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

Macrophages count on two O ₂ -consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O ₂ ^{• -} ) and nitric oxide ( ^• NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O ₂ ^{• -} and ^• NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO ₂ ) normally ranges within 2-14 %, with a typical average pO ₂ value for most tissues ca. 5 %. Given that O ₂ is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O ₂ ^{• -} and ^• NO production. Also, O ₂ 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 O ₂ ^{• -} and ^• NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO ₂ ; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O ₂ ) which are far from the physiological pO ₂ . Here, the role of O ₂ as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO ₂ and O ₂ ^{• -} and ^• NO formation rates were measured, obtaining a K _M of 26 and 42 μM O ₂ for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO ₂ , reaching a maximum at ≥ 10 % O ₂ , but even at levels as low as 2 % O ₂ , 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 pO ₂ values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.
Competing Interests: Declaration of competing interest The authors have no competing interests to declare.
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