INTRODUCTION

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Cigarette smoking is the major causative agent of chronic bronchitis (1). Chronic bronchitis is diagnosed clinically as persistent cough with the production of sputum on most days of the month for at least 3 mo/yr of 2 consecutive yr (2). Pathological evidence leaves no doubt that smokers with chronic bronchitis have an inflammatory process in the airway mucosa (3), characterized by an increased number of mononuclear cells and by an increased expression of mononuclear cell activation markers and adhesion molecules (5, 6). Neural mechanisms may be involved in the pathophysiology of chronic bronchitis, contributing to the symptoms (cough and sputum production) and, possibly, to the inflammatory response (7, 8). Inflammatory mediators may influence the release of neurotransmitters via activation of sensory nerves leading to reflex responses and via stimulation of prejunctional receptors influencing the release of neurotransmitters (9). In turn, neural mechanisms may modulate the inflammatory response, either reducing or aggravating it.

To investigate the effect of symptoms of chronic bronchitis on airway nerves, we performed a quantitative immunohistochemical analysis of surgical specimens (lobar and segmental bronchi) obtained both from smokers with chronic bronchitis and from smokers without symptoms of chronic bronchitis. These specimens were processed immediately upon sampling to avoid handling artefact and then analyzed to assess the number of inflammatory cells and the density of PGP 9.5-, VIP-, SP-, and CGRP-immunoreactive nerves.

	METHODS

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Subjects

We studied 19 patients undergoing thoracotomy for localized nonobstructing peripheral malignant lung lesions (Table 1). The study population was composed of two groups: 12 subjects with symptoms of chronic bronchitis (including eight current smokers and four exsmokers), and seven subjects without symptoms of chronic bronchitis (including two smokers and five exsmokers). Chronic bronchitis was diagnosed as cough and sputum production on most days of the month for at least 3 mo/yr during the previous 2 yr (2). Subjects selected for inclusion in the study did not have exacerbations, defined as increased dyspnea associated with a change in quality and quantity of sputum that had led the subject to seek medical attention, within the preceding month. No subjects had acute upper respiratory tract infections, or had taken glucocorticoids or antibiotics within the month preceding the study. They had no past history of asthma or allergic rhinitis. They were nonatopic (i.e., they had negative skin prick tests for common allergen extracts).

The study conformed to the Declaration of Helsinki, and informed written consent was obtained from each subject. Each patient underwent interview, chest radiography, ECG, routine blood tests, skin prick tests with common allergen extracts, and pulmonary function tests one week before surgery.

Pulmonary Function Tests

Pulmonary function tests (Biomedin Spirometer, Padova, Italy) included measurements of FEV₁ and FVC in all subjects examined. The predicted normal values used were those from the European Community for Coal and Steel (CECA) (10). The majority of the subjects had normal FEV₁ (FEV₁ greater than 80% predicted). In order to assess the reversibility of the airway obstruction in the three subjects with FEV₁ less than 80% predicted, the FEV₁ measurement was repeated 15 min after the inhalation of 0.2 mg salbutamol.

Sample Processing: Histology

We obtained lung tissue from patients who underwent surgical operation for limited lung lesions. After excision, rings of bronchi (lobar or segmental bronchus) were taken at least 2 cm from the tumor and were then fixed in 4% formaldehyde. After dehydration, they were embedded in paraffin. Tissue specimens were oriented and 6-µm-thick serial sections were cut for histological analysis.

Light microscopic analysis was performed on the coded slides with a Jenamed 30G0040 microscope at magnification of ×190 using a ×10 objective. Mononuclear cells and neutrophils were quantified in the wall of central airways as already reported (11) using a modification of the method described by Cosio and colleagues (12). A score was assigned to each microscopic field; the scores of all the fields (8 to 18) were summed and expressed as percentage of the maximum possible score for each bronchus.

The Reid Index

The ratio between bronchial gland thickness to bronchial wall thickness is referred to as the Reid index of mucous gland size. We measured the maximum thickness of every gland and then measured the basement membrane to inner perichondrium distance on exactly the same line as suggested by Thurlbeck (13). We used lobar or segmental bronchi.

