Lack of involvement of type 7 phosphodiesterase in an experimental model of asthma.

Type 7 phosphodiesterases (PDE7) are responsible for the decrease of intracellular cyclic AMP (cAMP) in many cells involved in allergic asthma by suppressing their potential to respond to many activating stimuli. The elevation of intracellular cAMP has been associated with immunosuppressive and anti-inflammatory activities and represents a potential treatment of asthma. Our aim was to evaluate the impact of the deletion of the murine phosphodiesterase (PDE)7B gene and then to evaluate the efficacy of a newly described selective PDE7A and -B inhibitor on an ovalbumin (OVA)-induced airway inflammation and airway hyperreactivity (AHR) model in mice. Inflammation was determined 72 h after single OVA challenge or 24 h after multiple challenges by the relative cell influx and cytokine content in bronchoalveolar lavage fluid. AHR and immunoglobulin E levels in serum were determined after multiple challenges. For the first time, we have demonstrated that the deletion of the PDE7B gene or the pharmacological inhibition of PDE7A and -B had no effect on all the parameters looked at in this model. These results highlight the absence of any implication of the PDE7 enzyme in our model.

Involvement of MMP-12 and phosphodiesterase type 4 in cigarette smoke-induced inflammation in mice.

The aim of the present study was to characterise a mouse model of airways inflammation induced by cigarette smoke and to compare it with a lipopolysaccharide (LPS) model with regards to the efficacy of a PDE4 inhibitor (cilomilast), a corticosteroid (dexamethasone) and macrophage metalloelastase (MMP)-12 gene deletion. Cigarette smoke exposure for 3 days induced a time-dependent airway neutrophilia associated with an increased level of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, MIP-1alpha and MMP-9 in the bronchoalveolar lavage (BAL). LPS exposure also induced an increase in the number of neutrophils in BAL. Studies in MMP-12-/- mice showed that in contrast to the smoking model, MMP-12 did not have a critical role in LPS-induced inflammation. Both cilomilast and dexamethasone blocked LPS-induced neutrophilia in a dose-dependent manner. Cilomilast inhibited cigarette smoke-induced neutrophilia and MIP-1alpha, but only 10 mg.kg(-1) of dexamethasone was effective. Both anti-inflammatory treatments had no effect on the levels of KC and MIP-2 in the BAL. Although the inflammatory response was very similar in the smoking model and LPS, the pharmacological modulation and the MMP-12 gene deletion highlighted the differences in the mechanisms involved. Furthermore, the cigarette smoke model seemed to better represent the situation described in chronic obstructive pulmonary disease patients. In conclusion, these differences underline the importance of using an acute smoke-exposure model to investigate potential new treatments for chronic obstructive pulmonary disease.

Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease.

OBJECTIVE AND DESIGN: Chronic obstructive pulmonary disease (COPD) is characterized by an inflammatory process and airway remodeling involving matrix metalloproteinases (MMPs). Thus, we analyzed the expression and release of MMP-12 (macrophage metalloelastase) in bronchoalveolar lavage (BAL) and lung tissue from COPD patients and control subjects. METHODS: Immunocytochemistry and immunochemistry were performed to analyze the expression of MMP-12 in BAL cells and bronchial biopsies. Western blotting was used for the evaluation of MMP-12 in BAL fluids. RESULTS: The number of MMP-12 expressing macrophages together with the staining intensity was higher in BAL samples from COPD patients than in controls subjects. Similar results were noted in bronchial biopsies with higher MMP-12 expression in COPD subjects than in controls. Enhanced MMP-12 level was also observed in BAL fluids from patient with COPD in comparison to control subjects. CONCLUSION: This study demonstrated that COPD patients produce greater quantities of MMP-12 than controls, which may be a critical step in the pathogenesis of COPD and emphysema.

Eotaxin and CCR3 are up-regulated in exacerbations of chronic bronchitis.

BACKGROUND: Eosinophils and T lymphocytes represent constant features in the airways of subjects with exacerbated chronic bronchitis. Eotaxin is the most potent and selective eosinophil chemoattractant which can also attracts lymphocytes. The aim of the study was to evaluate the expression of eotaxin and its receptor, CCR3, in bronchial airways during exacerbation of chronic bronchitis. METHODS: By immunohistochemistry we studied eotaxin and CCR3 expression in the lamina propria of 14 subjects with acute exacerbation of chronic bronchitis. 20 asthmatics, and 8 healthy subjects. We determined the cell types expressing the CCR3 receptor by colocalization experiments. We finally studied the relationship between eotaxin and CCR3 and eosinophils and T lymphocytes. RESULTS: The number of eotaxin+ and CCR3+ cells was significantly higher in exacerbated chronic bronchitis (P<0.003 and P<0.002) and asthma (P<0.002 and P<0.0001) when compared to healthy subjects. CCR3 was mainly expressed by eosinophils and to a lesser extent by CD4+ and CD8+ lymphocytes. In exacerbated chronic bronchitis the number of CCR3+ cells was strongly correlated to the number of eosinophils (P<0.0002. r=0.85) and to the number of CD4+ lymphocytes (P<0.05, r=0.57). CONCLUSION: Our study suggests that eotaxin and CCR3 are up-regulated and could be involved in the eosinophil and CD4+ lymphocyte recruitment into the airways which occur during acute exacerbations of chronic bronchitis.

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation.

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.

CCR3 blockade as a new therapy for asthma.

Among the inflammatory cells infiltrating the lungs of asthmatic patients, eosinophils and Th2 cells are thought to play a central role in the pathogenesis of this disease. Several studies have implicated that chemokines are prime candidates for being responsible for the selective recruitment of the leukocyte subsets found in atopic diseases. Regulated upon activation, normal T-cell-expressed and secreted (RANTES), monocyte chemoattractant protein-3 (MCP-3), MCP-4 and the eotaxins, for example, have been shown in vitro to potently induce eosinophil chemotaxis as well as initiate several other pro-inflammatory activities such as integrin activation, lipid mediator biosynthesis and degranulation. Ligand binding and chemotaxis experiments with these chemokines demonstrated that a G-protein coupled-receptor (GPCR) cloned from eosinophils, termed CCR3, was responsible for producing a chemokine selectivity profile identical to that of eosinophils. In addition, blocking CCR3 on eosinophils, with a monoclonal antibody, completely abolished eosinophil responses to these chemokines. Together these studies strongly suggest a central role for this receptor in eosinophil trafficking. CCR3 has also been found on in vitro derived Th2 cells and on T-cells co-localising with eosinophils in diseased tissue, thus revealing a possible pathogenic mechanism for T-cell recruitment into the airways. Therefore, blockade of CCR3 represents a highly attractive and innovative strategy for asthma therapy.

Asthma: where beyond steroids?

The prevalence of asthma is increasing dramatically despite major changes in monitoring and treatment of this disease. Currently available bronchodilators and anti-inflammatory drugs are effective in most patients, although these can have side effects and are mainly symptomatic. Many drugs are now in development for the treatment of asthma. Most of these new therapies are aimed at inhibition of the inflammatory components, with better safety profiles than steroids.

Airway inflammation driven by antigen-specific resident lung CD4(+) T cells in alphabeta-T cell receptor transgenic mice.

CD4(+) T cells are thought to play a major role in the initiation and perpetuation of T helper cell, type 2 (Th2)-like allergic airway inflammation. However, it is not clear whether activation of resident antigen-specific CD4(+) T cells is in itself sufficient to induce such a phenotype. Using ovalbumin (OVA)-specific alphabeta-T cell receptor transgenic Balb/c DO11.10 mice, we were able to test this hypothesis. Nonsensitized DO11.10 mice but not wild-type mice responded to a primary OVA aerosol with a rapid and impressive bronchoalveolar lavage (BAL) neutrophilia followed by a smaller but significant eosinophilia. Responses in DO11.10 mice were mediated by OVA-specific activation of CD4(+) T cells because in vivo depletion of CD4(+) but not CD8(+) T cells abrogated inflammatory cell influx. Cytokines measured in BAL fluid (BALF) after OVA aerosol exposure of DO11.10 mice were indicative of a T helper cell, type 1 (Th1)-like immune response. Further, neutralization of interferon gamma (IFN-gamma) with antibody enhanced eosinophil influx, suggesting that IFN-gamma production was limiting the development of a Th2 response. Despite this, an increased prevalence of cells staining for mucus was seen in the bronchial epithelium, a feature more commonly associated with a Th2-immune response. Unlike what was seen in OVA-sensitized wild-type mice, multiple OVA aerosol exposures of DO11.10 mice failed to induce airway hyperresponsiveness (AHR) to inhaled methacholine. In conclusion, in vivo stimulation of resident lung CD4(+) T cells with antigen caused lung inflammation with characteristics of both a Th1- and Th2-immune response but was insufficient to directly induce AHR.