Chest ACCP Career Connection
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arjomandi, M.
Right arrow Articles by Balmes, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arjomandi, M.
Right arrow Articles by Balmes, J.
(Chest. 2005;128:416-423.)
© 2005 American College of Chest Physicians

Sputum Induction and Bronchoscopy for Assessment of Ozone-Induced Airway Inflammation in Asthma*

Mehrdad Arjomandi, MD; Isabelle Schmidlin, PhD; Peter Girling, BA; Kevin Boylen; Ron Ferrando, BA and John Balmes, MD, FCCP

* From the Lung Biology Center, Northern California Center for Occupational and Environmental Health, and Medical Service, San Francisco General Hospital, University of California, San Francisco, CA.

Correspondence to: John R. Balmes, MD, University of California, San Francisco, Box 0843, San Francisco, CA 94143-0843; e-mail: jbalmes{at}itsa.ucsf.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Neutrophilic airway inflammation, as defined by cell counts in respiratory tract lining fluid (RTLF), is a key end point in many studies of respiratory toxicity in both healthy and asthmatic subjects. BAL and sputum induction (SI) are the most common methods of sampling RTLF in such studies. However, the comparability of these methods (BAL and SI) after experimental treatment has not been investigated in a head-to-head controlled trial.

Methods: To determine whether BAL and SI are comparable and can be used in place of each other in the assessment of neutrophilic airway inflammation after ozone (O3) exposure, we exposed 13 asthmatic subjects to either 0.2 ppm of O3 or filtered air (FA) followed by either BAL or SI. Subjects then underwent the alternate (O3 or FA) exposure followed by the same method of RTLF sampling. Next, subjects repeated the same exposure protocol with the alternate method of RTLF sampling. Differences in inflammatory indexes including the percentage of polymorphonuclear neutrophils (%PMNs) between the exposures were then correlated by regression analysis.

Results: The %PMNs in sputum was poorly correlated with that in BAL fluid (R = 0.12). The correlation between the %PMNs in sputum and in the bronchial fraction of BAL (BFx) fluid, however, was somewhat higher (R = 0.50). Furthermore, the uncertainty of the estimate of %PMN values in BFx fluid and BAL fluid based on those of sputum values, using regression models, was almost as great as the magnitude of the O3 effect itself (ie, 9.7% and 5.5% estimate errors for O3 effects of 17.0% and 7.5%, respectively).

Conclusion: We concluded that SI and BAL indexes are not directly interchangeable in the assessment of O3-induced airway inflammation in asthmatic subjects.

Key Words: airway inflammation • asthma • bronchoscopy • ozone • sputum


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies of human respiratory tract inflammation have traditionally relied on the analysis of samples of airway and alveolar lining fluid and bronchial tissue obtained by bronchoscopy. Although the analysis of these samples has contributed much to our current understanding of various pulmonary disease processes, bronchoscopy is invasive, technically difficult, and not always acceptable to volunteer subjects.

Sputum induction (SI) has been demonstrated to be a noninvasive alternate method of assessing airway inflammation that is reproducible, valid, and responsive to changes induced by proinflammatory and antiinflammatory stimuli.1234 While both SI and bronchoscopy appear to be valid methods for assessing airway inflammation, it is not clear whether the results obtained by these two different methods have the same biological meaning. It has been shown that similar qualitative information may be obtained from both induced sputum and bronchoscopy samples (ie, bronchial wash and BAL fluid) in both asthmatic and healthy subjects.5 Changes in inflammatory cells and mediators in samples of induced sputum also have been shown to be qualitatively similar to those reported in BAL fluid after aerosolized whole-lung allergen challenge6 and after ozone (O3) exposure.78 Cell differential counts in induced sputum also have been compared with those in bronchial wash and BAL fluid samples before and after therapy with inhaled corticosteroids (ICSs) or ß-agonists in asthmatic subjects.9 To our knowledge, however, no direct quantitative comparisons have been performed to demonstrate the degree of association between the changes in inflammatory indexes in induced sputum and those in BAL fluid after an experimental protocol.

To determine whether changes in SI and BAL indexes of airway inflammation are predictive of each other, we performed a randomized crossover study in which asthmatic subjects were exposed either to O3 or filtered air (FA). The respiratory tract lining fluid (RTLF) was then sampled using both SI and bronchoscopy. We then compared the indexes of inflammation in bronchial wash or BAL fluid samples after O3 and FA exposures with those in induced sputum samples.

We chose O3 as the experimental treatment in this study because it produces a significant neutrophilia in airway and alveolar lining fluid, which is readily measurable in induced sputum samples781011121314151617 and bronchial wash or BAL fluid samples.18192021 We chose to study asthmatic subjects because we and others have previously shown that O3 causes greater airway inflammation in asthmatic subjects compared to healthy subjects.2122


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Design
The study had a randomized crossover design. Subjects were exposed either to O3 or FA for 4 h followed by either bronchoscopy or SI 18 h later. Subjects then underwent the alternate exposure (O3 or FA) followed by the same method of airway lining fluid sampling. In the crossover part of the study, subjects repeated the same exposure protocol with the alternate method of airway lining fluid sampling.

Subjects
Subjects were initially recruited for this study by advertisements on bulletin boards at the University of California, San Francisco (UCSF), and on those of other colleges and universities in the San Francisco Bay area, and by advertisements in local newspapers. The inclusion criteria were physician-diagnosed asthma, airway hyperresponsiveness to inhaled methacholine (provocative concentration of methacholine causing a 20% fall in FEV1 from baseline [PC20], ≤ 8.0 mg/mL), and the ability to perform moderately strenuous exercise. All subjects were nonsmokers who denied any history of cardiac or pulmonary diseases other than asthma, or any respiratory infections within 6 weeks of the start of each exposure. They were required to have not been receiving oral steroid medications for at least 3 months prior to their first visit, and inhaled steroids for at least 2 weeks prior to their first visit. No subject used supplemental vitamin C or E during the study. The subjects were informed of the risks of the experimental protocol and signed a consent form that had been approved by the Committee on Human Research of the UCSF. All of the subjects received financial compensation for their participation. Although 21 subjects were recruited for this study, only 13 subjects completed the experimental protocol. The protocol required a substantial time commitment requiring nine different visits over a minimum of 12 weeks, during which the subjects had to continue not to receive most of their asthma medications. The characteristics of the 13 individual study participants are listed in Table 1 .


View this table:
[in this window]
[in a new window]

  		Table 1. Subject Characteristics*

 
Pulmonary Function Measurements
Spirometry was performed with a dry rolling-seal spirometer using American Thoracic Society performance criteria.23 Airway responsiveness was determined by the FEV1 response to the inhalation of nebulized phosphate-buffered saline (PBS) solution followed by doubling concentrations (0.125, 0.25, 0.5, 2.5, 5, and 10 mg/mL) of methacholine in PBS solution delivered via a dosimeter at the rate of 0.01 mL per breath following a protocol modified from the Lung Health Study.24 PC20 compared to the post-PBS baseline was calculated by log-linear interpolation (Table 1).

Exposure Chamber and Atmospheric Monitoring
All exposures took place in a chamber ventilated with FA at 20°C and 50% relative humidity to which O3 was added. The stainless steel-and-glass chamber, 2.5 x 2.5 x 2.4 m in size, was custom-built and designed to maintain chamber temperature and relative humidity within 2.0°C and 4%, respectively, of the set points. Relative humidity and temperature were recorded every 30 s and were averaged over each exposure. O3 was produced with a corona-discharge O3 generator (model T408; Polymetrics, Inc; San Jose, CA), and its concentration was monitored with an ultraviolet light photometer (model 1004AH; Dasibi; Glendale, CA). The O3 analyzer was calibrated biannually with an O3 transfer standard (model 1003PC; Dasibi) by the California Air Resources Board and was precision-checked on a monthly basis. The mean (± SD) O3 concentration was 0.21 ± 0.007 ppm for SI and bronchoscopy O3 exposures, and was < 0.007 ± 0.003 ppm for both FA exposures. The mean temperature ranged from 19.9 to 20.0°C, relative humidity ranged from 47 to 55%, and the subjects’ exercise minute ventilation (E) ranged from 41.4 to 44.3 L/min for the four exposures. There were no significant differences in mean temperature, relative humidity, or E among any of the exposures.

