Pulmonary ischemia/reperfusion lung injury is an important phenomenon encountered in many clinical situations, including lung transplantation and thrombolysis after pulmonary embolism. Pulmonary IR results in endothelial damage and dysfunction leading to the development of high permeability pulmonary edema. Reactive oxygen species (ROS) have been implicated in the pathogenesis of lung ischemia/reperfusion (IR) injury, although their source(s) remains unclear.We studied the role of phagocyte-type NADPH oxidase in this process, using a model of isolated buffer-perfused mouse lungs. Ischemia was induced by stopping perfusion for 90 min. Lung injury was quantified by assessment of lung vascular permeability and fluid accumulation during reperfusion. Production of ROS was measured during reperfusion in perfusate samples from venous outflow by electron spin resonance spectroscopy using the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine. The relative contribution of NADPH oxidase expressed on resident leukocytes versus NADPH oxidase on endothelial cells was further studied in bone-marrow (BM)-transplanted chimeras.In wild-type mouse lungs, IR resulted in increased vascular permeability and edema formation that was associated with increased intravascular ROS release. Pretreatment with superoxide dismutase or apocynin, an NADPH oxidase inhibitor, significantly attenuated lung injury and ROS generation. Nox2 is the catalytic subunit of phagocyte-type NADPH oxidase. Decreased tissue injury in lungs from Nox2 knockout mice was associated with reduced ROS production.The relative contribution of NADPH oxidase expressed by resident leukocytes versus endothelial cells was further studied in bone marrow-transplanted chimeras. When NADPH oxidase in resident leukocytes was selectively inactivated by transplanting bone marrow cells from Nox2 knockout mice into wild-type animals, IR-induced tissue injury was comparable to that in wild-type mouse lungs. Conversely, transplanting bone marrow cells from wild-type mice into Nox2 knockout animals selectively reconstituted NADPH oxidase from resident leukocytes and offered protection against IR-induced lung injury.Collectively, these findings suggest that endothelial NADPH oxidase contributes to lung injury in early reperfusion, but leukocyte NADPH oxidase does not.
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