Specific Role of Neutrophil Inducible Nitric Oxide Synthase in Murine Sepsis-Induced Lung Injury In Vivo

Abstract

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes importantly to acute lung injury (ALI), but the specific contribution of neutrophil iNOS has not been defined. Thus, we defined the role of neutrophils and specifically neutrophil iNOS in a murine model of septic ALI. Four hours after cecal ligation/perforation, ALI was characterized by increases in pulmonary neutrophil infiltration (tissue myeloperoxidase activity, bronchoalveolar lavage neutrophils), microvascular leak of Evans blue (EB) dye-labeled albumin, and oxidant stress (8-isoprostane levels). Septic ALI was neutrophil dependent, as pretreatment with anti-CD18 before cecal ligation/perforation significantly (P < 0.05) attenuated septic increases in pulmonary myeloperoxidase (39 ± 11 vs. 85 ± 14 mU/mg protein), bronchoalveolar lavage neutrophils (0.5% ± 0.2% vs. 2.1% ± 0.6%), microvascular EB-albumin leak (1.3 ± 0.3 vs. 2.6 ± 0.7 μg EB/g per minute), and 8-isoprostane content (74 ± 15 vs. 115 ± 16 pg/mg protein). The role of neutrophil iNOS was assessed by creation of neutrophil-iNOS chimeric mice: iNOS(+/+) versus iNOS(-/-) mice were bone marrow depleted by irradiation and selectively reconstituted with iNOS(+/+) versus iNOS(-/-) neutrophils. Cecal ligation/perforation resulted in significant septic ALI in + to - neutrophil-iNOS chimeric mice (iNOS(+/+) neutrophils in iNOS(-/-) mice), but not in - to + neutrophil depleted-reconstituted mice (iNOS(-/-) neutrophils in iNOS(+/+) mice). There were no significant differences between iNOS(+/+) and iNOS(-/-) neutrophils in phagocytosis, respiratory burst, or CD11a/b/CD18 surface expression, although septic shedding of CD62L was blunted in iNOS(-/-) neutrophils. Neutrophil iNOS contributes importantly to murine septic ALI in vivo, but not simply through a change in neutrophil phenotype. We speculate that neutrophil iNOS may modulate neutrophil-endothelial interactions in complex fashion, including regulation of transendothelial neutrophil migration and pulmonary neutrophil infiltration.