Experimental Protocol
After a telephone interview, subjects were scheduled for an initial visit to the laboratory, where a medical history questionnaire was completed. Baseline spirometry, methacholine challenge test, and a 15-min exercise test designed to determine a workload that generated the target ventilatory rate were also completed on the initial visit. The experimental protocol involved two randomly ordered arms (ie, an SI arm and a bronchoscopy arm). Each arm involved two randomly ordered exposures, 0.2 ppm of O3 or FA, with a minimum interval of 3 weeks between the exposures. Each subject performed spirometry immediately before and after each exposure. Exposures were for 4 h on each study day, with subjects exercising for the first 30 min of each hour and then resting for the following 30 min of each hour. The exercise consisted of either walking or running on a treadmill or pedaling a cycle ergometer. The exercise intensity was adjusted for each subject to achieve a target expired E of 25 L/min/m2 body surface area. During exercise, the E was calculated from tidal volume, and breathing frequency was measured using a pneumotachograph at the 10-min and 20-min intervals of each 30-min exercise period. Peak expiratory flow was measured 10 min into each 30-min rest period to monitor for possible bronchoconstriction. Subjects remained inside the chamber for the entire 4-h exposure period.

Bronchoscopy and BAL Procedures
Bronchoscopies were performed a mean time of 18 ± 2 h after the two exposures in the bronchoscopy arm of the protocol. The mean 18 ± 2 h postexposure time was chosen because previous studies192025 by both our laboratory and other investigators have documented the presence of an O3-induced inflammatory response in many subjects at this time point. The performance of bronchoscopy and BAL in our laboratory has been discussed in detail previously.2025 Briefly, IV access was established, supplemental O2 was delivered, and the upper airways were anesthetized with topical lidocaine. Sedation with IV midazolam was used as needed for subject comfort. The bronchoscope was introduced through the mouth and vocal cords into the airways. The bronchoscope was then directed into the right middle lobe where BAL was performed with three 50-mL aliquots 0.9% saline solution that had been warmed to 37°C. The first 15 mL of fluid returned from the first 50-mL aliquot was collected separately and labeled as the bronchial fraction of BAL (BFx) fluid, whereas the remaining fluid returned was labeled BAL. Both lavage samples were immediately put on ice. After bronchoscopy, each subject was observed during a recovery period of approximately 2 h.

The total number of cells were counted on uncentrifuged aliquots of BFx and BAL fluid using a hemocytometer. Differential cell counts were obtained from slides prepared using a cytocentrifuge (25g for 5 min) and were stained (Diff-Quik; American Scientific Products; Astmoor, UK) as previously described.20 Two hundred cells were counted independently by two individuals, and the mean of these counts was used in the data analysis. BFx and BAL fluids were then centrifuged at 180g for 15 min, and the supernatant was separated and recentrifuged at 1,200g for 15 min to remove any cellular debris prior to freezing at –80°C.

