known to exert neuroprotective effects by direct action on neurons and indirect action on neurons through astrocytes. Interleukin (IL)-6 plays a key role in neuroinflammation, which accompanies infection, traumatic brain injury, ischemia, neurodegenerative disorders, as both a pro-inflammatory cytokine and an anti-inflammatory cytokine. Dexmedetomidine suppresses immune function. However, the effects of dexmedetomidine on cytokine synthesis in the central nervous system (CNS) remain elusive. We previously reported that IL-1β stimulates IL-6 synthesis in the rat C6 glioma cell line through the phosphorylation of p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and IκB. In the present study, we investigated the effects of dexmedetomidine on the IL-1β-induced IL-6 synthesis in C6 cells. Dexmedetomidine inhibited the IL-1β-stimulated IL-6 release and mRNA expression in C6 cells. 8-Bromo-adenosine-3',5'-cyclic monophosphate, but not 8-bromo-guanosine 3',5'-cyclic monophosphate, significantly enhanced the IL-1β-induced IL-6 release and mRNA expression. However, dexmedetomidine failed to affect cAMP accumulation in the cells treated with IL-1β or forskolin, an activator of adenylyl cyclase. Yohimbine, an α2-adrenoceptor antagonist, did not reverse the suppressive effects of dexmedetomidine on the IL-1β-induced IL-6 release. Dexmedetomidine did not affect the IL-1β-induced phosphorylation of p38 MAP kinase, SAPK/JNK, IκB, nuclear factor (NF)-κB or c-Jun. Our findings strongly suggest that dexmedetomidine inhibits the IL-1β-induced IL-6 synthesis independently of the adenylyl cyclase-cAMP pathway through α2-adrenoceptors in C6 glioma cells. It is possible that dexmedetomidine may affect the immune system in the CNS by regulating the production of IL-6.

Anti-inflammatory effects of 5-HT3 receptor antagonists in interleukin-1beta stimulated primary human chondrocytes.

Chondrocytes are one of the main cell types involved in rheumatoid inflammation, releasing mediators which add to cartilage destruction, bone damage and consequently disability. Current evidence suggests that serotonin 5-HT(3) receptor antagonists (5-HT(3)RA) show anti-inflammatory and antioxidant properties in vitro and in vivo. Yet, the mechanisms of the anti-inflammatory effects of 5-HT(3)RA have not been elucidated in detail. Therefore, we examined in detail the effects of 5-HT(3)RA on inflammatory parameters in human primary chondrocytes in vitro by studying prostaglandin E2 (PGE2) and 8-isoprostane (8-iso-PGF2α) levels by EIA and interleukin-6 (IL-6) synthesis by ELISA. Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) protein levels were analyzed by Western blot. We found a significant reduction of IL-1β induced PGE2, 8-iso-PGF2β and IL-6 chondrocytes by 5-HT(3)RA especially by dolasetron. This study provides additional support to the potential use of 5-HT(3)RAs as therapeutic agents to reduce joint inflammation.

The association between sex-related interleukin-6 gene polymorphisms and the risk for cerebral palsy.

BackgroundThe relationship between genetic factors and the development of cerebral palsy (CP) has recently attracted much attention. Polymorphisms in the genes encoding proinflammatory cytokines have been shown to be associated with susceptibility to perinatal brain injury and development of CP. Interleukin-6 (IL-6) is a proinflammatory cytokine that plays a pivotal role in neonatal brain injury, but conflicting results have been reported regarding the association between IL-6 single nucleotide polymorphisms (SNPs) and CP. The purpose of this study was to analyze IL-6 gene polymorphisms and protein expression and to explore the role of IL-6 in the Chinese CP population.MethodsA total of 753 healthy controls and 713 CP patients were studied to detect the presence of five SNPs (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL-6 locus. Of these, 77 healthy controls and 87 CP patients were selected for measurement of plasma IL-6 by Luminex assay. The SHEsis program was used to analyze the genotyping data. For all comparisons; multiple testing on each individual SNP was corrected by the SNPSpD program.ResultsThere were no differences in allele or genotype frequencies between the overall CP patients and controls among the five genetic polymorphisms. However, subgroup analysis found significant sex-related differences in allele and genotype frequencies. Differences were found between spastic CP and controls in males for rs2069837; between CP with periventricular leukomalacia and controls in males for rs1800796 and rs2066992; and between term CP and controls in males for rs2069837. Plasma IL-6 levels were higher in CP patients than in the controls, and this difference was more robust in full-term male spastic CP patients. Furthermore, the genotype has an effect on IL-6 synthesis.ConclusionsThe influence of IL-6 gene polymorphisms on IL-6 synthesis and the susceptibility to CP is related to sex and gestational age.

