Induced Interleukin-6 Release Research Articles

NMO-IgG Induce Interleukin-6 Release via Activation of the NF-κB Signaling Pathway in Astrocytes

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disorder of the central nervous system (CNS) that frequently affects the optic nerve and spinal cord. Interleukin-6 (IL-6) is considered a key cytokine in the pathogenesis of NMOSD, and the level of IL-6 is significantly increased in the sera and cerebrospinal fluid (CSF) of patients with NMOSD. We have reported that the production of IL-6 depends on the JAK/STAT3 signaling pathway. However, it is not clear whether the NF-κB-dependent inflammatory response stimulated by neuromyelitis optica IgG (NMO-IgG) could also drive the production of IL-6 in astrocytes. In this study, we used an in vitro model of primary rat astrocytes stimulated by NMO-IgG to study the role of the NF-κB signaling pathway in mediating the release of IL-6. First, we confirmed that the level of IL-6 was significantly higher in the sera of NMOSD patients than that of healthy people by humoral fluid analysis and that NMO-IgG can significantly induce the release of IL-6 from astrocytes by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Then, Western blotting and immunocytochemistry showed that NMO-IgG can activate the intracellular NF-κB signaling pathway. Finally, it was found that S3633, an inhibitor of the NF-κB signaling pathway, can effectively inhibit the increase in IL-6 levels. These results prove that the production of IL-6 is partly mediated by the NF-κB signaling pathway, providing a potential effective strategy for targeted treatment of NMOSD.

Deletion of Superoxide Dismutase 1 Blunted Inflammatory Aortic Remodeling in Hypertensive Mice under Angiotensin II Infusion.

Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of two superoxide anions (O2·−) into hydrogen peroxide (H2O2) and oxygen (O2) and is generally known to protect against oxidative stress. Angiotensin II (AngII) causes vascular hypertrophic remodeling which is associated with H2O2 generation. The aim of this study is to investigate the role of cytosolic SOD (SOD1) in AngII-induced vascular hypertrophy. We employed C57/BL6 mice (WT) and SOD1 deficient mice (SOD1−/−) with the same background. They received a continuous infusion of saline or AngII (3.2 mg/kg/day) for seven days. The blood pressures were equally elevated at 1.5 times with AngII, however, vascular hypertrophy was blunted in SOD1−/− mice compared to WT mice (WT mice 91.9 ± 1.13 µm versus SOD1−/− mice 68.4 ± 1.41 µm p < 0.001). The elevation of aortic interleukin 6 (IL-6) and phosphorylation of pro-inflammatory STAT3 due to AngII were also blunted in SOD1−/− mice’s aortas. In cultured rat vascular smooth muscle cells (VSMCs), reducing expression of SOD1 with siRNA decreased AngII induced IL-6 release as well as phosphorylation of STAT3. Pre-incubation with polyethylene glycol (PEG)-catalase also attenuated phosphorylation of STAT3 due to AngII. These results indicate that SOD1 in VSMCs plays a role in vascular hypertrophy due to increased inflammation caused by AngII, probably via the production of cytosolic H2O2.

Bitter Sensing TAS2R50 Mediates the trans-Resveratrol-Induced Anti-inflammatory Effect on Interleukin 6 Release in HGF-1 Cells in Culture.

