BACKGROUND:Respiratory viral infection causes chronic obstructive pulmonary disease (COPD) exacerbations. We previously reported increased bronchial mucosa eosinophil and neutrophil inflammation in patients with COPD experiencing naturally occurring exacerbations. But it is unclear whether virus per se induces bronchial mucosal inflammation, nor whether this relates to exacerbation severity.OBJECTIVES:We sought to determine the extent and nature of bronchial mucosal inflammation following experimental rhinovirus (RV)-16-induced COPD exacerbations and its relationship to disease severity.METHODS:Bronchial mucosal inflammatory cell phenotypes were determined at preinfection baseline and following experimental RV infection in 17 Global Initiative for Chronic Obstructive Lung Disease stage II subjects with COPD and as controls 20 smokers and 11 nonsmokers with normal lung function. No subject had a history of asthma/allergic rhinitis: all had negative results for aeroallergen skin prick tests.RESULTS:RV infection increased the numbers of bronchial mucosal eosinophils and neutrophils only in COPD and CD8+ T lymphocytes in patients with COPD and nonsmokers. Monocytes/macrophages, CD4+ T lymphocytes, and CD20+ B lymphocytes were increased in all subjects. At baseline, compared with nonsmokers, subjects with COPD and smokers had increased numbers of bronchial mucosal monocytes/macrophages and CD8+ T lymphocytes but fewer numbers of CD4+ T lymphocytes and CD20+ B lymphocytes. The virus-induced inflammatory cells in patients with COPD were positively associated with virus load, illness severity, and reductions in lung function.CONCLUSIONS:Experimental RV infection induces bronchial mucosal eosinophilia and neutrophilia only in patients with COPD and monocytes/macrophages and lymphocytes in both patients with COPD and control subjects. The virus-induced inflammatory cell phenotypes observed in COPD positively related to virus load and illness severity. Antiviral/anti-inflammatory therapies could attenuate bronchial inflammation and ameliorate virus-induced COPD exacerbations.

Background: Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting beta(2)-adrenoceptor (beta R-2) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs).Materials and methods: ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]). Cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+](i)) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction.Results: VI and salmeterol (10(-12)-10(-6) M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. beta R-2 antagonism by propranolol or ICI 118.551 (10(-12)-10(-4) M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5x10(-6) M, 30 minutes) attenuated VI-induced cAMP production (P < 0.05), whereas pretreatment with UMEC (10(-8) M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10(-11)-5x10(-6) M) resulted in a concentration-dependent increase in [Ca2+](i), which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+](i) release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression.Conclusion: These data indicate that UMEC reverses cholinergic inhibition of VI-induced cAMP production, and is a more potent muscarinic receptor antagonist when in combination with VI versus either alone.

Long acting beta2 agonist/corticosteroids (LABA/ICS) change breathing pattern and chest wall parasternal muscle (PARA) activity in severe COPD (Chest 2010 137:558). However, the global effect on diaphragm and other respiratory muscles, and on exercise performance, is unknown. Aim: Examine effects of ultra-LABA/ICS VFF (Relvar) on respiratory muscle output and walking performance, as well as breathing pattern, dyspnea and PARA EMG in severe COPD. Methods: In N=21 severe Gold IV (2001) COPD (mean FEV1 0.85L;30% Pred), implanted with fine-wire PARA EMG, at baseline, 2hr and 3hr, after 2 puffs VFF, we measured: pulmonary function, breathing pattern, dyspnea (BORG), PARA EMG, global respiratory muscle output by Sniff Nasal Inspiratory Pressure (SNIP), and walking performance during 6 Minute Walk test. Results: With VFF, pulmonary function change was extremely minimal – FEV1 +1.8%, FRC -2.5%, IC +2.8%. Yet, tidal volume increased and respiratory rate decreased (P<0.05), less dyspnea (Borg 13.5 to 9.2 (P<0.05)), peak tidal PARA EMG decreased (P<0.01). Global respiratory muscle output and walk performance increased very significantly: 30.8% and 32.8% respectively, (P<0.01) (Fig1). Conclusion: Even without pulmonary function change, inhaled VFF (Relvar) induced significant improvement in respiratory muscle output and walking performance in severe COPD.

