Abstract
To characterize the inflammatory process in the bronchial glands of smokers with chronic sputum production, we examined lobar bronchi from 18 subjects undergoing lung resection for localized pulmonary lesions, all with a history of cigarette smoking. Nine of the subjects had symptoms of chronic bronchitis and chronic airflow obstruction, and nine were asymptomatic, with normal lung function. The number of neutrophils, eosinophils, mast cells, macrophages, CD4+ and CD8+ T-lymphocytes, and the ratio of CD4+ to CD8+ cells were assessed in the bronchial glands, epithelium, and submucosa. Cells were identified through immunohistochemistry. Smokers with symptoms of chronic bronchitis had an increased number of neutrophils (p = 0.01) and macrophages (p = 0.03) and a decreased CD4+/CD8+ ratio (p = 0.01) in the bronchial glands as compared with asymptomatic smokers. Chronic bronchitic smokers also had an increased number of epithelial neutrophils (p = 0.04), whereas the numbers of macrophages and CD4+ and CD8+ T-lympohcytes in the epithelium and submucosa were similar in the two groups of smokers. No differences in numbers of eosinophils or mast cells were observed between bronchitic and asymptomatic smokers in any of the compartments examined. In conclusion, smokers with chronic sputum production have an increased infiltration of neutrophils and macrophages and an increased proportion of CD8+ T-lymphocytes in their bronchial glands, supporting the important role of bronchial-gland inflammation in the pathogenesis of chronic bronchitis.
Chronic bronchitis is a clinical syndrome defined by chronic sputum production (1). The major risk factor for the development of chronic bronchitis is cigarette smoking, but the precise pathogenetic mechanism of chronic sputum production is still unknown.
Several studies of bronchial biopsy specimens from subjects with chronic bronchitis have provided evidence for an inflammatory process, consisting predominantly of mononuclear cells (2-4), in the large airways of these subjects. These studies, however, have examined only the epithelial (2, 4) or submucosal (3, 4) compartments, whereas little information is available on the inflammatory process in these subjects' bronchial glands. Among all the studies performed on bronchial biopsies, only one (5) was able to investigate the bronchial-gland compartment, and demonstrated a mast-cell infiltration in subjects with chronic bronchitis. The investigators in that study, however, limited their analysis to mast cells, and did not attempt a complete characterization of the inflammatory cells infiltrating the bronchial glands. As compared with bronchial biopsy, which can sample only a small portion of the bronchial wall, specimens obtained at surgery allow analysis of the whole bronchial wall, and therefore a better examination of the bronchial-gland compartment. By investigating surgical specimens, Mullen and coworkers (6) demonstrated an inflammatory process in the bronchial glands of subjects with chronic bronchitis. Their report, however, was based on classic histologic examination, which does not allow the clear distinction between different inflammatory cells that would be better achieved with immunohistochemical methods. To the best of our knowledge, no information is available on the immunohistochemical characterization of the inflammatory cells infiltrating the bronchial glands in smokers with symptoms of chronic bronchitis.
To investigate whether the inflammatory process in the bronchial glands is different in smokers with and those without chronic bronchitis, we examined lobar bronchi from 18 subjects undergoing lung resection for localized pulmonary lesions, all with a history of cigarette smoking. Nine of the subjects had symptoms of chronic bronchitis and chronic airflow obstruction, and nine were asymptomatic, with normal lung function. Lobar bronchi were examined with immunohistochemical techniques to identify the number of neutrophils, eosinophils, mast cells, macrophages, CD4+ T-lymphocytes, and CD8+ T-lymphocytes in the bronchial glands. The same inflammatory cells were also quantified in the epithelium and submucosa.
The study population was composed of 18 subjects with a history of cigarette smoking, who were undergoing lung resection for a solitary peripheral carcinoma. Nine had symptoms of chronic bronchitis and fixed airway obstruction (chronic bronchitic group) and nine were asymptomatic, with normal FEV1 (control group). 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 (1). Fixed airway obstruction was defined as an FEV1 < 80% predicted, with a reversibility of less than 15% after inhalation of 200 μg of salbutamol. Chronic bronchitic subjects had no exacerbations, which were defined as increased dyspnea associated with a change in the quality and quantity of sputum that led the subject to seek medical attention (7) during the month preceding the study.
All subjects in 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. The subjects 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 with the Declaration of Helsinki, and informed written consent was obtained for each subject undergoing surgery. Each subject underwent an interview, chest radiography, electrocardiography, routine blood tests, skin tests with common allergen extracts, and pulmonary function tests in the week before surgery.
Pulmonary function tests performed as previously described (7). Briefly, they included measurements of FEV1 and FVC in all the subjects examined. The predicted normal values used were those from Communité Europeenne du Carbon e de l'Acier (CECA) (8). In subjects with a baseline FEV1 > 80% predicted, inhalation challenge with methacholine was performed according to the previously described protocol (9), and results were expressed as the provocative dose that elicited a 20% decline in FEV1 (PD20 FEV1) (mg methacholine). In order to assess the reversibility of airway obstruction in subjects with a baseline FEV1 < 80% predicted, the FEV1 measurement was repeated 15 min after the inhalation of 200 μg of salbutamol.
Bronchial rings were taken from the lobar or segmental bronchus of the lobe obtained at surgery, away from the tumor site. One satisfactory ring was selected for each subject, fixed in 4% formaldehyde and, after dehydration, embedded in paraffin; bronchial rings were then oriented, and 6-μm-thick serial sections were cut for immunohistochemical analysis. Mouse monoclonal antibodies (MAbs) were used for identification of neutrophils (anti-elastase; M792 Dako Ltd, High Wycombe, UK), eosinophils (EG-2; Pharmacia Diagnostics, Fairfield, NJ), mast cells (antitryptase, M7058; Dako), macrophages (anti-CD68, M814; Dako), CD4+ T-lymphocytes (anti-CD4, M834; Dako), and CD8+ T-lymphocytes (anti-CD8, M7103; Dako). MAb binding was detected with the alkaline phosphatase–antialkaline phosphatase method (APAAP kit system K670; Dako) and fast-red substrate. To identify macrophages, tissue sections were pretreated with 0.1% trypsin (Sigma Chemical, St. Louis, MO) in 45 mM calcium chloride at pH 7.8 at 37° C for 20 min. To identify CD8+ T-lymphocytes, sections of bronchial rings, immersed in citrate buffer 0.5 mM at pH 6.0, were heated in a microwave oven (Philips M704; 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 anticytocheratin MAb (M717; Dako) as a negative control.
Light-microscopic analysis of the submucosa, bronchial glands, intact epithelium, in the coded slides was performed with a Jenamed 30G0040 microscope (Aus, Jena, Germany). The bronchial glands included the entire gland area, comprising acini plus interstitium between acini. The image of each gland was projected onto a computer-linked graphic tablet, and its area was measured. Cell counts were performed in this area and expressed as number of cells per square millimeter. The intact epithelium was defined by the presence of both basal and columnar cells, with no appearance of metaplasia; the submucosa was defined as the zone 100 μm deep to the limit of the reticular basement membrane, as defined with an eyepiece graticule. The cells in the epithelium and submucosa were counted in adjacent nonoverlapping high-power fields (hpf) until all of the available area was covered. Cell counts were expressed as number of cells per millimeter length of intact epithelium for the epithelial infiltrate, and as number of cells per square millimeter for the submucosal infiltrate.
Reid's index, which measures bronchial gland size, was calculated by measuring the maximum thickness of each bronchial gland and dividing this by the bronchial-wall thickness as measured from basement membrane to inner perichondrium along a single axis (10).
Group data were expressed as means ± SE, or as medians and ranges when appropriate. Differences between groups were analyzed with the unpaired Student's t test for clinical data and the Mann–Whitney U-test for morphologic data. Correlation coefficients were calculated using Spearman's rank method. Values of p < 0.05 were accepted as significant. At least three replicate measurements of morphometric parameters were performed by the same observer, and the intraobserver reproducibility was assessed with the coefficient of variation (CV) for repeated measurements. The interobserver reproducibility was assessed with Spearman's rank correlation and Student's t test for paired data.
The characteristics of subjects with chronic bronchitis and controls 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). Six of the control subjects and five of the chronic bronchitic subjects were ex-smokers. PaO2 values (85 ± 3 mm Hg versus 87 ± 3 mm Hg) and PaCO2 values (40 ± 2 mm Hg versus 39 ± 2 mm Hg) were similar in subjects with chronic bronchitis and in controls. As expected from the criteria of selection, subjects with chronic bronchitis had a significantly lower value of FEV1 (% predicted) and FEV1/FVC ratio (%) than did controls. In subjects with chronic bronchitis, whose FEV1 ranged from 56 to 79% predicted, the average response to bronchodilator was 5% (range: 0 to 13%). In control subjects, whose FEV1 ranged from 86 to 115% predicted, the inhalation challenge with methacholine showed a bronchial reactivity within the normal range (PD20FEV1 > 1.4 mg methacholine).
| Subjects | Sex (M/F ) | Age (yr) | Smoking History (packs/yr) | Smoking Starting Age (yr) | FEV1(% pred ) | FEV1/FVC (%) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Bronchitic subjects | ||||||||||||
| 1 | M | 55 | 61 | 20 | 71 | 76 | ||||||
| 2 | M | 70 | 49 | 16 | 56 | 61 | ||||||
| 3 | M | 66 | 78 | 14 | 68 | 67 | ||||||
| 4 | M | 68 | 43 | 10 | 62 | 72 | ||||||
| 5 | M | 63 | 16 | 13 | 78 | 75 | ||||||
| 6 | M | 84 | 61 | 17 | 66 | 69 | ||||||
| 7 | M | 68 | 50 | 18 | 73 | 64 | ||||||
| 8 | M | 63 | 45 | 18 | 79 | 69 | ||||||
| 9 | M | 72 | 100 | 13 | 57 | 54 | ||||||
| Means | 68 | 56 | 15 | 68* | 67* | |||||||
| SEM | 3 | 8 | 1 | 3 | 2 | |||||||
| Controls | ||||||||||||
| 10 | M | 65 | 48 | 18 | 86 | 76 | ||||||
| 11 | M | 70 | 54 | 16 | 86 | 82 | ||||||
| 12 | M | 72 | 65 | 20 | 108 | 72 | ||||||
| 13 | M | 81 | 60 | 20 | 116 | 91 | ||||||
| 14 | M | 69 | 40 | 15 | 86 | 73 | ||||||
| 15 | M | 66 | 45 | 16 | 108 | 76 | ||||||
| 16 | M | 58 | 16 | 27 | 94 | 87 | ||||||
| 17 | M | 56 | 29 | 15 | 101 | 75 | ||||||
| 18 | M | 68 | 35 | 18 | 102 | 70 | ||||||
| Means | 67 | 44 | 18 | 99 | 78 | |||||||
| SEM | 2 | 5 | 1 | 4 | 2 |
Quantification of inflammatory cells was satisfactory in all the subjects except Subjects 1 and 4 (Table 1), in whom quantification of CD8+ T-lymphocytes could not be performed because of unsatisfactory CD8 immunoreactivity. In Subject 7, quantification of the inflammatory cells in the epithelium could not be performed because of complete epithelial denudation.
The mean CV for three repeated measurements by the same observer ranged from 8 to 14% for the cells studied, and the interobserver correlation coefficient varied from 0.89 to 0.96. Furthermore, no significant differences were found between observers with Student's t test for paired data.
In the bronchial glands, the number of neutrophils and macrophages was increased in subjects with chronic bronchitis as compared with controls (p = 0.01 and p = 0.03, respectively) (Figures 1 and 2). Although the absolute numbers of CD4+ T-lymphocytes and CD8+ T-lymphocytes were not significantly different in the two groups of subjects, the CD4+/ CD8+ ratio was decreased in subjects with chronic bronchitis as compared with controls (p = 0.01) (Figure 1). No significant differences were observed in the number of eosinophils and mast cells in bronchitic and control subjects (Table 2). Reid's index was not significantly different in the two groups of subjects examined (37 ± 4% in controls and 48 ± 4% in chronic bronchitic subjects).
Fig. 1. Individual counts for neutrophils, CD4+ T-lymphocytes, CD8+ T-lymphocytes, and macrophages, and the CD4+/CD8+ ratio in the bronchial glands of chronic bronchitic and control subjects. Horizontal bars represent median values.
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Fig. 2. Microphotograph showing neutrophil infiltration in the bronchial glands of a subject with chronic bronchitis (A) and of a control subject (B). Arrows indicate neutrophils. Immunostaining was done with antihuman neutrophil elastase.
[More] [Minimize]| Controls | Bronchitic Subjects | p Value | ||||
|---|---|---|---|---|---|---|
| Glands | ||||||
| Eosinophils/mm2 | 2 (0–11) | 2 (0–27) | n.s. | |||
| Mast cells/mm2 | 15 (1–87) | 18 (10–42) | n.s. | |||
| Epithelium | ||||||
| Eosinophils/mm | 0.28 (0–1.88) | 1.03 (0–6.84) | n.s. | |||
| Mast cells/mm | 0.59 (0–7.44) | 0.36 (0.16–13.34) | n.s. | |||
| Submucosa | ||||||
| Eosinophils/mm2 | 5 (1–98) | 30 (2–166) | n.s. | |||
| Mast cells/mm2 | 104 (4–189) | 70 (18–490) | n.s. |
In the epithelium, the number of neutrophils was increased in subjects with chronic bronchitis as compared with controls (p = 0.04), whereas the number of eosinophils, mast cells, macrophages, CD4+ T-lymphocytes, and CD8+ T-lymphocytes, and the CD4+/CD8+ ratio were similar in the two groups of subjects (Table 2 and Figure 3).
Fig. 3. Individual counts for neutrophils, CD4+ T-lymphocytes, CD8+ T-lymphocytes, and macrophages, and the CD4+/CD8+ ratio in bronchial epithelium of chronic bronchitic and control subjects. Horizontal bars represent median values.
[More] [Minimize]In the submucosa, no significant differences were observed in the number of neutrophils, eosinophils, mast cells, macrophages, CD4+ T-lymphocytes, CD8+ T-lymphocytes, or in the CD4+/CD8+ ratio between subjects with chronic bronchitis and controls (Table 2 and Figure 4).
Fig. 4. Individual counts for neutrophils, CD4+ T-lymphocytes, CD8+ T-lymphocytes, and macrophages, and the CD4+/CD8+ ratio in the submucosa of chronic bronchitic and control subjects. Horizontal bars represent median values.
[More] [Minimize]When the population was divided according to smoking history, no significant differences between current smokers and ex-smokers were found for any of the cells examined.
When all the subjects were considered together, the number of neutrophils in the bronchial glands were negatively correlated with the values of FEV1 (p = 0.05, ρ = −0.46), and the CD4+/CD8+ ratio in the bronchial glands was positively correlated with the values of FEV1 (p = 0.018, ρ = +0.61) and FEV1/FVC (p = 0.012, ρ = +0.65).
This study shows that smokers with symptoms of chronic bronchitis have an increased number of inflammatory cells in their bronchial glands when compared with asymptomatic smokers. This inflammatory process consists predominantly of neutrophils and macrophages, and of an increased proportion of CD8+ T-lymphocytes.
Previous studies of the inflammatory changes in the large airways of subjects with chronic bronchitis have shown a predominance of mononuclear cells in the airway wall (2-4, 11) and a predominance of neutrophils in the airway lumen (12– 14). This discrepancy has led to the hypothesis that the inflammation in the lumen may differ from that in the bronchial wall in patients with chronic bronchitis. However, the majority of the studies examining the bronchial wall in these subjects have been focused on the bronchial submucosa (3, 4, 11), and have not analyzed the bronchial-gland compartment. The present study, by showing an increased number of neutrophils in the bronchial glands of subjects with chronic bronchitis, provides evidence for a neutrophilia not only in the airway lumen but also in the airway wall of these subjects, contributing to a better understanding of the apparent discrepancy between luminal and parenchymal findings in this disease.
Since neutrophil elastase is a remarkably potent secretagogue for cultured airway submucosal glands (19), it is possible that the location of neutrophils within the bronchial glands is crucial for activation of the secretory function of gland cells, and therefore for induction of the chronic sputum production in subjects with chronic bronchitis. In our study, neutrophils were increased not only in the bronchial glands but also in the bronchial epithelium of subjects with chronic bronchitis, in agreement with previous observations (15). Such findings suggest a possible recruitment of these cells by epithelium-derived factors such as interleukin-8 (IL-8) (16), a potent neutrophil chemoattractant, which has been found to be increased in the sputum (17) and bronchial washings (18) of subjects with chronic bronchitis.
Along with the increase in neutrophil numbers, there was an increase in the number of macrophages and in the proportion of CD8+ T-lymphocytes in the bronchial glands of subjects with chronic bronchitis. The increased number of macrophages confirms and extends previous observations (3, 4, 11) by showing that the inflammatory process in the large airways, consisting predominantly of mononuclear cells, involves not only the submucosa but also the bronchial glands. The increased proportion of CD8+ T-lymphocytes, which was significantly correlated with the functional parameters of airway obstruction, supports the recent observations of a relative prevalence of CD8+ over CD4+ T-lymphocytes in both bronchial biopsy (2, 20) and surgical specimens (21) from smokers with chronic bronchitis and chronic airflow obstruction.
The finding of a similar number of mast cells in the bronchial glands of bronchitic and control subjects may appear to be in contrast with the report of Pesci and coworkers (5), who found an increased number of mast cells in patients with chronic bronchitis. This apparent discrepancy may reflect the different populations examined in the two studies: nonsmoking controls versus chronic bronchitic subjects with and without airflow obstruction in the report of Pesci and coworkers (5), and smoking controls versus chronic bronchitic subjects with airflow obstruction in our study.
Bronchitic subjects in the present study were selected to be in stable condition, with their disease far from clinical exacerbation. The absence of eosinophilia in the bronchial glands of these subjects extends our previous observations (4, 7), by showing that a low number of eosinophils is present not only in the epithelium (4) and submucosa (4, 7), but also in the bronchial glands of chronic bronchitic subjects examined under baseline conditions.
Interestingly, Reid's index was similar in subjects with chronic bronchitis and in controls, supporting the observations of Nagai and coworkers (22), who found no correlations between sputum production and mucous-gland enlargement in smokers. These findings are in agreement with those of Mullen and coworkers (6), who demonstrated that in contrast to airway inflammation, Reid's index was unable to distinguish smokers with chronic bronchitis from those without chronic bronchitis, supporting the hypothesis that airway inflammation provides a better morphologic indication of chronic bronchitis than does hypertrophy of mucous glands.
Although the mechanisms underlying the relationship between inflammation, hypertrophy of mucous glands, and hypersecretion of mucus is still unknown, cigarette smoking appears to be the common etiologic factor. Since in the present study both subjects with chronic bronchitis and controls had similar exposure to cigarettes (including packs/year and smoking starting age) as well as similar mucous-gland size, the presence of gland inflammation and chronic mucus hypersecretion only in a subgroup of subjects suggests a different response to the same injury.
The role of chronic mucus hypersecretion in the development of chronic airflow obstruction is still controversial. Although the two conditions may coexist in the same subject, several reports have suggested that chronic bronchitis and chronic airflow obstruction should be considered as separate entities rather than as manifestations of a single disease (23, 24). However, a recent study of a large random population sample (25) showed that chronic mucus hypersecretion was significantly associated with both an excessive decline of FEV1 and an increased risk of subsequent hospitalization because of chronic obstructive pulmonary disease (COPD), supporting the concept of a causal role of chronic mucus hypersecretion in the development of chronic airflow obstruction. In the present study, the chronic bronchitic subjects were selected for both chronic mucus hypersecretion and chronic airflow obstruction, and therefore represent a subset of all chronic bronchitic individuals. Whether bronchial-gland inflammation is also a characteristic feature of chronic bronchitis without airflow obstruction remains to be investigated.
All of the patients examined in the present study had smoked cigarettes at some point in their life, but six of the control subjects and five of the chronic bronchitic subjects had quit smoking prior to the study. No significant differences between current smokers and ex-smokers were found for any of the cells examined, in accord with previously reported findings (26, 27).
A confusing element in any study performed on specimens surgically resected from patients with lung cancer is that the cancer itself may influence the results. However, as compared with bronchial biopsies, which sample only a small portion of the bronchial wall, specimens obtained at surgery allow analysis of the entire bronchial wall, and therefore a better examination of the bronchial-gland compartment, in subjects with preoperatively measured pulmonary function. 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 bronchial-gland inflammation in subjects with chronic bronchitis are valid.
In conclusion, smokers with chronic sputum production have an increased infiltration of neutrophils and macrophages, and an increased proportion of CD8+ T-lymphocytes, in their bronchial glands, supporting the important role of bronchial-gland inflammation in the pathogenesis of chronic bronchitis.
The writers thank Drs. G. Cavalesco and G. Azzena for their expert collaboration; P. Bortolami, I. Adinolfi, and L. Zedda for their technical assistance; and M. Galliani and G. Fulgeri for revising and typing the manuscript.
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