Epithelial Desquamation in Asthma Artifact or Pathology?

Epithelial Desquamation in Asthma Artifact or Pathology? CLAUDIA ORDOÑEZ, RON FERRANDO, DALLAS M. HYDE, HOFER H. WONG, and JOHN V. FAHY Departments of Pediatrics and Medicine and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California; and University of California, Davis, Davis, California To determine whether the denudation of the bronchial epithelium observed in endobronchial biopsies from asthmatic subjects is a true pathologic feature or an artifact of tissue sampling, we analyzed epithelial integrity in bronchial biopsies from 14 subjects with mild and moderate asthma and 12 healthy subjects. In each subject, 4 to 8 bronchial biopsies were taken from large airways during bronchoscopy, fixed in 4% paraformaldehyde, embedded in glycomethacrylate, cut into 2- M sections, and stained with toluidine blue. A 4 image of each biopsy was copied to a computer file using a video camera, and lines were drawn and measured along the basement membrane underlying areas completely denuded of overlying epithelium, areas covered by a single layer of basal cells, and areas of intact epithelium. We found that the percentage of basement membrane that was denuded of epithelium was similar in the healthy and asthmatic subjects (14.8 11.8 versus 11.4 9.8% respectively, p 0.38); the percentage of basement membrane that was covered by a single layer of basal cells was also similar in the two groups (46.4 11.0 versus 54.5 9.8%, respectively, p 0.11). In the asthmatic subjects, we found no significant correlation between the percentage of basement membrane covered by denuded epithelium or by a single layer of basal cells and the FEV 1 percentage of predicted or the PC 20 methacholine. We conclude that denudation of bronchial epithelium in endobronchial biopsies from asthmatic subjects with stable mild and moderate disease is an artifact of tissue sampling and is not a true pathologic feature of the disease, and that the extent of airway epithelial denudation is not correlated with the severity of airway narrowing or the severity of bronchial hyperresponsiveness. One of the consequences of airway inflammation in asthma is thought to be desquamation of airway epithelial cells resulting from the action of inflammatory mediators such as eosinophil granule proteins (1 3). Indeed, the loss of airway epithelium in asthma has been hypothesized to result in loss of epithelium-derived relaxing factor, in turn inducing airway narrowing and airway hyperreactivity (4, 5). Support for the hypothesis that desquamation of the airway epithelium is a pathologic feature of asthma arises from observations of epithelial desquamation in postmortem specimens from asthmatics dying of acute severe asthma (6, 7), and in endobronchial biopsies from chronic stable asthmatics (1) (2). The hypothesis is further supported by finding clumps of epithelial cells (creola bodies) in sputum from asthmatic subjects (8) and increased numbers of epithelial cells in bronchoalveolar lavage (BAL) from asthmatic subjects (9 11). In addition, (Received in original form January 13, 2000 and in revised form May 17, 2000) Supported by RO1 HL61662 and P50 HL 56385 from the National Institutes of Health. Correspondence and requests for reprints should be addressed to John V. Fahy, M.D., Box 0111, University of California, San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143. E-mail: jfahy@itsa.ucsf.edu Am J Respir Crit Care Med Vol 162. pp 2324 2329, 2000 Internet address: www.atsjournals.org some of these studies have found correlations between airway epithelial desquamation or epithelial cell number in (BAL) and the degree of airway hyperreactivity (2, 9, 10). Despite these findings, the hypothesis that epithelial desquamation is a pathologic feature of asthma is far from proven. Although some postmortem studies describe epithelial cells mixed with mucus in the airway lumen (6, 12), other studies that have quantitated the integrity of the airway epithelium have found no increase in the degree of epithelial desquamation in patients who die from asthma (13). Although some analyses of endobronchial biopsies from asthmatic subjects have found increased epithelial desquamation, more recent studies, with larger numbers of control subjects, have not (14, 15). Furthermore, analysis of endobronchial biopsies from healthy subjects has highlighted the potential for epithelial damage as an artifact of tissue sampling (16). Finally, many studies of the cellular composition of BAL and sputum from healthy and asthmatic subjects do not describe increased numbers of epithelial cells (17 23). Because of continued uncertainty about whether epithelial desquamation is a true pathologic feature of asthma or an artifact of tissue sampling, we set out to determine if epithelial desquamation is greater than normal in asthmatic subjects, and to determine if the degree of epithelial desquamation is related to the severity of baseline airway narrowing or airway hyperreactivity. Specifically, we obtained endobronchial biopsies from healthy and asthmatic subjects using fiberoptic bronchoscopy, and we quantitated epithelial integrity in these biopsies using video- and computer-linked microscopy. METHODS Subjects Twelve healthy and 14 asthmatic subjects were studied (Table 1). Inclusion criteria for the asthmatic subjects were a history of symptoms of asthma and bronchial hyperreactivity to methacholine (provocative concentration of methacholine causing a 20% reduction in FEV 1 [PC 20 ] 8 mg/ml). Exclusion criteria were inhaled or oral steroid use or history of an upper respiratory infection in the previous 6 wk, and tobacco use within the past 10 yr or greater than 5 pack-years total smoking history. Healthy subjects had no history of lung disease or of allergic rhinitis. Healthy subjects with one positive skin test to an aeroallergen were enrolled; subjects with more than one positive skin test were excluded. All asthmatic and healthy subjects were either lifetime nonsmokers or had not smoked in more than 10 yr and had smoked a maximum of 5 pack-years. All subjects signed consent forms approved by the Committee on Human Research at the University of California, San Francisco. Protocol The study involved two visits to the laboratory. The first visit was for consent, spirometry, allergen skin testing, and methacholine challenge. The second visit (1 to 2 wk after the first visit) was for bronchoscopy.

Ordoñez, Ferrando, Hyde, et al.: Asthma and Epithelial Desquamation 2325 Subject TABLE 1 CLINICAL CHARACTERISTICS OF THE STUDY SUBJECTS Sex Age (yr) FEV 1 (% pred) Spirometry and Methacholine Challenge Peak Flow Variability PC 20 * (mg/ml) Healthy subjects 1 F 34 103 15.3 2 F 34 97 80.0 3 M 25 110 80.0 4 F 23 118 80.0 5 F 23 110 80.0 6 M 27 102 80.0 7 F 27 103 21.5 8 F 24 106 80.0 9 F 25 91 17.0 10 F 44 103 80.0 11 F 25 103 80.0 12 F 34 116 15.2 Asthmatic subjects 1 M 30 65.5 39.3 0.259 2 M 37 88.5 20.4 0.412 3 M 35 110 4.2 0.250 4 F 26 93.0 15.7 0.359 5 M 34 77.0 19.0 0.147 6 M 30 72.0 18.8 1.210 7 F 26 67.0 27.2 0.090 8 M 34 63.5 7.6 0.354 9 M 37 74.0 2.3 0.412 10 M 35 76.5 26.6 0.154 11 M 28 117.0 7.0 2.680 12 F 39 85.5 9.0 0.281 13 M 30 96.5 0.359 14 F 34 100.0 51.4 2.176 * The maximal dose of methacholine delivered was 64 mg/ml. A PC 20 64 mg/ml indicates that the subject s FEV 1 did not decrease by 20% upon inhaling 5 breaths of this concentration of methacholine. Bronchodilators were withheld for 8 h before testing. Spirometry and methacholine challenge were performed using methods previously described (24). Allergen Skin Tests Skin-prick testing was performed using standardized disposable allergy prick test needles (Morrow Brown, Topeka, KS). Eleven common inhalant allergens were used (Alternaria, cat hair, dog hair, Dermatophagoides pteronyssinus, D. farinae, American cockroach, Cladosporium, Aspergillus mix, plantain sorrel mix, short ragweed, Bermuda and Johnson grass mix) a diluent control, and histamine control, all of which were obtained from Greer Lab, Inc., Lenoir, NC. If the skin wheal diameter caused by the allergen was 3 mm larger than that of the diluent control, the skin test was considered positive. Peak Flow Monitoring During Visit 1 subjects were instructed to record twice-daily peak flow measurements for 1 wk using a MiniWright Peak Flow Meter (Clement Clarke, Columbus, OH). Bronchoscopy Bronchoscopy was performed in accordance with guidelines for research bronchoscopy in asthmatic subjects (25) using a method described previously (26). One bronchoscopist experienced in research bronchoscopy (J.V.F.) performed all but two of the bronchoscopy procedures; another bronchoscopist in our research group, also experienced in research bronchoscopy, performed the other two procedures (in one healthy subject and one asthmatic subject). The bronchoscope (Pentax FB-19D; Pentax Precision Instrument Corporation, Orangeburg, NY) was introduced orally and wedged in a subsegmental bronchus of the lingula, and a bronchial wash was performed by instilling and quickly aspirating 50 ml of normal saline. The bronchoscope was then moved to the right side where six mucosal biopsies were taken from the carina of the right upper lobe and the right middle lobe using a spiked cupped biopsy forceps (Pentax KH2411S and Pentax 8228). The biopsy forceps were used an average of five times. Six biopsies were embedded in glycomethacrylate. Total and Differential Cell Counts in the Bronchial Wash The bronchial wash sample was placed on ice in the bronchoscopy laboratory and transferred within 5 min to the processing laboratory. Ten microliters of the wash was loaded on a hemacytometer for determination of the total cell count. Aliquots of 250 l of the bronchial wash (diluted in saline to prevent cell crowding on the slide) were loaded in a cytocentrifuge (Model 7 cytospin; Shandon Scientific, Sewickley, PA) to make cytocentrifuged slides. These slides were then stained using the Leukostat stain (Fisher Scientific, Pittsburgh, PA) and at least 200 cells on each slide were read by an investigator blinded to the clinical details of the subjects. Processing, Staining, and Analysis of the Endobronchial Biopsies The biopsies were transferred from the biopsy forceps directly to a solution of 4% paraformaldehye (PFA) in 0.1 M Na phosphate buffer (ph 7.2) and allowed to fix for 2 h. The biopsies were then dehydrated in graded alcohol solutions as follows: 70% acetone in ddh 2 O with 2% DMSO for 15 min, 95% acetone in ddh 2 O with DMSO for 15 min, and 100% acetone in ddh 2 O for 30 min. Next, the biopsies were infiltrated in glycomethacrylate (Polysciences, Inc., Warrington, PA) as follows: 100% acetone and JB-4 A (1:1) followed by 100% JB-4 A solution overnight under vacuum at 4º C. Biopsies infiltrated in JB-4 A were then placed in an embedding mold filled with a mixture of JB-4 A with benzoyl peroxide (0.18 g in 20 ml) and JB-4 B (25:1). The molds were then placed under vacuum at 4 C overnight to allow the resin to harden. Once cured, the blocks were removed from the mold, labeled, and stored at 20 C. Each biopsy was cut into 2- M sections and stained with toluidine blue (Sigma, St. Louis, MO). The method for measuring epithelial integrity in the endobronchial biopsies is demonstrated in Figure 1. Four to seven endobronchial biopsies per subject were available for analysis (a total of 134 biopsies). For each subject, the results from the 4 to 7 biopsies were averaged to yield the data on epithelial integrity. Statistics Data was entered on computer spreadsheet (Microsoft Excel; Microsoft, Redmond, WA) and exported to a statistics program (Statview; Abacus Concepts Inc., Berkeley, CA) for descriptive and comparative statistics. Data are presented as mean SEM or as the median, as appropriate. Because most of the data were not normally distributed, the Mann-Whitney U test was used in most instances to compare data from asthmatic and healthy subjects. An exception was the cell differential data in bronchial wash which was analyzed using the unpaired t test. Spearman s rank order test was used to determine correlations between data. A probability value of 0.05, using twotailed tests, was considered significant. RESULTS Two distinct patterns of epithelial damage were noted in the biopsies. The most common appearance was that basement membrane was covered by a single layer of basal cells with no intact ciliated cells or goblet cells (Figure 2). Less commonly observed was complete denudation of the epithelial cells, including basal cells (Figure 2). Both of these appearances of the epithelium were observed in both healthy and asthmatic subjects (Figure 2). The percentage of basement membrane covered by no epithelial cells (completely denuded epithelium) was similar in the endobronchial biopsies from healthy and asthmatic subjects (Figure 3). The percentage of basement membrane covered by a single layer of basal cells was also similar in the endobronchial biopsies from healthy and asthmatic subjects (Figure 3). In the asthmatic subjects there was no correlation

2326 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 162 2000 Figure 1. Method for measuring epithelial integrity in the endobronchial biopsies. Using NIH Image software, lines are drawn on the basement membrane (BM) of three identical computer images (original magnification 260) of an entire section from an endobronchial biopsy (a, b, and c). One line is drawn on the BM that is covered with a single layer of basal cells (a). Another line is drawn on the BM that is covered with no epithelial cells (b). A third line is drawn on the BM that is covered with intact epithelium (c). The lengths of these lines in millimeters was measured using NIH Image. The total length of the BM (total BM) was determined by adding the lengths of the three lines. Thus, for the calculation of percentage of BM covered by a single layer of basal cells, the numerator was the length of the (interrupted) line drawn on the BM covered with a single layer of basal cells and the denominator was the length of total BM. Similarly, for the calculation of percentage of BM covered by no cells, the numerator was the length of the (interrupted) line drawn on the BM covered with no cells and the denominator was the length of total BM. Figure 2. Appearance of the airway epithelium in healthy and asthmatic subjects. Two distinct patterns of epithelial damage were noted in the biopsies. The most common appearance was that basement membrane was covered by a single layer of basal cells with no intact ciliated cells or goblet cells (panels 1 and 4). Less commonly observed was complete denudation of the epithelial cells, including basal cells (panels 2 and 5). Both of these appearances of the epithelium were observed in both healthy (panels 1 and 2) and asthmatic subjects (panels 4 and 5). Intact epithelium was found in biopsies from healthy subjects (panel 3) and asthmatic subjects (panel 6).

Ordoñez, Ferrando, Hyde, et al.: Asthma and Epithelial Desquamation 2327 between the percent predicted FEV 1 and the percentage of basement membrane that was completely denuded of epithelium or covered by a single layer of basal cells (Table 2). In addition, there was no significant correlation between the PC 20 methacholine in the asthmatic subjects and the percentage of basement membrane completely denuded of epithelium or the percent that was covered by a single layer of basal cells (Table 2). The median length of basement membrane in the endobronchial biopsies was similar in the healthy and asthmatic subjects (5.4 [0.8 21.5] versus 5.5 [1.2 29.7] mm). The length of the basement membrane in each biopsy did not correlate with the percentage of the basement membrane that was completely denuded of epithelium or covered by a single layer of basal cells (r s 0.08, p 0.34 and r s 0.08, p 0.38, respectively). There was a trend for a higher than normal percentage of epithelial cells in the bronchial wash samples from the asthmatic subjects (p 0.09) (Table 3). The percentage of eosinophils was significantly higher than normal in the bronchial wash from the asthmatic subjects. In addition, there was no significant correlation between the PC 20 methacholine in the asthmatic subjects and the percentage of epithelial cells in the bronchial wash samples (Table 2). DISCUSSION This study shows that the percentage of basement membrane covered by no epithelial cells (completely denuded epithelium) or covered by a single layer of basal cells is similar in endobronchial biopsies from healthy and asthmatic subjects and that the degree of epithelial desquamation in asthmatic subjects is not related to the severity of baseline airway narrowing or of airway hyperreactivity. Our findings suggest that epithelial desquamation in endobronchial biopsies in asthmatic subjects is an artifact of tissue sampling and is not a true pathologic feature of the disease. We found that approximately 50% of the basement membrane in both healthy and asthmatic subjects was covered by a single layer of basal cells; an additional 10 to 15% of the basement membrane was completely denuded of any epithelial cells. Thus, the majority of the basement membrane in these biopsy samples was not covered by a pseudostratified epithelial layer. Our data demonstrate that the plane of cleavage of the columnar epithelial cells from the basement membrane is at the level of the basal cell and not usually at the level of the basement membrane. This observation is consistent with data showing that the ratio of columnar cells to basal cells in BAL is far greater than the ratio of these cell types observed in intact bronchial epithelium (27). This observation also confirms TABLE 2 CORRELATION BETWEEN AIRWAY REACTIVITY TO METHACHOLINE, FEV 1 % PREDICTED, AND DEGREE OF EPITHELIAL DESQUAMATION IN AIRWAY BIOPSIES FROM ASTHMATIC SUBJECTS % Basement Membrane Covered by a Single Layer of Basal Cells Correlation Coefficient (rs) % Basement Membrane Completely Denuded of Epithelium % Epithelial Cells in Bronchial Wash PC 20 methacholine 0.36 0.06 0.28 Percent predicted FEV 1 0.26 0.30 0.47 * None of the correlations was statistically significant (all p values 0.10). the important role of the basal cell in anchoring columnar epithelial cells to the basement membrane. The anchoring of columnar epithelial cells to the basement membrane is thought to depend entirely on desmosomal connections to basal cells (28). Basal cells are themselves anchored to the lamina densa of the basement membrane by hemidesmosomal connections and by other adhesive mechanisms involving integrins and anchoring filaments (29). Our data suggest that desmosomal connections of columnar cells to basal cells represent a more fragile and a more easily disrupted attachment than the adhesive mechanisms responsible for the attachment of basal cells to the basement membrane. We found no difference in the degree of epithelial desquamation in endobronchial biopsies from healthy and asthmatic subjects. Although this finding is not in agreement with previous studies proposing epithelial desquamation as a pathologic feature of asthma, we believe that our data are valid for at least three reasons. First, we used quantitative methodology. By using video- and computer-linked microscopy we were able to quantify the percentage coverage of basement membrane with intact and nonintact epithelium in an entire biopsy. Not all previous studies that have proposed epithelial desquamation as a pathologic feature of asthma have used quantitative methods. Second, we decreased sampling bias by analyzing up to seven endobronchial biopsies per subject (the median number of biopsies analyzed per subject was five). This is a larger number of biopsies analyzed per subject than the number reported in previous studies, which have usually analyzed 1 to 3 biopsies per subject (1, 2). Third, we studied biopsies from a relatively large number of healthy control subjects compared with earlier studies in which as few as one healthy control subject was studied (1). Although our finding that epithelial desquamation is similar in endobronchial biopsies from healthy and asthmatic subjects contrasts with the findings of some investigators, it is in agreement with that of others. For example, Boulet and co- TABLE 3 CELL DIFFERENTIAL OF BRONCHIAL WASH FROM HEALTHY AND ASTHMATIC SUBJECTS Healthy Asthma p Value Figure 3. The percentage of basement membrane covered by a single layer of basal cells (left panel) or completely denuded of epithelium (right panel ) in endobronchial biopsies from 12 healthy and 14 asthmatic subjects. Epithelial cells 20.1 9.7 26.4 9.1 0.09 Macrophages 64.6 10.8 60.1 11.9 0.31 Neutrophils 8.1 7.2 4.7 3.6 0.12 Eosinophils 0.2 0.2 1.2 0.9 0.0007 Lymphocytes 5.0 2.0 5.3 6.2 0.86

2328 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 162 2000 workers (14) and Lozewicz and coworkers (15) found no significant difference in the extent of epithelial desquamation in biopsies from healthy and asthmatic subjects. Similarly, Carroll and coworkers (13) found no significant difference in the degree of epithelial desquamation in whole lung specimens from cases of fatal asthma, asthmatics dying for reasons other than acute asthma, and control cases. In addition, Soderberg and coworkers (16) have documented extensive epithelial damage in endobronchial biopsies from healthy subjects, highlighting the potential for artifactual damage to the airway epithelium during biopsy sampling, and raising doubts about the cause of epithelial desquamation in asthma. Bronchial biopsy technique, the size and type of biopsy forceps, and the methods of fixation and embedding of biopsy tissue are all variables that might influence epithelial integrity in endobronchial biopsies. Indeed, one recent study has suggested that the type of biopsy forceps can influence epithelial morphology (30). In our study, both healthy and asthmatic subjects underwent the same bronchoscopy protocol to control for these factors. The influence of different tissue embedding protocols and tissue fixation protocols is more difficult to evaluate. However, a recent study of bronchial biopsies from healthy and asthmatic subjects, which used a different fixation and embedding protocol than ours, found epithelial desquamation in the biopsies in a range similar to that reported here (14). We found no correlation in the asthmatic subjects between the percent predicted FEV 1 or the PC 20 methacholine and the percent of basement membrane that was completely denuded of epithelium or covered by a single layer of basal cells. Although this finding contrasts with that of Jeffery and coworkers (2), it is in agreement with that of Boulet and coworkers (14) who also found no significant correlation between the degree of epithelial desquamation and airway hyperresponsiveness or airflow obstruction in asthmatic subjects. We also found no significant correlation in the asthmatic subjects between the percent predicted FEV 1 or the PC 20 methacholine and the percent of epithelial cells in bronchial wash fluid. This finding contrasts with that of Beasely and coworkers (10) who did find a significant inverse correlation between the percentage of epithelial cells in BAL and airway hyperresponsiveness. The percentage of epithelial cells in BAL may reflect trauma during the lavage procedure and may be an indicator of epithelial fragility rather than of epithelial desquamation. In addition, the technique of BAL, specifically the volume instilled (26, 31), can influence the percentages of epithelial cells in the lavage and may influence the relationship between PC 20 values and values for epithelial cells in the lavage fluid. It is important to acknowledge that the lack of significant differences in measurements of epithelial desquamation in this group of healthy and asthmatic subjects does not argue against epithelial cell dysfunction in asthma. Asthma is associated with significant abnormalities of epithelial structure and function (32 35), but these abnormalities may not necessarily result in desquamation. For example, activation markers of inflammation are unregulated in the airway epithelium in asthma (35), and structural changes such as goblet cell hyperplasia have been shown (36). Our results here do not question these established characteristics of asthma, but they do question whether they are associated with epithelial cell desquamation. 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