Purpose To compare pulmonary and systemic inflammatory mediator release, pre- and poststimulation, ex vivo, in cells from Japanese patients with chronic obstructive pulmonary disease (COPD), non-COPD smoking controls, and non-COPD nonsmoking controls (NSC). Patients and methods This was a nontreatment study with ten subjects per group. Inflammatory biomarker release, including interleukin (IL)-6 and -8, matrix metalloproteinase-9, and tumor necrosis factor (TNF)-α, was measured in peripheral blood mononuclear cells (PBMC) and sputum cells with and without lipopolysaccharide or TNF-α stimulation. Results In PBMC, basal TNF-α release (mean ± standard deviation) was significantly different between COPD (81.6±111.4 pg/mL) and nonsmoking controls (9.5±5.2 pg/mL) (P<0.05). No other significant differences were observed. Poststimulation biomarker release tended to increase, with the greatest changes in the COPD group. The greatest mean increases were seen in the lipopolysaccharide-induced release of matrix metalloproteinase-9, TNF-α, and IL-6 from PBMC. Pre- and poststimulation data from sputum samples were more variable and less conclusive than from PBMC. In the COPD group, induced sputum neutrophil levels were higher and macrophage levels were lower than in either control group. Significant correlations were seen between the number of sputum cells (macrophages and neutrophils) and biomarker levels (IL-8, IL-6, and TNF-α). Conclusion This was the first study to compare cellular inflammatory mediator release before and after stimulation among Japanese COPD, smoking controls, and nonsmoking controls populations. Poststimulation levels tended to be higher in patients with COPD. The results suggest that PBMC are already preactivated in the circulation in COPD patients. This provides further evidence that COPD is a multicomponent disease, involving both airway and systemic inflammation.

PURPOSE:To assess the importance of inflammation in chronic obstructive pulmonary disease (COPD) by measuring airway and systemic inflammatory biomarkers in Japanese patients with the disease and relevant control groups.PATIENTS AND METHODS:This was the first study of its type in Japanese COPD patients. It was a non-treatment study in which 100 participants were enrolled into one of three groups: nonsmoking controls, current or ex-smoking controls, and COPD patients. All participants underwent standard lung function assessments and provided sputum and blood samples from which the numbers of inflammatory cells and concentrations of biomarkers were measured, using standard procedures.RESULTS:The overall trends observed in levels of inflammatory cells and biomarkers in sputum and blood in COPD were consistent with previous reports in Western studies. Increasing levels of neutrophils, interleukin 8 (IL-8), surfactant protein D (SP-D), and Krebs von den Lungen 6 (KL-6) in sputum and clara cell 16 (CC-16), high-sensitivity C-reactive protein (hs-CRP), and KL-6 in serum and plasma fibrinogen were seen in the Japanese COPD patients compared with the non-COPD control participants. In sputum, significant correlations were seen between total cell count and matrix metalloproteinase 9 (MMP-9; P<0.001), neutrophils and MMP-9 (P<0.001), macrophages and KL-6 (P<0.01), total cell count and IL-8 (P<0.05), neutrophils and IL-8 (P<0.05), and macrophages and MMP-9 (P<0.05). Significant correlations were also observed between some inflammatory cells in sputum and biomarkers in serum, with the most significant between serum CC-16 and both total cell count (P<0.005) and neutrophils (P<0.005) in sputum.CONCLUSION:These results provide evidence for the first time that COPD in Japanese patients is a multicomponent disease, involving both airway and systemic inflammation, in addition to airway obstruction. Therefore, intervention with anti-inflammatory therapy may provide additional benefit in disease management of COPD in Japan.

