Simon Lea

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

Synergistic Effects of p38 Mitogen-Activated Protein Kinase Inhibition with a Corticosteroid in Alveolar Macrophages from Patients with Chronic Obstructive Pulmonary Disease

Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage–colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38 MAPK inhibitor with a corticosteroid synergistically enhances the anti-inflammatory effects on LPS-mediated cytokine production by alveolar macrophages from patients with COPD and controls.

Effects of cigarette smoke on Toll-like receptor (TLR) activation of chronic obstructive pulmonary disease (COPD) macrophages.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll‐like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR‐2, ‐4 and ‐5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non‐typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR‐induced tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, IL‐10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi‐induced TNF‐α but not NTHi‐induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS‐induced TNF‐α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS‐induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.

Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

CD8 cells may contribute towards an autoimmune process in COPD. Down regulation of T cell receptor (TCR) signalling molecules occurs in autoimmune diseases with consequent T cell dysfunction. We hypothesise that TCR signalling is abnormal in COPD pulmonary CD8 cells. Micro-array gene expression analysis of blood and pulmonary COPD CD8 samples was performed and compared to pulmonary CD8 cells from smoker controls (S). We focused on the TCR signalling pathway, with validation of key findings using polymerase chain reaction and immunofluorescence. TCR signalling molecules in COPD pulmonary CD8 cells were down regulated compared to blood CD8 cells (CD247: fold change (FC) −2.43, Q = 0.001; LCK: FC −2.25, Q = 0.01). Micro-array analysis revealed no significant differences between COPD and S pulmonary CD8 cells. However, PCR revealed significantly lower gene expression levels of CD247 (FC −1.79, p = 0.04) and LCK (FC −1.77, p = 0.01) in COPD compared to S pulmonary CD8 cells. CD247 down regulation in COPD CD8 cells was confirmed by immunofluorescent staining of bronchoalveolar lavage cells: Significantly fewer COPD CD8 cells co-expressed CD247 compared to healthy non-smoker CD8 cells (mean 88.9 vs 75.2%, p<0.05) There is down regulation of TCR signalling molecules in COPD pulmonary CD8 cells. This may cause T cell dysfunction.

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

BackgroundThere 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.MethodsData 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.ResultsThere 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.ConclusionsWe 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.

Evaluation of Glucocorticoid Receptor Function in COPD Lung Macrophages Using Beclomethasone-17-Monopropionate

Previous studies of glucocorticoid receptor (GR) function in COPD lung macrophages have used dexamethasone to evaluate inhibition of cytokine production. We have now used the clinically relevant corticosteroid beclomethasone-17-monopropionate (17-BMP) to assess GR function in COPD lung macrophages, and investigated the transactivation of glucocorticoid sensitive genes and GR phosphorylation in addition to cytokine production. Lung macrophages were purified from surgically acquired lung tissue, from patients with COPD, smokers, and non-smokers. The transactivation of glucocorticoid sensitive genes (FKBP51 and GILZ) by 17-BMP were analysed by polymerase chain reaction. 17-BMP suppression of LPS-induced TNFα, IL-6 and CXCL8 was measured by ELISA and GR phosphorylation was measured by immunohistochemistry and Western blot. 17-BMP reduced cytokine release in a concentration dependent manner, with >70% inhibition of all cytokines, and no difference between COPD patients and controls. Similarly, the transactivation of FKBP51 and GILZ, and GR phosphorylation was similar between COPD patients and controls. In this context, GR function in COPD lung macrophages is unaltered. 17-BMP effectively suppresses cytokine production in COPD lung macrophages.

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

BackgroundThere 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.MethodsWe 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.ResultsThe 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.ConclusionsGW3965 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.

Corticosteroid insensitive alveolar macrophages from asthma patients; synergistic interaction with a p38 mitogen-activated protein kinase (MAPK) inhibitor

