Rosalia Gagliardo

Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis

BACKGROUND Apoptosis regulates inflammatory cell survival, and its reduction contributes to the chronicity of an inflammatory process. Apoptosis is controlled by suppressing or inducing genes, such as bcl-2 and p53, respectively. OBJECTIVE We sought to assess apoptosis of eosinophils, macrophages, and T lymphocytes in bronchial biopsy specimens from asthmatic subjects and to examine its regulation by evaluating the expression of B-cell lymphoma leukemia-2 (Bcl-2) and P53 proteins. We also sought to explore the relationships between cell apoptosis and GM-CSF, a cytokine able to increase eosinophil and macrophage survival. METHODS Apoptosis in eosinophils, macrophages, and T lymphocytes was evaluated in bronchial biopsy specimens obtained from 30 asthmatic subjects, 26 subjects with chronic bronchitis, and 15 control subjects by combining the terminal deoxynucleotidyl transferase-mediated dNTP nick end-labeling technique and immunohistochemistry. The expression of P53, Bcl-2, and GM-CSF was studied through immunohistochemistry by using specific mAbs. RESULTS The number of apoptotic eosinophils and macrophages was lower in subjects with asthma than in those with chronic bronchitis (P <.007 and P <.001, respectively) and inversely correlated with the clinical severity of asthma (P <.001 and P <.002, respectively). Few T lymphocytes were apoptotic in all groups studied. In asthma GM-CSF+ cells correlated with the number of nonapoptotic eosinophils and macrophages (P =.0001) and with the severity of the disease (P <.003). In asthma Bcl-2+ cells were higher than in control subjects and subjects with chronic bronchitis (P <.002 and P <.015, respectively), they outnumbered P53+ cells, and they correlated with the number of T lymphocytes (P <.001) and with the severity of the disease (P <.003). CONCLUSION Airway inflammation in asthma is associated with an enhanced survival of different cell types caused by reduced apoptosis.

Biochemical interaction between effects of beclomethasone dipropionate and salbutamol or formoterol in sputum cells from mild to moderate asthmatics

Background:  Several in vitro studies demonstrate that corticosteroids and long‐acting β2 agonists may have a complementary and synergistic mode of action on the inflammatory processes in asthma.

Apoptosis and airway inflammation in asthma

Asthma is a disease characterized by a chronic inflammation of the airways and by structural alterations of bron-chial tissues, often referred to as airway remodelling. The development of chronic airway inflammation in asthma depends upon the continuous recruitment of inflammatory cells from the bloodstream towards the bronchial mucosa and by their subsequent activation. It is however increasingly accepted that mechanisms involved in the regulation of the survival and apoptosis of inflammatory cells may play a central role in the persistent inflammatory process characterizing this disease. Increased cellular recruitment and activation, enhanced cell survival and cell:cell interactions are therefore the key steps in the development of chronic airway inflammation in asthma, and represent the major causes for tissue damge, repair and remodelling.

Non-invasive markers of airway inflammation and remodeling in childhood asthma.

To evaluate the relationship between pro‐inflammatory and pro‐remodeling mediators and severity and control of asthma in children, the levels of IL‐8, MMP‐9, TIMP‐1 in induced sputum supernatants, the number of sputum eosinophils, as well as FeNO, were investigated in 35 asthmatic children, 12 with intermittent (IA) and 23 with moderate asthma (MA), and 9 controls (C). The patients with asthma were followed for 1 yr and sputum was obtained twice during the follow‐up. Biomarker levels were correlated with the number of exacerbations. We found that IL‐8, MMP‐9, TIMP‐1 and the numbers of eosinophils in induced sputum, as well as FeNO, were increased in children with IA and MA in comparison to C. The ongoing inflammation was confirmed by increased nuclear p65 NF‐κB subunit localization in sputum cells. In MA, FeNO measurements, sputum eosinophils and IL‐8 levels, positively correlated with the occurrence of disease exacerbations during a 1‐yr follow‐up. According to FeNO, sputum eosinophils and IL‐8 sputum concentrations, and the number of exacerbations, two distinct phenotypes of MA were identified. This study shows that the presence of bronchial inflammation is detectable in the airways of some IA, as well as in the airways of MA, despite the regular ICS treatment. This study also proposes the need to perform large prospective studies to confirm the importance of measuring specific biomarkers in induced sputum, concomitantly to FeNO analyses, to assess sub‐clinical airway inflammation and disease control in children with asthma.

