Induced Interleukin-6 mRNA Expression Research Articles

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 μg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

Canonical NF-κB p65, but Not p105, Contributes to IL-1β-Induced IL-8 Expression in Cardiac Fibroblasts.

Cardiac fibroblasts participate in the inflammatory process of heart diseases as sentinel cells of the cardiac tissue. In this study, we investigated the effect of the proinflammatory cytokine, interleukin 1β (IL-1β), on the expression of interleukin 8 (IL-8), which contributes to the induction of innate immunity via the activation and recruitment of innate immune cells, such as neutrophils, to the site of inflammation in canine cardiac fibroblasts. IL-1β mediates IL-8 mRNA expression and protein release in a dose- and time-dependent manner. The IL-β-mediated IL-8 protein release and mRNA expression were inhibited by 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide, an inhibitor of the transcription factor, nuclear factor (NF)-κB. In cells treated with IL-1β, NF-κB p65 and p105 were transiently phosphorylated, indicating the activation of NF-κB. However, IL-1β failed to induce IL-8 mRNA expression in the cells transfected with p65 small interfering RNA (siRNA), but not in those transfected with p105 siRNA. These observations suggest that IL-1β induces IL-8 expression via the activation of NF-κB p65 in canine cardiac fibroblasts.

Interleukin-1β promotes interleulin-6 expression via ERK1/2 signaling pathway in canine dermal fibroblasts.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of the immune response and inflammation. In this study, we investigated effect of the proinflammatory cytokine interleukin-1β (IL-1β) on IL-6 expression in canine dermal fibroblasts. IL-1β induced IL-6 mRNA expression and protein release in a time- and dose-dependent manner. When cells were treated with inhibitors of mitogen-activated protein kinases (MAPKs), the extracellular signal-regulated kinase (ERK) inhibitor FR180240 inhibited IL-1β-induced IL-6 mRNA expression, but not SP600125 or SKF86002, which are c-Jun N-terminal kinase (JNK) and p38 MAPK inhibitors, respectively. In cells treated with U0126, an inhibitor of MAPK/ERK kinase (MEK), which activates ERK, IL-1β-induced IL-6 mRNA expression was also inhibited. IL-1β stimulated ERK1/2 phosphorylation. In cells transfected with ERK1 and ERK2 isoform siRNAs, IL-1β-induced IL-6 mRNA expression was reduced. These observations suggest that IL-1β induces IL-6 expression via ERK1/2 signaling pathway in canine dermal fibroblasts.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection.

Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10−7 M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and chorion cells.

The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle.

In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic pathways occurring during heat shock.

P120-Catenin modulating nuclear factor-κB activation is partially RhoA/ROCKdependent in scratch injury.

p120-catenin (p120) is known as a cadherin-associated protein that participates in tumor metastasis and invasion, as well as an anti-inflammatory mediator. Recently, its anti-inflammatory role is drawing increasing attention, but the regulatory mechanisms are still unknown. Here, we report that p120 modulated inflammatory responses partially depends on RhoA/ROCK pathway in scratch-induced injury in human bronchial epithelial cells (BECs). For the first time, we found that p120 was significantly reduced in BECs after scratching, which could induce interleukin-8 (IL-8) production through nuclear factor-κB (NF-κB) activation accompanied with IκBα phosphorylation. Over-expression of p120 3A could inhibit NF-κB activation and IL-8 mRNA expression and protein synthesis after scratching, while p120 knockdown by small interfering RNA could promote NF-κB activation and IL-8 mRNA expression and protein synthesis after scratching. Furthermore, we found that RhoA was the binding partner of p120 in BECs. Although total RhoA and p120-binded RhoA remained unchanged, the RhoA activity was increased after scratching. Chemical blockade of RhoA/ROCK signaling (Y27632) inhibited scratch-induced nuclear translocation of NF-κB p65. Over-expression of p120 3A attenuated scratch-induced RhoA activation, whereas silence of p120 significantly elevated scratch-induced RhoA activation in BCEs. Conclusively, these results indicate an anti-inflammatory effect of p120 in bronchial epithelial cells through its modulation of NF-κB signaling depending on RhoA/ROCK pathway.

Angiotensin III induces signal transducer and activator of transcription 3 and interleukin-6 mRNA levels in cultured rat astrocytes.

