Alveolar macrophage induced septic pulmonary microvascular endothelial cell injury is inducible nitric oxide synthase‐dependent

Abstract

We previously found that alveolar macrophage (AM) inducible nitric oxide synthase (iNOS) appeared essential for pulmonary microvascular protein leak in septic mice. However, the direct role of AM iNOS in septic lung injury or endothelial injury has not been addressed. Thus, we assessed the effects of AM iNOS on pulmonary microvascular endothelial cell (PMVEC) injury under septic conditions in vitro AM-PMVEC co-culture. Murine PMVEC were utilized as protein leak and neutrophil migration predominantly occur in the pulmonary microvasculature during septic lung injury. PMVEC were isolated and seeded on Transwell inserts (3.0ìm pore) and allowed to grow to confluence. In AM-PMVEC co-cultures stimulated with 3ng/ml cytomix (equal concentrations of TNF-á, IL-â and IFN-ã) for 9 hours, the permeability of the PMVEC monolayer was assessed by the degree of Evans blue labeled albumin leak across the monolayer in the final hour of stimulation. The presence of wildtype (iNOS+/+) AM in the upper compartment dose-dependently (1:1 and 3:1) increased trans-PMVEC albumin leak (80.7 ± 18% of control in 1:1 vs. 265.1 ± 28% of control in 3:1). In contrast, presence of AM in the lower compartment had no effect on cytomix-stimulated PMVEC albumin leak (128 ± 30% of control). When compared to co-culture of PMVEC with iNOS+/+ AM, presence of iNOS knockout (−/−) AM, in a 3:1 ratio of AM to PMVEC, had no effect on cytomix-stimulated trans-PMVEC albumin leak. These data are consistent with a direct role of AM iNOS in the septic injury of PMVEC. This suggests an important role for AM iNOS in septic lung injury, and a possible new therapeutic approach.