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

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.