Jonathan Plumb

Identification of Cells Expressing IL-17A and IL-17F in the Lungs of Patients With COPD

BACKGROUND Lymphocytes secrete IL-17A and IL-17F, which enhance innate immune responses. IL-17 expression has not been studied in COPD small airways. The aim of this study was to quantify IL-17A and IL-17F expression in the peripheral lung tissue of patients with COPD compared with control subjects and to identify inflammatory cells that express IL-17. METHODS IL-17 expression was assessed using immunohistochemistry in peripheral lung tissue (18 patients with COPD and 10 smokers and 10 nonsmokers with normal lung function) and induced sputum (12 patients with COPD and six nonsmokers). Alveolar macrophages from eight patients with COPD, eight smokers, and seven nonsmokers were used for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. RESULTS The number of inflammatory cells expressing IL-17A in the small airway subepithelium was higher in patients with COPD than in smokers (P = .01) and nonsmokers (P = .02). IL-17A expression was higher than IL-17F in this region. IL-17A was expressed by lymphocytes, neutrophils, and macrophages (confirmed by RT-PCR). The expression of IL-17F was greater than IL-17A in epithelial cells and lymphoid follicles, although there were no differences among subject groups. CONCLUSIONS Our findings indicate different roles for IL-17A and IL-17F in the pathogenesis of COPD. IL-17A plays a role in small airway subepithelial inflammation, whereas IL-17F appears to play a more prominent role within lymphoid follicles.

Circadian timing in the lung; a specific role for bronchiolar epithelial cells.

In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology.

Increased T-regulatory cells within lymphocyte follicles in moderate COPD

Lymphoid follicles in the lung parenchyma are a characteristic feature of chronic obstructive pulmonary disease (COPD). There are reports of altered CD4 T-regulatory cell numbers in COPD lungs, but the location of these cells within COPD lung tissue specific follicles has not been investigated. The presence of CD4+FOXP3+ T-regulatory cells was assessed in surgically resected lung tissue from 12 COPD patients, 11 smokers with normal lung function and seven nonsmokers by combined immunofluorescence and immunohistochemistry. Organised lymphoid follicles were observed in all three groups of patients, as well as lymphoid clusters lacking organisation. The percentage of CD4 cells that were T-regulatory cells were significantly increased (p = 0.02) within COPD (16%) follicles compared with smokers (10%) and nonsmokers (8%). In contrast, there was no change (p>0.05) in the percentage of T-regulatory cells in clusters or the subepithelium between groups. Lymphoid follicles in COPD patients have increased T-regulatory cells. Therefore, T-regulatory activity may be altered within COPD lymphoid follicles.

Inhibition of Lipopolysaccharide-Stimulated Chronic Obstructive Pulmonary Disease Macrophage Inflammatory Gene Expression by Dexamethasone and the p38 Mitogen-Activated Protein Kinase Inhibitor N-cyano-N′-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d] pyrimidin-2-yl]amino}ethyl)guanidine (SB706504)

p38 mitogen-activated protein kinase (MAPK) signaling is known to be increased in chronic obstructive pulmonary disease (COPD) macrophages. We have studied the effects of the p38 MAPK inhibitor N-cyano-N′-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]-pyrimidin-2-yl]amino}ethyl)guanidine (SB706504) and dexamethasone on COPD macrophage inflammatory gene expression and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. Lipopolysaccharide (LPS)-stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AMs) were cultured with dexamethasone and/or SB706504. MDMs were used for gene array and protein studies, whereas tumor necrosis factor (TNF) α protein production was measured from AMs. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared with SB706504 alone (-2.04) or dexamethasone (-3.39). Twenty-three genes were insensitive to the effects of both drugs, including interleukin (IL)-1β, IL-18, and chemokine (CC motif) ligand (CCL) 5. In addition, the chromosome 4 chemokine cluster members, CXCL1, CXCL2, CXCL3, and CXCL8, were all glucocorticoid-resistant. SB706504 significantly inhibited LPS-stimulated TNFα production from COPD and smoker AMs, with near-maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS-regulated genes, including IL-1β, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.

