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

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.

 

Pulmonary Function Measurements

Spirometry was performed with a dry rolling-seal spirometer using American Thoracic Society performance criteria. 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. 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 (Ve) ranged from 41.4 to 44.3 L/min for the four exposures. There were no significant differences in mean temperature, relative humidity, or Ve 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 Ve of 25 L/min/m2 body surface area. During exercise, the Ve 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

Watch video representing bronchoscopy technique worked out by Bronchoscopy International (http://www.bronchoscopy.org/):

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 studies 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. 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. 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. 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. The SI procedure of our laboratory has been described in detail before. Briefly, subjects were pretreated with 360 |j,g 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, 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 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. 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. For SI, a sample size of 9 was calculated based on a mean increase in sputum neutrophilia of 17.5 ± 15.9% from a study 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.

Previously published article will tell you introduction to this research – “Sputum Induction and Bronchoscopy for Assessment of Ozone-Induced Airway Inflammation in Asthma“.
Table 1—Subject Characteristics

Subject No.	Sex	Age, yr	BMI, kg/m2	FEVjt		FVC		PC20mg/mL
				L	I% Predicted	L	I% Predicted
1	M	31	23.8	4.68	106	6.03	111	3.40
2	M	33	20.3	3.24	74	5.19	96	0.02
3	F	27	22.3	3.08	91	4.26	103	2.47
4	F	21	25.6	3.01	98	3.34	91	2.03
5	M	22	22.5	4.64	105	5.49	107	0.16
6	F	45	24.3	3.55	125	4.17	119	1.84
7	F	36	19.6	2.37	93	3.10	101	0.79
8	F	28	21.2	2.52	78	3.74	96	0.02
9	F	35	28.4	3.02	108	3.74	111	4.29
10	F	24	21.3	3.21	96	3.95	98	0.81
11	F	36	39.6	2.96	99	3.32	91	1.59
12	F	27	22.0	3.56	110	4.15	106	1.92
13	M	37	28.4	3.63	112	4.09	101	7.48
Meanj		30.9	24.6	3.34	99.6	4.20	102.4	2.06
SD		6.9	5.3	0.69	13.9	0.88	8.3	2.08