Synthesis of IL-6 by hepatocytes is a normal response to common hepatic stimuli.

Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response.

The biology and medical implications of interleukin-6.

Cytokines are soluble mediators, which aid cell-to-cell communication in immune responses, and interleukin-6 (IL-6) is a prototypical cytokine featuring redundant and pleiotropic activity. The complete elucidation of the IL-6-mediated signal transduction system has provided a molecular basis for the characteristic features of cytokines. When tissue damage or inflammation due to infections or injuries occurs, IL-6 synthesis is promptly induced, contributing to the host defense through the stimulation of acute-phase immune reactions and hematopoiesis. The production of IL-6 is terminated when tissue homeostasis is restored. The synthesis of IL-6 is tightly regulated transcriptionally and posttranscriptionally. However, the dysregulated continual synthesis of IL-6 has been implicated in the development of various diseases, including autoimmune and chronic inflammatory diseases and cancers. Clinical trials using the humanized anti-IL-6 receptor monoclonal antibody tocilizumab have demonstrated the efficacy of IL-6 blockade for the treatment of refractory inflammatory diseases, such as rheumatoid arthritis, systemic juvenile idiopathic arthritis, and Castleman disease. Moreover, favorable results from the off-label use of tocilizumab strongly suggest that it may be applicable for the treatment of other refractory immune-mediated diseases, including cancer. Therefore, the mechanisms for the dysregulated synthesis of IL-6 need to be elucidated to understand the pathogenesis of the resultant diseases and to facilitate the development of effective therapeutic strategies.

An imbalance in the production of proinflammatory and anti-inflammatory cytokines is observed in whole blood cultures of preeclamptic women in comparison with healthy pregnant women

Objective: To compare the production of pro- and anti-inflammatory cytokines by whole blood samples stimulated with lipopolysaccharide from normotensive and preeclamptic women. Methods: The synthesis of tumor necrosis factor-α, interleukyn-1β, interleukin-6 and interleukin-10 were assayed by immunosorbent assays in 56 and 50 preeclamptic and normotensive women, respectively. Results: Preeclamptic women showed a higher synthesis (p < 0.05) of interleukin-1β and interleukin-6 versus normotensive group. Interleukin-10 synthesis from samples of normotensive women showed a higher production versus preeclamptic group (p < 0.05). Conclusion: Preeclamptic women showed an imbalance in the production of pro and anti-inflammatory cytokines in comparison with normotensive women.

Interleukin 6 and rheumatoid arthritis.

Interleukin-6 (IL-6) is a representative cytokine featuring pleiotropic activity and redundancy. A transient synthesis of IL-6 contributes to host defense against infectious agents and tissue injuries by inducing acute phase reactions and immunological and hematopoietic responses. However, uncontrolled persistent production of IL-6 may lead to the development of several immune-mediated diseases. Rheumatoid arthritis (RA) is a chronic disease with joint and systemic inflammation resulting from immunological abnormalities and it has been found that IL-6 plays a key role in the development of this disease. Clinical trials in various parts of the world of tocilizumab, a humanized anti-IL-6 receptor antibody, have proved its efficacy and tolerable safety either as monotherapy or in combination with disease-modifying antirheumatic drugs. As a result, it is currently used as a first-line biologic for the treatment of moderate-to-severe RA in more than 100 countries. Clarification of the mechanism(s) through which tocilizumab exerts its effect on RA and of the reason(s) why IL-6 is continuously produced in RA can be expected to lead to the best use of this agent for RA patients and aid in investigations into the pathogenesis of RA.