Recently we established that pro-inflammatory actions of angiotensin (Ang) II in astrocytes involved Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and interleukin-6 (IL-6). In our current study, we determined in brainstem and cerebellum whether Ang III also activates STAT3 leading to IL-6 mRNA expression and astrocyte proliferation. Ang III induced STAT3 phosphorylation in a concentration- and time-dependent manner. Significant STAT3 phosphorylation was rapid and was maximal within 10 min, and with 100 nM Ang III. The Ang AT1 receptor was shown to mediate this action of Ang III. Ang III also significantly induced IL-6 mRNA expression within an hour, and maximal Ang III-mediated IL-6 mRNA expression occurred in the presence of 100 nM Ang III. Ang III-mediated IL-6 mRNA expression occurred by the interaction of the peptide with the Ang AT1 receptor and was mediated by STAT3. In addition, STAT3 was shown to mediate Ang III astrocyte proliferation. These findings suggest that Ang III, similar to Ang II, has pro-inflammatory effects since it induces STAT3 leading to an induction of IL-6 mRNA expression, outcomes that lend relevance to the physiological importance of central Ang III.

Anti‐inflammatory Properties of Gastric‐derived Lactobacillus plantarum XB7 in the Context of Helicobacter pylori Infection

Helicobacter pylori colonization of the gastric epithelium induces interleukin-8 (IL-8) production and inflammation leading to host cell damage. We searched for gastric-derived Lactobacillus with the ability to suppress H. pylori-induced inflammation. Conditioned media from gastric-derived Lactobacillus spp. were tested for the ability to suppress H. pylori-induced IL-8 production in AGS gastric epithelial cells. IL-8 protein and mRNA levels were measured by ELISA and qPCR, respectively. The changes on host cell signaling pathway were analyzed by Western blotting and the anti-inflammatory effect was tested in a Sprague-Dawley rat model. Conditioned media from L. salivarius B101, L. rhamnosus B103, and L. plantarum XB7 suppressed IL-8 production and IL-8 mRNA expression in H. pylori-induced AGS cells without inhibiting H. pylori growth. Conditioned media from LS-B101, LR-B103, and LP-XB7 suppressed the activation of NF-κB in AGS cells, while strain LP-XB7 also suppressed c-Jun activation. The anti-inflammatory effect of LP-XB7 was further assessed in vivo using a H. pylori-infected Sprague-Dawley rat model. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology. Additionally, the administration of LP-XB7 correlated with the suppression of TNF-α and CINC-1 in sera, and suppression of CINC-1 in the gastric mucosa of H. pylori-infected rats. These results suggest that L. plantarum XB7 produces secreted factors capable of modulating inflammation during H. pylori infection, and this probiotic Lactobacillus strain shows promise as an adjunctive therapy for treating H. pylori-associated disease.

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.

Palmitate induces interleukin‐8 expression in human aortic vascular smooth muscle cells via Toll‐like receptor 4/nuclear factor‐κB pathway (TLR4/NF‐κB通路介导了软脂酸诱导人主动脉血管平滑肌细胞白细胞介素‐8的基因表达)

Recent evidence demonstrates that saturated free fatty acids (FFAs) induce the inflammatory response via the Toll-like receptor 4 (TLR4) pathway. Interleukin-8 (IL-8) is a proinflammatory cytokine that induces vascular smooth muscle cell proliferation and migration in vitro. However, the regulation of IL-8 expression by palmitate in human vascular smooth muscle cells (HVSMCs) has not been clarified. The aim of this study was to investigate the regulation of IL-8 expression by free fatty acids and determine the underlying mechanisms in HVSMCs. Human vascular smooth muscle cells were cultured and treated with palmitate, various signaling inhibitors or TLR4 shRNA adenovirus, and the mRNA and protein expression levels of IL-8, nuclear factor κB (NF-κB) luciferase activity and NF-κB p65 binding activity were studied. Palmitate induced IL-8 mRNA expression and secretion in a dose-dependent manner. Palmitate significantly stimulated both nuclear factor κB (NF-κB) luciferase activity and NF-κB p65 binding activity, which were markedly diminished by pretreatment with the NF-κB inhibitor, parthenolide. Parthenolide pretreatment also abolished IL-8 mRNA and protein induction by palmitate. By contrast, disrupting the ceramide and phosphoinositide-3 kinase (PI3K) pathways with myriocin and wortmannin did not affect palmitate-induced IL-8 expression. Inhibition of protein kinase C (PKC) activation with calphostin C and chelerythrine partially suppressed palmitate-stimulated IL-8 expression, but it had no effect on palmitate-induced NF-κB activation. Finally, knockdown of TLR4 markedly abolished palmitate-induced NF-κB activation and IL-8 expression. Palmitate induces IL-8 gene expression in HVSMCs through the TLR4/NF-κB pathway.

Angiotensin II induces interleukin-6 synthesis in osteoblasts through ERK1/2 pathway via AT1 receptor

Background Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteoblast secretion of IL-6 plays an important role in the regulation of bone metabolism. Angiotensin II (Ang II) has been shown to regulate the expression of potent inflammatory factors, including MCP-1 and IL-6, by stimulating endothelia cells, vascular smooth muscle cells (VSMC) and monocytes. However, of the mechanism by which Ang II regulates IL-6 expression in osteoblasts is unknown. Aims The present study was designed to investigate the effect of Ang II on IL-6 expression in osteoblasts isolated from mice. The receptor(s) required and the potential role of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in Ang II-induced IL-6 synthesis was also examined in these cells. Methods The osteoblasts were isolated from the calvaria of mice and cultured in α-MEM medium. IL-6 mRNA expression and protein synthesis was determined by qPCR and ELISA analyses. ERK1/2 kinase activation was determined by western blot. Results The results indicate that Ang II induced IL-6 mRNA expression and protein synthesis in cultured osteoblasts. However, these effects were abolished by pre-treatment with Ang II type 1 (AT1) receptor antagonist, losartan, and the ERK1/2 inhibitor, U0126, inhibited Ang II-mediated IL-6 expression and the phosphorylation of ERK1/2. Conclusion Ang II induces the synthesis of IL-6 in osteoblasts through activation of the ERK1/2 pathway via the AT1 receptor.

ERK1/2 is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells

We previously reported that cyclic compressive force (CCF) induced interleukin-6 mRNA expression in osteocyte-like MLO-Y4 cells. But little is known about how the stimuli are converted into the biochemical signals in MLO-Y4 cells. The aim of this research was to study the effect of CCF on the IL-6 secretion and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in this process. The cells were exposed to CCF with different magnitudes (1000, 2000 and 4000 μstrain), frequencies (0.5, 1.0 and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) by a four-point bending system. The IL-6 secretion and ERK1/2 phosphorylation of the cells were determined by ELISA and Western blotting, respectively. The results showed that IL-6 protein secretion was significantly up-regulated in response to CCF in a magnitude-, frequency- and duration-dependent fashion. The phosphorylation of ERK1/2 also increased in all cases but not depended on the magnitude, frequency or duration of CCF. Furthermore, the inhibition of the ERK1/2 pathway by its specific inhibitor PD098059 decreased but not completely abrogated the IL-6 secretion from stressed MLO-Y4 cells. These findings demonstrate that CCF-induced IL-6 secretion occurs via a mechanism that involves ERK1/2 signaling pathway and suggest that modulation of this event contributes to the pathogenesis of osteoporosis and stress-induced pathological bone resorption as well.

Interleukin-8 production and interleukin-8 mRNA expression induced by lipopolysaccharides fromPrevotella intermediaandPrevotella nigrescensin monocyte-derived macrophages

Purpose: Interleukin-8 (IL-8) is an important mediator of immune and inflammatory reactions and is produced by a variety of different cell types. This study was undertaken to investigate the effects of lipopolysaccharides (LPSs) from Prevotella intermedia and Prevotella nigrescens, the major causes of inflammatory periodontal disease, on the production of IL-8 and the expression of IL-8 mRNA in differentiated THP-1 cells, a human monocytic cell line. Methods: LPSs from P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 were prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. Results: We found that LPS preparations from P. intermedia and P. nigrescens can induce IL-8 mRNA expression and stimulate the release of IL-8 in differentiated THP-1 cells without additional stimuli. Conclusions: There are no previous reports of the ability of P. intermedia and P. nigrescens LPS to stimulate the release of IL-8, and the present study clearly shows, for the first time, that LPSs from P. intermedia and P. nigrescens fully induced IL-8 mRNA expression and IL-8 production in differentiated human monocytic cell line THP-1. The ability of P. intermedia and P. nigrescens LPS to promote the production of IL-8 may be important in the pathogenesis of inflammatory periodontal disease. (J Korean Acad Periodontol 2009;39:177-184)

