Oxidative Stress Modulates the Expression Pattern of Peroxiredoxin-6 in Peripheral Blood Mononuclear Cells of Asthmatic Patients and Bronchial Epithelial Cells.

PURPOSE: Reduction-oxidation reaction homeostasis is vital for regulating inflammatory conditions and its dysregulation may affect the pathogenesis of chronic airway inflammatory diseases such as asthma. Peroxiredoxin-6, an important intracellular anti-oxidant molecule, is reported to be highly expressed in the airways and lungs. The aim of this study was to analyze the expression pattern of peroxiredoxin-6 in the peripheral blood mononuclear cells (PBMCs) of asthmatic patients and in bronchial epithelial cells (BECs). METHODS: The expression levels and modifications of peroxiredoxin-6 were evaluated in PBMCs from 22 asthmatic patients. Phosphorylated and acetylated peroxiredoxin-6 in hydrogen peroxide-treated human BECs was detected using immunoprecipitation analysis. The expression level of peroxiredoxin-6 was also investigated in BECs treated with hydrogen peroxide. Cycloheximide and proteasome inhibitors were used to determine whether peroxiredoxin-6 is degraded by proteasomes. RESULTS: Peroxiredoxin-6 expression was significantly reduced in the PBMCs of asthmatic patients compared to control subjects. Distinct modification patterns for peroxiredoxin-6 were observed in the PBMCs of asthmatic patients using 2-dimensional-electrophoresis. The levels of phosphorylated serine and acetylated lysine in peroxiredoxin-6 were significantly increased in the BECs following hydrogen peroxide treatment. The level of peroxiredoxin-6 expression was reduced in hydrogen peroxide-stimulated BECs, presumably due to proteasomes. CONCLUSIONS: The expression of peroxiredoxin-6, which is down-regulated in the immune cells of asthmatic patients and BECs, can be modified by oxidative stress. This phenomenon may have an effect on asthmatic airway inflammation.

Macrophage-derived progranulin promotes allergen-induced airway inflammation.

BACKGROUND: Progranulin (PGRN), mainly produced by immune and epithelial cells, has been known to be involved in the development of various inflammatory diseases. However, the function of PGRN in allergic airway inflammation has not been clearly elucidated, and we investigated the role of PGRN in allergic airway inflammation. METHODS: Production of PGRN and various type 2 cytokines was evaluated in mouse airways exposed to house dust mite allergen, and main cellular sources of these molecules were investigated using macrophage, airway epithelial cell, and NKT cell lines. We elucidated the role of PGRN in allergic airway inflammation in mouse models of asthma using macrophage-derived PGRN-deficient mice and NKT cell knockout mice by evaluating cytokine levels in bronchoalveolar lavage fluids and histopathology. We also supplemented recombinant PGRN in the mouse models to confirm the role of PGRN in allergic airway inflammation. RESULTS: PGRN production preceded other cytokines, mainly from macrophages, in the airway exposed to allergen. PGRN induced IL-4 and IL-13 production in NKT cells and IL-33 and TSLP in airway epithelial cells. PGRN-induced Th2 cytokine production was abolished in NKT-deficient mice. Finally, allergic inflammation was significantly attenuated in allergen-exposed PGRN-deficient mice, but inflammation was restored when recombinant PGRN was supplemented during the allergen sensitization period. CONCLUSION: The presence of macrophage-derived PGRN in airways in the early sensitization period may be critical for mounting a Th2 immune response and for following an allergic airway inflammation pathway via induction of type 2 cytokine production in NKT and airway epithelial cells.

Endothelial Sox17 promotes allergic airway inflammation.

BACKGROUND: IL-33, levels of which are known to be increased in patients with eosinophilic asthma and which is suggested as a therapeutic target for it, activates endothelial cells in which Sry-related high-mobility-group box (Sox) 17, an endothelium-specific transcription factor, was upregulated. OBJECTIVE: We investigated the relationship between Sox17 and IL-33 and the possible role of Sox17 in the pathogenesis of asthma using a mouse model of airway inflammation. METHODS: We used ovalbumin (OVA) to induce airway inflammation in endothelium-specific Sox17 null mutant mice and used IL-33 neutralizing antibody to evaluate the interplay between IL-33 and Sox17. We evaluated airway inflammation and measured levels of various cytokines, chemokines, and adhesion molecules. We also carried out loss- or gain-of-function experiments for Sox17 in human endothelial cells. RESULTS: Levels of IL-33 and Sox17 were significantly increased in the lungs of OVA-challenged mice. Anti-IL-33 neutralizing antibody treatment attenuated not only OVA-induced airway inflammation but also Sox17 expression in pulmonary endothelial cells. Importantly, endothelium-specific deletion of Sox17 resulted in significant alleviation of various clinical features of asthma, including airway inflammation, immune cell infiltration, cytokine/chemokine production, and airway hyperresponsiveness. Sox17 deletion also resulted in decreased densities of Ly6chigh monocytes and inflammatory dendritic cells in the lungs. In IL-33-stimulated human endothelial cells, Sox17 showed positive correlation with CCL2 and intercellular adhesion molecule 1 levels. Lastly, Sox17 promoted monocyte adhesion to endothelial cells and upregulated the extracellular signal-regulated kinase-signal transducer and activator of transcription 3 pathway. CONCLUSION: Sox17 was regulated by IL-33, and its genetic ablation in endothelial cells resulted in alleviation of asthma-related pathophysiologic features. Sox17 might be a potential target for asthma management.

IL-32γ attenuates airway fibrosis by modulating the integrin-FAK signaling pathway in fibroblasts.

BACKGROUND: Fibrosis in severe asthma often leads to irreversible organ dysfunction. However, the mechanism that regulates fibrosis remains poorly understood. Interleukin (IL)-32 plays a role in several chronic inflammatory diseases, including severe asthma. In this study, we investigated whether IL-32 is involved in fibrosis progression in the lungs. METHODS: Murine models of chronic airway inflammation induced by ovalbumin and Aspergillus melleus protease and bleomycin-induced pulmonary fibrosis were employed. We evaluated the degree of tissue fibrosis after treatment with recombinant IL-32γ (rIL-32γ). Expression of fibronectin and α-smooth muscle actin (α-SMA) was examined and the transforming growth factor (TGF)-ß-related signaling pathways was evaluated in activated human lung fibroblasts (MRC-5 cells) treated with rIL-32γ. RESULTS: rIL-32γ significantly attenuated collagen deposition and α-SMA production in both mouse models. rIL-32γ inhibited the production of fibronectin and α-SMA in MRC-5 cells stimulated with TGF-ß. Additionally, rIL-32γ suppressed activation of the integrin-FAK-paxillin signaling axis but had no effect on the Smad and non-Smad signaling pathways. rIL-32γ localized outside of MRC-5 cells and inhibited the interaction between integrins and the extracellular matrix without directly binding to intracellular FAK and paxillin. CONCLUSIONS: These results demonstrate that IL-32γ has anti-fibrotic effects and is a novel target for preventing fibrosis.

Progranulin protects lung epithelial cells from cigarette smoking-induced apoptosis.

BACKGROUND AND OBJECTIVE: Emphysema is characterized by irreversible destruction of alveolar walls with distal air space enlargement. Cigarette smoke (CS) is considered a major risk factor for emphysematous changes in COPD. Progranulin (PGRN), a glycoprotein induced by CS, has been reported to participate in apoptosis. However, the precise role of PGRN in emphysema is currently unknown. This study aimed to evaluate the role of PGRN in human alveolar epithelial cells (AECs) in response to CS. METHODS: First, PGRN expression was assessed in a mouse model of CS-induced emphysema and in AECs after exposure to CS extract (CSE). Then, the effect of PGRN on CSE-mediated apoptosis was determined under PGRN silencing or overexpressing conditions. To investigate the functional mechanism of PGRN, endoplasmic reticulum (ER) stress markers and the mitogen-activated protein kinase (MAPK) pathway were also evaluated in the CSE-exposed cells. Finally, PGRN expression levels in sera and peripheral blood mononuclear cells (PBMCs) were measured and compared between patients with COPD and healthy subjects. RESULTS: Our results revealed that PGRN expression was elevated in CS-exposed mouse lungs and CSE-treated AECs. CSE-induced cellular apoptosis was significantly increased in PGRN-knockdown AECs and decreased in PGRN-overexpression cells. The activation of ER stress-associated molecules correlated with PGRN expression levels. Compared with healthy controls, COPD patients exhibited significantly lower PGRN serum levels and higher PBMC intracellular PGRN levels. CONCLUSION: PGRN in airway epithelial cells may regulate CS-induced AEC apoptosis and may be involved in the development of COPD.