Caffeic acid phenethyl ester attenuates pro-inflammatory and fibrogenic phenotypes of LPS-stimulated hepatic stellate cells through the inhibition of NF-κB signaling

Hepatic stellate cells (HSCs) are the major cell type involved in liver fibrosis. Lipopolysaccharide (LPS)-mediated signaling through Τoll-like receptor 4 (TLR4) in HSCs has been identified as a key event in liver fibrosis, and as the molecular link between inflammation and liver fibrosis. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, on the pro-inflammatory and fibrogenic phenotypes of LPS-stimulated HSCs. HSCs from rats were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM). Following treatment with LPS, HSCs showed a strong pro-inflammatory phenotype with an upregulation of pro-inflammatory mediators, and a fibrogenic phenotype with enhanced collagen synthesis, mediated by transforming growth factor-β1 (TGF-β1). CAPE significantly and dose-dependently reduced LPS-induced nitrite production, as well as the transcription and protein synthesis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and enzyme-linked immunosorbent assays (ELISA). CAPE further reduced the TGF-β1-induced transcription and translation (protein synthesis) of the gene coding for collagen type I α1 (col1A1), in LPS-stimulated HSCs. Following LPS stimulation, the phosphorylation of the nuclear factor-κB (NF-κB) inhibitor IκBα and consequently, the nuclear translocation of NF-κB, were markedly increased in the HSCs, and these changes were reversed by pre-treatment with CAPE. In conclusion, CAPE attenuates the pro-inflammatory phenotype of LPS-stimulated HSCs, as well as the LPS-induced sensitization of HSCs to fibrogenic cytokines by inhibiting NF-κB signaling. Our results provide new insight into the treatment of hepatic fibrosis through regulation of the TLR4 signaling pathway.

Genetic Polymorphism of Toll-like Receptor 4 — Predictor of Susceptibility to Recurrent Course of Chronic Pyelonephritis in Children

Objective of investigation: to study the role of Toll-like receptor 4 Asp299Gly gene polymorphism in the implementation of susceptibility to chronic pyelonephritis, to analyze its association with the major manifestations of the disease and to assess the impact on the synthesis of interleukin-6 in the blood of sick children.Methods. We examined a group of children aged 1–15 years with chronic pyelonephritis in the active stage (n = 60). Control group consisted of 95 apparently healthy people. Results. There were revealed significantly higher prevalence of Toll-like receptor 4 Asp299Gly gene polymorphism among children with chronic pyelonephritis (n = 60), identified a statistically significant correlation of mutant genotypes Asp299Gly, Gly299Gly with manifestation of the disease before the age of 3, and frequent episodes of acute respiratory viral infections, torpid urinary syndrome, unstable remission and increased secretion of interleukin-6 (p &lt; 0.001). Thus, genetically determined Toll-like receptor 4 Asp299Gly gene polymorphism is a marker of high risk of early manifestation of chronic inflammation in the kidney interstitium and prolonged recurrent clinical course of the disease in children.

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.

Human Bladder Uroepithelial Cells Synergize with Monocytes to Promote IL-10 Synthesis and Other Cytokine Responses to Uropathogenic Escherichia coli

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

(-)-Epigallocatechin gallate amplifies interleukin-1-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells

We previously reported that interleukin-1 (IL-1), a potent bone resorptive cytokine, stimulates the synthesis of interleukin-6 (IL-6) via activation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through the inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway. On the other hand, it is recognized that catechin possesses a beneficial property for bone metabolism. Among them, (-)-epigallocatechin gallate (EGCG) is an abundant and major bioactive component. In the present study, we investigated the effect of EGCG on the IL-1 stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. EGCG significantly enhanced the IL-1-stimulated IL-6 synthesis in a dose-dependent manner in the range between 50 and 100μM. EGCG increased the mRNA levels of IL-6 stimulated by IL-1. IL-1-induced phosphorylation of IκB and NF-κB were suppressed by EGCG. On the other hand, EGCG failed to affect the IL-1-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and AMPK. These results strongly suggest that EGCG enhances IL-1-stimulated IL-6 synthesis through inhibiting the AMPK-IκB/NF-κB pathway at the point between AMPK and IκB/NF-κB in osteoblasts.