title: Modulation of neutrophil apoptosis by murine pulmonary microvascular endothelial cell inducible nitric oxide synthase.
abstract: Neutrophils contribute significantly to ALI (acute lung injury) through adhesion to pulmonary microvascular endothelial cells (PMEC), trans-PMEC migration and alveolar infiltration. Trans-PMEC migration delays expression of neutrophil apoptosis, which promotes intra-alveolar neutrophil survival and neutrophil mediated ALI. We assessed the role of neutrophil vs PMEC inducible nitric oxide (NO) synthase (iNOS) in modulating neutrophil apoptosis. Apoptosis of wild-type vs iNOS-/- neutrophils was quantified by microscopy and FACS annexin-V binding. In a murine model of ALI, neutrophils isolated by BAL(broncho-alveolar lavage) from iNOS-/- mice had increased expression of apoptosis after 24h culture ex vivo than wild-type neutrophils (15.2±3.3 vs 3.0±0.4%, mean±sd, p<0.01). Apoptosis rates of isolated bone marrow iNOS+/+ vs iNOS-/- neutrophils were similar under basal and LPS/IFN-γ stimulation, and following LPS/IFN-γ-stimulated trans-PMEC migration. Apoptosis of both iNOS+/+ and iNOS-/- neutrophils was inhibited by trans-PMEC migration only across iNOS+/+ PMEC (1.6±0.3 and 1.5±0.3%, respectively; p<0.05 for each vs non-migrated neutrophils) but not across iNOS-/- PMEC (4.3±1 and 3.1±0.6%, respectively). PMEC iNOS-dependent inhibition of neutrophil apoptosis was independent of changes in neutrophil caspase-3 activity. We conclude that PMEC iNOS, but not neutrophil iNOS, has an important inhibitory effect on neutrophil apoptosis during trans-PMEC neutrophil migration, which is independent of caspase-3 activity. Further studies will define the mechanism of PMEC iNOS-dependent inhibition of neutrophil apoptosis and assess the potential relevance of this phenomenon in human neutrophils and ALI.
authors.affiliation: Centre for Critical Illness Research, Lawson Health Research Institute, London Health Sciences Center, London, Ontario, Canada.
authors.forename: Lefeng
authors.initials: L
authors.lastname: Wang
authors.forename: Sanjay
authors.initials: S
authors.lastname: Mehta
authors.forename: Chris
authors.initials: C
authors.lastname: Gillis
authors.forename: Cedrin
authors.initials: C
authors.lastname: Law
authors.forename: Ravi
authors.initials: R
authors.lastname: Taneja
mesh_terms: D000818:Animals; D017209:Apoptosis; D018893:Bronchoalveolar Lavage; D053148:Caspase 3 / Q000378:metabolism; D002465:Cell Movement; D004730:Endothelium, Vascular / Q000201:enzymology*; D008168:Lung / Q000098:blood supply*; D051379:Mice; D008817:Mice, Mutant Strains; D055806:Microvessels / Q000166:cytology / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*
chemical_list: D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D053148:Caspase 3