Impaired Inhibition by Dexamethasone of Cytokine Release by Alveolar Macrophages from Patients with Chronic Obstructive Pulmonary Disease

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom

	ABSTRACT

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Chronic obstructive pulmonary disease (COPD) is characterized byinflammation of the respiratory tract in which macrophages are the predominant inflammatory cell and for which the efficacy of treatment with corticosteroids is controversial. We investigated the effect of dexamethasone on basal and interleukin (IL)-1ß or cigarette smoke media (CSM)–stimulated release of IL-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) by bronchoalveolar lavage macrophages from cigarette smokers and patients with COPD (n = 15). Basal release of IL-8 was approximately fivefold greater in patients with COPD than smokers, whereas GM-CSF was similar for each group. IL-1ß and CSM increased IL-8 and GM-CSF release by macrophages from both smokers and patients with COPD. Dexamethasone did not inhibit basal or stimulated IL-8 release from macrophages from patients with COPD but inhibited release in smokers. In contrast, basal and IL-1ß–stimulated GM-CSF release, but not CSM-stimulated release, was inhibited by dexamethasone. We conclude that the lack of efficacy of corticosteroids in COPD might be due to the relative steroid insensitivity of macrophages in the respiratory tract.

　

Key Words: alveolar macrophage • cigarette smoke • chronic obstructive pulmonary disease • corticosteroid • dexamethasone

	INTRODUCTION

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Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory condition that is characterized by a progressive and largely irreversible airflow limitation (1). Cigarette smoking is the major risk factor for development of COPD, and smoking cessation is the only intervention that slows disease progression (2, 3). The pathophysiology of COPD is multifactorial with an inflammatory cell profile that includes macrophages, neutrophils, and T lymphocytes (4–6).

Macrophages are suggested to be the orchestrators of the chronic inflammatory response and tissue destruction associated with COPD (7). For example, macrophages contribute to airway inflammation in smokers and patients with COPD by secreting neutrophil and macrophage chemotactic factors and related chemokines such as interleukin (IL)-8 (8, 9), and by the generation of reactive oxygen species (10). Bronchoalveolar lavage (BAL) from asymptomatic smokers and patients with COPD yields higher numbers of macrophages than BAL from nonsmokers (11). Cigarette smoking affects the cellular composition of BAL, and markers of inflammation (12), and macrophages recovered from smokers secrete increased levels of chemotactic factors (13), cytokines (14), and proteases (15) compared with nonsmokers.

Cigarette smoke medium (CSM), produced by bubbling smoke through cell culture medium (16), induces IL-8 release from cultured human bronchial epithelial cells (9). CSM constituents in vitro also increase cytokine mRNA expression (17) and reduce surfactant secretion by alveolar type II cells (16). However, the effects of CSM on cytokine secretion by alveolar macrophages have not been evaluated.

Glucocorticosteroids inhibit cytokine release from inflammatory and other cell types mainly by suppressing the expression of inflammatory genes (18). Dexamethasone inhibits in vitro release of tumor necrosis factor- from human airway smooth muscle cells (19) and granulocyte macrophage-colony stimulating factor (GM-CSF) from monocytes (20). Dexamethasone also inhibits IL-8 release by human airway epithelial cells (21), U937 monocytic cells (22), and porcine alveolar macrophages (23). The inhaled corticosteroids fluticasone propionate and budesonide inhibit tumor necrosis factor-, IL-6, and IL-8 release by alveolar macrophages from nonsmokers (24). GM-CSF release from alveolar macrophages is reduced in subjects with asthma who are treated with inhaled steroids (25), and oral prednisolone reduces leukotriene B₄ release by macrophages in subjects with nocturnal asthma (26). The effect of corticosteroids on macrophage function in COPD is not reported. Although corticosteroids are an effective treatment in asthma (27), their clinical efficacy in COPD is controversial (28, 29). Neither high-dose inhaled nor oral corticosteroids reduce the inflammatory response, concentrations of IL-8, nor proteases in induced sputum of patients with COPD (30–32). Consequently, reduced corticosteroid efficacy in COPD could be due to a decreased effect on macrophage function.

The aim of this study was to determine the effects of a corticosteroid on cytokine release by alveolar macrophages from patients with COPD. Consequently, we examined the effects of dexamethasone on IL-8 and GM-CSF release by alveolar macrophages from smokers and patients with COPD under basal conditions and after stimulation with IL-1ß or CSM. We chose to evaluate IL-8 because its concentration is elevated in BAL fluid of smokers and patients with COPD (12, 33–35). Similarly, we chose to evaluate GM-CSF because it is elevated in BAL fluid of patients with chronic bronchitis (36). GM-CSF also enhances neutrophil survival (37), and in patients with chronic bronchitis, it localizes to monocytes–macrophages in sputum (38). We also evaluated the effect of lipopolysaccharide (LPS) on macrophage function to account for possible LPS contamination of CSM.

	METHODS

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Patients
Fifteen patients with COPD (smokers) diagnosed according to American Thoracic Society criteria (39) and 15 current smokers without airway obstruction (FEV₁ of more than 80% predicted) were recruited  . All subjects had a smoking history of more than 20 pack years. COPD subjects maintained their current therapy (ß₂-agonists, n = 14; anticholinergics, n = 15; inhaled corticosteroids, n = 6). Smokers were unmedicated. The study was approved by the Riverside Ethics Committee and the Ethics Committee of the Royal Brompton and Harefield National Health Service Trust. All subjects, including those undergoing diagnostic bronchoscopy, gave informed, written consent.

fig.ommitted TABLE 1. Clinical characteristics and pathology of smokers and patients with chronic obstructive pulmonary disease

 
BAL
BAL was collected according to standard protocols (15) from the right middle lobe or the contralateral lobe to pathology . Sixty milliliters of warmed 0.9% (wt/vol) normal saline was instilled to a maximum of 240 ml. Subjects were monitored with digital oximetry. BAL differential cell counts were performed (40).

Isolation and Culture of Alveolar Macrophages
BAL was filtered and centrifuged, and the washed cells were resuspended in culture medium (RPMI-1640 containing 10% vol/vol fetal calf serum, 2 mM glutamine, 100 i.u./ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphoteracin) at a concentration of a million cells per milliliter (15). For consistency, alveolar macrophages were seeded for all experiments in 24-well Falcon cell culture plates (Becton Dickinson, Cowley, UK) at a density of 250,000 cells/well and were incubated (37°C, 5% CO₂, humidified air) for 2 hours to allow the macrophages to adhere, after which the medium was replaced, removing nonadherent cells. After 24-hour culture, the medium was replaced, and cells were cultured for a further 24 hours under experimental conditions. Cell viability was assessed using Trypan Blue dye exclusion.

CSM
CSM was produced using the method of Wirtz and Schmidt (16). Briefly, smoke from two cigarettes (12-mg tar, 0.9-mg nicotine) was bubbled through a 20-ml culture medium. Absorbance was measured spectrophotometrically, after which the media were diluted approximately 12-fold to give an absorbance of 0.15 at 320 nm. This concentration (nominally one) was serially diluted with untreated media (0.001-fold to 1-fold) and applied to cells. Freshly prepared CSM was used in all experiments.

Cytokine Measurements
GM-CSF and IL-8 were measured in cell-free macrophage culture supernatants using paired antibody quantitative enzyme-linked immunosorbent assays and appropriate blanks (R&D Systems, Abingdon, UK) (31, 41). The lower limit of detection was 15.6 pg/ml for both assays.

Statistical Analysis
Data are presented as means ± SEM. Changes in macrophage secretory products were compared with control subjects using analysis of variance. Comparisons between experimental groups were performed using the Mann-Whitney U test. The concentration of inhibitor causing 50% inhibition of stimulated cytokine release was calculated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

	RESULTS

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Data from all subjects are included. There was no significant difference in the total number of inflammatory cells recovered in BAL from smokers and patients with COPD . There were no significant differences in the number of macrophages and neutrophils in the BAL fluid from these subjects  None of the treatments had any effect on the viability of the macrophages. There were no significant differences in basal or stimulated cytokine release by alveolar macrophages from patients with or without a diagnosis of lung cancer (see Table E1 in the online supplement) or in their response to dexamethasone (see Table E2 in the online supplement).

fig.ommitted TABLE 2. Bronchoalveolar lavage fluid total cell counts and differential cell counts

 
Basal Cytokine Release and the Effect of Stimulation
Basal IL-8 release by alveolar macrophages from smokers was approximately fivefold less than that from patients with COPD  . In contrast, basal GM-CSF release was similar between the two groups . IL-1ß increased IL-8 release by macrophages from smokers in a concentration-dependent manner with a maximal increase of 77% above control at 10 ng/ml . In contrast, maximal IL-1ß–induced IL-8 release by macrophages from patients with COPD was 28% . IL-1ß increased GM-CSF by macrophages from smokers and patients with COPD in a concentration-dependent manner with a maximal increase of 80 and 86%, respectively, above control at 10 ng/ml .

fig.ommitted Figure 1. Effect of IL-1ß, CSM, or LPS on cytokine release by alveolar macrophages from smokers (open squares) and patients with COPD (closed circles). Data are mean ± SEM concentration of IL-8 (A, C, and E) and GM-CSF (B, D, and F) for 15 subjects in each group. For some data points, SEMs are within the symbol. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control subjects, ##p < 0.01, ###p < 0.001 compared with patients with COPD. In panels A, C, and E, all data points for smokers are significantly different from the equivalent values in patients with COPD (p < 0.001). Note that the ordinate is split.

 
CSM increased IL-8 release by macrophages from smokers and patients with COPD in a concentration-dependent manner with maximal increases of 187 and 106%, respectively, above control at a 1x dilution . CSM increased GM-CSF by macrophages from smokers and patients with COPD in a concentration-dependent manner with a maximal increase of 103 and 173%, respectively, above control at a 1x dilution .

Concentrations of IL-8 were significantly higher in macrophages from patients with COPD at all concentrations of IL-1ß and dilutions of CSM . For GM-CSF, there was no significant difference in basal concentrations (discussed earlier here), and this was maintained during stimulation with IL-1ß. In contrast, stimulation with CSM led to a divergence between smokers and patients with COPD in GM-CSF release, with patients with COPD producing significantly more .

LPS (Escherichia coli 055:B5) did not significantly change the release of either IL-8 or GM-CSF by macrophages from either smokers or patients with COPD .

Effect of Dexamethasone on Basal Cytokine Release
Dexamethasone inhibited basal IL-8 release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 27% at 10 µM  . In contrast, dexamethasone had no effect on basal release of IL-8 by macrophages from patients with COPD  . Dexamethasone also inhibited basal GM-CSF release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 57% at 10 µM, and in contrast to its lack of effect on IL-8 release, also inhibited GM-CSF release (by 44%) from patients with COPD . The concentration causing 50% inhibition (IC₅₀) for inhibition by dexamethasone of GM-CSF release by macrophages from smokers was significantly different to that for release by patients with COPD , with the curve shifted significantly to the left .

fig.ommitted Figure 2. Effect of dexamethasone on basal cytokine release by alveolar macrophages from smokers (open squares) and patients with COPD (closed circles). Data are mean ± SEM concentration of IL-8 (A) and GM-CSF (B) for 15 subjects in each group. For some data points, SEMs are within the symbol. ***p < 0.001 compared with control subjects; ##p < 0.01 compared with patients with COPD. In A, all data points for smokers are significantly different to the equivalent values in patients with COPD (p < 0.001); the ordinate is split.

 

fig.ommitted TABLE 3. Inhibition by dexamethasone of basal or stimulated cytokine release from alveolar macrophages

 
Effect of Dexamethasone on IL-1ß–stimulated Cytokine Release
Dexamethasone inhibited IL-1ß–stimulated IL-8 release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 50% at 10 µM . In contrast, dexamethasone had no effect on stimulated IL-8 release by macrophages from patients with COPD . Dexamethasone also inhibited stimulated GM-CSF release by macrophages from smokers in a concentration-dependent manner to below basal levels, and again, in contrast to its lack of effect on IL-8 release (discussed previously here), reduced GM-CSF release to basal levels by macrophages from patients with COPD .

fig.ommitted Figure 3. Effect of dexamethasone on IL-1ß–stimulated cytokine release by alveolar macrophages from smokers (open squares) and patients with COPD (closed circles). Data are mean ± SEM concentration of IL-8 (A) and GM-CSF (B) for 15 subjects in each group. For some data points, SEMs are within the symbol. ***p < 0.001 compared with control subjects (IL-1ß-stimulated, 10 ng/ml), #p < 0.05, ###p < 0.001 compared with patients with COPD. In A, all data points for smokers are significantly different to the equivalent values in patients with COPD (p < 0.001); the ordinate is split. Dashed line is the basal value for smokers, and the dotted line is basal value for patients with COPD.

 
Effect of Dexamethasone on CSM-stimulated Cytokine Release
Dexamethasone inhibited CSM-stimulated IL-8 release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 25% at 10 µM . In contrast, dexamethasone had no effect on stimulated IL-8 release by macrophages from patients with COPD . In contrast to its concentration-dependent inhibition of IL-8 release in smokers, dexamethasone only significantly inhibited (by 42%) stimulated GM-CSF release at the highest concentration used (10 µM) in these subjects . Dexamethasone also had no inhibitory effect on GM-CSF release by macrophages from patients with COPD .

fig.ommitted Figure 4. Effect of dexamethasone on CSM-stimulated cytokine release by alveolar macrophages from smokers (open squares) and patients with COPD (closed circles). Data are mean ± SEM concentration of IL-8 (A) and GM-CSF (B) for 15 subjects in each group. For some data points, SEMs are within the symbol. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control subjects (CSM-stimulated, 1x dilution). In A and B, all data points for smokers are significantly different from the equivalent values in patients with COPD (p < 0.001); the ordinate is split. Dashed line is the basal value for smokers, and the dotted line is the basal value for patients with COPD.

 

	DISCUSSION

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In this study, we found that alveolar macrophages from patients with COPD release approximately fivefold more IL-8 than macrophages from cigarette smokers. This is consistent with the observation that IL-8 is elevated in induced sputum and BAL fluid from patients with COPD (42–44). The data for IL-8 release, as well as that for GM-CSF, under different experimental conditions showed limited variability, as indicated by small SEM values . The precise reason for this is unclear but may be due to the number of patients studied or to selection of homogeneous populations, as indicated by the small variability in clinical parameters between subjects (SEM 10% or less of the mean value; ). Intracellular levels of IL-8 in neutrophils and epithelial cells from patients with COPD are elevated compared with control subjects (45). IL-8 release from epithelial cells in patients with COPD has not been measured. Increased IL-8 release could lead to chemoattraction of lymphocytes, monocytes, and neutrophils into the lungs of patients with COPD (1). In contrast to the difference in basal IL-8 release by macrophages from smokers and patients with COPD, there was no difference in this study in GM-CSF release between the two subject groups. Conversely, baseline concentrations of GM-CSF are elevated in BAL from patients with chronic bronchitis and are further elevated during exacerbations (36). However, in contrast to this study, the control group in the latter study comprised predominantly nonsmokers. The reasons for the difference in profile of basal release of IL-8 and GM-CSF in this study are unclear but may reflect differential regulation and release of inflammatory gene products by macrophages in COPD. In addition, although not measured specifically in this study, possible differences in BAL cytokine and inflammatory mediator profile between the patients with COPD and the smoking control subjects may also affect subsequent macrophage responses in vitro.

In this study, IL-1ß stimulated both IL-8 and GM-CSF release by alveolar macrophages from smokers and patients with COPD. We used IL-1ß as an inflammatory stimulus because its levels are elevated in the BAL fluid of cigarette smokers compared with nonsmokers (46). Although IL-1ß stimulates IL-8 release by BAL macrophages from cigarette smokers (47), IL-1ß–stimulated IL-8 release by macrophages from patients with COPD has not been reported previously. Similarly, although IL-1ß stimulates GM-CSF release by macrophages from patients with asthma and control subjects (25), this study is the first to compare IL-1ß–stimulated GM-CSF release between smokers and patients with COPD. In this study, CSM also stimulated the release of IL-8 and GM-CSF. Stimulation by CSM is unlikely to be due to contamination by LPS because herein LPS alone did not stimulate cytokine release in these cells. The latter observation is in contrast to a number of studies showing that LPS stimulates cytokine release from human alveolar macrophages (48, 49). The reason for this discrepancy is unclear but may be related to a number of factors. For example, responses to LPS can be variable between patients with the same diagnosis (50). In addition, the response by macrophages from smokers is less than that in nonsmokers (51). In a number of cases, the concentrations of LPS exceed that used in this study (48, 49, 52). Also, LPS elicits an increased secretion of cytokines that is inverse to the basal secretion (53). In this study, we observed a marked basal secretion of IL-8 and GM-CSF. Finally, there is inconsistency in the serotype of LPS used between published studies, which hinders a comparison with our present observations. We do not know the serotype of the LPS that may be present in our samples of CSM. Therefore, we cannot exclude a contribution of LPS to our CSM data.

Stimulation by CSM is consistent with release of tumor necrosis factor- and IL-6 by alveolar macrophages from normal subjects after exposure to tobacco smoke (54). The mechanism(s) of CSM-mediated cytokine release by macrophages is not investigated in this study. Cigarette smoke contains 4,700 compounds, including radicals, hydrogen peroxide, peroxynitrite, and acrolein (55). A number of these are found in aqueous solutions of smoke, including hydrogen peroxide (56) and semiquinone radicals that can react with oxygen to produce O₂^.- (57). Reactive oxygen species activate transcription factors, including nuclear factor-B (NF-B) and activator protein-1, which regulate expression of inflammatory genes such as IL-8 and GM-CSF (58). However, the reactive oxygen species content of our CSM was not determined, and in addition, soluble CS particulates may also have contributed to the increased macrophage activity observed herein. There is scant literature on the validity of comparison between CSM and in vivo exposure to cigarette smoke. In this study, the relationship between CSM and exposure of macrophages to cigarette smoke in vivo is not known. However, in rats, cigarette smoke condensates in vitro and cigarette smoke in vivo induce similar patterns of DNA damage (59).

The profile of release of IL-8 by IL-1ß and CSM was similar for both subject groups. The profile of IL-1ß–stimulated GM-CSF release was also similar. In contrast, CSM-stimulated release of GM-CSF was elevated in macrophages from patients with COPD compared with smokers. The reason for selective elevation of CSM-induced GM-CSF release is unknown but may be due to increased oxidant sensitivity of macrophages from patients with COPD.

In this study, dexamethasone had different inhibitory effects on cytokine release by alveolar macrophages from cigarette smokers and patients with COPD. The most striking difference was the lack of inhibitory effect of dexamethasone on IL-8 release by macrophages from patients with COPD compared with the inhibition by macrophages from smokers. In macrophages from normal volunteers, fluticasone proprionate or budesonide inhibit LPS-induced IL-8 release by alveolar macrophages by approximately 33% and approximately 60%, respectively (24). In addition, dexamethasone inhibits IL-1ß–induced IL-8 release by 64% in macrophages from normal subjects but only by 29% in cigarette smokers (47). Our present observation extends these findings and demonstrates a trend to increased resistance to steroids by macrophages from normal subjects to smokers to patients with COPD. The mechanisms underlying the relative steroid insensitivity of macrophages from patients with COPD in this study are not investigated but include altered glucocorticoid receptor function and apoptosis. There is no difference in glucocorticoid receptor expression in mononuclear cells in bronchial biopsies from patients with chronic bronchitis compared with nonsmoking control subjects (60). In contrast, there are more apoptotic macrophages in bronchial biopsies from patients with chronic bronchitis than from patients with asthma or healthy control subjects (61). Specific studies are required to determine the functional significance of these observations to macrophage corticosteroid insensitivity in COPD.

In contrast to its lack of inhibitory effect on IL-8 release by macrophages from patients with COPD, dexamethasone inhibited basal and IL-1ß–stimulated GM-CSF release and. However, macrophages from patients with COPD were less responsive than those from smokers, and the concentration–response curve was shifted to the right. These observations indicate a differential cytokine-specific effect of dexamethasone. This suggestion is consistent with the observation that dexamethasone inhibits IL-8 release by only approximately 50% compared with complete inhibition of GM-CSF release from human primary airway epithelial cells (41), which indicates differential corticosteroid sensitivity of inflammatory genes. In this study, in contrast to IL-1ß stimulation, GM-CSF release after CSM exposure was steroid insensitive . Similarly, dexamethasone did not inhibit IL-1ß–stimulated tumor necrosis factor- release by alveolar macrophages from cigarette smokers compared with nonsmokers (47). This lack of inhibitory effect of dexamethasone on cytokine release was mimicked by hydrogen peroxide treatment of a macrophage-like cell line (47). These combined observations suggest that oxidative mechanisms contribute, at least in part, to CSM stimulation of cytokine release by alveolar macrophages. This further indicates that steroid responsiveness of cytokine release by macrophages is both stimulus and cytokine-dependent. This proposal is consistent with the observation that human rhinovirus-induced respiratory epithelial cell expression of IL-8 and IL-6 is via an NF-B–independent pathway, whereas induction of GM-CSF is partially dependent on NF-B activation (62). Because glucocorticoids inhibit NF-B activity (63), the greater inhibition seen herein by dexamethasone on GM-CSF production compared with IL-8 production may reflect a greater relative contribution of NF-B activity to GM-CSF gene transcription rather than IL-8 gene transcription. However, this is not likely to be a general rule and may be an oversimplification, as IL-8 responses can also be mediated via NF-B (64).

The clinical efficacy of corticosteroids in COPD is controversial (28, 29). However, neither high doses of inhaled nor oral corticosteroid treatment reduces markers of airway inflammation, including IL-8, in induced sputum in patients with COPD (30, 31, 44). Any lack of efficacy could be due to a reduced steroid sensitivity by macrophages, the predominant inflammatory cell in COPD (7). In this study, we show that alveolar macrophages from patients with COPD display reduced sensitivity to dexamethasone compared with macrophages from smokers. Specifically, inhibition of the neutrophil chemotactic factor, IL-8, and the cell survival cytokine, GM-CSF, was reduced. It should be noted that the difference in inhibition by dexamethasone of cytokine release between smokers and patients with COPD in this study was relatively small. However, when combined with the comparative lack of effect of corticosteroids on other aspects of COPD pathophysiology, for example, reduced inhibition of neutrophil apoptosis (1), small reductions in efficacy could be additive and become clinically significant. Thus, any lack of efficacy of steroids on macrophage activity in COPD could lead to reduced inhibition of neutrophil chemoattractants and increased survival, with perpetuation of pulmonary neutrophilic inflammation. It is difficult to predict at this stage of understanding of the pathophysiology of COPD the relative impact on disease progression of individual experimental observations.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

1. Barnes PJ. Chronic obstructive pulmonary disease. N Engl J Med 2000;343:269–280.

2. Scanlon PD, Connett JE, Waller LA, Altose MD, Bailey WC, Buist AS. Smoking cessation and lung function in mild-to-moderate chronic obstructive pulmonary disease: the Lung Health Study. Am J Respir Crit Care Med 2000;161:381–390.

3. Culpitt SV, Rogers DF. Evaluation of current pharmacotherapy of chronic obstructive pulmonary disease. Expert Opin Pharmacother 2000;1:1007–1020.

4. Jeffery PK. Comparison of the structural and inflammatory features of COPD and asthma: Giles F. Filley Lecture. Chest 2000;117:251S–260S.

5. Saetta M, Turato G, Zuin R. Structural basis for airflow limitation in chronic obstructive pulmonary disease. Sarcoidosis Vasc Diffuse Lung Dis 2000;17:239–245.

6. Saetta M, Turato G, Baraldo S, Zanin A, Braccioni F, Mapp CE, Maestrelli P, Cavallesco G, Papi A, Fabbri LM. Goblet cell hyperplasia and epithelial inflammation in peripheral airways of smokers with both symptoms of chronic bronchitis and chronic airflow limitation. Am J Respir Crit Care Med 2000;161:1016–1021.

7. Shapiro SD. The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:S29–S32.

8. Lohmann-Matthes ML, Steinmuller C, Franke-Ullmann G. Pulmonary macrophages. Eur Respir J 1994;7:1678–1689.

9. Mio T, Romberger DJ, Thompson AB, Robbins RA, Heires A, Rennard SI. Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells. Am J Respir Crit Care Med 1997;155:1770–1776.

10. Fantone JC, Ward PA. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1982;107:395–418.

11. Linden M, Rasmussen JB, Piitulainen E, Tunek A, Larson M, Tegner H, Venge P, Laitinen LA, Brattsand R. Airway inflammation in smokers with nonobstructive and obstructive chronic bronchitis. Am Rev Respir Dis 1993;148:1226–1232.

12. Riise GC, Ahlstedt S, Larsson S, Enander I, Jones I, Larsson P, Andersson B. Bronchial inflammation in chronic bronchitis assessed by measurement of cell products in bronchial lavage fluid. Thorax 1995;50:360–365.

13. Hunninghake GW, Crystal RG. Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 1983;128:833–838.

14. Costabel U, Guzman J. Effect of smoking on bronchoalveolar lavage constituents. Eur Respir J 1992;5:776–779.

15. Lim S, Roche N, Oliver BG, Mattos W, Barnes PJ, Chung KF. Balance of matrix metalloprotease-9 and tissue inhibitor of metalloprotease-1 from alveolar macrophages in cigarette smokers: regulation by interleukin-10. Am J Respir Crit Care Med 2000;162:1355–1360.

16. Wirtz HR, Schmidt M. Acute influence of cigarette smoke on secretion of pulmonary surfactant in rat alveolar type II cells in culture. Eur Respir J 1996;9:24–32.

17. Francus T, Romano PM, Manzo G, Fonacier L, Arango N, Szabo P. IL-1, IL-6, and PDGF mRNA expression in alveolar cells following stimulation with a tobacco-derived antigen. Cell Immunol 1992;145:156–174.

18. Barnes PJ. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci (Lond) 1998;94:557–572.

19. John M, Au BT, Jose PJ, Lim S, Saunders M, Barnes PJ, Mitchell JA, Belvisi MG, Chung KF. Expression and release of interleukin-8 by human airway smooth muscle cells: inhibition by Th-2 cytokines and corticosteroids. Am J Respir Cell Mol Biol 1998;18:84–90.

20. Seldon PM, Stevens DA, Adcock IM, O'Connor BJ, Barnes PJ, Giembycz MA. Albuterol does not antagonize the inhibitory effect of dexamethasone on monocyte cytokine release. Am J Respir Crit Care Med 1998;157:803–809.

21. Kwon OJ, Au BT, Collins PD, Baraniuk JN, Adcock IM, Chung KF, Barnes PJ. Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells. Immunology 1994;81:389–394.

22. Deaton PR, McKellar CT, Culbreth R, Veal CF, Cooper JA Jr. Hyperoxia stimulates interleukin-8 release from alveolar macrophages and U937 cells: attenuation by dexamethasone. Am J Physiol 1994;267:L187–L192.

23. Lin G, Pearson AE, Scamurra RW, Zhou Y, Baarsch MJ, Weiss DJ, Murtaugh MP. Regulation of interleukin-8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide. J Biol Chem 1994;269:77–85.

24. Ek A, Larsson K, Siljerud S, Palmberg L. Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages. Allergy 1999;54:691–699.

25. John M, Lim S, Seybold J, Jose P, Robichaud A, O'Connor B, Barnes PJ, Chung KF. Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma. Am J Respir Crit Care Med 1998;157:256–262.

26. Wenzel SE, Trudeau JB, Westcott JY, Beam WR, Martin RJ. Single oral dose of prednisone decreases leukotriene B4 production by alveolar macrophages from patients with nocturnal asthma but not control subjects: relationship to changes in cellular influx and FEV1. J Allergy Clin Immunol 1994;94:870–881.

27. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987;136:225–244.

28. Calverley PM. Inhaled corticosteroids are beneficial in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;161:341–342.

29. Barnes PJ. Inhaled corticosteroids are not beneficial in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;161:342–344.

30. Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997;155:542–548.

31. Culpitt SV, Maziak W, Loukidis S, Nightingale JA, Matthews JL, Barnes PJ. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:1635–1639.

32. Loppow D, Schleiss MB, Kanniess F, Taube C, Jorres RA, Magnussen H. In patients with chronic bronchitis a four week trial with inhaled steroids does not attenuate airway inflammation. Respir Med 2001;95:115–121.

33. Soler N, Ewig S, Torres A, Filella X, Gonzalez J, Zaubet A. Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease. Eur Respir J 1999;14:1015–1022.

34. Rutgers SR, Timens W, Kaufmann HF, van der Mark TW, Koeter GH, Postma DS. Comparison of induced sputum with bronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J 2000;15:109–115.

35. Tanino M, Betsuyaku T, Takeyabu K, Tanino Y, Yamaguchi E, Miyamoto K, Nishimura M. Increased levels of interleukin-8 in BAL fluid from smokers susceptible to pulmonary emphysema. Thorax 2002;57:405–411.

36. Balbi B, Bason C, Balleari E, Fiasella F, Pesci A, Ghio R, Fabiano F. Increased bronchoalveolar granulocytes and granulocyte/macrophage colony-stimulating factor during exacerbations of chronic bronchitis. Eur Respir J 1997;10:846–850.

37. Lopez AF, Williamson DJ, Gamble JR, Begley CG, Harlan JM, Klebanoff SJ, Waltersdorph A, Wong G, Clark SC, Vadas MA. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J Clin Invest 1986;78:1220–1228.

38. Hoshi H, Ohno I, Honma M, Tanno Y, Yamauchi K, Tamura G, Shirato K. IL-5, IL-8 and GM-CSF immunostaining of sputum cells in bronchial asthma and chronic bronchitis. Clin Exp Allergy 1995;25:720–728.

39. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1995;152:S77–S120.

40. Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2002;26:602–609.

41. Donnelly LE, Barnes PJ. Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells. Am J Respir Cell Mol Biol 2002;26:144–151.

42. Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153:530–534.

43. Nocker RE, Schoonbrood DF, van de Graaf EA, Hack CE, Lutter R, Jansen HM, Out TA. Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease. Int Arch Allergy Immunol 1996;109:183–191.

44. Yamamoto C, Yoneda T, Yoshikawa M, Fu A, Tokuyama T, Tsukaguchi K, Narita N. Airway inflammation in COPD assessed by sputum levels of interleukin-8. Chest 1997;112:505–510.

45. de Boer WI, Sont JK, van Schadewijk A, Stolk J, van Krieken JH, Hiemstra PS. Monocyte chemoattractant protein 1, interleukin 8, and chronic airways inflammation in COPD. J Pathol 2000;190:619–626.

46. Ekberg-Jansson A, Andersson B, Bake B, Boijsen M, Enanden I, Rosengren A, Skoogh BE, Tylen U, Venge P, Lofdahl CG. Neutrophil-associated activation markers in healthy smokers relates to a fall in DL(CO) and to emphysematous changes on high resolution CT. Respir Med 2001;95:363–373.

47. Ito K, Lim S, Caramori G, Chung KF, Barnes PJ, Adcock IM. Cigarette smoking reduces histone deacetylase 2 expression, enhances cytokine expression, and inhibits glucocorticoid actions in alveolar macrophages. FASEB J 2001;15:1110–1112.

48. Goodman RB, Strieter RM, Frevert CW, Cummings CJ, Tekamp-Olson P, Kunkel SL, Walz A, Martin TR. Quantitative comparison of C-X-C chemokines produced by endotoxin-stimulated human alveolar macrophages. Am J Physiol 1998;275:L87–L95.

49. Abe Y, Hashimoto S, Horie T. Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. Pharmacol Res 1999;39:41–47.

50. Gosset P, Wallaert B, Tonnel AB, Fourneau C. Thiol regulation of the production of TNF-alpha, IL-6 and IL-8 by human alveolar macrophages. Eur Respir J 1999;14:98–105.

51. Ohta T, Yamashita N, Maruyama M, Sugiyama E, Kobayashi M. Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages. Respir Med 1998;92:922–927.

52. Ek A, Larsson K, Siljerud S, Palmberg L. Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages. Allergy 1999;54:691–699.

53. Losa Garcia JE, Rodriguez FM, Martin de Cabo MR, Garcia Salgado MJ, Losada JP, Villaron LG, Lopez AJ, Arellano JL. Evaluation of inflammatory cytokine secretion by human alveolar macrophages. Mediators Inflamm 1999;8:43–51.

54. Dubar V, Gosset P, Aerts C, Voisin C, Wallaert B, Tonnel AB. In vitro acute effects of tobacco smoke on tumor necrosis factor alpha and interleukin-6 production by alveolar macrophages. Exp Lung Res 1993;19:345–359.

55. Church DF, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspect 1985;64:111–126.

56. Nakayama T, Church DF, Pryor WA. Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts. Free Radic Biol Med 1989;7:9–15.

57. Pryor WA, Stone K. Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann NY Acad Sci 1993;686:12–27.

58. MacNee W, Rahman I. Oxidants and antioxidants as therapeutic targets in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:S58–S65.

59. Randerath E, Danna TF, Randerath K. DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo. Mutat Res 1992;268:139–153.

60. Vachier I, Chiappara G, Vignola AM, Gagliardo R, Altieri E, Terouanne B, Vic P, Bousquet J, Godard P, Chanez P. Glucocorticoid receptors in bronchial epithelial cells in asthma. Am J Respir Crit Care Med 1998;158:963–970.

61. Vignola AM, Chanez P, Chiappara G, Siena L, Merendino A, Reina C, Gagliardo R, Profita M, Bousquet J, Bonsignore G. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J Allergy Clin Immunol 1999;103:563–573.

62. Kim J, Sanders SP, Siekierski ES, Casolaro V, Proud D. Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection. J Immunol 2000;165:3384–3392.

63. Newton R. Molecular mechanisms of glucocorticoid action: what is important? Thorax 2000;55:603–613.

64. Friedland JS, Constantin D, Shaw TC, Stylianou E. Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells. J Leukoc Biol 2001;70:447–454.