Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice.
A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO) production secondary to induction of inducible NO synthase (iNOS) is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium. C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle. Twelve hours later, leakage of FITC-dextran (M(r) 4 kDa; FD4) from blood into bronchoalveolar lavage fluid was significantly increased in endotoxemic but not control mice. This decrease in bronchoalveolar barrier function was associated with upregulation of iNOS protein expression and NF-kappaB activation in lung tissue. Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluorescence, decreased in lung after the injection of mice with LPS. Treatment of endotoxemic mice with an isoform-selective iNOS inhibitor [l-N(6)-(1-iminoethyl)lysine; l-NIL] ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function. Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1,000 U/ml IFN-gamma, 10 ng/ml TNF-alpha, and 1 ng/ml IL-1beta) increased permeability to FD4, but adding l-NIL prevented this effect. These results suggest that decreased expression and mistargeting of TJ proteins in lung after systemic inflammation may be NO dependent.
Han
Xiaonan
X
Department of Critical Care Medicine, 616 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261, USA.
Fink
Mitchell P
MP
Uchiyama
Takashi
T
Yang
Runkuan
R
Delude
Russell L
RL
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D003911:Dextrans / Q000493:pharmacokinetics; D019446:Endotoxemia / Q000378:metabolism* / Q000503:physiopathology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000201:enzymology* / Q000503:physiopathology; D019108:Tight Junctions / Q000378:metabolism*; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D002352:Carrier Proteins; D003911:Dextrans; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; C569047:Tjp2 protein, mouse; C491774:Tjp3 protein, mouse; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C015219:fluorescein isothiocyanate dextran; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D016650:Fluorescein-5-isothiocyanate