NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION USING EXHALED NITRIC OXIDE AND INDUCED SPUTUM

THE PATHOBIOLOGY OF ASTHMA: IMPLICATIONS FOR TREATMENT

0272-5231 / 0 0 $15.00

+ .OO

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION USING EXHALED NITRIC OXIDE AND INDUCED SPUTUM Current Status and Future Use Philip E. Silkoff, MBBS, MRCP

Support for the central role of airway inflammation in the pathogenesis of asthma has stimulated much research into the underlying mechanisms of this inflammation, and therapy has been directed toward the anti-inflammatory class of asthma medication^.^^ The standard diagnosis and monitoring of asthma include symptom questionnaires; home peak expiratory flow rate recording; and, more recently, spirometry; hospitalbased pulmonary function testing; and bronchial challenge. These tools, however, principally assess airway patency and smooth muscle reactivity, which only indirectly, and not always consistently, reflect changes in airway inflammation. GOLD-STANDARD TESTS FOR ASSESSING AIRWAY INFLAMMATION

The gold standard for the objective assessment of airway inflammation remains sampling of airway fluid and lung tissue using bronchoscopy or, in special circumstances,

even macroscopic lung biopsy, with characterization of inflammatory cells, mediators, and structural change^.'^ Bronchial biopsy can convey important information about the nature of the cellular inflammation and airway structure but is limited to more central airways. More recently, transbronchial biopsies have revealed that the inflammatory process affects the lung parenchyma itself, with dynamic influx of inflammatory cells into the distal airways and alveoli at night in subjects with nocturnal asthma.47The invasiveness and expense of bronchoscopy, however, has precluded its routine and repeated application in the clinical setting as a tool for monitoring airway inflammation. Bronchoalveolar lavage (BAL) samples cells and mediators from proximal and distal airways, but carries the disadvantages of dilutional inaccuracies, sampling errors, and variable return of fluid. THE IDEAL TEST FOR AIRWAY INFLAMMATION

Ideally, a test for airway inflammation should be noninvasive, safe, simple to per-

From the National Jewish Medical and Research Center, Denver, Colorado

CLINICS IN CHEST MEDICINE VOLUME 21 * NUMBER 2 * JUNE 2000

345

346

SILKOFF

form, and inexpensive, thereby allowing its repeated application in subjects of all ages with all severities of disease. The test maneuver should be standardized, and should correlate with indices of inflammation obtained using the gold standard tests-i.e., bronchoscopy; additional correlation with other outcome measures, such as pulmonary function, bronchial reactivity, and disease severity, would allow airway inflammation to be more closely linked to airway function and prognosis. Repeated application in the same subject at the same session should show good reproducibility. Although no test will ever be ideal, there have been exciting developments in the field of noninvasive assessment of airway inflammation-namely, induced sputum analysis (IS) and exhaled breath testing (e.g., nitric oxide [NO]). In addition, studies are appearing measuring nonvolatile molecules in the liquid phase of breath (breath condensate). The characteristics of these new tools are compared critically to those of an "ideal test" in this review. This review focuses on methodologic issues rather than the findings in asthma.

SPUTUM ANALYSIS

Initially, sputum induction was used successfully to obtain material from the lower respiratory tract for the diagnosis of pulmonary tubercul~sis~~ and Pneurnocystis curinii in patients with acquired immunodeficiency syndrome.86Since then, the analysis of cells and mediators in sputum has been applied successfully to the investigation and to some extent the clinical monitoring of airway inflammationzl,57, in asthma and other diseases (e.g., COPD). In cases in which sputum is not produced spontaneously, it can be induced in the majority of individuals by nebulization of hypertonic saline. Expectorated sputum contains material that originates in the lower respiratory tract, although it traverses the oral cavity and therefore is subject to contamination with material originating in both the oropharyngeal and nasal cavities (e.g., if subjects have postnasal drip).

Sputum has been generally regarded to reflect inflammatory processes in the more central airways, and to be less sensitive to processes in more distal airways. Recent work by Gershman et a1,26however, has shown that the fraction of sputum obtained during the first 12 minutes of the induction procedure may be more central in origin, as evidenced by more eosinophils and neutrophils and fewer macrophages, whereas the fraction obtained after 12 minutes of induction contains more macrophages and surfactant proteins, suggesting that the alveolar region is being sampled. In the future, standardization of sputum induction duration may be necessary. The Cellular and Soluble Phases of Induced Sputum

Analysis of sputum gives information about cellular profiles and assessment of salivary contamination from the percent of squamous cells. It is also possible to perform special tests on cells (e.g., immunohistochemistry). Induced sputum has the advantage over BAL fluid of higher density of cell recovery, and a stronger signal for fluid-phase markers; the list of measured substances in induced sputum is extensive. These include eosinophil products such as eosinophil cationic protein66(ECP) and tryptase, fibrinogen, and cytokines such as interleukin-8 (IL-8) and tumor necrosis f a ~ t 0 r - a . ~ ~ Uses of Induced Sputum in Asthma Diagnosis of Asthma

A recent search of the literature revealed 153 articles on IS in asthma, indicating the intense interest and activity in this area. In the realm of diagnosis, Hsu et aI3l examined spontaneously produced or induced sputum in 114 subjects with episodic wheezing, dyspnea, or cough of unknown origin. An increased percentage of sputum eosinophils was seen in 92% of asthmatics (n = 52), 36% of patients with COPD ( n = 25), and 28% of chronic coughers ( n = 25), but not in patients with congestive heart failure (n = 12). The use of IS in subjects with respiratory symp-

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

toms has revealed a subgroup with sputum eosinophilia who do not have airway obstruction or bronchial hyperresponsiveness. This is now regarded as a new entity-nonasthmatic eosinophilic bronchitis.8Park et a160examined the characteristics of IS percent eosinophil count and ECP concentration in the diagnosis and assessment of the variability of airway inflammation in 68 patients with asthma and other diagnoses. The asthmatic group ( n = 41) showed a higher percentage of sputum eosinophilia (24.5 k 7.6 % versus 2.2 k 2.9%, P<.OOl) and a higher level of sputum ECP (198.2 versus 90.6 kg/L, R . 0 5 ) than the nonasthmatic group (n = 27). The sensitivity and specificity of sputum eosinophilia equal to or greater than 5% for the diagnosis of asthma were 85.4% and 92.6%, compared with 68.3% and 55.5%, respectively, for sputum ECP equal to or greater than 100 kg/L. Patients with moderate to severe persistent asthma (n = 23) had a higher percent sputum eosinophilia (34.6 k 10.6%)than that found in mild, persistent asthma ( n = 18; 10.7 k 5.2%, P<.Ol). Eosinophilic cationic protein levels did not differ significantly between the mild and moderate to severe groups, however. These results suggest that sputum eosinophil count and ECP level are helpful in the diagnosis of asthma, although sputum eosinophilia is not restricted to asthma alone. Induced sputum also helped diagnose occupational asthma (OA) in a study by Lemisre et who examined the change in sputum cell counts and ECP assays after work exposure. Occupational asthma was defined as (1) worse symptoms and (2) a more than 20% drop in forced expiratory volume in 1 second (FEV,) or a fourfold change in the dose of methacholine causing a 20% fall in FEV, (PC,,) at work compared with away from work. Controls were subjects with increased symptoms but with a change in FEV, of less than 20% and in methacholine PCz0of less than fourfold. Patients with OA (n = 10) had a significant increase in median (interquartile range) sputum eosinophils and ECP when at work compared with the periods out of work (10.0 [17.05] versus 0.8 [1.6]%; P = 0.007 and 3840 [6076] versus 116 [NO] pg/L; P = 0.01). The authors concluded that sputum percent eosinophils and ECP could sup-

