interleukin-8 and diphenyleneiodonium

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.

Topics: Antibodies, Blocking; Bronchi; Cells, Cultured; Cigarette Smoking; Extracellular Traps; HMGB1 Protein; Humans; Inflammation; Interleukin-6; Interleukin-8; Neutrophils; Onium Compounds; Ornithine; Proteinase Inhibitory Proteins, Secretory; Pulmonary Alveoli; Respiratory Mucosa; Uric Acid

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.

Topics: Acetophenones; Amiloride; Epithelial Sodium Channel Blockers; Gene Expression Regulation; Humans; Interleukin-8; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; NF-kappa B; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Primary Cell Culture; Protein Transport; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2 O2 ) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2 O2 and interleukin 8 (IL-8) after PM exposure.. Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2 O2 assays were performed.. LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2 O2 into the medium. Exposure to PM (20 μg/cm(2) ) increased H2 O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2 O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2 O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 μg/cm(2) = 5002 ± 1327 MFI; 40 μg/cm(2) = 8219 ± 1090 MFI; p = 0.022).. PM increases the quantity of H2 O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2 O2 and mucin production in sinonasal epithelium.

Topics: Adult; Cells, Cultured; Female; Humans; Hydrogen Peroxide; Immunity, Innate; Interleukin-8; L-Lactate Dehydrogenase; Male; Middle Aged; Models, Biological; Mucins; NADP; Nasal Mucosa; Onium Compounds; Paranasal Sinus Diseases; Particulate Matter; Prevalence; Primary Cell Culture

Glycated serum albumin (GSA) promotes vascular complications in diabetes. The aim of this study was to determine if GSA induces chemokine, particularly CXCL8 (IL-8), and to determine intracellular signaling pathways activated by GSA in vascular smooth muscle cells (VSMCs). GSA increased IL-8 transcription via promoter activation and enhanced CXCL8 release from VSMCs. GSA-induced promoter activation of the IL-8 gene was suppressed by dominant-negative mutants of TLR-4, MyD88, and TRIF, but not by a dominant-negative form of TLR-2. In addition, IL-8 up-regulation in response to GSA was inhibited by resveratrol, curcumin, diphenyleneiodium, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed GSA-induced promoter activation. Moreover, gene delivery of IkappaB suppressed CXCL8 release. This study suggests that GSA induces expression of IL-8 in VSMCs and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and NADPH oxidase are involved in that process.

Topics: Butadienes; Cells, Cultured; Curcumin; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Interleukin-8; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; NF-kappa B; Nitriles; Onium Compounds; Promoter Regions, Genetic; Resveratrol; Serum Albumin; Stilbenes; Toll-Like Receptor 4; Transcriptional Activation; Up-Regulation

LOX-1, a lectin-like receptor on endothelial cells, facilitates the uptake of oxidized low-density lipoprotein (oxLDL). Expression of LOX-1 is involved in the pathobiological effects of oxLDL in endothelial cells, including reactive oxygen species (ROS) generation, suppression of endothelial nitric oxide synthase (eNOS) activity, and leukocytic adhesion. Moderate consumption of phenolic-enriched food may have a protective effect against the development of atherosclerosis via the antioxidant capacity of phenolic compounds at the endothelial level. In this study, we determined whether ellagic acid, a polyphenolic compound widely distributed in fruits and nuts, protects against oxLDL-induced endothelial dysfunction by modulating the LOX-1-mediated signaling pathway.. Human umbilical vein endothelial cells (HUVECs) were pretreated with ellagic acid at doses of 5, 10, 15, and 20 μM for 2 hours and then incubated with oxLDL (150 μg/mL) for an additional 24 hours.. LOX-1 protein expression was markedly lower after exposure to oxLDL in HUVECs pretreated with ellagic acid or diphenyleneiodonium, a well-known inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, than in HUVECs exposed to oxLDL alone, suggesting that ellagic acid deactivates NADPH oxidase. We also found that oxLDL activated the membrane assembly of p47phox, Rac1, gp91 and p22phox, and the subsequent induction of ROS generation; however, ROS generation was markedly suppressed in cells pretreated with ellagic acid or anti-LOX-1 monoclonal antibody. In addition, oxLDL down-regulated eNOS and up-regulated inducible NO synthase (iNOS), thereby augmenting the formation of NO and protein nitrosylation. Furthermore, oxLDL induced the phosphorylation of p38 mitogen-activated protein kinase, activated the NF-κB-mediated inflammatory signaling molecules interleukin-(IL) 6 and IL-8 and the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin, and stimulated the adherence of THP-1 (a human acute monocytic leukemia cell line) to HUVECs. Pretreatment with ellagic acid, however, exerted significant cytoprotective effects in all events.. Findings from this study may provide insight into a possible molecular mechanism by which ellagic acid inhibits LOX-1-induced endothelial dysfunction. Our data indicate that ellagic acid exerts its protective effects by inhibiting NADPH oxidase-induced overproduction of superoxide, suppressing the release of NO by down-regulating iNOS, enhancing cellular antioxidant defenses, and attenuating oxLDL-induced LOX-1 up-regulation and eNOS down-regulation.

Topics: Anti-Inflammatory Agents; Antioxidants; Cell Adhesion Molecules; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Ellagic Acid; Endothelial Cells; Enzyme Inhibitors; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Lipoproteins, LDL; NADPH Oxidases; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Scavenger Receptors, Class E; Signal Transduction; Superoxides

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Topics: Animals; Cell Death; Chemokine CXCL2; Cystic Fibrosis; Enzyme Inhibitors; Extracellular Space; Humans; Inflammation; Interleukin-8; Lung Diseases; Mice; NADPH Oxidases; Neutrophil Activation; Neutrophils; Onium Compounds; Receptors, Interleukin-8B

Cholesterol and its oxidation products, namely oxysterols, have very recently been shown to potentially interfere with homeostasis of the human digestive tract, by promoting and sustaining irreversible damage of the colonic epithelial layer. This report concerns the strong proinflammatory action that a dietary oxysterol mixture and, to a lesser extent, an identical concentration of unoxidized cholesterol exert on CaCo-2 colonic epithelial cells by up-regulating both expression and synthesis of interleukin 8. The oxysterol mixture and its most effective component, 7β-hydroxycholesterol, are also shown to markedly enhance the expression of key inflammatory and chemotactic cytokines in colonic epithelial cells, more efficiently than unoxidized cholesterol. The sterols' proinflammatory effect seems to be mediated by enhanced activation of NOX1, because it is prevented by pretreatment of the cells with DPI, a selective inhibitor of this oxidase. Importantly, NOX1 hyperactivation by the oxysterol mixture or cholesterol was fully prevented by CaCo-2 cell preincubation with epigallocatechin-3-gallate. Consistently, supplementation with this compound fully protected colonic epithelial cells against overexpression of inflammatory and chemotactic genes induced by the sterols investigated.

Topics: Antioxidants; Apoptosis; Caco-2 Cells; Catechin; Cholesterol; Enterocytes; Enzyme Activation; Humans; Hydroxycholesterols; Inflammation Mediators; Interleukin-8; Ketocholesterols; NADPH Oxidases; Onium Compounds; Up-Regulation

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation.

Topics: Animals; Arrestins; beta-Arrestin 1; beta-Arrestins; Cell Death; Cell Line; Cell Membrane; Chemokine CXCL12; Chemokines, CXC; Cytoplasm; Endocytosis; Enzyme Inhibitors; Escherichia coli; Genes, Dominant; Humans; Immunoblotting; Immunoprecipitation; Interleukin-8; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NADPH Oxidases; Onium Compounds; Oxidants; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plasmids; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-raf; Reactive Oxygen Species; Receptors, Interleukin-8B; Respiratory Burst; Subcellular Fractions; Time Factors; Transfection

Homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are major chemokines for leukocyte trafficking and have been identified in atheromatous plaques. MCP-1 and IL-8 have been found to express mainly by macrophages in human lesion. We undertook this study to determine whether Hcy could induce the secretion of chemokines from human monocytes and, if so, to explore the mediating mechanism. We found that clinically relevant levels of Hcy (10 to 1000 micromol/L) increased the protein secretion and mRNA expression as well as activity of MCP-1 and IL-8 in cultured primary human monocytes. These effects of Hcy were primarily mediated by reactive oxygen species (ROS) through NAD(P)H oxidase, because Hcy could upregulate the production of ROS and the inhibitors of protein kinase C, calmodulin, free radical scavengers, or NAD(P)H oxidase abolished Hcy-induced ROS production and MCP-1 and IL-8 secretion in these cells. Furthermore, the inhibitors of mitogen-activated protein kinase (p38 and extracellular signal-regulated kinase 1/2) and nuclear factor-kappaB or the activator of peroxisome proliferator-activated receptor gamma (PPARgamma) significantly decreased Hcy-induced MCP-1 and IL-8 secretion in these cells. These data indicate that pathophysiological levels of Hcy can alter human monocyte function by upregulating MCP-1 and IL-8 expression and secretion via enhanced formation of intracellular ROS originated from NAD(P)H oxidase source via calmodulin or protein kinase C signaling pathways and that Hcy-induced ROS subsequently activates mitogen-activated protein kinase (p38 and ERK1/2) and nuclear factor-kappaB in a PPARgamma activator-sensitive manner. Thus, activation of PPARgamma may become a therapeutic target for preventing Hcy-induced proatherogenic effects.

Topics: Antioxidants; Cells, Cultured; Chemokine CCL2; Chromans; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Genistein; Homocysteine; Humans; Imidazoles; Indoles; Interleukin-8; Monocytes; Naphthalenes; Onium Compounds; Pyridines; Pyrrolidines; Reactive Oxygen Species; RNA, Messenger; Sulfonamides; Thiazoles; Thiazolidinediones; Thiocarbamates; Time Factors; Troglitazone; Tyrphostins

The results of this study show that recombinant interleukin-8 (IL-8) enhances the intracellular killing of Mycobacterium fortuitum by human granulocytes. This chemokine did not stimulate the phagocytosis of M. fortuitum by granulocytes at various bacterium-to-cell ratios. The killing process was not affected by the NADPH oxidase inhibitor diphenyleneiodonium bisulfate, which indicates that recombinant IL-8 stimulates oxygen-independent mycobactericidal mechanisms of granulocytes. IL-8 did not stimulate H2O2 production in granulocytes but primed the cells for enhanced H2O2 production upon stimulation with preopsonized M. fortuitum. In sum, the chemokine IL-8 not only is involved in the recruitment of granulocytes to the site of infection but also facilitates the elimination of microorganisms by increasing the efficiency of the bactericidal activity of granulocytes.

Topics: Blood Bactericidal Activity; Granulocytes; Humans; Hydrogen Peroxide; Interleukin-8; Nontuberculous Mycobacteria; Onium Compounds; Phagocytosis; Recombinant Proteins