Remodeling in Response to Infection and Injury
Airway Inflammation and Hypersecretion of Mucus in Smoking Subjects with Chronic Obstructive Pulmonary Disease

PIERO MAESTRELLI, MARINA SAETTA, CRISTINA E. MAPP, and LEONARDO M. FABBRI

University of Padua, Padua, Italy; University of Ferrara, Ferrara, Italy; and University of Modena and Reggio Emilia, Modena, Italy

	ABSTRACT

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Airway epithelium represents the first line of defense against toxic inhalants. In some subjects, cigarette smoking causes airway inflammation, hypersecretion of mucus, and poorly reversible airflow limitation through mechanisms that are still largely unknown. Likewise, it is unclear why only some smokers develop chronic obstructive pulmonary disease (COPD). Two cell types consistently result in relation to chronic airflow limitation in COPD: neutrophils and CD8⁺ cells. Neutrophils are compartmentalized in the mucosal surface of the airways and air spaces, that is, the epithelium and lumen, whereas CD8⁺ cells exhibit a more extensive distribution along the subepithelial zone of the airways and lung parenchyma, including alveolar walls and arteries. This pattern of inflammatory cell distribution is observed in mild or moderate COPD, and in patients who have developed COPD, it is not modified by smoking cessation. The number of neutrophils further increases in the submucosa of patients with severe COPD, suggesting a role for these cells in the progression of the disease. Hypersecretion of mucus is a major manifestation in COPD. Mucus is produced by bronchial glands and goblet cells lining the airway epithelium. Unlike mucous gland enlargement, greater mucosal inflammation is associated with sputum production. Whereas neutrophil infiltration of submucosal glands occurs only in smokers with COPD, goblet cell hyperplasia in peripheral airways occurs both in smokers with or without COPD, suggesting that the major determinant of goblet cell hyperplasia is cigarette smoke itself.

	INTRODUCTION

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Keywords: histology; sputum cytology; glands; goblet cells; biopsies

Cigarette smoke is a complex mixture of chemical compounds, including free radicals and other oxidants that have the potential to induce tissue damage (1). The epithelial surface of the airways and of the air spaces is the first line of defense in the lungs against toxic inhalants. The events occurring in these respiratory tract compartments may be relevant to the pathogenesis of smoking-induced respiratory diseases, as chronic obstructive pulmonary disease (COPD). It has long been recognized that exposure to cigarette smoke is associated with airway inflammation (2, 3), but only more recently have the type and extent of inflammation been characterized more precisely (4). However, the mechanism by which cigarette smoke induces hyperproduction of mucus and airflow limitation is still poorly understood, as well as the reason why only a relatively small proportion of smokers develop overt disease. Changes observed in the airways of smokers susceptible to developing COPD may shed some light on the pathogenesis of the disease. Increased concentrations of tumor necrosis factor  (TNF-) and interleukin 8 (IL-8) were detected in induced sputum of patients with COPD compared with smoking and nonsmoking control subjects (7). Epithelial cells and macrophages present in the lumen of the airways may be the source of IL-8, a potent chemotactic factor for neutrophils, whereas TNF- is known to upregulate the expression of adhesion molecules.

	AIRWAY INFILTRATION BY INFLAMMATORY CELLS

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Neutrophils seem to play a significant role in the development and progression of COPD in smokers. Epithelial neutrophilia has been reported in smokers with an excessive decline in FEV₁ over time (8, 9). In particular, the longitudinal study by Stanescu and coworkers (8) examined whether the airway inflammatory process is different in smokers susceptible to developing COPD as compared with "resistant" smokers by induced sputum. The percentages of sputum neutrophils were greater in smokers with COPD, compared with asymptomatic smokers, and correlated with the annual decline in FEV₁ (Figure 1). In addition, in smokers with COPD, sputum neutrophils exhibited increased expression of adhesion molecule CD11b/CD18, the ICAM-1 (intercellular adhesion molecule type 1) ligand, and this expression was related to the degree of airway obstruction (8). A number of subsequent studies confirmed increased airway neutrophilia in COPD as compared with healthy smokers (7, 10, 11).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Significant correlation between percentages of neutrophils in sputum and decline in FEV1 in smokers. Data are presented as medians and interquartile range. Spearman rank correlation coefficient = 0.43, p < 0.01. Reprinted by permission from Reference 8.