The effect of peroxisome proliferator-activated receptor-γ ligands on in vitro and in vivo models of COPD

Peroxisome proliferator-activated receptor (PPAR)-&ggr; is expressed in alveolar macrophages. The anti-inflammatory potential of the PPAR-&ggr; ligands rosiglitazone and pioglitazone were investigated using in vitro alveolar macrophage models and in vivo animal models relevant to chronic obstructive pulmonary disease (COPD). PPAR-&ggr; protein and gene expression in COPD alveolar macrophages was compared with control smokers and never-smokers. COPD macrophages were used to investigate the effects of PPAR-&ggr; ligands and corticosteroids on lipopolysaccharide-induced cytokine production, alternative macrophage activation (M2) gene expression and efferocytosis. The effects of PPAR-&ggr; ligands in a subchronic tobacco smoke model in mice were investigated. PPAR-&ggr; protein expression was similar in COPD patients compared to controls, although increased gene expression levels were observed in COPD patients and control smokers compared to never-smokers. PPAR-&ggr; ligands reduced tumour necrosis factor-&agr; and CC chemokine ligand-5, but not CXC chemokine ligand-8, in COPD alveolar macrophages; these effects were generally less than those of the corticosteroid dexamethasone. Rosiglitazone increased M2 gene expression and enhanced efferocytosis of apoptotic neutrophils. Rosiglitazone and pioglitazone attenuated airway neutrophilia in a corticosteroid-resistant mouse model of pulmonary inflammation. We show biological actions of PPAR-&ggr; agonists on corticosteroid-resistant disease, tobacco smoke-induced pulmonary inflammation, skewing of macrophage phenotype and clearance of apoptotic neutrophils. Biological actions of a PPAR-&ggr; agonist shown in COPD-relevant models may affect progression and side-effects in patients http://ow.ly/r50DF

Induced sputum genes associated with spirometric and radiological disease severity in COPD ex-smokers

Background Induced sputum is used to sample inflammatory cells, predominantly neutrophils and macrophages, from the airways of COPD patients. The authors aim was to identify candidate genes associated with the degree of airflow obstruction and the extent of emphysema by expression profiling, and then to confirm these findings for selected candidates using PCR and protein analysis. Methods Two sputum studies were performed in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 2–4 COPD ex-smokers from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort. First, gene array profiling at baseline in samples from 148 patients. The findings were replicated in a separate population of 176 patients using real-time PCR. The findings for one selected gene IL-18R were further analysed using immunohistochemistry in lung tissue and induced sputum from patients outside the ECLIPSE cohort. Results Gene expression profiling revealed changes in 277 genes associated with GOLD stage 2 versus 3 and 4, and 198 genes with changes associated with the degree of emphysema (p<0.01 for each gene). Twelve of these candidate genes were analysed by PCR in the replication cohort, with significant changes (p<0.05) observed for 11 genes. IL-18R protein expression was higher on alveolar macrophages in lung tissue of COPD patients (mean 23.2%) compared to controls (mean ex-smokers 2% and non-smokers 2.5%). Conclusion Gene expression profiling in sputum cells identified candidate genes that may play roles in molecular mechanisms associated with COPD. The replication by PCR and protein in different studies confirms these findings, and highlights a potential role for IL-18R upregulation in severe COPD.

Increased phosphorylated p38 mitogen-activated protein kinase in COPD lungs.

The p38 mitogen-activated protein kinase (MAPK) pathway is upregulated in chronic obstructive pulmonary disease (COPD). To date, dual labelling to identify cell-type-specific presence of phosphorylated (phospho-)p38 MAPK has not been carried out. Phospho-p38 MAPK was quantified in a variety of cell types in the lung tissue of 20 COPD patients, 12 smokers and 12 nonsmokers using immunohistochemistry. Paired blood and sputum neutrophils (from seven subjects with COPD), and CD8 and epithelial cells (from three subjects with COPD) were cultured with a p38 MAPK inhibitor. Supernatant tumour necrosis factor-α and CXCL8 levels were analysed by ELISA. Sputum and blood neutrophil cytospins were analysed for phospho-p38 MAPK. Phospho-p38 MAPK was increased in bronchial epithelial cells, macrophages and CD20+ and CD8+ lymphocytes in COPD lungs. Sputum and lung tissue neutrophils were devoid of phospho-p38 in all patient groups. The p38 MAPK inhibitor SB100 attenuated pro-inflammatory mediator release in COPD lung CD8 cells and airway epithelia, but there was no effect on COPD sputum neutrophils. Our data indicate cell-specific anti-inflammatory effects of p38 MAPK inhibition in the lung.

IFN-γ synergistically enhances LPS signalling in alveolar macrophages from COPD patients and controls by corticosteroid-resistant STAT1 activation.

BACKGROUND AND PURPOSE IFN‐γ levels are increased in chronic obstructive airway disease (COPD) patients compared with healthy subjects and are further elevated during viral exacerbations. IFN‐γ can ‘prime’ macrophages to enhance the response to toll‐like receptor (TLR) ligands, such as LPS. The aim of this study was to examine the effect IFN‐γ on corticosteroid sensitivity in alveolar macrophages (AM).

Reduced glucocorticoid receptor expression and function in airway neutrophils

Chronic Obstructive Pulmonary Disease (COPD) is a glucocorticoid resistant condition characterised by airway neutrophilia. Reduced glucocorticoid receptor (GR) expression in COPD airway neutrophils may be a mechanism that contributes to glucocorticoid resistance. Our objective was to investigate the expression and function of GR within COPD airway neutrophils. Dual-label immunofluorescence was used to analyse airway neutrophil expression of GR within peripheral lung tissue samples (11 COPD patients, 7 healthy non-smokers [NS]) and induced sputum (7 COPD patients, 7 NS). TNFα and CXCL8 release were measured in neutrophils isolated from induced sputum and peripheral blood (7 COPD patients) in the presence of dexamethasone. In lung tissue, GR was abundantly expressed in macrophages and lymphocytes, but very low expression was observed in neutrophils (means 6.8% and 4.3% in COPD patients and NS respectively). Similarly low expression was observed in sputum neutrophils (means 3.8% and 6.9% in COPD patients and NS respectively). In contrast, GR was expressed by 100% of blood neutrophils. Dexamethasone had less suppressive effect on TNFα and CXCL8 production in vitro by neutrophils from induced sputum compared to neutrophils from paired blood samples. Airway neutrophils have low expression of GR in both COPD patients and controls. The effects of glucocorticoids on cytokine production from airway neutrophils are reduced. Increased numbers of airway neutrophils lacking GR may contribute to glucocorticoid resistance in COPD patients.

LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

LPS inhalation was used to investigate whether sputum supernatant post-LPS challenge increases neutrophil chemotactic activity and to elucidate the role of CXCR1/CXCR2 signalling in this process. 14 healthy non-smoking subjects inhaled 30μg of LPS. Sputum was induced at baseline, 6 and 24h post-LPS challenge. Differential cell counts were determined and supernatants CXCL8, CXCL1, IL-6 and CCL2 levels measured. Peripheral blood neutrophils obtained from healthy volunteers were used for chemotaxis experiments using sputum supernatant. To delineate signalling mechanisms, the effects of a CXCR2/CXCR1 (dual) antagonist (Sch527123) and a CXCR2 specific antagonist (SB656933) were tested. LPS inhalation significantly increased sputum neutrophil counts from 45.3% to 76.7% and 69.3% at 6 and 24h respectively. LPS increased CXCL8, IL-6 and CCL2 levels but not CXCL1. Neutrophil chemotaxis significantly increased (2.7 fold) at 24h compared to baseline. Chemotaxis was inhibited by 79.0% with Sch527123 and 52.0% with SB656933. We conclude that LPS challenge increases sputum supernatant CXCL8 levels, which is associated with increased chemotactic activity which is dependent on both CXCR1 and CXCR2.