Cysteinyl Leukotriene-1 Receptor Activation in a Human Bronchial Epithelial Cell Line Leads to Signal Transducer and Activator of Transcription 1-Mediated Eosinophil Adhesion

We studied the effect of leukotriene D4 (LTD4) on a human bronchial epithelial cell line (16HBE) overexpressing the cysteinyl leukotriene (CysLT) 1 receptor (HBECysLT1R), looking at the associated signal transduction mechanisms as well as at effects on inflammatory cell adhesion. The results obtained showed that LTD4 increases the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 and of the signal transducer and activator of transcription 1 (STAT-1) in serine 727 (STAT-1Ser727), resulting in increased eosinophil adhesion to HBECysLT1R, associated with enhanced surface expression of intercellular adhesion molecule (ICAM) 1. Pretreatment with a CysLT1R-selective antagonist or with a selective inhibitor of protein kinase C (PKC) or with a selective inhibitor of the mitogen-activated protein kinase kinase (MEK) successfully suppressed both LTD4-induced STAT-1Ser727 phosphorylation and the associated increase in eosinophil adhesion. The use of the MEK inhibitor and of the selective CysLT1R antagonist in electrophoretic mobility shift assay experiments showed that LTD4 promotes the nuclear translocation of STAT-1 through the activation of ERK1/2 pathway. The key role of STAT-1 in leukotriene D4 transduction signaling was confirmed by RNA interference experiments, where silencing of STAT-1 expression abolished the effect of leukotriene D4 on eosinophil adhesion. In conclusion, for the first time, we provide evidence of the involvement of STAT-1 in the signal transduction mechanism of the CysLT1 receptor; phosphorylation of STAT-1, through PKC and ERK1/2 activation, causes enhanced ICAM-1 surface expression and eosinophil adhesion. Effective CysLT1R antagonism may therefore contribute to the control of the chronic inflammatory condition that characterizes human airways in asthma.

Th17 Immunity in Children with Allergic Asthma and Rhinitis: A Pharmacological Approach

Th17 cells and IL-17A play a role in the development and progression of allergic diseases. We analyzed the IL-17A levels in sputum supernatants (Ss), nasal wash (NW) and plasma (P) from Healthy Controls (HC) and children with Asthma/Rhinitis. We tested the expression of IL-17A, RORγ(t) and FOXP3 in peripheral blood T-lymphocytes from intermittent and mild-moderate asthma. The effect of Budesonide and Formoterol was tested “in vitro” on IL-17A, RORγ(t) and FOXP3 expression in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis patients, and on nasal and bronchial epithelial cells stimulated with NW and Ss from mild-moderate asthma/persistent rhinitis. Further, the effect of 12 weeks of treatment with Budesonide and Formoterol was tested “in vivo” in T-lymphocytes from mild-moderate asthma/persistent rhinitis patients. IL-17A was increased in Ss, NW and P from children with mild-moderate asthma compared with intermittent and HC. In cultured T-lymphocytes IL-17A and RORγ(t) expression were higher in mild-moderate asthma/persistent rhinitis than in mild-moderate asthma/intermittent rhinitis, while FOXP3 was reduced. Budesonide with Formoterol reduced IL-17A and RORγ(t), while increased FOXP3 in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis, and reduced the IL-8 release mediated by IL-17A present in NW and Ss from mild-moderate asthma/persistent rhinitis in nasal and bronchial epithelial cells. Finally, Budesonide with Formoterol reduced IL-17A levels in P and Ss, CD4+IL-17A+T-cells, in naïve children with mild-moderate asthma/persistent rhinitis after 12 weeks of treatment. Th17 mediated immunity may be involved in the airway disease of children with allergic asthma and allergic rhinitis. Budesonide with Formoterol might be a useful tool for its therapeutic control.

Is there a role for glucocorticoid receptor beta in asthma

Glucocorticoids (GCs) are routinely used as anti-inflammatory drugs in the treatment of asthma. They act through binding to glucocorticoid receptor α (GRα), which represses numerous genes encoding pro-inflammatory mediators. A hormone binding deficient GR isoform named GRβ has been isolated in humans. When overexpressed by transfection, GRβ may function as a dominant negative modulator of GRα. However, to act as such, GRβ has to be more abundant than GRα, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. Moreover, the dominant negative effect was not confirmed by independent laboratories. In GC-resistant asthmatics, GRβ was expressed by an increased number of peripheral blood mononuclear cells (PBMCs), airway T cells, and cells found in skin biopsies of tuberculin responses. However, the relative amounts of GRα and GRβ in these cells were not determined. In GC-dependent asthmatics, PBMCs expressed GRα predominantly. No cells containing higher levels of GRβ than GRα have yet been reported in asthmatics. Even if the existence of such cells is demonstrated, the role of GRβ in asthma will remain a matter of controversy because functional studies have given discrepant data.

IL-33/ST2 axis controls Th2/IL-31 and Th17 immune response in allergic airway diseases.

IL-33 targeting ST2 receptor (T1/ST2), expressed on Th2 cell surface, regulates the production of cytokines like IL-17A and IL-31. We studied the role of IL-33/ST2 axis in IL-31 and IL-17A production in patients with allergic rhinitis (AR) and with concomitant allergic asthma and rhinitis (AAR). 20 healthy control subjects (HC), 14 AR and 17 AAR subjects were recruited and blood samples collected. IL-33, soluble ST2 (sST2), IL-17A and IL-31 plasma concentrations were measured by ELISA method. T1/ST2, IL-31 and IL-17A cellular expression were studied in peripheral blood mononuclear cells (PBMC) from HC, AR and AAR (n=6 for each group) by flow-cytometry. In vitro, we also evaluated the effect of beclomethasone dipropionate (BDP) on T1/ST2, IL-31 and IL-17A expression in CD3(+)T-cells from PBMC of AAR (n=6). Plasma levels of IL-33, IL-31 and IL-17A were significantly higher and sST2 was lower in patients with AR and AAR than in HC. IL-31 and IL-17A intracellular levels significantly increased, whereas T1/ST2 expression was significantly lower, in CD3(+)T-cells from AR and AAR compared to HC. Positive correlations were observed between plasmatic components of IL-33/ST2 axis and IL-31 in both AR and AAR and IL-17A in AAR. In vitro IL-31 and IL-17A intracellular levels decreased after BDP treatment, whereas T1/ST2 expression increased in cultured CD3(+)T-cells obtained from AAR. IL-33/ST2 axis is involved in Th2/IL-31 and Th17 immune response during the progression of allergic airway disease. In vitro BDP is able to control Th2/IL-31 and Th17 immune response in PBMC from allergic patients.

ICAM‐1 and α3β1 expression by bronchial epithelial cells and their in vitro modulation by inflammatory and anti‐inflammatory mediators

Background: Adhesion molecules are involved in inflammatory and repair processes of the bronchial epithelium. ICAM‐1 is mainly involved in inflammatory reactions, whereas integrins, such as α3β1, are mainly involved in repair processes.

In vitro effects of flunisolide on MMP‐9, TIMP‐1, fibronectin, TGF‐β1 release and apoptosis in sputum cells freshly isolated from mild to moderate asthmatics

Background:  Corticosteroids play an important role in inflammation and remodelling of airways and are considered an important therapeutic target in asthma. Inflammation in asthma is characterized by a dysregulation of eosinophil apoptosis and of markers of airways remodelling. We evaluated the ability of flunisolide to inhibit in vitro the release of metalloproteinases‐9 (MMP‐9), tissue inhibitor metalloproteinases‐1 (TIMP‐1), transforming growth factor (TGF‐β) and fibronectin by sputum cells (SC) as well as to induce sputum eosinophil apoptosis.