RESUMO

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.

Assuntos

Interleucina-8/metabolismo , Glicoproteínas de Membrana/genética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/genética , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/imunologia , Transdução de Sinais/efeitos dos fármacos , Acetofenonas/farmacologia , Amilorida/farmacologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Oniocompostos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia