Nitric oxide and peroxynitrite in ozone-induced lung injury.

One of the hallmarks of the inflammatory response associated with tissue injury is the accumulation of macrophages at sites of damage. These cell types release proinflammatory cytokines and cytotoxic mediators to destroy invading pathogens and initiate wound repair. However, when produced in excessive amounts, these macrophage-derived mediators may actually contribute to tissue injury. This process involves both direct damage to target tissues and amplification of the inflammatory response. One group of macrophage-derived mediators of particular interest are reactive nitrogen intermediates including nitric oxide and peroxynitrite which have been implicated in tissue injury induced by a variety oftoxicants. Our laboratory has been investigating the role of reactive nitrogen intermediates in lung injury induced by oxidants such as ozone. Inhalation of ozone causes epithelial cell damage and Type II cell hyperplasia. This is associated with an accumulation of activated macrophages in the lower lungs which we have demonstrated contribute to toxicity. To analyze the role of macrophage-derived reactive nitrogen intermediates in ozone toxicity, we used transgenic mice lacking the gene for inducible nitric oxide synthase (NOSII). Treatment of wild type control animals with ozone (0.8 ppm) for 3 hr resulted in an increase in bronchoalveolar lavage (BAL) fluid protein reaching a maximum 24-48 hr after exposure. This was correlated with increased expression of NOSII protein and mRNA by alveolar macrophages and increased production of nitric oxide as well as peroxynitrite. Ozone inhalation also resulted in the appearance of nitrotyrosine residues in the lungs, an in vivo marker of peroxynitrite-induced damage. In contrast, in NOSII knockout mice, BAL protein was not increased demonstrating that these mice were protected from ozone-induced epithelial injury. Moreover, alveolar macrophages from the transgenic mice did not produce nitric oxide or peroxynitrite even after ozone inhalation. There was also no evidence for the formation of nitrotyrosine in lung tissue. These data indicate that ozone-induced lung injury is mediated by reactive nitrogen intermediates.

Inhaled nitric oxide primes lung macrophages to produce reactive oxygen and nitrogen intermediates.

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.