Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype

Background

A major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.

Methods

To this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.

Results

Both CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.

Conclusions

Direct exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.     

Cigarette Smoke-Induced Collagen Destruction; Key to Chronic Neutrophilic Airway Inflammation?

Background

Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.

Methodology/Principal Findings

Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.

Conclusions

This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study

Background

Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD.

Methods

The extent of dendritic cell infiltration in COPD tissue specimens was determined using immunohistochemical localization of CD83+ cells (marker of matured myeloid dendritic cells), and CD1a+ cells (Langerhans cells). The extent of tissue infiltration with Langerhans cells was also determined by the relative expression of the CD207 gene in COPD versus control tissues. To determine mechanisms by which dendritic cells accumulate in COPD, complimentary studies were conducted using monocyte-derived human dendritic cells exposed to cigarette smoke extract (CSE), and dendritic cells extracted from mice chronically exposed to cigarette smoke.

Results

In human COPD lung tissue, we detected a significant increase in the total number of CD83+ cells, and significantly higher amounts of CD207 mRNA when compared with control tissue. Human monocyte-derived dendritic cells exposed to CSE (0.1-2%) exhibited enhanced survival in vitro when compared with control dendritic cells. Murine dendritic cells extracted from mice exposed to cigarette smoke for 4 weeks, also demonstrated enhanced survival compared to dendritic cells extracted from control mice. Acute exposure of human dendritic cells to CSE induced the cellular pro-survival proteins heme-oxygenase-1 (HO-1), and B cell lymphoma leukemia-x(L) (Bcl-xL), predominantly through oxidative stress. Although activated human dendritic cells conditioned with CSE expressed diminished migratory CCR7 expression, their migration towards the CCR7 ligand CCL21 was not impaired.

Conclusions

These data indicate that COPD is associated with increased numbers of cells bearing markers associated with Langerhans cells and mature dendritic cells, and that cigarette smoke promotes survival signals and augments survival of dendritic cells. Although CSE suppressed dendritic cell CCR7 expression, migration towards a CCR7 ligand was not diminished, suggesting that reduced CCR7-dependent migration is unlikely to be an important mechanism for dendritic cell retention in the lungs of smokers with COPD.     

Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression

Background

Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a.

Methods

RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed.

Results

Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects.

Conclusions

Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.     

Upregulation of IL-17A/F from human lung tissue explants with cigarette smoke exposure: implications for COPD

Background

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved.

Methods

Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media.

Results

No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects.

Conclusions

We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-κB and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.     

Role of aberrant metalloproteinase activity in the pro-inflammatory phenotype of bronchial epithelium in COPD

Background

Cigarette smoke, the major risk factor for COPD, is known to activate matrix metalloproteinases in airway epithelium. We investigated whether metalloproteinases, particularly A Disintegrin and Metalloproteinase (ADAM)17, contribute to increased pro-inflammatory epithelial responses with respect to the release of IL-8 and TGF-α, cytokines implicated in COPD pathogenesis.

Methods

We studied the effects of cigarette smoke extract (CSE) and metalloproteinase inhibitors on TGF-α and IL-8 release in primary bronchial epithelial cells (PBECs) from COPD patients, healthy smokers and non-smokers.

Results

We observed that TGF-α was mainly shed by ADAM17 in PBECs from all groups. Interestingly, IL-8 production occurred independently from ADAM17 and TGF-α shedding, but was significantly inhibited by broad-spectrum metalloproteinase inhibitor TAPI-2. CSE did not induce ADAM17-dependent TGF-α shedding, while it slightly augmented the production of IL-8. This was accompanied by reduced endogenous inhibitor of metalloproteinase (TIMP)-3 levels, suggesting that CSE does not directly but rather indirectly alter activity of ADAM17 through the regulation of its endogenous inhibitor. Furthermore, whereas baseline TGF-α shedding was lower in COPD PBECs, the early release of IL-8 (likely due to its shedding) was higher in PBECs from COPD than healthy smokers. Importantly, this was accompanied by lower TIMP-2 levels in COPD PBECs, while baseline TIMP-3 levels were similar between groups.

