Smoking and COPD increase sputum levels of extracellular superoxide dismutase

Extracellular superoxide dismutase (ECSOD) is the major superoxide-scavenging enzyme in the lung. Certain ECSOD polymorphisms are protective against COPD. We postulated that smokers and COPD subjects would have altered levels of ECSOD in the lung, airway secretions, and/or plasma. Lung tissue ECSOD was evaluated from nonsmokers, smokers, and subjects with mild to very severe COPD by Western blot, immunohistochemistry, and ELISA. ECSOD levels in plasma, bronchoalveolar lavage fluid (BALF), and induced-sputum supernatants were analyzed by ELISA and correlated with smoking history and disease status. Immunohistochemistry identified ECSOD in extracellular matrix around bronchioles, arteries, and alveolar walls, with decreases seen in the interstitium and vessels of severe COPD subjects using digital image analysis. Plasma ECSOD did not differ between COPD subjects and controls nor based on smoking status. ECSOD levels in induced sputum supernatants were elevated in current smokers and especially in COPD subjects compared to nonsmokers, whereas corresponding changes could not be seen in the BALF. ECSOD expression was reduced around vessels and bronchioles in COPD lungs. Substantial increases in sputum ECSOD in smokers and COPD is interpreted as an adaptive response to increased oxidative stress and may be a useful biomarker of disease activity in COPD.     

Proteomic study of human bronchoalveolar lavage fluids from smokers with chronic obstructive pulmonary disease by combining surface-enhanced laser desorption/ionization-mass spectrometry profiling with mass spectrometric protein identification

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate cellular and molecular changes in the course of lung disorders. The pattern of soluble proteins in BALF obtained from patients at different stages of respiratory disorders may provide deeper insights in the molecular mechanisms of the disease. We used surface-enhanced laser desorption/ionization mass spectrometry (MS) for differential protein display combined with reversed-phase chromatography and subsequent matrix-assisted laser desorption/ionization-MS or nanoliquid chromatography MS/MS analysis for protein identification to compare the protein pattern of BALF samples obtained from ten smokers suffering from chronic obstructive pulmonary disease (COPD), eight clinically asymptomatic smokers, and eight nonsmokers without pulmonary disease. In this context, we were able to identify small proteins and peptides, either differentially expressed or secreted in the course of COPD or in a direct response to cigarette smoke. The concentrations of neutrophil defensins 1 and 2, S100A8 (calgranulin A), and S100A9 (calgranulin B) were elevated in BALFs of smokers with COPD when compared to asymptomatic smokers. Increased concentrations in S100A8 (Calgranulin A), salivary proline-rich peptide P-C, and lysozyme C were detected in BALFs of asymptomatic smokers when compared to nonsmokers, whereas salivary proline-rich peptide P-D and Clara cell phospholipid-binding protein (CC10) were reduced in their concentration. The identified proteins and peptides might be useful in the future as diagnostic markers for smoke-induced lung irritations and COPD.     

Vascular endothelial growth factor: an angiogenic factor reflecting airway inflammation in healthy smokers and in patients with bronchitis type of chronic obstructive pulmonary disease?

BackgroundPatients with bronchitis type of chronic obstructive pulmonary disease (COPD) have raised vascular endothelial growth factor (VEGF) levels in induced sputum. This has been associated with the pathogenesis of COPD through apoptotic and oxidative stress mechanisms. Since, chronic airway inflammation is an important pathological feature of COPD mainly initiated by cigarette smoking, aim of this study was to assess smoking as a potential cause of raised airway VEGF levels in bronchitis type COPD and to test the association between VEGF levels in induced sputum and airway inflammation in these patients.Methods14 current smokers with bronchitis type COPD, 17 asymptomatic current smokers with normal spirometry and 16 non-smokers were included in the study. VEGF, IL-8, and TNF-α levels in induced sputum were measured and the correlations between these markers, as well as between VEGF levels and pulmonary function were assessed.ResultsThe median concentrations of VEGF, IL-8, and TNF-α were significantly higher in induced sputum of COPD patients (1,070 pg/ml, 5.6 ng/ml and 50 pg/ml, respectively) compared to nonsmokers (260 pg/ml, 0.73 ng/ml, and 15.4 pg/ml, respectively, p < 0.05) and asymptomatic smokers (421 pg/ml, 1.27 ng/ml, p < 0.05, and 18.6 pg/ml, p > 0.05, respectively). Significant correlations were found between VEGF levels and pack years (r = 0.56, p = 0.046), IL-8 (r = 0.64, p = 0.026) and TNF-α (r = 0.62, p = 0.031) levels both in asymptomatic and COPD smokers (r = 0.66, p = 0.027, r = 0.67, p = 0.023, and r = 0.82, p = 0.002, respectively). No correlation was found between VEGF levels in sputum and pulmonary function parameters.ConclusionVEGF levels are raised in the airways of both asymptomatic and COPD smokers. The close correlation observed between VEGF levels in the airways and markers of airway inflammation in healthy smokers and in smokers with bronchitis type of COPD is suggestive of VEGF as a marker reflecting the inflammatory process that occurs in smoking subjects without alveolar destruction.     

Levels of angiopoietins 1 and 2 in induced sputum supernatant in patients with COPD

Pathological features of chronic obstructive pulmonary disease (COPD) include lung vascular remodeling and angiogenesis. Angiopoietin-1 (Ang-1), is an essential mediator of angiogenesis by establishing vascular integrity, whereas angiopoietin-2 (Ang-2) acts as its natural inhibitor. We determined the levels of angiopoietins in sputum supernatants of patients with COPD and investigated their possible association with mediators and cells involved in the inflammatory and remodeling process. Fifty-nine patients with COPD, 25 healthy smokers and 20 healthy non-smokers were studied. All subjects underwent lung function tests, sputum induction for cell count identification and Ang-1, Ang-2, VEGF, TGF-β1, MMP-2, LTB4, IL-8, albumin measurement in sputum supernatants. Airway vascular permeability (AVP) index was also assessed. Ang-2 levels were significantly higher in patients with COPD compared to healthy smokers and healthy non-smokers [median, interquartile ranges pg/ml, 267 (147-367) vs. 112 (67-171) and 98 (95-107), respectively; p<0.001]. Regression analysis showed a significant association between Ang-2 levels and AVP index, VEGF, IL-8 and MMP-2 levels in COPD, the strongest being with VEGF. Our results indicate that induced sputum Ang-2 levels are higher in COPD compared to healthy smokers and healthy non-smokers. Moreover, Ang-2 is associated with AVP, IL-8, MMP-2, and VEGF, indicating a possible role for Ang-2 in the pathogenesis of the disease.     

Chronic Bronchitis and Current Smoking Are Associated with More Goblet Cells in Moderate to Severe COPD and Smokers without Airflow Obstruction

BackgroundGoblet cell hyperplasia is a classic but variable pathologic finding in COPD. Current literature shows that smoking is a risk factor for chronic bronchitis but the relationship of these clinical features to the presence and magnitude of large airway goblet cell hyperplasia has not been well described. We hypothesized that current smokers and chronic bronchitics would have more goblet cells than nonsmokers or those without chronic bronchitis (CB), independent of airflow obstruction.MethodsWe recruited 15 subjects with moderate to severe COPD, 12 healthy smokers, and 11 healthy nonsmokers. Six endobronchial mucosal biopsies per subject were obtained by bronchoscopy and stained with periodic acid Schiff-Alcian Blue. Goblet cell density (GCD) was quantified as goblet cell number per millimeter of basement membrane. Mucin volume density (MVD) was quantified as volume of mucin per unit area of basement membrane.ResultsHealthy smokers had a greater GCD and MVD than nonsmokers and COPD subjects. COPD subjects had a greater GCD than nonsmokers. When current smokers (healthy smokers and COPD current smokers, n = 19) were compared with all nonsmokers (nonsmoking controls and COPD ex-smokers, n = 19), current smokers had a greater GCD and MVD. When those with CB (n = 12) were compared to those without CB (n = 26), the CB group had greater GCD. This finding was also seen in those with CB in the COPD group alone. In multivariate analysis, current smoking and CB were significant predictors of GCD using demographics, lung function, and smoking pack years as covariates. All other covariates were not significant predictors of GCD or MVD.ConclusionsCurrent smoking is associated with a more goblet cell hyperplasia and number, and CB is associated with more goblet cells, independent of the presence of airflow obstruction. This provides clinical and pathologic correlation for smokers with and without COPD.     

Persistence of Smoking-Induced Dysregulation of MiRNA Expression in the Small Airway Epithelium Despite Smoking Cessation

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10^th to 12^th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.     

Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF

The current report describes the use of CapLC-ESI-Q/TOF-MS for investigating the proteome profiles of hypertonic saline-induced sputum samples from 56 smokers. The severity of their lung disease ranged from normal (healthy smokers) to chronic bronchitis, chronic obstructive pulmonary disease (COPD), and COPD with emphysema. This pilot study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype and cigarette smoking illness severity. A total of 203 unique proteins were identified. These may represent the most highly expressed proteins in induced sputum. Our results provide evidence that different proteins are expressed, as the disease progresses from health to more advanced stages, and support our contention that a proteomic approach would be beneficial in discovering selective molecules linked to specific COPD stages.     

Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

Lipocalin 2 is protective against E. coli pneumonia

Background

Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD.

Methods

To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects.

Results

Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02).

Conclusions

Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.     

Repeatability of and Relationship between Potential COPD Biomarkers in Bronchoalveolar Lavage,Bronchial Biopsies,Serum, and Induced Sputum

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, primarily affecting the airways. Stable biomarkers characterizing the inflammatory phenotype of the disease, relevant for disease activity and suited to predict disease progression are needed to monitor the efficacy and safety of drug interventions. We therefore analyzed a large panel of markers in bronchoalveolar lavage, bronchial biopsies, serum and induced sputum of 23 healthy smokers and 24 smoking COPD patients (GOLD II) matched for age and gender. Sample collection was performed twice within a period of 6 weeks. Assays for over 100 different markers were validated for the respective matrices prior to analysis. In our study, we found 51 markers with a sufficient repeatability (intraclass correlation coefficient >0.6), most of these in serum. Differences between groups were observed for markers from all compartments, which extends (von-Willebrand-factor) and confirms (e.g. C-reactive-protein, interleukin-6) previous findings. No correlations between lung and serum markers were observed, including A1AT. Airway inflammation defined by sputum neutrophils showed only a moderate repeatability. This could be improved, when a combination of neutrophils and four sputum fluid phase markers was used to define the inflammatory phenotype.In summary, our study provides comprehensive information on the repeatability and interrelationship of pulmonary and systemic COPD-related markers. These results are relevant for ongoing large clinical trials and future COPD research. While serum markers can discriminate between smokers with and without COPD, they do not seem to sufficiently reflect the disease-associated inflammatory processes within the airways.     

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n?=?18) or slow (n?=?18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.     

吸烟对肺组织D4-GDI表达的影响及其与慢性阻塞性肺疾病相关性的蛋白质组学研究

慢性阻塞性肺疾病(COPD)是环境因素与遗传因素共同作用的结果,而吸烟是导致COPD发生的最主要危险因素,然而,吸烟导致COPD的机制及COPD的遗传易感机制目前尚未完全阐明．蛋白质组学研究方法具有高效和信息含量丰富的特点,为COPD研究提供了有力的帮助,被认为在COPD的研究领域具有广阔的前景．运用二维凝胶电泳和基质辅助激光解吸电离飞行时间质谱蛋白质组学研究方法结合生物信息学技术,对不吸烟者、非COPD吸烟者和吸烟COPD患者的肺组织进行蛋白质组比较,共鉴定出24种表达差异蛋白．研究发现,非COPD吸烟者肺组织ρ-GDP解离抑制因子2(D4-GDI)表达水平为不吸烟者的1.7倍,吸烟COPD患者肺组织D4-GDI表达水平接近非COPD吸烟者的2倍．通过免疫组织化学染色和Western-blotting检测对肺组织D4-GDI表达水平进行验证,获得了与蛋白质组学研究相一致的结果．结果首次说明：吸烟能够导致肺组织D4-GDI表达水平升高,D4-GDI参与了COPD的发病机制而且可能与COPD易感性相关．     

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD

Introduction

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.

Methods

Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.

Results

MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.

Conclusion

MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.     

Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial

Background

KL-6 is a high-molecular-weight glycoprotein classified as a human MUC1 mucin. It was hypothesized that KL-6 could be detectable in the circulating blood and especially in airway secretions in lung diseases associated with mucus production such as chronic obstructive pulmonary disease (COPD). Additional aims of this study were to investigate whether the levels of KL-6 in plasma and sputum are related to ageing and smoking history.

Methods

The concentrations of KL-6 in plasma and induced sputum supernatants from young and/or middle aged/elderly non-smokers, smokers and patients with COPD were assayed by ELISA (n = 201). The subjects were classified into five groups according to age, smoking status and presence of COPD. In addition, KL-6 expression in control and diseased lung i.e. samples from patients with COPD (n = 28), were analyzed by immunohistochemistry and digital image analysis.

Results

The plasma levels of KL-6 increased with age both in non-smokers and smokers. Among middle aged/elderly subjects, plasma KL-6 levels in all smokers regardless of COPD were significantly higher than in non-smokers, whereas sputum levels of KL-6 were significantly higher in COPD compared not only to non-smokers but also to smokers. KL-6 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs. Plasma and sputum KL-6 levels correlated inversely with obstruction and positively with smoking history and ageing. The linear multiple regression analysis confirmed that age and cigarette smoking had independent effects on plasma KL-6.

Conclusions

KL-6 increases with ageing and chronic smoking history, but prospective studies will be needed to elucidate the significance of KL-6 in chronic airway diseases.     

Aging-Related Systemic Manifestations in COPD Patients and Cigarette Smokers

Rationale

Chronic obstructive pulmonary disease (COPD) is often associated with age-related systemic abnormalities that adversely affect the prognosis. Whether these manifestations are linked to the lung alterations or are independent complications of smoking remains unclear.

Objectives

To look for aging-related systemic manifestations and telomere shortening in COPD patients and smokers with minor lung destruction responsible for a decline in the diffusing capacity for carbon monoxide (DL_CO) corrected for alveolar volume (K_CO).

Methods

Cross-sectional study in 301 individuals (100 with COPD, 100 smokers without COPD, and 101 nonsmokers without COPD).

Measurements and Main Results

Compared to control smokers, patients with COPD had higher aortic pulse-wave velocity (PWV), lower bone mineral density (BMD) and appendicular skeletal muscle mass index (ASMMI), and shorter telomere length (TL). Insulin resistance (HOMA-IR) and glomerular filtration rate (GFR) were similar between control smokers and COPD patients. Smokers did not differ from nonsmokers for any of these parameters. However, smokers with normal spirometry but low K_CO had lower ASMMI values compared to those with normal K_CO. Moreover, female smokers with low K_CO, had lower BMD and shorter TL compared to those with normal K_CO.

Conclusions

Aging-related abnormalities in patients with COPD are also found in smokers with minor lung dysfunction manifesting as a K_CO decrease. Decreased K_CO might be useful, particularly among women, for identifying smokers at high risk for aging-related systemic manifestations and telomere shortening.     

Decreased Histone Deacetylase 2 (HDAC2) in Peripheral Blood Monocytes (PBMCs) of COPD Patients

Background

Histone deacetylase 2 (HDAC2) is a class I histone deacetylase family member that plays a critical role in suppressing inflammatory gene expression in the airways, lung parenchyma, and alveolar macrophages in patients with chronic obstructive pulmonary disease (COPD). However, the expression of HDAC2 in peripheral blood monocytes (PBMCs), nuclear factor kappa B (NF-κB) p65, and serum inflammatory cytokine levels in COPD patients, smokers, and non-smokers remains unclear.

