Respiratory Research - Latest Articles

Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells

Amir Zeki — 2012-05-14T00:00:00Z

Background: Asthma causes significant morbidity worldwide in adults and children alike, and incurs largehealthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases,have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, antiinflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergicmouse model of asthma, we previously demonstrated a benefit of statins in reducingperibronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia,and lung IL-4 and IL-13 production.ObjectivesIn this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatorygene expression of asthma-related cytokines in well-differentiated primary mouse trachealepithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression ofIL-13-inducible genes in MTE cells. Methods: We harvested tracheal epithelial cells from naive BALB/c mice, grew them under air-liquidinterface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTEcells using a quantitative real-time PCR mouse gene array kit. Results: We found that simvastatin had differential effects on IL-13-mediated gene expression(inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 andCCL20 (MIP-3alpha)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10,CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible orstatin effects on gene expression. Conclusions: Simvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatorycytokines and chemokines in primary mouse tracheal epithelial cells. The airway epitheliummay be a viable target tissue for the statin drugs. Further research is needed to assess themechanisms of how statins modulate epithelial gene expression.

Effect of beta2-adrenergic receptor gene (ADRB2) 3'-untranslated region polymorphisms on inhaled corticosteroid/long-acting beta2-adrenergic agonist response

Helen Ambrose — 2012-05-04T00:00:00Z

Background: Evidence suggests that variation in the length of the poly-C repeat in the 3'-untranslated region (UTR) of the beta2-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in beta-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the beta2-adrenergic receptor gene (ADRB2) 3' UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting beta2-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response. Methods: In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (NCT00242775; AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed. Results: Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients' pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype. Conclusions: The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3' UTR did not predict therapeutic response to ICS/LABA therapy.

Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1

Kai You — 2012-05-04T00:00:00Z

Background: Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury. Methods: Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1-7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology. Results: We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury. Conclusions: These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.

Bacteria in sputum of stable severe asthma and increased airway wall thickness

Qingling Zhang — 2012-04-18T00:00:00Z

Background: Patients with chronic asthma have thicker intrapulmonary airways measured on high resolution computed tomography (HRCT). We determined whether the presence of lower airway bacteria was associated with increased airway wall thickness. Methods: In 56 patients with stable severe asthma, sputum specimens obtained either spontaneously or after induction with hypertonic saline were cultured for bacteria and thoracic HRCT scans obtained. Wall thickness (WT) and area (WA) expressed as a ratio of airway diameter (D) and total area, respectively, were measured at five levels. Results: Positive bacterial cultures were obtained in 29 patients, with H. influenzae, P. aeruginosa and S. aureus being the commonest strains. Logistic regression analysis showed that this was associated with the duration of asthma and the exacerbations during the past year. In airways > 2 mm, there was no significant difference in WA (67.5 ± 5.4 vs 66.4 ± 5.4) and WT/D (21.6 ± 2.7 vs 21.3 ± 2.4) between the culture negative versus positive groups. Similarly, in airways (≤ 2 mm), there were no significant differences in these parameters. The ratio of √wall area to Pi was negatively correlated with FEV1% predicted (p < 0.05). Conclusions: Bacterial colonization of the lower airways is common in patients with chronic severe asthma and is linked to the duration of asthma and having had exacerbations in the past year, but not with an increase in airway wall thickness.

Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis.

William Chang — 2012-04-18T00:00:00Z

Background: Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools. Methods: Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) -2 and -9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured. Results: Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median = 1174 vs. 332 pg/ml p < 0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93-1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p = 0.017) but not the presence of angiomyolipomas. Conclusions: Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.

Evidence for local dendritic cell activation in pulmonary sarcoidosis

Bregje Ten Berge — 2012-04-18T00:00:00Z

Background: Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation. Methods: We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c+DCs was assessed in mucosal airway biopsies. Results: mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4+ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86+ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients. Conclusion: Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.

The prevalence and identity of Chlamydia-specific IgE in children with asthma and other chronic respiratory symptoms

Katir Patel — 2012-04-18T00:00:00Z

Background: Recent studies have confirmed the presence of viable Chlamydia in the bronchoalveolar lavage (BAL) fluid of pediatric patients with airway hyperresponsiveness. While specific IgG and IgM responses to C. pneumoniae are well described, the response and potential contribution of Ag-specific IgE are not known. The current study sought to determine if infection with Chlamydia triggers the production of pathogen-specific IgE in children with chronic respiratory diseases which might contribute to inflammation and pathology. Methods: We obtained BAL fluid and serum from pediatric respiratory disease patients who were generally unresponsive to corticosteroid treatment as well as sera from age-matched control patients who saw their doctor for wellness checkups. Chlamydia-specific IgE was isolated from BAL and serum samples and their specificity determined by Western blot techniques. The presence of Chlamydia was confirmed by species-specific PCR and BAL culture assays. Results: Chlamydial DNA was detected in the BAL fluid of 134/197 (68%) patients. Total IgE increased with age until 15 years old and then decreased. Chlamydia-specific IgE was detected in the serum and/or BAL of 107/197 (54%) patients suffering from chronic respiratory disease, but in none of the 35 healthy control sera (p < 0.0001). Of the 74 BAL culture-positive patients, 68 (91.9%, p = 0.0001) tested positive for Chlamydia-specific IgE. Asthmatic patients had significantly higher IgE levels compared to non-asthmatics (p = 0.0001). Patients who were positive for Chlamydia DNA or culture had significantly higher levels of serum IgE compared to negative patients (p = 0.0071 and p = 0.0001 respectively). Only 6 chlamydial antigens induced Chlamydia-specific IgE and patients with C. pneumoniae-specific IgE had significantly greater levels of total IgE compared to C. pneumoniae-specific IgE negative ones (p = 0.0001). Conclusions: IgE antibodies play a central role in allergic inflammation; therefore production of Chlamydia-specific IgE may prove significant in the exacerbation of chronic, allergic airway diseases, thus highlighting a direct role for Chlamydia in asthma pathogenesis.