BACKGROUND:Corticosteroids (CS) have limited efficacy in the treatment of chronic obstructive pulmonary disease (COPD). p38 mitogen-activated protein kinase (MAPK) activation is increased in lung macrophages of COPD. We investigated whether p38 MAPK inhibition can modulate CS insensitivity of peripheral blood mononuclear cells (PBMCs) from patients with COPD.METHODS:PBMCs from patients with COPD (n=8) or healthy smokers (n=8) were exposed to lipopolysaccharide (LPS) with a selective p38 MAPK inhibitor (GW856553; 10(-10)-10(-6) M), with dexamethasone (10(-10)-10(-6) M), or with both. Phosphorylated glucocorticoid receptor (GR) was measured by Western blot.RESULTS:Baseline (P<0.01) and LPS-induced (P<0.05) CXCL8 release was greater in PBMCs from COPD compared to healthy smokers. Inhibition of LPS-induced CXCL8 release by dexamethasone (10(-6) M) was reduced, and baseline and LPS-induced p38 MAPK activation increased in PBMCs of COPD. GW856553 (10(-9) and 10(-10) M) synergistically increased the inhibitory effect of dexamethasone (10(-8) and 10(-6) M) on LPS-induced CXCL8 release in COPD. Similar results were obtained for IL-6 release. GW856553 inhibited dexamethasone- and LPS-activated phosphorylation of serine 211 on GR. CS insensitivity in COPD PBMCs is reversed by inhibition of p38 MAPK activity, partly by preventing phosphorylation of GR at serine 211.CONCLUSION:p38 MAPK inhibition may be beneficial in COPD by restoring CS sensitivity.

Purpose: Using sputum neutrophils as the primary measure, and other inflammation biomarkers, this study evaluated the anti-inflammatory effects of the combination salmeterol 50 mcg and fluticasone propionate 250 mcg (SFC 250) in Japanese patients with chronic obstructive pulmonary disease (COPD).Patients and methods: Patients were treated in a randomized, double-blind, parallel group, placebo-controlled trial with SFC 250 twice daily (n=26) or placebo (n=26) for 12 weeks. At the start and end of treatment, inflammation biomarkers (sputum and serum), lung function, and health status (COPD Assessment Test [ CAT] questionnaire) were measured.Results: Although a numerical decrease in differential neutrophil count was observed from baseline, SFC 250 did not significantly reduce sputum neutrophils compared with placebo, nor were there significant changes from baseline in the other biomarkers (sputum or serum), lung function, or CAT, versus placebo. Squamous epithelial cell contamination in some sputum samples rendered them unacceptable for analysis, which reduced the sample size to n=19 (SFC 250) and n=10 (placebo). However, inclusion of contaminated samples did not affect the overall trend of the outcome. Ad hoc bootstrap statistical analysis showed a 27.9% (SFC 250) and 1.3% (placebo) decrease in sputum neutrophils. Sputum IL-8 decreased by 43.2% after SFC 250 but increased by 48.3% with placebo. Responder analyses showed 42% of patients had. 20% decrease in neutrophils from baseline; and 47% of patients had a. 200 pg/mL change in sputum IL-8 following SFC 250 versus 20% after placebo; both changes are considered clinically relevant.Conclusion: This study provides additional information about inflammation in Japanese COPD patients and is the first to study the anti-inflammatory effects of SFC 250 in this context and population. In the primary analysis, SFC 250 did not produce significant changes from baseline in sputum neutrophil levels or other sputum or serum inflammatory markers compared with placebo. Secondary ad hoc statistical analysis showed that SFC 250 reduced the number of sputum neutrophils and IL-8 compared with placebo.

