Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils

Objective and design Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47 phox phosphorylation-dependent translocation of cytosolic NADPH oxidase compon...

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Published in:	Inflammation research 2017-10, Vol.66 (10), p.891-899
Main Authors:	Creed, T. Michael, Tandon, Shweta, Ward, Richard A., McLeish, Kenneth R.
Format:	Article
Language:	eng
Subjects:	Allergology Biomedical and Life Sciences Biomedicine Chemiluminescence Chlorpromazine Clathrin Dermatology Endocytosis Enzyme-linked immunosorbent assay Exocytosis Ferrocytochrome c Gelatinase Granular materials Immunology Inflammation Inhibition Leukocytes (neutrophilic) MAP kinase NAD(P)H oxidase Neurology Neutrophils Original Research Paper Oxidase Pharmacology/Toxicology Phosphorylation Priming Reactive oxygen species Respiratory burst Rheumatology Statistical analysis Stimulation Sucrose Sugar Superoxide Translocation
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Summary:	Objective and design Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47 phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Materials and methods Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47 phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey–Kramer multiple-comparison test. Results Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47 phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Conclusions Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.
ISSN:	1023-3830 1420-908X