Bronchoalveolar Lavage in Stable Asthmatics Does Not Cause Pulmonary Inflammation 1- 3

NIZAR N. JARJOUR and WILLIAM J. CALHOUN With the technical assistance of Steven M. Salisbury and Carol A. Stevens

Introduction

Chronic asthma is now recognized as an inflammatory disease of the airways; inflammation is particularly prominent in late-phase reactions and contributes importantly to the chronicity and severity of asthma symptoms (1, 2). Airway hyperresponsiveness, the cardinal feature of asthma, is closely linked to airway inflammation (3-5). Bronchoalveolar lavage (BAL) has emerged as a valuable research tool to evaluate and study mechanisms of inflammatory lung diseases. Studies focusing on BAL in asthma have helped to establish its feasibility and safety in this group (6). General guidelines have been developed to ensure the proper and safe use of BAL in subjects with asthma (7). Because studies of human asthma often require a paired design (8, 9), investigators have used repeated BAL studies in a given individual to assess the influence of an intervention such as bronchial challenge. However, the influence of airway instrumentation, which might itself contribute to changes in air-space cellsand proteins, has not been addressed in a rigorous study. To examine the effect of BAL on airway biology and to establish further the safety of repetitive BAL and airway instrumentation in subjects with mild asthma, we performed paired lavages separated by 24 h in eight subjects with stable asthma and evaluated the response by spirometry, cellular influx, reactive oxygen species metabolism of air-space cells, and by BAL total protein. Our findings form the basis of this report. Methods Subjects Eight asthmatics 18 to 45 yr of age (three women, five men) participated in the study, which was approved by the University of Wisconsin Committee for Human Subjects. Informed consent was obtained from each subject prior to each bronchoscopy and BAL. All had mild asthma (FEV 1 > 70% pre100

SUMMARY Bronchoalveolar lavage (BAL) has become an important tool for evaluating changes in airway cells and fluid In asthma, and It may give insights into mechanisms of bronchial inflammation. Many factors contribute to airway inflammation In asthma Including, possibly, airway Instrumentation. To establish whether BAL leads to diffuse airway Inflammation In stable asthmatics, we performed paired BAL studies (24 h apart) in eight subjects with mild asthma whose prebronchoscopy spirometric results were similar on both days. Airflow limitation did not occur in any subject after bronchoscopy. Weobserved no significant changes in BAL volume return, cell differential, lymphocyte subsets, reactive oxygen species metabolism by air-space cells, or BAL total protein. There was a slight Increase in second-day BAL total cell return. We conclude that bronchoscopy and BAL In stable asthmatics with mild disease is not associated with evidence of diffuse airway AM REV RESPIR DIS 1990; 142:100-103 inflammation.

dieted in all cases, FEV 1 86 ± 4070 predicted, mean ± SEM) that was stable (no exacerbation in the preceding month) and controlled by a ~-agonist inhaler alone (six of eight subjects) or with oral theophylline (four of eight). Smokers and those receiving cromolyn or steroids were excluded. Screening physical examination and spirometry were done prior to enrollment. Methacholine POlOwas determined by graded inhalation from a nebulization dosimeter, 2 h after the second day BAL. Our subjects demonstrated mild airway hyperresponsiveness, with a POlOof 19.1 ± 7.0 cumulative breath units (mean ± SEM) and a range of 1.85 to 41 U, which is in the asthmatic range in our laboratory. Any effect that the bronchoscopy premedication might have had would have increased POlO(decreased hyperresponsiveness).

Bronchoscopy and Bronchoalveolar Lavage Subjects were instructed to stop their oral theophylline (24 h) and ~-agonist inhaler (12 h) prior to the BAL. They were admitted to the University of Wisconsin General Clinical Research Center (GCRC) at approximately 7:00 A.M. A brief physical examination and spirometry were performed. Intravenous access was established with an intermittent infusion device in a forearm vein. All bronchoscopies were done by one of us (NNJ) between 7:30 and 8:30 A.M., as previously described (10, 11),and in accordance with the published guidelines for BAL in asthma (7). Thirty minutes before BAL, the subjects were premedicated intramuscularly with atropine sulfate 0.6 mg and midazolam 1.25mg. Albuterol (2 puffs) was given 10min prior to bronchos-

copy. Local anesthesia of the airways and control of cough were achieved with 1070 lidocaine. The fiberoptic bronchoscope (Olympus Corp. of America, New Hyde Park, NY) was wedged into a subsegment of the right middle lobe (Day 1) or lingula (Day 2). Lavage was performed using four aliquots (60 ml each) of sterile, nonpyrogenic 0.9070 NaCI at 37° C that was instilled and withdrawn immediately by hand suction. The first and second BAL , studies were separated by 24 ± 0.5 h.

BAL Initial Analysis The lavage return was pooled, and BAL cells were recovered by centrifugation at 400 g for 10min at room temperature. Supernatant was pooled and frozen at - 70° C until analyzed for total protein. The cell pellet was washed once in phosphate-buffered saline (PBS) and resuspended in Hank's balanced salt solution (HBSS). Cells were counted using a hemacytometer, and were suspended at a final concentration of 2 x lQ6 cells/ml, which was used

(Received in originalform August 18, 1989 and in revised form January 8, 1990) I From the Section of Pulmonary and Critical. Care, Department of Medicine, University of Wisconsin, School of Medicine, Madison, Wisconsin. 2 Supported in part by grants from the Department of Medicine, University of Wisconsin, and by Grant No. RR-03186 from the General Clinical Research Center, Grant No. AI-I0404 from the National Institutes of Health, and by Clinical Investigator Award No. K08-0l828. 3 Correspondence and requests for reprints should be addressed to N. Jarjour, M.D., 600 Highland Avenue (H6/380 CSC), Madison, WI 53792.

101

SAL DOES NOT CAUSE PULMONARY INFLAMMATION IN STABLE ASTHMATICS

for cytofuge preparation and cell functional assays. Cytofuge studies were air-dried, fixed in methanol, and stained (Diff-Quik®; Scientific Products, Chicago, IL). Differential cell counts were done by identifying ~ 300 cells as lymphocytes, neutrophils, eosinophils, or macrophages. Lymphocyte subsets bearing specific surface antigens were identified using commercially available monoclonal antibodies (OK Series; Ortho Diagnostics, Raritan, NJ) and flow cytometric analysis (FACStar; Becton-Dickinson, Mountain View, CA).

Total Proteins BAL fluid total protein was measured using a Lowry assay (12) modified for microtiter equipment with a sensitivity of 20 ug/ml (about one-third of the normal BAL protein concentration).

Superoxide Assay Superoxide production by BAL cells was quantified by superoxide-dismutase-(SOD)-inhibitable reduction of ferricytochrome c, adapted for 96-well microtiter assay (13, 14). Briefly, each well contained lOS cells, 0.1070 gelatin, 1.2 mg/ml (::::: 100 ~M) cytochrome c, and HBSS. Paired wells were established with 25 ug/ml SOD, and each cell preparation was assayed under three conditions: (1) no activator, (2) opsonized zymosan (0.5 mg/ml), and (3) phorbol myristate acetate (PMA) (20 ug/ml).

Chemiluminescence Assay Chemiluminescence (CL) responses of adherence-purified air-space cells were determined at 37° C in a Picolite model 6500 luminometer interfaced to a laboratory computer (13-15). Briefly, 2 x lOS cells were al-

lowed to adhere in a glass vial in the presence of HBSS and autologous serum (10070). Nonadherent cells were aspirated, and the adherent cells were refed with HBSS and gelatin. Luminol-enhanced (10 ~M) CL determinations were made after the addition of PMA (20 ug/ml), opsonized zymosan (0.5 mg/ml), or buffer. Samples were run in duplicate, and light output was measured starting immediately after adding the activator and every 5 min for 60 min. The mean of duplicate basal and peak stimulated CL measurements were used for calculations.

Spirometry A Puritan-Bennett spirometer (Model PS 600; Puritan-Bennett, Kansas City, KS) was used to determine baseline and postbronchoscopy spirometry. An PVC maneuver was performed, and the best of three trials was accepted according to ATS guidelines (16). Spirometry was measured prior to BAL and every hour after the completion of the procedure for at least 2 h.

