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Published on: October 22, 2012
fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils
María A Hidalgo1, María D Carretta1, Stefanie E Teuber1
1Laboratory of Molecular Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Sciences, Universidad Austral de Chile, Independencia 631, 5110566 Valdivia, Chile.
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.
Insights
N-Formyl-methionyl-leucyl-phenylalanine (fMLP) triggers interleukin-8 (IL-8) release in neutrophils via NADPH oxidase. Reactive oxygen species (ROS) play a key role in activating PI3K/Akt and NF-κB signaling pathways.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) elicit comparable intracellular signaling, yet only fMLP stimulates interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils.
- The precise role of reactive oxygen species (ROS) in fMLP-induced IL-8 secretion and neutrophil signaling remains debated.
Purpose of the Study:
- To investigate the involvement of NADPH oxidase in fMLP-induced IL-8 secretion.
- To determine the role of NADPH oxidase and ROS in activating PI3K/Akt, MAPK, and NF-κB pathways in neutrophils stimulated by fMLP.
Main Methods:
- Neutrophils from healthy volunteers were utilized.
- Quantification of IL-8 (ELISA), IL-8 mRNA (qPCR), and ROS production (chemiluminescence, ferricytochrome c reduction, FACS).
- Analysis of intracellular pH, Akt, ERK1/2, p38 MAPK phosphorylation (immunoblotting), and NF-κB translocation (immunocytochemistry).
Main Results:
- Inhibition of NADPH oxidase (using HMAP, DPI, siRNA Nox2) reduced both ROS and IL-8 release in fMLP-treated neutrophils.
- HMAP, DPI, and amiloride inhibited Akt phosphorylation, while p38 MAPK and ERK1/2 activity remained unaffected.
- DPI and HMAP diminished fMLP-induced NF-κB translocation.
Conclusions:
- fMLP-induced IL-8 release from neutrophils is dependent on NADPH oxidase activity.
- ROS generated by NADPH oxidase may act redundantly in cell signaling, ultimately activating PI3K/Akt and NF-κB pathways.