Methods

PBMCs were obtained from COPD patients, healthy smokers, and healthy nonsmokers. The HDAC2 and NF-κB p65 expression were quantified by Western Blot. HDAC activity was assessed by an HDAC fluorometric immunoprecipitation activity assay kit. Serum tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) levels were measured by ELISA.

Results

HDAC2 expression and HDAC activity were decreased in PBMCs in COPD patients compared with smokers and non-smokers. Increased NF-κB p65 expression, serum TNF-α and IL-8 levels were observed in COPD patients compared with nonsmokers. The FEV₁%pred was positively correlated with HDAC2 expression and HDAC activity in COPD patients. Smokers had decreased HDAC activity, increased NF-κB p65 expression and serum TNF-α compared with nonsmokers.

Conclusions

HDAC2 expression was decreased in PBMCs of COPD patients and was correlated with disease severity. The reduction of HDAC2 expression not only directly enhances the expression of inflammatory genes, but may account for the activation of NF-κB mediated inflammation. Decreased HDAC2 may serve as a potential biomarker of COPD and predict the decline of lung function.     

Glutathione S-transferase omega in the lung and sputum supernatants of COPD patients

Background

The major contribution to oxidant related lung damage in COPD is from the oxidant/antioxidant imbalance and possibly impaired antioxidant defence. Glutathione (GSH) is one of the most important antioxidants in human lung and lung secretions, but the mechanisms participating in its homeostasis are partly unclear. Glutathione-S-transferase omega (GSTO) is a recently characterized cysteine containing enzyme with the capability to bind and release GSH in vitro. GSTO has not been investigated in human lung or lung diseases.

Methods

GSTO1-1 was investigated by immunohistochemistry and Western blot analysis in 72 lung tissue specimens and 40 sputum specimens from non-smokers, smokers and COPD, in bronchoalveolar lavage fluid and in plasma from healthy non-smokers and smokers. It was also examined in human monocytes and bronchial epithelial cells and their culture mediums in vitro.

Results

GSTO1-1 was mainly expressed in alveolar macrophages, but it was also found in airway and alveolar epithelium and in extracellular fluids including sputum supernatants, bronchoalveolar lavage fluid, plasma and cell culture mediums. The levels of GSTO1-1 were significantly lower in the sputum supernatants (p = 0.023) and lung homogenates (p = 0.003) of COPD patients than in non-smokers.

Conclusion

GSTO1-1 is abundant in the alveolar macrophages, but it is also present in extracellular fluids and in airway secretions, the levels being decreased in COPD. The clinical significance of GSTO1-1 and its role in regulating GSH homeostasis in airway secretions, however, needs further investigations.     

Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.     

Midkine Is Expressed and Differentially Processed during Chronic Obstructive Pulmonary Disease Exacerbations and Ventilator-Associated Pneumonia Associated with Staphylococcus aureus Infection

Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.     

Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases,cysteine, and serine proteases

Chronic obstructive pulmonary disease (COPD) is a common lung disease with cigarette smoking as the major etiological factor, but only 15% of smokers develop COPD. Destruction of lung elastin observed in COPD is mediated by many enzymes, including cysteine, serine, and matrix metalloproteinases (MMP). The contribution of these enzymes to the lung elastolytic load, released from alveolar macrophages collected from nonsmokers, healthy smokers, and COPD patients, was examined by radiolabeled elastin as substrate in the presence of specific enzyme inhibitors. The activity of MMP was further examined by zymography and Western blotting. COPD macrophages degraded more elastin than either of the other groups. Elastolysis was greatest in the initial 24 h. Through the 72-h culture period, the contribution to elastolysis of serine elastases decreased, MMP increased, and cysteine elastases remained constant. The increased release of elastolytic enzymes in COPD subjects may explain why some smokers develop COPD. This difference may be due to unknown susceptibility factors. Serine proteases play a significant role; however, other enzymes, particularly the MMP, deserve further investigation.