PERSPECTIVE
Nitric Oxide in Asthma
Pathogenic, Therapeutic, or Diagnostic?

Scherer P. Sanders

Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland

Nitrogen monoxide, more commonly called nitric oxide or simply NO, is a fascinating free radical molecule, which participates in a broad range of important physiologic processes, including vasodilation, neurotransmission, and host defense. NO is generated from L-arginine by the enzyme nitric oxide synthase (NOS), of which there are three known isoforms. Types I and III NOS, found predominantly in neurons and endothelium, respectively, are constitutively expressed and are dependent upon calcium for activity. The third isoform, NOS II, can be expressed by a wide range of cells primarily after it has been induced by certain cytokines, microbes, or microbial products (1, 2). All three isoforms are dimeric, with each subunit containing iron protoporpyrin IX, tetrahydrobiopterin, flavin adenine dinucleotide, flavin mononucleotide, and binding sites for L-arginine, calmodulin, and nicotinamide adenine dinucleotide phosphate (NADPH). In situ hybridization and immunohistochemical techniques have localized NOS in one isoform or another to a variety of cells in the human lung, including epithelium, endothelium, neurons, eosinophils, neutrophils, macrophages, and smooth-muscle cells (3).

The role of NO in the pathogenesis of asthma is currently under intense debate. Initial interest stems from observations in the early 1990s that subjects with asthma had elevated expression of NOS II immunoreactive protein in airway cells (4) and significantly higher levels of NO gas in exhaled air, compared with normal subjects (3, 5). Since then, numerous studies have confirmed these findings and have demonstrated that in allergic asthmatics challenged with allergen, the levels of NO in exhaled air can be increased beyond the already elevated basal levels. Technical concerns were initially raised regarding the origin of the NO in exhaled breath because of the possible contribution of NO from the NO-rich nasal passages. Several approaches, including the use of bronchoscopy to sample lower airway gas directly and techniques to exclude nasal NO, such as exhaling against positive pressure to close the velum, have determined that the increased NO measured in exhaled air from asthmatics is derived from the lower airways (6). Additional studies have shown that NO in exhaled air can be lowered by steroid therapy and inhibitors of NOS. But what does this mean for disease pathogenesis and the development of new therapeutics for asthma? In analyzing these concepts and the role of NO in asthma, it is important to understand that the physiologic consequences of NO produced in the airways will be critically dependent upon the timing, amount, and sites of synthesis (or drug delivery) of this reactive molecule, as well as on the chemical milieu of the surrounding environment (1).

There are two central issues to consider regarding the role of NO in the pathogenesis of asthma. First, how does it relate to lung function and bronchial reactivity and, second, what is its role in inflammation? Certainly, one of the potentially beneficial effects of NO derives from its bronchodilating actions, which are mediated via the interaction of NO with guanylate cyclase in smooth-muscle cells (7). As reviewed by Gaston and colleagues, NO donor compounds were used over a century ago to treat patients with bronchospasm (3). There is evidence in animal models and humans that inhaled NO can modulate bronchial tone (7, 8), but NO gas or NO donor compounds are not routinely included in the current therapeutic strategies for asthma. Moreover, at first glance it may seem paradoxical that asthmatics have elevated levels of exhaled NO in the context of a disease characterized by bronchial hyperreactivity (9). The relevance of this exhaled NO to disease pathogenesis, however, is currently unclear. Although the cellular source of exhaled NO has not yet been unequivocally identified, studies suggest that the NO in exhaled air is primarily produced by cells in the airway surface, such as epithelial cells rather than the cells of the pulmonary circulation (10). It may be that NO in exhaled breath represents an overflow of NO that has not reacted with specific targets in airway cells. Consequently, it is important to determine the source of exhaled NO in the context of bronchial reactivity. For example, if NO is released apically from NOS II in epithelial cells, it would be reasonable to suggest that it does not relate to bronchial reactivity.

Indeed, studies from knockout mice targeted at defining the role of NO in the airways suggest that exhaled NO is primarily synthesized by NOS II (inducible NOS) (11), which appears to be upregulated in mice lacking NOS I or III. It is intriguing that mice lacking NOS II have significantly reduced inflammatory responses to allergen challenge in the lung with little difference in the levels of airway hyperreactivity (12). By contrast, other studies in these murine models suggest that it is neuronal NOS, NOS I, that is the predominant isoform of NOS involved in modulation of the severity of airway hyperreactivity (13). Consistent with these findings are the observations that NO released from nerves mediates, at least in part, the nonadrenergic noncholinergic bronchodilator response in human airways (14).

Collectively, given the bronchodilating capacity of NO, there is potential for NO inhalation, NO-donating compounds, agents that augment NO release from airway nerves, or perhaps even targeted overexpression of genes for specific isoforms of NOS to have therapeutic benefit in diseases characterized by bronchial reactivity. But before these approaches become new therapies for asthma, the pivotal observations that have been made in animal models must be extended to humans, and the second key issue pertaining to NO in the airways must be addressed (i.e., its role in inflammation).

