Respiratory Research - Latest Articles

Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro

Xiao-Yun Fan — 2009-07-03T00:00:00Z

Background: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this also results in exaggerated production of IL-6 and IL-8. Methods: NCI-H292 airway epithelial-like cells and primary bronchial epithelial cells in medium with or without arginine were exposed to TNF-alpha, LPS or no stimulus to induce IL-6 and IL-8, which were determined by ELISA. Arginine deficiency may also occur due to poly-L-arginine or major basic protein (MBP) that can block arginine uptake. Epithelial cells were exposed to these polycations and L-14C-arginine uptake was assessed as well as IL-6 and IL-8 production. Subsequently, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation. Results: Arginine concentrations of [less than or equal to]20 microM enhanced basal epithelial IL-6 and IL-8 production and synergized with stimulus-induced IL-6 and IL-8 production. MBP and poly-L-arginine enhanced the stimulus-induced but not basal IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The IL-6 and IL-8 production under these conditions was not mediated by inducible nitric oxide synthase (iNOS) as shown with the iNOS inhibitor, L-NAME. Conclusions: We conclude that both reduced arginine availability ([less than or equal to]20 microM) per se and the presence of polycations may promote inflammation in asthma by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

Does unrestrained single-chamber plethysmography provide a valid assessment of airway responsiveness in allergic BALB/c mice?

Qingling Zhang — 2009-07-03T00:00:00Z

Background: Unrestrained plethysmography has been used to monitor bronchoconstriction because of its ease of use and ability to measure airway responsiveness in conscious animals. However, its reliability remains controversial.ObjectiveTo investigate if unrestrained plethysmography could provide a valid interpretation of airway responsiveness in allergic BALB/c mice. Methods: Ovalbumin sensitized BALB/c mice were randomized to receive either a single-dose Ovalbumin challenge (OVA-1D group) or a three-dose Ovalbumin challenge (OVA-3D group). The OVA-1D group was further divided into OVA-1D-I (measured invasively, using lung resistance as the index of responsiveness) and OVA-1D-N group (measured non-invasively, using Penh as the index of responsiveness). Similarly the OVA-3D group was divided into OVA-3D-I and OVA-3D-N groups based on the above methods. The control groups were sensitized and challenged with normal saline. Bronchial alveolar lavage fluid was taken and airway histopathology was evaluated for airway inflammation. Nasal responsiveness was tested with histamine challenge. Results: Compared with controls, a significant increase in airway responsiveness was shown in the OVA-1D-N group (P<0.05) but not in the OVA-1D-I group. Both OVA-3D-I and OVA-3D-N groups showed higher responsiveness than their controls (P<0.05). The nasal mucosa was infiltrated by eosinophic cells in all Ovalbumin immunized groups. Sneezing or nasal rubbing in allergic groups appeared more frequent than that in the control groups. Conclusion: Penh can not be used as a surrogate for airway resistance. The invasive measurement is specific to lower airway. Penh measurement (done as a screening procedure), must be confirmed by a direct invasive measurement specific to lower airway in evaluating lower airway responsiveness.

Mitogen-activated protein kinases and NFkappaB are involved in SP-A-enhanced responses of macrophages to mycobacteria

Joseph Lopez — 2009-07-01T00:00:00Z

Background: Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A. Methods: Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 ug of SP-A to 5x105 BCG for 30 min at 37 degrees Celsius. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined. Results: Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively. Conclusion: These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.

Efficacy of salmeterol/fluticasone propionate by GOLD stage of chronic obstructive pulmonary disease: analysis from the randomised, placebo-controlled TORCH study

Christine Jenkins — 2009-06-30T00:00:00Z

Background: The efficacy of inhaled salmeterol plus fluticasone propionate (SFC) in patients with severe or very severe COPD is well documented. However, there are only limited data about the influence of GOLD severity staging on the effectiveness of SFC, particularly in patients with milder disease. Methods: TORCH was a 3-year, double-blind, placebo-controlled trial of 6112 patients with moderate/severe COPD with pre-bronchodilator FEV1 < 60% predicted (mean age 65 years, 76% male, mean 44% predicted FEV1, 43% current smokers). To understand the relative efficacy of SFC and its components by GOLD stages, we conducted a post-hoc analysis of the TORCH dataset using baseline post-bronchodilator FEV1 to segment patients into three groups: moderate COPD (GOLD stage II and above: [greater than or equal to] 50%; n = 2156), severe COPD (GOLD stage III: 30% to < 50%; n = 3019) and very severe COPD (GOLD stage IV: < 30%; n = 937). Results: Compared with placebo, SFC improved post-bronchodilator FEV1: 101 ml (95% confidence interval [CI]: 71, 132) in GOLD stage II, 82 ml (95% CI: 60, 104) in GOLD stage III and 96 ml (95% CI: 54, 138) in GOLD stage IV patients, and reduced the rate of exacerbations: 31% (95% CI: 19, 40) in GOLD stage II, 26% (95% CI: 17, 34) in GOLD stage III and 14% (95% CI: -4, 29) in GOLD stage IV. SFC improved health status to a greater extent than other treatments regardless of baseline GOLD stage. Similarly, SFC reduced the risk of death by 33% (hazard ratio [HR] 0.67; 95% CI: 0.45, 0.98) for GOLD stage II, 5% (HR 0.95; 95% CI: 0.73, 1.24) for GOLD stage III, and 30% (HR 0.70; 95% CI: 0.47, 1.05) for GOLD stage IV. The rates of adverse events were similar across treatment arms and increased with disease severity. Overall, there was a higher incidence of pneumonia in the fluticasone propionate and SFC arms, compared with other treatments in all GOLD stages. Conclusions: In the TORCH study, SFC reduced moderate-to-severe exacerbations and improved health status and FEV1 across GOLD stages. Treatment with SFC may be associated with reduced mortality compared with placebo in patients with GOLD stage II disease. The effects were similar to those reported for the study as a whole. Thus, SFC is an effective treatment option for patients with GOLD stage II COPD.Trial registrationClinicaltrial.gov registration NCT00268216; Study number: SCO30003

Protection from pulmonary ischemia-reperfusion injury by adenosine A2A receptor activation

Ashish Sharma — 2009-06-26T00:00:00Z

Background: Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A2A receptor (A2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model. Methods: To assess the protective effects of A2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2hr with no ischemia), an IR group (1hr ischemia + 1hr reperfusion) and an IR+ATL313 group where ATL313, a specific A2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/ chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid. Results: After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-alpha, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice. Conclusion: Specific activation of A2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A2AAR activation on resident lung cells such as alveolar macrophages. Specific A2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.

Lymphocyte apoptosis in murine Pneumocystis pneumonia

Xin Shi — 2009-06-26T00:00:00Z

Background: Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. Methods: Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. Results: In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. Conclusions: Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Soy consumption and risk of COPD and respiratory symptoms: a case-control study in Japan

Fumi Hirayama — 2009-06-26T00:00:00Z

Background: To investigate the relationship between soy consumption, COPD risk and the prevalence of respiratory symptoms, a case-control study was conducted in Japan. Methods: A total of 278 eligible patients (244 men and 34 women), aged 50-75 years with COPD diagnosed within the past four years, were referred by respiratory physicians, while 340 controls (272 men and 68 women) were recruited from the community. All participants underwent spirometric measurements of respiratory function. Information on demographics, lifestyle characteristics and habitual food consumption was obtained using a structured questionnaire. Results: Total soy consumption was positively correlated with observed lung function measures. The mean soy intake was significantly higher among controls (59.98, SD 50.23 g/day) than cases (44.84, SD 28.5 g/day). A significant reduction in COPD risk was evident for highest versus lowest quartile of daily intake of total soybean products, with adjusted odds ratio (OR) 0.392, 95% CI 0.194-0.793, p for trend 0.001. Similar decreases in COPD risk were associated with frequent and higher intake of soy foods such as tofu and bean sprouts, whereas respiratory symptoms were inversely associated with high consumption of soy foods, especially for breathlessness (OR 0.989, 95% CI 0.982-0.996). Conclusions: Increasing soy consumption was associated with a decreased risk of COPD and breathlessness.

Blockade of advanced glycation end product formation attenuates bleomycin-induced pulmonary fibrosis in rats

Lei Chen — 2009-06-24T00:00:00Z

Background: Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation. Methods: Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFbeta1 and its downstream Smad proteins were analyzed by Western blot. Results: AGEs level in lung tissues, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissue, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p<0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFbeta1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissue. Conclusions: These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFbeta/Smads signaling.

Alveolar hypoxia, alveolar macrophages, and systemic inflammation

Jie Chao — 2009-06-22T00:00:00Z

Diseases featuring abnormally low alveolar PO2 are frequently accompanied by systemic effects. The common presence of an underlying inflammatory component suggests that inflammation may contribute to the pathogenesis of the systemic effects of alveolar hypoxia. While the role of alveolar macrophages in the immune and defense functions of the lung has been long known, recent evidence indicates that activation of alveolar macrophages causes inflammatory disturbances in the systemic microcirculation. The purpose of this review is to describe observations in experimental animals showing that alveolar macrophages initiate a systemic inflammatory response to alveolar hypoxia. Evidence obtained in intact animals and in primary cell cultures indicate that alveolar macrophages activated by hypoxia release a mediator(s) into the circulation. This mediator activates perivascular mast cells and initiates a widespread systemic inflammation. The inflammatory cascade includes activation of the local renin-angiotensin system and results in increased leukocyte-endothelial interactions in post-capillary venules, increased microvascular levels of reactive O2 species; and extravasation of albumin. Given the known extrapulmonary responses elicited by activation of alveolar macrophages, this novel phenomenon could contribute to some of the systemic effects of conditions featuring low alveolar PO2.

Altered effector function of peripheral cytotoxic cells in COPD

Richard Urbanowicz — 2009-06-22T00:00:00Z

Background: There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3-) cells and NKT-like (CD56+CD3+) cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results: The proportion of peripheral blood NKT-like (CD56+CD3+) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p<0.001) and non-smoking healthy participants (HNS) (3.3%, p<0.001). NK (CD56+CD3-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p<0.001) as were NKT-like (CD56+CD3+) cells (16.7% vs 52.4% specific lysis, p<0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells from smokers and HNS. Conclusion: In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.