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Cooperation of SRC-1 and p300 With NF-κB and CREB in Angiotensin II-Induced IL-6 Expression in Vascular Smooth Muscle Cells

The purpose of this study was to evaluate the role of coactivator histone acetyltransferases (HATs) p300 and SRC-1 in angiotensin II (Ang II)-induced interleukin-6 (IL-6) gene expression in vascular smooth muscle cells (VSMCs). Ang II increased IL-6 mRNA expression via NF-kappaB and CREB in an extracellular signal-regulated kinase (ERK)-dependent manner in rat VSMCs. It was also significantly enhanced by the histone deacetylase inhibitor, Trichostatin A. Chromatin immunoprecipitation (ChIP) assays showed that Ang II increased Histone H3 Lysine (K9/14) acetylation on the IL-6 promoter. Ang II-induced IL-6 promoter transactivation was significantly enhanced by p300 and SRC-1, with maximal activation in cells cotransfected with NF-kappaB (p65) and SRC-1. Nucleofection of VSMCs with either an ERK phosphorylation site mutant of SRC-1 or p300/CBP HAT deficient mutants significantly blocked Ang II-induced IL-6 expression. ChIP assays revealed that Ang II enhanced coordinate occupancy of p65, CREB, p300, and SRC-1 at the IL-6 promoter. An ERK pathway inhibitor blocked Ang-induced IL-6 promoter SRC-1 occupancy and histone acetylation. Ang II-induced IL-6 expression requires NF-kappaB and CREB as well as ERK-dependent histone acetylation mediated by p300 and SRC-1. These results provide new insights into nuclear chromatin mechanisms by which Ang II regulates inflammatory gene expression.

Green Tea Extract Induces Interleukin-8 (IL-8) mRNA and Protein Expression but Specifically Inhibits IL-8 Secretion in Caco-2 Cells

The chemokine interleukin (IL)-8 is a cytokine involved in neutrophil attraction and activation and elevated levels have been observed in intestinal inflammation. Anti-inflammatory activities have been attributed to green tea or its major constituent (-)-epigallocatechin gallate (EGCG). In this study, we investigated the effects of a defined green tea extract (GTE) or EGCG on basal or IL-1beta-induced IL-8 expression and secretion in the human gastrointestinal epithelial cell line Caco-2. mRNA expression levels were determined by quantitative RT-PCR. GTE significantly induced IL-8 mRNA expression, which was not mediated indirectly via an induction of IL-1beta mRNA expression. EGCG only exerted a weak although significant induction of IL-8 mRNA expression at the highest concentration. Intracellular and extracellular protein levels were analyzed by an enzyme-linked immunosorbent assay. GTE and EGCG significantly decreased secreted IL-8 concentrations. Determination of intracellular and secreted IL-8 concentrations after 24 h, 48 h, and 72 h of incubation suggested that GTE specifically inhibited IL-8 secretion while inducing DE NOVO synthesis of IL-8. The IL-1beta-mediated increase of IL-8 secretion was significantly inhibited by GTE in a dose-dependent manner. At the highest concentration, GTE inhibited IL-1beta-induced IL-8 secretion to a similar extent as found for brefeldin A, an inhibitor of vesicular transport. These results suggest that GTE may exert an anti-inflammatory activity in enterocytes, which may be useful for the treatment of intestinal inflammation.