Recent data have shown anti-inflammatory effects for trans-resveratrol (RSV) and rosmarinic acid (RA) in various immune-competent cell models through reduction of lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release. Because both compounds have been reported to taste bitter, we hypothesized an involvement of human bitter taste sensing receptors (TAS2Rs) on IL-6 release in LPS-treated human gingival fibroblasts (HGF-1). First, the bitter taste intensity of RSV and RA was compared in a sensory trial with 10 untrained panelists, of whom 90% rated a 50 ppm of RSV in water solution more bitter than 50 ppm of RA. A mean 19 ± 6% reduction of the RSV-induced bitter taste intensity was achieved by co-administration of 50 ppm of the bitter-masking, TAS2R43 antagonist homoeriodictyol (HED). Mechanistic experiments in a stably CRISPR-Cas9-edited TAS2R43ko gastric cell model revealed involvement of TAS2R43 in the HED-evoked effect on RSV-induced proton secretion, whereas the cellular response to RSV did not depend upon TAS2R43. Next, the IL-6 modulatory effect of 100 μM RSV was studied in LPS-treated immune-competent HGF-1 cells. After 6 h of treatment, RSV reduced the LPS-induced IL-6 gene expression and protein release by -46.2 ± 12.7 and -73.9 ± 2.99%, respectively. This RSV-evoked effect was abolished by co-administration of HED. Because real-time quantitative polymerase chain reaction analyses revealed a regulation of TAS2R50 in RSV with or without HED-treated HGF-1 cells, an siRNA knockdown approach of TAS2R50 was applied to verify TAS2R50 involvement in the RSV-induced reduction of the LPS-evoked IL-6 release in HGT-1 cells.

Lipopolysaccharide activates microglia via neuraminidase 1 desialylation of Toll-like Receptor 4.

Most cell surface receptors are sialylated, i.e. have sialic acid as the terminal residue of their sugar chains, but can be desialylated by sialidases, such as neuraminidase 1 (Neu1). Desialylation by Neu1 can activate immune cells, such as neutrophils, macrophages and monocytes. We investigated the role of Neu1 in activation of microglia using BV-2 cells (a murine microglial cell line) by cytokine ELISAs, enzyme activity assays, antibody/lectin binding and proximity labelling. We found that lipopolysaccharide (LPS) activation caused an increase in Neu1 protein on the cell surface, and an increase in surface sialidase activity that was prevented by Neu1 knockdown. Moreover, LPS induced interleukin 6 (IL-6) and MCP-1 release, which was reduced by Neu1 knockdown and increased by Neu1 over-expression. Neu1 knockdown also prevented the maintenance of IL-6 release by microglia after LPS was removed. Sialidase treatment of the cells was sufficient to induce IL-6 release, prevented by inhibiting toll-like receptor 4 (TLR4). Neu1 was found in close proximity to TLR4 on the surface of cells, and LPS induced desialylation of TLR4 on the cell surface, prevented by Neu1 knockdown. Sialic acid-binding immunoglobulin-like lectin E was found to bind to TLR4 via sialic acid residues and inhibit IL-6 release by BV-2 cells. We conclude that LPS causes Neu1 to translocate to the cell surface, where it desialylates TLR4, releasing inhibitory sialic acid-binding immunoglobulin-like lectin E, enhancing and maintaining inflammatory activation of the microglia. Thus, sialylation is a potent regulator of microglial activation, and Neu1 may be a target to reduce activation of microglia.

Cerebrospinal fluid of chronic osteoarthritic patients induced interleukin-6 release in human glial cell-line T98G

BackgroundChronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored.MethodsFifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1β and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression.ResultsSignificant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients’ CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3–15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase.ConclusionHigher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of “unknown molecule” in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

In vitro lung epithelial cell transport and anti-interleukin-8 releasing activity of liposomal ciprofloxacin

As a promising long-acting inhaled formulation, liposomal ciprofloxacin (Lipo-CPFX) was characterized in the in vitro human lung epithelial Calu-3 cell monolayer system, compared to ciprofloxacin in solution (CPFX). Its modulated absorptive transport and uptake, and sustained inhibitory activity against induced pro-inflammatory interleukin-8 (IL-8) release were examined. The absorptive transport and uptake kinetics for Lipo-CPFX and CPFX were determined at 0.1–50 mg/ml in the Transwell system. The Lipo-CPFX transport was then challenged for mechanistic exploration via cell energy depletion, a reduced temperature, endocytosis and/or lipid fusion inhibition, and addition of excess non-loaded liposomes. The inhibitory activities of Lipo-CPFX and CPFX against lipopolysaccharide (LPS)-induced IL-8 release were assessed in a co-incubation or pre-incubation mode. In the tight Calu-3 cell monolayers, Lipo-CPFX yielded 15-times slower ciprofloxacin flux of absorptive transport and 5-times lower cellular drug uptake than CPFX. Its transport appeared to be transcellular; kinetically linear, proportional to encapsulated ciprofloxacin concentration; and consistent with the cell energy-independent lipid bilayer fusion mechanism. Lipo-CPFX was equipotent to CPFX in the anti-IL-8 releasing activity upon 24 h co-incubation with LPS. Additionally, Lipo-CPFX, but not CPFX, retained the anti-IL-8 releasing activity even 24 h after pre-incubation. In conclusion, Lipo-CPFX enabled slower absorptive lung epithelial cell transport and uptake of ciprofloxacin, apparently via the lipid bilayer fusion mechanism, and the sustained inhibitory activity against LPS-induced IL-8 release, compared to CPFX.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Insights on wood combustion generated proinflammatory ultrafine particles (UFP)

This study aimed to collect, characterize ultrafine particles (UFP) generated from the combustion of wood pellets and logs (softwood and hardwood) and to evaluate their pro-inflammatory effects in THP-1 and A549 cells. Both cell lines responded to UFP producing interleukin-8 (IL-8), with wood log UFP being more active compared to pellet UFP. With the exception of higher effect observed with beech wood log UFP in THP-1, the ability of soft or hard woods to induce IL-8 release was similar. In addition, on weight mass, IL-8 release was similar or lower compared to diesel exhaust particles (DEP), arguing against higher biological activity of smaller size particles. UFP-induced IL-8 could be reduced by SB203580, indicating a role of p38MAPK activation in IL-8 production. The higher activity of beech wood log UFP in THP-1 was not due to higher uptake or endotoxin contamination. Qualitatively different protein adsorption profiles were observed, with less proteins bound to beech UFP compared to conifer UFP or DEP, which may provide higher intracellular availability of bioactive components, i.e. levoglucosan and galactosan, toward which THP-1 were more responsive compared to A549 cells. These results contribute to our understanding of particles emitted by domestic appliances and their biological effects.

Importance of the C-terminal histidine residues of Helicobacter pylori GroES for Toll-like receptor 4 binding and interleukin-8 cytokine production.

Helicobacter pylori infection is associated with the development of gastric and duodenal ulcers as well as gastric cancer. GroES of H. pylori (HpGroES) was previously identified as a gastric cancer-associated virulence factor. Our group showed that HpGroES induces interleukin-8 (IL-8) cytokine release via a Toll-like receptor 4 (TLR4)-dependent mechanism and domain B of the protein is crucial for interactions with TLR4. In the present study, we investigated the importance of the histidine residues in domain B. To this end, a series of point mutants were expressed in Escherichia coli, and the corresponding proteins purified. Interestingly, H96, H104 and H115 were not essential, whereas H100, H102, H108, H113 and H118 were crucial for IL-8 production and TLR4 interactions in KATO-III cells. These residues were involved in nickel binding. Four of five residues, H102, H108, H113 and H118 induced certain conformation changes in extended domain B structure, which is essential for interactions with TLR4 and consequent IL-8 production. We conclude that interactions of nickel ions with histidine residues in domain B help to maintain the conformation of the C-terminal region to conserve the integrity of the HpGroES structure and modulate IL-8 release.

The 2-Pore Domain Potassium Channel TREK-1 Regulates Cytokine Secretion from Human Alveolar Epithelial Cells Independently of Potassium Currents

