Inducible NO synthase (iNOS) in human neutrophils but not pulmonary microvascular endothelial cells (PMVEC) mediates septic protein leak in vitro

Sepsis-induced acute lung injury (ALI) is characterized by injury of the pulmonary microvascular endothelial cells (PMVEC) leading to high-protein pulmonary edema. Inducible NO synthase (iNOS) mediates trans-PMVEC protein leak in septic mice in vivo and in murine PMVEC under septic conditions in vitro, but the role of iNOS in human PMVEC protein leak has not been addressed. We hypothesized that iNOS in human neutrophils, but not human PMVEC, mediates septic trans-PMVEC protein leak in vitro. We isolated human PMVEC from lung tissue using magnetic bead-bound anti-PECAM antibody and assessed Evans blue albumin leak across human PMVEC monolayers under septic conditions in the presence/absence of human neutrophils. PMVEC were used at passages 3-4, seeded on 3 mu m Transwell inserts and grown to confluence. Cytomix-stimulated trans-PMVEC albumin leak was not attenuated by pre-treatment with 1400 W, a selective iNOS inhibitor, or L-NAME, a nonselective NOS inhibitor. In neutrophil-PMVEC co-culture, basal unstimulated trans-EB-albumin leak was 0.6 +/- 0.3%, which was increased by cytomix stimulation to 11.5 +/- 4.4%, p < 0.01. Cytomix-stimulated EB-albumin leak in neutrophil-PMVEC co-cultures was inhibited by pre-treatment with 1400 W (3.8 +/- 1.0%,p < 0.05) or L-NAME (4.0 +/- 1.1%,p < 0.05). Pre-treatment of neutrophil-PMVEC co-cultures with PEG-SOD (superoxide scavenger) and FeTPPS (peroxynitrite scavenger) also significantly attenuated neutrophil-dependent cytomix-stimulated leak (4.7 +/- 3.0%,p < 0.05; 0.5 +/- 1.0%,p < 0.01, respectively). In conclusion, trans-human PMVEC albumin leak under septic conditions is dependent on iNOS activity specifically in neutrophils, but not in PMVEC themselves. Septic neutrophil-dependent trans-PMVEC albumin leak may be mediated by peroxynitrite. (c) 2007 Elsevier Inc. All rights reserved.