Induction Of Interleukin-8 Gene Expression Research Articles

Role of type VI secretion system protein TssJ-3 in virulence and intracellular survival of Burkholderia pseudomallei

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3-complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3, an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei-derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections.

Effect of Dietary Acidolysis-Oxidized Konjac Glucomannan Supplementation on Serum Immune Parameters and Intestinal Immune-Related Gene Expression of Schizothorax prenanti.

The present study was conducted to investigate the effects of dietary acidolysis-oxidized konjac glucomannan (A-OKGM) (0%, 0.4%, 0.8%, and 1.6%) supplementation on the immunity and expression of immune-related genes in Schizothorax prenanti. After feeding for eight weeks, the serum and guts were used for measurement of biochemical parameters, and immune-related gene expression in the gut were also analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). C-reactive protein and IgM levels were significantly higher in the A-OKGM fed groups than in the control group, regardless of the dosage. The 0.4% and 1.6% A-OKGM groups showed significant up-regulation of tumor necrosis factor α (TNFα) in the anterior gut. The 0.8% and 1.6% A-OKGM groups also showed significantly enhanced TNFα expression in the mid- and distal guts. Interleukin-1β (IL-1β) expression in the anterior gut of fish fed with 0.4% and 1.6% A-OKGM diets was significantly enhanced. The 0.8% and 1.6% A-OKGM diets resulted in significantly increased the expression of IL-1β in the distal gut. Similarly, the interleukin-6 (IL-6) messenger RNA (mRNA) levels in the 0.4% and 1.6% diet groups were significantly higher in the anterior gut. The 0.8% and 1.6% A-OKGM diet groups showed significant induction of IL-6 gene expression in the distal gut. A-OKGM modified from KGM can act as an immunostimulant to enhance the immunity of S. prenanti.

Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells.

BackgroundPersistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells.MethodsThe effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay.ResultsDust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract.ConclusionsOur studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0455-z) contains supplementary material, which is available to authorized users.

Regulation of IL-8 gene expression in gliomas by microRNA miR-93.

BackgroundDifferent strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas.MethodsHere we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G.ResultsIL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3′UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis).ConclusionIn conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1659-1) contains supplementary material, which is available to authorized users.

Oxidative stress and endocytosis are involved in upregulation of interleukin-8 expression in airway cells exposed to PM2.5.

Inhaled PM2.5 (particulate matter with an aerodynamic diameter of 2.5 μm or less) can induce lung inflammation through released inflammatory mediators from airway cells, such as interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α). However, the mechanisms underlying PM2.5-induced IL-8 gene expression have not been fully characterized. BEAS-2B cells (a human bronchial epithelial cell line) and THP-1 cells (a human macrophage-like cell line) were used as the in vitro models to investigate the underlying mechanism in this study. IL-8 expression was increased in the cells treated with PM2.5 in a dose-dependent manner. The water-soluble and insoluble fractions of PM2.5 suspension were both shown to induce IL-8 expression. PM2.5 exposure could obviously induce ROS (reactive oxygen species) generation, indicative of oxidative stress. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) potently inhibited PM2.5-induced IL-8 expression. Employment of the transition metal chelators including TPEN (N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine) or DFO (desferrioxamine) inhibited IL-8 expression induced by PM2.5 by over 20% in BEAS-2B cells, but had minimal effect in THP-1 cells. Pretreatment with the endocytosis inhibitor CytD markedly blocked IL-8 expression induced by PM2.5 in both BEAS-2B and THP-1 cells. In summary, exposure to PM2.5 induced IL-8 gene expression through oxidative stress induction and endocytosis in airway cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1869-1878, 2016.

Sjögren's Syndrome Antigen B Acts as an Endogenous Danger Molecule to Induce Interleukin-8 Gene Expression in Polymorphonuclear Neutrophils.

