Inhaled calcium salts inhibit tobacco smoke-induced inflammation by modulating expression of chemokines and cytokines.

Tobacco smoke-induced lung inflammation in patients with chronic obstructive pulmonary disease (COPD) worsens with disease progression and acute exacerbations caused by respiratory infections. Chronic therapies to manage COPD center on bronchodilators to improve lung function and inhaled corticosteroids (ICS) to help reduce the risk of exacerbations. Novel therapies are needed that reduce the underlying inflammation associated with COPD and the inflammation resulting from respiratory infections that worsen disease. The lung is lined with airway surface liquid (ASL), a rheologically active material that provides an innate defense for the airway against inhaled particulate and is continuously cleared from the airways by mucociliary clearance. The rheological properties of the ASL can be altered by changes in airway hydration and by cations, such as calcium, that interact with electronegative glycoproteins. The effect of inhaled salts on inflammation resulting from tobacco smoke exposure was studied to determine if cations could be used to alter the properties of the ASL and reduce inflammation. Inhaled calcium salts, but not sodium or magnesium salts, reduced cellular inflammation and key chemokines and cytokines that were induced by tobacco smoke exposure. Similar anti-inflammatory effects of calcium salts were observed using in vitro cultures of human monocyte derived macrophages and human bronchial epithelial cells. The data suggest that inhaled calcium salts may act broadly on both biophysical and biological pathways to reduce pulmonary inflammation.

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

Peroxisome proliferator-activated receptor (PPAR)-γ is expressed in alveolar macrophages. The anti-inflammatory potential of the PPAR-γ 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-γ 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-γ ligands and corticosteroids on lipopolysaccharide-induced cytokine production, alternative macrophage activation (M2) gene expression and efferocytosis. The effects of PPAR-γ ligands in a subchronic tobacco smoke model in mice were investigated. PPAR-γ 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-γ ligands reduced tumour necrosis factor-α 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-γ agonists on corticosteroid-resistant disease, tobacco smoke-induced pulmonary inflammation, skewing of macrophage phenotype and clearance of apoptotic neutrophils.

p38alpha-selective mitogen-activated protein kinase inhibitor SD-282 reduces inflammation in a subchronic model of tobacco smoke-induced airway inflammation.

Chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, which is relatively insensitive to inhaled corticosteroids. The extent of the pulmonary inflammation in COPD correlates with disease severity, and it is thought to play a significant role in disease progression. We have evaluated a selective p38alpha-selective mitogen-activated protein kinase (MAPK) inhibitor, indole-5-carboxamide (ATP-competitive inhibitor of p38 kinase) (SD-282), in an 11-day model of tobacco smoke (TS)-induced pulmonary inflammation in A/J mice, by using dexamethasone as a reference steroid. Two oral treatment paradigms were evaluated in this TS model: prophylactic with daily pretreatment before each daily exposure, and therapeutic with daily treatment for 6 days commencing after 5 days of smoke exposure. Bronchoalveolar lavage and histological evaluation of lung sections taken after exposure to TS revealed an inflammatory response composed of increased numbers of macrophages and neutrophils and enhanced mucin staining. Phospho-p38 staining in macrophages and type II epithelial cells after TS exposure was also observed. Given prophylactically or therapeutically, dexamethasone failed to inhibit any of the TS-induced inflammatory changes. By contrast, SD-282 inhibited TS-induced increases in macrophages and neutrophils. Furthermore, SD 282 reduced TS-induced increases in cyclooxygenase-2 and interleukin-6 levels, and phospho-p38 expression in the lungs. In conclusion, SD-282 markedly reduced TS-induced inflammatory responses when given prophylactically or therapeutically whereas dexamethasone was ineffective. This is the first evidence that a p38alpha-selective MAPK inhibitor can exert pulmonary anti-inflammatory activity in a TS exposure model when given in a therapeutic mode, establishing the potential of p38 MAPK inhibitors as a therapy for COPD.

Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways.

BACKGROUND: The link between eosinophils and the development of airway hyperresponsiveness (AHR) in asthma is still controversial. This question was assessed in a murine model of asthma in which we performed a dose ranging study to establish whether the dose of steroid needed to inhibit the eosinophil infiltration correlated with that needed to block AHR. METHODS: The sensitised BALB/c mice were dosed with vehicle or dexamethasone (0.01-3 mg/kg) 2 hours before and 6 hours after each challenge (once daily for 6 days) and 2 hours before AHR determination by whole-body plethysmography. At 30 minutes after the AHR to aerosolised methacholine the mice were lavaged and differential white cell counts were determined. Challenging with antigen caused a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid and lung tissue, and increased AHR. RESULTS: Dexamethasone reduced BAL and lung tissue eosinophilia (ED50 values of 0.06 and 0.08 mg/kg, respectively), whereas a higher dose was needed to block AHR (ED50 of 0.32 mg/kg at 3 mg/ml methacholine. Dissociation was observed between the dose of steroid needed to affect AHR in comparison with eosinophilia and suggests that AHR is not a direct consequence of eosinophilia. CONCLUSION: This novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR by using steroids that are the mainstay of asthma therapy. These data suggest that eosinophilia is not associated with AHR and questions the rationale that many pharmaceutical companies are adopting in developing low-molecular-mass compounds that target eosinophil activation/recruitment for the treatment of asthma.

A gene-driven ENU-based approach to generating an allelic series in any gene.

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F1 ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.