NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages

TY - JOUR

T1 - NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages

AU - Ejlerskov, Patrick

AU - Christensen, Dan Ploug

AU - Beyaie, David

AU - Burritt, James B

AU - Paclet, Marie-Helene

AU - Gorlach, Agnes

AU - van Deurs, Bo

AU - Vilhardt, Frederik

PY - 2012/2/10

Y1 - 2012/2/10

N2 - 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 3cytochemistry 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 558depends 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.

AB - 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 3cytochemistry 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 558depends 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.

KW - Adenosine Triphosphate

KW - Animals

KW - CD40 Ligand

KW - Calcium-Binding Proteins

KW - Cells, Cultured

KW - Clathrin

KW - Clathrin-Coated Vesicles

KW - Cytochrome b Group

KW - Endosomes

KW - Exocytosis

KW - Intracellular Signaling Peptides and Proteins

KW - Macrophage Activation

KW - Macrophages

KW - Membrane Glycoproteins

KW - Microglia

KW - NADPH Oxidase

KW - Rats

KW - Superoxides

KW - Tumor Necrosis Factor-alpha

KW - rab GTP-Binding Proteins

U2 - 10.1074/jbc.M111.293696

DO - 10.1074/jbc.M111.293696

M3 - Journal article

C2 - 22157766

SN - 0021-9258

VL - 287

SP - 4835

EP - 4852

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 7

ER -