INTRODUCTION

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It is a well established fact that cigarette smoking causes an inflammatory process in peripheral airways (1). The majority of studies examining the peripheral airways in smokers have been focused in the subepithelial portion of the airway wall (2), while only a few investigations have examined the epithelium (11, 12), and these studies reported conflicting results. In addition, to the best of our knowledge, a precise quantification of the mucus-secreting cells in the epithelium of peripheral airways has never been attempted, despite the fact that a rise in goblet cells has been reported in smokers by several investigators (2, 13). Investigating goblet cells in smokers might be of interest because these cells could potentially contribute to the development of smoking-induced airway obstruction in at least two ways: first, by producing an excess of mucus which could alter the surface tension of the airway lining fluid, rendering the peripheral airways unstable and facilitating their closure (16); second, by inducing lumenal occlusion through the formation of mucous plugs in peripheral airways (2).

The aim of our study was to quantify the number of goblet cells and inflammatory cells in the epithelium of peripheral airways in smokers with both symptoms of chronic bronchitis and chronic airflow limitation. For this purpose we examined surgical specimens obtained from 25 subjects undergoing lung resection for localized pulmonary lesions: 10 smokers with both symptoms of chronic bronchitis and chronic airflow limitation, six asymptomatic smokers with normal lung function, and nine nonsmoking control subjects.

	METHODS

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Subjects

We recruited to the study three groups of subjects undergoing lung resection for a solitary peripheral carcinoma: 10 smokers with both symptoms of chronic bronchitis and fixed airway obstruction (smokers with chronic obstructive pulmonary disease [COPD]), six asymptomatic smokers with normal lung function, and nine asymptomatic nonsmoking subjects with normal lung function. 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 before the study (17). Fixed airway obstruction was defined as a FEV₁ less than 80% predicted, with a reversibility of less than 15% after inhalation of 200 µg of salbutamol. Smokers with symptoms of chronic bronchitis and chronic airflow limitation had had no exacerbations, which are defined as increased dyspnea associated with a change in the quality and quantity of sputum leading the subject to seek medical attention (18), during the month preceding the study.

All the subjects 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, 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 within the week before surgery as previously described (18). Briefly, they included measurements of blood gas analysis, FEV₁ and FVC. The predicted normal values used were those from Communité Européenne du Carbon et de l'Acier (CECA) (19). To assess the reversibility of the airway obstruction in subjects with a baseline FEV₁ less than 80% predicted, the FEV₁ measurement was repeated 15 min after the inhalation of 200 µg of salbutamol.

Histology

Four to six randomly selected tissue blocks (template size 2 × 2.5 cm) were taken from the subpleural parenchyma of the lobe obtained at surgery, avoiding areas involved by tumor. Samples were fixed in 4% formaldehyde in phosphate-buffered saline at pH 7.2 and, after dehydration, embedded in paraffin wax. Tissue specimens were oriented and 5-µm-thick serial sections were cut for histochemical and immunohistochemical analysis.

Periodic acid-Schiff staining (PAS) was performed to identify mucus-secreting cells (goblet cells). Mouse monoclonal antibodies were used for identification of total leukocytes (antileukocyte common antigen CD45, M0701; Dako Ltd., High Wycombe, UK), neutrophils (anti-elastase, M0752; Dako), macrophages (anti-CD68, M0814; Dako), CD4⁺ cells (anti-CD4, M0834; Dako), and CD8⁺ cells (anti-CD8, M7103; Dako).

Monoclonal antibody binding was detected with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method (Dako) and fast-red substrate. To expose the immunoreactive epitopes of cell markers, the sections to be stained for CD45 and CD8, immersed in citrate buffer 5 mM at pH 6.0, were incubated in a microwave oven (model M704; Philips, Eindhoven, The Netherlands) on high power, while the sections to be stained for CD68 were incubated with 0.1% trypsin (Sigma Chemical, St. Louis, MO) in 0.1% calcium chloride at pH 7.8 at 37° C for 20 min. Control slides were included in each staining run, using human tonsil as a positive control and mouse monoclonal anticytokeratin antibody (M0717 Dako) as a negative control.

Light microscopic analysis of peripheral airways was performed using a light microscope (Leica DMLB; Leica, Cambridge, UK) connected to a video recorder linked to a computerized image system (software: Casti Imaging, Venezia, Italy). The cases were coded and the measurements made without knowledge of clinical data.

At least four noncartilaginous peripheral airways with an internal perimeter less than 6 mm were selected for each patient. To avoid measurements in tangentially cut airways, bronchioles with a short/ long diameter ratio less than one-third were excluded from the study. In each airway we measured the internal perimeter along the subepithelial basement membrane and the luminal diameter as the greater distance in a plane perpendicular to the long axis of the lumen.

In each peripheral airway we quantified goblet cells, CD45⁺ cells, neutrophils, macrophages, CD4⁺ and CD8⁺ cells in the intact epithelium, defined by the presence of both basal and columnar cells above the luminal edge of the basement membrane. All 25 cases had peripheral airways suitable for inflammatory cell counts in the epithelium, and 23 cases had peripheral airways suitable for goblet cell counts in the epithelium. Results were expressed as number of positive cells per millimeter of basement membrane.

Although our study was focused on the epithelium, for CD45⁺ cells we extended the analysis to evaluate the distribution of total leukocytes within the entire wall of peripheral airways. For this purpose, airway wall area was subdivided in two regions: the "inner" area (submucosa) which extended from the distal edge of the basement membrane to the internal edge of the smooth muscle, and the "outer" area (adventitia) which extended from the outer edge of the smooth muscle to the alveolar attachments (20). In the peripheral airways demonstrating smooth muscle surrounding at least 30% of the perimeter and having well-demarcated alveolar attachments, we quantified CD45⁺ cells in the "inner" and in the "outer" areas. Twenty of 25 cases had peripheral airways suitable for analysis of cell distribution. Results were expressed as: (1) inner leukocyte density: number of CD45⁺ cells per square mm of "inner" submucosal area, and (2) outer leukocyte density: number of CD45⁺ cells per square mm of "outer" adventitial area.

Statistical Analysis

Group data were expressed as means and standard error (SEM), or as medians and interquartile range when appropriate. Differences between groups were analyzed using the following tests for multiple comparisons: the analysis of variance (ANOVA) for clinical data, and the Kruskall-Wallis test for histologic data. The Mann-Whitney U-test was carried out after Kruskall-Wallis test when appropriate. Comparison between inner and outer leukocyte density within each group of subjects was made using Student's paired t test. Spearman's rank correlation test was used to examine the association between histologic parameters and clinical data. Probability values of p  0.05 were accepted as significant. At least three replicate measurements of goblet and inflammatory cells were performed by the same observer in 10 randomly selected slides, and the intraobserver reproducibility was assessed with the coefficient of variation and with the intraclass correlation coefficient. The intraobserver coefficient of variation was 11% for goblet cells and ranged from 4 to 15% for the inflammatory cells examined. The intraobserver correlation coefficient was 0.96 for goblet cells and ranged from 0.59 to 0.96 for the inflammatory cells examined.

	RESULTS

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Clinical Findings

Table 1 shows the characteristics of the subjects examined. The three groups of subjects were similar with regard to age, Pa_O2 and Pa_CO2 values. There was no significant difference in the smoking history between smokers with COPD and smokers with normal lung function. As expected from the selection criteria, smokers with COPD had a significantly lower value of FEV₁ (percentage of predicted) and FEV₁/FVC ratio (percent), than did smokers with normal lung function (p < 0.05) and nonsmokers (p < 0.05). In smokers with COPD, whose FEV₁ ranged from 54 to 79% predicted, the average response to bronchodilator was 5%.

Histologic Findings

The number of airways examined was 67 in smokers with COPD, 40 in smokers with normal lung function, and 63 in nonsmokers. The airway internal perimeter was similar in smokers with COPD (median and interquartile range: 1,441 and 1,010 to 2,001 µm), smokers with normal lung function (1,362 and 1,003 to 1,678 µm), and nonsmokers (1,457 and 1,207 to 1,621 µm), indicating that we compared airways of similar size. The airway diameter was also similar in the three groups of subjects examined (smokers with COPD: 196 and 112 to 386 µm; smokers with normal lung function: 170 and 119 to 274 µm; nonsmokers: 188 and 120 to 243 µm).

The results of the cell counts in the epithelium of peripheral airways are shown in Figures 1-3. The number of goblet cells was increased in smokers with COPD (Figures 1 and 4) when compared with nonsmokers (p < 0.01), but the difference was not significant when compared with smokers with normal lung function. Smokers with normal lung function had a number of goblet cells not significantly different from that of nonsmokers (Figure 1). The number of CD45⁺ cells was increased in both smokers with COPD and smokers with normal lung function as compared with nonsmokers (p < 0.01 and p < 0.05, respectively), whereas it was not significantly different between the two groups of smokers (Figure 2). The number of macrophages was increased in both smokers with COPD and smokers with normal lung function as compared with nonsmokers (p < 0.05), whereas it was not significantly different between the two groups of smokers (Figure 3). The number of CD8⁺ cells was increased in smokers with COPD when compared with nonsmokers (p < 0.01), but the difference was not significant when compared with smokers with normal lung function. Smokers with normal lung function had a number of CD8⁺ cells not significantly different from that of nonsmokers (Figure 3). No significant differences among the three groups of subjects examined were observed in the number of neutrophils and CD4⁺ cells (Figure 3). When the ratio CD4/CD8 was computed, no significant differences were observed between smokers with COPD (median and interquartile range: 0.31, 0.19 to 0.66), smokers with normal lung function (1.21, 0.39 to 2.36), and nonsmokers (1.31, 0.91 to 2.23).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Individual counts for goblet cells in the epithelium of peripheral airways of nonsmokers (NS), smokers with normal lung function (S), and smokers with both symptoms of chronic bronchitis and chronic airflow limitation (COPD). The results are expressed as number of cells per millimeter of basement membrane. Horizontal bars represent median values.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Individual counts for CD45+ cells in the epithelium of peripheral airways of nonsmokers (NS), smokers with normal lung function (S), and smokers with both symptoms of chronic bronchitis and chronic airflow limitation (COPD). The results are expressed as number of cells per millimeter of basement membrane. Horizontal bars represent median values.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Individual counts for neutrophils, macrophages, CD4+ cells, and CD8+ cells in the epithelium of peripheral airways of nonsmokers (NS), smokers with normal lung function (S), and smokers with both symptoms of chronic bronchitis and chronic airflow limitation (COPD). The results are expressed as number of cells per millimeter of basement membrane. Horizontal bars represent median values.

View larger version (134K): [in this window] [in a new window]   Figure 4.   (A) Microphotograph of a peripheral airway of a smoker with both symptoms of chronic bronchitis and chronic airflow limitation, showing goblet cells in the epithelium. Bar represents 80 µm. (B) Detail of panel A. Bar represents 32 µm. Periodic acid-Schiff staining.

When all the smokers (those with COPD and those with normal lung function) were considered together, they showed an increased number of neutrophils (p < 0.05) along with an increased number of goblet cells (p < 0.05), CD45⁺ cells (p < 0.01), macrophages (p < 0.05), and CD8⁺ cells (p < 0.05), as compared with nonsmokers. The number of CD4⁺ cells was similar in smokers and nonsmokers (Table 2).

When we examined the distribution of CD45⁺ cells within the peripheral airway wall, we observed that, in smokers with COPD, the inner leukocyte density (median and interquartile range: 2,008 and 1,860 to 2,158 cells/mm²) was greater than the outer leukocyte density (1,041 and 1,004 to 2,228 cells/mm², p = 0.05), whereas no differences between inner and outer leukocyte density were observed in smokers with normal lung function (1,652 and 1,265 to 2,026 versus 2,149 and 1,114 to 2,452 cells/mm²) nor in nonsmokers (1,003 and 443 to 1,830 versus 1,254 and 506 to 2,019 cells/mm²).

Correlations

When all the subjects were considered together, the number of goblet cells showed a negative correlation with the values of FEV₁/FVC (%) (r = 0.61, p = 0.002). This correlation did not remain significant when nonsmokers were excluded from analysis. When all the subjects were considered together, the inner leukocyte density showed a negative correlation with the values of FEV₁ (percentage of predicted) (r = 0.49; p = 0.02). This correlation did not remain significant when nonsmokers were excluded from analysis. No other significant correlations were observed between cellular counts and functional data, nor between goblet cells and inflammatory cells.

No significant correlations were observed between smoking history and cellular counts, nor between wall and epithelial inflammation.

	DISCUSSION

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In this study we have shown that smokers with both symptoms of chronic bronchitis and chronic airflow limitation have an increased number of goblet cells and inflammatory cells in the epithelium of peripheral airways.

The majority of studies examining peripheral airways in smokers were focused in the subepithelial portion of the airway wall (2) and reported the presence of an important inflammatory process in this region. Our results extend these observations by showing that an infiltration of inflammatory cells is present in the epithelium as well.

In the normal lung, goblet cells are found regularly in central airways but rarely in peripheral airways (21). Although a precise quantification has not been attempted, some researchers reported the appearance of goblet cells in peripheral airways of smokers (2, 13) whereas other investigators did not confirm this observation (7, 22, 23). Our study, by using quantitative methods, demonstrated the presence of goblet cell hyperplasia in smokers with both symptoms of chronic bronchitis and chronic airflow limitation. The fact that these subjects had an increased number of goblet cells and inflammatory cells when compared with nonsmokers but not when compared with smokers with normal lung function, suggests that the major determinant factor for epithelial inflammation and goblet cells hyperplasia is smoking itself, and not airway obstruction. However, when we examined the relationship between number of goblet cells (or inflammatory cells) and smoking history, we failed to find significant correlations. A possible explanation for this lack of correlation is the narrow range of smoking history in our population of smokers. They all were heavy smokers and they all started smoking at a very young age.

The potential interaction between goblet cells and inflammatory cells, which are both increased in number in the epithelium of smokers, remains speculative. In the present study, when all smokers were grouped together, they showed an increased number of neutrophils in the epithelium of peripheral airways. Because neutrophil elastase is a remarkably potent secretagogue (24), it is possible that the location of neutrophils within the epithelium is crucial for activation of the secretory function of goblet cells in smokers. This hypothesis is supported by the fact that, in a previous study (25), a prominent neutrophilia was observed even in the bronchial glands, which are the site responsible for mucus hypersecretion in the central airways of smokers.

Our findings of an increased epithelial infiltration of CD8⁺ cells in smokers with both symptoms of chronic bronchitis and chronic airflow limitation extend previous results obtained in the subepithelium of peripheral airways (9), as well as in the epithelium (26) and subepithelium (27) of central airways, suggesting a consistent inflammatory process along the entire tracheobronchial tree of these subjects. Along with the increase in CD8⁺ cells, there was an increase in the number of macrophages, in agreement with the results of Grashoff and colleagues (12). These findings may appear to be in contrast with those of Bosken and colleagues (11), who found no increase in epithelial inflammatory cells in subjects with COPD. The authors, however, did not include nonsmoking control subjects in their populations, therefore making the comparison with the present report difficult.

The fact that, in our study, an epithelial infiltration of macrophages and CD45⁺ cells was present even in smokers with normal lung function is consistent with the results of Niewoehner and colleagues (4), who showed that a marked inflammatory process is already present in the peripheral airways of young smokers who experienced sudden death outside the hospital. Taken together, these findings suggest that the inflammation of peripheral airways may represent an early event in smokers.

Because the internal perimeter has been shown to remain constant despite changes in smooth muscle tone and lung volume (28), we used the internal perimeter as a marker of airway size, and we related the epithelial cell counts to this parameter. In our study, the internal perimeters of peripheral airways were similar in smokers with both symptoms of chronic bronchitis and chronic airflow limitation, smokers with normal lung function, and nonsmokers. This indicates that, despite the possible different lung volumes caused by tissue preparation and the possible different smooth muscle tone caused by bronchoconstriction in the three groups of subjects examined, we were comparing bronchioles of similar size.

Although our study was focused on the epithelium, for CD45⁺ cells we extended the analysis to evaluate the distribution of total leukocytes within the entire wall of peripheral airways (20). We observed that smokers with symptoms of chronic bronchitis and chronic airflow limitation had a greater density of inflammatory cells in the "inner" submucosal region compared with the "outer" adventitial region. This regional difference in inflammatory cell density was not observed in smokers with normal lung function nor in nonsmoking subjects, suggesting that this "inner" versus "outer" pattern is not part of a nonspecific inflammatory response, but may rather be related to the disease. Interestingly, Haley and coworkers (20) have recently examined the distribution of inflammatory cells in the peripheral airways of asthmatic subjects. At variance with our study, these investigators found a greater density of total leukocytes in the "outer" compared with the "inner" airway wall region, suggesting different mechanisms for airway narrowing in asthma and COPD. In asthma the increased cellular density in the adventitia would promote airway constriction by decreasing the tethering effects of the parenchyma, whereas in COPD the increased cellular density in the submucosa would promote airway constriction by amplifying the effect of airway smooth muscle shortening on the caliber of the airways (29). The correlation observed in the present study between submucosal cellular density and reduced expiratory flow supports this hypothesis.

In conclusion, smokers with both symptoms of chronic bronchitis and chronic airflow limitation have an increased number of goblet cells and inflammatory cells in the epithelium of peripheral airways. In addition, they have an increased cellular density in the submucosal as compared with the adventitial region. The functional role of these cells located in the epithelium and in the submucosa still remains to be investigated.