Emphysema distribution and annual changes in pulmonary function in male patients with chronic obstructive pulmonary disease

Naoya Tanabe — 2012-04-18T00:00:00Z

Background: The progression of chronic obstructive pulmonary disease (COPD) considerably varies among patients. Those with emphysema identified by quantitative computed tomography (CT) are associated with the rapid progression assessed by forced expiratory volume in one second (FEV1). However, whether the rate of the decline in lung function is independently affected by the regional distribution or the severity of emphysema in the whole lung is unclear. Methods: We followed up 131 male patients with COPD for a median of 3.7 years. We measured wall area percent (WA%) in right apical segmental bronchus, total lung volume, percent low attenuation volume (LAV%), and the standard deviation (SD) of LAV% values from CT images of 10 isovolumetric partitions (SD-LAV) as an index of cranial-caudal emphysema heterogeneity. Annual changes in FEV1 were then determined using a random coefficient model and relative contribution of baseline clinical parameters, pulmonary function, and CT indexes including LAV%, SD-LAV, and WA% to annual changes in FEV1 were examined. Results: The mean (SD) annual change in FEV1 was 44.4 (10.8) mL. Multivariate random coefficient model showed that higher baseline FEV1, higher LAV%, current smoking, and lower SD-LAV independently contributed to an excessive decline in FEV1, whereas ratio of residual volume to total lung capacity, ratio of diffusing capacity to alveolar ventilation, and WA% did not, after adjusting for age, height, weight, and ratio of CT-measured total lung volume to physiologically-measured total lung capacity. Conclusions: A more homogeneous distribution of emphysema contributed to an accelerated decline in FEV1 independently of baseline pulmonary function, whole-lung emphysema severity, and smoking status. In addition to whole-lung analysis of emphysema, CT assessment of the cranial-caudal distribution of emphysema might be useful for predicting rapid, progressive disease and for developing a targeted strategy with which to prevent disease progression.

The ion channel transient receptor potential melastatin-2 does not play a role in inflammatory mouse models of chronic obstructive pulmonary diseases

Liz Hardaker — 2012-04-04T00:00:00Z

Background: There is strong evidence that oxidative stress is associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). The transient receptor potential melastatin-2 (TRPM2) is an oxidative stress sensing channel that is expressed in a number of inflammatory cells and therefore it has been suggested that inhibition of TRPM2 could lead to a beneficial effect in COPD patients. In this study, we have investigated the role of TRPM2 in a variety of mouse models of oxidative stress and COPD using TRPM2-deficent mice. Methods: Mice were exposed to ozone (3 ppm for 4 h) or lipopolysaccharide (LPS, 0.3 mg/kg, intranasaly). In another model, mice were exposed to tobacco smoke (750 μg/l total wet particulate matter) for 30 min twice a day on three consecutive days. For the exacerbation model, the smoke exposure on the morning of day 3 animals was replaced with intranasal administration of LPS (0.3 mg/kg). Animals were killed 3 and 24 h after the challenge (ozone and LPS model) or 18 h after the last tobacco smoke exposure. In vitro neutrophil chemotaxis and monocyte activation were also studied using cells isolated from wild type and TRPM2-deficient animals. Statistical significance for the in vivo data (P < 0.05) was determined using analysis of variance with Kruskal-Wallis and Dunns multiple comparison test. Results: In all models studied, no difference in the bronchoalveolar lavage inflammation could be evidenced when comparing wild type and TRPM2-deficient mice. In addition, no difference could be seen in the lung inflammation as assessed by the measurement of various cytokines/chemokines. Similarly in various in vitro cellular activation assays using isolated neutrophils and monocytes no significant differences could be observed when comparing wild type and TRPM2-deficient mice.DiscussionWe have shown, in all the models tested, no difference in the development of airway inflammation or cell activation between TRPM2-deficient mice and their wild type counterparts. These results would suggest that inhibiting TRPM2 activity in COPD would have no anti-inflammatory effect.

Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury

Wang Deng — 2012-03-30T00:00:00Z

Background: Stimulation of epithelial sodium channel (ENaC) increases Na+ transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo. Methods: A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of alpha-,beta-,and gamma-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Results: In vivo,insulin decreased TLW, enchanced AFC,increased the expressions of alpha-,beta-,and gamma-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI,the effects of which were blocked by wortmannin. Amiloride,a sodium channel inhibitor,significantly reduced insulin-induced increase in AFC. In vitro,insulin increased the expressions of alpha-,beta-,and gamma-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway. Conclusions: Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.