Sample Processing: Histochemistry

Rings of bronchi (lobar or segmental bronchus) were taken at least 2 cm from the tumor and were immediately immersed in a solution of ice-cold 1% paraformaldehyde in phosphate-buffered saline (PBS, 0.001 M phosphate buffer in 0.15 M NaCl; pH 7.4) for 4-6 h. Tissues were then immersed and rinsed in PBS containing 0.45 M sucrose and 0.01% sodium azide, for at least 12 h, and frozen.

Cryostat sections (10-µm thick) were taken up on poly-L-lysine- coated microscope slides and were left to dry for 1 h at room temperature. Sections were stained with antisera to neural antigens and neuropeptides (Table 2) by the streptavidin-biotin complex peroxidases method and the peroxidase activity was revealed using the nickel enhancement method (14). One section per block was stained for each antigen. Briefly, endogenous peroxidase activity was blocked by immersing slides in 0.03% hydrogen peroxide in methanol for 1 h. After washing in PBS (3 × 10-min changes), nonspecific binding was blocked by incubating in 3% normal swine serum in PBS containing 0.05% bovine serum albumin (BSA) and 0.1% sodium azide (BSA solution) for 30 min. The sections were then incubated overnight at 4° C with primary antisera in BSA solution at predetermined optimal dilutions. Negative controls were performed by omission of the primary antibody. After washing in PBS (3 × 10-min changes), the sections were incubated for 30 min with biotinylated swine anti-rabbit IgG (1:200) and streptavidin-biotin-complex reagent for 60 min. The sections were rinsed in PBS (3 × 10-min changes) and then in acetate buffer (0.1 M, pH 6.0). Sections were dehydrated, mounted in Eukitt and examined with a light microscope (Zeiss, Germany).

Quantification

For each bronchus, we examined 8-10 fields of smooth muscle layer and of mucous glands. Images of appropriate fields were captured and digitized by an IBAS 2000 image analysis system (Kontron; Eching, Monaco, Germany) via a video camera attached to a microscope (Zeiss, Germany) at magnification of ×256. Digital images (average of 5) were 512 × 512 pixels (picture points), each pixel represented by a gray value from 0 (= black) to 255 (= white). Area was measured in the image by interactive delineation. The number of pixels contained between the boundaries, traced using the computer cursor, was converted to the tissue area using a factor determined by calibrating the image analyzer with a series of area standards viewed using the same microscope and objective used for the study. The image was segmented to separate nerves from non-nerve tissue by selecting a threshold gray value that was intermediate between the intensities of each. The image analyzer highlighted, in an artificial color, the pixels having a gray value smaller than that of the threshold. The operator then selected all the nerves and calculated their area. The data are expressed as the area density of nerves, i.e., area nerve/area tissue.

Statistical Analysis

Group data were expressed as mean ± SEM or as median and range for normally and nonnormally distributed data, respectively. Differences between the two groups were analyzed using analysis of variance for clinical data (normally distributed) and the Mann-Whitney U-test for morphological data (nonnormally distributed). Probability 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 for repeated measurements.

	RESULTS

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Clinical Findings

Though the two groups showed no significant differences with respect to age, gender, and smoking history (packs/yr), bronchitic subjects had significantly lower mean (± SEM) resting FEV₁% predicted (86.0 ± 3.5%) (p < 0.05) than nonbronchitic subjects (100.0 ± 3.8%) (as reported in Table 1). The three subjects with FEV₁ less than 80% of predicted showed no significant response to bronchodilator. All the subjects were nonatopic, i.e., they had negative skin prick tests to common allergen extracts. Exsmokers with symptoms of chronic bronchitis had stopped smoking on average 4 yr (range: 1 to 6 yr) before the study, whereas asymptomatic smokers had stopped 11 yr (range: 2 to 30 yr) prior to the study. Bronchitic subjects started to smoke earlier (average 12 yr, range: 8 to 18 yr) than nonbronchitic subjects (average 18, range: 14 to 27 yr).

Surgical Specimen Findings

Smokers with symptoms of chronic bronchitis had an increased number of mononuclear cells compared with smokers without symptoms of chronic bronchitis, but the number of neutrophils was similar in the two groups of subjects examined (Figure 1). The two groups had equivalent proportions of mucous glands, and Reid index in central airways was similar in the two groups (bronchitics: 0.36 ± 0.03; nonbronchitics: 0.31 ± 0.04).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Individual scores for mononuclear cell and neutrophil infiltration in bronchi of bronchitic (B) and nonbronchitic (NB) subjects. Horizontal bars represent median values.

Nerves staining positively for PGP 9.5 were identified in all the specimens from both subject groups, and were localized to epithelium, smooth muscle, glands, blood vessels, and neuroendocrine cells. There was no significant difference in the density of PGP 9.5-positive nerves both in smooth muscle and in glands (Figure 2) between the bronchitic and nonbronchitic subjects. A negative relationship was found between the presence of airway inflammation, as indexed by mononuclear tissue infiltration and the density of PGP 9.5-immunoreactive nerves both in smooth muscle (Rho = 0.62, p < 0.001) and in glands (Rho = 0.59, p < 0.01).

View larger version (103K): [in this window] [in a new window]   View larger version (97K): [in this window] [in a new window]   Figure 2.   Immunostaining of bronchial surgical specimens for PGP 9.5 (glands) in bronchitic (a) and nonbronchitic (b) subjects. PGP 9.5 immunoreactivity is localized around the acini (A) and within gland cells (arrows). Bar = 50 µm. Cryostat sections, peroxidase-labeled streptavidin-biotin complex, light microscopy.

Nerves staining positively for substance P were found in most of the specimens, being localized to smooth muscle and glands. In a few sections, SP-positive nerves were seen below the epithelium. Similarly, nerves staining positively for CGRP were confined to smooth muscle layer and glands of most of the specimens.

Nerve staining positively for VIP were likewise confined to smooth muscle layer and glands (Figure 3), while no staining could be localized to epithelium in any subject group.

View larger version (81K): [in this window] [in a new window]   View larger version (81K): [in this window] [in a new window]   Figure 3.   Immunostaining of bronchial surgical specimens for VIP (glands) in bronchitic (a) and nonbronchitic subjects (b). VIP immunoreactivity is localized around the acini (arrows). Bar = 50 µm. Cryostat sections, peroxidase-labeled streptavidin-biotin complex, light microscopy.

There was no significant difference in the density of SP- and CGRP-positive nerves at the level of smooth muscle between the bronchitic and nonbronchitic subjects. Although there was a trend toward an increased density of VIP-positive nerves at the location of smooth muscle in bronchitic subjects, the difference failed to reach significance.

There was no significant difference in the density of SP- and CGRP-positive nerves at the level of glands between the two groups, but the density of VIP-positive nerves was significantly increased (p < 0.05) in bronchitic as compared with nonbronchitic subjects (Figure 4). When bronchitics with airflow obstruction (FEV₁ < 80%; n = 3) were excluded from analysis, this significant increase was maintained (p < 0.05). A relationship was found between the smoking history (packs/yr) and the densities of CGRP- or VIP-immunoreactive nerves in the glands (respectively Rho = 0.57, p < 0.01 and 0.58, p < 0.05). A negative relationship was also found between the age of onset of smoking and the density of VIP-immunoreactive nerves in the glands (Rho = 0.57, p < 0.01).

View larger version (10K): [in this window] [in a new window]   Figure 4.   SP-, CGRP-, VIP-nerve density in bronchi (mucous glands) of bronchitic and nonbronchitic subjects.

No age-related change in the density of PGP 9.5-, SP-, CGRP-, or VIP-immunoreactive nerves was found in this study.

	DISCUSSION

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This investigation of bronchial surgical specimens found an increased density of mucous gland nerve immunostaining for VIP (mucous gland size remaining constant) in bronchitic as compared with nonbronchitic subjects. A relationship was found between smoking and density of VIP-positive nerves in the glands. We also found an inflammatory process, represented by mononuclear cell infiltration in the airway mucosa of smokers with chronic bronchitis, which was negatively correlated with the density of nerves immunostaining for PGP 9.5 both in smooth muscle and in glands. This last finding contrasts with a previous report (15) that indicates no relationship between airway inflammation and PGP 9.5- or VIP-immunoreactive nerves. However, important differences exist between the two studies: we studied bronchial surgical specimens of bronchitics, and airway inflammation was assessed as mononuclear cell infiltration; whereas in the other study, bronchial biopsies obtained from asthmatics were analyzed, and airway inflammation was assessed as eosinophil tissue infiltration. It is possible that the site of biopsy or the amount of tissue investigated could have led to an underestimate of nerve fiber presence.

Both age and cigarette smoking may diminish TK-immunoreactive staining and they may account for the deficiency of SP in lung tissue obtained from elderly smokers (16). Recently, it has been confirmed that the innervation of the airways changes with age (17). In fact, the relative number of peptide-containing nerves within the respiratory unit decreases from the child lung to the adult lung (17 to 24 yr). The subjects investigated in this study were current or exsmokers and ranged from 34 to 84 yr of age. No age-related change in the PGP 9.5-, SP-, CGRP-, or VIP-immunoreactive nerves was found. The lack of changes in SP-immunoreactive nerves in bronchi of symptomatic compared to asymptomatic smokers was unexpected since SP is one of the most potent stimulants of mucus secretion (18). However, this may be due to several factors. First, SP concentrations decrease with age and cigarette smoking (16, 17), and in our elderly smokers SP-immunoreactive nerves were in fact one-tenth as compared to VIP-immunoreactive nerves. Second, they may be localized to a lesser extent in smooth muscle and glands compared with other airway structures. Finally, we studied lobar or segmental bronchi and not peripheral airways. It is possible that SP may play a role in mucus secretion in peripheral airways of cigarette smokers. Globet cells are the only source of mucus in peripheral airways and SP is the most potent in stimulating globet cell secretion (18).

VIP-immunoreactive nerves were present in all the specimens from the subjects studied. We used a well characterized antibody that was specific for VIP. VIP is the most abundant peptide isolated from human lung (18). VIP-immunoreactive nerves prominent in large airways and virtually absent from bronchioles, are localized in the smooth muscle layer and in bronchial glands (19). VIP is an effective dilator of systemic and pulmonary vessels, a powerful stimulant of chloride ion transport, a potent stimulant of mucus secretion, and may have antiinflammatory action in airways (18). In isolated rat lung (20) and in isolated guinea pig lung (21), it has been shown that VIP has antioxidant activity, enabling it to protect against injury caused by xanthine, xanthine oxidase and paraquat.

Conflicting results have been reported on the presence of an anatomical deficiency of VIP-containing nerves and an excess of SP-immunoreactive nerves as features of asthma (22- 24). Although it seems unlikely that a primary abnormality in VIP innervation exists in the airways of asthmatics, it is possible that an increased degradation of this peptide may result from the inflammatory process in the airways of these patients. If the role of airway neuropeptides is far from being clear in asthma, it is largely unknown in chronic bronchitis. This study shows that VIP could play a role in the excess secretion of mucus present in chronic bronchitis. To the best of our knowledge, no other quantitative immunohistochemical studies of surgical bronchial specimens examining nerve density in bronchitics and nonbronchitics have so far been reported. The acini of submucosal glands are surrounded by a network of VIP-containing nerves. VIP, a potent stimulator of mucus secretion, stronger than isoproterenol, stimulates the production of a mucus secretion rich in glycoproteins (25). VIP is released from nerve terminals and produces its effects by activation of specific cell surface receptors. Binding of VIP to its receptor activates adenylate cyclase and VIP itself then stimulates cAMP formation. The action of VIP is therefore similar to that of beta-adrenoceptor agonists. VIP receptors are found in high density in pulmonary vascular smooth muscle, in airway smooth muscle of large airways, in airway epithelium and submucosal glands and in the alveolar walls (26). Mucus secretion at all airway sites is caused by parasympathetic nerves. However, VIP and nitric oxide may also be involved in neuroregulation, as they can cause or modulate secretion from submucosal glands (27). Some evidence argues against VIP as a stimulant of mucus secretion. VIP has an inhibitory effect on glycoprotein secretion from human tracheal explants (28). This finding is surprising because agonists that stimulate cyclic AMP formation would be expected to stimulate and not to inhibit secretion. However, the study shows the inhibitory effect of VIP only in normal human airway mucosa and not in explants from the airways of patients with chronic bronchitis, suggesting a significant difference in sensitivity to VIP inhibition between normal and bronchitic airway explants. It is possible that the reduction of VIP inhibition in the bronchitic airway could contribute to the mucus hypersecretion characteristic of this disease. It is also likely that the effects of VIP on mucus secretion are more complex and may depend on the drive to gland secretion (18). In other diseases in which secretion is important, such as cystic fibrosis (CF), it has been shown that patients with CF exhibited a marked deficiency of VIP-immunoreactive nerves in both acini and ducts of eccrine sweat glands (29). However, the study did not clarify whether the decreased VIP innervation was a primary abnormality or the result of an excessive depletion.

VIP positive nerves were increased in the glands of smokers with chronic bronchitis, but the size of the glands was similar among bronchitic and nonbronchitic subjects. Some authors found that mucous glands were significantly enlarged in chronic bronchitis (30), whereas others did not find any association between sputum production in chronic bronchitis and bronchial submucosal gland enlargement (31). In fact, an increased number and size of glandular elements may or may not be associated with increased secretion of mucus (32).

Epidemiological studies have shown that only half of heavy cigarette smokers develop mucus secretion, and 10 to 15% develop airflow obstruction (33). It is possible that the response of human lung to cigarette smoking is heterogenous and that the increase in sputum production may be mediated by a cigarette smoke-related inflammatory process (3). The majority of the published literature has dealt with the knowledge of the pathogenesis of chronic obstructive pulmonary disease (COPD), and more emphasis has been placed on the presence of airflow obstruction rather than the presence of symptoms. This choice has been made on the basis of numerous studies that did not show any association between chronic mucus hypesecretion and FEV₁ decline (34) or mortality in COPD (35), and leading to a diminution of interest in mucus hypersecretion in the natural history of COPD. However, a recent study of a large random population sample showed that chronic mucus hypersecretion was significantly associated with both an excess FEV₁ decline and an increased risk of subsequent hospitalization because of COPD (36). The authors suggested that chronic mucus hypersecretion should not be regarded as a marker of recurrent airway infection, but as a marker of airway inflammation, triggered by exogeneous stimuli, such as smoking. Mullen and coworkers (37) found that, unlike Reid's index, inflammation of cartilaginous airways separated those subjects with chronic bronchitis from those without. In fact, the two subject groups had equivalent proportions of mucous glands in central airways, but bronchitics had greater inflammation on mucosal surface of all bronchi larger than 2-mm luminal diameter and around glands and gland ducts in bronchi larger than 4-mm diameter.

The present study supports these findings and indicates that in smokers, chronic mucus hypersecretion, in the absence of airflow obstruction, is associated with an inflammatory process in the large airways and that VIP may play a role. When we considered only smokers with symptoms of chronic bronchitis without the presence of airflow obstruction (FEV₁ > 80%), we again observed an increase in VIP-positive nerves in the glands of bronchitic as compared with nonbronchitic subjects. In conclusion, this study of bronchial surgical specimens reveals a significant difference between bronchitic and nonbronchitic subjects in the density of nerve fibers immunoreactive for VIP in the mucous glands, and in the presence of airway inflammation assessed by mononuclear cell infiltration, and shows no differences between the two groups in the overall innervation as indexed by the PGP 9.5 nerve density localized to smooth muscle and glands of large airways, and in the size of mucous glands. These findings support the hypothesis that VIP is a potent stimulator of mucus secretion not only in animals but also in humans. Furthermore, these results suggest that, even in the absence of airflow obstruction, chronic mucus hypersecretion is related to airway inflammation. In addition, this study suggests that the technique of surgical specimens is suitable for studying changes in airway neuropeptides in chronic bronchitis. Further studies are required to investigate the role of chronic mucus hypersecretion in the natural history of chronic obstructive pulmonary disease (COPD).