Statistical Analysis Data were analyzed preliminarily in an electronic spreadsheet (SuperCale 4; Computer Associates, San Jose, CA). Subsequent analysis was done using a microcomputer statistics package (Abstat 5; Anderson-Bell, Parker, CO). All data were expressed as mean ± SEM. All comparisons used Student's independent and paired t test unless otherwise noted (17).

Results

BAL Cell Count Differential and Lymphocyte Subsets Total BAL cell recovery was slightly high-

er on the second day (11.7 ± 2.3 versus 14.9 ± 2.7 million cells, p = 0.05, MannWhitney test), but BAL cell differential was similar on both days (table 1). No granulocytic or lymphocytic cell influx was seen in the second -day HAL. The proportion of BAL lymphocytes bearing OKT4 (helper subsets) surface antigen and OKT8 (suppressor) antigen, and the ratio ofT4:T8 were also similar both days (table 1).The asthmatic group had a higher percentage of HAL eosinophils than did the normal volunteers studied in our laboratory (normal value: 0.08 ± 0.05, n = 21, p < 0.05).

Air-space Cell Functional Studies There was no significant change in airspace cell release of superoxide at baseline or after stimulation with either PMA or zymosan between the two days (table 2). Spontaneous and stimulated CL of adherence-purified alveolar macrophages were also similar on both days (table 2).

BAL Volume Return and Total Protein BAL volume return was similar on both study days (166 ± 10versus 176 ± 7 mI). There was a tendency toward a higher return on the second day, which paralleled an increase in FEV 1 and FEV 1/FVC compared with those on the first day. Total protein was similar on both days (72.4 ± 9.5 versus 72.5 ± 6.4 ug/rnl),

TABLE 1 BAL PROFILE Subject No.

Day

Vol

Cells

AM

Lys

PMN

EOS

T4

T8

1 2

177 159

6.7 16.0

93.4 93.2

5.4 6.2

1.0 0.0

0.2 0.6

32.1 26.1

37.5 35.6

2

1 2

140 180

11.9 12.5

94.6 91.3

4.3 6.6

0.3 1.3

0.6 0.6

14.2 33.8

18.6 14.8

3

1 2

140 170

7.9 14.3

90.3 93.3

9.3 5.6

0.0 1.0

0.0 0.0

47.4 46.2

35.3 21.5

4

1 2

180 175

23.8 28.6

89.6 88.0

10.0 10.0

0.3 0.3

0.0 1.6

26.8 27.5

72.4 73.5

5

1 2

130 143

3.0 5.8

75.0 83.3

22.0 15.3

1.3 0.0

1.6 1.3

6

1 2

199 198

12.4 8.4

94.7 95.3

5.0 2.0

0.3 2.7

0.0 0.0

42

7

1 2

190 200

14.8 19.4

99.0 93.7

0.3 6.0

0.7 0.3

0.0 0.0

68 64.5

18.7 20.6

8

1 2

175 178

12.9 14.1

95.3 95.3

3.3 4.7

0.7 0.0

0.7 0.0

56 56

29 18

166 ± 10 175 ± 7

11.7 ± 2.3 14.9 ± 2.6

91.5 ± 2.7 91.7 ± 1.6

7.5 ± 2.5 7.1 ± 1.5

0.6 ± 0.15 0.7 ± 0.3

0.4 ± 0.20 0.5 ± 0.23

Day 1" Day 2"

Not done Not done 28 Not done

40.8 ± 6.9 42.4 ± 6.5

34.2 ± 6.4 30.7 ± 9.0

Definition of abbreviations: Vol = volume return in ml (240 ml injectate); AM = alveolar macrophages; Lys = lymphocytes; PMN = polymorphonuclear cells; EOS = eosinophils; T4 = % Lys bearing OKT4; T8 = % Lys bearing OKTB. • Values are mean ± SEM. No significant differences between Day 1 and Day 2 were discerned.

JARJOUR AND CALHOUN

102 TABLE 2

FEF25 - 75 postbronchoscopy was 79 ± 6 and 82 ± 6% of predicted on Days 1 and 2, respectively.

AIR-SPACE CELL FUNCTIONS Chemiluminescencet

Superoxide * Subject No.

B

P

1 2

5.3 2.1

9.4 4.1

9.0 9.0

2

1 2

6.4 3.8

16.1 11.8

3

1 2

3.5 2.3

4

1 2

5

Day

B

Z

P

Z

98 107

253 280

1,263 1,614

15.2 7.2

217 233

832 583

5,773 1,314

9.3 8.8

14.6 12.5

43 19

183 65

268 138

5.7 5.4

10.3 13.9

10.8 15.7

50 115

205 302

934 1,310

1 2

0.4 6.5

5.7 14.1

5.6 11.9

82 92

298 406

3,497 1,552

6

1 2

3.6 1.4

6.5 3.6

7.2 2.6

7

1 2

1.2 2.8

12.3 9.5

7.8 9.2

176 74

3,128 227

13,746 1,709

8

1 2

a.1 5.5

9.9 11.2

7.6 10.9

125 63

4,699 278

2,020 198

3.5 ± 0.8 3.7 ± 0.7

9.9 ± 1.2 9.6 ± 1.5

9.7 ± 1.3 9.9 ± 1.5

113 ± 25 100 ± 25

1,371 ± 684 306 ± 60

3,929 ± 1,780 1,119 ± 252

Day 1:t Day 2:t

Not done Not done

Definition of abbreviations: B = basal; P = phorbol-myristate-acetate-driven; Z = zymosan-driven . • Superoxide data are expressed in nmol/SOO k cells/50 min. t Chemiluminescence data are in thousand counts per minute. :j:Values are mean ± SEM. No significant differences between Day 1 and Day 2 were discerned.

Spirometry Baseline spirometry results from Day 1 and Day 2, as shown in Table 3, were very similar, with FEV 1 higher than 70070 predicted in all the subjects on both study days. Post bronchoscopy spirome-

try showed no worsening compared with that at baseline. In contrast, the FEV 1 significantly increased compared with prebronchoscopy values (p < 0.05), even when the worst post-HAL spirometry result was used for analysis (table 3).

TABLE 3 SPIROMETRY BEFORE AND AFTER BRONCHOSCOPY FEV,

FEV,/FVC

Day

Before

After

Before

1 2

80 76

95 88

64 61

75 72

2

1 2

84 102

88 98

81 93

88 89

3

1 2

90 85

89 84

75 78

84 81

4

1 2

105 107

112 106

66 64

79 78

5

1 2

81 90

91 88

66 72

80 79

6

1 2

73 74

94 114

60 56

79 84

7

1 2

78 79

120 115

69 70

81 82

8

1 2

93 112

83 114

68 68

60 73

86 ± 4 91 ± 5

97 ± 4t 101 ± 5

69 ± 2 70 ± 4

NS

NS

Subject

Day 1* Day 2* p Value, Day 1 versus Day 2 * Values are mean

NS:t ± SEM.

t p < 0.05 before versus after bronchoscopy. :j: Not significant, p > 0.10.

After

78 ± 80 ± NS

at zt

Procedure Safety Subjects were observed in GCRC for at least 3 h after the completion of BAL. We encountered no episodes of bronchospasm or worsening pulmonary functions after BAL in contrast to those in some earlier studies (8, 18, 19). No subject required prolonged observation or overnight admission. We attributed this to careful selection of subjects with mild, stable asthma, premedication with atropine and albuterol, meticulous upper airwayanesthesia, and subject cooperation. Discussion

