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Relationship of Cellular Transmigration and Airway Response after Allergen Challenge IKEADI MAURICE NDUKWU, EDWARD T. NAURECKAS, CHRISTOPHER MAXWELL, MICHAEL WALDMAN, and ALAN R. LEFF Section of Pulmonary and Critical Care Medicine, and the Committees on Cellular Physiology and Clinical Pharmacology, Division of the Biological Sciences, The University of Chicago, Chicago, Illinois

We examined the relationship between eosinophil migration into the bronchoalveolar space and change in FEV1 after endobronchial allergen challenge (EBAC) in atopic asthmatic (AA) and atopic nonasthmatic (ANA) subjects. The purpose of this study was to obtain continuous, intrasubject controlled assessment of the relationship between cell migration in control and allergen-challenged segments in the same individuals over 96 h. In AA subjects, the eosinophil (Eos) count in the bronchoalveolar lavage fluid (BALF) increased from a baseline of 7,896 6 3,865 to 416,476 6 231,012 Eos/ml by 72 h (p 5 0.001) in the challenged segment post-EBAC. For ANA subjects, the postsegmental challenge count was 29,874 6 474 Eos/ml (p 5 0.03 versus baseline and p , 0.05 AA peak versus ANA peak). In both groups, there was a comparable decrease in peripheral blood eosinophil count beginning 5 h after challenge, which resolved at 24 h. In AA subjects, 416,476 6 231,012 Eos/ml was obtained from the allergen-challenged segment and 23,522 6 8,298 Eos/ml was obtained from the sham-challenged segment (p , 0.001) at 72 h. In contrast, there was no difference in the Eos count obtained from the BALF between the antigen- and sham-challenged segments of ANA subjects. We also found that increased airway neutrophils were present in equal numbers in allergen-challenged and sham-challenged segments in both AA and ANA subjects. We conclude that augmented eosinophil migration after EBAC is a characteristic of atopic asthma and is not present in atopic subjects who do not have asthma. We find that BAL eosinophilia in ANA patients as well as neutrophilia in both ANA and AA subjects are nonspecific consequences of bronchoscopy. Finally, we find no relationship between specific airway eosinophil migration into the BALF and FEV1 , 72 h after challenge; however, at 96 h, there is a substantial decrease in FEV1 that accompanies BALF eosinophilia. Ndukwu IM, Naureckas ET, Maxwell C, Waldman M, Leff AR. Relationship of cellular transmigration and airway response after allergen challenge. AM J RESPIR CRIT CARE MED 1999;160:1516–1524.

Bronchoalveolar lavage fluid (BALF) after antigen challenge is a widely used tool for assessment of related events of airway inflammation in subjects with asthma. Although some previous investigations have demonstrated at baseline a relationship between the presence of inflammatory cells and enhanced airway responsiveness (1–3), others have shown little or no relationship between the cellular response in BALF and the airway physiological response (4, 5). Haley and Drazen (6) suggested that this apparent discordance among investigators should be expected because multiple factors modulate chronic

(Received in original form December 23, 1998 and in revised form May 6, 1999) Supported by General Clinic Research Center Grant M01 RR00055, the Foundation for Fellows in Asthma Research (funded by an educational grant from Forest Pharmaceuticals and UAD Laboratories), Olympus America, Inc., HL46368, SCOR HL56399, and Glaxco Wellcome, Inc. Address correspondence and reprint requests to Ikeadi M. Ndukwu, M.D., M.P.H., Assistant Professor of Medicine, The University of Chicago, Department of Medicine, Section of Pulmonary and Critical Care Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637. E-mail: [email protected] Am J Respir Crit Care Med Vol 160. pp 1516–1524, 1999 Internet address: www.atsjournals.org

