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 mum 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 non-selective 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.
Shelton
Jennifer L
JL
Centre for Critical Illness Research, Lawson Health Research Institute, Division of Respirology, Department of Medicine, London Health Sciences Center-Victoria Hospital, University of Western Ontario, 800 Commissioner's Road, London, Ontario, Canada.
Wang
Lefeng
L
Cepinskas
Gediminas
G
Sandig
Martin
M
Scott
Jeremy A
JA
North
Michelle L
ML
Inculet
Richard
R
Mehta
Sanjay
S
D000418:Albumins / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D008168:Lung / Q000098:blood supply*; D008833:Microcirculation / Q000187:drug effects / Q000201:enzymology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D018805:Sepsis / Q000201:enzymology*; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology
D000418:Albumins; D016166:Free Radical Scavengers; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019331:NG-Nitroarginine Methyl Ester