BackgroundSjögren’s syndrome antigen B is expressed in the nucleus and surface membrane of human polymorphonuclear neutrophils and is released after cell death. However, its biological role is not clear. This study is aimed to investigate the effect of Sjögren’s syndrome antigen B on human polymorphonuclear neutrophils.MethodsHuman recombinant Sjögren’s syndrome antigen B (rSSB) purified from E. coli was incubated with human polymorphonuclear neutrophils as well as retinoid acid-induced granulocytic differentiated HL-60 cells, HL-60 (RA). Interleukin (IL)-8 protein production and mRNA expressions were measured by enzyme-linked immunosorbent assay and quantitative-polymerase chain reaction, respectively. Uptake of fluorescein isothiocyanate (FITC)-rSSB was assessed by flow cytometry and fluorescence microscopy. Moreover, mitogen-activated protein kinase (MAPK) pathways and nuclear factor-kappaB activation were investigated.ResultsHuman rSSB stimulated IL-8 production from normal human neutrophils and HL-60 (RA) cells in a time- and dose-dependent manner. This IL-8-stimulated activity was blocked by chloroquine and NH4Cl, indicating that endosomal acidification is important for this effect. We found rSSB activated both MAPK pathway and nuclear factor-kappaB signaling to transcribe the IL-8 gene expression of cells. Furthermore, tumor necrosis factor-α exerted an additive effect and rSSB-anti-SSB immune complex exhibited a synergistic effect on rSSB-induced IL-8 production.ConclusionsSjögren’s syndrome antigen B might act as an endogenous danger molecule to enhance IL-8 gene expression in human polymorphonuclear neutrophils.

Transcriptional and posttranscriptional regulation and endocytosis were involved in zinc oxide nanoparticle-induced interleukin-8 overexpression in human bronchial epithelial cells

Inhaled zinc oxide nanoparticles (ZnO-NPs) can induce lung inflammation through released inflammatory mediators, such as interleukin 8 (IL-8), from airways. However, the mechanisms underlying ZnO-NP-induced IL-8 gene expression have not been fully characterized. The transcription inhibitor actinomycin D (Act D) and the BEAS-2B cells stably overexpressing wild-type or mutated IL-8 promoter at the NFκB or C/EBPβ binding site were used to determine the involvement of transcriptional mechanisms. The effect of ZnO-NPs on IL-8 mRNA stability was examined using mRNA decay assay. The phagocytosis inhibitor cytochalasin B (CB) was utilized to define the role of endocytosis in ZnO-NP-induced IL-8 expression. In addition, the solubility of ZnO-NPs in culture medium was assessed using atomic absorption spectroscopy. Exposure to ZnO-NPs significantly increased the expression of IL-8 mRNA and protein in a dose-dependent manner. Pretreatment with Act D blocked ZnO-NP-induced IL-8 expression. Both NFκB and C/EBPβ transcription factors were required for ZnO-NP-induced IL-8 transcription. mRNA decay assay showed that ZnO-NP stimulation delayed IL-8 mRNA degradation in BEAS-2B cells. Pretreatment of BEAS-2B cells with CB blocked ZnO-NP-induced IL-8 expression by 30%. Exposure to ZnO-NPs induced IL-8 gene expression through transcriptional activation and mRNA stabilization. Internalization of nanoparticles was partially involved in ZnO-NP-induced IL-8 expression.

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.

Hypoxia Antagonizes Glucose Deprivation on Interleukin 6 Expression in an Akt Dependent, but HIF-1/2α Independent Manner

Although both glucose deprivation and hypoxia have been reported to promote cascades of biological alterations that lead to induction of inflammatory mediators, we hypothesized that glucose deprivation and hypoxia might show neutral, synergistic or antagonistic effects to each other on gene expression of inflammatory mediators depending on the regulatory components in their promoters. Gene expression of interleukin 6 (IL-6) was analyzed by real-time PCR, ELISA, or Western blot. Effects of glucose deprivation and/or hypoxia on activation of signaling pathways were analyzed by time-dependent phosphorylation patterns of signaling molecules. We demonstrate that hypoxia antagonized the effects of glucose deprivation on induction of IL-6 gene expression in microglia, macrophages, and monocytes. Hypoxia also antagonized thapsigargin-induced IL-6 gene expression. Hypoxia enhanced phosphorylation of Akt, and inhibition of Akt was able to reverse the effects of hypoxia on IL-6 gene expression. However, inhibition of HIF-1/2α did not reverse the effects of hypoxia on IL-6 gene expression. In addition, phosphorylation of p38, but not JNK, was responsible for the effects of glucose deprivation on IL-6 gene expression.

Simvastatin prevents the induction of interleukin-6 gene expression by titanium particles in human osteoblastic cells

One of the most important complications of total joint arthroplasty is failure associated with periprosthetic osteolysis, a process mainly initiated by the biological response to wear-derived products from the biomaterials in service. The inflammatory mediator interleukin-6 (IL-6) plays a key role in the establishment and progression of aseptic loosening. Metal particles specifically up-regulate IL-6 production in bone-forming cells and implant–bone interfacial tissues. The use of statins has been recently associated with a significantly reduced risk of revision in patients that undergo total hip arthroplasty. We hypothesized that simvastatin (Simv) could modulate the osteoblastic response to titanium particles (Ti) by attenuating the production of IL-6. Pre-treatment of human osteoblastic cells with Simv down-regulated Ti particle-induced IL-6 gene expression at mRNA and protein levels. The effect of Simv on Ti-induced IL-6 production in osteoblastic cells could not be explained by inhibition of the internalization of metal particles. The mechanism involved in this down-regulation is based in the inhibition of the HMG-CoA/GGPP/RhoA/ROCK pathway, independently of Simv effects in the cholesterol synthesis. The cytokine-lowering property of Simv has been observed in Saos-2 cells and human primary osteoblasts (hOBs) exposed to Ti particles, and was further enhanced when hOBs were co-cultured with macrophages.

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.

Bacterial Factors Other Than Injected CagA and Those Mediating IL-8 Induction Participate in H. pylori-Mediated Gastric H,K-ATPase Repression

We previously reported that acute H. pylori infection of gastric epithelial (AGS) cells and human gastric biopsies causes H,K-ATPase α subunit (HKα) gene repression and inhibits acid secretion, events that may facilitate gastric H. pylori colonization and underlie transient clinical hypochlorhydria (Gastroenterology, 139:239, 2010; Gut, 59:874, 2010). In those studies, HKα repression was abrogated by infection with H. pylori isogenic mutant strains deficient in the cag pathogenicity island genes cagL, cagE or cagM, each of which fails to inject CagA into host cells or to induce interleukin-8 (IL-8) expression; HKα repression was partially abrogated by a cagA-deficient strain that induces IL-8. To test the hypothesis that bacterial factors other than those mediating CagA injection or IL-8 induction participate in HKα repression, AGS cells transfected with HKα promoter-Luc reporter constructs were infected (8 h, multiplicity of infection=50) with wild-type (WT) H. pylori strain 7.13 or isogenic mutants previously reported to be competent for CagA injection and IL-8 induction (cagζ, cage, cagN, cagZ or cagD), or competent only for IL-8 induction (cagβ [virD4]). AGS cell fractions were immunoblotted with anti-CagA antibodies, and IL-8 in cell-conditioned media was measured by ELISA. CagA was detected in cytoplasmic fractions of cells infected with WT H. pylori or cagζ, cage, cagN, cagZ or cagD mutants, but not with the virD4 mutant. AGS cell IL-8 secretion induced by the cagζ and cage mutants was comparable to WT; the virD4 mutant induced 22% less IL-8 than WT (P<0.01); and the cagN, cagZ and cagD mutants induced 32%, 68%, and 81% less IL-8 respectively than WT (all P<0.01). AGS cell infection by WT H. pylori inhibited HKα promoter activity (measured by luminometry) by 63% (P<0.01); the cagA-deficient mutant had minor effects on activity. The virD4 mutant inhibited HKα promoter activity by 23% (P<0.002), cagζ, cagN, cagZ and cagD mutants inhibited HKα by 40-50% (all P<0.01), and the cage mutant inhibited HKα by 87% (P<0.001). These data indicate that (i) CagA injection is associated with varying degrees of HKα repression, which is not abrogated by complete absence of CagA; (ii) HKα repression is poorly correlated with H. pylori-mediated induction of IL-8 gene expression; (iii) virD4, which encodes a cytoplasmic type IV secretion system nucleoside triphosphatase coupling protein, is necessary for bacterial signals culminating in HKα repression and consequent acid secretory inhibition; and (iv) neither CagA nor perturbation of signaling pathways leading to IL-8 induction are solely responsible for HKα repression. These findings are significant because hypochlorhydria is a predisposing factor for gastric epithelial progression to neoplasia, andH. pylori factors other than CagA and/or signaling to IL-8 are thus implicated in gastric carcinogenesis.

Differential Effects of Multiplicity of Infection on Helicobacter pylori-Induced Signaling Pathways and Interleukin-8 Gene Transcription

Interleukin-8 (IL-8) plays a central role in the pathogenesis of Helicobacter pylori infection. We used four different H. pylori strains isolated from patients with gastritis or duodenal ulcer disease to examine their differential effects on signaling pathways and IL-8 gene response in gastric epithelial cells. IL-8 mRNA level is elevated in response to high (100) multiplicity of infection (MOI) independent of cagA, vacA, and dupA gene characteristics. By lower MOIs (1 or 10), only cagA ( + ) strains significantly induce IL-8 gene expression. This is based on differential regulation of IL-8 promoter activity. Analysis of intracellular signaling pathways indicates that H. pylori clinical isolates induce IL-8 gene transcription through NF-κB p65, but by a MOI-dependent differential activation of MAPK pathways. Thus, the major virulence factors of H. pylori CagA, VacA, and DupA might play a minor role in the level of IL-8 gene response to a high bacterial load.

Induction of interleukin-8 gene expression and protein secretion by C-reactive protein in ARPE-19 cells

C-reactive protein (CRP) is an acute phase reactant and its level rises rapidly during inflammation. Recent studies have suggested the potential involvement of CRP in the pathogenesis of age-related macular degeneration (AMD). To delineate the functional roles of CRP in inflammatory response by the ocular posterior segments, the effects of CRP on ARPE-19, an immortalized human retinal pigment epithelia (hRPE) cell line, were investigated in the present study. Treatment of ARPE-19 cells with CRP resulted in enhanced NF-kB nuclear translocation and dose-dependent transient induction of IL-8 mRNA synthesis and protein secretion. Stimulated expression of VEGF, but not MCP-1 by CRP was also observed. The induced IL-8 expression was transient and peaked at 12 h post stimulation. In the presence of inhibitors for NF-kB, p38, MEK and JNK, the CRP-induced IL-8 production was abolished by 99.5 ± 2.3, 97.8 ± 2.1, 55.3 ± 2.5 and 37.3 ± 1.3%, respectively. Neutralization of Fc gamma receptors by anti-CD32 and CD64 antibodies produced 39.9 ± 1.6 and 59.5 ± 2.6% reduction, respectively, of CRP-stimulated IL-8 secretion, whereas that by anti-CD16 antibody had no effect. This study suggests that the pro-inflammatory effects of CRP in ARPE-19 cells may contribute to the inflammatory retinal diseases by induction of pro-inflammatory cytokines such as IL-8. This induction is mediated by NF-kB and multiple MAPK pathways through Fc gamma receptors.

Prothrombin fragments containing kringle domains induce migration and activation of human neutrophils

The cross-talk between inflammatory and coagulation cascades has been demonstrated. Prothrombin processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: prothrombin fragments 1 (F1) and 2 (F2). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of prothrombin fragments on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F1 and F2 prothrombin fragments, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, fragment 1 and fragment 2 induce human neutrophil chemotaxis. The effect of fragment 2, but not fragment 1, was partially inhibited by pertussis toxin, an inhibitor of G αi-signaling. The pre-treatment of cells with fragment 2 inhibited thrombin-induced chemotaxis, while both fragments impaired neutrophil migration induced by interleukin-8. F1 and F2 increased the expression and activation of G-protein-coupled receptor kinase-2, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, prothrombin fragments activated extracellular signal-regulated kinase 1/2, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both prothrombin fragments induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing prothrombin fragments may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses.

Corticosteroids and β2 Agonists Differentially Regulate Rhinovirus-induced Interleukin-6 via Distinct Cis-acting Elements

Interleukin-6 (IL-6) is a proinflammatory cytokine up-regulated by rhinovirus infection during acute exacerbations of asthma and chronic obstructive pulmonary disease. The role of IL-6 during exacerbations is unclear; however, it is believed IL-6 could contribute to airway and systemic inflammation. In this study we investigate the effects of common asthma treatments fluticasone propionate and beta(2) agonists salmeterol and salbutamol on IL-6 production in BEAS-2B and primary bronchial epithelial cells. Salmeterol and salbutamol enhanced rhinovirus- and IL-1beta-induced IL-6 production; however, fluticasone treatment caused a reduction of IL-6 protein and mRNA. Combined activity of salmeterol and fluticasone at equimolar concentrations had no effect on rhinovirus or IL-1beta induction of IL-6. The induction of IL-6 by salmeterol was dependent upon the beta(2) receptor and could also be induced by cAMP or cAMP-elevating agents forskolin and rolipram. Using transfection of IL-6 promoter reporter constructs, dominant negative mutants, and electromobility shift assays, it was found that NF-kappaB was the only transcription factor required for rhinovirus induction of IL-6 gene expression. Salmeterol caused an augmentation of rhinovirus-induced promoter activation via a mechanism dependent upon the c/EBP and/or CRE (cyclic AMP response element) cis-acting sites. The suppressive effect of FP was dependent upon distinct glucocorticoid response element sequences proximal to the transcriptional start site within the IL-6 promoter. The data demonstrate that beta(2) agonists can augment IL-6 expression by other stimuli in an additive manner via cyclic AMP and that the negative effect of steroids is mediated by glucocorticoid response elements within the IL-6 promoter.

The role of sphingosine 1-phosphate in the TNF-α induction of IL-8 gene expression in lung epithelial cells

Tumor necrosis factor-α (TNF-α) is an important cytokine involved in the pathogenesis of inflammatory diseases of the lung. Interleukin-8 (IL-8), a C-X-C chemokine, is induced by TNF-α and initiates injury by acting as a chemoattractant for neutrophils and other immune cells. Although sphingolipids such as ceramide and sphingosine 1-phosphate (S1-P) have been shown to serve as signaling molecules in the TNF-α inflammatory response, their role in the TNF-α induction of IL-8 gene expression in lung epithelial cells is not known. We investigated the role of sphingolipids in the TNF-α induction of IL-8 gene expression in H441 lung epithelial cells. We found that TNF-α induced IL-8 mRNA levels by increasing gene transcription, and the stability of IL-8 mRNA was not affected. Exogenous S1-P but not ceramide or sphingosine increased IL-8 mRNA levels and IL-8 secretion. Dimethylsphingosine, an inhibitor of sphingosine kinase, partially inhibited TNF-α induction of IL-8 mRNA levels indicating the importance of intracellular increases in S1-P in the IL-8 induction. S1-P induction of IL-8 mRNA was due to an increase in gene transcription, and the stability of IL-8 mRNA was not affected. S1-P induction of IL-8 mRNA was associated with an increase in the binding activity of AP-1 but the activities of NF-κB and NF IL-6 were unchanged. S1-P induced the phosphorylation of ERK, p38 and JNK MAPKs. Pharmacological inhibitors of ERK and p38 but not JNK partly inhibited S1-P induction of IL-8 mRNA levels. These data show that increases in the intracellular S1-P partly mediate TNF-α induction of IL-8 gene expression in H441 lung epithelial cells via ERK and p38 MAPK signaling pathways and increased AP-1 DNA binding.

Ozone Enhances Diesel Exhaust Particles (DEP)-Induced Interleukin-8 (IL-8) Gene Expression in Human Airway Epithelial Cells through Activation of Nuclear Factors- κB (NF-κB) and IL-6 (NF-IL6)

Ozone, a highly reactive oxidant gas is a major component of photochemical smog. As an inhaled toxicant, ozone induces its adverse effects mainly on the lung. Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals. Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM[2.5-10]), including diesel exhaust particles (DEP) has been correlated with increased acute and chronic respiratory morbidity and exacerbation of asthma. Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems. Ozone and particulate matter may be chemically coupled in the ambient air. In the present study we determined whether ozone exposure enhances DEP effect on interleukin-8 (IL-8) gene expression in human airway epithelial cells. We report that ozone exposure (0.5 ppm x 1 hr) significantly increased DEP-induced IL-8 gene expression in A549 cells (117 +/- 19 pg/ml, n = 6, p < 0.05) as compared to cultures treated with DEP (100 microg/ml x 4 hr) alone (31 +/- 3 pg/ml, n = 6), or cultures exposed to purified air (24 +/- 6 pg/ml, n = 6). The increased DEP-induced IL-8 gene expression following ozone exposure was attributed to ozone-induced increase in the activity of the transcription factors NF-kappaB and NF-IL6. The results of the present study indicate that ozone exposure enhances the toxicity of DEP in human airway epithelial cells by augmenting IL-8 gene expression, a potent chemoattractant of neutrophils in the lung.

Differential regulation of interleukin-8 gene transcription by death receptor 3 (DR3) and type I TNF receptor (TNFRI)

TL1A induces interleukin-8 (IL-8) secretion in human peripheral blood monocyte-derived macrophage in a dose- and time-dependent manner. Overexpression of its cognate receptor DR3 can induce a higher amount of IL-8 protein secretion than that induced by TNFRI even though both receptors activate IL-8 gene transcription in a similar fashion. The underlying mechanism for the regulation of the IL-8 gene transcription by DR3 has not been investigated yet. Here, we used HEK293 cells as a model system to dissect the possible signaling components that are involved in the regulation of DR3-mediated IL-8 gene expression. Although both DR3 and TNFRI activated TRAF2 and NF-kappaB to induce IL-8 gene transcription, the kinase cascades that transduce signals for DR3- and TNFRI-induced IL-8 gene transcription are different. The axis TAK1/ASK1-MKK4/MKK7-JNK2 is responsible for DR3-mediated IL-8 gene expression whereas the axis ASK1-MKK4-JNK1/JNK2/p38MAPK is the choice for TNFRI-mediated activation of IL-8 gene expression. This indicates that the downstream signaling pathways of DR3 and TNFRI for IL-8 secretion are divergent even though both receptors contain death-domain and induce IL-8 secretion via TRAF2.

Dengue Virus Nonstructural Protein NS5 Induces Interleukin-8 Transcription and Secretion

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.