Beta agonists have been shown to change ventilation and chest wall activity in COPD (Chest 2010 137:558-65). We studied the new ultra long acting beta 2 agonist-steroid combination, vilanterol trifenatate/fluticasone furoate (Relvar, VFF) to determine its effects on breathing pattern, dyspnea and respiratory muscle function in severe COPD. We studied 6 subjects with severe COPD (mean FEV 1 0.68), very minimally reversible, implanted with fine-wire EMG in parasternal (PARA) muscles, at baseline and 2hr after 2 puffs VFF. We measured: breathing pattern, dyspnea scores (BORG), PARA activity, sniff inspiratory nasal pressure (SNIP), and pulmonary function tests. Minute ventilation, frequency and mean inspiratory flow decreased (pl0.01). This breathing pattern was associated with much lower dyspnea scores (pl0.01). Mean PARA also decreased after VFF, while SNIP increased (pl0.01). We conclude that inhaled vilanterol trifenatate/fluticasone furoate (Relvar) induces a significant change in breathing pattern, which is associated with less dyspnea and increased global respiratory muscle function.

BACKGROUND:COPD is associated with increased numbers of T cells in the lungs, particularly CD8+ T cells. The mechanisms of increased T cells are unknown but may be related to repeated virus infections in COPD patients. We analysed lymphocyte subsets in blood and bronchoalveolar lavage in smokers and COPD subjects during experimental rhinovirus infections.METHODS:Lymphocytes were isolated from blood and bronchoalveolar lavage from COPD subjects and non-obstructed smokers prior to, and following experimental rhinovirus infection. Lymphocyte surface markers and intracellular cytokines were analysed using flow cytometry.RESULTS:Following rhinovirus infection CD4+ and CD8+ T cell numbers in the COPD subjects were significantly reduced in blood and CD3+ and CD8+ T cells increased in bronchoalveolar lavage compared to baseline. T cells did not increase in BAL in the control subjects. CD3+ T cells correlated with virus load.CONCLUSIONS:Following rhinovirus infection T cells move from the circulation to the lung. Repeated virus infections may contribute to T cell accumulation in COPD patients.

Inhaled vilanterol trifenatate/fluticasone furoate (VFF), an ultra long acting beta2 agonist/ICS combination (Relvar) may change respiratory muscle activation and diaphragm contractility. Beta agonist effects on respiratory muscle activity have been shown in normal mammals and COPD patients (Resp Phys 2008 161:253-60,Chest 2010 137:558-65). Magnetic supra maximal phrenic nerve stimulation (MagStim) was utilized to assess diaphragm contraction via measurement of mouth pressure. 6 patients with severe COPD (mean FEV1 0.68), very minimally reversible (no change in FRC, IC or lung volumes) were studied before and 2 hrs after 2 puffs of VFF while breathing quietly, recording mouth pressure, and airflow through a pneumotach. MagStim was discharged at FRC (end expiratory zero airflow) and at selected points in inspiration and expiration, unanticipated by the subject. For all subjects, pressure from maximum diaphragm contraction by MagStim increased significantly after VFF (p<0.01). Similarly at matching points during the breathing cycle, lengths shorter than FRC, maximal contraction pressure increased after VFF (p<0.01). In severe COPD subjects, diaphragm contraction at matching lengths is increased after vilanterol trifenatate/fluticasone furoate (Relvar). ![Figure][1] [1]: pending:yes

Background: Inhaled beta2-agonist and corticosteroid (LABA/ICS), salmeterol-fluticasone propionate (Advair), changes chest wall activity in severe COPD (Chest 2010 137:558-65). To elucidate the inspiratory muscle changes, we examined raw parasternal (PARA) EMG in severe COPD utilizing a frequency domain measure, turns counting analysis, before and after the ultra long acting LABA/ICS vilanterol trifenatate/fluticasone furoate (Relvar, VFF). Methods: Fine wire EMG was implanted in PARA muscles of subjects (N=5, mean FEV1 0.68) with severe minimally reversible COPD (no change: FRC, IC). At baseline and 2 hrs after 2 puffs of VFF, we measured pulmonary function, airflow and PARA raw EMG (12Khz sampling). We computed breath-by-breath values for breathing pattern, peak PARA Mavg EMG, and raw EMG total turns and mean amplitude per turn per breath. Results: For all subjects, minute ventilation, rate and mean inspiratory flow decreased significantly after VFF (P<0.01). Peak PARA Mavg EMG decreased significantly (P<0.01). There was a marked reduction in raw PARA EMG total turns per breath (mean 423 to 267) with significant decrease in mean amplitude per turn (mean 5.2 to 2.5) (P<0.01). Summary: Vilanterol trifenatate/fluticasone furoate (Relvar) induces significant changes in raw parasternal muscle EMG activity in severe COPD - a fundamental alteration in the population of motor units within this inspiratory muscle. ![Figure][1] [1]: pending:yes

In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting β2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.