A dissociation between a predominance of neutrophils in the airway lumen, assessed by bronchoalveolar lavage (BAL) or sputum (7), and a lack of increase of these cells in the subepithelium of bronchial biopsies, was observed in mild to moderate COPD. It is likely that neutrophils accumulate in the airway lumen by recruitment from circulation. Upregulation of E-selectin on vessels in the submucosa and increased expression of epithelial ICAM-1 on basal epithelial cells in patients with COPD, in addition to the chemotactic activity of IL-8, suggest a mechanism for recruitment of these cells from the circulation and for their migration to the airway lumen (13). Indeed, increased numbers of neutrophils were detected in the bronchial epithelium of the central and peripheral airways of patients with COPD (14, 15). An imbalance between pro- and anti-inflammatory cytokine may favor this process. In fact, the inhibitory IL-10 was reduced in the sputum of subjects with COPD in contrast to the elevated concentrations of IL-8 and TNF- (7, 16).

The bronchopulmonary inflammation in COPD differs from that seen in asthma, even if some patients with COPD exhibit some degree of airway eosinophilia (10, 11, 17). It is controversial whether eosinophilia in COPD is related to concomitant features of asthma. Chanez and coworkers (17) reported that patients with COPD who responded to corticosteroid treatment had a significantly larger number of eosinophils in their airways and thicker reticular basement membrane. In contrast, patients with exacerbated chronic bronchitis who exhibited airway eosinophilia were undistinguishible from other COPD patients without eosinophilia by common diagnostic criteria and pathology findings (18, 19). It should be pointed out that studies including biopsies at exacerbation have been performed in patients with symptoms of chronic bronchitis and generally mild airflow obstruction. Patients with more severe COPD may not show mucosal eosinophilia when their condition is exacerbated. It has been suggested that eosinophilia in COPD is related to the intensity of the inflammatory process in the airways, leading to aspecific recruitment and activation of eosinophils (10, 20, 21). This suggestion is supported by findings that both sputum neutrophilia and sputum eosinophilia were more intense in subjects with greater impairment in FEV₁ (7, 8, 11), with a direct relationship between neutrophil and eosinophil numbers (11).

The airway wall beneath the epithelial surface in mild/moderate COPD displays a mononuclear cell inflammation with increased macrophages, T lymphocytes, and their activation markers CD25, very late antigen 1, and HLA-DR (4, 22). In particular, a shift in the balance of the CD4⁺/CD8⁺ cell ratio in favor of CD8⁺ cells was observed in a number of studies. The CD8⁺ cell infiltrate was present in several compartments of the central airways (6, 14, 23), peripheral airways (15, 24), and lung parenchyma (25). At variance with the neutrophilic inflammation at the mucosal surface of the airways, the CD8⁺ cell inflammatory process appears to be extensively distributed in the lung. The role of these cells, which are likely T lymphocytes, is not yet clear, but the correlation between the number of infiltrating CD8⁺ cells in large and small airways and lung parenchyma with the degree of airflow limitation suggests that they may be related to the progression of the disease (Figure 2). In addition to their known function as cytotoxic cells, CD8⁺ cells are capable of inducing lung damage when present in excess in response to respiratory viral infections (26). There is evidence that the CD8⁺ lymphocytes exhibit different functions depending on the different profile of cytokines (27) and are able to produce cytokines associated with either type 1 or type 2 responses (28, 29). CD8⁺ cells may therefore have a role in the regulation of the inflammatory response to cigarette smoke through the release of their products (30). Some of these chemotactic lymphokines may be implicated in the pathogenesis of COPD by recruiting neutrophils (31, 32). It is interesting that the IL-13 observed in animal models causes emphysema (33) and mucin production (34). Human data presented in abstract form are consistent with a role of IL-13 in smokers susceptible to hypersecretion of mucus. Mapp and coworkers (35) showed increased expression of IL-13 immunoreactivity on cells in the bronchial walls of smokers with chronic bronchitis compared with asymptomatic smokers.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Relationship between the number of CD8+ cells in the peripheral airways and values of FEV1% pred. Spearman rank correlation coefficient = 0.63, p < 0.01. Reprinted by permission from Reference 1.

On the basis of a study that demonstrated genetic control of the ratio between CD4⁺ and CD8⁺ cells, with a small (5%) percentage of the population having a CD4⁺/CD8⁺ cell ratio of < 1 (36), O'Shaughnessy (6) suggested that those with a genetically determined increase in the CD8⁺ subset could be more susceptible to a further increase in CD8⁺ cells when exposed to cigarette smoke. This hypothesis may explain the reason why the differences in subepithelial CD8⁺ cells found by Lams and coworkers (23) were more evident between smokers with COPD and asymptomatic smokers than between smokers and nonsmokers. In fact, the former group would include both genetic variants with high and low numbers of CD8⁺ cells.

These data together show that two cell types are consistently related to chronic airflow limitation in COPD: neutrophils and CD8⁺ cells. Neutrophils are compartmentalized in the mucosal surface of large airways, that is, the epithelium and lumen, whereas CD8⁺ cells exhibit a more extensive distribution along the subepithelial zone of the large and small airways and in the lung parenchyma, including alveolar walls and arteries. This pattern of inflammatory cell distribution refers to COPD of mild or moderate severity. In contrast, an increased number of neutrophils in the submucosa was reported during exacerbation of chronic bronchitis (18, 19) and in stable COPD with severe airflow limitation (37). As the severity of airflow limitation increases, the number of neutrophils in the subepithelium increases, reinforcing the hypothesis that these cells play a role in the progression of the disease.

A relevant question related to the airway remodeling in COPD is whether pathologic changes detected in smokers susceptible to developing the disease are reversible on cessation of cigarette smoke injury. This was investigated by Turato and coworkers (38) in bronchial biopsies by comparing current smokers and ex-smokers who had stopped smoking on average 13 yr before the study, all with symptoms of chronic bronchitis. The number of inflammatory cells in the bronchial mucosa, the markers of mononuclear cell activation, and the expression of adhesion molecules and cytokines were not significantly different between current and ex-smokers. These data indicate that the airway inflammation process characteristic of chronic bronchitis may persist in subjects who continue to have chronic cough and sputum production despite smoking cessation, suggesting that when the disease is established the pathology is not reversible with the avoidance of injury. Longitudinal studies demonstrated an improvement of lung function in COPD after smoking cessation. In the absence of longitudinal data on morphology, it is unknown whether ex-smokers had reduced airway inflammation from the time when they were still smoking.

	GOBLET CELL HYPERPLASIA, BRONCHIAL GLAND INFLAMMATION, AND HYPERSECRETION OF MUCUS

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Chronic hypersecretion of mucus is a major manifestation in COPD. However, its role in the development of chronic airflow obstruction is controversial (39). The results of the Copenhagen City Heart Study Group indicated that chronic sputum production is associated with both an excessive FEV₁ decline and increased risk of hospitalization due to COPD, supporting a role for hypersecretion of mucus in the development of chronic airflow limitation (40). Mucus is produced by bronchial glands and goblet cells lining the airway epithelium. In healthy subjects, peripheral airways contain few goblet cells, but goblet cell metaplasia may occur in respiratory disorders. Two conditions are responsible for hyperproduction of mucus: an increase in mucus-producing cells and an increase in degranulation of mucus content.

Mucous gland hypertrophy was suggested to play a pathogenetic role in the induction of chronic bronchitis. However, the descriptions of enlarged submucosal glands made in the 1950s (41) were not confirmed by quantitative histology performed in a number of subsequent studies. In the 1980s, both Nagai and coworkers (42) and Mullen and coworkers (2) found no correlation between sputum production and mucous gland enlargement. In contrast, smokers with chronic bronchitis had greater inflammation around gland ducts in bronchi larger than 4 mm in diameter (4). The characterization of inflammatory cells in submucosal glands showed that, unlike Reid's index, increased infiltration of neutrophils in close association with gland cells was a feature of smokers with COPD compared with healthy smokers (Figure 3)(14). Neutrophil infiltration of the airway epithelium may mediate hypersecretion by direct interaction with mucus-producing cells. In the animal model, it has been demonstrated that neutrophil-associated elastase, rather than the released enzyme, is involved in degranulation. Furthermore, neutrophils binding through ₂-integrin (CD11b/CD18) to epithelial ICAM-1 was required to induce mucous cells to degranulate in human trachea (43). Neural mechanisms may also be involved in the pathophysiology of COPD by contributing to the symptoms (cough and sputum production) and, possibly, to the inflammatory response. Mediators from inflammatory cells may influence the release of neurotransmitters, which in turn may modulate the inflammatory response (44). The hypothesis that neuropeptides are involved in hypersecretion of mucus in humans is supported by a study of bronchial surgical specimens, which revealed a significant increase in the density of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP) in the mucous glands of smokers with chronic bronchitis (45).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Individual counts of inflammatory cells in the bronchial glands of smokers with both symptoms of chronic bronchitis and chronic airflow limitation (bronchitics) and healthy smokers (controls). Horizontal bars represent median values. Reprinted by permission from Reference 14.

Whereas hypersecretion of mucus deriving from the large conducting airways is associated with symptoms of chronic bronchitis, that is, cough and sputum, excessive production of mucus in the peripheral airways may contribute to airflow obstruction. Secretions can alter the surface tension of airway lining fluid, rendering peripheral airways unstable and more prone to dynamic closure (46) or mechanically occlude their lumen through the formation of mucous plugs (47). In normal subjects, goblet cells are localized in central airways. The appearance of goblet cells in the peripheral airways of smokers was reported by some investigators (41, 47, 48), but not consistently (3, 49). Only more recently has a precise quantification of mucus-secreting cells in the epithelium of peripheral airways been performed. In surgical specimens of subjects undergoing lung resection for localized pulmonary lesions, the smokers with both symptoms of chronic bronchitis and airflow obstruction exhibited an increased number of goblet cells in airways with a diameter of less than 1 mm. The finding that the subjects with COPD had an increased number of goblet cells when compared with nonsmokers, but not when compared with healthy smokers, and the lack of correlation with FEV₁ suggests that the major determinant of goblet cell hyperplasia is cigarette smoke itself (Figure 4). The mechanism of goblet cell hyperplasia in smoking subjects is unknown. Activation of epidermal growth factor receptor (EGF-R) was proposed in the animal model (50). Multiple stimuli may upregulate EGF-R and stimulate hypersecretion, including oxidants present in cigarette smoke or produced by inflammatory cells, and cytokines (TNF-, IL-8, and IL-13) (34, 51).

View larger version (10K): [in this window] [in a new window]   Figure 4.   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. Reprinted by permission from Reference 15.

In conclusion, the two major effects of lung damage induced by cigarette smoke in susceptible subjects, that is, chronic airflow limitation and hypersecretion of mucus, result in association with airway inflammation. In the early stage of COPD, two cell types characterize the inflammatory process: neutrophils and CD8⁺ cells. Neutrophils are compartmentalized in the epithelium and lumen of the airways, and in submucosal glands, whereas CD8⁺ cells exhibit a more extensive distribution along the subepithelial zone of the airways and lung parenchyma, including alveolar walls and arteries.

	Footnotes

Correspondence and requests for reprints should be addressed to Piero Maestrelli, Respiratory Pathophysiology Unit, Department of Environmental Medicine and Public Health, University of Padua, Via Giustiniani 2, 35136 Padua, Italy. E-mail: piero.maestrelli{at}unipd.it

(Received in original form June 15, 2001 and accepted in revised form August 28, 2001).

Acknowledgments: Supported by the Italian Ministry of University and Research (MURST 40%); by the Universities of Padua, Ferrara, and Modena and by a special research unrestricted grant by Glaxo Wellcome, Verona, Italy.

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