Effects of PDE4 inhibitors on lipopolysaccharide-induced priming of superoxide anion production from human mononuclear cells.

AIMS: Phosphodiesterase 4 (PDE4) inhibitors have been described as potent anti-inflammatory compounds, involving an increase in intracellular levels of cyclic 3',5'-adenosine monophosphate (AMP). The aim of this study was to compare the effects of selective PDE4 inhibitors, rolipram and RP 73-401 with the cell permeable analogue of cyclic AMP, dibutyryl-cyclic AMP (db-cAMP) and the anti-inflammatory cytokine interleukin-10 (IL-10) on superoxide anion production from peripheral blood mononuclear cells preincubated with lipopolysaccharide (LPS). MAJOR FINDINGS: We report that, after incubation of the cells with LPS, a large increase in superoxide anion production was observed. Rolipram or RP 73-401 (10(-8) to 10(-5) M) induced significant reductions of fMLP-induced superoxide anion production in cells incubated with or without LPS. The db-cAMP (10(-5) to 10(-3) M) also elicited dose-dependent inhibitions of the fMLP-induced superoxide anion production. In contrast, IL-10 (1 or 10 ng/ml) did not elicit a reduction in fMLP-induced superoxide anion production in both conditions. PRINCIPAL CONCLUSION: These results suggest that the inhibitory activity of PDE4 inhibitors on fMLP-induced production of superoxide anion production is mediated by db-cAMP rather than IL-10

Phosphodiesterase 4 inhibitors and db-cAMP inhibit TNF-α release from human mononuclear cells. Effects of cAMP and cGMP-dependent protein kinase inhibitors

We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-α release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10−5 to 10−3 M), rolipram (10−9 M to 10−5 M) or Ro 20-1724 (10−9 M to 10−5 M) significantly inhibited LPS-induced TNF-α release. When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10−4 M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10−4 M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10−9 to 10−5 M) on LPS-induced TNF-α release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-α release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation

Bronchial responses to substance P after antigen challenge in the guinea-pig: in vivo and in vitro studies

The effect of antigen challenge on the airway responses to substance P and on the epithelial neutral endopeptidase (NEP) activity was investigated in aerosol sensitized guinea-pigs. In vivo, bronchial responses to aerosolized substance P were similar to the responses observed in antigen-challenged guinea-pigs and in the control groups. In contrast, when the guinea-pigs were pretreated with the NEP inhibitor, phosphoramidon, a significant increase in the airway responses to substance P was observed after antigen challenge in vivo. However, in vitro, the contractile responses of the tracheal smooth muscle to substance P were similar between groups of guinea-pigs, in respect to the presence or absence of the epithelium and/or phosphoramidon. Histological studies showed an accumulation of eosinophils in the tracheal submucosa after antigen challenge and intact epithelial cells. These results show that in vivo bronchial hyperresponsiveness to substance P after antigen challenge in the guinea-pig is not associated with increased responses of the smooth muscle to exogenous SP in vitro. In addition, the results with phosphoramidon suggest that loss of NEP activity cannot account for the in vivo bronchial hyperresponsiveness to substance P presently observed

Role of ADAM and ADAMTS metalloproteinases in airway diseases

Lungs are exposed to the outside environment and therefore to toxic and infectious agents or allergens. This may lead to permanent activation of innate immune response elements. A Disintegrin And Metalloproteinases (ADAMs) and ADAMs with Thrombospondin motifs (ADAMTS) are proteinases closely related to Matrix Metalloproteinases (MMPs). These multifaceted molecules bear metalloproteinase and disintegrin domains endowing them with features of both proteinases and adhesion molecules. Proteinases of the ADAM family are associated to various physiological and pathological processes and display a wide spectrum of biological effects encompassing cell fusion, cell adhesion, "shedding process", cleavage of various substrates from the extracellular matrix, growth factors or cytokines... This review will focus on the putative roles of ADAM/ADAMTS proteinases in airway diseases such as asthma and COPD

Models of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a major global health problem and is predicted to become the third most common cause of death by 2020. Apart from the important preventive steps of smoking cessation, there are no other specific treatments for COPD that are as effective in reversing the condition, and therefore there is a need to understand the pathophysiological mechanisms that could lead to new therapeutic strategies. The development of experimental models will help to dissect these mechanisms at the cellular and molecular level. COPD is a disease characterized by progressive airflow obstruction of the peripheral airways, associated with lung inflammation, emphysema and mucus hypersecretion. Different approaches to mimic COPD have been developed but are limited in comparison to models of allergic asthma. COPD models usually do not mimic the major features of human COPD and are commonly based on the induction of COPD-like lesions in the lungs and airways using noxious inhalants such as tobacco smoke, nitrogen dioxide, or sulfur dioxide. Depending on the duration and intensity of exposure, these noxious stimuli induce signs of chronic inflammation and airway remodelling. Emphysema can be achieved by combining such exposure with instillation of tissue-degrading enzymes. Other approaches are based on genetically-targeted mice which develop COPD-like lesions with emphysema, and such mice provide deep insights into pathophysiological mechanisms. Future approaches should aim to mimic irreversible airflow obstruction, associated with cough and sputum production, with the possibility of inducing exacerbations

Roflumilast partially reverses smoke-induced mucociliary dysfunction

BACKGROUND: Phosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion. Since PDE4 predominates in airway epithelial cells, PDE4 inhibitors can stimulate Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by increasing cAMP. Tobacco smoking and COPD are associated with decreased CFTR function and impaired mucociliary clearance (MCC). However, the effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored. METHODS: Primary normal human bronchial epithelial cells (NHBE) from non-smokers, cultured at the air-liquid interface (ALI) were used for most experiments. Cultures were exposed to cigarette smoke in a Vitrocell VC-10 smoking robot. To evaluate the effect of roflumilast on intracellular cAMP concentrations, fluorescence resonance energy transfer (FRET) between CFP- and YFP-tagged protein kinase A (PKA) subunits was recorded. Airway surface liquid (ASL) was measured using light refraction scanning and ciliary beat frequency (CBF) employing infrared differential interference contrast microscopy. Chloride conductance was measured in Ussing chambers and CFTR expression was quantified with qPCR. RESULTS: While treatment with 100 nM roflumilast had little effect alone, it increased intracellular cAMP upon stimulation with forskolin and albuterol in cultures exposed to cigarette smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ß-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF stimulation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke exposed cell cultures. CONCLUSIONS: Our results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function