airway responsiveness. They further suggested that studies performed at a single arbitrary time point might not detect reliably the relationship between cellular and airway responses. Accordingly, some prior reports have shown that an increase in airway inflammatory cells in BALF correlated with enhanced airway responsiveness after allergen challenge (7, 8), but others have not consistently demonstrated this relationship (9). Prior investigations have studied subjects only at one or two time points after allergen challenge, and the methods of allergen challenge (whole lung inhalation, endobronchial instillation, or a combination of both) have varied among investigators (7, 10, 11). This study was undertaken to answer four major questions, using a highly standardized endobronchial allergen challenge: (1) Which cell type migrates in a site-specific manner during allergen challenge? (2) Will there be a difference in the number of a specific inflammatory cell that distinguishes an atopic asthmatic (AA) from an atopic nonasthmatic (ANA) patient after endobronchial allergen challenge (EBAC)? (3) Over what time period does a site-specific migration occur after EBAC? (4) What is the relationship between eosinophil migration and (a) physiological function and (b) asthma symptom score after EBAC in AA subjects?

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Ndukwu, Naureckas, Maxwell, et al.: Cellular Profile of Patients with Asthma after EBAC TABLE 1 SUBJECT DEMOGRAPHICS Age (yr) Group

n

Mean 6 SEM

Range

Sex (M:F)

Atopic asthmatics (AA) Atopic nonasthmatics (ANA) Nonatopic nonasthmatics (NANA)

12 6 4

22.2 6 1.3 22.67 6 1.3 24.00 6 3.49

18–35 19–26 18–33

8:4 1:5 1:3

PC20 (mg/ml ) Methacholine

FEV1 (% pred )

Mean 6 SEM

Range

89 6 3 108 6 6 109 6 3

1.14 6 0.64 21.25 6 1.96 25.00*

0.043–7.959 12.97–25.00

* A 20% reduction in FEV1 was not achieved.

Our data demonstrated that eosinophilia in BALF after allergen challenge occurs specifically in atopic asthmatic subjects, but not substantially in atopic nonasthmatic subjects. However, eosinophilia corresponding to a change in FEV1 did not occur at , 96 h. We further found that neutrophilia was comparable in both AA and ANA subjects as well as in allergen-challenged and sham-challenged segments, suggesting that this response is a nonspecific consequence of the procedure.

METHODS Subjects The study was performed with 22 volunteers (10 men and 12 women) divided into three groups. The atopic asthmatic (AA, n 5 12) subjects had a positive skin allergen prick test, a positive methacholine challenge test (see below), and a history of asthma with atopy. The atopic nonasthmatic (ANA, n 5 6) subjects had positive skin tests, a negative methacholine challenge study, and a history of atopy but no history of asthma. The nonatopic nonasthmatic (NANA, n 5 4) subjects had negative skin tests, a negative methacholine challenge, and no history of asthma. The AA and ANA subjects were further divided into two groups. Group I (AA, n 5 6 and ANA, n 5 3) underwent baseline bronchoscopy for BALF followed by EBAC with repeat bronchoalveolar lavage (BAL) at 5, 48, and 72 h after the challenge. Group II (AA, n 5 6 and ANA, n 5 3) underwent baseline bronchoscopy for BALF followed by EBAC with repeat BAL at 24, 48, and 96 h after

the challenge. The characteristics of the study subjects are shown in Table 1. The institutional review board for human studies at the University of Chicago approved this protocol. All volunteers were informed of the nature of the study and gave written consent.

Atopic State Determination The skin allergen test was performed using a modification of techniques previously described (12, 13). All subjects underwent a screening skin prick test with 14 allergen extracts (Greer Laboratories, Lenoir, NC) placed on the skin with a sterile Wyeth bifurcated needle (Allergy Laboratories of Ohio, Columbus, OH). Each wheal was measured and graded as follows: negative 5 no reaction except for needle mark, < 2 mm, or without definite wheal; Grade 1 5 reaction 2.0 to 3.0 mm, slightly raised definite wheal; Grade 2 5 reaction . 3.0mm wheal and well defined. The allergen that produced the most reaction (greater diameter) was chosen as the endobronchial allergen challenge agent. This chosen allergen was used in a skin wheal dose– response series, using 10-fold dilutions in 0.9% saline. The final concentration chosen for the endobronchial challenge was the most diluted allergen extract concentration to cause a Grade 3 skin wheal response (Table 2).

Methacholine Challenge Test After spirometry, each subject underwent standard bronchoprovocation testing with methacholine in the Pulmonary Function Laboratory of the University of Chicago. A provocation dose that caused a 20%

TABLE 2 DETERMINATION OF DOSE OF ALLERGEN FOR ENDOBRONCHIAL CHALLENGE

ID No.

Allergen Type

Original Concentration

Skin Prick Dilution Used for Challenge

01 02 03 04 05 06 07 08 09 10† 11 12† 13 14 15† 16† 17† 18†

Rye grass (Lolium perenne) HDM (Dermatophagoides farinae) Short ragweed (Ambrosia artemisifolia) Short ragweed (A. artemisifolia) HDM (D. farinae) Mold (Aspergillus fumigatus) Short ragweed (A. artemisifolia) European HDM (Dermatophagoides pteronyssinus) Short ragweed (A. artemisifolia) European HDM (D. pteronyssinus) HDM (D. farinae) HDM (D. farinae) HDM (D. farinae) Rye grass (L. perenne) HDM (D. farinae) Rye grass (L. perenne) Rye grass (L. perenne) HDM (D. farinae)

51,500 PNU/ml 10,000 AU/ml 76,500 PNU/ml 76,500 PNU/ml 10,000 AU/ml 32,650 PNU/ml 76,500 PNU/ml 10,000 AU/ml 76,500 PNU/ml 10,000 AU/ml 10,000 AU/ml 10,000 AU/ml 10,000 AU/ml 51,500 PNU/ml 10,000 AU/ml 51,500 PNU/ml 51,500 PNU/ml 10,000 AU/ml

1:1 3 109 1:1 3 107 1:1 3 109 1:1 3 108 1:1 3 109 1:1 3 105 1:1 3 107 1:1 3 107 1:1 3 107 1:1 3 107 1:1 3 107 1:1 3 107 1:1 3 108 1:1 3 108 1:1 3 106 1:1 3 107 1:1 3 109 1:1 3 109

Challenge Concentration

Amount of Allergen/25 ml*

5.150 3 1025 PNU/ml 1.000 3 1023 AU/ml 7.650 3 1025 PNU/ml 7.650 3 1024 PNU/ml 1.000 3 1023 AU/ml 3.265 3 1021 PNU/ml 7.650 3 1025 PNU/ml 1.000 3 1023 AU/ml 7.650 3 1023 PNU/ml 1.000 3 1023 AU/ml 1.000 3 1023 AU/ml 1.000 3 1023 AU/ml 1.000 3 1024 AU/ml 5.150 3 1024 PNU/ml 1.000 3 1022 AU/ml 5.150 3 1023 PNU/ml 5.150 3 1025 PNU/ml 1.000 3 1025 AU/ml

1.288 3 1023 PNU 2.500 3 1022 AU 1.913 3 1023 PNU 1.913 3 1022 PNU 2.500 3 1024 AU 8.163 PNU 1.913 3 1023 PNU 2.500 3 1023 AU 1.913 3 1021 PNU 2.500 3 1022 AU 2.500 3 1022 AU 2.500 3 1022 AU 2.500 3 1023 AU 1.288 3 1022 PNU 2.500 3 1021 AU 1.288 3 1021 PNU 1.288 3 1023 PNU 2.500 3 1024 AU

Definition of abbreviations: AU 5 antigen unit; HDM 5 house dust mite; PNU 5 protein nitrogen unit. * To calculate the amount per 25 cm3: (1) (original concentration)/(skin prick dilution used for challenge) 5 challenge concentration; (2) (challenge concentration)(25 ml) 5 amount per 25 cm3. Example (with volunteer 8516-01): (1) (51,500 PNU/ml)/(1,000,000,000) 5 0.0000515 PNU/ml; (2) (0.0000515 PNU/ml)/(25 ml) 5 0.0012875 PNU. † Atopic nonasthmatic subjects.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

decrease in FEV1 (PD20), if < 8 mg/dl, was considered to be positive for airway hyperresponsiveness.

Preparation for Bronchoscopy Morphine sulfate and midazolam were given intravenously approximately 30 min before bronchoscopy. Topical anesthesia was achieved with aerosolized 4% lidocaine hydrochloride and 2% viscous lidocaine HCl (Roxane Laboratories, Columbus, OH). Fifteen minutes before bronchoscopy, each subject received aerosolized albuterol (0.5 ml) mixed with 3 ml of 1% lidocaine HCl topical solution for 5 min. During the bronchoscopy supplemental oxygen was given, and continuous monitoring of cardiac rhythm, oxygen saturation, and blood pressure was performed. All subjects had FEV1 . 2 L before bronchoscopy (. 60% predicted). The fiberoptic bronchoscopy and BAL technique were modified from prior investigations (14–21). The baseline BAL was performed in the lingular (superior [LB4]) segment by advancing a large-channel video therapeutic bronchoscope (Olympus BF-1T200; Olympus America, Lake Success, NY) into wedge position. Two 60-ml aliquots of warmed (378 C) sterile normal saline solution (total of 120 ml) were injected into the airway distal to the wedged bronchoscope, followed by suction into a sterile container. Isolated endobronchial segmental chaIlenges. The bronchoscope was positioned at the entrance of the target airway segment. An occlusion balloon pulmonary catheter (Pro-Bal [Mill-Rose Laboratories, Mentor, OH] or balloon catheter B5-2C [Olympus America]) was passed into the lumen of the bronchoscope and positioned distal to the bronchoscope tip. The balloon on the catheter was inflated with 1.5 ml of air, and a portion of the airway segment thus was isolated distally by the catheter and proximally by the bronchoscope. For the sham control site, the bronchoscope was positioned in the inferior segmental (LB5) bronchus of the lingula. Five aliquots of 5 ml each of warmed (378 C) normal saline (total volume of 25 ml) were instilled at 1.5-min intervals.

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For the allergen-challenge site, the bronchoscope was positioned in the right middle lobe. Increasing doses of allergen were instilled through the bronchoscope in 5-ml aliquots at 1.5-min intervals. The dose of allergen extract used in the endobronchial challenge was comparable among AA and ANA subjects. The endobronchial allergen extract challenge dose ranged from 7.650 3 1025 to 3.265 3 1021 protein nitrogen units (PNU)/ml or 1.000 3 1025 to 1.000 3 1022 allergen unit (AU)/ml (Table 2). The local airway challenge was terminated when the airway decreased in diameter as estimated by direct visual observation and/or after instillation of 25 ml (total volume) of the allergen. All subjects tolerated the instillation of the final volume of 25 ml during endobronchial allergen challenge. Specimen collection and processing. Twenty milliliters of blood was withdrawn through a heparinized syringe before each bronchoscopy. The lavage fluid was centrifuged for 5 min at 200 3 g. The supernatants were decanted and frozen quickly at 2708 C in aliquots for later analysis. Cell counts. Blood leukocytes were counted with a Coulter (Hialeah, FL) counter, and blood eosinophil counts were determined by Wright–Giemsa-stained smear. BAL cells were quantified using cytocentrifuge preparations stained with Wright-Giemsa. At least 500 cells were enumerated from each cytospin preparation of the BALF to determine cell population, i.e., macrophage, neutrophils, eosinophils, and lymphocytes. Measure of clinical response. We used a modification of the first domain component of the validated quality of life in asthma instrument developed by Juniper and coworkers (22) to assess daily asthma symptoms before and after EBAC. Daily asthma symptom score (DASS) was determined before each bronchoscopy for the AA group. Symptoms were graded as 0 5 no symptoms; 1 5 mild symptoms (which did not interfere with activities); 2 5 moderate symptoms (which interfered with some activities); and 3 5 severe symptoms (which interfered with many activities). Allergen challenge-induced airflow limitation was determined by spirometry before each bronchoscopy and presented as change in FEV1.

