Previous studies have shown an increased number of inflammatory cells and, in particular, CD8 + ve cells in the airways of smokers with chronic obstructive pulmonary disease (COPD). In this study we investigated whether a similar inflammatory process is also present in the lungs, and particularly in lung parenchyma and pulmonary arteries. We examined surgical specimens from three groups of subjects undergoing lung resection for localized pulmonary lesions: nonsmokers (n = 8), asymptomatic smokers with normal lung function (n = 6), and smokers with COPD (n = 10). Alveolar walls and pulmonary arteries were examined with immunohistochemical methods to identify neutrophils, eosinophils, mast cells, macrophages, and CD4 + ve and CD8 + ve cells. Smokers with COPD had an increased number of CD8 + ve cells in both lung parenchyma (p < 0.05) and pulmonary arteries (p < 0.001) as compared with nonsmokers. CD8 + ve cells were also increased in pulmonary arteries of smokers with COPD as compared with smokers with normal lung function (p < 0.01). Other inflammatory cells were no different among the three groups. The number of CD8 + ve cells in both lung parenchyma and pulmonary arteries was significantly correlated with the degree of airflow limitation in smokers. These results show that an inflammatory process similar to that present in the conducting airways is also present in lung parenchyma and pulmonary arteries of smokers with COPD.
Previous reports have shown that smokers with chronic obstructive pulmonary disease (COPD) have an increased number of inflammatory cells and, in particular, CD8+ve cells in central (1) and peripheral (2) airways. Whether, in these patients, a similar inflammatory infiltrate is also present in lung parenchyma and pulmonary arteries still remains to be investigated.
To the best of our knowledge, there are only a few morphologic studies examining the inflammatory process in the lung parenchyma of smokers (3, 4), despite the fact that an inflammation in the alveolar spaces has been shown by several studies performed on bronchoalveolar lavage (5-7). Indeed, a localization of inflammatory cells within the alveolar walls might contribute to smoking-induced parenchymal destruction and the subsequent chronic airflow limitation (3). Morphologic studies examining the inflammatory process in pulmonary arteries are also lacking, even though involvement of these vessels can be hypothesized because of the anatomic contiguity of airways and pulmonary arteries and the possible spreading of the inflammatory process from the bronchiolar wall (2, 8-15) to the adjacent vessel. Indeed, inflammatory cells might cause structural changes in pulmonary arteries of smokers (16-18) and contribute to the development of pulmonary hypertension present in severe COPD (19, 20).
In this study, we investigated whether an inflammatory process similar to that present in the airways is also present in lung parenchyma and pulmonary arteries of smokers with COPD. We obtained surgical specimens from three groups of subjects undergoing lung resection for localized pulmonary lesions: nonsmokers (n = 8), asymptomatic smokers with normal lung function (n = 6), and smokers with COPD (n = 10). Lung parenchyma and pulmonary arteries were examined with immunohistochemical methods to identify neutrophils, eosinophils, mast cells, macrophages, and CD4+ve and CD8+ve cells.
We recruited to the study three groups of subjects: 10 smokers with COPD (smokers with symptoms of chronic bronchitis and fixed airway obstruction), six asymptomatic smokers with normal lung function, and eight asymptomatic nonsmoking subjects with normal lung function. All the subjects underwent lung resection for a solitary peripheral carcinoma. 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 (21). 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. Subjects with COPD had had no exacerbations, defined as increased dyspnea associated with a change in the quality and quantity of sputum that led the subject to seek medical attention (22), 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 from each subject undergoing surgery. Each patient underwent interview, chest radiography, ECG, routine blood tests, skin tests with common allergen extracts, and pulmonary function tests in the week before surgery.
Pulmonary function tests were performed within the week before surgery as previously described (22). Briefly, they included measurements of blood gas analysis, FEV1, FVC, and forced midexpiratory flow (FEF25–75) in all the subjects examined, whereas residual volume (RV) and TLC were performed only in 13 subjects. The predicted normal values used were those from Communité Europeenne du Carbon e de l'Acier (CECA) (23). In order 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.
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 serial sections 5 μm thick were cut for immunohistochemical analysis of lung parenchyma and pulmonary arteries.
Mouse monoclonal antibodies were used for identification of neutrophils (anti-elastase, M752; Dako Ltd., High Wycombe, UK), eosinophils (anti-EG-2; Pharmacia Diagnostics, Fairfield, NJ), mast cells (antitryptase, M7052; Dako), macrophages (anti-CD68, M814; Dako), CD4+ve cells (anti-CD4, M834; Dako) and CD8+ve cells (anti-CD8, M7103; Dako). Monoclonal antibody binding was detected with the alkaline phosphatase, antialkaline 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, eosinophils, and mast cells 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+ve cells, immersed in citrate buffer 5 mM at pH 6.0, were incubated in a microwave oven (Philips M704 Eindhoven, The Netherlands) on high 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 analysis of lung parenchyma and pulmonary arteries was performed by one single observer using a light microscope (Leica DMLB; Leica, Cambridge, UK) connected to a video recorder linked to a computerized image system (Software: Casti Imaging SC processing, Italy). The cases were coded and the measurements made without knowledge of clinical data.
For the cell counts in the lung parenchyma, we examined only the alveolar walls with a single layer of cells to avoid bias caused by technical artifacts such as adjacent alveolar walls. The number of inflammatory cells within the alveolar walls was computed as previously described (3). Briefly, at a magnification ×630, we measured the length of the alveolar walls, and the number of positive cells within these alveolar walls was counted. Ten fields randomly distributed across the slide were studied per subject, and the result was expressed as the number of positive cells/mm of alveolar wall. We decided to examine 10 fields per subject since this number of fields was sufficient to obtain a mean value per subject that remained rather constant after further increasing the number of fields examined.
In pulmonary arteries we performed both morphometric and immunohistochemical analysis. Fifteen muscular pulmonary arteries with a perimeter less than 1,500 μm (corresponding to a diameter of about 500 μm), and a double elastic lamina visible for at least half the circumference (24) were selected for each patient. To avoid measurements in tangentially cut vessels, muscular pulmonary arteries with a short/long diameter ratio less than one-third were excluded from the study.
Morphometric measurements were performed on sections stained with elastic van Gieson stain as previously described (24). Briefly, the arterial perimeter was measured along the internal elastic lamina while the intimal, medial, and adventitial thicknesses were computed in a line perpendicular to the long axis of each artery, as follows. The intimal thickness was measured as the distance between the inner edge of the internal elastic lamina and the vessel lumen, the medial thickness was measured as the distance between the inner edge of the external elastic lamina and the outer edge of the internal elastic lamina, and the adventitial thickness was measured as the distance between the external edge of the external elastic lamina and the external edge of the adventitia. Results were expressed as the average thickness of intima, media, and adventitia on opposite sides of each artery relative to arterial perimeter (%).
Immunohistochemical analysis of inflammatory cells was performed in the adventitia since most of the cells were located in this layer of the arterial wall, and the final result was expressed in two ways: (1) as the number of positive cells per mm2 of the entire adventitia, and (2) as percent of arteries infiltrated by positive cells over the total number of arteries examined.
To analyze the relative distribution of pulmonary arteries as opposed to airways, pulmonary arteries close (n = 4 ± 2) and distant (n = 11 ± 2) to bronchioles were counted separately. In arteries adjacent to bronchioles the external limit of the adventitia was extrapolated by visually connecting the two opposite adventitial edges with a straight line.
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 the Kruskall-Wallis test when appropriate. Spearman's rank correlation coefficient test was used to examine the association between histologic parameters and clinical data. Wilcoxon's signed-rank test was used to compare cell infiltration in pulmonary arteries distant and close to bronchioles. 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. The intraobserver coefficient of variation for measurements ranged from 6 to 14% for the inflammatory cells examined, and from 6 to 10% for morphometric parameters. For the cell counts in the lung parenchyma, the field-to-field variation ranged between 0 and 2 cells/ mm of alveolar wall in the subject with the lowest variability, and between 0 and 22 cells/mm of alveolar wall in the subject with the highest variability.
The characteristics of the subjects studied are shown in Table 1. The three groups of subjects were similar with regard to age, PaO2 , PaCO2 , and aaPo 2 values. There was no significant difference in the smoking history (packs/year and smoking starting age) 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 (% predicted), FEV1/FVC ratio (%), and FEF25–75 (% predicted) than did smokers with normal lung function (p < 0.01) and nonsmokers (p < 0.01). In smokers with COPD, whose FEV1 ranged from 54 to 79% predicted, the average response to bronchodilator was 5%. The average value of RV (% predicted) was higher in smokers with COPD than in the other two groups of subjects; however, since the measurements of residual volume were performed in only 13 subjects, the statistical analysis could not be applied for this parameter.
|Nonsmokers||Smokers with Normal Lung Function||Smokers with COPD|
|Age, yr||68 ± 3||68 ± 3||69 ± 2|
|Smoking starting age, yr||—||20 ± 2||15 ± 1|
|Smoking history, packs/years||—||45 ± 7||57 ± 8|
|FEV1, % pred||104 ± 7||102 ± 4||68 ± 3†|
|FEV1/FVC, %||79 ± 2||78 ± 3||64 ± 2†|
|FEF25–75, % pred||122 ± 15||90 ± 10||43 ± 6†|
|TLC, % pred‡||96 ± 6||100 ± 13||99 ± 5|
|RV, % pred‡||96 ± 7||93 ± 19||133 ± 17|
|PaO2 , mm Hg||83 ± 3||89 ± 4||82 ± 2|
|PaCO2 , mm Hg||39 ± 1||37 ± 2||41 ± 1|
|aaPo 2, mm Hg||16 ± 4||14 ± 4||17 ± 2|
Lung parenchyma. The results of the cell counts in the lung parenchyma are shown in Figures 1 and 2. The average length of alveolar walls examined per patient was 5.4 ± 0.3 mm in smokers with COPD, 6.4 ± 0.6 mm in smokers with normal lung function, and 5.7 ± 0.4 mm in nonsmokers. The number of CD8+ve cells per mm of alveolar wall was significantly increased in smokers with COPD (Figure 3) when compared with nonsmokers (p < 0.05), but it was not significantly increased when compared with smokers with normal lung function. Smokers with normal lung function had a number of CD8+ve cells per mm of alveolar wall not significantly different from that of nonsmokers (Figure 1). No significant differences among the three groups of subjects examined were observed in the number of neutrophils, eosinophils, mast cells, macrophages, and CD4+ve cells per mm of alveolar wall (Figures 1 and 2). When the ratio CD4:CD8 was computed, no significant differences were observed between smokers with COPD (median and interquartile range: 0.5 and 0.3–0.7), smokers with normal lung function (0.6 and 0.4–1.1), and nonsmokers (0.9 and 0.5–4.7), even though there was a trend toward a decreased ratio CD4/CD8 in smokers with COPD as compared with nonsmokers. On average, a predominance of CD8+ve over CD4+ve cells was observed in all three groups of subjects examined.
Pulmonary arteries. The results of the morphometric measurements in pulmonary arteries are illustrated in Table 2. The arterial perimeter was similar in the three groups of subjects examined, indicating that we were comparing arteries of similar size. The intimal, medial, and adventitial thicknesses were not significantly different in smokers with COPD, smokers with normal lung function, and nonsmokers.
|Nonsmokers||Smokers with Normal Lung Function||Smokers with COPD|
|Perimeter, μm||563 (474–668)||506 (450–566)||529 (404–587)|
|Intimal thickness, %||2.0 (1.7–2.5)||2.1 (1.6–2.4)||2.2 (2.0–2.7)|
|Medial thickness, %||4.3 (4.1–4.6)||4.5 (4.3–5.8)||5.2 (3.7–5.7)|
|Adventitial thickness, %||6.4 (6.1–7.1)||6.7 (6.3–8.4)||7.4 (6.2–9.1)|
The percentage of arteries infiltrated by positive cells over the total number of arteries examined is illustrated in Table 3. The percentage of arteries infiltrated by CD8+ve cells was increased in smokers with COPD as compared with both smokers with normal lung function and nonsmokers (p < 0.01), whereas the percentage of arteries infiltrated by neutrophils, eosinophils, mast cells, macrophages, and CD4+ve cells was not significantly different in the three groups of subjects examined.
|Nonsmokers||Smokers with Normal Lung Function||Smokers with COPD|
|Percentage of arteries infiltrated by:|
|CD4+ve cells||50 (29–73)||60 (53–73)||76 (60–86)|
|CD8+ve cells||11 (4–27)||33 (13–40)||56 (47–60)†|
|Macrophages||32 (14–53)||16 (9–20)||37 (20–53)|
|Neutrophils||8 (7–23)||7 (7–13)||7 (7–20)|
|Eosinophils||0 (0–10)||0 (0–13)||7 (0–13)|
|Mast cells||50 (25–54)||33 (27–40)||26 (0–67)|
The results of the cell counts in pulmonary arteries, expressed as number of positive cells per mm2 of adventitia, are illustrated in Figures 4 and 5. The number of CD8+ve cells was increased in smokers with COPD (Figure 6) as compared with both smokers with normal lung function (p < 0.01) and nonsmokers (p < 0.001). The number of CD8+ve cells was also increased in smokers with normal lung function as compared with nonsmokers (p < 0.05) (Figure 4). No significant differences among the three groups of subjects examined were observed in the number of neutrophils, eosinophils, mast cells, macrophages, and CD4+ve cells in the adventitia (Figures 4 and 5). The CD4:CD8 ratio was decreased in smokers with COPD (1.1 and 0.8–2.7) when compared with nonsmokers (8.6 and 3.3–35.9; p < 0.01), but it was not different when compared with smokers with normal lung function (2.1 and 1.4–6.0). On average, a predominance of CD4+ve over CD8+ve cells was observed in all three groups of subjects examined.
To verify the possible spreading of the inflammatory process from the bronchiolar wall to the adjacent vessel, the numbers of CD8+ve cells in pulmonary arteries distant and in those close to bronchioles were counted separately. In smokers with COPD there was a trend toward an increased number of CD8+ve cells in the pulmonary arteries close to bronchioles (250 and 207–288 cells/mm2) as compared with pulmonary arteries distant to bronchioles (95 and 84–166 cells/mm2; p = 0.06). No differences in CD8+ve cell counts were observed between arteries close and distant to bronchioles both in smokers with normal lung function (45 and 0–148 cells/mm2 versus 43 and 26–68 cells/mm2) and in nonsmokers (0 and 0–23 cells/mm2 versus 11 and 1–36 cells/mm2).
When all the subjects were considered together, the number of CD8+ve cells in both lung parenchyma (p < 0.01, r = −0.57) and pulmonary arteries (p < 0.001, r = −0.70) showed a negative correlation with the values of FEV1 (% predicted) (Figures 7 and 8). The percent of pulmonary arteries infiltrated by CD8+ve cells was also negatively correlated with the values of FEV1 (p < 0.001 and r = −0.74). All these correlations remained significant when nonsmokers were excluded from analysis (p < 0.05 and r = −0.59 for the number of CD8+ve cells in the lung parenchyma; p < 0.05 and r = −0.57 for the number of CD8+ve cells in pulmonary arteries, and p < 0.01, r = −0.69 for the percentage of pulmonary arteries infiltrated by CD8+ve cells).
This study has shown that smokers with COPD have an infiltration of CD8+ve cells in both lung parenchyma and pulmonary arteries.
Previous studies have shown that an important inflammatory process is present in central and peripheral airways of subjects with COPD (1, 2, 8-15) and have demonstrated that it is mainly the CD8+ve cell subset that increases in number in these patients (1, 2). Our results confirm and extend these observations by showing that lung parenchyma and pulmonary arteries are also involved by an inflammatory process with similar cellular characteristics.
Eidelman and coworkers (3) demonstrated that the number of inflammatory cells localized within the alveolar walls is increased in smokers and is correlated with parenchymal destruction and airflow limitation. However, a clear distinction between different types of inflammatory cells was not assessed. The present report, by using immunohistochemical methods, extends these observations by showing that it is the CD8+ve cell subset that increases in number in smokers with COPD. It is possible that CD8+ve cells, because of their localization within the alveolar walls, may contribute to parenchymal destruction and therefore to the development of chronic airflow limitation in these subjects. The significant correlation observed in our population of smokers between increased number of CD8+ve cells in the alveolar walls and reduced expiratory airflow supports this hypothesis.
It is noteworthy that we did not observe an increased number of neutrophils in the alveolar walls of smokers with COPD. This finding may appear to be counter to reports using bronchoalveolar lavage (6), showing neutrophilia in smokers with chronic airflow limitation. A possible explanation for the discrepancy between lavage and parenchymal tissue is the rapid migration of neutrophils through the alveolar tissue to the alveolar spaces, so that the effect of accumulation of these cells is undetectable by tissue analysis but quite detectable by lavage analysis. On the other hand, it is possible that lavage findings reflect the pathology of other compartments of the bronchial tree such as submucosal glands, where a prominent neutrophilia has been demonstrated (25).
The lack of a significant increase in macrophages in our subjects with COPD is somehow surprising. This observation contrasts with the findings of Finkelstein and coworkers (4), who demonstrated an increased number of macrophages in the lung parenchyma of smokers. A possible explanation for this discrepancy is that, at variance with our study, these investigators included in their counts not only the alveolar walls but also the alveolar spaces, where the macrophages could be preferentially located.
An increased infiltration of CD8+ve cells was present in pulmonary arteries both when the results were expressed as number of CD8+ve cells per mm2 of adventitia and as percent of arteries infiltrated by CD8+ve cells over the total number of arteries examined, indicating that not only the severity but also the extent of vascular inflammation was increased in subjects with COPD.
The finding that CD8+ve cells are increased in both peripheral airways (2) and pulmonary arteries of subjects with COPD suggests that the inflammation present in the bronchiolar wall of these subjects may spread over the adjacent vessel (24, 26). This hypothesis is supported by the findings that: (1) the inflammatory infiltrate was localized in the adventitia, i.e., the layer of the arterial wall adjacent to bronchioles, and (2) when pulmonary arteries both distant and close to bronchioles were examined separately, there was a trend toward an increased number of CD8+ve cells in the pulmonary arteries adjacent to bronchioles. However, since an inflammatory cell infiltrate is also present in arteries far from the airways, it can be hypothesized that these inflammatory cells could have originated in the bronchial circulation, which supplies the adventitia of pulmonary arteries both close and distant to the airways.
In our population of smokers, the vascular infiltration of CD8+ve cells was correlated with the degree of airflow limitation. Because reduced expiratory flow has been shown to correlate with infiltration of CD8+ve cells in the peripheral airways of smokers (2), it is possible that the correlations observed in the present study simply reflect the vessel involvement in the inflammatory process present in the airways of these subjects as the disease progresses.
The lack of structural changes in our smokers with COPD may appear to be in contrast with the results of Peinado and coworkers (17), who reported a thicker intima in smokers with COPD than in control subjects. There are, however, several differences between our study and that of Peinado and coworkers, which included both differences in the characteristics of the subjects and differences in the methods used to assess the intimal thickening. At variance with our study, their subjects with COPD had a lower PaO2 than their control subjects, and this difference in blood gases may have contributed to the intimal thickening in their study. Furthermore, their patients were selected on the basis of airway obstruction, regardless of the presence of symptoms of chronic bronchitis. Finally, they measured the area occupied by the intimal layer, whereas we measured the linear thickness of the intimal layer. Because areas and linear measurements are differently affected by cutting orientation, this may have contributed to the different findings of the two studies.
Our observation of an increased infiltration of CD8+ve cells in pulmonary arteries of smokers when the intimal and medial layers are not thickened, suggests that the inflammatory process may represent an early event in these subjects. The fact that, in our study, an infiltration of CD8+ve cells was present even in smokers with normal lung function supports this hypothesis and suggests a pivotal role for tobacco smoking in vascular inflammation.
There is a recent interesting observation by O'Shaughnessy and coworkers (1) of relevance to our present report. These investigators demonstrated an increased number of CD8+ve cells in bronchial biopsies obtained from subjects with COPD, suggesting an infiltration of these cells not only in the periphery of the lung but also in the central airways.
We believe that CD8+ve cells are likely to represent T-lymphocytes, although a precise confirmation of this point will require double immunostaining of lung tissue. Traditionally, the major activity of CD8+ve T-lymphocytes has been considered the rapid resolution of acute viral infections (27, 28), viral infections being a frequent occurrence in patients with COPD. It is possible that an excessive recruitment of CD8+ve T-lymphocytes may occur in response to repeated viral infections in some smokers, and that this excessive response may damage the lungs in these subjects. In the present study we took great care in selecting patients clinically free of respiratory infections in the month before surgery. However, a latent viral infection cannot be excluded in patients with COPD (29), and thus we cannot completely rule out the possibility of an underlying respiratory infection.
In conclusion, smokers with COPD have an increased number of CD8+ve cells, not only in the conducting airways but also in lung parenchyma and pulmonary arteries. The functional role of these cells located within the alveolar walls and pulmonary arteries still remains to be investigated.
The writers thank Dr. G. Azzena and M. P. Ruggieri for their expert collaboration; A. Zanin, P. Bortolami, I. Adinolfi, and L. Zedda for their technical assistance; and C. A. Drace-Valentini and G. Fulgeri for editing the manuscript.
Supported by the Italian Ministry of University and Research; by the Regione Veneto, Giunta Regionale - Ricerca Sanitaria Finalizzata - Venice, Italy; by ENFUMOSA grant no. BMH4-CT96 1471; by European Commission BMH4-CT98-3951, and by a special Grant to A Ciaccia by Azienda Arcispedale S. Anna, Ferrara, Italy.
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