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HIGHLIGHTED TOPICS
Airway Hyperresponsiveness: From Molecules to Bedside
SELECTED CONTRIBUTION
1Pulmonary and Critical Care Medicine, 2Biostatistics, and 3Cancer Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Submitted 6 December 2002 ; accepted in final form 4 February 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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NO/t = (NO15 - NO0)/15, where denotes a change and t is time. The NO accumulation rates in asthmatic and control subjects were similar at baseline; however, NO accumulation at 24 h increased threefold from baseline in asthmatic compared with control subjects (asthmatic subjects, 0.6 ± 0.2 ppb/s; control subjects, 0.2 ± 0.1 ppb/s; P = 0.01). Our study suggests that increased NO during an asthma exacerbation is due to increased synthesis, perhaps by increased expression of NO synthases. allergy; peak flows; spirometry; whole lung aerosolized allergen challenge
Increased NO may represent active airway inflammation through a number of mechanisms. NO can act as a vasodilator, causing airway edema and airway narrowing. NO may also inhibit T-helper (TH)-1 cell IFN-
production, promoting TH-2 cell proliferation and interleukin-4 and 5 synthesis, thereby increasing IgE synthesis and eosinophil recruitment (15). NO can also facilitate direct toxic effects on airway epithelium via oxygen radicals (5). Other deleterious effects of NO in asthma include the formation of reactive nitrogen species, such as peroxynitrite (12). These species decrease protein function through nitration of lung cell proteins and may also contribute to airway and parenchymal inflammation and epithelial cytotoxicity (19). Peroxynitrite also increases airway hyperreactivity in guinea pigs, suggesting a similar role in humans (16). Sustained high levels of NO in the airway of asthmatic subjects may induce increased blood flow and permeability, which would allow mediators of inflammation to accumulate locally. In support of this, studies suggest that increased exhaled NO is associated with active asthma under suboptimal control (3). Furthermore, exhaled NO levels in asthmatic subjects decrease after treatment with inhaled or oral glucocorticoids to levels similar to normal volunteers (21). The decreased NO parallels improvement in lung function [forced expiratory volume in 1 s (FEV1)], bronchial hyperresponsiveness, and other markers of airway inflammation, such as sputum eosinophilia (23).
Conversely, increased NO may have a regulatory and beneficial role. NO may cause bronchodilation from smooth muscle relaxation. This is supported by the studies that demonstrated that NO synthase inhibitors increase bronchoconstriction in response to histamine and bradykinin stimulation (9). There is also an inverse correlation between the proinflammatory transcription factor NF-
B in bronchoalveolar lavage fluid and airway NO after localized, bronchoscopic allergen challenge (26). NO-related increases in the NF-B inhibitor is one mechanism through which NO may curb the inflammatory response in asthma (24). Another potential mechanism for a protective role for NO in asthma may be through its ability to consume harmful reactive oxygen species (12).
Although it is fairly certain that exhaled NO is a marker of airway inflammation, the etiology of higher levels of NO during asthma exacerbation is not clear (4, 8, 19). Understanding of the role of NO in asthma has been further complicated by the variable and sometimes normal NO levels in asthma reported in the literature. We hypothesized that acute asthmatic flares are associated with increased NO levels due to increased synthesis of NO. Because asthma is characterized by cycles of spontaneous exacerbations and remissions, this increased production of NO with exacerbations may be responsible for the variability of NO levels seen in asthma. To evaluate this hypothesis, we performed whole lung allergen challenge on asthmatic and control subjects to study the effect of an acute asthma exacerbation on exhaled NO levels in vivo. In addition to confirming our hypothesis, the findings also offer an explanation for the variable levels of exhaled NO seen in asthma.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Pulmonary function studies. Spirometry was performed consistent with American Thoracic Society (ATS) standards. The FVC, FEV1, and FEV1/FVC ratio were collected for each of three efforts before and after the administration of two puffs of albuterol via aerochamber. Reference equations for spirometry are those of Crapo et al. (7). Methacholine provocation testing was performed according to published standards by the method of Chai et al. (6). Methacholine chloride solution was prepared in five concentrations (ranging from 0.025 to 25 mg/ml), and the subject inhaled five breaths at each stage. The test was considered positive if a ≥20% drop in FEV1 occurred at the highest concentration administered. FEV1 was also measured in asthmatic and healthy control subjects before allergen challenge (baseline), immediately after whole lung challenge, and at 2, 5, and 24 h after the challenge.
