FIGURE 8.

ATP, TNF-α, and CD40L induce exocytosis of cyt b₅₅₈-containing vesicles to the cell surface, which is followed by re-internalization of cyt b₅₅₈. A, Ra2 microglia were stimulated with 1 mm ATP for 15 min and then chased in the absence of ATP for a further 10, 20, or 40 min. Then cell surface levels of gp91^phox were determined by a FACS protocol on fixed unpermeabilized cells using 8G11, an anti-gp91^phox mAb with an extracellular epitope on gp91^phox. The graph depicts background-subtracted (isotype-matched control) mean fluorescence intensity of 8G11 staining normalized to unstimulated control Ra2 cells and represents mean ± S.E. (n = 3). RFLU, relative fluorescent light units. B, Ra2 cells expressing CFP-gp91^phox were stimulated with 1 mm ATP, and live cell imaging was performed. Shown are 1.0-μm confocal medial sections of a single cell over time (indicated in seconds) before and after ATP stimulation (at 250 s). Note that CFP-gp91^phox fluorescence is redistributed in part from cytosol to the cell surface. A histogram of CFP fluorescence intensity as a function of linear distance across the cell (red line in images) is depicted below each confocal image. Bar, 10 μm. C, cell surface expression of gp91^phox in Ra2 microglia after stimulation with 100 ng/ml CD40L or 20 ng/ml TNFα was followed over time (indicated in minutes) by cell surface biotinylation with NHS-biotin. The anti-gp91^phox Western blot shown is representative of three independent experiments and shows streptavidin-precipitate (upper lanes) and cell lysate load control (C, lower lanes). D, mean ± S.E. (n = 3) densitometric values for gp91^phox Western blots as shown above. The ordinate in arbitrary units represents gp91^phox band optical density of streptavidin-precipitates normalized to load control. E, PMA-evoked production of superoxide by Ra2 microglia or primary microglia stimulated with CD40L or TNFα was estimated by NBT reduction assay with or without inclusion of 400 units/ml SOD. The ordinate represents the fraction of SOD-sensitive (extracellular) to total NBT (no SOD) absorbance at 570 nm after 15 or 30 min of PMA stimulation for CD40L and TNFα, respectively. Mean ± S.E. (n = 3) is shown.