title: Human neutrophil-pulmonary microvascular endothelial cell interactions in vitro: differential effects of nitric oxide vs. peroxynitrite.
abstract: Sepsis-induced acute lung injury is characterized by activation and injury of pulmonary microvascular endothelial cells (PMVEC), increased neutrophil-PMVEC adhesion and migration, and trans-PMVEC high-protein edema. Inducible NO synthase (iNOS) inhibits septic murine neutrophil migration in vivo and in vitro. The effects of NO in human neutrophil-PMVEC interactions are not known. We isolated human PMVEC using magnetic bead-bound anti-PECAM antibody. Confluent PMVEC at passage 3-4 were co-cultured with human neutrophils for assessment of neutrophil-PMVEC adhesion, and trans-PMVEC neutrophil migration and Evans-Blue dye-labeled albumin leak. Two NO donors (spermine-NONOate, S-nitroso-N-acetylpenicillamine) attenuated both cytomix-enhanced neutrophil-PMVEC adhesion by 64+/-14% (p<0.01) and 32+/-3% (p<0.05), respectively, and cytomix-induced trans-PMVEC neutrophil migration by 85+/-16% (p<0.01) and 43+/-5% (p<0.01), respectively. Correspondingly, iNOS inhibition with 1400W enhanced cytomix-stimulated neutrophil migration by 52+/-3% (p<0.01), but had no effect on neutrophil-PMVEC adhesion. Conversely, a peroxynitrite donor (SIN-1) increased both neutrophil-PMVEC adhesion (38+/-2% vs. 14+/-1% control, p<0.01) and trans-PMVEC neutrophil migration; with both effects were completely inhibited by scavenging of NO, superoxide, or peroxynitrite (p<0.05 for each). Scavenging of peroxynitrite also eliminated cytomix-induced neutrophil adhesion and migration. Blocking CD18-dependent neutrophil adhesion prevented cytomix-stimulated trans-PMVEC EB-albumin leak (p<0.05), while inhibiting neutrophil migration paradoxically enhanced cytomix-stimulated EB-albumin leak (11+/-1% vs. 7+/-0.5%, p<0.01). FMLP-induced neutrophil migration had no effect on trans-PMVEC EB-albumin leak. In summary, we report differential effects, including the inhibitory action of NO and stimulatory effect of ONOO(-) on human neutrophil-PMVEC adhesion and trans-PMVEC migration under cytomix stimulation. Moreover, neutrophil-PMVEC adhesion, but not trans-PMVEC migration, contributes to human PMVEC barrier dysfunction.
authors.affiliation: Department of Medicine, Division of Respirology, London Health Sciences Center, Centre for Critical Illness Research, Lawson Health Research Institute University of Western Ontario, London, Ontario Canada.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Shelton
authors.forename: Lefeng
authors.initials: L
authors.lastname: Wang
authors.forename: Gediminas
authors.initials: G
authors.lastname: Cepinskas
authors.forename: Richard
authors.initials: R
authors.lastname: Inculet
authors.forename: Sanjay
authors.initials: S
authors.lastname: Mehta
mesh_terms: D000071443:12E7 Antigen; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000276:immunology; D018821:CD18 Antigens / Q000276:immunology; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000276:immunology; D002450:Cell Communication / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology
chemical_list: D000071443:12E7 Antigen; C466420:5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D016207:Cytokines; D007097:Imines; D008665:Metalloporphyrins; C496401:N-((3-(aminomethyl)phenyl)methyl)ethanimidamide; D020030:Nitric Oxide Donors; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D009240:N-Formylmethionine Leucyl-Phenylalanine; C002385:linsidomine; D008981:Molsidomine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II