Allergy skin testing. Allergy skin testing was performed with the skin prick method. Allergens used were cat allergens, dog hair, D. farinae, cockroach, tree mix, grass mix, ragweed mix, Alternaria mold, Aspergillus mold, Cladosporium mold, normal saline (negative control), and histamine (positive control). Allergens were purchased from Hollstier Stier (Spokane, WA). Prick needle was a 2-prong lancet from Allergy Labs of Ohio. Skin tests were read after 15 min. A positive reaction was a 3-mm-diameter wheal with 10-mm erythematous flare. Atopy was defined as two or more positive skin tests.
WLAC. Subjects with two or more positive skin tests underwent (skin test relevant) aerosol allergen bronchoprovocation as described above in Pulmonary function studies for methacholine. A stock solution of allergen [10,000 protein nitrogen units (PNU)/ml] was diluted with saline to produce eight concentrations (1, 3.16, 10, 31.6, 100, 316, 1,000, and 3,162 PNU/ml). The patient inhaled five breaths at each concentration range. The concentration of allergen producing a 20% fall in FEV1 was determined. At the end of the challenge, two puffs of inhaled albuterol were administered via a metered dose inhaler.
Exhaled NO levels. Exhaled NO levels were measured in mixed exhaled breath from asthmatic and healthy control subjects by the off-line method. Mixed exhaled breath from each individual was first collected into a Mylar balloon, and then NO levels were measured in the balloon by using a chemiluminescent analyzer (NOA 280, Sievers) as previously described (12, 14). The mixed exhaled breath was obtained by collecting a single exhaled breath from total lung capacity into a mylar balloon against 10 cmH2O pressure without breath holding (NO0), according to ATS guidelines (2), and again after a 15-s breath hold (NO15) at total lung capacity. The rate of NO accumulation (parts/billion per second) was calculated as follows: NO/t = (NO15 - NO0)/15, where denotes change and t is time. Exhaled NO levels were measured in asthmatic and healthy control subjects before allergen challenge (baseline), immediately after WLAC (before bronchodilator was administered), and at 2, 5, and 24 h after the challenge.
Statistical analyses. The NO and accumulation rate measurements were modeled over time by using a repeated-measurements ANOVA that allowed for different standard deviations at each of the time points. This was useful since there appeared to be greater variability at times further from baseline. The model allowed estimation of mean values at the individual time points within each group (asthmatic vs. control subjects), as well as the mean differences from baseline to each time point within each group. The models also provided standard errors for the mean estimates, and group differences were evaluated by Wald tests. P values for the analyses were reported.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Peak flow rates and FEV1. Exhaled NO levels and peak flows were obtained before (baseline), immediately after (time 0), as well as 2, 5, and 24 h after WLAC by using a cybermedic spirometer. Peak flow rates were lower in the asthmatic subjects compared with healthy control subjects at all time points, reaching statistical significance at baseline, immediately after, and 24 h after allergen (Ag). Immediately after whole lung allergen challenge, peak expiratory flow rate decreased significantly in asthmatic but not in control subjects (Fig. 1A).
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Baseline FEV1 values in asthmatic and control subjects were similar at baseline but were significantly different immediately after Ag (post-WLAC FEV1 in asthmatic subjects 62 ± 6% vs. control subjects 95 ± 3%; P = 0.03). The decrease in FEV1 in asthmatic subjects immediately after challenge was also significant (asthmatic FEV1 at baseline 84 ± 6% and post-WLAC 62 ± 6%; P < 0.001).