SI Procedures
SIs were performed a mean time of 18 ± 2 h after two of the exposures in the SI arm of the protocol and following a modified procedure previously described by the Asthma Clinical Research Network of the National Heart, Lung, Blood Institute.226 The mean 18 ± 2 h postexposure time was the same as that for the bronchoscopy arm of the study. We have previously shown that a significant O3-induced inflammatory response can be detected in sputum samples obtained at this time point.10 The SI procedure of our laboratory has been described in detail before.10 Briefly, subjects were pretreated with 360 µg of albuterol, and spirometry was performed before and 15 min after the administration of albuterol to ensure that the post-albuterol FEV1 was > 60% of the predicted value for each subject. Subjects then underwent SI in an isolation booth to control for any possible airborne infections. SI was performed by the inhalation of nebulized 3% sterile saline solution for 20 min. At each 2-min interval, subjects were asked to clear saliva from their mouths by spitting into a sterile plastic container and then cough up sputum into a second such container. We chose a 20-min SI time to obtain respiratory samples from peripheral airways and the distal lung,27 which are more likely comparable to samples obtained by bronchoscopy (BFx and BAL fluid). All subjects tolerated 20 min of the SI procedure without difficulty. For quality control, a sputum sample was considered to be inadequate if its volume was < 1 mL or if its percentage of squamous cells was > 80%.

The volume of the induced sputum sample was determined and an equal amount of 0.1% dithiothreitol was added. The sample was homogenized by mixing gently by a vortex and then was placed in a shaking water bath at 37°C for a minimum of 15 min. After the sample was homogenized, it was removed from the water bath, and a 1-mL aliquot was placed on ice for the determination of total and differential cell counts. The total number of cells was counted in uncentrifuged aliquots of induced sputum using a hemocytometer. Differential cell counts were obtained from slides prepared using a cytocentrifuge (25g for 5 min) and were stained (Diff-Quik) as previously described.20 Two hundred cells were counted independently by two individuals, and the mean of these counts was used in the data analysis. The remainder of the sample was centrifuged at 180g for 15 min, and the supernatant was separated and recentrifuged at 1,200g for 15 min to remove any cellular debris before freezing at –80°C.

Statistical Analysis
All data were initially collected on paper and were then entered into a database (Microsoft Access 2000; Microsoft; Redmond, WA). Processed data were then analyzed using a statistical software package (Stata, version 7.0; StataCorp; College Station, TX) with consultative assistance from the UCSF Department of Biostatistics and Epidemiology. The sample size for the experiment was chosen from the greater of sample sizes needed to show neutrophilia in BAL fluid or in SI samples after O3 exposure. We used previous data from our laboratory to calculate these sample sizes (two-sided type I error, 0.05; power, 0.8). For bronchoscopy, a sample size of 11 subjects was calculated based on a mean increase in BAL fluid neutrophilia of 12.0 ± 12.5% from a study done with a similar O3 exposure protocol in asthmatic subjects.21 For SI, a sample size of 9 was calculated based on a mean increase in sputum neutrophilia of 17.5 ± 15.9% from a study10 performed in healthy nonasthmatic individuals using the identical exposure protocol. Our final sample size of 13 subjects, therefore, should provide an adequate number of subjects for the observation of statistically significant O3-induced neutrophilia in asthmatic subjects by either BAL or SI methods.

For statistical comparisons, if the measured variable had a normal distribution, the Student paired t test was used to compare paired data between exposure arms. If the variable did not have a normal distribution, the Wilcoxon signed rank test was used. A p value of 0.05 was considered to be statistically significant in all data analyses. The assumption of equal variance was valid for all of the analyzed variables. The data on the percentage of polymorphonuclear neutrophils (%PMNs) followed a normal distribution. Therefore, the change in the %PMNs between FA and O3 exposures for each subject was calculated by simple subtraction to obtain comparable data. The FA-O3 differences in the bronchoscopy and SI arms were then analyzed in a linear regression model, in which induced sputum data were used as the independent variable, and BFx and BAL data were used as dependent variables. The measured magnitudes of dependent variables were then compared with their estimated magnitude based on the regression model, and the residuals were used to determine the accuracy of the dependent variable estimates from the independent variable data.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spirometry
The mean (± SD) values of the spirometry indexes are shown in Table 2 . The preexposure FEV1 values for all subjects were similar in all arms of the study. Across FA exposures, there was a slight increase in FEV1. Across O3 exposures, there was a significant decrease in FEV1. The post-FA exposure FEV1 values for all subjects were similar in the SI and bronchoscopy arms of the study, as they were after O3. FVC and maximal mid-expiratory flow rate followed a pattern of change that was similar to that of FEV1.


View this table:
[in this window]
[in a new window]

  		Table 2. Spirometry Indices Before and After Each Exposure Arm*

 
O3-Induced Neutrophilia in Induced Sputum, BFx Fluid, and BAL Fluid
In comparison with FA exposure, O3 exposure significantly increased the %PMNs in induced sputum (mean increase in the %PMNs, 10.1%; 95% confidence interval [CI], 0.2 to 20.0), BFx fluid (mean increase in %PMNs, 17.0%; 95% CI, 8.8 to 25.3), and BAL fluid (mean increase in %PMNs, 7.5%; 95% CI, 2.3 to 12.6) [Table 3 ]. The O3-induced increases in %PMNs in BFx and BAL fluid were associated with much smaller p values than those in induced sputum because of the smaller variation associated with %PMNs in BFx and BAL samples. O3 exposure also increased the concentration of neutrophils in induced sputum, BFx fluid, and BAL fluid, but the increase in sputum was not statistically significant (p = 0.149).


View this table:
[in this window]
[in a new window]

  		Table 3. Leukocyte and Neutrophil Counts in RTLF Samples*

 
Correlation of Induced Sputum Data With Lavage Data
To determine how well the inflammatory indexes in induced sputum correlated with those in BFx and BAL fluid, we constructed linear regression models of %PMNs in BFx and BAL fluid vs that in induced sputum, with induced sputum values as the independent variables and BFx and BAL values as the dependent variables (Fig 1 ). These regression models showed that the changes in %PMNs in sputum samples were not well-correlated with those in BFx and BAL fluid samples (BFx sample, R = 0.50; and BAL fluid sample, R = 0.12). We subsequently used this model to estimate the change in the %PMNs of BFx and BAL fluid samples from that in induced sputum samples, and then compared these estimated values with the actual measured values of BFx and BAL fluid samples. The differences between the estimated and measured values of BFx and BAL fluid samples were then considered as the estimate errors. The estimate error values were then compared with the actual measured changes (ie, the actual O3 effects) in the %PMNs in BFx and BAL (Table 4 ). The comparison showed that the estimate errors were similar to the actual measured changes in the %PMNs with a substantial overlap between their 95% CIs, making these estimates quite inaccurate (Fig 2 ).



View larger version (7K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Linear regressions of changes in the %PMNs in BFx and BAL fluid samples vs those in induced sputum (IS) samples. • = actual values of change in %PMNs in BFx and BAL fluid samples; {triangleup} = predicted values of %PMNs for BFx and BAL fluid samples.

 

View this table:
[in this window]
[in a new window]

  		Table 4. The Errors in Estimating the Effect of Ozone on the %PMNs in BAL Fluid or BFx Fluid From the Corresponding Changes in Induced Sputum*

 


View larger version (6K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. The measured change in the %PMNs after O3 exposure in BFx and BAL fluid samples, and the errors in their estimates using the linear regression model based on induced sputum values. • = mean value of the change or the mean value of the error in the %PMNs in respiratory samples; bars = the corresponding 95% CIs.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we used a model of irritant-induced nonspecific inflammation to compare these two methods of sampling RTLF. Inhaled O3 at high ambient concentrations can induce the recruitment of neutrophils into the airway lumen. This has been demonstrated by increases in both the concentration of polymorphonuclear neutrophils and the %PMNs in RTLF samples obtained by bronchoscopy18192021 as well as by SI.781011121314151617 In our study, O3 exposure increased the %PMNs in BAL fluid and the BFx fluid samples by 7.5% and 17.0%, respectively, which is consistent with the values reported in previous studies from other laboratories and ours.18192021 O3 exposure also significantly increased the %PMNs in induced sputum samples by 10.1%, which is a similar value to those previously reported1011 using a similar effective dose and SI time point. Although the overall neutrophilic response was qualitatively similar in both induced sputum and BFx/BAL fluid samples, the individual responses to O3, as assessed by induced sputum, were quite variable, with some subjects showing paradoxical responses compared to their responses as assessed by BFx/BAL.

The greater variability seen in sputum samples compared to BFx/BAL samples may be due to several factors. First, SI may be sampling different parts of the RTLF compared to bronchoscopy. The distal portion of the airways (terminal bronchioles) are particularly sensitive to injury by O3.28 Compared to BAL, SI may be less sensitive in detecting the inflammatory response to O3-induced injury as there may be preferential sampling of more proximal airways than distal lung, in effect obtaining a different composition of RTLF. The correlation in our data between induced sputum samples and BFx samples was somewhat higher (BFx samples, R = 0.50) than that between these samples and BAL fluid samples (BAL fluid samples, R = 0.12), suggesting that induced sputum and BFx samples overlap more in terms of the airway compartments that they sample. Despite the better correlation, the values of induced sputum samples still explained only a small fraction of the total variability in BFx fluid (ie, 25% of BFx variance). Clearly, there is mixing of RTLF from different parts of the airways with any of these methods, and a pure sample from any specific compartment cannot be obtained using any of these three methods.

Second, the SI procedure itself is much more subject-dependent than bronchoscopy. During SI, the subjects are asked to inhale a hypertonic saline mist, clear their mouth of saliva, and then cough up sputum into a container. Even with strict coaching of the subjects and monitoring of the procedure, there may be significant between-subject differences in following the instructions or in the effort to cough up sputum. In addition, sputum samples are more difficult to process due to their viscosity. Furthermore, slides of sputum that have been stained for differential cell counting are generally of lower quality and have a higher number of epithelial cells, making them more difficult to score. With bronchoscopy, the procedure of lavage is more operator-dependent, and the samples are easier to process and score.

In a previous study, Hiltermann and colleagues29 reported an O3 effect on the %PMNs in BAL fluid samples, although they were unable to show a statistically significant effect in induced sputum samples. In our study, we were able to show a statistically significant increase in the %PMNs from the airway after O3 exposure in both induced sputum and BAL fluid samples. The inability of Hiltermann and colleagues29 to show an O3-induced neutrophilia in induced sputum samples may have been due to the performance of a methacholine challenge test before SI (but not before bronchoscopy). Methacholine has been shown to induce neutrophil recruitment to the airways in one study30 and thus may have obscured the effect of O3. In any case, the results of the study by Hiltermann et al29 are consistent with our finding that it is more difficult to show an O3 effect in airway lining fluid samples obtained by SI than in those obtained by bronchoscopy.

Our study only investigated O3-induced airway inflammation, and thus our results may not be generalized to other exposures or treatments. Specifically, we studied a model of nonspecific neutrophilic inflammation, and our results may not be applicable to a more eosinophilic inflammatory response to an allergen challenge in specifically sensitized subjects. Our results, however, certainly raise the question of whether induced sputum and BAL fluid can be used interchangeably to assess inflammatory responses in distal lung RTLF samples after other exposures or treatments. Nocker and colleagues9 investigated the equivalency of SI and BAL fluid samples before and after treatment with ICSs in two parallel groups of 15 subjects (a non-repeated-measures design). They found a statistically significant change in the percentage of eosinophils with the use of ICSs, and a correlation (R = 0.50) between induced sputum samples and BFx fluid samples, and between induced sputum and BAL fluid samples (R = 0.70) in terms of the percentage of eosinophils. These investigators did not proceed to analyze the accuracy of the predictions made using one method by using the other method. A review of the reported interquartile ranges for the baseline measurement of eosinophils in induced sputum sample, BFx fluid samples, and BAL fluid samples (the posttreatment values for BFx and BAL fluid samples were not reported), however, reveals that there was a substantially larger variation in the percentage of eosinophils in sputum samples compared to BFx and BAL fluid samples (95% CI: sputum samples, 2.0 to 15.5 vs 0.9 to 3.5 and 0.6 to 2.9, respectively).

Another caveat to our study is that some investigators use a different method of SI in which mucus conglomerations were separated from induced sputum, homogenized, and then used for the assessment of inflammation (eg, with cell counts and determination of biochemical parameters). We used the homogenized, whole induced sputum method that was recommended by the Asthma Clinical Research Network2 and used by Fahy et al,56 Hiltermann et al,29 and Nocker et al9 in the studies discussed above. It is unclear whether our results using the method recommended by the Asthma Clinical Research Network can be extrapolated to the mucus conglomeration method.

In conclusion, although SI and bronchoscopy are both accepted methods for sampling RTLF after O3 exposure in asthmatic subjects, the O3-induced inflammatory parameters measured by these methods did not correlate with each other. Extrapolating the inflammatory parameters observed in samples obtained by one method to those expected in the samples obtained by the other method may result in substantial errors.


   Acknowledgements
 
We thank Drs. Peter Bacchetti and David Glidden for their assistance with the statistical analysis, Allyson Witten for her invaluable help with data management, and Robert Montanti for his assistance with bronchoscopy.


   Footnotes
 
Abbreviations: BFx = bronchial fraction of BAL; CI = confidence interval; FA = filtered air; ICS = inhaled corticosteroid; O3 = ozone; PBS = phosphate-buffered saline; PC20 = provocative concentration of methacholine causing a 20% fall in FEV1; %PMN = percentage of polymorphonuclear neutrophil; RTLF = respiratory tract lining fluid; SI = sputum induction; UCSF = University of California, San Francisco; E = minute ventilation

This research was supported by the National Institutes of Health (grants R01 ES08970 and M01RR00083–41) and the American Lung Association (Research Training Fellowship).

Received for publication September 17, 2004. Accepted for publication December 2, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thomas, PS, Yates, DH, Barnes, PJ (1999) Sputum induction as a method of analyzing pulmonary cells: reproducibility and acceptability. J Asthma 36,335-341[ISI][Medline]
  2. Fahy, JV, Boushey, HA, Lazarus, SC, et al Safety and reproducibility of sputum induction in asthmatic subjects in a multicenter study. Am J Respir Crit Care Med 2001;163,1470-1475[Abstract/Free Full Text]
  3. Fireman, E Induced sputum: opening a new window to the lung. Sarcoidosis Vasc Diffuse Lung Dis 2001;18,263-271[Medline]
  4. Belda, J, Leigh, R, Parameswaran, K, et al Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000;161,475-478[Abstract/Free Full Text]
  5. Fahy, JV, Wong, H, Liu, J, et al Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects. Am J Respir Crit Care Med 1995;152,53-58[Abstract]
  6. Fahy, JV, Liu, J, Wong, H, et al Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: a method for studying allergic airway inflammation. J Allergy Clin Immunol 1994;93,1031-1039[CrossRef][ISI][Medline]
  7. Fahy, JV, Wong, HH, Liu, JT, et al Analysis of induced sputum after air and ozone exposures in healthy subjects. Environ Res 1995;70,77-83[Medline]
  8. Hiltermann, TJ, Stolk, J, Hiemstra, PS, et al Effect of ozone exposure on maximal airway narrowing in non-asthmatic and asthmatic subjects. Clin Sci (Lond) 1995;89,619-624[Medline]
  9. Nocker, RE, Out, TA, Weller, FR, et al Induced sputum and bronchoalveolar lavage as tools for evaluating the effects of inhaled corticosteroids in patients with asthma. J Lab Clin Med 2000;136,39-49[CrossRef][Medline]
  10. Criqui, GI, Solomon, C, Welch, BS, et al Effects of azithromycin on ozone-induced airway neutrophilia and cytokine release. Eur Respir J 2000;15,856-862[Abstract]
  11. Holz, O, Mucke, M, Paasch, K, et al Repeated ozone exposures enhance bronchial allergen responses in subjects with rhinitis or asthma. Clin Exp Allergy 2002;32,681-689[Medline]
  12. Liu, L, Leech, JA, Urch, RB, et al A comparison of biomarkers of ozone exposure in human plasma, nasal lavage, and sputum. Inhal Toxicol 1999;11,657-674[Medline]
  13. Vagaggini, B, Carnevali, S, Macchioni, P, et al Airway inflammatory response to ozone in subjects with different asthma severity. Eur Respir J 1999;13,274-280[Abstract]
  14. Nightingale, JA, Rogers, DF, Barnes, PJ Effect of inhaled ozone on exhaled nitric oxide, pulmonary function, and induced sputum in normal and asthmatic subjects. Thorax 1999;54,1061-1069[Abstract/Free Full Text]
  15. Newson, EJ, Krishna, MT, Lau, LC, et al Effects of short-term exposure to 0.2 ppm ozone on biomarkers of inflammation in sputum, exhaled nitric oxide, and lung function in subjects with mild atopic asthma. J Occup Environ Med 2000;42,270-277[Medline]
  16. Nightingale, JA, Rogers, DF, Fan Chung, K, et al No effect of inhaled budesonide on the response to inhaled ozone in normal subjects. Am J Respir Crit Care Med 2000;161,479-486[Abstract/Free Full Text]
  17. Vagaggini, B, Taccola, M, Conti, I, et al Budesonide reduces neutrophilic but not functional airway response to ozone in mild asthmatics. Am J Respir Crit Care Med 2001;164,2172-2176[Abstract/Free Full Text]
  18. Seltzer, J, Bigby, BG, Stulbarg, M, et al O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans. J Appl Physiol 1986;60,1321-1326[Abstract/Free Full Text]
  19. Koren, HS, Devlin, RB, Graham, DE, et al Ozone-induced inflammation in the lower airways of human subjects. Am Rev Respir Dis 1989;139,407-415[ISI][Medline]
  20. Aris, RM, Christian, D, Hearne, PQ, et al Ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy. Am Rev Respir Dis 1993;148,1363-1372[ISI][Medline]
  21. Scannell, C, Chen, L, Aris, RM, et al Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med 1996;154,24-29[Abstract]
  22. Basha, MA, Gross, KB, Gwizdala, CJ, et al Bronchoalveolar lavage neutrophilia in asthmatic and healthy volunteers after controlled exposure to ozone and filtered purified air. Chest 1994;106,1757-1765[Abstract/Free Full Text]
  23. American Thoracic Society. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152,1107-1136[ISI][Medline]
  24. Kanner, RE, Connett, JE, Altose, MD, et al Gender difference in airway hyperresponsiveness in smokers with mild COPD: the Lung Health Study. Am J Respir Crit Care Med 1994;150,956-961[Abstract]
  25. Aris, R, Christian, D, Tager, I, et al Effects of nitric acid gas alone or in combination with ozone on healthy volunteers. Am Rev Respir Dis 1993;148,965-973[ISI][Medline]
  26. Gershman, NH, Wong, HH, Liu, JT, et al Comparison of two methods of collecting induced sputum in asthmatic subjects. Eur Respir J 1996;9,2448-2453[Abstract]
  27. 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 1999;104,322-328[CrossRef][ISI][Medline]
  28. Castleman, WL, Dungworth, DL, Schwartz, LW, et al Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in rhesus monkeys exposed to ozone. Am J Pathol 1980;98,811-840[Abstract]
  29. Hiltermann, JT, Lapperre, TS, van Bree, L, et al Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma. Free Radic Biol Med 1999;27,1448-1454[CrossRef][ISI][Medline]
  30. Gershman, NH, Fahy, JV The effect of methacholine challenge on the cellular composition of induced sputum. J Allergy Clin Immunol 1999;103,957-959[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arjomandi, M.
Right arrow Articles by Balmes, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arjomandi, M.
Right arrow Articles by Balmes, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS