CD8 T-Lymphocytes in Peripheral Airways of Smokers with Chronic Obstructive Pulmonary Disease

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1 CD8 T-Lymphocytes in Peripheral Airways of Smokers with Chronic Obstructive Pulmonary Disease MARINA SAETTA, ANTONINO DI STEFANO, GRAZIELLA TURATO, FABRIZIO M. FACCHINI, LAURA CORBINO, CRISTINA E. MAPP, PIERO MAESTRELLI, ADALBERTO CIACCIA, and LEONARDO M. FABBRI Institute of Occupational Medicine, University of Padova; Fondazione S. Maugeri, Centro Medico Veruno; and Institute of Respiratory Diseases, University of Ferrara, Italy To investigate whether the inflammatory process in peripheral airways is different in smokers who develop symptoms of chronic bronchitis and chronic airflow limitation and in asymptomatic smokers who do not develop chronic airflow limitation, we examined surgical specimens obtained from 16 smokers undergoing lung resection for localized pulmonary lesions. Nine had symptoms of chronic bronchitis and chronic airflow limitation and seven were asymptomatic with normal lung function. In peripheral airways, immunohistochemical methods were performed to identify neutrophils, macrophages, CD4 and CD8 T-lymphocytes infiltrating the airway wall, and morphometric methods were used to measure the internal perimeter, the airway wall area, and the smooth muscle area. The number of CD8 T-lymphocytes and the smooth muscle area were increased in smokers with symptoms of chronic bronchitis and chronic airflow limitation as compared with asymptomatic smokers with normal lung function, while the number of neutrophils, macrophages, and CD4 T-lymphocytes were similar in the two groups of subjects examined. We concluded that smokers who develop symptoms of chronic bronchitis and chronic airflow limitation have an increased number of CD8 T-lymphocytes and an increased smooth muscle area in the peripheral airways as compared with asymptomatic smokers with normal lung function, supporting the important role of CD8 T-lymphocytes and airway remodeling in the pathogenesis of chronic obstructive pulmonary disease. Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM. CD8 T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. AM J RESPIR CRIT CARE MED 1998;157: Cigarette smoking is a major risk factor for the development of chronic obstructive pulmonary disease (COPD), but only 15 to 20% of heavy smokers actually develop chronic airflow limitation (1, 2). The concept that this airflow limitation is due to an inflammatory process in the peripheral airways is well established (3), however the characteristics that differentiate smokers who develop COPD from those who do not develop COPD remain unclear. The present study was designed to investigate whether the inflammatory process in peripheral airways is different in smokers who develop symptoms of chronic bronchitis and chronic airflow limitation and in asymptomatic smokers who (Received in original form September 9, 1997 and in revised form November 5, 1997) Supported by the Italian Ministry of University and Research; the Regione Veneto, Giunta Regionale, Ricerca Sanitaria Finalizzata, Venice, Italy; ENFUMOSA grant BMH4-CT ; and Azienda Arcispedale S. Anna, Ferrara, Italy. Correspondence and requests for reprints should be addressed to Marina Saetta, M.D., Istituto di Medicina del Lavoro, Università degli Studi di Padova, Via Giustiniani 2, Padova, Italy. Am J Respir Crit Care Med Vol 157. pp , 1998 do not develop chronic airflow limitation. Surgical specimens were obtained from 16 smokers undergoing lung resection for localized pulmonary lesions. Nine had symptoms of chronic bronchitis and chronic airflow limitation and seven were asymptomatic with normal lung function. Peripheral airways (internal perimeter less than 6 mm) were examined with immunohistochemical methods to identify neutrophils, macrophages, CD4 and CD8 T-lymphocytes infiltrating the airway wall, and with morphometric methods to measure the internal perimeter, the airway wall area, and the smooth muscle area. METHODS Subjects The study population was composed of 16 subjects with a history of cigarette smoking, undergoing lung resection for a solitary peripheral carcinoma. Nine had symptoms of chronic bronchitis and fixed airway obstruction (COPD) and seven control subjects were asymptomatic with normal FEV 1. Chronic bronchitis was defined as cough and sputum production occurring on most days of the month for at least 3 mo a year during the 2 yr prior to the study (4). Fixed airway obstruction

2 Saetta, Di Stefano, Turato, et al.: Peripheral Airway Inflammation in COPD 823 TABLE 1 CHARACTERISTICS OF THE SUBJECTS* Controls COPD p Value Subjects examined, n 7 9 Age, yr NS Sex, M/F 7/0 9/0 Smoking starting age, yr NS Smoking history, packs/yr NS FEV 1, % of predicted FEV 1 /FVC, % Pa O2, mm Hg NS Pa CO, mm Hg NS * Values are expressed as means SEM. Values are expressed as absolute numbers. was defined as a FEV 1 less than 80% predicted, with a reversibility of less than 15% after inhalation of 200 g of salbutamol. COPD subjects had no exacerbations, defined as increased dyspnea associated with a change in the quality and quantity of sputum that led the subject to seek attention (5), during the month preceding the study. All subjects of both groups had been free of acute upper respiratory tract infections and none had received glucocorticoids or antibiotics within the month preceding surgery, or bronchodilators