Exhaled NO0 levels. Exhaled NO0 levels were significantly higher in the asthmatic subjects at baseline, immediately after Ag, and 24 h after Ag challenge (Fig. 1B). Exhaled NO0 levels showed significant decreases from baseline to 2 and 5 h after Ag, and NO0 levels in asthmatic subjects at the 2- and 5-h time points were not significantly different from controls. There was no significant change in exhaled NO0 from baseline to 24 h after Ag.
Exhaled NO levels with breath hold. Baseline and immediately post-Ag NO15 levels were significantly higher in asthmatic than in control subjects. NO levels in asthmatic subjects with breath hold at the 2- and 5-h time points were not significantly different from controls. By 24 h, asthmatic NO levels with breath hold started rising again to levels significantly higher than control levels and higher than asthmatic levels at baseline (Fig. 1C).
NO net accumulation. The rate of NO accumulation in asthma and controls were similar at baseline, immediately post-Ag, and 2 and 5 h post-Ag. At 24 h post-Ag, the rate of NO accumulation in asthmatic subjects was significantly higher than in control subjects at 24 h and higher than asthma at baseline (Fig. 1D).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Previous work in asthmatic patients after bronchoscopic segmental allergen challenge demonstrated that baseline exhaled NO and lower airway measurements of NO were not significantly different between asthmatic and control subjects. However, by 48 h after segmental challenge, both exhaled and lower airway NO levels were significantly higher in asthmatic than in control subjects, with exhaled NO levels, however, underestimating the intrapulmonary levels (12). In support of many such earlier studies, NO levels in our present study were higher in asthmatic subjects compared with control subjects at all time points and reached statistical significance at baseline, immediately after, and 24 h after challenge. This supports prior studies documenting elevated NO levels in the airways of asthmatic subjects (5, 12, 20).
During the acute exacerbation (immediately after allergen challenge), NO0 levels declined over the following 2 h to levels close to controls (Fig. 1B). During the same time frame, NO accumulation rates did not change significantly and remained similar to controls. The reduced NO levels immediately after challenge with a preserved NO accumulation rate suggest consumption of NO in the airways of asthmatic subjects. To characterize this decrease as solely due to decreased production or changes in airflow may be an oversimplification. Prior studies have documented significant reductions in exhaled NO after spirometry or hypertonic saline challenge in asthmatic subjects (11). These studies proposed that decreased NO levels in exhaled air were due to a decrease in luminal airway surface from constriction of airways after stimulation (10, 25). In our model, it is unlikely that this reduction in exhaled NO levels can be explained completely by changes in flow, because NO levels continue to decrease despite the rapid resolution of airflow limitation (Fig. 1, A and B). In addition, the preserved NO accumulation rate speaks against decreased NO production. Therefore, the most likely explanation for the immediate reduction in NO levels is consumption of NO by reactive oxidant species to form peroxynitrite and eventually nitrate (12).
We propose this new and simple concept of NO accumulation rate as a sensitive marker for changes in NO levels. As explained previously, this marker is obtained by measuring NO levels with two different breath hold times and calculating NO/t. In our study, one of the time points for measurement was 0 s, i.e., no breath hold, which is the method presently recommended by the ATS (2). The NO accumulation rate may be more sensitive than a single NO measurement to discern small changes in airway NO levels, thereby offering more insight into changes in the lower airway. Prior studies have shown increases in the NO synthetic machinery in asthmatic subjects after Ag challenge (17, 18). By using the presently recommended method (i.e., without breath hold) for measuring exhaled NO, however, we did not see the expected increase in asthmatic NO from baseline to 24 h. This is in sharp contrast to the increased NO levels with breath hold from baseline to 24 h as well as increased NO accumulation rate. These data suggest that production of NO is increased, likely through mechanisms such as induction of NO synthase II transcription or the accelerated breakdown of nitrosothiols to NO and other nitrates (18, 22). The increased production and/or release of NO appears to eventually outpace consumption by 24 h after challenge (Fig. 1, B–D). Thus asthmatic subjects not only have higher levels of NO than control subjects at baseline but are also primed to increase synthesis by several times in the event of an exacerbation.