Conclusions

Our data indicate that IL-8 secretion is regulated independently from ADAM17 activity and TGF-α shedding and that particularly its early release is differentially regulated in PBECs from COPD and healthy smokers. Since TIMP-2-sensitive metalloproteinases could potentially contribute to IL-8 release, these may be interesting targets to further investigate novel therapeutic strategies in COPD.     

The Effect of Azithromycin in Adults with Stable Neutrophilic COPD: A Double Blind Randomised,Placebo Controlled Trial

Background

Chronic Obstructive Pulmonary Disease (COPD) is a progressive airway disease characterised by neutrophilic airway inflammation or bronchitis. Neutrophilic bronchitis is associated with both bacterial colonisation and lung function decline and is common in exacerbations of COPD. Despite current available therapies to control inflammation, neutrophilic bronchitis remains common. This study tested the hypothesis that azithromycin treatment, as an add-on to standard medication, would significantly reduce airway neutrophil and neutrophils chemokine (CXCL8) levels, as well as bacterial load. We conducted a randomised, double-blind, placebo-controlled study in COPD participants with stable neutrophilic bronchitis.

Methods

Eligible participants (n = 30) were randomised to azithromycin 250 mg daily or placebo for 12 weeks in addition to their standard respiratory medications. Sputum was induced at screening, randomisation and monthly for a 12 week treatment period and processed for differential cell counts, CXCL8 and neutrophil elastase assessment. Quantitative bacteriology was assessed in sputum samples at randomisation and the end of treatment visit. Severe exacerbations where symptoms increased requiring unscheduled treatment were recorded during the 12 week treatment period and for 14 weeks following treatment. A sub-group of participants underwent chest computed tomography scans (n = 15).

Results

Nine participants with neutrophilic bronchitis had a potentially pathogenic bacteria isolated and the median total bacterial load of all participants was 5.22×10⁷ cfu/mL. Azithromycin treatment resulted in a non-significant reduction in sputum neutrophil proportion, CXCL8 levels and bacterial load. The mean severe exacerbation rate was 0.33 per person per 26 weeks in the azithromycin group compared to 0.93 exacerbations per person in the placebo group (incidence rate ratio (95%CI): 0.37 (0.11,1.21), p = 0.062). For participants who underwent chest CT scans, no alterations were observed.

Conclusions

In stable COPD with neutrophilic bronchitis, add-on azithromycin therapy showed a trend to reduced severe exacerbations sputum neutrophils, CXCL8 levels and bacterial load. Future studies with a larger sample size are warranted.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12609000259246     

Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.

Methodology and Principal Findings

Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1 ^−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.

Conclusions

We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.     

Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

Background

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway.

Methods

Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure.

Results

CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects.

Conclusions

The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.     

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

Backround

Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR''s). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers.

Methods

The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative.

Results

The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients.

Conclusion

Our data suggest a smoke related change in the phenotype of AM''s and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.     

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses

Background

Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.

Methods

To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×10⁶ plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.

Results

RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.

Conclusions

Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.     

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle

Background

Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.

Methods

The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M₂ and M₃ receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.

Results

Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.

Conclusions

We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.     

Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Background

Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists.

Methods

Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was measured in cell cultures after TLR3 stimulation with poly I:C 10 μg/mL.

Results

We found that TLR3 expression was increased by 1.95 fold (protein) and 2.5 fold (mRNA) in lung tissues from smokers with COPD and inversely correlated with lung function. The TLR3 agonist poly I:C 10 μg/mL increased the IL-8 release in HBECs that was poorly inhibited by dexamethasone in smokers (24.5%) and smokers with COPD (21.6%). In contrast, roflumilast showed similar inhibitory effects on IL-8 release in healthy (58.8%), smokers (56.6%) and smokers with COPD (50.5%). The combination of roflumilast N-oxide and dexamethasone showed additive inhibitory effects. Mechanistically, roflumilast N-oxide when combined with dexamethasone increased the expression of MKP1, and enhanced the inhibitory effects on phospho-p38, AP1 and NFκB activities which may explain the additive anti-inflammatory effects.

Conclusions

Altogether, our data provide in vitro evidence for a possible clinical utility to add roflumilast on top of inhaled corticosteroid in COPD.     

The role of the liver X receptor in chronic obstructive pulmonary disease

Background

There is a need for novel anti-inflammatory therapies to treat COPD. The liver X receptor (LXR) is a nuclear hormone receptor with anti-inflammatory properties.

Methods

We investigated LXR gene and protein expression levels in alveolar macrophages and whole lung tissue from COPD patients and controls, the effect of LXR activation on the suppression of inflammatory mediators from LPS stimulated COPD alveolar macrophages, and the effect of LXR activation on the induction of genes associated with alternative macrophage polarisation.

Results

The levels of LXR mRNA were significantly increased in whole lung tissue extracts in COPD patients and smokers compared to non-smokers. The expression of LXR protein was significantly increased in small airway epithelium and alveolar epithelium in COPD patients compared to controls. No differences in LXR mRNA and protein levels were observed in alveolar macrophages between patient groups. The LXR agonist GW3965 significantly induced the expression of the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. In LPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10 and CCL5, whilst stimulating IL-10 production.

Conclusions

GW3965 did not significantly suppress the production of TNFα, IL-1β, or CXCL8. Our major finding is that LXR activation has anti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolar macrophages.     

The effects of corticosteroids on COPD lung macrophages: a pooled analysis

Background

There is large variation in the therapeutic response to inhaled corticosteroids (ICS) in COPD patients. We present a pooled analysis of our previous studies investigating the effects of corticosteroids on lung macrophages, in order to robustly determine whether corticosteroid sensitivity in COPD cells is reduced compared to controls, and also to evaluate the degree of between individual variation in drug response.

Methods

Data from 20 never smokers (NS), 27 smokers (S) and 45 COPD patients was used. Lung macropahges had been stimulated with lipopolysaccharide (LPS), with or without the corticosteroid dexamethasone, and tumour necrosis factor (TNF)-α, interleukin (IL)-6 and chemokine C-X-C motif ligand (CXCL) 8 production was measured.

Results

There was no difference in the anti-inflammatory effects of corticosteroids when comparing group mean data of COPD patients versus controls. The inhibition of TNF-α and IL-6 was greater than CXCL8. The effects of corticosteroids varied considerably between subjects, particularly at lower corticosteroid concentrations.

Conclusions

We confirm that overall corticosteroid sensitivity in COPD lung macrophages is not reduced compared to controls. The varied effect of corticosteroids between subjects suggests that some individuals have an inherently poor corticosteroid response. The limited suppression of lung macrophage derived CXCL8 may promote neutrophilic inflammation in COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0260-0) contains supplementary material, which is available to authorized users.     

The role of uPAR in epithelial-mesenchymal transition in small airway epithelium of patients with chronic obstructive pulmonary disease

Background

Epithelial-mesenchymal transition (EMT) plays a crucial role in small airway fibrosis of patients with chronic obstructive pulmonary disease (COPD). Increasing evidence suggests that the urokinase plasminogen activator receptor (uPAR) is involved in the pathogenesis of COPD. Increased uPAR expression has been implicated in the promotion of EMT in numerous cancers; however the role of uPAR in EMT in small airway epithelial cells of patients with COPD remains unclear. In this study, we investigated the degree of EMT and uPAR expression in lung epithelium of COPD patients, and verified the effect of uPAR on cigarette smoke extract (CSE)-induced EMT in vitro.

Methods

The expression of EMT biomarkers and uPAR was assessed in lung epithelium specimens from non-smokers (n = 25), smokers (n = 25) and non-smokers with COPD (n = 10) and smokers with COPD (n = 18). The role of uPAR on CSE-induced EMT in human small airway epithelial cells (HSAEpiCs) was assessed by silencing uPAR expression in vitro.

Results

Markers of active EMT and uPAR expression were significantly increased in the small airway epithelium of patients with COPD compared with controls. We also observed a significant correlation between uPAR and vimentin expression in the small airway epithelium. In vitro, CSE-induced EMT in HSAEpiCs was associated with high expression of uPAR, and targeted silencing of uPAR using shRNA inhibited CSE-induced EMT. Finally, we demonstrate that the PI3K/Akt signaling pathway is required for uPAR-mediated EMT in HSAEpiCs.

Conclusions

A uPAR-dependent signaling pathway is required for CSE-induced EMT, which contributes to small airway fibrosis in COPD. We propose that increased uPAR expression in the small airway epithelium of patients with COPD participates in an active EMT process.