We previously identified the 2-pore domain potassium (K2P) channel TREK-1 as an important regulator of tumor necrosis factor-α (TNF-α)-induced interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) secretion from alveolar epithelial cells (AECs). Furthermore, in an in vivo model of Acute Lung Injury (ALI) we found that TREK-1 deficiency led to increased lung damage but decreased broncho-alveolar lavage cytokine levels. Unfortunately, the mechanisms underlying TREK-1 regulation of inflammatory cytokine secretion remain poorly understood.This study was designed to determine whether the changes in cytokine secretion from TREK-1 deficient cells are due to the absence of TREK-1 protein or the lack of TREK-1-mediated potassium currents. For this purpose, we stimulated control and TREK-1 deficient human AECs (A549) with TNF-α and measured IL-6 and MCP-1 release by ELISA in the presence and absence of three TREK-1 blockers, spadin (0.5mM), amlodipine (2μM) and charybdotoxin (100nM). While spadin is recognized as a specific TREK-1 inhibitor, amlodipine and charybdotoxin at these concentrations also inhibit TREK-1.We found that TREK-1 deficient AECs released less IL-6 and more MCP-1 upon TNF-α stimulation but this cytokine release was unaffected by any of the three TREK-1 blockers. To determine whether cytokine secretion occurred independently of potassium currents in general, we raised the extracellular potassium concentration (100mM) to minimize the electrochemical driving force for potassium. Interestingly, we found that TNF-α-induced IL-6 and MCP-1 secretion were significantly impaired in the absence of a potassium gradient.In summary, TREK-1 protein, but not TREK-1 mediated potassium current, is required for IL-6 and MCP-1 secretion from AECs, although other potassium conductances appear important. These data suggest that TREK-1 may alter cytokine secretion from AECs by functioning as a regulatory molecule rather than a potassium channel.

Partial purification and functional characterization of Ts19 Frag-I, a novel toxin from Tityus serrulatus scorpion venom.

BackgroundThe yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.MethodsThe fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman’s degradation and tested on macrophages.ResultsThe previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).ConclusionsWe partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

Soluble and immobilized graphene oxide activates complement system differently dependent on surface oxidation state

Graphene oxide (GO) is believed to become applicable in biomedical products and medicine, thereby necessitating appropriate safety evaluation dependent on their applications and the route of administration. We have examined the effect of GO form (in solution versus immobilized) and oxidation state on two related elements of innate immunity: the complement system and interleukin-6 (IL-6) release in human blood. In solution, there was a decrease in GO-mediated complement activation with decreasing surface oxygen content (and altered oxygen functionality), whereas with immobilized GO complement response were reversed and increased with decreasing oxygen content. GO solutions, at concentrations below complement activating threshold, did not induce IL-6 release from human blood leukocytes, and further dampened lipopolysaccharide-induced IL-6 release in the whole blood. The latter effect became more profound with GO's having higher oxygen content. This protective role of GO solutions, however, disappeared at higher concentrations above complement-activating threshold. We discuss these results in relation to GO surface structure and properties, and implications for local administration and development of GO-based implantable devices.

FMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

Sphingosine 1-Phosphate (S1P) Induced Interleukin-8 (IL-8) Release Is Mediated by S1P Receptor 2 and Nuclear Factor κB in BEAS-2B Cells

The airway epithelium may release pro-inflammatory cytokines and chemokines in the asthmatic airway. Sphingosine 1-phosphate (S1P) is a bioactive lipid, increased in the airways of asthmatics, that may trigger the release of the potent neutrophil chemoattractant Interleukin-8 (IL-8) by epithelial cells. S1P is a ligand for 5 G protein-coupled receptors, S1PR1-5. We wished to explore the mechanisms of S1P induced IL-8 secretion with regard to the receptor(s) and downstream signaling events involved. Our results indicate that S1P induced IL-8 release is mediated by S1PR2 and the transcription factor NF-κB. Since the Epidermal Growth Factor Receptor (EGFR) and reactive oxygen species (ROS) have been implicated in IL-8 release in response to activation of other G protein-coupled receptors, we examined their importance in S1P induced IL-8 release and established that they are not involved. This study reveals S1PR2 and NF-κB as potential therapeutic targets in neutrophilic airway diseases such as severe asthma.

Induction of Regulator of G-Protein Signaling 2 Expression by Long-Acting β2-Adrenoceptor Agonists and Glucocorticoids in Human Airway Epithelial Cells

In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting β2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.

Cerebrospinal fluid of postherpetic neuralgia patients induced interleukin-6 release in human glial cell-line T98G

Chronic intractable pain caused by postherpetic neuralgia (PHN) can be alleviated by intrathecal (i.t.) steroid therapy. We investigated the possibility that interleukin-6 (IL-6) release in an in vitro system could be a potential marker for evaluating the effectiveness of i.t. steroid therapy in PHN patients. We studied 32 patients who received a course of i.t. injection of water-soluble dexamethasone. Their therapeutic index was calculated as such: ((Pain score before treatment – Pain score after treatment)÷Pain score before treatment)×100%, and they were divided into two groups, therapy effective (index>50%) and ineffective (index<50%). Cerebrospinal fluid (CSF) from the patients was used to stimulate cultures of T98G glioblastoma cells, and the subsequent IL-6 release was measured by enzyme-linked immunosorbent assay (ELISA). Our results showed that the CSF triggered IL-6 release from T98G cells in a volume-dependent manner. IL-6 release was significantly lower when using CSF from the therapy effective patient group (p<0.001) compared to the therapy ineffective group. In particular, therapy effective patients had less IL-6 release even before treatment as compared to therapy ineffective patients. In the therapy effective group, in vitro steroid treatment suppressed the CSF’s IL-6 releasing effect almost completely, whereas in the therapy ineffective group, the IL-6 release was significantly reduced but remained detectable. These in vitro tests may provide an objective evaluation on the efficacy of i.t. steroid therapy administered to PHN patients.

Regulation of interleukin-6 secretion by the two-pore-domain potassium channel Trek-1 in alveolar epithelial cells

We recently proposed a role for the two-pore-domain K(+) (K2P) channel Trek-1 in the regulation of cytokine release from mouse alveolar epithelial cells (AECs) by demonstrating decreased interleukin-6 (IL-6) secretion from Trek-1-deficient cells, but the underlying mechanisms remained unknown. This study was designed to investigate the mechanisms by which Trek-1 decreases IL-6 secretion. We hypothesized that Trek-1 regulates tumor necrosis factor-α (TNF-α)-induced IL-6 release via NF-κB-, p38-, and PKC-dependent pathways. We found that Trek-1 deficiency decreased IL-6 secretion from mouse and human AECs at both transcriptional and translational levels. While NF-κB/p65 phosphorylation was unchanged, p38 phosphorylation was decreased in Trek-1-deficient cells, and pharmacological inhibition of p38 decreased IL-6 secretion in control but not Trek-1-deficient cells. Similarly, pharmacological inhibition of PKC also decreased IL-6 release, and we found decreased phosphorylation of the isoforms PKC/PKDμ (Ser(744/748)), PKCθ, PKCδ, PKCα/βII, and PKCζ/λ, but not PKC/PKDμ (Ser(916)) in Trek-1-deficient AECs. Phosphorylation of PKCθ, a Ca(2+)-independent isoform, was intact in control cells but impaired in Trek-1-deficient cells. Furthermore, TNF-α did not elevate the intracellular Ca(2+) concentration in control or Trek-1-deficient cells, and removal of extracellular Ca(2+) did not impair IL-6 release. In summary, we report the expression of Trek-1 in human AECs and propose that Trek-1 deficiency may alter both IL-6 translation and transcription in AECs without affecting Ca(2+) signaling. The results of this study identify Trek-1 as a new potential target for the development of novel treatment strategies against acute lung injury.

HuR post-transcriptionally regulates TNF-α-induced IL-6 expression in human pulmonary microvascular endothelial cells mainly via tristetraprolin

HuR and tristetraprolin (TTP) are both RNA-binding proteins, which are characterized as binding to the AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTRs) of target mRNAs. Studies have shown that some ARE-containing mRNAs are stabilized by HuR, whereas are destabilized by TTP. Our previous study showed that HuR upregulated tumor necrosis factor-α (TNF-α)-induced interleukin-6 (IL-6) expression by stabilizing its mRNA in human pulmonary microvascular endothelial cells (HPMECs). Considering IL-6 mRNA has AREs, we decided to examine whether TTP was also involved in the regulation of TNF-α-induced IL-6 expression in HPMECs and whether HuR and TTP influenced each other at protein and mRNA level. Here, we report that TTP silencing increased IL-6 levels. HuR silencing increased TTP expression. TTP had no effect on HuR expression and subcellular localization. Compared to TTP silencing alone, double knockdown of HuR and TTP did not significantly reduce IL-6 release. The RNA-binding protein immunoprecipitation (RIP) results further showed that TTP but not HuR bound to intracellular IL-6 mRNA in HPMECs. We demonstrate for the first time that HuR post-transcriptionally regulates IL-6 expression mainly via TTP.

Synergistic induction of interleukin-6 expression by endothelin-1 and cyclic AMP in adipocytes

We have demonstrated previously that endothelin-1 (ET-1) may stimulate interleukin-6 (IL-6) release from 3T3-L1 adipocytes. In this study, we further examined the combined effect of ET-1 and cyclic adenosine monophosphate (cAMP) on IL-6 release. IL-6 release was measured by enzyme-linked immuosorbent assay. Reverse transcriptase-PCR and real-time PCR analyses were used to determine cellular mRNA levels. A luciferase reporter driven by promoter (-1310/+198) of mouse IL-6 gene was transfected into 3T3-L1 adipocytes to monitor IL-6 transcription. ET-1 and cAMP induced IL-6 release in a synergistic manner that can be attributed to their synergistic induction of IL-6 gene expression, as evidenced by IL-6 mRNA analysis and the IL-6 promoter reporter assay. Both ET(A) and ET(B) receptors seem to be involved. In addition, enhanced IL-6 promoter activity can be similarly induced by ET-1 and catecholamines (epinephrine and norepinephrine). The cooperative interaction between ET-1 and cAMP on IL-6 expression seems distinctive, as no other proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1β, are similarly affected. In fact, cAMP inhibited ET-1-stimulated TNF-α and IL-1β expressions in adipocytes. Furthermore, injection of mice with epinephrine and ET-1 induced a tremendously synergistic increase in serum IL-6 levels. Nevertheless, whereas cAMP induced IL-6 expression in RAW264.7 mouse macrophages, ET-1 had no effect on either the basal or the cAMP-induced IL-6 expression. ET-1 and epinephrine may boost plasma IL-6 levels in mice in a synergistic manner, probably through their synergistic induction of IL-6 expression in adipocytes. This study should provide a new perspective for treating IL-6-related diseases, especially those accompanied with elevated ET-1 and catecholamine levels.

Methylmercury-induced IL-6 release requires phospholipase C activities

Methylmercury (MeHg) is a neurotoxin capable of causing severe damage to the CNS, especially in the developing fetus. Glia in the CNS release a number of cytokines that are important for proper CNS development and function. We reported earlier that MeHg could induce interleukin-6 (IL-6) release in primary mouse glia. This finding is significant considering previous reports indicating that sustained IL-6 exposure could be detrimental to cerebellar granule neurons, one of the major cellular targets of MeHg cytotoxicity. By using pharmacological antagonists against phophatidycholine- and phosphoinositol-specific phospholipase C, the current study indicated that phospholipase C activity was necessary for MeHg-induced IL-6 release. Results from pharmacological antagonists further suggested that the calcium signaling initiated by phospholipase C appeared essential for this event. In contrast, protein kinase C activity did not appear to be important. Even though mitogen-activated protein kinases were important for IL-6 release in some experimental systems, these enzymes did not appear to be required for MeHg-induced IL-6 release in glia. Based on these data and those reported by us and others, there is a possibility that MeHg-induced phospholipase C activation initiates a calcium signaling that causes phospholipase A2 activation. This, in turn, leads to arachidonic acid and lysophosphatidyl choline generation, both of which are potent inducers for IL-6 release.