within the previous 48 h. They were nonatopic (i.e., they had negative skin tests for common allergen extracts), and had no past history of asthma or allergic rhinitis. The study conformed to the Declaration of Helsinki, and informed written consent was obtained for each subject undergoing surgery. Each patient underwent interview, chest radiography, electrocardiogram (ECG), routine blood tests, skin tests with common allergen extracts, and pulmonary function tests in the week before surgery. Pulmonary Function Tests Pulmonary function tests were performed as previously described (5). Briefly, they included measurements of forced expiratory volume in one second (FEV 1 ) and forced vital capacity (FVC) in all the subjects examined. The predicted normal values used were those from the European Coal and Steel Community (CECA) (6). In order to assess the reversibility of the airway obstruction in subjects with a baseline FEV 1 less than 80% predicted, the FEV 1 measurement was repeated 15 min after the inhalation of 200 g of salbutamol. Histology Four to six randomly selected tissue blocks (template size cm) were taken from the subpleural parenchyma of the lobe obtained at surgery, avoiding areas involved by tumor. Samples were fixed in 4% formaldehyde and, after dehydration, embedded in paraffin. Tissue specimens were oriented and serial sections 5 m thick were cut for morphometric and immunohistochemical analysis. Morphometric Analysis Morphometric measurements were performed on sections stained with hematoxylin eosin. At least four intact airways with an internal perimeter of less than 6 mm were identified for each patient. Airways with a short/long diameter ratio less than 0.3 were excluded from the study, as being tangentially cut. Internal perimeter rather than airway diameter was selected as a criterion to describe airway size because it remains constant irrespective of airway constriction or relaxation (7). The internal perimeter (defined by the basement membrane), the total wall area (everything between the basement membrane and the outer border of the airway wall, and the smooth muscle area were measured, as previously described (8), using a light microscope (Leitz Biomed, Leica, Cambridge, UK) (magnification: 200) connected to a video recorder linked to a computerized image system (quantimet 500 Image Processing and Analysis System, Software Qwin V0200B; Leica, Cambridge). The cases were coded and the measurements made without knowledge of clinical data. Total wall area and muscle area were normalized by the internal perimeter as previously reported (9). Immunohistochemical Analysis Mouse monoclonal antibodies were used for identification of neutrophils (anti-elastase, M752; Dako Ltd., High Wycombe, UK), macrophages (anti-cd68, M814; Dako), CD4 T-lymphocytes (anti-cd4, M834; Dako), and CD8 T-lymphocytes (anti CD8, M7103; Dako). Monoclonal antibody binding was detected with the alkaline phosphatase anti-alkaline phosphatase method (APAAP kit system K670; Dako) and fast-red substrate. To expose the immunoreactive epitopes of cell markers, the sections to be stained for macrophages were pretreated with an aqueous solution of 0.1% trypsin (Sigma Chemical, St. Louis, MO) in 0.1% calcium chloride at ph 7.8 and at 37 C for 20 min. The sections to be stained for CD8 T-lymphocytes, immersed in citrate buffer 0.5 mm at ph 6.0, were heated in a microwave oven (M704; Philips, Eindhoven, The Netherlands) at maximal power for 1 h. Control slides were included in each staining run, using human tonsil as a positive control and mouse monoclonal anticytokeratin antibody (M717; Dako) as a negative control. The cellular infiltrate was quantified in the airway wall excluding smooth muscle, because the smooth muscle was notable for the absence of inflammatory cells even in severely inflamed airways. The final results were expressed as number of cell per square millimeter of tissue examined. Statistical Analysis Group data were expressed as means and standard error (SE), or as medians and range when appropriate. Differences between groups were analyzed using the nonparametric Mann-Whitney U test for morphological data, and the unpaired Student s t test for clinical data. Correlation coefficients were calculated using Spearman s rank method. Probability values of p 0.05 were accepted as significant. At least three replicate measurements of inflammatory cells and morphometric parameters were performed by the same observer in 10 randomly selected slides, and the intraobserver reproducibility was assessed with the coefficient of variation for repeated measurements. RESULTS Clinical Findings The characteristics of control and COPD subjects are reported in Table 1. The two groups of subjects were similar with regard to age, sex, smoking history (packs/year and smoking starting age), and PaO 2 and Pa CO2 values. As expected from the selection criteria, COPD subjects had a significantly lower value of FEV 1 (% predicted) and FEV 1 /FVC ratio (%) than did control subjects. In COPD subjects, whose FEV 1 ranged from TABLE 2 MORPHOMETRIC CHARACTERISTICS OF THE AIRWAYS* Controls COPD p Value Airways examined, n Perimeter, m 2,108 (1,475 2,793) 1,989 (1,192 3,350) NS Wall area/perimeter, m 77 (72 102) 95 (59 155) NS Muscle area/perimeter, m 11 (8 14) 17 (12 36) Muscle area/wall area, % 14 (11 19) 20 (12 37) 0.05 * Values are expressed as medians (range). Values are expressed as absolute numbers.

3 824 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL area normalized by internal perimeter (17, 12 to 36 versus 11, 8 to 14 m) was increased in COPD subjects as compared with control smokers (Figure 1), and was still increased when expressed as percent of total area (20, 12 to 37 versus 14, 11 to 19%). Figure 2 illustrates the results of the differential cell counts in the peripheral airways of COPD subjects and control smokers. The number of CD8 T-lymphocytes was increased in COPD subjects as compared with control smokers (median, range: 470, 204 to 723 cells mm 2 versus 163, 89 to 526 cells/ mm 2 ; p 0.02) while the numbers of neutrophils (266, 70 to 992 cells/mm 2 versus 113, 67 to 593 cells/mm 2 ), macrophages (168, 68 to 572 cells/mm 2 versus 143, 66 to 306 cells/mm 2 ), and CD4 T-lymphocytes (396, 268 to 946 cells/mm 2 versus 293, 218 to 592 cells/mm 2 ) were not significantly different in the two groups of subjects (Figure 2). When all the smokers were considered together, the number of CD8 T-lymphocytes showed a significant negative correlation with FEV 1 (p 0.01, 0.63) (Figure 3) as did the value of smooth muscle area normalized by internal perimeter (p 0.009, 0.67) (Figure 4). The intraobserver coefficient of variation for computerassisted measurements was 2.31% and 2.34% for perimeters and areas, respectively, and ranged from 8% to 10% for the cells studied. Figure 1. Individual values for smooth muscle area normalized by internal perimeter in the peripheral airways of COPD subjects and control smokers. Horizontal bars represent median values. 54 to 79% predicted, the average response to bronchodilator was 5%. Histological Findings Table 2 illustrates the morphometric characteristics of the peripheral airways examined. The airway internal perimeter (median, range: 1,989, 1,192 to 3,350 versus 2,108, 1,475 to 2,793 m) was not significantly different in COPD subjects and controls. The total wall area, normalized by internal perimeter (95, 59 to 155 versus 77, 72 to 102 m) was not significantly different in the two groups of subjects, while the muscle DISCUSSION This study shows that smokers with symptoms of chronic bronchitis and chronic airflow limitation have an increased number of CD8 T-lymphocytes and an increased smooth muscle area in the peripheral airways as compared with asymptomatic smokers with normal lung function. The majority of studies on peripheral airway inflammation in COPD subjects have been based on classic histologic examination (10 16), not allowing for a clear distinction between different inflammatory cells, which instead can be achieved with immunohistochemical methods. Several studies have used these methods to investigate the inflammatory cell types infiltrating the mucosa of central airways in COPD subjects, and have demonstrated that the inflammatory process consists Figure 2. Individual counts for neutrophils, macrophages, CD4 T-lymphocytes, and CD8 T-lymphocytes in the peripheral airways of COPD subjects and control smokers. The results are expressed as the number of cells per mm 2 of tissue examined. Horizontal bars represent median values. Figure 3. Relationship between the number of CD8 T-lymphocytes in the peripheral airways and values of FEV 1 % predicted (Spearman rank correlation 0.63, p 0.01).

4 Saetta, Di Stefano, Turato, et al.: Peripheral Airway Inflammation in COPD 825 Figure 4. Relationship between the values of smooth muscle area normalized by internal perimeter and values of FEV 1 % of predicted (Spearman rank correlation 0.67, p 0.01). predominantly of mononuclear cells (17 20), and in particular of CD8 T-lymphocytes (21). Our findings confirm and extend these observations (17, 21) by showing that the increased number of CD8 T-lymphocytes observed in central airways of COPD subjects is also present in peripheral airways, which are the site responsible for chronic airflow limitation in smokers (3). Although we are well aware that correlations do not imply cause-effect relationships, we believe that the significant correlation observed in the overall population of smokers between increased CD8 T-lymphocytes in peripheral airways and reduced expiratory airflow suggests a possible role for these cells in the pathogenesis of smoking-induced airflow limitation. The increase in CD8 T-lymphocytes observed in peripheral airways of smokers with COPD may appear to be in contrast with the results of Bosken and coworkers (22) who reported no differences in CD8 T-lymphocyte infiltration of peripheral airways between smokers with airway obstruction and smokers without airway obstruction. However, there are several methodological differences between our study and that of Bosken and coworkers. Their patients were selected on the basis of airway obstruction, regardless of the presence of symptoms of chronic bronchitis, and the specimens were frozen before immunohistochemical analysis. In our study, the specimens were fixed and embedded in paraffin before immunohistochemical analysis, and the patients were selected on the basis of both airway obstruction and symptoms of chronic bronchitis. The role of symptoms of chronic bronchitis in the development of chronic airflow limitation is still controversial. In fact, chronic sputum production, which characterizes chronic bronchitis, has traditionally been considered to be irrelevant to the development of chronic airflow limitation (23 25). However, a recent study (26) has shown that chronic sputum production was significantly associated with both an excess of FEV 1 decline and an increased risk of subsequent hospitalization because of COPD, suggesting a causal role for chronic sputum production in the development of chronic airflow limitation. Because the COPD subjects in the present study were selected on the basis of both chronic airflow limitation and chronic sputum production, the relative contribution of these two conditions to the increase in CD8 T-lymphocytes still remains to be investigated. Traditionally, the major activity of CD8 T-lymphocytes has been considered the rapid resolution of acute viral infections (27), viral infections being a frequent occurrence in patients with COPD. As suggested by O Shaughnessy and coworkers (21), it is possible that an excessive recruitment of CD8 T-lymphocytes may occur in response to repeated viral infections in some smokers, and that this excessive response may play a crucial role for the development of pulmonary damage in these subjects (28, 29). Our finding of a nonsignificant increase of neutrophils in smokers with airflow limitation compared with control smokers extends the results obtained in central airways (18 20, 30) to the peripheral airways. The disparity between the relatively low neutrophil number in the airway wall and high numbers of neutrophils reported in the bronchoalveolar lavage of subjects with COPD (30, 31) could be due to their rapid migration across the tissues into the lumen, such that at any time point their numbers in tissue are low (21). The increased smooth muscle area observed in peripheral airways of smokers with COPD is in agreement with the results of previous reports (32, 33). In the present study, when all the smokers were grouped together, a significant correlation was observed between increased smooth muscle area and reduced expiratory airflow, supporting the hypothesis that airway remodeling in peripheral airways may play an important role in the development of chronic airflow limitation. The mechanism by which cigarette smoke causes smooth muscle hypertrophy remains speculative. It is possible that the inflammatory process present in the peripheral airways of these subjects could act on the smooth muscle either directly by the release of growth factor, or indirectly by inducing a chronic increase in muscle tone (16). A confusing element in any study performed on surgical resected specimens of patients with lung cancer is that the presence of cancer itself may influence the results. However, surgical specimens are the only specimens that allow for the examination of peripheral airways in subjects with preoperative pulmonary function, and peripheral airways are the site responsible for the development of chronic airflow limitation in smokers (3). Moreover, as a result of our having examined only tissue away from the tumor site, and having included subjects with lung cancer in our control group, we feel rather confident that our findings of increased infiltration of CD8 T-lymphocytes and increased smooth muscle area in the peripheral airways of COPD subjects are valid. Because the internal perimeter has been shown to remain constant despite changes in smooth muscle tone and lung volume (7), we used the internal perimeter as a marker of airway size, and we normalized the wall area and the smooth muscle area by this parameter. In our study, the internal perimeters of peripheral airways were similar in COPD subjects and control smokers, indicating that, despite the possible different lung volumes caused by tissue preparation and the possible different smooth muscle tone in the two groups of subjects, we were comparing bronchioles of similar size. In conclusion, smokers who develop symptoms of chronic bronchitis and chronic airflow limitation have an increased number of CD8 T-lymphocytes and an increased smooth muscle area in the peripheral airways as compared with asymptomatic smokers who do not develop chronic airflow limitation, supporting the important role of CD8 T-lymphocytes and airway remodeling in the pathogenesis of COPD.

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