NO in the airways has been touted as a proinflammatory mediator in asthma, and in fact it has been suggested that elevated levels of NO in exhaled breath may reflect ongoing airways inflammation (5, 15). Studies have shown that NO reacts rapidly with superoxide anions to form the potent cytotoxic species peroxynitrite anion (16). At sites of inflammation where cogeneration of NO and superoxide occurs, the prooxidant versus antioxidant outcome of these reactions is critically dependent on the relative concentrations of the individual reactive species (16). In theory, NO may have effects on a myriad of cell functions, including alterations in DNA integrity, mitochondrial respiration, apoptosis, leukocyte adherence, mast cell reactivity, and eosinophil recruitment. Whether these NO-mediated effects are involved in asthma pathogenesis is not clear. Limited studies in murine cells suggest that NO may amplify inflammation by altering the balance between T-helper (Th)1 and Th2 cell types, leading to the proliferation of Th2 lymphocytes, which putatively produce a cytokine profile that has been associated with exacerbations of asthma (17). These observations, however, have not yet been extended to humans where the Th1/Th2 paradigm is less defined (18).

If cytotoxicity is confined to the host response to protect against invading microorganisms and tumor cells, then the "toxic" effects of NO may, in fact, be protective. Given the increasing evidence that upper respiratory viruses, particularly rhinoviruses, are a major cause of acute exacerbations of asthma, NO may play an important beneficial role through its potent antiviral properties (10). A wide range of both DNA and RNA viruses are inhibited by the induction of NOS II or by the addition of chemical donors of NO (19). Inhibitors of NOS have been shown to increase viral load and decrease survival in virus-infected mice, and mice deficient in NOS II have been reported to be more susceptible to infection (20). We have recently shown that NO can inhibit rhinovirus replication, as well as virus- induced production of several proinflammatory cytokines, including interleukin (IL)-8, IL-6, regulated on activation, normal T cells expressed and secreted (RANTES), and granulocyte macrophage colony-stimulating factor (GMCSF) in a dose-dependent fashion (10, 21, and unpublished observations). Parenthetically, it is of interest to note that NO has been shown to inhibit other inflammatory pathways as well, including the production of arachidonate metabolites by both lipoxygenase (22) and cyclooxygenase (23) pathways.

Because all isoforms of NOS depend upon the amino acid L-arginine as the nitrogen donor for NO, regulation of the availability of this substrate may determine cellular rates of NO synthesis and subsequent physiologic responses. The arginine biosynthetic pathway, which is linked to the citric acid cycle, is highly regulated and provides a source of substrate for NO synthesis in a wide variety of cells (1, 24). As discussed by Hammermann and colleagues in this issue (25), the availability of substrate for arginine-requiring enzymes is regulated, at least in part, by specific transmembrane proteins called cationic amino acid transporters. Interestingly, these authors have shown that cationic proteins, such as the eosinophil product major basic protein, inhibit the cellular uptake of L-arginine and consequently reduce NO synthesis in alveolar macrophages and tracheal epithelial cells. These results have interesting implications for asthma pathogenesis, given the central role of the eosinophil and its products in asthma and the possibility that inhibition of NO synthesis may relate to airway reactivity. Indeed, it has been shown that inhaled L-arginine induces a significant increase in the amount of exhaled NO in subjects with or without asthma, suggesting that NO production in the airways relates directly to increased availability of substrate (15). The same study further showed that there was a negative correlation between the increase in exhaled NO and the fall in FEV₁, which the authors attributed to the proinflammatory effects of NO. The specificity of these effects may be in question, however, as inhalation of alanine, an amino acid which is not a substrate for NOS, also caused a significant fall in FEV₁ without altering levels of exhaled NO (15).

While the mechanistic issues regarding the therapeutic and pathogenic effects of NO are being debated, NO may emerge as a diagnostic indicator of airway disease. One of the first clinical uses of NO gas was for measurements of alveolar-capillary diffusing capacity (3). Use of inhaled NO as a test gas to study diffusion limitation in the lung has been hindered by the instability and toxicity of NO. Recent observations regarding NO in asthmatics, however, have sparked renewed interest in the diffusion characteristics of NO in the airways. Studies have demonstrated that the output of endogenously generated NO in exhaled air is critically dependent upon expiratory flow rate. Silkoff and associates have reported that under conditions of low expiratory flows, the relationship between expired NO output and concentration of NO in exhaled breath are linear (26). As a result, the diffusing capacity of NO and the difference in concentration of NO between the airway wall and the lumen can be estimated from the slope and the concentration-axis intercept, respectively (26). Using this intriguing relationship, these authors have speculated that the measurement of the diffusing capacity of endogenously generated NO may be diagnostic in airway diseases such as asthma. Several recent studies, but not all, suggest that levels of exhaled NO relate to asthma severity and symptoms (9, 27). Since elevated levels of NO have been reported in other airway diseases, however, the application of measurements of exhaled NO as a diagnostic tool may be best suited for tracking the degree of inflammation and airway reactivity within individual patients rather than for discriminating one airway disease from another.

Although asthma is clearly a multifactorial disease, the studies outlined previously provide growing evidence that NO may play an important role in disease pathogenesis. Molecular genetic approaches using targeted disruption of genes in mice for the three isoforms of NOS have provided important insights into the role of NO in the airways. As we better understand the factors regulating NO production and the cellular targets of this reactive molecule, we will determine whether modulation of NO in the airways, either by genetic or chemical means, will be a worthwhile therapeutic strategy for asthma.

	Footnotes

Address correspondence to: Scherer P. Sanders, Ph.D., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6901. E-mail: ssanders{at}welch.jhu.edu

(Received in original form June 11, 1999).

Acknowledgments: The author gratefully acknowledges helpful discussions with Drs. David Proud and Mark Liu and support from Grant HL-61011 from the National Institutes of Health.

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