Abstract
The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.
Highlights
Gen to the superoxide anion (O2. ), which is the precursor of the other reactive oxygen species (ROS)
The main findings of this study reveal that IL-8 induced in a sequential manner the phosphorylation of cytosol phox components and the recruitment of the NADPH oxidase components into lipid rafts, suggesting that this pre-assembly could play at least some part in the priming effect of IL-8 on the neutrophil oxidative burst triggered in response to N-formyl peptides
As previously reported [21], these findings suggest that IL-8 has a dose- and time-dependent priming effect on the O2. production induced by formyl peptides (fMLP)
Summary
Gen to the superoxide anion (O2. ), which is the precursor of the other ROS. In resting cells, this multicomponent enzyme system is inactive, and its components are dispersed between the cytosol and the membranes. Proinflammatory cytokines, such as interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor ␣, are known to modulate NADPH oxidase activity by means of a priming phenomenon that strengthens the bactericidal capacity of the neutrophils (8 –12) These cytokines do not stimulate oxidative burst activity on their own, but strongly enhance ROS production in response to exposure to a secondary applied stimulus, such as fMLP. The main findings of this study reveal that IL-8 induced in a sequential manner the phosphorylation of cytosol phox components and the recruitment of the NADPH oxidase components (including flavocytochrome b558) into lipid rafts, suggesting that this pre-assembly could play at least some part in the priming effect of IL-8 on the neutrophil oxidative burst triggered in response to N-formyl peptides
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