TNF-α induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

We investigated the effect of TNF-α on interleukin-8 (IL-8) and endothelin-1 (ET-1) expression, and their different signal transduction pathways. Human dermal microvascular endothelial cells (HMECs) were treated with TNF-α. By Northern blot analysis, TNF-α at 50, 100, 200, and 400 U/ml significantly induced IL-8 mRNA expression by 206%, 252%, 211%, and 158%, respectively, as compared to controls ( p < 0.05). Overexpression of human superoxide dismutase (SOD) by adenovirus-mediated gene transfer or addition of exogenous hydrogen peroxide (H 2O 2) significantly enhanced TNF-α-induced IL-8 mRNA expression. Furthermore, HMECs treated with TNF-α at 50, 100, and 200 U/ml significantly increased ET-1 mRNA expression by 71%, 82%, and 66%, respectively ( p < 0.05). By contrast, SOD gene transfer and exogenous H 2O 2 significantly inhibited TNF-α-induced ET-1 mRNA expression. Thus, TNF-α significantly induces both IL-8 and ET-1 gene expression in HMECs possibly through different redox signaling pathways. H 2O 2 enhances TNF-α-induced IL-8 expression, but inhibits TNF-α-induced ET-1 expression.

Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappaB.

Circulating interleukin-6 (IL-6), insulin, and free fatty acid (FFA) concentrations are associated with impaired insulin action in obese and type 2 diabetic individuals. However, a causal relationship between elevated plasma FFAs and IL-6 has not been shown. Because skeletal muscle represents a major target of impaired insulin action, we studied whether FFAs may affect IL-6 expression in human myotubes. We demonstrate that specifically saturated FFAs, e.g. palmitate (0.25 mm), induce IL-6 mRNA expression and protein secretion by a proteasome-dependent mechanism that leads to a rapid and chronic activation of nuclear factor-kappaB. Insulin, high glucose concentrations, or unsaturated FFAs did not activate IL-6 expression. In fact, the unsaturated FFA linoleate inhibited palmitate-induced IL-6 production. Because inhibition of palmitate metabolism by the acyl-CoA synthetase inhibitor triacsin C did not abolish IL-6 expression, it appears that the palmitate molecule per se exerts the observed effects. Furthermore, we show that in human myotubes, IL-6 activates the phosphorylation of signal transducer and activator of transcription 3 in concentrations similar to hepatocytes. However, no inhibitory effect of IL-6 on insulin action, determined as phosphatidylinositol 3-kinase association with insulin receptor substrate-1, Akt phosphorylation, and glycogen synthesis, was detected. We conclude that IL-6 expression may be modulated by the composition of circulating FFA, e.g. by diet, and that skeletal muscle cells could be target cells for IL-6.

Interferon gamma downregulates IL-8 production in primary human colonic epithelial cells without induction of apoptosis.

In acute or chronic inflammatory bowel disease (IBD) interferon gamma (IFNgamma) is still considered to be an important pro-inflammatory mediator. In the present study we investigated the impact of IFNgamma on interleukin-8 (IL-8) production as a read-out for cell activation in intestinal epithelial cell (IEC) lines and primary human colonic epithelial cells (CEC). Primary cultures of human CEC were established from the mucosa of patients without inflammatory disease. CEC, HT-29 or Caco-2 cells were incubated with either IFNgamma, tumor necrosis factor (TNF)alpha or IL-10. IL-8 and IL-1Ra secretion was determined by ELISA. Competicon PCR was used for quantification of IL-8mRNA. Apoptosis was quantified by propidium iodine incorporation and fluorescence activated cell sorting (FACS) analysis. In contrast to HT-29 cells in primary human CEC 100 U/ml IFNgamma inhibited IL-8 secretion significantly to 70+/-15% of unstimulated primary CEC (p<0.005) more effectively than IL-10 (87+/-21% versus unstimulated cells, n.s.). In HT-29 cells, IL-8 secretion was induced to 405+/-101% of unstimulated cells. In Caco-2 cells, IFNgamma had no significant effect on IL-8 secretion. The effect in HT-29 and CEC was concentration dependent. In primary CEC, 200 U/ml IFNgamma further reduced IL-8 secretion to 48+/-18% of unstimulated CEC (p<0.05). Whereas IL-8 mRNA was strongly upregulated in HT-29 cells, no upregulation or even a downregulation was found in CEC. Pre-incubation with 100 U/ml IFNgamma did not increase the susceptibility to apoptosis mediated by anti-Fas antibody (CH-11) in primary CEC, whereas HT-29 cells showed increased rates of apoptosis after priming with IFNgamma. In contrast to HT-29, IFNgamma downregulated IL-8 secretion and did not induce IL-8 mRNA expression in primary human CEC. This effect was not due to induction of apoptosis.