347

plement other physiologic outcomes in establishing the diagnosis of OA. Assessing Response to Therapy in Asthma

Many studies have shown improvement in markers of inflammation in IS after therapy with inhaled steroids.34,37,39, 84 Furthermore, in one study, it was possible to demonstrate a dose response for the effect of inhaled budesonide using IS eosinophilia, exhaled NO, and methacholine PC,,, although sputum eosinophils separated the effects of a wider dose range of steroid than exhaled NO.35This study suggests that IS eosinophila could be used to titrate the minimal dose of inhaled steroids. Withdrawal of therapy also resulted in a rise of markers of inflammation in IS in a study in severe asthma by in’t Veen et who compared 13 patients with difficult-tocontrol asthma and 15 patients with severe but stable asthma. Exacerbations were induced by double-blind, controlled tapering of inhaled corticosteroids (fluticasone propionate) at weekly intervals. Steroid tapering caused a decrease (mean k SEM) in FEV, (12.1 k 3.1% predicted; P = 0.045) and methacholine PCz0(2.1 k 0.4 doubling dose; P = 0.004), accompanied by an increase in sputum eosinophils (10 k 3%; P = 0.008) and soluble markers (ECP, albumin, IL-6, IL-8) for the two groups combined, with no significant differences between groups. Interestingly, theophylline withdrawal has also resulted in increases in sputum eosinophilia, suggesting an anti-inflammatory effect of this m e d i ~ a t i o n . ~ ~ The steroid-tapering studies suggest that adherence to therapy could also be monitored with IS. Diagnosis of Causes of Asthma Exacerbation

Induced sputum has also been used to examine the airway inflammatory effects of acute viral infections in asthmatics and contro1s.68Both groups, but the asthmatics in particular, demonstrated increasing neutrophilia, but not eosinophilia, accompanied by increases in IL-8 levels. The neutrophilia re-

348

SILKOFF

solved by 21 days postinfection. Induced sputum could perhaps be used to distinguish infectious exacerbations from asthma deterioration. These studies and many others suggest that IS can be used to diagnose asthma, monitor the effects, titrate the minimal effective dose of anti-inflammatory therapy, and investigate causes of acute (infection) versus asthma exacerbation.

Method of Sputum Induction and Processing Techniques for Induction of Sputum

There is no nationally or internationally accepted methodology for sputum induction, although there is broad agreement on general principles.M Different methods appear to be equally successful in yielding sputum with similar qualities. General Principles

Subjects inhale an aerosol of hypertonic saline, commonly generated by an ultrasonic nebulizer. Usually, the total saline inhalation time is up to 30 minutes, divided into periods (e.g., 2 minutes). Expectoration is attempted and peak expiratory flow or spirometry is monitored between each period. Different investigators have used concentrations of saline that vary from 3% to 5%; the concentration may be held constant with subjects inhaling for progressively longer durations, or the saline concentration increases as the test proceeds. After inhaling for each determined period, subjects (some methods require subjects to wear a nose clamp) blow their nose, rinse their mouth (not universally employed) and then expectorate into a receptacle. The test is terminated when an adequate volume of sputum has been obtained or significant side effects ensue (see section on safety). When subjects spontaneously produce sputum, induction may be unnecessary, but spontaneous sputum may demonstrate decreased cell viability compared with freshly induced samples, as reported in a study on chronic obstructive pulmonary disease.6

lnvasiveness and Safety Sputum induction, although generally safe, carries significant risks, particularly in the moderate-to-severe category of patients. The main risks are bronchospasm, which occurs in a significant percentage of subjects, and oxygen desaturation,'O as has been reported recently. Wong et alS7reported that, despite pretreatment with inhaled albuterol (180 Fg), FEV, fell by more than 20% (range 20%-69%) in 11 of 74 asthmatic subjects; furthermore, the incidence and severity of bronchospasm was greater in those with lower baseline FEV, and greater bronchial reactivity. This has led to measures to prevent and monitor bronchospasm. Regarding prevention of bronchospasm, some investigators apply lower limits for baseline postbronchodilator FEV, (e.g., 1 L or 60% predicted, whichever is higher), although a recent paper by de la Fuente et all4reported safely testing subjects with severe asthma (FEV, as low as 40% predicted). Similarly, Pizzichini et aP7 reported on successful sputum induction in subjects with severe asthma exacerbations, some of whom had a baseline FEV, less than 40%. Interestingly, in the latter study, the induction procedure started with normal saline, which was adequate in seven subjects to produce sputum. Certainly, the usefulness of sputum induction would be increased if we could test sicker patients. In a recent study by Hunter et al:2 use of a low-output ultrasonic nebulizer resulted in less bronchospasm in their asthmatic subjects, in whom FEV, fell by only 5.4% on average, and adequate sputum was obtained in more than 90% of subjects. Most investigators pretreat with a bronchodilator (e.g., albuterol, 200400 pg); this appears to be only partially protective against bronchospasm. Most protocols require monitoring of PEFR or FEV, after each inhalation period or if symptoms develop, and have "end-of-test" criteria based on the readings. In general, emergent bronchospasm appears to be easily reversible with bronchodilators and does not appear to result in admission for prolonged asthma exacerbation. Subjects should stay in the laboratory until FEV, returns to greater than 90% of baseline values, however.

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

Castagnaro et allomeasured pulse oximetry during sputum induction, and reported a significant mean drop in oxygen saturation in asthmatic patients (6%), smokers (5.3%), and healthy subjects (6.0%). The mean ( 2 SD) drop in FEV, was not statistically different among the three groups. The results suggest that subjects who are hypoxemic before sputum induction could experience significant hypoxemia and therefore require pulse oximetry monitoring during the procedure, with oxygen supplementation if needed. In view of potential infection of personnel or other subjects with undiagnosed pulmonary tuberculosis or other infectious agents, sputum induction requires an induction facility (e.g., a hood with continuous removal of aerosols); each laboratory should consult with their local infectious diseases department so that appropriate measures can be taken. Processing of Sputum

The processing of sputum should be immediate, when possible, and at most within 2 hours to preserve cellular viability. Samples should be refrigerated until processing. Two methods of processing have been widely adopted. In that used by Hargreave and colleagues,63,65 dense or viscid portions ("sputum plugs") are selected out of the collected samples for processing; Fahy and colleaguesz1 process the entire expectorate, although they take measures to exclude saliva by asking subjects to collect saliva before expectoratingZ7Kips et a145have critically compared the Ilargreave and Fahy methods and report that they yielded comparable results. Following induction, similar processing is used by both groups. Although detailed methods will not be described here, the main steps are: (1)The sample is liquefied to disperse the cells by adding 0.1% dithiothreitol in a fixed proportion to the volume of sputum obtained. (2) The sample is centrifuged to separate supernatant, which is stored at - 70°C for future analysis. And (3) cytospins for differential cell count are prepared. Expert processing of the sputum is critical for valid and reproducible results. A welltrained technician who can prepare the Samples and read the slides accurately is essen-

349

tial. In a recent editorial, FahyZ0reports that inadequate homogenization of the sample is the most common cause of poor cell quality on slide preparations. Reproducibility

Hypertonic saline administered as part of an initial induction could affect airway inflammation itself, thereby resulting in changes in subsequent inductions performed in the short term. Pizzichini and colleagues" have examined the reproducibility of two IS samples taken within 6 hours in 10 healthy subjects, 19 stable asthmatics, and 10 smokers with nonobstructive bronchitis for the following parameters: sputum cells and fluid-phase ECP, major basic protein, eosinophil-derived neurotoxin, albumin, fibrinogen, tryptase, and IL-5. The reproducibility of measurements, calculated by the intraclass correlation coefficient, was high for all indices measured, with the exception of total cell counts and proportion of lymphocytes. The reproducibility of most cellular and soluble-phase parameters of two sputum inductions performed in the same week was also good, as reported by Spanevello et a1.81 In contrast to the previous two studies, Holz et a130 compared IS results between two inductions performed 24 hours apart in 10 subjects with mild asthma and 19 healthy subjects. Sputum was obtained and analyzed separately, during three consecutive 10-minute periods of hypertonic saline inhalation. In all three consecutive inhalation periods, mean percentages of neutrophils increased from day 1 to day 2 in both healthy (17.4%) and asthmatic (14.6%) subjects. The percentage of macrophages decreased from day 1 to day 2, whereas eosinophil and lymphocyte percentages did not change significantly. This study suggests that the induction procedure itseg causes a change in the composition of sputum detectable after 24 hours. This effect may need to be taken into account when repeated sputum induction is performed. Effect of Other Test Procedures on Induced Sputum Parameters

Bronchial challenge with methacholine 1 hour prior to induction does not appear to

350

SILKOFF

significantly affect sputum cell counts.82This is important because protocols often include both tests. Importantly, albuterol administration does not affect the differential cell count.44 Comparison with “Gold Standard Investigations

Remarkably few studies have examined the correlation between IS and bronchoscopic material. Bronchoalveolar Lavage Fluid. Ideally, sputum should reflect qualitatively the findings in lavage of the lung, although BAL fluid is diluted several-fold by the lavage fluid. In 1995, Fahy et a1= published a comparison of markers of inflammation in BAL fluid (4 x 60 mL lavages), bronchial washing (50 mL; BW), and SI in mild asthmatics and normal controls. In both groups, sputum contained significantly total higher numbers of inflammatory cells compared with both BAL fluid and BW ( P = .OOOl) and ECP ( P = .0001). Induced sputum eosinophil percent and ECP showed high correlations to BW ( r = .67, P = .005; r = .69, P = .0008 respectively) but only weak correlation with BAL fluid (r = 0.5, P = .03; r = .37, P = .11 respectively), probably because of the marked dilutional factors in the latter. This important study confirmed that IS reflects qualitatively, the findings from the markedly invasive bronchoscopy but, more importantly, contains more concentrated material from the lower respiratory tract. In a similar vein, Pizzichini et aP6 compared cellular and fluid-phase indices of inflammation in IS, BAL fluid, and peripheral blood (the latter is not reviewed here). Median results of sputum compared with BAL showed a higher number of inflammatory cells (53 versus 0.8 X lo6 cells/mL, P = .003), more neutrophils (34.3 versus LO%, P<.OO1), fewer macrophages (60.3 versus 95.0%, P= .002) and markedly higher levels of ECP (264 versus 2.0 Fg/L, P<.OOl), tryptase (17.6 versus 2.2 U/L, P<.OOl), and fibrinogen (1400 versus 150 Fg/L, P=.OOl). Sputum and BAL neutrophils and CD4+ T cells were strongly correlated. These studies suggest that, in mild

asthmatics, sputum and BAL measure different compartments of inflammation. Bronchial Biopsy. Grootendorst et alZ8compared the cellular compositions of IS, BW, BAL fluid, and endobronchial biopsies (EBBX) in 18 clinically stable patients with mild to moderate atopic asthma (eight subjects were on inhaled steroids). The percent eosinophils in sputum significantly correlated with their percentage in BW ( r = 0.52, P = .03) and in BAL ( r = 0.55, P = .02), whereas there was a trend toward such a correlation between the number of eosinophils per milliliter sputum and the number of EG2 + eosinophils per millimeter squared lamina propria in bronchial biopsies (Y = 0.44, P = .07). In summary, inflammatory indices in IS broadly correlate with those obtained by bronchoscopy (BW, BAL, and EBBX); importantly, however, IS contains more nonsquamous cells and much higher concentrations of soluble-phase markers, probably because of less dilutional artifact. Correlation with Other Outcome Measures

Studies comparing IS parameters with pulmonary function and bronchial reactivity are only just appearing in the literature. Pulmonary Function. Ohnishi et a158induced sputum in 10 patients with atopic asthma and examined the bronchial hyperresponsivity, pulmonary function, and differential cell counts in induced sputum of the patients before and after inhaled beclomethasone dipropionate (BDe therapy. The percentage of eosinophils in induced sputum from asthmatic patients was significantly higher than that of nonasthmatic subjects. The mean percent eosinophils in IS fell from 22.9 k 7.2% to 13.9 k 8.3% ( R . 0 5 ) after 3 months of therapy. The percent eosinophils in IS before BDP treatment was significantly correlated with percent predicted FEV, at baseline (r= -.75, R . 0 5 ) but not with methacholine reactivity at baseline ( P = .18). Furthermore, the change in percent predicted FEV, after treatment correlated significantly with the change in the sputum eosinophil percentage (r = - .79, R . 0 1 ) . Jatakanon et a138reported a

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

significant correlation between sputum eosinophils (percent) and methacholine PC,, (r = - 0.4) in steroid-free mild asthmatics. Lim et a150examined the effect of inhaled budesonide (800 pg twice daily) on lung function and markers of airway inflammation (methacholine PC,, exhaled nitric oxide (FENO),IS percent eosinophils, BAL, and EBBX from 14 patients with mild asthma on P2-agonist therapy only. After treatment, FEV, and PC,, both significantly increased; F E fell. ~ Eosino~ phils in IS and airway biopsy sections also significantly decreased, although BAL eosinophi1 counts remained unchanged. Before treatment only, significant correlations were observed between IS eosinophils and both FEV, ( r = -0.63, P=.O5), and log (r = - 0.67, R.05). In summary, there appears to be a reasonable but variable correlation between sputum eosinophilia and both FEV, and methacholine PC,,. This suggests that IS is assessing airway inflammation in a valid manner that is relevant to asthma.

Future Application in Asthma Management

Induced sputum can provide information regarding which cells and mediators participate in a particular subject’s disease and may help the clinician confirm the diagnosis of asthma, assess the severity of inflammation, and track the effects of therapy directed toward inflammation. Certainly, there is a need to increasingly apply IS to subjects with severe whose disease is difficult to control, and who may have sputum eosinophilia despite systemic steroid therapy.

EXHALED BREATH TESTING

Undoubtedly, a most exciting recent development is the measurement of volatile mediators in exhaled breath and, in particular, NO as potential noninvasive markers of airway inflammation. There are in excess of 350 publications on exhaled NO (FENO)in asthma and other diseases. Other volatile substances such as ethane, pentane,46and recently exhaled car-

351

bon monoxide (CO),s9are also being studied. Nonvolatile mediators (e.g., hydrogen peroxide) are detectable in the liquid-phase of breath, which is termed breath condensate (BC). It is curious that it took so long to realize the potential for breath tests in pulmonary disease; other fields of medicine (e.g., gastroenterology) have employed hydrogen breath tests in the diagnosis of malabsortion for many years and, more recently, radiolabeled carbon dioxide (CO,) in the diagnosis of ulcer disease related to Heliobacter pylori.

Exhaled Nitric Oxide Nitric Oxide

Endogenous enzymatic NO formation was confirmed relatively recently, leading to the recognition that many physiologic and pathologic processes are mediated by NO, which has both local and systemic effects. The smooth-muscle-relaxant action of endothelialderived relaxing factors was found to depend on NO synthesis; this has led to thousands of publications about the role of NO in both physiologic and pathologic processes, culminating in the award of the Nobel Prize for Medicine to three NO investigators in 1998. Constitutive Nitric Oxide Production

Nitric oxide is produced by NO synthases (NOS) of both constitutive and inducible forms.8sConsitutive forms are calcium dependent, a n d secrete small parcels of NO that diffuse locally to activate soluble guanylate cyclase on target organs. Binding of NO to the heme portion of the receptor results in enzyme activation, resulting in the production of cyclic guanosine monophosphate, which mediates the physiologic effect (e.g., vasorelaxation). Constitutive NOS forms include endothelial NOS, which controls vascular tone in the pulmonary and systemic circulations, and neuronal NOS, which produces NO acting as a central and peripheral neurotransmitter. The nonadrenergic, noncholinergic inhibitory airway nerves (iNANC), the only bronchodilator nerves in humans, have NO as their neurotransmitter? Ciliary motion

352

SILKOFF

is mediated by NO, and NO stimulates airway secretion^.^^ Inducible Nitric Oxide Synthase and Inflammation

Inducible NOS is calcium independent and produces large amounts of NO relative to constitutive forms. Inflammatory cytokines such as interferon-?, tumor necrosis factor-a, and interleukin-1p can induce the expression of iNOS in other cells. Inducible NOS is of great pathogenic importance in inflammation of many kinds (e.g. sepsis, inflammatory bowel disease) and of relevance to asthmatic airway i n f l a m m a t i ~ n The . ~ ~ expression of iNOS is also important in host defense against bacteria because NO is toxic to microorganisms; exhaled NO therefore is high in pulmonary t u b e r c ~ l o s i s .Biopsy ~~ studies show that iNOS is increasingly expressed in bronchial epithelial cells in nonsteroid-treated asthmaz9;this expression is significantly reduced after inhaled steroids.70 The large amounts of iNOS-derived NO may have both pro- and anti-inflammatory actions. The proinflammatory effects of NO include vasodilatation, chemoattraction of inflammatory cells, increased airway secretions, and, of great importance, oxidative damage from metabolites such as peroxynitrite, which is formed from the reaction between NO and superoxide and is increased in asthma.70Peroxynitrite damages lipid membranes; oxidizes proteins, affecting enzymatic function; and can corrupt DNA. Anti-inflammatory actions of NO include the antibacterial action and immunomodulatory actions on other cells (e.g., macrophages).

ship was reported; FENO fell significantly after even 100 kg/day of inhaled budesonide and a maximal response was seen at 400 kg/ day.35 A recent study in children showed a signifi~ treatment ~ with montelcant drop in F E after ukast odium.^ Allergen challenge has been reported to cause an increase in F E N O at the time of the late response, compatible with worsening inflammation at that time.41In contrast, two studies have not shown any increase in FENO at night in subjects with nocturnal asthma, who are known to have wors83 ening airway and alveolar inflamrnati~n.’~, In both these studies, however, F E was ~ ~ higher in subjects with NA compared with those without NA. Several authors have reported that F E was ~ ~high in acute asthma exacerbations and fell after anti-inflammatory therapy.’*,48, 54 Exhaled NO is even increased in nonasthmatic atopic children compared with healthy Recent evidence suggests that NO output from the airway is increased in asthmatics compared with normal subjects even ufter steroid therapy because of N O S enhanced ability to diffuse in the airway in asthma.78,79 On the presumption that FENO after steroids derives from constitutive NOS production, this suggests that the airway diffusiveness of NO derived from cNOS isoforms is upregulated. This is most likely because of an increase in the surface area of airway epithelium occupied by cells containing cNOS isoforms. Because NO is the neurotransmitter of the nonadrenergic iNANC in h ~ r n a n sthis ,~ system may be upregulated as a compensatory mechanism to overuome bronchospasm. In support of this, airway NO output after steroids showed a positive correlation with the dose of methacholine that would cause a 20% drop in FEV, (Fig. l).78,79

Exhaled Nitric Oxide in Asthma

The demonstration of a raised F E in~ ~The Measurement of Lower Respiratory Tract Exhaled Nitric asthma has been reported by many investigaOxide tors in both adults and children.’ 42, 52, Exhaled NO falls rapidly after treatment with Exogenous Sources of Exhaled Nitric inhaled or oral corticosteroids and rises Oxide equally rapidly after steroid ~ i t h d r a w a lEx.~~ haled NO rose and fell with modulation of These include ambient NO, which can inhaled steroid dose in one study.43In another reach levels in excess of endogenous NO, and study, a significant dose-response relationNO in inspired gases (e.g., wall oxygen gas

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

50

6E J

h

10I'

-

353

50

-

o l/: 1

0

v

0

0.1 =

$ 0.01

0.001

-

.01= 0.01

0 I

I

Illrm

-08

0.001

I

I I I I l r n

Figure 1. The relationship between maximum NO output by diffusion in the airway (the product of exhaled NO concentration and expiratory flow rate) after inhaled steroids, and methacholine P&. The NO output after steroids, presumably in the main a constitutive NOS product, showed a direct correlation to methacholine reactivity and FEV,/FVC ratio, before and after inhaled steroids. The cNOS isoform of interest may be neuronal NOS in the nonadrenergic noncholinergic inhibitory bronchodilating system (iNANC) which has NO as its neurotransmitter.

supplies). Gastric NO, formed from the action of gastric acid on nitrates in food, can reach extremely high levelss3; similarly, nitrates in the oral cavity from ingestion of certain foods can result in increased FENo.90Standardized measurement techniques should exclude these exogenous sources of NO. Endogenous Sources of Exhaled Nitric Oxide

This refers to NO produced by NOS in the respiratory tract or from systemic sources and excreted in the lung. The upper airway and the paranasal sinuses contain extremely high levels of NO. This may help maintain sinus ~ t e r i l i t y .Acceptable ~~ measurement techniques must exclude nasal NO. This can be achieved by exhaling against expiratory resistance with a positive mouth pressure, which closes the velum, isolating the nasal cavity from the orally exhaled gas.77The oral cavity, through which exhaled gas passes, has been shown to contribute a small proportion of NO in normal after a nitrate-rich meal, this proportion may rise?O Nitric oxide synthases in diverse cells present in conducting airways (e.g., bronchial epithelial cells) pro-

duce NO that diffuses into the exhaled gas under a concentration gradient. This airway NO is primarily that which we wish to measure in airway inflammation. Finally, systemic NO production could result in increased excretion from pulmonary capillaries to alveolar gas. Nitric oxide is avidly bound by hemoglobin, however, which minimizes the transfer of NO from blood to alveoli." Indeed, most studies have shown that alveolar NO levels are very low (<5 ppb).'* ~ the ~ lack of a stanThe novelty of F E and daraized technique of measurement has led to the adoption of diverse measurement tech~ ~ reniques, resulting in variable F E levels ported by investigators and difficulty in comparing studies. A task force of the European Respiratory Society published European guidelines in 1997,40 and, more recently, a workshop sponsored by the American Thoracic Society and American Lung Association established principles for reproducible and standardized measurement.' Nitric Oxide Detection

The most widely used method of measurement uses the principle of chemiluminesence.

354

SILKOFF

The Restricted Breath Technique One commonly used method to measure FENO is the restricted breath technique.77Subjects inhale gas that has a low (<5 ppb) NO concentration. Then exhalation proceeds from total lung capacity (TLC) via a high expiratory resistance while subjects target a fixed mouthpiece pressure. The maneuver has been shown to close the velum, thereby isolating nasal NO from the sample, and the fixed mouthpiece pressure and resistance create a The Flow Dependence of Exhaled constant expiratory flow rate.77 The singleNitric Oxide breath NO profile when exhaling from TLC Alveolar NO levels are low because of the shows a washout phase followed by a steady intimate contact with hemoglobin in pulmoNO plateau that represents the F E value. ~ ~ nary capillaries. As alveolar gas ascends the Repeated exhalations are performed until airway, it is conditioned with NO that difthree plateau values agree at the 5% level. A fuses from conducting airway walls to the low expiratory flow (e.g., 50 mL/s) is prebronchial lumen under a concentration gradiferred because recent evidence suggests that ent. The flow in the conducting airways critilow flows (
Here, NO in the sample is drawn into a reaction cell, where it is reacted with ozone, emitting light that is detected by a photomultiplier tube. Modern instruments can measure fractional concentrations in parts per billion and even parts per trillion. Daily calibration with NO free gas and a NO standard gas, available from commercial companies, is essential.

40 1 I

450

350 h

300

n

g 250 4 200 W v

LL

150 100 50 0

1

10

100 1000 10000 Loglo Flow Rate mUs

100000

Figure 2. The flow dependence of F Ein ~ two~subjects with asthma. This shows the importance of tightly controlling exhalation flow rate when making single breath measurements.

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

lnvasiveness and Safety

Sampling of exhaled breath is noninvasive and extremely safe because no extraneous substances are used. The restricted breath technique just described has been safely applied in the author's laboratory to subjects with severe asthma and chronic obstructive pulmonary disease, and in the emergency room in severe asthma (R. Silverman MD, personal communication).Subjects may experience lightheadedness with repeated TLC inhalations and exhalations. Adequate rest therefore should be allowed between exhalations. Exhalation against expiratory pressure could cause syncope in susceptible subjects, so, in general, mouth pressures are kept below 20 CMS of water, which should minimize this risk. Another risk to subjects is infection from mouthpieces and valves, as can occur in pulmonary function testing. The infectious disease department in each hospital should address the procedure and recommend appropriate preventive measures. Commercially available NO analysis equipment should undergo periodic safety checks; exhaust gases from the analyzer and fumes from the pumps should be vented to reduce exposure of subjects. Reproducibility of F E ~ ~ Measurements

Exhaled NO plateau levels from Apeated exhalations at the same session are highly 71, 77 In normal subjects FENO reprodu~ible.~~, levels are also reproducible when measured repeatedly throughout the day and over a period of several weeks.n In one study (Sil~ ~rekoff et al, unpublished work), F E was markably stable in a cohort of 14 stable asthmatics, when measured at 0 and 7 days. Exhaled NO would not be necessarily reproducible over time in unstable disease, however.

355

inflammation, and with other standard outcome measures in asthma. Because FENO is a marker of airway inflammation, however, the relationship to markers of airway obstruction might not be close. In addition, the depressant effect of anti-inflammatory medication on F E could ~ ~potentially mask any relationship. Exhaled Nitric Oxide and Bronchoscopic Markers of Airway Inflammation

As with IS, there are remarkably few pub~ lished studies comparing F E to~ bronchoscopic indices of airway inflammation. The raised FENO in asthma, however, goes handin-hand with the enhanced expression of iNOS in human asthmatic bronchial epithelium in nonsteroid-treated asthma, as shown by Hamid et alZ9and Saleh et aL7OThe administration of inhaled steroids significantly reduced the iNOS expression in the second study. This clearly indicates that the increased F E in~ asthma ~ results from increased iNOS expression in the bronchial epithelium; the drop in FENO after steroids was also accompanied by reduced iNOS expression. In a recent study at the author's center, FENO showed no significant correlation with BAL cell counts in mild and severe asthma, but did correlate with activated eosinophils (EG2 + ) in endobronchial biopsies from the severe group (Silkoff et al; unpublished work). Exhaled Nitric Oxide and Induced Sputum

Several authors have reported a correlation between F E and ~ sputum ~ percent eosinophils in nonsteroid-treated asthma.3s, This is support for the validity of F E as~a ~ marker of eosinophilic airway inflammation, at least in nonsteroid-treated asthma.

The Correlation of Exhaled Nitric Oxide with Other Outcome Measures in Asthma

Exhaled Nitric Oxide and Airway Caliber

As with IS, it would be good if F E corre~ ~ lated with "gold standard indices of airway

Asthma severity is reflected in the degree of airway obstruction and its reversibility. One

356

SILKOFF

might expect that FEN0 would be higher in a more obstructed subject. Several studies have attempted to examine this issue. Of great importance in interpreting such studies is the fact that the FENO measurement is influenced by airway caliber, probably because the latter affects the mucosal surface area available for NO diffusion into the airway lumen. Bronchoconstriction, whether spontaneous or induced, therefore results in a drop in FENO, whereas bronchodilation increases F E levels ~ ~ by about Similarly, exhalation from functional residual capacity results in FENO levels that are lower than the same exhalations from TLC.= This can be attributed to a larger mucosal surface area at TLC, facilitating NO diffusion. Different studies have reported varying correlations between FENO and airway caliber. Silkoff et a176found no correlation between FENO and FEV, in 10 mild asthmatics while on or off inhaled beclomethasone. Furthermore, F E fell ~ significantly ~ on inhaled beclomethasone whereas pulmonary function was unchanged.76

Exhaled Nitric Oxide and Bronchial Reactivity

Increased airway inflammation could increase both bronchial reactivity and F E ~Sev~ . eral studies have demonstrated that FENOdoes show moderate correlations with bronchial reactivity. Dupont et all7 reported that FENO correlated with PC,, histamine in steroid-naive subjects (Y = - 0.65, P<.OOOl); the correlation disappeared in the same subjects after steroid administration. Similar findings have been reported by Lim etisowho found a significant correlation between FENO and PC,, methacholine (Y = - 0.64, R . 0 5 ) at baseline. No such relationship between FEN0 and methacholine was shown in another study, however, either on or off inhaled steroids.76

The Effects of Other Test Procedures on Exhaled Nitric Oxide

Exhaled NO is sensitive to other respiratory maneuvers. Spirometric maneuvers result in

~ lasts ~ up to 1 a temporary drop in F E that 8o Methacholine challenge or the bronchoconstriction that accompanies it results in a similar drop in F E , ~that returns to baseline after bronch~dilators.'~, 76 In a similar light, the administration of a bronchodilator results in a small but significant rise in FEN^.^' The common theme underlying these phenomena is that changes in airway caliber affect F E ~ ~ to a minor degree, probably by altering the mucosal surface area available for diffusion of NO from wall to lumen. The implication ~ ~ be meafor measurement is that F E should sured before or at least l hour after spirometry and that protocols that include other tests should be designed appropriately. Exhaled Nitric Oxide in the Future Management of Asthma

Whereas F E measurement ~ ~ has been a research tool, there is sufficient evidence to begin to define its use in the clinical management of asthma. The first step has been the standardization of measurement techniques in adults and children, including equipment specifications. A taskforce of the European Respiratory Society published guidelines in 199740and more recently a workshop of the American Thoracic Society established an international consensus on guidelines for the NO in adults measurement of F E and ~ nasal ~ t and children, including required equipment specifications.' The use of a standardized technique will allow the establishment of normative values. A further important step should be the establishment of an expert panel to decide how to use exhaled NO in clinical management. Issues to be addressed will include the clinical indications for the test, the integration of the test with other standard outcome efficacy measures, such as pulmonary function, and interpretation of the results. Current evidence suggests that exhaled NO may be useful in diagnosis," monitoring severity, and assessing therapeutic response and adherence in asthma, but more studies are required. BREATH CONDENSATE

The liquid phase of exhaled breath, BC, can be collected in a variety of manners. The most

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION

common employs low temperatures to condense out the moisture. Studies on breath condensate are starting to appear in the literature and the next section deals briefly with this new area of research. In 1993, Scheideler et al," reported that BC in eight healthy individuals contained between 4 pg and 1.4 mg/mL of protein, which originated partially from the naso-oropharyngeal tract and partially from lower regions of the airways. In patients with respiratory disease, BC contained up to 370 pg IL-1 p, 120 pg tumor necrosis factor-a, and 2159 U/ mL of soluble IL-2 receptor protein light chain. These nonvolatile molecules aerosolize into the exhaled gas from the lining fluid of the lower and upper respiratory tract. There is increasing interest in the use of BC to sample nonvolatile mediators of the lower respiratory tract. These include hydrogen peroxide (H202),16 and l e ~ k o t r i e n e sIn . ~ asthma, BC H,02 has been reported to be increased.16 Breath condensate thiobarbituric acid-reactive products and 8-isoprostanes may allow assessment of oxidative stress in inflammatory 56 airway di~ease.~,

357

There are major issues that require study, however, before BC can be reliably used as a noninvasive sampling technique in airway inflammation. For this reason, this article does not elaborate on BC. We need to determine whether a certain mediator arises from the lower respiratory tract or from the oral and nasal cavities. The influence of breathing pattern on BC has not yet been determined. Faster exhalation flow rates could theoretically result in the aerosolization of more material. Standardized sampling techniques will be essential to determine whether reproducible measurements are possible, and to determine the correlation with other indices of inflammation. SUMMARY

The recent use of IS and the analysis of exhaled mediators such as NO are important steps forward in our ability to noninvasively assess airway inflammation without the need to resort to bronchoscopy. Exhaled NO and IS are complementary techniques that provide different information (Table 1). Induced spu-

Table 1. A COMPARISON OF INDUCED SPUTUM AND EXHALED NITROUS OXIDE AS NONINVASIVE MARKERS OF AIRWAY INFLAMMATION Induced Sputum

Safety Test time

Information provided

Correlation with other parameters Practicality cost Application

Can r e h t in significant bronchospasm and oxygen desaturation during induction. Induction procedure takes approximately 1 hour. Processing sputum takes approximately 90 minutes. Total cell count, differential cell count, immunohistochemistry of cells; diverse mediators in soluble phase (e.g., ECP, IL-8, etc.).

Sputum cell differential correlates well with those seen in BAL and endobronchial biopsy. Time consuming and requires expert processing of sputum. Low capital cost but considerable technician time required.

Exhaled Nitric Oxide

No known significant associated hazards. Calibration procedures and testing require 2&30 minutes.

F E ~a ~nonspecific , marker of airway inflammation. F E determination ~ ~ at multiple flow rates allows calculation of airway NO diffusion factor, airway NO wall concentration, and alveolar NO levels. F E , ~correlates with induced sputum eosinophilia and methacholine PCz0 in steroid-naive asthma. Test is simple to learn and perform.

High initial capital cost of NO analyzer and computer, etc., but low technician and maintenance cost. Investigation of mechanisms of airway inflammation, diagnosis of asthma, periodic monitoring, assessment of response to therapeutic intervention, adherence monitoring.

ECP = eosinophil cationic protein; IL = interleukin; BAL = bronchoalveolar lavage; FE,, oxide; PC, = the dose of methacholine causing a 20% fall in FEV,.

=

expiratory nitric oxide; NO

=

nitric

358

SILKOFF

tum can provide knowledge regarding the cells and mediators participating in the inflammatory response, but is time consuming and expensive. Exhaled NO measurement is performed simply and quickly, and is a nonspecific marker of an inflammatory process. The initial capital costs of equipment for NO analysis are high, however. Once the problems of standardized collection and oropharyngeal contamination have been dealt with, BC may also prove to be an additional tool for the assessment of airway inflammation. It is likely that the next 10 years will see the establishment of these noninvasive tools for the clinical assessment of airway inflammation and oxidative stress, and change the entire way we manage asthma.

References 1. American Thoracic Society/American Lung Association: Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children. Am J Respir Crit Care Med 1602104, 1999 2. Alving K, Weitzberg E, Lundberg JM: Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 61368, 1993 3. Baraldi E, Azzolin NM, Cracco A, et ak Reference values of exhaled nitric oxide for healthy children 6-15 years old. Pediatr Pulmonol2754, 1999 4. Becher G, Winsel K, Beck E, et al: Breath condensate as a method of noninvasive assessment of inflammation mediators from the lower airways. Pneumologie 51(supp1)2456, 1997 5. Belvisi MG, Stretton CD,Yacoub M, et al: Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in humans. Eur J Pharmacol210221,1992 6. Bhowmik A, Seemungal TA, Sapsford RJ, et al: Comparison of spontaneous and induced sputum for investigation of airway inflammation in chronic obstructive pulmonary disease. Thorax 53:953, 1998 7. Bisgaard H, Loland L, Oj JA: NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am J Respir Crit Care Med 160:1227, 1999 8. Brightling CE, Ward R, Goh KL, et al: Eosinophilic bronchitis is an important cause of chronic cough. Am J Respir Crit Care Med 160:406, 1999 9. Carpenter CT, Price PV, Christman BW: Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest 1142653, 1998 10. Castagnaro A, Chetta A, Foresi A, et ak Effect of sputum induction on spirometric measurements and arterial oxygen saturation in asthmatic patients, smokers, and healthy subjects. Chest 116:941, 1999 11. Chatkin JM, Ansarin K, Silkoff PE, et al: Exhaled nitric oxide as a noninvasive assessment of chronic cough. Am J Respir Crit Care Med 1591810,1999

12. Crater SE, Peters EJ, Martin ML, et al: Expired nitric oxide and airway obstruction in asthma patients with an acute exacerbation. Am J Respir Crit Care Med 159:806, 1999 13. de Gouw HW, Hendriks J, Woltman AM, et al: Exhaled nitric oxide (NO) is reduced shortly after bronchoconstriction to direct and indirect stimuli in asthma. Am J Respir Crit Care Med 158:315, 1998 14. de la Fuente PT, Romagnoli M, Godard P,et al: Safety of inducing sputum in patients with asthma of varying severity. Am J Respir Crit Care Med 1571127, 1998 15. Deykin A, Halpem 0, Massaro AF, et al: Expired nitric oxide after bronchoprovocation and repeated spirometry in patients with asthma. Am J Respir Crit Care Med 157769, 1998 16. Dohlman AW, Black HR, Royal1 JA: Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma. Am Rev Respir Dis 148955, 1993 17. Dupont LJ, Rochette F, Demedts MG, et al: Exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma. Am J Respir Crit Care Med 157894, 1998 18. Dweik RA, Laskowski D, Abu-Soud HM, et ak Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 101:660, 1998 19. Fabbri LM, Durham S, Holgate ST, et al: Assessment of airway inflammation: An overview. Eur Respir J Suppl26:6S, 1998 20. Fahy JV A safe, simple, standardized method should be used for sputum induction for research purposes. Clin Exp Allergy 28:1047, 1998 21. Fahy JV, Liu J, Wong H, et ak Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis 1471126, 1993 22. Fahy JV, Wong H, Liu J, et a1 Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects. Am J Respir Crit Care Med 15253,1995 23. Franklin PJ, Taplin R, Stick SM: A community study of exhaled nitric oxide in healthy children. Am J Respir Crit Care Med 159:69, 1999 24. Gabbay E, Fisher AJ, Small T, et al: Exhaled singlebreath nitric oxide measurements are reproducible, repeatable and reflect levels of nitric oxide found in the lower airways. Eur Respir J 11:467, 1998 25. Georges G, Bartelson BB, Martin RJ, et al: Circadian variation in exhaled nitric oxide in nocturnal asthma. J Asthma 36:467, 1999 26. Gershman NH, Liu H, Wong HH, et al: Fractional analysis of sequential induced sputum samples during sputum induction: Evidence that different lung compartments are sampled at different time points. J Allergy Clin Immunol 104322, 1999 27. Gershman NH, Wong HH, Liu JT, et al: Comparison of two methods of collecting induced sputum in asthmatic subjects. Eur Respir J 9:2448, 1996 28. Grootendorst DC, Sont JK, Willems LN, et al: Comparison of inflammatory cell counts in asthma: Induced sputum vs bronchoalveolar lavage and bronchial biopsies. Clin Exp Allergy 27769, 1997 29. Hamid Q, Springall DR, Riveros-Moreno V, et al: Induction of nitric oxide synthase in asthma. Lancet 342:1510,1993 30. Holz 0, Richter K, Jorres RA, et al: Changes in sputum composition between two inductions performed on consecutive days. Thorax 53:83, 1998

NONINVASIVE MEASUREMENT OF AIRWAY INFLAMMATION 31. Hsu JY, Huang CM, King SL, et al: Importance of sputum differential cell counting in the diagnosis of airway diseases. J Formos Med Assoc 96330, 1997 32. Hunter CJ, Ward R, Woltmann G, et al: The safety and success rate of sputum induction using a low output ultrasonic nebuliser. Respir Med 93:345, 1999 33. in't Veen JC, Smits HH, Hiemstra PS, et al: Lung h c t i o n and sputum characteristics of patients with severe asthma during an induced exacerbation by double-blind steroid withdrawal. Am J Respir Crit Care Med 160:93, 1999 34. Inoue H, Aizawa H, Fukuyama S, et al: Effect of inhaled glucocorticoid on the cellular profile and cytokine levels in induced sputum from asthmatic patients. Lung 17753, 1999 35. Jatakanon A, Kharitonov S, Lim S, et al: Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma. Thorax 54108, 1999 36. Jatakanon A, Lalloo UG, Lim S, et al: Increased neutrophils and cytokines, TNF-a and IL-8, in induced sputum of non-asthmatic patients with chronic dry cough. Thorax 54234, 1999 37. Jatakanon A, Lim S, Chung W, et al: An inhaled steroid improves markers of airway inflammation in patients with mild asthma. Eur Respir J 12:1084, 1998 38. Jatakanon A, Lim S, Kharitonov SA, et al: Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 53:91, 1998 39. Keatings VM, Jatakanon A, Worsdell YM, et al: Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 155:542, 1997 40. Kharitonov SA, Alving K, Bames PJ: Exhaled and nasal nitric oxide measurements: Recommendations. The European Respiratory Society Task Force. Eur Respir J 103683, 1997 41. Kharitonov SA, OConnor BJ, Evans DJ, et al: Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am J Respir Crit Care Med 151:1894, 1995 42. Kharitonov SA, Yates D, Springall DR, et al: Exhaled nitric oxide is increased in asthma. Chest 107156S, 1995 43. Kharitonov SA, Yates DH, Chung KF, et al: Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur Respir J 9:196, 1996 44. Kips JC, Fahy JV,Hargreave FE, et al: Methods for sputum induction and analysis of induced sputum: A method for assessing airway inflammation in asthma. Eur Respir J Suppl 26:9S, 1998 45. Kips JC, Peleman RA, Pauwels RA: Methods of examining induced sputum: Do differences matter? Eur Respir J 11:529, 1998 46. Kneepkens CM, Ferreira C, Lepage G, et al: The hydrocarbon breath test in the study of lipid peroxidation: Principles and practice. Clin Invest Med 15:168, 1992 47. Kraft M, Djukanovic R, Wilson S, et al: Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1541505, 1996 48. Lanz MJ, Leung DY, White C W Comparison of exhaled nitric oxide to spirometry during emergency treatment of asthma exacerbations with glucocorticoids in children. Ann Allergy Asthma Immunol 82:161, 1999 49. Lemiere C, Pizzichini MM, Balkissoon R, et al: Diag-

359

nosing occupational asthma: Use of induced sputum. Eur Respir J 13:482, 1999 50. Lim S, Jatakanon A, John M, et a1 Effect of inhaled budesonide on lung function and airway inflammation. Assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 159:22,1999 51. Lundberg JO, Farkas-Szallasi T, Weitzberg E, et al: High nitric oxide production in human paranasal sinuses. Nat Med 1:370, 1995 52. Lundberg JO, Nordvall SL, Weitzberg E, et al: Exhaled nitric oxide in paediatric asthma and cystic fibrosis. Arch Dis Child 75:323, 1996 53. Lundberg JO, Weitzberg E, Lundberg JM, et al: Intragastric nitric oxide production in humans: Measurements in expelled air. Gut 35:1543, 1994 54. Massaro AF, Gaston 8, Kita D, et al: Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 152:800, 1995 55. Minoguchi K, Kohno Y, Oda N, et al: Effect of theophylline withdrawal on airway inflammation in asthma. Clin Exp Allergy 28(suppl3):57,1998 56. Nowak D, Kasielski M, Antczak A, et al: Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: No significant effect of cigarette smoking. Respir Med 93:389, 1999 57. OByme PM, Inman MD: Induced sputum to assess airway inflammation in asthma [editorial; comment]. Eur Respir J 9:2435, 1996 58. Ohnishi H, Maeda H, Nishimura Y, et al: Benefits of induced sputum for the evaluation of therapeutic efficacy in patients with bronchial asthma. Kobe J Med Sci 44:149, 1998 59. Ong EL, Mandal BK Tuberculosis in patients infected with the human immunodeficiency virus. Q J Med 80:613, 1991 60. Park JW, Whang YW, Kim CW, et al: Eosinophil count and eosinophil cationic protein concentration of induced sputum in the diagnosis and assessment of airway inflammation in bronchial asthma. Allergy Asthma Proc 19:61, 1998 61. Persson MG, Zetterstrom 0, Agrenius V, et al: Singlebreath nitric oxide measurements in asthmatic patients and smokers. Lancet 343:146, 1994 62. Piacentini GL, Bodini A, Costella S, et al: Exhaled nitric oxide and sputum eosinophil markers of inflammation in asihmatic children. Eur Respir J 13:1386, 1999 63. Pin I, Gibson PG, Kolendowicz R, et al: Use of induced sputum cell counts to investigate airway inflammation in asthma. Thorax 4725, 1992 64. Pizzichini E, Pizzichini MM, Efthimiadis A, et a1 Indices of airway inflammation in induced sputum: Reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 154:308, 1996 65. Pizzichini E, Pizzichini MM, Efthimiadis A, et al: Measurement of inflammatory indices in induced sputum: Effects of selection of sputum to minimize salivary contamination. Eur Respir J 9:1174, 1996 66. Pizzichini E, Pizzichini MM, Kidney JC, et al: Induced sputum, bronchoalveolar lavage and blood from mild asthmatics: Inflammatory cells, lymphocyte subsets, and soluble markers compared. Eur Respir J 11:828, 1998 67. Pizzichini MM, Pizzichini E, Clelland L, et al: Sputum in severe exacerbations of asthma: Kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med 155:1501, 1997

360

SILKOFF

68. Pizzichini MM, Pizzichini E, Efthimiadis A, et al: Asthma and natural colds. Inflammatory indices in induced sputum: A feasibility study. Am J Respir Crit Care Med 158:1178, 1998 69. Runer T, Lindberg S: Effects of nitric oxide on blood flow and mucociliary activity in the human nose. Ann Otol Rhino1 Laryngol 10740, 1998 70. Saleh D, Ernst P, Lim S, et a1 Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: Effect of inhaled glucocorticoid. FASEB J 12929, 1998 71. Salome CM, Roberts AM, Brown NJ, et al: Exhaled nitric oxide measurements in a population sample of young adults. Am J Respir Crit Care Med 159:911, 1999 72. Sartori C, Lepori M, Busch T, et al: Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans. Am J Respir Crit Care Med 160:879, 1999 73. Scheideler L, Manke HG, Schwulera U, et al: Detection of nonvolatile macromolecules in breath. A possible diagnostic tool? Am Rev Respir Dis 148:778, 1993 74. Shelhamer JH, Levine SJ, Wu T, et al: NIH conference. Airway inflammation. Ann Intern Med 123:288, 1995 75. Silkoff PE, McClean PA, Caramori M, et al: A significant proportion of exhaled nitric oxide arises in large airways in normal subjects. Respir Physiol 113:33, 1998 76. Silkoff PE, McClean PA, Slutsky AS, et a1 Exhaled nitric oxide and bronchial reactivity during and after inhaled beclomethasone in mild asthma. J Asthma 35:473, 1998 77. Silkoff PE, McClean PA, Slutsky AS, et al: Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med 155260, 1997 78. Silkoff PE, Sylvester JT, Zamel N, et al: Mechanism of elevated exhaled nitric oxide concentration in asthma [abstract]. Am J Respir Crit Care Med 159:A409, 1999

79. Silkoff PE, Sylvester JT, Zamel N, et al: Modulation of baseline pulmonary function and airways responsiveness by endogenous NO in asthma. Am J Respir Crit Care Med 159:A409, 1999 80. Silkoff PE, Wakita S, Chatkin J, et al: Exhaled nitric oxide after p,-agonist inhalation and spirometry in asthma. Am J Respir Crit Care Med 159:940, 1999 81. Spanevello A, Migliori GB, Sharara A, et al: Induced sputum to assess airway inflammation: A study of reproducibility. Clin Exp Allergy 271138, 1997 82. Spanevello A, Vignola AM, Bonanno A, et al: Effect of methacholine challenge on cellular composition of sputum induction. Thorax 54:37, 1999 83. ten Hacken NH, van der Vaart H, van der Mark TW, et al: Exhaled nitric oxide is higher both at day and night in subjects with nocturnal asthma. Am J Respir Crit Care Med 158:902, 1998 84. van Rensen EL, Straathof KC, Veselic-Charvat MA, et al: Effect of inhaled steroids on airway hyperresponsiveness, sputum eosinophils, and exhaled nitric oxide levels in patients with asthma. Thorax 54403, 1999 85. Wang CH, Liu CY, Lin HC, et al: Increased exhaled nitric oxide in active pulmonary tuberculosis due to inducible NO synthase upregulation in alveolar macrophages. Eur Respir J 11:809, 1998 86. Wehner JH, Jensen WA, Kirsch CM, et al: Controlled utilization of induced sputum analysis in the diagnosis of Pneurnocystis carinii pneumonia. Chest 105:1770, 1994 87. Wong HH, Fahy JV: Safety of one method of sputum induction in asthmatic subjects. Am J Respir Crit Care Med 156299, 1997 88. Zapol WM, Rimar S, Gillis N, et al: Nitric oxide and the lung. Am J Respir Crit Care Med 149:1375, 1994 89. Zayasu K, Sekizawa K, Okinaga S, et al: Increased carbon monoxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1561140, 1997 90. Zetterquist W, Pedroletti C, Lundberg JO, et al: Salivary contribution to exhaled nitric oxide. Eur Respir J 13:327, 1999

Address reprint requests to Philip E. Silkoff, MBBS, MRCP 1400 Jackson Street Denver. CO 80206 e-mail: [email protected]