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

Abstract

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)