Figure 6. ATRA-induced oxidative ability.

A. Induction of Nox2 activation during differentiation. Cells were allowed to phagocytose IgG-opsonized BMJ71 bacteria for 5 min (bacteria/cells, 2∶1), or were stimulated with 160 nM PMA, in the presence of 1 mg/ml NBT. Measurement of absorbance was used to quantitate the intracellular respiratory burst. Data are presented as the ratio between formazan formation in ATRA-treated cells compared to control cells. B. Localization of formazan formation. Differentiated HL-60 cells interacting with IgG-opsonized Oregon Green-labeled BMJ71 bacteria were incubated with 1 mg/ml NBT. A bacteria/cell ratio of 10∶1 was used. Respiratory burst activity was indicated by blue-black formazan precipitates, visible by light microscopy (iii, vi). Oregon Green fluorescence (i, iv) shows the total number of bacteria, and anti-streptococcal staining (ii, v) shows the location of extracellular bacteria. In the upper panel, precipitate formation on cell-adherent bacteria is illustrated. The lower panel shows that NBT precipitates may also be found on internalized bacteria. Size bar: 10 µm. C. Quantitation of respiratory burst-positive cells. The diagram shows the proportion of bacteria-interacting cells that display formazan precipitates and the effect of DPI (10 µM). At least 50 cells per sample were analyzed. Error bars show SEM, based on a total of three experiments.