Ladan Fakhrzadeh

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.

Regulation of Caveolin-1 Expression, Nitric Oxide Production and Tissue Injury by Tumor Necrosis Factor-α following Ozone Inhalation

Alveolar macrophages (AM) and inflammatory mediators including nitric oxide and peroxynitrite contribute to ozone-induced lung injury. The generation of these mediators is regulated, in part, by the transcription factor NF-kappaB. We previously demonstrated a critical role for NF-kappaB p50 in ozone-induced injury. In the present studies mechanisms regulating NF-kappaB activation in the lung after ozone inhalation were analyzed. Treatment of wild type (WT) mice with ozone (0.8 ppm, 3 h) resulted in a rapid increase in NF-kappaB binding activity in AM, which persisted for at least 12 h. This was not evident in mice lacking TNFalpha which are protected from ozone-induced injury; there was also no evidence of nitric oxide or peroxynitrite production in lungs from these animals. These data demonstrate that TNFalpha plays a role in NF-kappaB activation and toxicity. TNFalpha signaling involves PI-3-kinase (PI3K)/protein kinase B (PKB), and p44/42 MAP kinase (MAPK) which are important in NF-kappaB activation. Ozone Inhalation resulted in rapid and transient increases in p44/42 MAPK and PI3K/PKB in AM from WT mice, which was evident immediately after exposure. Caveolin-1, a transmembrane protein that negatively regulates PI3K/PKB and p44/42 MAPK signaling, was downregulated in AM from WT mice after ozone exposure. In contrast, ozone had no effect on caveolin-1, PI3K/PKB or p44/42 MAPK expression in AM from TNFalpha knockout mice. These data, together with our findings that TNFalpha suppressed caveolin-1 expression in cultured AM, suggest that TNFalpha and downstream signaling mediate activation of NF-kappaB and the regulation of inflammatory genes important in ozone toxicity, and that this process is linked to caveolin-1.

Macrophages, reactive nitrogen species, and lung injury

Evidence has accumulated over the past several years demonstrating that lung injury following inhalation of irritants like ozone is due, not only to direct effects of the chemical, but also indirectly to the actions of inflammatory mediators released by infiltrating macrophages. Among the mediators involved in the cytotoxic process, reactive nitrogen species (RNS) are of particular interest because of their well‐documented cytotoxic potential. Findings that macrophage suppression blocks RNS production and ozone‐induced toxicity provide strong support for a role of these cells and inflammatory mediators in lung injury. Recent investigations have focused on understanding pathways by which macrophages become activated to release RNS. One protein that has attracted considerable attention is caveolin‐1, a membrane scaffolding molecule that functions to negatively regulate cell signaling. The fact that expression of caveolin‐1 is down‐regulated in macrophages after ozone inhalation suggests a mechanism controlling the release of cytotoxic mediators by these inflammatory cells.

Mice Lacking Inducible Nitric Oxide Synthase Exhibit Reduced Responsiveness to the Irritant-Inducing Effects of Inhaled Nitric Oxide † 1772

Mice Lacking Inducible Nitric Oxide Synthase Exhibit Reduced Responsiveness to the Irritant-Inducing Effects of Inhaled Nitric Oxide † 1772

INCREASED PEROXYNITRITE PRODUCTION BY ALVEOLAR MACROPHAGES (AM) FOLLOWING INHALATION OF NITRIC OXIDE (NO). • 1621

Inhaled NO is a selective pulmonary vasodilator that is used for the treatment of pulmonary hypertension. In the present studies, we examined the effects of inhalation of NO on AM. These cells regulate host defense, in part, through the production of reactive nitrogen and oxygen intermediates. Overproduction of these reactive mediators has been implicated in tissue damage. Balb/c mice were exposed to 80 ppm NO for 5 h; control animals received room air. AM were isolated 0, 24, or 48 h after exposure and measurements made of NO and superoxide anion production. Unstimulated AM from both control and NO-treated animals exhibited low levels of inducible NO synthase expression, as determined by Western blotting and immunofluorescence, and negligible NO production, quantified as nitrite accumulation in the culture medium. Treatment of the cells for 24-72 hr with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ), known physiologic activators of AM, resulted in a time-dependent induction of NO synthase and NO production. AM from NO-exposed mice produced significantly more NO than AM from control animals. This was correlated with increased NO synthase expression. We also found that AM from NO-exposed animals produced significantly more superoxide anion in response to 12-O-tetradecanoyl- phorbol-13-acetate (TPA) than cells from control animals. This was evident in vitro in isolated cells and in situ in histologic sections. NO rapidly reacts with superoxide anion to form peroxynitrite, a potent oxidizing agent. Peroxynitrite induces lipid peroxidation, nitration and oxidation of proteins, and damage to DNA. Using dihydrorhodamine 123 and fluorescent image analysis, we measured peroxynitrite production by AM cultured with LPS and IFN-γ and then stimulated with TPA (30 min). Whereas minimal peroxynitrite was detected in AM from control animals, peroxynitrite production was significantly increased in AM from NO-exposed animals. Taken together, these data demonstrate that inhaled NO activates AM to release reactive oxygen and nitrogen intermediates. Increased production of these intermediates by AM may lead to tissue damage and long term toxicity, including potential genotoxicity. (Supported by N.J. Thoracic Society)

INHIBITION OF NF-κB p-50 NUCLEAR TRANSLOCATION REGULATES APOPTOSIS AND EXPRESSION OF BCL-2 IN MACROPHAGES. ♦ 62

INHIBITION OF NF-κB p-50 NUCLEAR TRANSLOCATION REGULATES APOPTOSIS AND EXPRESSION OF BCL-2 IN MACROPHAGES. ♦ 62