The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS−/−, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to − (iNOS+/+ donor bone marrow-transplanted into iNOS−/− recipient mice) and − to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS−/− mice and in − to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to − chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.

Nitric oxide (NO) derived from inducible NO synthase (iNOS) contributes to the pathophysiology of acute lung injury (ALI). The effect of iNOS on pulmonary neutrophil infiltration in ALI is not known. Thus, we assessed pulmonary microvascular neutrophil sequestration through intravital videomicroscopy and pulmonary neutrophil infiltration, reflected by myeloperoxidase activity and lavage neutrophil counts, after induction of sepsis by cecal ligation/perforation in wild-type (iNOS+/+) versus iNOS−/− mice. Pulmonary microvascular neutrophil sequestration was attenuated in septic iNOS−/− versus iNOS+/+ mice (15 ± 1 vs. 20 ± 1 leukocytes per field, p < 0.05), but lavage neutrophil counts were greater in iNOS−/− mice (5.7 ± 1.5% vs. 0.7 ± 0.1%, p < 0.05) between 6 and 18 hours after cecal ligation and perforation. When iNOS+/+ bone marrow was transplanted into bone marrow–depleted iNOS−/− mice (+ to − chimeras; iNOS limited to marrow-derived inflammatory cells), septic pulmonary microvascular neutrophil sequestration and lavage neutrophil counts were restored to levels seen in septic iNOS+/+ mice. In contrast, in − to + chimeras, pulmonary neutrophil trafficking was similar to iNOS−/− mice. In vitro cytokine-stimulated neutrophil transendothelial migration was significantly greater for iNOS−/− versus iNOS+/+ neutrophils (7.9 ± 0.7% vs. 3.8 ± 0.6%, p < 0.05) but was independent of endothelial iNOS. Thus, neutrophil iNOS-derived NO is an important autocrine modulator of pulmonary neutrophil infiltration in murine sepsis.

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS−/− AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 ± 0.1 vs. 1.4 ± 0.1 μg EB·g lung⁻¹·min⁻¹; P < 0.05) and MPO activity (37 ± 4 vs. 67 ± 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 ± 0.3 μg EB·g lung⁻¹·min⁻¹) but not with iNOS−/− donor AM. In iNOS−/− mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS−/− mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.

Although inhaled nitric oxide transiently improves oxygenation in patients with acute lung injury, it has not affected clinical outcomes. As well, the effects of inhaled nitric oxide on the pathophysiologic features of acute lung injury have not been well defined. Therefore, we assessed the effects of inhaled nitric oxide on the degree of pulmonary inflammation and injury in a mouse model of sepsis-induced acute lung injury. Randomized, controlled animal study. Research laboratory of an academic institution. Male C57Bl/6 mice. Sepsis was induced by cecal ligation and perforation. At the time of surgery, septic and naïve mice were randomized to exposure to either 40 ppm inhaled nitric oxide or room air for 24 hrs before they were killed. Sepsis-induced acute lung injury was characterized by increased pulmonary myeloperoxidase (68 ± 13 vs. 13 ± 3 mU/mg protein in naïve mice, p < .01), pulmonary 8-isoprostane content (627 ± 51 vs. 88 ± 20 pg/mg protein in naïve mice, p < .01), and protein in bronchoalveolar lavage fluid (p < .05). Inhaled nitric oxide exposure in septic mice completely abrogated the septic increases in myeloperoxidase activity (p < .05) and pulmonary 8-isoprostane content (p < .05) but had no effect on bronchoalveolar lavage protein. The induction of sepsis also was associated with an increase in pulmonary inducible NO synthase activity (2.8 ± 0.5 vs. 0.4 ± 0.1 pmol·min−1·mg−1 protein in naïve mice, p < .05), and inhaled nitric oxide attenuated this increase in pulmonary inducible NO synthase activity (p < .05). Exposure to inhaled nitric oxide early in the course of sepsis-induced acute lung injury is associated with reduced pulmonary leukocyte infiltration and less oxidative injury. Decreased lung inflammation and injury with inhaled nitric oxide is associated with decreased pulmonary inducible NO synthase activity. Therefore, inhaled NO may have greater clinical benefit if administered earlier in the natural history of acute lung injury in patients.

Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS. Randomized, controlled animal study. Research laboratory of an academic institution. Male iNOS+/+, iNOS−/− C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to− (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS−/− recipient mice) and the reciprocal −to+ chimeras. Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naïve groups (n = 138). In septic iNOS−/− vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 ± 11 [mean ± sem] vs. 365 ± 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 ± 0.0 vs. 0.9 ± 0.4 μmol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 ± 20 vs. 83 ± 12 mU/mg protein, p = .78). In +to− iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 ± 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 ± 0.2 μmol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic −to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS−/− mice. In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.

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.