NOS-2 Inhibition in Phosgene-Induced Acute Lung Injury

Abstract

Phosgene exposure via an industrial or warfare release produces severe acute lung injury (ALI) with high mortality, characterized by massive pulmonary edema, disruption of epithelial tight junctions, surfactant dysfunction, and oxidative stress. There are no targeted treatments for phosgene-induced ALI. Previous studies demonstrated that nitric oxide synthase 2 (NOS-2) is upregulated in the lungs after phosgene exposure; however, the role of NOS-2 in the pathogenesis of phosgene-induced ALI remains unknown. We previously demonstrated that NOS-2 expression in lung epithelium exacerbates inhaled endotoxin-induced ALI in mice, mediated partially through downregulation of surfactant protein B (SP-B) expression. Therefore, we hypothesized that a selective NOS-2 inhibitor delivered to the lung epithelium by inhalation would mitigate phosgene-induced ALI. Inhaled phosgene produced increases in bronchoalveolar lavage fluid protein, histologic lung injury, and lung NOS-2 expression at 24 h. Administration of the selective NOS-2 inhibitor 1400 W via inhalation, but not via systemic delivery, significantly attenuated phosgene-induced ALI and preserved epithelial barrier integrity. Furthermore, aerosolized 1400 W augmented expression of SP-B and prevented downregulation of tight junction protein zonula occludens 1 (ZO-1), both critical for maintenance of normal lung physiology and barrier integrity. We also demonstrate for the first time that NOS-2-derived nitric oxide downregulates the ZO-1 expression at the transcriptional level in human lung epithelial cells, providing a novel target for ameliorating vascular leak in ALI. Our data demonstrate that lung NOS-2 plays a critical role in the development of phosgene-induced ALI and suggest that aerosolized NOS-2 inhibitors offer a novel therapeutic strategy for its treatment.

Keywords: acute lung injury; lung epithelium; nitric oxide synthase 2; phosgene; surfactant protein B; tight junction protein 1.

FIG. 1.

Lung NOS-2 expression is elevated during phosgene-induced ALI. Mice were exposed to filtered air or phosgene and assessed for lung injury by measuring BAL fluid protein levels (A) and NOS-2 expression (relative to the 18S control) was measured by real time qPCR 24 h after phosgene exposure (B) (n = 5; mean ± SEM, P-value * < 0.05, *** < 0.001). Lungs of mice exposed to filtered air (C) or phosgene (D) were stained for NOS-2 protein 24 h after exposure (positive epithelial staining is indicated by arrows and positive inflammatory cell staining is indicated by arrowheads, scale bar corresponds to 50 µm).

FIG. 2.

Inhaled 1400  W mitigates phosgene-induced lung injury, while there is no beneficial effect after systemic 1400 W delivery. Mice were exposed to filtered air or phosgene, with administration of systemic 1400 W or vehicle (PBS) (A, C) or inhaled 1400 W or vehicle (PBS) (B, D), and harvested 24 h after exposure. The level of protein in BAL fluid (upper panel) and total BAL cell number (middle panel) were analyzed (n = 5; mean ± SEM, P-value * < 0.05). Mice exposed to filtered air (E) versus phosgene+aerosolized vehicle (F) versus phosgene + aerosolized 1400 W (G) were analyzed for histological signs of lung injury (scale bar corresponds to 50 µm).

FIG. 3.

Inhaled 1400 W inhibits nitrotyrosine accumulation within the lung injured with phosgene. Mice exposed to filtered air, phosgene + inhaled vehicle (PBS) or phosgene + inhaled 1400 W were euthanized 24 h after exposure, and the lungs were stained for nitrotyrosine. Images of the alveolar parenchyma (C) and the airways (D) were captured (scale bar corresponds to 50 µm), and the intensity of nitrotyrosine staining for alveolar parenchyma (A) and airways (B) was assessed using ImageJ (n = 30 pictures captured of the lungs from 3 mice per experimental condition were analyzed, mean ± SEM, P-value *** < 0.001).

FIG. 4.

The NOS-2 inhibitor 1400 W augments levels of the critical protein SP-B. Mice were exposed to phosgene or filtered air, administered with 1400 W or vehicle (PBS) systemically (A) or by inhalation (B), and euthanized 24 h later. The mice were analyzed for SP-B mRNA levels by real time qPCR (n = 5, mean ± SEM, P-value * < 0.05, ** < 0.01, *** < 0.001). Lungs harvested from mice subjected to filtered air (C), phosgene + aerosolized vehicle (D) or phosgene + aerosolized 1400 W (E) were stained for SP-B (examples of positive staining in lung epithelial cells indicated by arrows, scale bar corresponds to 50 µm).

FIG. 5.

Inhalation of 1400 W restores expression of the tight junction protein ZO-1 after phosgene-induced lung injury. Mice were exposed to phosgene or filtered air, administered with 1400 W or vehicle (PBS) systemically (A) or by inhalation (B), and euthanized 24 h later. The lung tissues were analyzed for ZO-1 mRNA levels by real time qPCR (n = 4, mean ± SEM, P-value * < 0.05, *** < 0.001). Lung sections from animals exposed to filtered air, phosgene + aerosolized vehicle or phosgene + aerosolized 1400 W were stained for ZO-1 protein by immunohistochemistry and counterstained with hematoxylin (C, scale bar corresponds to 150 µm), or stained by immunofluorescence (red) and counterstained with DAPI (blue) (D, scale bar corresponds to 50 µm).

FIG. 6.

NOS-2-derived NO downregulates ZO-1 gene expression in human lung epithelial cells in vitro. A549, HBEC-2 and HBEC-3 lung epithelial cells were incubated with the NO donor DETA NONOate (0.1, 0.5, or 1 mM) for 24 h, harvested for RNA isolation and analyzed for ZO-1 mRNA levels using real time qPCR (A) (n = 4, mean ± SEM). A549, HBEC-2, and HBEC-3 cells were transfected with empty, GFP-coding or NOS-2-coding plasmids. Transfection efficacy was estimated by microscopic observation of the GFP-coding plasmid transfected cells (B). Cells transfected with empty or NOS-2-coding plasmids were harvested for RNA isolation and analyzed for the ZO-1 mRNA levels using real time qPCR (C) (n = 4, mean ± SEM). For both (A) and (C) statistical significane is defined as a P-value * < 0.05, ** < 0.01, or *** < 0.001.