title: Alveolar macrophage inducible nitric oxide synthase-dependent pulmonary microvascular endothelial cell septic barrier dysfunction.
abstract: Inducible nitric oxide (NO) synthase (iNOS) from neutrophils and alveolar macrophages (AM) contributes to the pathophysiology of murine septic acute lung injury (ALI). It is not known if AM iNOS has a direct effect on septic pulmonary microvascular endothelial cell (PMVEC) permeability. We hypothesized that AM iNOS mediates PMVEC permeability in vitro under septic conditions through NO and peroxynitrite. 100,000 confluent PMVEC on cell-culture inserts were co-incubated with iNOS+/+ vs. iNOS-/- AM, in various ratios of AM to PMVEC. PMVEC injury was assessed by trans-PMVEC Evans Blue-labelled albumin flux in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta and IFN-gamma). Cytomix stimulation dose-dependently increased trans-PMVEC EB-albumin flux, which was exaggerated (1.4+/-0.1% vs. 0.4+/-0.1% in unstimulated PMVEC, p<0.05) in the presence of iNOS+/+, but not iNOS-/-, AM in the upper compartment. Similarly, iNOS+/+, but not iNOS-/-, AM in the lower compartment also enhanced septic trans-PMVEC albumin leak. The mechanism of iNOS-dependent septic PMVEC permeability was pursued through pharmacologic studies with inhibitors of NOS, and scavengers of NO, superoxide, and peroxynitrite, and treatment of PMVEC with the NO donor, DETA-NONOate. Septic iNOS+/+ AM-dependent trans-PMVEC albumin leak was significantly attenuated by pharmacologic iNOS inhibition (L-NAME and 1400W), and scavenging of either NO (oxyhemoglobin), superoxide (PEG-SOD), or peroxynitrite (FeTPPS). Exogenous NO (DETA-NONOate) had no effect on PMVEC permeability. These data are consistent with a direct role of AM iNOS in septic PMVEC barrier dysfunction, which is likely mediated, in part, through peroxynitrite.
authors: Centre for Critical Illness Research, Division of Respirology, Lawson Health Research Institute, London Health Sciences Center, London, Ontario, Canada.
authors: K S
authors: KS
authors: Farley
authors: L F
authors: LF
authors: Wang
authors: C
authors: C
authors: Law
authors: S
authors: S
authors: Mehta
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D042783:Endothelial Cells / Q000502:physiology; D008168:Lung / Q000098:blood supply* / Q000503:physiopathology*; D016676:Macrophages, Alveolar / Q000201:enzymology* / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055806:Microvessels / Q000503:physiopathology; D052247:Nitric Oxide Synthase Type II / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000378:metabolism; D018805:Sepsis / Q000503:physiopathology*; D013481:Superoxides / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D013481:Superoxides; D030421:Peroxynitrous Acid; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse