INTRODUCTION

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The majority of patients with occupational asthma have persistent symptoms and airway hyperresponsiveness despite removal from exposure (1). Airway remodeling and persistent airway inflammation are likely to be the reasons for the persistence of airway hyperresponsiveness (2, 3).

The persistence of symptoms and airway hyperresponsiveness in patients with occupational asthma has led to compensation issues for permanent disability. In 1993, the American Thoracic Society (ATS) published a guideline on "Disability/ Impairment Evaluation of Patients with Asthma" recognizing that patients with asthma are different from patients with chronic irreversible lung disease (4). This guideline was based on the consensus of a group of experts and has not been validated.

Sputum examination has been shown to be a useful noninvasive method to study airway inflammation (5). Recently, exhaled nitric oxide (NO) was suggested to be a usable marker of airway inflammation becuase it was found to be significantly increased in patients with inflammatory airway diseases such as asthma (6, 7).

The purpose of this study was to utilize the above two new techniques to: (1) relate the degree of respiratory impairment as determined by the ATS guidelines in patients with Western red cedar asthma and the degree of airway inflammation as reflected in cell counts in induced sputum and the level of exhaled NO, and (2) explore the relationship between the level of exhaled NO and the degree of airway inflammation.

	METHODS

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Subjects

Seventy-one consecutive patients with asthma caused by Western red cedar (Thuja plicata), diagnosed by specific challenge test using plicatic acid (the agent responsible for red cedar asthma) (8), participated in a follow-up study.

Study Protocol

The follow-up examination consisted of the following: a questionnaire interview, spirometry, and methacholine challenge test or measurement of bronchodilator response. In addition, the level of exhaled NO was measured and sputum induction was carried out. The subjects were told to refrain from using short-acting ₂-agonists for at least 6 h and oral bronchodilators for 12 h before testing. The study was approved by our institutional ethics committee, and informed consent was obtained from all the subjects.

Lung Function and Methacholine Challenge Tests

FEV₁ and FVC were measured using a rolling-seal spirometer (Model VRS 2000; S and M Instruments, Doylestown, PA). The results were expressed as percent predicted using the normal values of Crapo and colleagues (9). Methacholine inhalation tests were carried out in subjects with FEV₁ more than 70% predicted (n = 51). For those with FEV₁ less than 70% predicted, spirometry was repeated after inhalation of salbutamol 200 µg (n = 21). The details of methacholine inhalation tests have been described previously (10). Briefly, the diluent control solution and solutions of methacholine were aerosolized in a Bennett twin nebulizer with oxygen at a flow rate of 5 L/min (Bennett Respiration Products, Los Angeles, CA). The patients inhaled the solutions at tidal breathing for 2 min, starting with the diluent and followed by increasing concentrations of methacholine at intervals of 5 min until the highest concentration of 32 mg/ml was reached. Measurements of FEV₁ and FVC were made before the test and at 30 s and at 3 min after each inhalation. The provocative concentration of methacholine that induced a 20% fall in FEV₁ (PC₂₀) was obtained by interpolation of the last two points of the dose-response curve drawn on a semilogarithmic noncumulative scale.

Measurement of Exhaled NO

Exhaled NO was measured by a rapid-response chemiluminescent method using the Sievers 280 NOA analyzer (Sievers, Boulder, CO), with sensitivity from 5 to 500,000 ppb, according to Silkoff and coworkers (11). Briefly, the subject with no noseclip inhaled through the mouth to total lung capacity, and immediately exhaled through an expiratory resistance to control expiratory flow. During exhalation, the subject was asked to maintain for a period of 10 to 15 s a constant mouth pressure of 20 mm Hg, which was displayed on the pressure monitor, and to support his or her cheeks manually to achieve a good mouthpiece seal to ensure vellum closure. Nitric oxide was sampled via a side port close to the mouth. Nitric oxide and mouth pressure signals were simultaneously displayed on the screen of a computer. The end point for the measurement was defined when a plateau at least 5 s in duration was reached. Repeated exhalations were recorded to give three values of NO plateau within 10% of each other. A rest of 30 s was given between exhalations. Before use each day, the analyzer was calibrated with zero NO gas and 50 ppm NO. We measured the ambient NO before testing each subject, and the levels varied between 0.7 and 376 ppb. However, there was no correlation between the levels of ambient NO and the levels of exhaled NO (r = 0.05, p = 0.68); the ambient NO level could be high when the subject's exhaled NO was low and vice versa.

Sputum Induction

Sputum was induced with an aerosol of inhaled hypertonic saline using a modification of the method of Pin and colleagues (12). Before sputum induction, inhaled salbutamol 200 µg was given to inhibit possible airway constriction. FEV₁ and FVC were measured three times before induction. The best FEV₁ value was used as the baseline. The Fisoneb ultrasonic nebulizer (Clement Clarke International Ltd., Harlow, Essex, UK) with an output of 0.87 ml/min and an aerodynamic mass median diameter of 5.58 µm was used. Concentrations of saline at 0.85, 3, 4, and 5%, each for 7 min, were given by inhalation through a mouthpiece without a valve or noseclip. After each inhalation period, spirometry was repeated. The patient was then asked to rinse his mouth with water and blow his nose (to reduce contamination of the sputum specimen with saliva and postnasal drip) before coughing sputum into a container. The nebulization was stopped if a sputum sample of good quality was obtained. If the FEV₁ fell by > 10% from the baseline the same concentration of saline was used for the next inhalation. If the FEV₁ fell by > 20%, or if troublesome symptoms occurred, nebulization was discontinued.

Sputum Examination

Sputum was collected in a sterile container and analyzed within 2 h using the method described by Pizzichini and coworkers (13). The whole sample of sputum was transferred to a Petri dish. The weight and macroscopic characteristics of the sample were recorded. All visible portions of sputum with little or no squamous cell contamination were carefully selected, placed in a preweighed conical tube, and weighed. Freshly prepared Dithiothreitol (DTT) (Spitolysin; Calbiochem Corp., San Diego, CA) in a dilution of 1 in 10, was added in a volume (in microliters) equal to four times the weight of sputum (in milligrams), votexed for 15 s, and rocked on a bench rocker for 15 min. It was then mixed with an equal volume of Dulbecco's phosphate-buffered saline (D-PBS), rocked for another 5 min, and filtered through 48-µm nylon gauze. The suspension was centrifuged at 790 × g for 10 min, and the supernatant was aspirated. The pellet was resuspended in a volume of 500 to 1,000 µl of D-PBS. The total cell count was determined using a Neubauer hemocytometer, and the number of cells per milliliter of processed sputum was calculated. The viability of cells was evaluated by trypan blue exclusion method. The cell suspension was adjusted to 1 × 10⁶ cells/ml. Two cytospins (each coded) were prepared by placing 75 µl cell suspension in cups of cytocentrifuge and centrifuged at 600 rpm for 5 min. The slides were air-dried and stained with Wright's stain. Four hundred nonsquamous cells were counted in the selected portion by two investigators blinded to the clinical status of the subjects. The nonsquamous differential cell counts were expressed as a percentage of the total nonsquamous counts.

Impairment and Disability Score

The rating of impairment for each subject was done according to ATS guidelines (4). The degree of impairment was calculated as the sum of the scores for lung function, airway hyperresponsiveness, or response to the bronchodilator and the medication used to control asthma. The class of impairment was expressed as Class 0 (total score, 0), Class 1 (total score, 1 to 3), Class 2 (total score, 4 to 6), Class 3 (total score, 7 to 9), and Class 4 (total score, 10 to 11).

Statistical Analysis

Of the 71 subjects, 67 successfully brought up sputum on induction and 64 subjects had reproducible exhaled NO measurements. Levels of exhaled NO, PC₂₀, total cell count, and percentages of eosinophils, epithelial cells, and macrophages were not normally distributed. The results were transformed into logarithms before analysis. Independent sample t tests and two-way ANOVA were used for comparison of variables between groups. The relationship between variables was analyzed by Pearson's correlation. A value of p < 0.05 was considered statistically significant.

	RESULTS

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The characteristics of the study subjects at the time of follow-up examination are shown in Table 1. Of the 71 subjects, 50 were no longer exposed to Western red cedar, whereas 21 continued to work with the wood. Those who were no longer exposed were older at the time of diagnosis than were those who continued to be exposed. There were no differences between the two groups in smoking habits, atopic status, and in the type of asthmatic reaction induced by an inhalation challenge test with plicatic acid (at the time of diagnosis). The average period of follow-up was 10 yr for both groups (range, 1 to 24 yr). Fifty-three percent of those who were no longer exposed were still receiving steroids compared with 76.1% of those who were still exposed (p = 0.07). The majority of subjects who required steroids took them by inhalation; three subjects required oral steroids (in the group no longer exposed) in addition to high dose inhaled steroids.

The results of lung function, airway hyperresponsiveness, exhaled NO, and sputum examination of these two groups of subjects, according to steroid requirement, are shown in Table 2. In both exposure groups, those who did not require any steroids had higher FEV₁ and PC₂₀ than did those requiring steroids for the control of their asthma. After adjusting for steroid usage, no difference in FEV₁ was found between those who were still exposed and those who were not exposed to Western red cedar, but PC₂₀ was significantly lower among those who were still exposed (p = 0.05). No differences were found in the level of exhaled NO and percentage of eosinophils in sputum between the exposed and the not exposed, irrespective of steroid requirement.

The percentage of eosinophils in sputum was inversely correlated with FEV₁ (r = 0.46, p < 0.001), but not with PC₂₀ (r = 0.01, p = 0.95). There was no correlation between the level of exhaled NO and FEV₁ (r = 0.03, p = 0.82) or exhaled NO and PC₂₀ (r = 0.11, p = 0.45). The above findings were similar when analyses were carried out separately for those receiving steroids and those not receiving steroids. Significant correlation was found between the percentages of eosinophils and the levels of exhaled NO (r = 0.42, p < 0.001) (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Relationship between percentage sputum eosinophils and exhaled nitric oxide (ppb) (r = 0.42, p < 0.001).

Of the 50 subjects, 12 had no impairment or Class 0, 17 belonged to Class 1, 12 to Class 2, five to Class 3, and four to Class 4. The results of FEV₁, PC₂₀, percent eosinophils in sputum, and levels of exhaled NO for each class of respiratory impairment, according to the ATS guidelines, are shown in Figure 2. The class of respiratory impairment (or the total impairment score) was inversely related to the level of FEV₁ and PC₂₀. There was a direct correlation between the class of respiratory impairment and the percentage of eosinophils in sputum (r = 0.52, p < 0.001): the higher the impairment class, the higher the percentage of eosinophils in sputum. No relationship was found between the class of respiratory impairment and the level of exhaled NO. The results were similar when the analysis was carried out on all subjects irrespective of exposure.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Class of respiratory impairment (according to the ATS guidelines) and FEV1 percentage predicted, PC20, percentage sputum eosinophils, and exhaled nitric oxide (ppb).

	DISCUSSION

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In this study, we have once again demonstrated that most of the patients with occupational asthma caused by Western red cedar still have not recovered completely many years after removal from exposure. Among those who left exposure, we found that there was a good correlation between the percentage of sputum eosinophils and the impairment class; those with the highest impairment class had the highest percentage of eosinophils in the sputum, indicating the persistence of airway inflammation in these subjects. The ATS guidelines for assessment of respiratory impairment in patients with asthma require three separate parameters, lung function, airway hyperresponsiveness, and the medication required for the control of asthma (4). It is an attempt to take into consideration that patients with asthma have variable airway obstruction; thus using lung function tests alone as for patients with irreversible lung disease is not appropriate. Patients with asthma also have airway hyperresponsiveness that makes it difficult for them to work in places with exposure to gases, fumes, or smoke and reduces their quality of life by restricting their daily living activities. This study provides objective evidence to support the method of rating of impairment in patients with asthma by demonstrating its relationship with airway inflammation.

We found an inverse relationship between the percentage of eosinophils in the sputum and the degree of airflow obstruction, but not the degree of airway hyperresponsiveness. The lack of correlation between sputum eosinophils and PC₂₀ in this study may be due to the fact that subjects with FEV₁ less than 70% did not have methacholine challenge tests, whereas FEV₁ was available in all subjects for correlation with percentages of eosinophils. Some previous studies have shown a strong relationship between eosinophilic inflammation and airway hyperresponsiveness (14), whereas others have failed to show such a relationship (18). Haley and Drazen (21) suggested that in some asthmatics, cellular infiltration may occur during hyperresponsiveness, whereas in others the cellular events leading to the hyperresponsive state may be dissipated by the time the hyperresponsive state manifests.

An interesting finding is the significant correlation between the percentage of eosinophils in sputum and the level of exhaled NO. NO has also been found to have a selective suppressive effect on the Th1 CD4+ T-cells and may shift the balance of T-cells to Th2 predominance (22) and may induce eosinophilic airway inflammation; on the other hand, the increased airway eosinophilia may lead to increased NO (6, 7).

We failed to find any correlation between the level of exhaled NO and FEV₁, PC₂₀, and the class of respiratory impairment irrespective of whether the subject was receiving steroids or not. Our finding in this aspect is at variance with those reported by Dupont and colleagues (23) who demonstrated a correlation between airway hyperresponsiveness and exhaled NO in steroid-naive patients but not in steroid-treated asthmatics. More recent studies failed to demonstrate any relationship between exhaled NO and airway hyperresponsiveness (24). Deykin and coworkers (27) and Therminarias and colleagues (28) have shown that ambient levels of NO (greater than 40 ppb) affect the exhaled NO response. It is possible that the lack of relationship between exhaled NO and other lung function parameters in this study was due to our failure to give the subjects NO-free air before the measurement of exhaled NO. The total lack of relationship between ambient and exhaled NO levels and the significant relationship between exhaled NO and percent sputum eosinophils render this explanation somewhat unlikely. Most previous studies have not taken air contamination by NO into consideration (7, 11, 23). The results of this study and others (24- 26) suggest that exhaled NO may be of limited clinical utility on its own in the monitoring of asthma severity.

In conclusion, we found persistent airway inflammation in subjects with occupational asthma caused by Western red cedar who continued to be exposed and in those who failed to recover after removal from exposure. There was an inverse relationship between the degree of eosinophilic airway inflammation and the degree of airflow obstruction as well as the class of respiratory impairment. We found a significant relationship between exhaled NO and sputum eosinophils but no relationship between the level of exhaled NO and the degree of respiratory impairment irrespective of steroid usage. This is the first study to provide objective evidence to support the impairment rating proposed by the ATS for assessment of patients with asthma.