Introduction: Inhaled corticosteroid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy proved to be effective in asthma management, but it was not rationally designed. In the present study, we describe using, the relationship between the intrinsic activities of GR ligands and the ability of the ultra-LABA, indacaterol, to enhance glucocorticoid (GC) dependent gene transcription. Methods: BEAS-2B airway epithelial cells were used as an experimental model. Expressions of glucocorticoid induced anti-inflammatory genes such as glucocorticoid-induced leucine zipper (GILZ), kinase inhibitor protein 2 of 57 kDa (p57kip2), cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and a side effect gene pyruvate dehydrogenase kinase 4 (PDK4) were examined by qPCR. Results: Concentration-response curves were constructed to six GR ligands (Fluticasone furoate [FF], Dexamethasone [dex], desisobutyryl-ciclesonide [DC], GW870086X [GW], mifepristone [Mif] and ORG34517). Relative to Dex, a reference full agonist (intrinsic activity (α) = 1), DC, GW and Mif were partial agonists (α = 0.55, 0.36 and 0.02 respectively). Indacaterol significantly augmented expression of p57kip2, PDK4 and luciferase genes depending on the intrinsic activity of the GC. However, augmentation of expression of GILZ, CRISPLD2 in presence of indacaterol was independent of the intrinsic activity of the GC. Conclusions: We submit that the generation of glucocorticoid-inducible, gene “fingerprints” will provide important information that could assist the rational design of optimised ICS/LABA therapy for asthma and allied inflammatory diseases that hitherto, has not been possible.

Background COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections. Methods Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points. Results At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects. Conclusion Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.

Background: Combination inhaled therapy with long-acting beta(2) agonists (LABAs) and corticosteroids is beneficial in treating asthma and chronic obstructive pulmonary disease (COPD).Objective: In asthma, LABAs enhance glucocorticoid receptor (GR) nuclear translocation in the presence of corticosteroids. Whether this biological mechanism occurs in COPD, a relatively corticosteroid-resistant disease, is uncertain.Methods: Eight patients with mild/moderate COPD participated in a double-blind, placebo-controlled, crossover study and inhaled single doses of fluticasone propionate (FP) 100 mu g, FP 500 mu g, salmeterol xinafoate (SLM) 50 mu g, and combination FP 100 mu g + SLM 50 mu g. One hour postinhalation, sputum was induced, nuclear proteins isolated from purified macrophages, and levels of activated nuclear GR quantified by using a GR-glucocorticoid response element ELISA-based assay.Results: Nuclear GR significantly increased after the inhalation of FP 500 mu g(P<.01), but not after the inhalation of FP 100 mu g or SLM 50 mu g, compared with placebo. Interestingly, SLM in combination with FP 100 mu g increased nuclear GR levels equivalent to those of FP 500 mu g alone. This was significantly greater than either FP 100 mg (P<.05) or SLM 50 mu g (P<.01) alone. In vitro in a human macrophage cell line, SLM(10(-8) mol/L) enhanced FP (10(-9) mol/L)-induced mitogen-activated protein kinase phosphatase-1 mRNA (5.8 +/- 0.6 vs 8.4 +/- 1.1 x 10(-6) copies, P<.05) and 2 x glucocorticoid response element-luciferase reporter gene activity (250.1 +/- 5.6 vs 103.1 +/- 23.6-fold induction, P<.001). Addition of SLM (10(-9) mol/L) to FP (10(-11) mol/L) significantly enhanced FP-mediated suppression of IL-1 beta-induced CXCL8 (P<.05).Conclusions: Addition of SLM 50 mu g to FP 100 mu g enhanced GR nuclear translocation equivalent to that seen with a 5-fold higher dose of FP in sputum macrophages from patients with COPD. This may account for the superior clinical effects of combination LABA/corticosteroid treatment compared with either as monotherapy observed in COPD.

RATIONALEChronic obstructive pulmonary disease (COPD) exacerbations are associated with virus (mostly rhinovirus) and bacterial infections, but it is not known whether rhinovirus infections precipitate secondary bacterial infections.OBJECTIVESTo investigate relationships between rhinovirus infection and bacterial infection and the role of antimicrobial peptides in COPD exacerbations.METHODSWe infected subjects with moderate COPD and smokers and nonsmokers with normal lung function with rhinovirus. Induced sputum was collected before and repeatedly after rhinovirus infection and virus and bacterial loads measured with quantitative polymerase chain reaction and culture. The antimicrobial peptides secretory leukoprotease inhibitor (SLPI), elafin, pentraxin, LL-37, α-defensins and β-defensin-2, and the protease neutrophil elastase were measured in sputum supernatants.MEASUREMENTS AND MAIN RESULTSAfter rhinovirus infection, secondary bacterial infection was detected in 60% of subjects with COPD, 9.5% of smokers, and 10% of nonsmokers (P < 0.001). Sputum virus load peaked on Days 5-9 and bacterial load on Day 15. Sputum neutrophil elastase was significantly increased and SLPI and elafin significantly reduced after rhinovirus infection exclusively in subjects with COPD with secondary bacterial infections, and SLPI and elafin levels correlated inversely with bacterial load.CONCLUSIONSRhinovirus infections are frequently followed by secondary bacterial infections in COPD and cleavage of the antimicrobial peptides SLPI and elafin by virus-induced neutrophil elastase may precipitate these secondary bacterial infections. Therapy targeting neutrophil elastase or enhancing innate immunity may be useful novel therapies for prevention of secondary bacterial infections in virus-induced COPD exacerbations.

BACKGROUND:Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.METHODS:The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).RESULTS:In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.CONCLUSIONS:This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.

Inhaled glucocorticoids (GCs) w/wo long-acting β 2 -adrenoceptor agonists (LABA) are the most effective treatment for asthma. However, GC insensitivity is a facet of severe asthma and COPD. We have examined TNFα-induced resistance to GC-dependent transcription by steroidal and non-steroidal GC receptor (GR) ligands in the absence and presence of a LABA, formoterol. METHODS: GC-dependent transcription was modeled by a simple 2×GRE-luciferase reporter in human bronchial epithelial BEAS-2B cells treated with TNFα (10 ng/ml) for 1 h prior to addition of GR ligands, and harvested after 6 h for luciferase assay. RESULTS: TNFα reduced by 43-54% the ability to drive 2×GRE-dependent transcription by dexamethasone, budesonide, fluticasone propionate or fluticasone furoate. The GCs des-ciclesonide, GW870086X, RU24858 and the non-steroidal GR agonist, GSK9027, all showed reduced maximal responses (E max ) with intrinsic activities 0.5-0.77 relative to dexamethasone. In each case, TNFα reduced E max by a further 39-55%. Conversely, formoterol enhanced GRE-dependent transcription by each ligand and rescued the resistance induced by TNFα; these effects were proportional to the E max of each drug. Statistically significant reversal of TNFα-induced resistance was observed with the c-jun N-terminal kinase inhibitor, JNK8, and PS-1145, an IκB kinase 2 (IKK2) inhibitor. CONCLUSIONS: TNFα induces GC resistance to steroidal GR ligands that are both full and partial agonists, as well as to a non-steroidal GR agonist. This effect is rescued by the addition of formoterol. It is possible that inhibition of inflammatory signalling may also reduce GC resistance.