Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions

Macrophages count on two O₂-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O₂^•-) and nitric oxide (^•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O₂^•- and ^•NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO₂) normally ranges within 2-14 %, with a typical average pO₂ value for most tissues ca. 5 %. Given that O₂ is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O₂^•- and ^•NO production. Also, O₂ is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O₂^•- and ^•NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO₂; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O₂) which are far from the physiological pO₂. Here, the role of O₂ as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO₂ and O₂^•- and ^•NO formation rates were measured, obtaining a K_M of 26 and 42 μM O₂ for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO₂, reaching a maximum at ≥ 10 % O₂, but even at levels as low as 2 % O₂, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO₂ values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.