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

María A Hidalgo,Stefanie E Teuber,María D Carretta,Cristian Zárate,Rafael A Burgos,Ilona I Concha,Leonardo Cárcamo

doi:10.1155/2015/120348

María A Hidalgo, Stefanie E Teuber + Show 5 more

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https://doi.org/10.1155/2015/120348

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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.

Highlights

Polymorphonuclear neutrophils (PMNs) are the first line of defence against microorganisms and are the main cellular component in the acute inflammatory response.Neutrophils are primarily activated by chemotactic factors such as fMLP [1] and platelet-activating factor (PAF) [2].Both compounds bind to the neutrophil cell surface via specific seven transmembrane domain G-protein coupled receptors [3, 4] and induce the activation of MAPK, PI3K, and NF-κB pathways in neutrophils [5, 6]
We showed that IL-8 release induced by fMLP is dependent on nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase, and radical oxygen species (ROS) could play a redundant role in cell signalling, activating the PI3K/Akt and NF-κB pathways in neutrophils
We present evidence that supports the role of NADPH oxidase in IL-8 release, the PI3K/Akt pathway, and NF-κB activity in human neutrophils treated with fMLP

Summary

Introduction

Polymorphonuclear neutrophils (PMNs) are the first line of defence against microorganisms and are the main cellular component in the acute inflammatory response. Neutrophils are primarily activated by chemotactic factors such as fMLP [1] and PAF [2]. It is widely known that fMLP induces a significant increase in superoxide production via NADPH oxidase; in contrast, basal levels of superoxide are unaltered in PAF activated neutrophils [9, 10]. ROS originate from the activation of NADPH oxidase, which is assembled at the plasma membrane This reaction produces two superoxide anions (O2−) and 2H+. It has been described that the superoxide anion induces NF-κB activation (IκBα degradation and p65 NF-κB translocation) and increases the expression of TNFα and macrophage inflammatory protein-2 in neutrophils [16]. We present evidence that supports the role of NADPH oxidase in IL-8 release, the PI3K/Akt pathway, and NF-κB activity in human neutrophils treated with fMLP

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