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

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 bronchoscopy as well as by SI. 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. O3 exposure also significantly increased the %PMNs in induced sputum samples by 10.1%, which is a similar value to those previously report-ed 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.

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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. 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.

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In a previous study, Hiltermann and colleagues 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 col-leagues 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 study and thus may have obscured the effect of O3. In any case, the results of the study by Hiltermann et al 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 col-leagues 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 Network and used by Fahy et al,> Hiltermann et al, and Nocker et al 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.

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