title: fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils.
abstract: 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.
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: María A
authors.initials: MA
authors.lastname: Hidalgo
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: María D
authors.initials: MD
authors.lastname: Carretta
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: Stefanie E
authors.initials: SE
authors.lastname: Teuber
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: Cristian
authors.initials: C
authors.lastname: Zárate
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Cárcamo
authors.affiliation: Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: Ilona I
authors.initials: II
authors.lastname: Concha
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Burgos
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000276:immunology; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000276:immunology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D009861:Onium Compounds / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000276:immunology; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000276:immunology
chemical_list: D000098:Acetophenones; C508606:CXCL8 protein, human; D062686:Epithelial Sodium Channel Blockers; D016209:Interleukin-8; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009861:Onium Compounds; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C085119:3-hydroxy-4-methoxyacetophenone; D009240:N-Formylmethionine Leucyl-Phenylalanine; C007517:diphenyleneiodonium; D000584:Amiloride; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases