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Published ahead of print on March 30, 2006, doi:10.1164/rccm.200505-725OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 41-50, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200505-725OC

Original Article

The Effect of Smoking on the Transcriptional Regulation of Lung Inflammation in Patients with Chronic Obstructive Pulmonary Disease

Patryk Szulakowski, Ann J. L. Crowther, Luis A. Jiménez, Kenneth Donaldson, Ruth Mayer, Thomas B. Leonard, William MacNee and Ellen M. Drost

ELEGI Colt Laboratories, MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; and GlaxoSmithKline, King of Prussia, Pennsylvania

Correspondence and requests for reprints should be addressed to Prof. William MacNee, M.D., ELEGI Colt Laboratories, MRC Centre for Inflammation Research, University of Edinburgh, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: w.macnee{at}ed.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Chronic obstructive pulmonary disease (COPD) is believed to result from an abnormal inflammatory response in the lungs to noxious particles and gases usually found in cigarette smoke.

Objectives: In this study, the molecular mechanisms for the enhanced proinflammatory cytokine gene transcription in COPD were investigated.

Methods: Lung tissue was examined from 56 subjects undergoing resection for peripheral lung tumors as follows: current smokers with (n = 14) and without COPD (n = 17), ex-smokers with COPD (n = 13), and nonsmokers (n = 12). The levels of inhibitor {kappa}B-{alpha} (I{kappa}B-{alpha}), histone deacetylase 2 (HDAC2), acetylated (ac-) histone H3 and H4, the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), proinflammatory cytokine messenger RNA, and 8-isoprostane were measured.

Measurements and Main Results: I{kappa}B-{alpha} levels were significantly decreased in healthy smokers and current and ex-smoking patients with COPD when compared with nonsmokers (p < 0.001), with an associated increase in NF-{kappa}B DNA binding in current smokers (p < 0.05). An increase in acetylated histone 4 (ac-H4; p < 0.01) was found in current smokers. Conversely, ex-smokers with COPD showed an increase in ac-H3 (p < 0.05). Decreased levels of cytoplasmic, but not nuclear, HDAC2 protein levels were detected. From the cytokine profiles, no significant differences were detected; however, interleukin-12p40 expression correlated with ac-H4 in current smokers with COPD (p < 0.01).

Conclusion: These data propose a role for modification of nucleosomal structure in inflammatory cytokine gene transcription in response to smoking. The imbalance between histone deacetylation and acetylation in favor of acetylation may contribute to the enhanced inflammation in smokers susceptible to the development of COPD.

Key Words: chronic obstructive pulmonary disease • histone acetylation • nuclear factor-{kappa} • B • oxidative stress • smoking

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide, affecting 4 to 6% of people over the age of 45 yr. The prevalence of this disease is expected to rise in the future and the World Health Organization predicts that COPD will be the third major cause of death by 2020 (1, 2).

COPD is characterized by progressive airflow obstruction, which is not fully reversible, and is believed to result from an abnormal inflammatory response in the lungs to noxious gases and particles largely in cigarette smoking (3). Relatively little is known about the inflammatory response in smokers' lungs and how this relates to disease susceptibility (4).

One hypothesis is that cigarette smoke–induced oxidative stress (5) initiates activation of signal transduction systems, such as the redox-sensitive transcription factors, nuclear factor-{kappa}B (NF-{kappa}B) and activator protein 1 (AP-1) (6), which have a central role in regulating a wide range of inflammatory responses through the expression of many proinflammatory genes. In unstimulated cells, NF-{kappa}B is found in the cytoplasm in an inactive non-DNA binding form, associated with its inhibitory protein {kappa}B{alpha} (I{kappa}B{alpha}) (7). I{kappa}B{alpha} degradation unmasks the nuclear localization signal present in NF-{kappa}B, allowing it to enter the nucleus, bind DNA, and initiate gene transcription. Gene transcription is, however, not only dependent on the binding of transcription factors but also on the modification of core histone proteins that regulate the accessibility of the genome to transcription factors and cofactors (8). DNA is tightly wrapped around an octamer containing two copies of each histone core protein, H2A, H2B, H3, and H4 (9). These histones contain many N-terminal side chains, which can undergo post-translational modification, such as acetylation, phosphorylation, and methylation. In interphase, lysine residues in the N-terminal tails are deacetylated, producing a condensed DNA configuration resulting in inhibition of gene transcription. Conversely, lysine residues can be acetylated by histone acetyltransferases (HATs), which neutralize their positive charge and result in chromatin relaxation. Such unwinding of DNA from chromatin increases the accessibility of transcription factors and RNA polymerase II to the transcriptional machinery, enhancing gene transcription (10). Disruption of the acetylation/deacetylation balance in favor of acetylation may lead to sustained gene transcription of proinflammatory genes controlled by NF-{kappa}B and hence chronic inflammation. Therefore, we hypothesized that histone acetylation/deacetylation imbalance accounts for the enhanced inflammatory response in "susceptible" smokers who develop COPD. We obtained lung tissue from nonsmokers and smokers with and without COPD who underwent lung resection for peripheral tumors. We show a clear effect of cigarette smoking on I{kappa}B{alpha} degradation, NF-{kappa}B translocation to the nucleus and acetylation of histone H4 as a marker of chromatin condensation and transcriptional accessibility. We demonstrate increased intracellular oxidative stress by the presence of increased levels of 8-isoprostane in current smokers. We also show that smoking cessation in patients with COPD is associated with increased histone H3 acetylation, which suggests that the persistence of the inflammation in the lungs in COPD after smoking cessation may be regulated by H3 acetylation. This study shows a clear impact of cigarette smoking on chromatin remodeling in the lungs. Some of the results of these studies have been previously reported in abstract form (11).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Peripheral (distal) lung tissue specimens were obtained from patients who underwent lung resection for peripheral lung cancers. All patients included in the study gave their consent after a full discussion of the nature of the study, which had been approved by the Lothian Region Ethics Committee. No patient in the study had chemotherapy or radiotherapy before lung resection. We included 56 subjects: current smokers with COPD (n = 14) and without COPD ("healthy smokers"; n = 17), ex-smokers with COPD (n = 13), and a nonsmoking control group (n = 12). COPD was defined according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (3). All patients with COPD had airflow limitation (FEV1 < 80% predicted, FEV1/FVC < 70%, GOLD stage 2). Spirometry was normal in the control subjects and healthy smokers. Patients with COPD had treatment with inhaled bronchodilators but not with inhaled corticosteriods or theophyllines. All subjects were characterized with respect to sex, age, smoking history, COPD symptoms, comorbidity, and current medical treatment. Patients with COPD and healthy smokers had a smoking history of 10 pack-years or more. Ex-smokers had stopped smoking at least 1 yr before surgery. Exclusion criteria included the following: other systemic diseases, other lung diseases apart from COPD and lung tumors, pulmonary infection and antibiotic treatment 4 wk before operation or inhaled or oral glucocorticoids in the 3 mo before operation, obstruction of central bronchi due to the tumor, and absence of tumor-free or pneumonia-free lung tissue specimens.

Tissue Homogenization
A total of 100 mg of human lung tissue per patient was homogenized for 1 min in lysis buffer containing 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM NaF, 50 mM beta-glycerophosphate, a protease inhibitor tablet, 0.2 mM Na-orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotenin, and 10% Nonidet P-40. Thereafter, the samples were incubated on ice for 15 min and then centrifuged at 13,000 x g for 30 s. The resulting supernatants (cytosolic fraction) were then used for the measurement of I{kappa}B{alpha}, phosphorylated and native p38 mitogen-activated protein kinase (MAPK), histone deacetylase 2 (HDAC2), and isoprostane levels.

The cell pellets containing nuclei were retained and resuspended in extracting buffer (50 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 50 mM KCl, 300 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM NaF, 0.2 mM Na-orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotenin, 50 mM beta-glycerophosphate, and a protease inhibitor tablet (Complete Mini; Roche Diagnostics, Mannheim, Germany). The samples were incubated on a rotating platform for 30 min at 4°C followed by centrifugation at 13,000 x g for 5 min. The resulting nuclear extract was used for the determination of HDAC2 and Ph-p38 MAPK levels and nuclear translocation of the transcription factor NF-{kappa}B. Integrity of the cytoplasmic and nuclear fractions was demonstrated by blotting for a nuclear protein, lamin A/C (see Figure E1 of the online supplement) shows a representative blot for seven random samples with minimal contamination of the cytoplasmic extract with nuclear proteins. Acid-soluble histones were purified from the retained pellets by a method modified from Ito and colleagues (12) and used to measure acetylated histones H3 and H4. Protein concentrations were measured using a BCA kit (Sigma-Aldrich, Irvine, Scotland).

Western Blotting
For Western blots, 8 to 40 µg of isolated soluble proteins were separated by polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes, and treated with antibodies to HDAC2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti–I{kappa}B{alpha} (Santa Cruz Biotechnology), anti–acetylated histones H3 and H4 (Upstate, Milton Keynes, UK), and anti–beta-actin (Abcam, Cambridge, UK). After washing, bound antibody was detected using respectively anti-rabbit (Santa Cruz Biotechnology), anti-goat (Santa Cruz Biotechnology), or anti-mouse antibody (Abcam) linked to horseradish peroxidase, and the bound complexes were detected using enhanced chemiluminescence (ECL) (Amersham-Pharmacia, Chalfont St. Gibes, UK). The constitutively expressed protein, beta-actin, served as an internal control, and the data were presented as a ratio to beta-actin.

Electrophoretic Mobility Shift Assay
A total of 10 µg of nuclear extract protein was incubated with {gamma}32P-ATP end-labeled double-stranded NF-{kappa}B or AP-1 consensus probe (Promega, Southampton, UK) as reported previously (13). Band density was determined using a PhosphorImager system (STORM; Molecular Dynamics, Sunnyvale, CA).

cDNA Preparation and Reverse Transcriptase–Polymerase Chain Reaction
RNA extraction was performed using the Trizol method as reported previously (13). Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to determine the expression of the following proinflammatory cytokines, using a Multiplex PCR kit (BioSource, Nivelles, Belgium): interleukin (IL)-8, IL-6, IL-1beta, tumor necrosis factor (TNF)-{alpha}, IL-12p40, IL-2, and IL-13.

8-Isoprostane Enzyme Immunoassay
8-Isoprostane concentrations in cytoplasmic extracts from lung tissue were measured by specific enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). The lysis reagent was added to the standard at the same concentration as for the samples. The samples were analyzed in duplicate. The detection limit of the assay was 4 pg/ml.

Statistical Analysis
The data are expressed as means ± SEM. Statistical significance was calculated using one-way analysis of variance (ANOVA) or Kruskal-Wallis nonparametric ANOVA followed by Tukey-Kramer's or Dunn's post hoc tests for multiple comparisons respectively.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Healthy smokers and non-smokers had normal lung function, whereas patients with COPD had airflow obstruction (30% <= FEV1 < 80% predicted, FEV1/FVC < 70%). The number of pack-years was not significantly different between the smoking groups. A summary of the lung function data is shown in Table 1.


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  		TABLE 1. SUBJECTS' CHARACTERISTICS

 
Inhibitory Protein I{kappa}B{alpha}
We measured I{kappa}B{alpha} protein levels to determine the role its degradation might have on the activation of proinflammatory gene transcription. A significant decrease in I{kappa}B{alpha} levels was found in current smokers with and without COPD (healthy smokers) when compared with nonsmokers (Figure 1), with an even greater decrease observed in ex-smokers with COPD (p < 0.001).


Figure 1
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  		Figure 1. I{kappa}B-{alpha} protein levels were detected in human lung tissue. (A) A representative Western blot for I{kappa}B-{alpha} expressed as a ratio to beta-actin. (B) Mean (± SEM) values showing a significant reduction in I{kappa}B{alpha} protein levels in smokers and patients with chronic obstructive pulmonary disease (COPD; analysis of variance [ANOVA], p = 0.0001). Subject numbers are shown in parentheses. (Comparisons shown: *p<0.05, **p<0.01, ***p<0.001).

 
Nuclear Translocation of the Transcription Factors NF-{kappa}B and AP-1
Translocation of NF-{kappa}B into the nucleus was significantly increased in the lungs of healthy smokers and current smokers with COPD, suggesting an effect of current cigarette smoking (Figure 2). NF-{kappa}B DNA binding in the lungs of ex-smokers with COPD was not significantly different from nonsmokers. Furthermore, nuclear binding of the transcription factor NF-{kappa}B correlated significantly with the number of pack-years (p = 0.032, r = 0.33; Figure 3). AP-1 nuclear translocation was not significantly different for the patient groups; however, the data showed a similar trend as that seen for NF-{kappa}B, with an increase in DNA binding for smokers but not ex-smokers (nonsmokers: 651.6 ± 125.3, n = 8; healthy smokers: 934.6 ± 142.7, n = 19; current COPD: 835.6 ± 140.8, n = 12; ex-smoker COPD: 493.9 ± 84.9, n = 6; p > 0.05).


Figure 2
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  		Figure 2. Nuclear translocation of the transcription factor nuclear factor (NF)-{kappa}B. (A) Electrophoretic mobility shift assay for NF-{kappa}B (neg = negative control; NC = noncompetitor; CC = cold competitor). (B) Significant increase of nuclear translocation of NF-{kappa}B was observed in healthy smokers and current smokers with COPD (ANOVA, p = 0.0044; comparisons shown, *p < 0.05).

 

Figure 3
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  		Figure 3. Positive correlation between NF-{kappa}B and the number of pack-years of cigarette smoking.

 
HDAC2
To determine whether the enhanced inflammatory response in patients with COPD was associated with reduced levels of HDAC2, we measured cytoplasmic and nuclear HDAC2 protein by Western blotting. Our study did not show any significant differences in nuclear HDAC2 protein levels between the groups (Figure 4A). However, there was a significant decrease observed in cytoplasmic HDAC2 levels in patients with COPD, particularly in those who were ex-smokers (Figure 4B). This may be partly explained by a smoking effect on HDAC2 protein levels because a reduction in cytoplasmic (p < 0.05), but not nuclear, protein levels (p = 0.296) was seen where more time had elapsed since the patient last smoked a cigarette (Figures 5A and 5B, respectively). However, in expressing the data relative to GOLD COPD severity criteria, we also found a decrease in HDAC2 levels with increasing disease severity (Figures 6A and 6B), suggesting a disease effect. Consistent with an effect of COPD, we found significant correlations between the level of cytoplasmic HDAC2 and FEV1/FVC % predicted in all subjects (p = 0.008, r = 0.45, n = 34; Figure 7A) and nuclear HDAC2 and FEV1% predicted in all subjects (p = 0.02, r = 0.34, n = 46; Figure 7B).


Figure 4
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  		Figure 4. Histone deacetylase 2 (HDAC2) expressed as a ratio to beta-actin in (A) nuclear extracts and (B) cytoplasmic extracts. A significant decrease in cytoplasmic levels of HDAC2 were found (ANOVA; nuclear HDAC2, p = 0.16; cytoplasmic HDAC2, p < 0.001; compared with nonsmokers: *p < 0.05, **p < 0.01; compared with healthy smokers: #p < 0.05).

 

Figure 5
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  		Figure 5. The levels of (A) cytoplasmic and (B) nuclear HDAC2 protein are shown relative to the time since last cigarette. An effect of cigarette smoking was seen for cytoplasmic (ANOVA, p = 0.03) but not nuclear (ANOVA, p = 0.3) HDAC2 levels (compared with nonsmokers [NS], *p < 0.05).

 

Figure 6
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  		Figure 6. The levels of HDAC2 protein were graphed according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) severity categories for (A) cytoplasmic and (B) nuclear levels of HDAC2 protein expressed as a ratio to beta-actin. A decrease in HDAC2 levels was associated with increasing disease severity, which was significant for cytoplasmic levels (ANOVA, p = 0.0113) but not for nuclear HDAC2 levels (ANOVA, p = 0.091; compared with nonsmokers, **p < 0.05).

 

Figure 7
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  		Figure 7. Positive correlations between the level of (A) cytoplasmic HDAC2 protein and FEV1/FVC % predicted (p = 0.0077, r = 0.45, n = 34) and (B) nuclear HDAC2 and FEV1% predicted in all subjects (p = 0.02, r = 0.341, n = 46).

 
Acetylated Histones H3 and H4
We examined the expression of acetylated H3 and H4 to establish whether increased acetylation of histones H3 and H4 occurs as a mechanism responsible for increased DNA accessibility for transcription factors in COPD. A significant increase in acetylated H4 levels (p < 0.05; Figure 8A) was observed in healthy smokers (231%) when compared with nonsmokers (100%). The level of H4 acetylation was significantly lower in ex-smokers than in healthy smokers (p < 0.01). These data suggest an effect of smoking, which was apparent, although not significant, when acetylated H4 levels were expressed relative to the time since last cigarette smoked (p = 0.091; Figure 8B). A significant increase in acetylated H3 was seen for ex-smokers with COPD (382%, when compared with nonsmokers, 100%; p < 0.05), but no significant differences were detected for healthy smokers and current smokers with COPD (120.6 and 121.5%, respectively; Figure 9).


Figure 8
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  		Figure 8. The level of acetylated histone H4 (ac H4) protein (A) expressed as a ratio to beta-actin and (B) illustrating the effect of smoking. (A) A significant increase in acetylated H4 was observed in healthy smokers and current smokers with COPD (ANOVA, p = 0.0035; compared with nonsmokers: *p < 0.05, **p < 0.001). Mean (± SEM) values are shown. Subject numbers are shown in parentheses. (B) Individual data points are plotted relative to time since last cigarette smoked (NS = nonsmokers; ANOVA, p = 0.0913).

 

Figure 9
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  		Figure 9. The level of acetylated histone H3 (ac H3) expressed as a ratio to beta-actin. A significant increase in acetylated H3 was measured in ex-smokers with COPD (ANOVA, p = 0.03). Mean (± SEM) values are shown. The number of individual subjects is shown in parentheses (compared with nonsmokers, *p < 0.05)

 
Proinflammatory Cytokine Gene Expression
An enhanced inflammatory response in the lungs in patients with COPD has been associated with increased cytokine levels in the airways. To screen the expression of genes coding proinflammatory cytokines, we performed Multiplex RT-PCR for IL-8, IL-6, IL-1beta, TNF-{alpha}, IL-12p40, IL-2, and IL-13. There were no significant differences in mRNA expression among the groups (Figure 10). However, there was a trend for an increase in mRNA expression in smokers with COPD for IL-12p40, and this cytokine was positively correlated with the levels of histone H4 acetylation (p = 0.01, r = 0.69, n = 13; Figure 11).


Figure 10
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  		Figure 10. Proinflammatory cytokine gene expression as a ratio to GAPDH. (A) Multipex reverse transcriptase–polymerase chain reaction for proinflammatory cytokine gene expression (bp = base pairs; MW = molecular weight standards; NC = negative control; PC = positive control). (B) There were no significant differences in mRNA expression between the groups. Mean (± SEM) values are shown.

 

Figure 11
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  		Figure 11. Positive correlation between the level of acetylated histone H4 (ac H4) and IL-12p40 mRNA in current smokers with COPD (p = 0.0095, r = 0.687, n = 13).

 
8-Isoprostane
To demonstrate the presence of an oxidative stress in the lung tissue, levels of 8-isoprostane were measured. No significant difference was found; however, a clear trend was present in smokers' lungs, suggesting the presence of an increased oxidant burden (p = 0.077; Figure 12A). Expressing the data relative to the time since the patient last smoked a cigarette demonstrates the presence of a smoking-generated oxidant burden (p < 0.05; Figure 12B).


Figure 12
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  		Figure 12. The levels of 8-isoprostane as (A) a marker of oxidative stress are shown and (B) graphed relative to last cigarette smoked. (A) No significant differences in the levels were detected (ANOVA, p = 0.077), although a clear trend is evident, suggesting cigarette smoking causes an oxidant burden in lung tissue. The number of individual subjects is shown in parenthesess. (B) 8-Isoprostane levels expressed relative to time since last cigarette smoked suggest the presence of an oxidative stress as a result of smoking (ANOVA, p = 0.014; comparison shown, p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An abnormal inflammatory response in the lungs to inhaled particles or gases, usually cigarette smoke, characterizes patients with COPD from smokers who have not developed the disease (3), and this is supported by studies assessing inflammation in lung or in bronchial biopsies (12, 14, 15). Cigarette smoking–induced oxidative stress plays a crucial role in the pathogenesis of COPD (5). Chronic lung inflammation persists despite smoking cessation (16), and there is a relationship between disease severity and inflammatory response intensity (12). The mechanism of the abnormal or enhanced inflammatory response to cigarette smoking in COPD is unknown.

To study the role of transcription factor activation and chromatin remodeling on lung inflammation, we examined human lung tissue from nonsmokers, healthy smokers, and patients with COPD. To assess the effect of smoking as distinct from COPD, patients were divided into two subgroups: current smokers and ex-smokers. We did not include a control group of healthy ex-smokers with normal lung function in this study. The inclusion of such a group would have provided an important evaluation in healthy subjects of the effects of smoking. Nonetheless, our data provide an important comparison of current and ex-smoker patients with COPD, which allows us to delineate the effects of smoking and disease.

Our data on 8-isoprostane, as a marker of oxidative stress in the lung tissue samples, show a cigarette smoking effect despite the lack of significance. This was confirmed when the data were expressed relative to the time since last cigarette and is consistent with the recent report on urinary 8-isoprostane levels in patients with COPD showing higher levels compared with control subjects and significant correlations with changes in FEV1 (17).

We found a clear effect of smoking on the regulation of NF-{kappa}B, with a significant reduction in I{kappa}B{alpha} protein expression in current healthy smokers and smokers with COPD, associated with a concomitant increase in the levels of NF-{kappa}B DNA binding. Surprisingly, we observed a greater depletion of I{kappa}B{alpha} levels in ex-smokers with COPD. However, the NF-{kappa}B DNA binding in these subjects was not different from that in nonsmokers. Nuclear translocation of NF-{kappa}B frequently results from activation by cytokines and/or oxidative stress of signal transduction cascades in cells (18, 19). Enhanced NF-{kappa}B activation in response to cigarette smoke has been shown by other investigators. Guinea pigs exposed to cigarette smoke showed increased NF-{kappa}B with subsequent IL-8 generation (20). Di Stefano and colleagues (15) analyzed segmental and subsegmental bronchial biopsies in healthy smokers and patients with COPD and found enhanced NF-{kappa}B activation in both groups associated with increased lipid peroxidation products as a marker of increased oxidative stress. However, no subdivision of patients with COPD into current smokers or ex-smokers was made. In addition, we observed a positive correlation between NF-{kappa}B translocation and number of pack-years smoked. Our data and these reports demonstrate the influence of active smoking on the nuclear translocation of NF-{kappa}B in lung cells.

We did not perform immunohistochemistry on our lung sections to identify the cells involved in the NF-{kappa}B activation. A localization study was performed by Di Stefano and colleagues (15), which showed increased p65 immunoreactivity and protein in bronchial epithelium but not in submucosa. However, no difference was found between healthy smokers and smokers with COPD. Likewise, Yagi and colleagues (21), examining I{kappa}B{alpha} expression, by immunostaining, in airway epithelial cells as an indirect measure of NF-{kappa}B activation, found ex-smokers with and without COPD to have increased levels of phosphorylated I{kappa}B{alpha}. Phosphorylation of I{kappa}B{alpha} is a prerequisite for the ubiquitination and degradation of the inhibitory subunit, allowing NF-{kappa}B to translocate to the nucleus. Also, with relevance to this study, Caramori and colleagues (22), examining p65 expression in sputum leucocytes from patients with exacerbated COPD, found p65 expression in macrophages but not in neutrophils.

The lack of increased NF-{kappa}B nuclear translocation in ex-smokers with COPD, despite increased I{kappa}B{alpha} degradation, is surprising because chronic, ongoing inflammation, despite smoking cessation, has been reported in smokers and patients with COPD (16). Although there are numerous reports in the literature of NF-{kappa}B activation without I{kappa}B{alpha} degradation, the converse, as found in this study, is not generally observed. A possible explanation for the decrease in NF-{kappa}B translocation in tissues, despite reports of increased inflammation in ex-smokers, may be due to NF-{kappa}B–independent signaling mechanisms. There are various reports in the literature of the involvement of NF-{kappa}B–independent mechanisms in the up-regulation of proinflammatory cytokines (23, 24), dependent on the type of stimulus. Also, Birrell and colleagues (25), in an animal model of emphysema, reported inflammation independent of NF-{kappa}B signaling.

An additional potential mechanism for the inhibition of NF-{kappa}B may be via a peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})–dependent pathway that is I{kappa}B{alpha} independent, as reported by Mishra and colleagues (26). This inhibitory effect of the nuclear receptor PPAR{alpha} could be through direct interaction with the p65 and cJun subunits of NF-{kappa}B (31) and AP-1, respectively, interfering with NF-{kappa}B and AP-1 transactivation and hence repressing the production of cytokines such as IL-6 (27). Thus, in the lungs of our ex-smoker patients with COPD, the lack of increased NF-{kappa}B translocation may be due to NF-{kappa}B inhibition rather than inactivation. Likewise, the fact that no difference in AP-1 DNA binding among the groups was found may also be due to PPAR inhibition of transactivation (27).

Although many proinflammatory genes are regulated by NF-{kappa}B binding to sites on their promotors, regulation of differential gene expression may also be determined by the status of histone acetylation in the chromatin structure. Increased histone acetylation by HATs, causing associated DNA unwinding, has been shown to be associated with increased gene transcription due to greater accessibility of the transcriptional machinery to the DNA template. HDACs, conversely, act by catalyzing the removal of acetyl groups on amino-terminal lysine residues of histones, producing DNA rewinding and a condensed chromatin structure, which results in transcriptional repression and gene silencing. Thus, the balance between HAT and HDAC activity may be influential in the gene expression of proinflammatory mediators.

In their recent publication, Ito and colleagues (28) examined the expression of HDAC gene (1 to 8) mRNA in lung tissue and alveolar macrophages from patients with COPD with GOLD stage 0 to 4. They observed a significant decrease relative to disease severity for HDAC2, HDAC5, and HDAC8 in lung tissue and HDAC2 and HDAC3 in alveolar macrophages. Because HDAC2 plays a role in the regulation of inflammation and has been implicated in the dysregulation in COPD (29, 30), we examined the protein levels of this HDAC in our lung tissue samples. Cytoplasmic HDAC2 protein levels were significantly reduced for all patients with COPD compared with nonsmokers, However, we observed no significant difference in nuclear HDAC2 protein between the groups. In addition, we found positive correlations for both cytoplasmic and nuclear HDAC2 and FEV1 as a percentage of the predicted values, suggesting an association between the levels of this HDAC2 enzyme, with the potential to cause greater unwinding of chromatin, and impaired lung function. In their report, Ito and coworkers also observed a significant reduction in HDAC2 levels and activity in bronchoalveolar macrophages and bronchial biopsies from smokers compared with nonsmokers (28–30), due to inactivation by oxidants (31). Likewise, work from our laboratory has shown decreased HDAC2 expression and activity in cigarette smoke–exposed rat lungs (32). The expression of HDAC2 levels relative to the time since last cigarette shows a cigarette smoking effect for cytoplasmic HDAC2, but not for nuclear HDAC2 levels. However, the fact that our ex-smoker COPD group had all ceased smoking for at least 1 yr before their lung resection and had the lowest HDAC2 levels of any of the groups suggests that the HDAC2 inhibition is possibly more influenced by the disease-derived oxidative stress rather than cigarette smoke–derived oxidants (i.e., a COPD rather than a smoking effect). Furthermore, expressing our HDAC2 values relative to GOLD severity categories, our data are consistent with that of Ito and colleagues (28) demonstrating decreasing levels of HDAC2 in COPD lung tissue associated with disease severity. In addition, their patients with category 4 GOLD stage, who showed the greatest reduction in HDAC2 levels, were also entirely an ex-smoking group.

The acetylation status of histone residues in lung tissue was investigated by measuring the protein levels of acetylated histones H3 and H4. The acetylation of both histones H3 and H4 has been associated with the promotion of transcription of proinflammatory genes in inflammation. In addition, constitutively expressed genes are associated with high levels of histone H4 acetylation (33). The data in this study show increased expression of acetylated histone H4 in smokers both with and without COPD, once again demonstrating a significant effect of smoking on the modification of the chromatin structure and, potentially, enhancing proinflammatory cytokine gene transcription. This was confirmed by graphing the data relative to the time since last cigarette. Increased histone H4 acetylation in response to cigarette smoking has previously been shown by our group in rat lung tissue (32) and a human lung cell line (34). Likewise, Tomita and colleagues found oxidative stress increased histone H4 acetylation, resulting in increased IL-8 generation (35). Moreover, Ito and colleagues found increased histone H4 acetylation at the IL-8 promotor in peripheral lung tissue samples relative to disease severity, such that patients with GOLD category 4 disease had the most extensive levels of acetylation (28). This hyperacetylation of H4 could not be related to cigarette smoking because the greatest acetylation was seen in a group of ex-smokers. However, their data suggest the considerable oxidant burden present in these patients with severe disease.

In contrast to histone H4, the acetylation of histone H3 was increased only in ex-smokers with COPD. Because gene promotors for proinflammatory mediators (e.g., IL-6, TNF-{alpha}, and cyclooxygenase 2 [COX2]) are located on both histones H3 and H4 (36), our observations support the hypothesis that subtle differences inferred by acetylation of lysines on these histones may regulate specific gene transcription.

Hence, the greater unwinding of chromatin, as suggested by the increased acetylation of histones H3 and H4, would allow increased transcription of inflammatory proteins. Indeed, Miao and coworkers (36) proposed acetylation of histone H3 to be a marker of the increased inflammatory state in diabetic monocytes.

In this study, beta-actin was used to normalize for protein loading for both cytoplasmic and nuclear extracts. Although beta-actin is present in the nucleus and associated with chromatin (37, 38), ideally a reference nuclear protein and nonacetylated histones should have been used. The integrity of cytoplasmic and nuclear fractions was demonstrated by Western blot using an antibody to the nuclear envelope protein lamin A/C (see Figure E1). Some contamination of the cytoplasmic extract with nuclear proteins was seen in one sample, but because such contamination would be random across the samples, we believe it would not affect the data presented.

The RT-PCR results in this study did not show a significant difference in cytokine gene expression between the subject groups, due to considerable variation among individuals within groups. Only IL-12p40 was consistently expressed in all subjects and showed a positive correlation with levels of acetylated histone H4, suggesting the IL-12p40 promotor is associated with histone H4 acetylation as reported by others (39). Nonetheless, for all the cytokines except TNF-{alpha}, increased levels of mRNA were seen in the healthy smokers and COPD patient groups, compared with nonsmokers. Thus, our data, although not significant, would at least seem to be consistent with the increased histone acetylation and decreased HDAC2 protein levels, shown here and by others, and the inflammation known to be present in the lungs of healthy smokers and current and ex-smoker patients with COPD.

In summary, in human lung tissue, we have found a smoking-related increase in NF-{kappa}B nuclear translocation associated with I{kappa}B{alpha} degradation. A greater degradation of I{kappa}B{alpha} was observed in ex-smokers with COPD, which, we hypothesize, may be associated with PPAR{alpha} activation. Recruitment of HATs to histone proteins by active smoking, as measured by acetylation of histones H3 and H4, may lead to transcription of those genes with promotors associated with histone H4, whereas smoking cessation in patients with COPD may promote transcription of genes directed through histone H3. Furthermore, we found that patients with COPD had decreased cytoplasmic, but not nuclear, HDAC2 protein levels, which was associated with disease severity. Moreover, decreased cytoplasmic HDAC2 levels appear to be mediated, in part, by a disease-associated mechanism as well as cigarette smoke–derived oxidative stress.

Given that the regulation of inflammatory genes is an integral feature of COPD, these studies suggest the molecular signaling pathways that may be important in this process.


   Acknowledgments
 
The authors thank Drs. Julie Wickenden, John Marwick, Robert Mróz, and Jane McNeilly and Kirsty A. Sherriffs for valuable comments and technical assistance.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200505-725OC on March 30, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 9, 2005; accepted in final form March 30, 2006


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