ABSTRACT
Chronic obstructive pulmonary disease (COPD) is a smoking related inflammatory airway disease in which macrophages play a key role. Previously we have shown that cigarette smoke extract (CSE) causes suppression of macrophage inflammatory mediators, with the exception of IL-8. We now investigate the effects of dexamethasone on these gene expression changes. Monocyte derived macrophages (MDMs) were cultured with CSE and dexamethasone. Microarray analysis was used to assess inflammatory mediator regulation, with qPCR and ELISA also performed for selected cytokines. The major effect of CSE was down-regulation of inflammatory genes (11 probe sets). For CSE regulated genes (n=13), the median fold change with CSE alone was -2.84 and with dexamethasone alone was -2.97. Both treatments combined caused the greatest suppression of gene expression; -4.47. qPCR also showed that IL-1beta, GM-CSF and IL-6 mRNA levels were significantly reduced by CSE and further suppressed by dexamethasone. qPCR and ELISA showed that IL-8 levels were increased by CSE, with suppression by dexamethasone. We show that CSE suppressed the expression of some inflammatory genes whilst up-regulating IL-8. Dexamethasone further suppressed gene expression when combined with CSE. The combined effect of GC and CSE causes suppression of the macrophage innate immune response.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Cytokines/metabolism , Dexamethasone/pharmacology , Gene Expression Regulation/drug effects , Macrophages/drug effects , Pulmonary Disease, Chronic Obstructive/drug therapy , Aged , Cells, Cultured , Cytokines/genetics , Gene Expression Profiling , Humans , Immunity, Innate/drug effects , Inflammation Mediators/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Pulmonary Disease, Chronic Obstructive/etiology , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Smoking/adverse effectsABSTRACT
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) alpha 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)-1beta, 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 TNFalpha 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-1beta, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.