Our study design also allowed us to offer an explanation for the widely varying and sometimes contradicting levels of NO in the exhaled breath of asthmatic subjects reported in the literature. By showing the changing levels of NO over time during an experimentally induced asthma attack, we demonstrate that levels of NO in asthma can vary significantly depending on the stage of the asthma attack. Previous studies have attributed the variability in NO levels reported in asthma primarily to technical differences among the measurement techniques. This was addressed by recent consensus statements on the standardization of measuring techniques (2). In this study, we used an off-line method similar to that recommended by the ATS and a modified method that utilizes an additional 15-s breath hold. Although the different measurement techniques can definitely contribute to some of the differences in the NO levels, we also show that the stage of asthma is another important factor that can contribute to this variability.
On the basis of the findings from this study, we conclude that asthmatic subjects not only have higher levels of NO than control subjects at baseline but are also ready to increase synthesis by several times in the case of an exacerbation. As the airway obstruction resolves, the up-regulated NO synthetic machinery in the asthmatic airway continues to generate even higher levels of NO. The rise in NO in the face of resolving airway obstruction suggests that NO may have a beneficial role in asthma. This also provides a new explanation for the known variability of NO levels in asthmatic individuals and groups. Calculation of NO accumulation rate may be more sensitive to change than a traditional single NO measurement. This concept may offer more insight into lower airway changes in NO chemistry that can otherwise only be known from more invasive methods. This makes the calculation of this NO accumulation rate potentially useful for monitoring effects of intervention or therapy.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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R. A. Dweik is supported by National Heart, Lung, and Blood Institute Grant HL-68863 and supported in part by Grant HL-04265.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address for S. Khatri: Emory University School of Medicine, 615 Michael St., Suite 205K, Atlanta, GA 30322.
Present address for J. Hammel: Cognigen Corporation, 395 Youngs Rd., Williamsville, NY 14221-5831.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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B activation in alveolar macrophages. Am J Respir Cell Mol Biol 21: 311-316, 1999.
2-agonist inhalation and spirometry in asthma. Am J Respir Crit Care Med 159: 940-944, 1999.This article has been cited by other articles:
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  A. Lara, S. B. Khatri, Z. Wang, S. A. A. Comhair, W. Xu, R. A. Dweik, M. Bodine, B. S. Levison, J. Hammel, E. Bleecker, et al. Alterations of the Arginine Metabolome in Asthma Am. J. Respir. Crit. Care Med., October 1, 2008; 178(7): 673 - 681. [Abstract] [Full Text] [PDF]
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ATS/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005 Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 912 - 930. [Full Text] [PDF]
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 R A Dweik Nitric oxide, hypoxia, and superoxide: the good, the bad, and the ugly! Thorax, April 1, 2005; 60(4): 265 - 267. [Full Text] [PDF]
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, Asthma data; 
, control data. *P
< 0.05, asthmatic vs. control subjects; **P < 0.001, asthmatic vs. control subjects; 
P < 0.05, asthmatic group change from baseline (BL) value. A: peak flow rates (PEFR) were lower in the asthmatic subjects compared with the healthy control subjects at all time points. Immediately after WLAC, peak flows significantly decreased further in asthmatic but not in control subjects. B: BL NO levels without breath hold (BH) were significantly higher in asthmatic than in control subjects at BL, immediately after WLAC, and 24 h after WLAC. Values in asthmatic subjects significantly decreased from baseline to 2 and 5 h after WLAC. C: BL NO levels with BH were significantly higher in asthmatic than in control subjects at BL, immediately after WLAC, and 24 h after WLAC. D: rate of NO accumulation in asthmatic and control subjects were similar at BL, immediately after WLAC, and at the 2- and 5-h time points, but asthmatic subjects were significantly higher than control subjects at 24 h. ppb, Parts/billion.