Figure 1. Baseline differential cell count. The percentage of leukocyte components in the blood and bronchoalveolar lavage (BAL) fluid was similar for all groups before allergen challenge. PMN 5 polymorphonuclear cells; EOS 5 eosinophils; LYMPH 5 lymphocytes; MONO 5 monocytes; MACRO 5 macrophages; EPI 5 epithelial cells.

Ndukwu, Naureckas, Maxwell, et al.: Cellular Profile of Patients with Asthma after EBAC Statistical Analysis Analysis of variance (ANOVA) was used to compare baseline data among subject groups. Repeat measures ANOVA was used to determine whether changes over time were significant. When this screening test was significant at the 5% level, multiple comparisons between time periods and groups were carried out by the Mann–Whitney–Wilcoxon test. The significance between changes in the mean number of cells over time was determined by longitudinal data analysis using generalized estimating equations (GEEs) (23, 24). Data are expressed as means 6 standard error of the mean (SEM). Significance between measurements was claimed if p < 0.05.

RESULTS Baseline Cell Counts

The mean peripheral blood white blood cell (WBC) count was slightly greater in the atopic asthmatic subjects [7.38 (6 0.82) 3 106 WBCs] compared with atopic nonasthmatics [5.68 (6 0.59) 3 106 WBCs, p , 0.05 versus AA] and nonatopic nonasthmatics [5.68 (6 0.47) 3 106 WBCs, p , 0.05 versus AA]. The total nucleated cells and leukocytes recovered from the BAL fluid were similar for all groups before endobronchial allergen challenge. Figure 1 shows the differential cell count of peripheral

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blood and BAL before endobronchial allergen challenge for all groups. The differential count for blood (Figure 1a) was similar for all groups, as was the differential cell count in the BAL fluid (Figure 1b). Cellular Profile after Endobronchial Challenge with Allergen

Endobronchial sham and allergen challenges followed by serial blood withdrawal and bronchoscopy for BAL fluid were performed on AA and ANA subjects only. Venous blood. Endobronchial allergen challenge caused an increase in peripheral WBCs from 7.39 (6 0.82) 3 103 to 10.76 (6 0.78) 3 103 cells/ml in AA subjects (p , 0.05). EBAC also caused an increase in peripheral WBCs in the ANA subjects from 6.30 (6 0.41) 3 103 to 9.87 (6 0.47) 3 103 cells/ml (p , 0.05 versus control and p . 0.05 for AA versus ANA) (Figure 2a). The peripheral WBC count remained increased in AA (p , 0.05 versus baseline) subjects but not in ANA subjects (p 5 0.340 versus baseline) at 24 h. Thereafter, peripheral WBC counts returned to basal levels in both groups. The early increase in venous WBC count after EBAC resulted from an increase in the number of polymorphonuclear cells (PMNs). The number of PMNs increased from 4.22 (6 0.67) 3 103 to

Figure 2. Venous blood cellular profile before and after endobronchial allergen challenge (EBAC). Changes in white blood cell (WBC), neutrophil, and eosinophil count after EBAC from baseline were similar in both atopic asthmatic (AA) and atopic nonasthmatic (ANA) subjects. EBAC-induced changes among these cells were not specific for AA. Note that the y-axis scale for eosinophils is different from that for WBC and neutrophils. *p , 0.05 compared with baseline.

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7.24 (6 0.77) 3 103 cells/ml in AA subjects (p , 0.05) and from 3.40 (6 0.38) 3 103 to 5.97 (6 0.54) 3 103 cells/ml in ANA subjects (p , 0.05) at 5 h (Figure 2b). In contrast, the number of venous blood eosinophils declined significantly 5 h after EBAC. The initial eosinophil count of 0.22 (6 0.05) 3 103 cells/ ml decreased to 0.13 (6 0.03) 3 103 cells/ml in AA subjects (p , 0.05) at 5 h; likewise, the initial eosinophil count of 0.17 (6 0.04) 3 103 cells/ml decreased to 0.08 (6 0.01) 3 103 cells/ml in ANA subjects (p , 0.05) at 5 h (Figure 2c). The venous blood eosinophil count increased significantly above baseline 24 h after EBAC and remained increased for 96 h after EBAC in both groups (p 5 0.02). Bronchoalveolar lavage fluid. Comparisons between AA and ANA subjects revealed that repeated BAL at the site of sham or allergen challenge resulted in a nonspecific increase in the recovered total leukocytes. The total leukocyte count in the BALF before challenge was 0.6 (6 0.08) 3 106 cells/ml for AA and 0.55 (6 0.08) 3 106 cells/ml for ANA (p 5 NS). At the allergen-challenged site, the total BALF leukocyte count increased to 1.6 (6 0.3) 3 106 cells/ml for AA subjects by 72 h after EBAC. EBAC also caused an increase in leukocytes at

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the allergen-challenged site to 1.1 (6 0.3) 3 106 cells/ml for ANA subjects by 72 h (p , 0.05 versus baseline, both groups). Total leukocyte count also increased similarly after sham (saline) challenge. For the AA group, the leukocyte count increased to 1.1 (6 0.4) 3 106 cells/ml at 72 h. For the ANA group, leukocyte count increased to 1.4 (6 0.7) 3 106 cells/ml at 72 h (p , 0.05 baseline, both groups). In both groups, the peak BALF leukocytosis occurred 72 h after allergen challenge and by 96 h returned to baseline values. Neutrophils and macrophages accounted predominantly for the changes in total leukocytes recovered in BALF in both groups and from both challenged sites. Neutrophil transmigration was not related to antigen challenge. Although the neutrophil count increased significantly above baseline after EBAC at all time periods, this increase occurred similarly in AA and ANA groups at both the sham- and allergen-challenged sites (Figures 3a and 4a). Similarly, the decrease (at 5 h) and subsequent (> 48 h) increase in macrophage counts over time occurred in both groups at both the sham- and allergen-challenged sites (Figures 3c and 4c). Changes in lymphocyte counts also did not differ among groups (Figures 3d and 4d).

Figure 3. Time-dependent transmigration of inflammatory cells into the lung after allergen challenge in atopic asthmatic subjects. Changes in the population of neutrophils, macrophages, and lymphocytes in the BAL over time were comparable in the sham- and allergen-challenged sites. In contrast, eosinophil count increased over time in the allergen-challenged site and not in the sham-challenged site. *p , 0.001 compared with baseline and time periods less than 24 h. Note that the y-axis scale is different for each cell population because a uniform scale did not allow for appreciation of the changes in each cell group.

Ndukwu, Naureckas, Maxwell, et al.: Cellular Profile of Patients with Asthma after EBAC

The eosinophil count in the BALF of AA subjects increased at the allergen-challenged site compared with the sham-challenged site over time. The early (5 and 24 h) eosinophil counts increased comparably in AA and ANA subjects (Figures 3b and 4b). At 48 h, eosinophil counts in AA subjects increased from a baseline of 7,896 6 3,865 eosinophils (Eos)/ml to 107,117 6 48,711 Eos/ml and to 416,476 6 231,012 Eos/ml at 72 h (p 5 0.01 versus baseline, early time period, and sham challenge). While the eosinophil count was significantly greater than baseline after endobronchial challenge at all time periods for the ANA subjects, this increase occurred at both the shamand allergen-challenged sites (Figure 4b). Comparison of AA and ANA at the allergen-challenged site (Figure 5a) revealed that the early eosinophil count (5 and 24 h) increased similarly. However, by the 48-h period the AA eosinophil count was 10-fold greater than ANA (p 5 0.001). In contrast, at the sham-challenged site (Figure 5b), the early eosinophil count was significantly greater in AA than ANA (p , 0.05). These changes in airway eosinophil suggest that eosinophil transmigration was specifically related to allergen challenge and to asthma. In addition, bronchoscopy and fluid instillation had greater effect on the asthmatic subjects. Clinical response to endobronchial allergen challenge. To assess the relationship between airway inflammation and re-

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sponsiveness, the DASS were determined and the prebronchoscopy FEV1 was measured in the AA group only. The relationship between values for DASS, FEV1, and airway eosinophil count over time is presented below. Daily Asthma Symptom Score

AA subjects were asymptomatic before the initial bronchoscopy for baseline BAL and EBAC. The highest asthma symptom score (Figure 6) was achieved 5 h after EBAC (p 5 0.001 baseline and , 0.05 versus other comparisons). BAL eosinophilia after endobronchial allergen challenge was not a marker for severity of asthma symptoms after EBAC. There was no relationship between DASS and airway eosinophil count (r 5 20.37 and p 5 0.475). Indeed, as DASS improved, the eosinophil count increased both in the blood and airway. Allergen-induced Airflow Limitation

EBAC induced significant decreases in FEV1 from baseline values (Figure 7). The greatest decrease in FEV1 (213.04 6 6.8% change from baseline, p , 0.05) occurred 24 h after EBAC. The changes in FEV1 were not significantly related to airway eosinophil count (r 5 0.357 and p 5 0.487) (see also Figure 5a).

Figure 4. Time-dependent transmigration of inflammatory cells into the lung after allergen challenge in atopic nonasthmatic subjects. Changes in the population of neutrophils, macrophages, lymphocytes, and eosinophils in the BALF over time were comparable in the sham- and allergen-challenged sites. *p , 0.05 compared with baseline. Note that the scale of the ordinate is different for each cell type.

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Figure 5. Eosinophil count after airway challenge: atopic asthmatic (AA) versus atopic nonasthmatic (ANA) subjects. ( a) The eosinophil count at the allergen-challenged site for both AA and ANA subjects. Before 48 h after EBAC, the changes in eosinophil count were comparable for AA and ANA subjects (p 5 NS). The eosinophil count > 48 h after EBAC is significantly increased for AA but not for ANA subjects (*p , 0.001). (b) The effect of bronchoscopy alone (i.e., sham infusion) in AA and ANA subjects (p 5 NS).

DISCUSSION This investigation was undertaken to determine precisely the time course and sequence of airway cellularity in the BAL fluid after endobronchial allergen challenge in subjects with asthma. To distinguish EBAC-induced features unique to asthma, we

Figure 6. Daily asthma symptom score over time. Atopic asthmatic subjects were most affected 5 h after EBAC. These subjects improved with time and by 96 h were also back to their pre-EBAC state.

compared cellular events occurring in AA subjects with those occurring in ANA subjects 5, 24, 48, 72, and 96 h after EBAC. All subjects were challenged both with allergen in the right middle lobe and saline (sham) in the lingular lobe and BALF was collected from both sites in all patients at all times. To evaluate the relationship between EBAC-induced cellular

Figure 7. Percent change in FEV1 after EBAC. Atopic asthmatic subjects experienced an early decrease in FEV 1, with the nadir occurring 24 h after EBAC. Recovery of FEV 1 back to prechallenge level occurred by 72 h after EBAC. However, a second decrease in FEV1 occurred 96 h after EBAC.

Ndukwu, Naureckas, Maxwell, et al.: Cellular Profile of Patients with Asthma after EBAC

changes and hyperresponsiveness in the AA group, we compared BALF cellularity with measures of activity limitations (DASS) and physiological function (FEV1). EBAC caused an early (, 5 h), significant decrease in the venous blood eosinophil count that was comparable in both AA and ANA groups (Figure 2). The timing of BAL was an important determinant of the number of Eos recovered in the BALF. EBAC induced a significant increase in the Eos count in the BALF of the AA group compared with the much smaller and nonspecific increase in ANA subjects (Figure 5). Symptom scores and changes in FEV1 after EBAC paradoxically did not correlate with BALF Eos count (Figures 6 and 7, respectively). However, at 96 h, a decrease in FEV1 in AA subjects was strongly and specifically associated with BALF eosinophilia (Figures 3 and 7). In this study of 12 asymptomatic AA, six asymptomatic ANA, and four nonatopic nonasthmatic subjects, the baseline cellular components of peripheral blood and BALF were similar in all subjects (Figure 1). Wardlaw and coworkers (25) and Bousquet and colleagues (3) have demonstrated similar findings for asymptomatic asthmatic groups compared with control subjects. After inhaled allergen challenge, Cookson and colleagues (26) found that the peripheral blood Eos count decreased below baseline values by 9 h. They also noted that a subsequent increase in Eos count occurred at 24 h, which was maintained for 48 h after challenge. They suggested that the decrease in Eos from the peripheral blood was associated with the development of a late asthmatic response and increased airway response. We also demonstrated a comparable peripheral blood Eos profile after allergen challenge in subjects with atopic asthma. However, our atopic nonasthmatic subjects responded comparably after airway challenge (Figure 2c). Therefore, the change in blood Eos profile appears to be related to allergen challenge rather than to asthmatic airway hyperresponsiveness per se. While most prior studies have demonstrated increased airway eosinophil counts after allergen challenge (16, 27, 28), only a few have reported a relationship between airway eosinophil influx and bronchoconstrictive response in some of the subjects (8, 29, 30). After endobronchial allergen challenge, we showed clearly that the increase in BALF Eos count was time dependent, with a peak at 72 h after EBAC. Prior to 48 h after EBAC, the increase in BALF Eos count was nonspecific (i.e., comparable at allergen-challenged and sham-challenged sites) (Figures 3b and 4b) and was also comparable in atopic asthmatic and atopic nonasthmatic subjects (Figure 5). We also did not observe a relationship between airway Eos count and the daily asthma symptom scores (Figure 6). In fact, as subjects improved, the eosinophil count increased both in the blood and airways. Crimi and colleagues (31) demonstrated that allergen inhalation produced an asthma symptom score that was predictive of the severity of seasonal asthmatic exacerbations. However, their data were not correlated with airway cellular infiltration. This observation that DASS in our AA subjects improved before the significant appearance of Eos in the BALF seems paradoxical. It is likely that allergeninduced bronchoconstriction is caused by mediators released before the appearance of eosinophils in the airway lumen (32– 35), i.e., to submucosal activity of infiltrating cells (36–38). There are some important limitations to the interpretation of our data. For safety and practical reasons, subjects were divided into two cohorts. Therefore, blood and BAL collections were not made at the same time from all subjects undergoing allergen challenge. However, the employment of the two cohorts allowed us to collect data over five time periods after allergen challenge. The role of inflammatory mediators such as

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cytokines and tissue cellular infiltration was not evaluated in this study. Tissue biopsy was specifically avoided to minimize nonspecific cellular recruitment and/or cytokine responses to tissue injury. Therefore, no cytokine assays or tissue biopsy examinations were performed for this study. We conclude that a decrease in peripheral blood eosinophil count caused by allergen challenge is not specific for asthma and also occurs in atopic subjects who do not have asthma. Our data suggest that recruitment of eosinophils into the lung is site specific and that recruitment of eosinophils into the airway of atopic nonasthmatic subjects is nonspecific. Similarly, we find in all cases that neutrophil recruitment into airways after challenge also is nonspecific and comparable in both challenged and unchallenged lung segments (i.e., a consequence of the procedure itself). Our data further suggest that the timing of cellular recruitment into the lung by allergen-induced inflammation can be clearly established, but does not correlate with either symptoms or FEV1. Acknowledgment : The authors thank Drs. J. P. Kress, Imre Noth, and Murray Favus for support and advice during the conduct of the study and Dr. Phil Schumm for statistical advice. The authors also thank the staff of the University of Chicago General Clinical Research Center.

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