Superoxide production by phagocytosing macrophages in relation to the intracellular distribution of oxygen

We simultaneously measured the concentration of oxygen ([O₂]) within the phagosomal and extracellular compartments of macrophages. By combining electron paramagnetic resonance (EPR) oximetry techniques with that of spintrapping, we found that a significant difference in oxygen concentration ([O₂]) exists between these two compartments and we were able to monitor (1) how [O₂] in the extracellular compartment and the rate of mitochondrial consumption affected this difference in [O₂], and (2) to what extent this gradient of [O₂] influenced production of reactive oxygen species by phagosomes. Under conditions where the [O₂] in the inflowing gas was high (210 μM; air), the [O₂] in the extracellular and phagosomal compartments was 180 and 141 μM, respectively. This was sufficient to maintain maximum superoxide production in these cells. When extracellular [O₂] was reduced to 84 or 36 μM, the [O₂] in phagosomes within the cells (31.7 and 7.7 μM, respectively) was too low to maintain superoxide production by the NADPH- oxidase system within the phagosomes. The [O₂] in the extracellular compartments of these samples, however, was always sufficient to maintain superoxide production by phagosomes at the cell surface. Our findings suggest that the distribution of oxygen surrounding and within macrophages can influence their ability to perform microbicidal and tumoricidal functions, even at an [O₂] in the media that appears to be adequate.