Abstract
Although cigarette smoking is of paramount importance in the development of chronic obstructive pulmonary disease (COPD), only a small proportion of smokers develop the disease. We tested the hypothesis that the response of the bronchial epithelium to cigarette smoke (CS) differs in patients with COPD. Such a difference might explain in part why only some cigarette smokers develop the disease. We established primary explant cultures of human bronchial epithelial cells (HBEC) from biopsy material obtained from never-smokers who had normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD, and exposed these for 20 min to CS or air. Measurements were subsequently made over a period of 24 h of transepithelial permeability and release of interleukin (IL)-1 β and soluble intercellular adhesion molecule-1 (sICAM-1). In addition, intracellular reduced glutathione (GSH) levels were measured after 24 h incubation. Exposure to CS increased the permeability of these cultures in all study groups, but the most marked effect was observed in cultures from patients with COPD (mean increase, 85.5%). The smallest CS-induced increase in the permeability was observed in HBEC cultured from smokers with normal pulmonary function (mean, 25.0%), and this was significantly lower than that of HBEC from never-smokers (mean, 53.4%) (P < 0.001). Compared with exposure to air, exposure to CS led to a significantly increased release of these mediators from cultures of the never-smoker group (mean 250.0% increase in IL-1 β and mean 175.3% increase in sICAM-1 24 h after exposure) and COPD group (mean 383.3% increase in IL-1 β and mean 97.4% increase in sICAM-1 24 h after exposure). In contrast, CS exposure did not influence significantly the release of either mediator from the cells of smokers with normal pulmonary function. Levels of intracellular GSH were significantly higher in cultures of HBEC derived from smokers, both those with normal pulmonary function and those with COPD, compared with cultures from healthy never-smokers. Exposure to CS significantly decreased the concentration of intracellular GSH in all cultures. However, the fall in intracellular GSH was significantly greater in cells from patients with COPD (mean 72.9% decrease) than in cells from never-smokers (mean 61.4% decrease; P = 0.048) or smokers with normal pulmonary function (mean 43.9% decrease; P = 0.02). These results suggest that whereas smokers with or without COPD demonstrate increased levels of GSH within bronchial epithelial cell cultures, those with COPD have a greater susceptibility to the effects of CS in reducing GSH levels and causing increased permeability and release of proinflammatory mediators such as IL-1 β and sICAM-1.
Several studies have suggested that although cigarette smoking is one of the most important causes of chronic obstructive pulmonary disease (COPD), due to the great variability in individual response to tobacco, only a small proportion of smokers develop the disease. However, of those who have COPD, over 90% are smokers (1, 2).
Exposure to cigarette smoke (CS) represents a considerable oxidant burden on the respiratory epithelium, which is the first line of defense to inhaled substances. CS, which is one of the most important indoor air pollutants, is a complex mixture of over 2,000 different compounds, including oxidants. It has been estimated that there are 1014 free radicals in each puff of cigarette smoke (3). Our previous studies using primary cultures of human bronchial epithelial cells (HBEC) showed that these cells can synthesize and release a wide variety of proinflammatory mediators that in vivo play a role in the initiation and maintenance of airway inflammation (4), and that ozone (O3), nitrogen dioxide (NO2), and particulates, which are all components of CS, are capable of increasing the release of inflammatory mediators such as interleukin (IL)-8, granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α (5-7). These pollutants also cause a decrease in ciliary beat frequency and an increase in cell damage and permeability of HBEC cultures.
Because the bronchial epithelium is the first line of defense to CS, differences in the response of epithelial cells to oxidant stress resulting from CS exposure may explain in part why COPD develops in only some smokers. To date, however, the functional differences between epithelial cells from smokers with and without COPD compared with never-smokers have not been investigated.
In this article we report our observations of the effect of CS exposure for 20 min on confluent primary cultures of HBEC obtained from healthy never-smokers, smokers with normal pulmonary function, and patients with COPD. We assessed the effect of CS exposure on the permeability and levels of intracellular glutathione (GSH) in these cultures. We also investigated the effect of CS on the release of IL-1β and soluble intercellular adhesion molecule-1 (sICAM-1) because these mediators have been implicated in the development of inflammatory changes affecting the airways.
All chemicals and reagents were of tissue culture grade and unless stated otherwise, were obtained from the Sigma Chemical Co. (Poole, UK).
Eighteen volunteers were recruited to the study, which was approved by our District Ethics Committee: six never-smokers with normal pulmonary function, six smokers with normal pulmonary function, and six smokers with COPD. Informed written consent was obtained from each subject. All volunteers were between 45 and 70 yr of age. They underwent skin-prick testing to common inhalant allergens and spirometry with reversibility to inhaled β2 agonist bronchodilators, as well as making serial recordings of peak expiratory flow rate for 2 wk in order to exclude individuals with evidence of atopy or asthma (Table 1). In addition, each volunteer had a chest radiograph in order to exclude any other significant pulmonary pathology. The patient characteristics of the three groups is shown in Table 2. The mean FEV1 was 57.3 ± 11.9% (standard deviation) predicted in the COPD group and normal in the other two groups. No individual had bronchodilator reversibility of greater than 10% or diurnal variation in peak expiratory flow rate greater than 5%. None of the volunteers had taken oral or inhaled corticosteroids for at least 6 mo before the study.
| Patient No. | Sex | Category | FEV1(%) | FVC (%) | Tl CO(%) | Reversibility (%) | Pack- Years | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | F | S | 111 | 136 | 84 | 5 | 25 | |||||||
| 2 | M | S | 122 | 129 | 86 | 0 | 40 | |||||||
| 3 | F | C | 72 | 101 | 79 | 0 | 45 | |||||||
| 4 | M | C | 56 | 79 | 68 | 5 | 40 | |||||||
| 5 | F | S | 100 | 113 | 97 | 7 | 50 | |||||||
| 6 | M | S | 120 | 116 | 94 | 0 | 40 | |||||||
| 7 | M | N | 120 | 113 | 101 | 0 | 0 | |||||||
| 8 | M | N | 113 | 116 | 95 | 7 | 0 | |||||||
| 9 | M | C | 66 | 79 | 58 | 7 | 60 | |||||||
| 10 | M | C | 61 | 84 | 64 | 1 | 60 | |||||||
| 11 | M | N | 96 | 106 | 115 | 6 | 0 | |||||||
| 12 | M | N | 90 | 90 | 90 | 3 | 0 | |||||||
| 13 | F | S | 120 | 121 | 79 | 4 | 40 | |||||||
| 14 | F | N | 121 | 120 | 83 | 0 | 0 | |||||||
| 15 | M | S | 95 | 112 | 76 | 6 | 40 | |||||||
| 16 | M | C | 43 | 91 | 66 | 4 | 40 | |||||||
| 17 | M | C | 42 | 92 | 60 | 2 | 50 | |||||||
| 18 | F | N | 110 | 133 | 92 | 6 | 0 |
| Never-Smokers | Smokers | COPD | ||||
|---|---|---|---|---|---|---|
| Sex | 4 M; 2 F | 3 M; 3 F | 5 M; 1 F | |||
| Age, yr | 50.7 ± 7.5 | 49.8 ± 5.3 | 56.8 ± 5.7 | |||
| FEV1, % | 108 ± 12.7 | 111 ± 11.5 | 57.3 ± 11.9 | |||
| FVC, % | 113 ± 14.4 | 121 ± 9.6 | 85.3 ± 10.1 | |||
| Tl CO, % | 96 ± 11 | 86 ± 8.2 | 63.2 ± 9.8 | |||
| Reversibility, % | 3.7 ± 3.1 | 3.7 ± 3.0 | 4.5 ± 3.3 | |||
| Pack-years | 0 ± 0 | 39.2 ± 3.3 | 49.2 ± 3.8 |
Each volunteer underwent fiberoptic bronchoscopy under light sedation, according to the American Thoracic Society/American College of Chest Physicians guidelines (8), at which time six bronchial mucosal biopsies were taken from segmental and subsegmental bronchi on the right side. Biopsies were immediately placed in prewarmed and gassed medium 199 and transported directly to the laboratory for processing for tissue culture.
Bronchial epithelial cells were cultured using the explant cell culture technique developed in our laboratory (9). Briefly, the epithelium was dissected away from the underlying lamina propria and after further dissection into smaller sections of approximately 0.5 mm3, the epithelium was washed three times with fresh, sterile medium 199. Single sections of the epithelium were explanted into 9-mm-diameter Falcon cell culture inserts (Becton Dickinson Ltd., Oxford, UK) and 100-μl aliquots of culture medium with the following supplements was added to each insert: 2.5% fetal calf serum and a variety of growth factors, including human transferrin (2.5 μg/ml), epidermal growth factor (20 ng/ml), bovine pancreatic insulin (2.5 μg/ml), hydrocortisone (0.36 μg/ml), L-glutamine (0.02 mg/ml), and 1.5% (vol/vol) antibiotic and antimycotic solution composed of penicillin, streptomycin, and amphotericin B.
The inserts were placed in 24-well culture plates (Becton Dickinson Ltd.) containing 400 μl of the complete culture medium, as described previously, in each well and incubated at 37°C in a humidified 5% CO2 in air atmosphere. The medium in the insert and the well was replaced with culture medium containing 2.5% NUSERUM IV Culture Supplement (Universal Biologicals Ltd., London, UK) after 3 d and then every 48 h. The cultures were observed for epithelial cell outgrowth until the cells had grown to confluency. The explants were then removed and the cultures incubated further until the area left barren by removal of the explant was overgrown with epithelial cells.
The identity of the epithelial cells was confirmed in all cultures by light microscopy and in randomly selected cultures by electron microscopy and immunocytochemical staining for cytokeratin using monoclonal antibody preparation CAM 5.2 (Becton Dickinson Ltd.) Contaminating cell types were analyzed by staining for T and B lymphocytes using monoclonal antibodies CD3 and CD37, respectively (Serotec Ltd., Oxford, UK), and fibroblasts, mast cells, neutrophils, and macrophages using monoclonal antibodies 5B5, AA1, NP57, and Ber-MAC3, respectively (Dako Ltd., Bucks, UK).
Exposure of HBEC cultures to CS was carried out in an exposure system that we have developed in our laboratory. A continuous, 850-ml/min flow of CS was generated by burning Superkings cigarettes (12 mg tar, 1.1 mg nicotine) and leading the smoke stream into an airtight polycarbonate exposure chamber with a capacity of 5.5 liters (Billups Rothenberg, Del Mar, CA) containing the HBEC cultures established on inserts. The continuous flow of CS was achieved by the use of an Airchek 50 sampling pump (SKC Ltd., Dorset, UK) capable of generating 5 to 3,000 ml/min flow. The pump was connected to the top of the exposure chamber while the lit cigarette was connected to the bottom outlet of the exposure chamber. The negative pressure created by the sampling pump kept the cigarettes burning with a constant speed. On average, it took 5 min for a cigarette to burn. To achieve even distribution of the CS within the exposure chamber, a small, solar energy–powered ventilator was placed and operated inside the chamber just below the HBEC cultures. The solar panel itself was placed outside of the exposure system and was illuminated by a 100 W light bulb positioned 5 cm from the sensor. The exposure chamber was tilted gently, at intervals of 2.5 s, to an angle of 10° from the horizontal in each quarter of the horizontal plane on a Luckham 4RT rocking table (Luckham Ltd., Burgess Hill, UK), thereby providing adequate mixing of the culture media covering the surface of the HBEC cultures during each tilt. To ensure that the temperature was kept stable at 37°C during exposure, the above system was placed in a 60-liter capacity SI.60 incubator (Stuart Scientific, Redhill, UK) made of acrylic material, thereby providing total visibility.
HBEC cultures were exposed to CS for 20 min. Appropriate controls were also prepared by exposing HBEC cultures to air for the same time period and then treating further for the test cultures.
Changes in permeability of HBEC cultures were investigated by measuring the passage of 14C-bovine serum albumin (BSA) (specific activity: 10 to 100 μCi/mg protein; Amersham International plc, Amersham, UK) across HBEC cultures established in cell culture inserts. Measurements of changes in electrical resistance of HBEC cultures were also made in each culture at each time point studied.
Passage of 14C-BSA across HBEC cultures. Changes in permeability of HBEC cultures were investigated by measuring the passage of 14C-BSA across HBEC established in cell culture inserts.
Immediately before exposure to CS or air, the cultures were washed gently with fresh culture medium and then incubated for 30 min in the presence of 0.025 μCi 14C-BSA added into the insert. At the end of this incubation, the medium in each insert well was collected and analyzed for total radioactivity by liquid scintillation counting in a Beckman LS6500 scintillation counter (Beckman-RIIC Ltd., High Wycombe, UK). When the total radioactivity passing across the culture was found to be less than 0.5% of the total 14C-BSA added to the inserts at the beginning of the experiment, the culture was deemed to be fully confluent and the actual experiment was started. HBEC cultures were then exposed to CS or air for 20 min. Immediately after exposure, the medium was collected from each insert well, and after addition of fresh medium, the cultures were incubated further at 37°C in 5% CO2 in air atmosphere. The culture medium from each insert well was collected at 1, 3, 6 and 24 h after exposure and 25-μl aliquots of each sample were analyzed for radioactivity. After correction for the total amount of radioactivity passing across the epithelial cultures at each time point, results were expressed as a percentage of the total added to the culture at the beginning of the experiment.
Measurement of electrical resistance (ER) of HBEC cultures. ER of HBEC cultures was measured before commencing the experiment and then immediately, 1, 3, 6, and 24 h after exposure, using an EVOM micro volt-ohm meter incorporating a fixed pair of electrodes (4 mm wide and 1 mm thick; World Precision Instruments, Owslebury, UK) that is specifically designed to facilitate measurements of membrane voltage and resistance of epithelial cells grown on semipermeable membranes. Each arm of this electrode set contains at its tip a voltage-sensing miniature electrode made of silver/silver chloride and a concentric spiral of silver wire that passes a current through the membrane sample. Measurements of ER were made by placing one electrode inside the insert and the other electrode in external bathing medium 199. After measurements, all ER results were calculated as ohms (Ω) according to the formula: ER(insert + HBEC + M199) − ER(insert + M199), where ER(insert + HBEC + M199) is the ER of a cell culture insert with an established confluent culture + medium 199 and ER(insert + M199) is the ER of a cell culture insert containing medium 199 only (mean of 25 inserts).
Data obtained from these experiments were analyzed after calculation of the percentage changes from baseline in each culture at each time point during the 24-h incubation period.
Confluent cell cultures that were 3 wk old were washed and incubated overnight in SF-1 supplement medium (serum-free medium; Hyclone Europe Ltd., Northumberland, UK). After this initial incubation, the cultures were gently washed three times with freshly prepared SF-1 medium, then 500 μl of SF-1 medium were added into each insert and well, and the cultures were exposed to CS or air for 20 min as described previously and then incubated further for up to 24 h at 37°C in 5% CO2 in air atmosphere. The culture medium collected from cultures immediately, 1, 3, 6, and 24 h after exposure were pooled, freeze-dried, and stored at −70°C until analysis for IL-1β and sICAM-1 using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Abingdon, UK). The insert membrane containing the attached epithelial cells was removed and stored by freezing at −20°C in 250 μl of 1 M NaOH until analysis for total cellular protein by the method of Lowry and coworkers (10). All results were expressed as picogram mediator/microgram cellular protein.
Measurement of GSH in all samples containing the epithelial cells was performed using a commercially available Bioxytech GSH-400 colorimetric assay for glutathione kit (Oxis International, Portland, OR) according to the protocol outlined by the manufacturer.
Data were tested for normality using a normal probability plot and the Shapiro-Wilk test (11). Because data followed a normal distribution, within group comparisons were performed using two-way analysis of variance and paired t test, and between group comparisons were performed by two sample t tests.
Microscopic examination of HBEC using Hoffman modulation contrast optics demonstrated large numbers of cells with polygonal morphology, typical of epithelial cells, growing out from the explants, which reached confluence normally after 3 wk. Indirect immunoperoxidase staining with CAM 5.2 revealed that all the cells in the cultures stained for cytokeratin confirmed the epithelial nature of the cells. Additionally, microscopic investigations also revealed that whereas in cultures derived from never-smokers around 25 to 30% of the cells were ciliated, in smokers with normal pulmonary function the percent of ciliated cells decreased to around 10%. Cultures from patients with COPD, however, were almost completely devoid of cilia.
Staining with specific monoclonal antibodies, as listed in Materials and Methods, against contaminating cell types, including endothelial cells, fibroblasts, muscle cells, macrophages, T and B lymphocytes, mast cells, eosinophils, and neutrophils, did not show any indication of these cells.
Passage of 14C-BSA across HBEC cultures. There was no difference between the permeability of HBEC cultured from healthy never-smokers with normal pulmonary function, smokers with normal pulmonary function, or smokers with COPD at any time after exposure to air up to the 24 h of incubation studied (Figure 1). In contrast, 20 min of exposure to CS of HBEC from healthy never-smoker individuals led to a significant 53.4% mean increase in the passage of 14C-BSA across HBEC cultures by 24 h after exposure when compared with the passage of 14C-BSA across these cells after exposure to air. In cultures from patients with COPD, exposure to CS increased the passage of 14C-BSA even further by 85.5% (mean). On the other hand, CS exposure led to only a mild, although statistically significant, mean 25.0% increase in the permeability of HBEC cultured from smokers with normal pulmonary function 24 h after exposure when compared with exposure to air. Interestingly, the CS–induced increase in the permeability of HBEC cultured from smokers with normal pulmonary function was significantly lower when compared with HBEC from healthy never-smokers (P < 0.001).
Fig. 1. The effect of 20 min exposure to CS on the passage of 14C-BSA across HBEC cultured from never-smokers with normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD over a period of 24 h (each datapoint represents the mean of data obtained from six different cultures from each individual at each timepoint; *P < 0.01, **P < 0.001 versus never-smokers+CS).
[More] [Minimize]Changes in ER of HBEC cultures. Similar to our findings investigating the passage of 14C-BSA, we found that whereas the ER of the HBEC cultures derived from each of the study groups was not significantly different from each other when exposed to air, they responded differently to CS exposure (Figure 2). Cultures from patients with COPD were found to be the most susceptible to the effect of CS, displaying a marked decrease in their ER 1 h after exposure onward. Cultures from smokers with normal pulmonary function were the most resistant as indicated by the least change in their ER after CS exposure. These measurements also showed that by 24 h after exposure to CS, cultures derived from smokers with normal pulmonary function regained their baseline ER, indicating that the detrimental effects of CS on the ER of these cultures were temporary (Figure 2). In the case of HBEC cultures obtained from never-smokers, there was only a partial recovery of the CS-induced changes, whereas in cultures derived from patients with COPD no such recovery was observed during the 24-h postexposure incubation period.
Fig. 2. The effect of 20 min exposure to CS on the electrical resistance of HBEC cultured from never-smokers with normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD over a period of 24 h (each datapoint represents the mean of data obtained from six different cultures from each individual at each timepoint).
[More] [Minimize]There was no significant difference between the study groups in the release of IL-1β (Figure 3) or sICAM-1 (Figure 4) from HBEC exposed to air. Exposure to CS, however, led to a significantly increased release of IL-1β in both the healthy never-smoker and COPD groups 24 h after exposure (250.0% mean increase [P = 0.043] and 383.3% mean increase [P = 0.016], respectively). Similarly, exposure to CS significantly increased the release of sICAM-1 in both the healthy never-smoker and COPD groups 24 h after exposure (175.3% mean increase [P = 0.032] and 97.4% mean increase [P = 0.035], respectively). In contrast, CS exposure did not influence significantly the release of either mediator from the cells of smokers with normal pulmonary function.
Fig. 3. The effect of 20 min exposure to CS on the release of IL-1β by HBEC cultured from never-smokers with normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD after a 24-h incubation (each datapoint represents the mean of data obtained from six different cultures from the same individual; horizontal lines represent the mean of these means).
[More] [Minimize]
Fig. 4. The effect of 20 min exposure to CS on the release of sICAM-1 by HBEC cultured from never-smokers with normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD after a 24-h incubation (each datapoint represents the mean of data obtained from six different cultures from the same individual; horizontal lines represent the mean of these means).
[More] [Minimize]After exposure to air, primary cultures of HBEC derived from both smokers with normal pulmonary function and patients with COPD contained significantly more GSH than did cultures from healthy never-smokers. Exposure to CS significantly decreased the concentration of intracellular GSH in all cultures when compared with exposure to air. However, the magnitude of decrease (mean percent change) was different in the study groups: 72.9% in cells from patients with COPD (P < 0.01), 61.4% in cells from healthy never-smokers (P < 0.05), and 43.9% in cells from smokers with normal pulmonary function (P < 0.05) (Figure 5). Additionally, analysis of the differences in the magnitude of decrease in cultures from each study group has shown that this observed decrease in intracellular GSH in cells from patients with COPD was significantly greater than that in cells from healthy never-smokers or smokers with normal pulmonary function (P = 0.048 and P = 0.015, respectively). There was no significant difference in the observed decrease of intracellular GSH between cultures from healthy never-smokers and smokers with normal pulmonary function.
Fig. 5. The effect of 20 min exposure to air (open bar) or CS (solid bar) on the levels of intracellular GSH in HBEC cultured from never-smokers with normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD after 24 h of incubation (each bar represents the mean of data obtained from six different cultures from six individuals in each study group; *P < 0.05 and **P < 0.01 versus air).
[More] [Minimize]In this study, we found that HBEC cultured from smokers with COPD showed greater susceptibility to the effects of CS in increasing transepithelial permeability and reducing intracellular GSH when compared with cultures from smokers with normal lung functions or healthy never-smokers. In addition, HBEC from smokers with COPD demonstrated greater CS-induced release of IL-1β and sICAM-1 compared with smokers without COPD.
We have previously demonstrated that HBEC cultured to confluence in vitro retain morphologic and biochemical characteristics similar to those found in vivo (9, 12). Characterization of the different epithelial phenotypes that constituted the HBEC cultures demonstrated that approximately 40 to 45% were ciliated epithelial cells, 35 to 40% glandular epithelial cells, 20% basal epithelial cells, and 0.5% goblet cells (12). Despite the lack of naturally occurring defense mechanisms such as a protective epithelial lining fluid that may limit the oxidant-induced cell damaging effect of CS, primary cultures from human airway epithelial cells offer a suitable in vitro model to study the effects of direct exposure to this agent and the mechanism(s) underlying these effects.
The changes observed in the present study are likely to be of pathophysiologic relevance, given their similarity in terms of magnitude to changes induced by other established inflammatory stimuli. For example, we have shown that exposure of bronchial epithelial cells to pollutants such as NO2, sulfur dioxide, O3, and diesel exhaust particulates caused increased epithelial permeability as well as release of proinflammatory cytokines (including IL-8, GM-CSF, and TNF-α) and adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) (13). In addition, purified endotoxin from Hemophilus influenzae caused the release of proinflammatory mediators from these cells (14), whereas activated eosinophils significantly increased airway epithelial permeability (15). In all of these studies, there were generally less than threefold changes in biologic markers of the effects of inflammatory stimuli compared with control values. A similar magnitude of change was observed in the present study and also in studies by other investigators using different epithelial cell culture systems (16-19).
We observed that primary cultures of HBEC derived from smokers, both those with normal pulmonary function and those who are COPD patients, contained significantly more GSH than did cultures from healthy never-smokers. This is in keeping with the observations of several other investigators who demonstrated differences in the antioxidant status of the airways of smokers compared with nonsmokers (20). For instance, cigarette smokers were found to have 80% higher levels of epithelial lining fluid total GSH, 98% of which was in the reduced form (3, 21). Cultured human type II alveolar cells exposed to CS condensate solution demonstrated increased GSH levels, gamma-glutamylcysteine synthetase (gamma-GCS) activity, and DNA binding of activator protein-1 and the human antioxidant response element (3). McCusker and Hoidal (22) found that in alveolar macrophages from cigarette smokers there were twofold increased activities of superoxide dismutase and catalase but no change in the activity of glutathione peroxidase compared with nonsmoking control subjects. They suggested that in cigarette smokers, augmented antioxidant activity may be important in restricting oxidant damage. Our observations are in keeping with this concept, in that augmented GSH levels in the HBEC of smokers with normal pulmonary function was associated with restriction of the CS-mediated increase in epithelial permeability and release of IL-1β and sICAM-1.
This restriction of CS effect did not apply to the epithelial cells of smokers with COPD. It is noteworthy that in this group, CS caused a significantly larger decrease in intracellular GSH than in cultures from either healthy never smokers or smokers with normal pulmonary function. Whether this differing decrease in intracellular GSH was due to different expression and or activity of the two main enzymes involved in GSH regulation, namely gamma-GCS and gamma-glutamine transpeptidase, remains to be determined.
MacNee and colleagues (23) and Rahman and coworkers (24) demonstrated that exposure of airway epithelial cell monolayers to CS condensate resulted in an initial decrease in intracellular GSH followed by a rebound increase 12 h after exposure. In the present study however, we did not observe a rebound increase. This may have been because we used mainstream CS rather than CS condensate, so that the observed decreased levels of intracellular GSH 24 h after exposure to CS resulted from not only the initial 20-min direct exposure but also from CS-derived oxidants solubilized in the culture medium that then remained in contact with the HBEC cultures during the entire incubation period.
The results of this study suggest that CS exposure may cause release of ICAM-1 from the surface of the epithelial cells because we observed that CS could increase local concentrations of sICAM-1. Barton and colleagues (25) showed that in cultures of rat type II alveolar epithelial cells, ICAM-1 remained associated with the cytoskeleton after detergent extraction, whereas other transmembrane molecules were completely removed. Additionally, ICAM-1 was redistributed on the cell surface of these cells after the disruption of actin filaments, suggesting interaction with the actin cytoskeleton. These observations suggest that the interaction of ICAM-1 with insoluble cytoskeletal elements in alveolar epithelial cells is likely to play a role in maintaining the normal anatomy of these cells and the cell layer formed by them. CS-induced loss of surface ICAM-1 might render the epithelial barrier less tight (26), leading to increased permeability of the epithelial layer and possibly an increased surface area interfacing with CS.
Taking together our present and previous (27-29) findings and the observations of others, we propose a model for COPD pathophysiology (Figure 6). CS exposure increases airway epithelial permeability (16, 17, 20, 23) by inducing damage to the epithelial cells themselves and the intercellular junctional complexes between them. Because the airway epithelium is the first line of defense against inhaled agents such as cigarette smoke, this increase in permeability of the epithelium facilitates the transepithelial passage of other inflammatory agents such as allergens, bacteria, and viruses, as well as pollutants. This process is further enhanced by the ability of CS exposure to markedly decrease the ciliary beat frequency of HBEC, which leads to impairment of airway mucociliary clearance (30).
Fig. 6. Putative mechanisms for the effects of cigarette smoke in the pathogenesis of COPD.
[More] [Minimize]In addition, CS exposure upregulates both the expression and release of proinflammatory cytokines such as IL-1β, which in turn has been shown to upregulate the expression by endothelial cells of adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1, a process which is a prerequisite for the recruitment and trafficking of inflammatory cells into the airway epithelium. CS also increases the release of IL-8 from bronchial epithelial cells (19, 28). IL-8 is a potent neutrophil chemotactic factor that plays a pivotal role in the activation of neutrophils, their adherence to endothelial cells, and their attraction to the epithelium. In the epithelium, activated neutrophils may release potent mediators such as myeloperoxidase and elastase, causing further damage to the epithelium.
We have previously shown that CS downregulates the expression of secretory component in bronchial epithelial cells (29). This might lead to decreased production of secretory immunoglobulin A and subsequent increased susceptibility to airway damage from bacterial infection. In keeping with this concept, recent data show that the expression of secretory component in the bronchial epithelium correlates negatively with bronchial obstruction in COPD (31).
Taken together, these pathophysiologic events would be likely to lead to sustained airway inflammation with functional and morphologic changes in the airway epithelium. However, it is not known why only a small proportion of the smokers exposed to these potential effects subsequently develop COPD. Given that oxidant-induced change in intracellular GSH is a likely contributing factor to the development of increased epithelial permeability after exposure to CS (23), it may be that interindividual variability in antioxidant capacity is a factor determining whether COPD develops in response to cigarette smoking. We have observed in the present study that smokers, both those with COPD as well as those with normal lungs, have increased intracellular GSH in bronchial epithelial cells, and it has been suggested (22) that such increased levels of antioxidant capacity may protect against oxidant-mediated damage. However, the present study has also demonstrated a larger CS-induced decrease in intracellular GSH levels in bronchial epithelium from smokers with COPD compared with smokers without COPD. In addition, HBEC of patients with COPD demonstrated a larger increase in permeability and release of sICAM-1 and IL-1β, compared with a control group of cigarette smokers without COPD.
These are novel observations and might explain in part why only a proportion of smokers develop COPD. However, it is important to recognize that our study does not allow assessment of whether in patients with COPD, the greater susceptibility of bronchial epithelial cells to CS exposure contributes to the development of COPD or is a secondary characteristic of this condition.
The writers thank the Joint Research Board of St. Bartholomew's Hospital, London (UK) for financial assistance.
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