In this study, paired HALs were performed in eight subjects with mild stable asthma 24 h apart, by the same individual, and using the same technique. BAL was obtained from the right middle lobe on the first day and from the lingula on the second day. We found that BAL did not cause diffuse alteration of airway biology, and did not lead to airway obstruction provided that subjects were premedicated with bronchodilators prior to bronchoscopy. In fact, the postbronchoscopy FEV 1 was significantly higher (p < 0.05) than at baseline on both study days, which likely reflects the bronchodilator effects of albuterol and atropine used as premedications. These data support the safety of repetitive HAL in mild asthma (8, 20). The total cell recovery was slightly higher on the second day. This increase could be related to the effect of a prior bronchoscopy and BAL or to differences between the right middle lobe and the lingula. However, there are few data to suggest intersegmental differences in BAL analysis in healthy or asthmatic subjects. It is also possible that these changes may simply reflect day-to-day variations that are normal physiologic fluctuations, as has been noted in normal subjects (21). It is unlikely that the increase in total cell count represents airway inflammation since HAL total protein was essentially unchanged. We found no significant changes in BAL cell differential, lymphocyte subsets (OKT4, OKT8), or air-space cell function as evaluated by superoxide release and CL assays. Our subjects had a higher percentage of BAL eosinophils than did the normal subjects; however, in other studies including our own (22), the percentage of HAL eosinophils in asthmatic subjects was found to be higher than what we noted in our current study.

BAL DOES NOT CAUSE PULMONARY INFLAMMATION IN STABLE ASTHMATICS

The relatively mild disease in our sub- second BAL was obtained from a segjects and its stability may explain this ment previously lavaged, as well as from difference. a segment not previously lavaged. The Studies in healthy dogs (23) showed return on the first 20-ml aliquot was rethat lobar BAL leads to recruitment of garded to be a "bronchial" lavage samneutrophils to the lungs and peripheral ple and the remaining four aliquots to leukocytosis 24 h after the initial BAL. represent the "alveolar" sample. They Earlier studies in rhesus monkeys showed noted a marked rise in neutrophils in the evidence of airway cellular influx, pre- "bronchial" lavage samples at the time dominantly polymorphonuclear cells, to of the second BAL, which was more the lung, starting at 4 h after whole-lung prominent in segments that were previlavage and persisting at 72 h (24). The ously lavaged; however, no changes were increase in BAL total cell recovery in our seen in the "alveolar" samples. Thus, losubjects was minimal and not associat- cal, and to some degree diffuse, bronchial ed with any increase in BAL neutrophils. inflammation may be seen 2 h after a Human studies have shown that in ac- BAL. In our study the two BALs were tive asthmatics (during the season or after done 24 h apart and obtained from differallergen challenge) there is a significant ent lobes since wewereinterested in evaludegree of cellular inflammation with a ating the presence of diffuse airway inrise in the proportions of eosinophils (1, flammation rather than local changes. 8), neutrophils (8), and enhanced alveo- Diffuse inflammation was not evident in lar macrophage metabolic activation (22, our group of subjects. 25). Metzger and coworkers (20) studied In conclusion, bronchoscopy and BAL allergic asthmatics by BAL at the same do not lead to diffuse airway inflammasite 48 and 96 h after local antigen chal- tion in stable asthmatics as assessed by lenge and found that local antigen chal- a subsequent BAL performed 24 h later. lenge caused significant local inflamma- BAL cell differentials were not different tion that was attributed to the challenges. on two consecutive days from two sepaThey inferred that BAL itself did not lead rate lung segments. Thus, it is reasonable to significant changes because in four to conclude that changes seen in BAL afsubjects in whom contralateral lavagewas ter a given challenge are primarily a reperformed 48 h later, they found no evi- sult of the challenge itself and are not dence of monocytic cell influx. artifacts of a prior BAL. Kirby and coworkers (26) found that Acknowledgment methacholine PD zo was unchanged 24 h after a BAL in a group of 10subjects with The writers would like to thank Dr. William stable asthma compared with baseline W. Busse for his critical review of the manuPD zo done prior to BAL. Their finding script, and Christie Lueck, Ramona Gasper, and the Secretarial Center for their secretariis consistent with the lack of generalized al help. airway inflammation after BAL seen in our study. References Ettenson and coworkers (21) investigat- 1. DeMonchy JG, Kauffman HF, Venge P, et a/. ed the consistency of results of repeated Bronchoalveolar eosinophilia during allergen-inBAL in normal volunteers obtained from duced late asthmatic reactions. Am Rev Respir Dis 1985; 131:373-6. the same segment (lingula) and found 2. Fabbri LM, Boschetto P, Zocca E, et a/. Brongenerally similar results in the same sub- choalveolar neutrophilia during late phase asthmatject on serial BALs. 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