PERSPECTIVE
Exhaled Nitric Oxide (NO), NO Synthase Activity, and Regulation of Nuclear Factor (NF)-B

Harvey E. Marshall and Jonathan S. Stamler

Divisions of Pulmonary and Critical Care Medicine and Cardiology, and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina

Nitric oxide (NO) and related molecules are produced in the lung by virtually all cellular constituents, including the epithelium, the endothelium, neurons, neuroendocrine cells, resident inflammatory cells, and alveolar macrophages (1). An increase in respiratory NO production, as measured by exhaled NO levels, has been touted as a marker of lung inflammation: exhaled NO is increased in patients with asthmatic flares, bronchiectasis, and active tuberculosis (2). In contrast, it is lowered in primary pulmonary hypertension (PPH) (5). The precise role of NO in lung inflammation is still under debate. It may contribute to injury in some instances. Increasing evidence, however, points to salutary functions in general, and in particular to significant immunomodulatory roles. One of the mechanisms by which NO might modulate lung inflammation is through its interaction with the transcription factor NF-B, which is activated by diverse inflammatory stimuli and has been causally linked to respiratory cell inflammation and pulmonary disease (6). In this issue, Raychaudhuri and colleagues (9), show an inverse correlation between exhaled levels of NO and NF-B activity in alveolar macrophages obtained from patients with asthma or PPH. What conclusions can we draw from these new insights?

NF-B regulates many of the genes involved in the immune response, including tumor necrosis factor- (TNF-), interleukin (IL)-8, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the cytokine-inducible NO-synthase (NOS 2) (10). The prototypic form of NF-B is the p50-p65 heterodimer. (Although other dimeric forms exist [e.g., p50-p50, p65-c-Rel], their function in transcriptional regulation is less clear.) In its inactive state, NF-B (p50-p65) is bound by an inhibitory protein, I-B and is sequestered in the cytoplasm. Upon stimulation by inflammatory mediators (e.g., TNF-, lipopolysaccharide [LPS]), NF-B is released from I-B and translocates to the nucleus where it can activate target gene transcription.

NF-B function is regulated by NO or related molecules. NO primarily inhibits NF-B activation, but the effects are not straightforward. For example, treatment of cytokine-stimulated endothelial or vascular smooth-muscle cells with S-nitrosothiols (SNOs) attenuates NF-B activation (11, 12), and inhibitors of nitric oxide synthase (NOS) augment NF-B activity in both resting endothelial cells and LPS-stimulated macrophages and microglial cells (11, 13, 14). It was originally suggested that NO increases the transcription and stability of the NF-B inhibitory protein, IB (11), but it may also inhibit NF-B directly through S-nitrosylation of the p50 subunit (15). This SNO modification of NF-B has been shown to prevent binding to its target DNA site (16). On the other hand, NO is an activator of NF-B in lymphocytes and neuronal cells (17, 18). S-nitrosylation of the G protein, p21ras, which stimulates nucleotide exchange and thereby activates NF-B, is the proposed mechanism in this case. It may be that NO can regulate NF-B both positively and negatively at multiple steps in the activating pathway, depending upon the cell type, cell stimulus, NO concentration, and NO-related species.

The state of NF-B activity must therefore be placed into a physiologic or pathophysiologic context. In this regard, the NO measurements taken by Raychaudhuri and associates from patients with asthma and PPH are an important step forward. There are, however, important caveats with the data, which should not be misapprehended to imply causality between the amount of NO in exhaled air and NOS activity in the lung or between the exhaled NO level and protection from NF-B activation. On the contrary, the source of expired NO in patients is unclear in general, and there is no clear relationship between endogenous NO-related activity and expired NO levels in patients with asthma (or PPH, for that matter) in particular (19). The amount of NO exhaled is, in fact, an indicator of the amount of NO eliminated by the lung, and not a direct measure of NOS activity. Exhaled NO might be more accurately analogized to sodium (or nitrate) excretion by the kidney: increased elimination does not imply larger amounts handled or produced, and decreased elimination does not imply a smaller load. This is even more apparent in disease states. Indeed, the level of expired NO has not been found to correlate well with NOS activity in the lung (20). Alternative measures of pulmonary NO production are the concentrations of SNO or nitrite in the bronchoalveolar lavage fluid. Airway SNO levels increase in acute inflammatory conditions and decrease in cystic fibrosis patients (B. Gaston, personal communication) and preterm infants on extracorporeal membrane oxygenation (ECMO) (21). These levels fall in asthma, but this may result from accelerated breakdown of SNO to NO rather than decreased NOS activity (19). None of these airway measurements, however, would accurately reflect the actual intracellular concentrations of NO/SNO that control NF-B activity and which vary from cell to cell.

The average concentration of NO in an exhaled breath is ~ 6 parts per billion (ppb), and levels may double in asthma (2). What is 10-ppb NO in terms of NO bioactivity and NOS output? Ten parts per billion of NO is equivalent to ~ 400 pM NOan amount far too low to inhibit NF-B. In fact, NF-B was not inhibited in LPS-stimulated alveolar macrophages, which generate their own NO at micromolar concentrations, unless they were also exposed to high concentrations of NO donors (9); that is, NO concentrations five orders of magnitude higher than present in exhaled air (3). (In contrast, SNO or nitrite concentrations in alveolar lavage or tracheal aspirates are in the micromolar range, and such activity may increase 20-fold [21].) There is something disconcerting about following an alleged marker of NOS activity that constitutes only a trivial fraction of the NO generated. At the very least, one would like to know the relationship between increases in NOS activity and increases in exhaled NO in diseases for which such a correlation can be causally established.

It is clear that very little is known of the interplay between NO and NF-B in vivo. Future studies in well defined patient populations should prove informative not only in the specific realm of inflammatory responses, but more generally in NF-B-mediated processes, such as apoptosis and cellular transformation. Ultimately, this understanding could provide a rational strategy for administering NO donors or NOS inhibitors, or both, to inhibit NF-B activation for therapeutic gain. Indeed, some studies indicate that inhaled NO can attenuate the inflammatory response in the lung (22, 23). Moreover, the mechanisms by which NO regulates NF-B may be shared by other transcription factors (e.g., OxyR, AP-1, p53), particularly in physiologic conditions characterized by nitrosative stress.

	Footnotes

Abbreviations: nuclear factor B, NF-B; nitric oxide, NO; nitric oxide synthase, NOS; primary pulmonary hypertension, PPH; S-nitrosothiols, SNOs.

(Received in original form July 1, 1999).

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