title: NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages.
abstract: Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.
authors.affiliation: Department of Cellular and Molecular Medicine, Panum Institute, Faculty of Health Sciences, University of Copenhagen, Copenhagen 2200N, Denmark.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ejlerskov
authors.forename: Dan Ploug
authors.initials: DP
authors.lastname: Christensen
authors.forename: David
authors.initials: D
authors.lastname: Beyaie
authors.forename: James B
authors.initials: JB
authors.lastname: Burritt
authors.forename: Marie-Helene
authors.initials: MH
authors.lastname: Paclet
authors.forename: Agnes
authors.initials: A
authors.lastname: Gorlach
authors.forename: Bo
authors.initials: B
authors.lastname: van Deurs
authors.forename: Frederik
authors.initials: F
authors.lastname: Vilhardt
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D000818:Animals; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000201:enzymology* / Q000235:genetics; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000201:enzymology / Q000235:genetics; D005089:Exocytosis / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008262:Macrophage Activation / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D017628:Microglia / Q000201:enzymology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
chemical_list: D002135:Calcium-Binding Proteins; D002966:Clathrin; D003573:Cytochrome b Group; C526966:Eps15 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D000075745:rab27 GTP-Binding Proteins; D013481:Superoxides; D023201:CD40 Ligand; D000255:Adenosine Triphosphate; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C512956:Rab27b protein, rat; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins