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Journal of Immunology Research
Volume 2015 (2015), Article ID 120348, 14 pages
http://dx.doi.org/10.1155/2015/120348
Research Article

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

1Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile
2Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile

Received 2 June 2015; Revised 30 September 2015; Accepted 7 October 2015

Academic Editor: Carlos Rosales

Copyright © 2015 María A. Hidalgo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.