AIMS Some asthma patients remain symptomatic despite using high doses of inhaled corticosteroids (ICS). We used alveolar macrophages to identify individual patients with insensitivity to corticosteroids and to evaluate the anti-inflammatory effects of a p38 mitogen-activated protein kinase (MAPK) inhibitor combined with a corticosteroid on these cells. METHODS Alveolar macrophages from 27 asthma patients (classified according to the Global Initiative for Asthma (GINA) treatment stage. Six GINA1, 10 GINA2 and 11 GINA3/4) were stimulated with lipoploysaccharide (LPS) (1 μg ml(-1)). The effects of dexamethasone (dex 1-1000 nm), the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2Hpyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796 1-1000 nm) and both drugs combined at all concentrations on supernatant TNFα, IL-6 and CXCL-8 concentrations were analyzed by ELISA. Dose-sparing and efficacy enhancing effects of combination treatment were determined. RESULTS Dexamethasone reduced LPS-induced TNFα, IL-6 and CXCL-8 in all groups, but maximum inhibition was significantly reduced for GINA3/4 compared with GINA2 and GINA1 (P < 0.01). A subgroup of corticosteroid insensitive patients with a reduced effect of dexamethasone on cytokine secretion were identified. BIRB-796 in combination with dexamethasone significantly increased cytokine inhibition compared with either drug alone (P < 0.001) in all groups. This effect was greater in corticosteroid insensitive compared with sensitive patients. There were significant synergistic dose-sparing effects (P < 0.05) for the combination treatment on inhibition of TNFα, IL-6 and CXCL-8 in all groups. There was also significant efficacy enhancing benefits (P < 0.05) on TNFα and IL-6. CONCLUSIONS p38 MAPK inhibitors synergistically enhance efficacy of corticosteroids in macrophages from asthma patients. This effect is greater in corticosteroid insensitive asthma patients, suggesting that this class of drug should be targeted to this patient phenotype.

Corticosteroid insensitive alveolar macrophages from asthma patients; synergistic interaction with a p38 MAPK inhibitor.

AIMS Some asthma patients remain symptomatic despite using high doses of inhaled corticosteroids (ICS). We used alveolar macrophages to identify individual patients with insensitivity to corticosteroids and to evaluate the anti-inflammatory effects of a p38 mitogen-activated protein kinase (MAPK) inhibitor combined with a corticosteroid on these cells. METHODS Alveolar macrophages from 27 asthma patients (classified according to the Global Initiative for Asthma (GINA) treatment stage. Six GINA1, 10 GINA2 and 11 GINA3/4) were stimulated with lipoploysaccharide (LPS) (1 μg ml(-1)). The effects of dexamethasone (dex 1-1000 nm), the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2Hpyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796 1-1000 nm) and both drugs combined at all concentrations on supernatant TNFα, IL-6 and CXCL-8 concentrations were analyzed by ELISA. Dose-sparing and efficacy enhancing effects of combination treatment were determined. RESULTS Dexamethasone reduced LPS-induced TNFα, IL-6 and CXCL-8 in all groups, but maximum inhibition was significantly reduced for GINA3/4 compared with GINA2 and GINA1 (P < 0.01). A subgroup of corticosteroid insensitive patients with a reduced effect of dexamethasone on cytokine secretion were identified. BIRB-796 in combination with dexamethasone significantly increased cytokine inhibition compared with either drug alone (P < 0.001) in all groups. This effect was greater in corticosteroid insensitive compared with sensitive patients. There were significant synergistic dose-sparing effects (P < 0.05) for the combination treatment on inhibition of TNFα, IL-6 and CXCL-8 in all groups. There was also significant efficacy enhancing benefits (P < 0.05) on TNFα and IL-6. CONCLUSIONS p38 MAPK inhibitors synergistically enhance efficacy of corticosteroids in macrophages from asthma patients. This effect is greater in corticosteroid insensitive asthma patients, suggesting that this class of drug should be targeted to this patient phenotype.

Corticosteroid effects on COPD alveolar macrophages: Dependency on cell culture methodology

It is unclear whether cell culture methodology affects the corticosteroid sensitivity of chronic obstructive pulmonary disease (COPD) alveolar macrophages. We compared the effect of a short and a long isolation procedure on corticosteroid inhibition of lipopolysaccharide (LPS) stimulated cytokine release from COPD alveolar macrophages. We also investigated signalling pathways associated with macrophage activation during cell isolation. Macrophages cultured using a short isolation protocol released higher unstimulated levels of tumour necrosis factor (TNF)-α and chemokine C–X–C motif ligand (CXCL) 8; these macrophages were less sensitive to corticosteroid inhibition of LPS stimulated TNF-α and CXCL8 release when compared to a long isolation procedure. This was associated with increased p38 mitogen activated kinase (MAPK) activation. The p38 MAPK inhibitor, BIRB-796, significantly reduced unstimulated cytokine release. A key finding of this study was that both cell culture methods showed no difference in the corticosteroid sensitivity between COPD and control macrophages. We conclude that the culture of alveolar macrophages using a short isolation procedure alters cytokine production through p38 MAPK activation; this is associated with a change in corticosteroid sensitivity.