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 microg 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 microg 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.