Multiple contributing roles for NOS2 in LPS-induced acute airway inflammation in mice

Abstract

Acute lung inflammation and injury were induced by intranasal instillation of lipopolysaccharide (LPS) in normal and type 2 nitric oxide synthase (NOS2)-deficient (NOS2 Ϫ/Ϫ ) C57BL/6 mice. LPS-induced increases in extravasated airway neutrophils and in lung lavage fluid of TNF-␣ and macrophage inflammatory protein-2 were markedly lower in NOS2 Ϫ/Ϫ than in wild-type mice, indicating that NOS2-derived nitric oxide (NO⅐) participates in inflammatory cytokine production and neutrophil recruitment. Instillation of LPS also increased total lung lavage protein and induced matrix metalloproteinase-9 and mucin 5AC, as indexes of lung epithelial injury and/or mucus hyperplasia, and increased tyrosine nitration of lung lavage proteins, a marker of oxidative injury. All these responses were less pronounced in NOS2 Ϫ/Ϫ than in wild-type mice. Inhibition of NOS activity also suppressed production of TNF-␣ and macrophage inflammatory protein-2 by LPS-stimulated mouse alveolar MH-S macrophages, and this was restored by NO⅐ donors, illustrating involvement of NO⅐ in macrophage cytokine signaling. Oligonucleotide microarray (GeneChip) analysis of global lung gene expression revealed that LPS inhalation induced a range of transcripts encoding proinflammatory cytokines and chemokines, stress-inducible factors, and other extracellular factors and suppressed mRNAs encoding certain cytoskeletal proteins and signaling proteins, responses that were generally attenuated in NOS2 Ϫ/Ϫ mice. Comparison of both mouse strains revealed altered expression of several cytoskeletal proteins, cell surface proteins, and signaling proteins in NOS2 Ϫ/Ϫ mice, changes that may partly explain the reduced responsiveness to LPS. Collectively, our results suggest that NOS2 participates in the acute inflammatory response to LPS by multiple mechanisms: involvement in proinflammatory cytokine signaling and alteration of the expression of various genes that affect inflammatory-immune responses to LPS.

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

“…Experiments utilizing NO synthase inhibitors or iNOS knockout mice in IT LPS or VILI models underscore the critical importance of iNOS in these injuries (24,31,33,42). For example, in IT LPS, NO production from iNOS was upstream of MIP-2 and TNF-␣ (31, 42) and contributed significantly to alveolar neutrophil influx (31,42), and alveolar-capillary barrier dysfunction (24,31,42). Indeed, NO synthase inhibition was more effective in blocking LPS-induced epithelial barrier dysfunction than either TNF-␣ antibody or neutrophil depletion (24).…”

“…ARDS is characterized by neutrophil infiltration, the generation of toxic cytokines and reactive oxygen species (ROS), increased NO production and the loss of endothelial and epithelial barrier function (4). These features can be reproduced in animal models by the installation of intratracheal (IT) LPS (10,24,31), a key component of the gram-negative bacterial cell wall. LPS, through Toll-like receptor-4 signaling, was shown to cause increased NO from phosphorylated endothelial (eNOS) (11) and inducible nitric oxide synthase (iNOS) (24,31) in alveolar macrophages, neutrophils, epithelium, and endothelium.…”

“…These features can be reproduced in animal models by the installation of intratracheal (IT) LPS (10,24,31), a key component of the gram-negative bacterial cell wall. LPS, through Toll-like receptor-4 signaling, was shown to cause increased NO from phosphorylated endothelial (eNOS) (11) and inducible nitric oxide synthase (iNOS) (24,31) in alveolar macrophages, neutrophils, epithelium, and endothelium. NO from both eNOS and iNOS was found to play a major pathogenic role in ALI from LPS (24,31,42) and in ventilator-induced lung injury (VILI) (33,39).…”

“…LPS, through Toll-like receptor-4 signaling, was shown to cause increased NO from phosphorylated endothelial (eNOS) (11) and inducible nitric oxide synthase (iNOS) (24,31) in alveolar macrophages, neutrophils, epithelium, and endothelium. NO from both eNOS and iNOS was found to play a major pathogenic role in ALI from LPS (24,31,42) and in ventilator-induced lung injury (VILI) (33,39). These injurious effects of endogenous NO have been attributed to a reaction with superoxide to generate peroxynitrite (31,33).…”

“…NO from both eNOS and iNOS was found to play a major pathogenic role in ALI from LPS (24,31,42) and in ventilator-induced lung injury (VILI) (33,39). These injurious effects of endogenous NO have been attributed to a reaction with superoxide to generate peroxynitrite (31,33). In both LPS-induced ALI (47) and VILI (39), however, NO also activates soluble guanylyl cyclase (sGC), increasing production of cGMP in multiple cell types including endothelium (39), epithelium (9,17), and macrophages (3).…”