hMSCs suppress neutrophil-dominant airway inflammation in a murine model of asthma.

Although chronic eosinophilic inflammation is a common feature in patients with asthma, some patients have neutrophil-dominant inflammation, which is known to be associated with severe asthma.Human mesenchymal stem cells (hMSCs) have shown promise in treating various refractory immunological diseases. Thus, hMSCs may represent an alternative therapeutic option for asthma patients with neutrophil-dominant inflammation, in whom current treatments are ineffective. BALB/c mice exposed to ovalbumin and polyinosinic:polycytidylic acid (Poly I:C) to induce neutrophilic airway inflammation were systemically treated with hMSCs to examine whether the hMSCs can modulate neutrophilic airway inflammation. In addition, cytokine production was evaluated in co-cultures of hMSCs with either anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asthmatic patients or cells of the human bronchial epithelial cell line BEAS-2B to assess the response to hMSC treatment. The total number of immune cells in bronchoalveolar lavage fluid (BALF) showed a dramatic decrease in hMSC-treated asthmatic mice, and, in particular, neutrophilic infiltration was significantly attenuated. This phenomenon was accompanied by reduced CXCL15 production in the BALF. BEAS-2B cells co-cultured with hMSCs showed reduced secretion of IL-8. Moreover, decreased secretion of IL-4, IL-13 and IFN-γ was observed when human PBMCs were cultured with hMSCs, whereas IL-10 production was greatly enhanced. Our data imply that hMSCs may have a role in reducing neutrophilic airway inflammation by downregulating neutrophil chemokine production and modulating T-cell responses.

Clusterin Modulates Allergic Airway Inflammation by Attenuating CCL20-Mediated Dendritic Cell Recruitment.

Recruitment and activation of dendritic cells (DCs) in the lungs are critical for Th2 responses in asthma, and CCL20 secreted from bronchial epithelial cells (BECs) is known to influence the recruitment of DCs. Because asthma is a disease that is closely associated with oxidative stress, we hypothesized that clusterin, an oxidative stress regulatory molecule, may have a role in the development of allergic airway inflammation. The aim of this study was to examine whether clusterin regulates CCL20 production from the BECs and the subsequent DC recruitment in the lungs. To verify the idea, clusterin knockout (Clu(-/-)), clusterin heterogeneous (Clu(+/-)), and wild-type mice were exposed intranasally to house dust mite (HDM) extract to induce allergic airway inflammation. We found that the total number of immune cells in bronchoalveolar lavage fluid and the lung was increased in Clu(-/-) and Clu(+/-) mice. Of these immune cells, inflammatory DCs (CD11b(+)CD11c(+)) and Ly6C(high) monocyte populations in the lung were significantly increased, which was accompanied by increased levels of various chemokines, including CCL20 in bronchoalveolar lavage fluid, and increased oxidative stress markers in the lung. Moreover, HDM-stimulated human BECs with either up- or downregulated clusterin expression showed that CCL20 secretion was negatively associated with clusterin expression. Interestingly, clusterin also reduced the level of intracellular reactive oxygen species, which is related to induction of CCL20 expression after HDM stimulation. Thus, the antioxidant property of clusterin is suggested to regulate the expression of CCL20 in BECs and the subsequent recruitment of inflammatory DCs in the airway.

Clusterin expression level correlates with increased oxidative stress in asthmatics.

BACKGROUND: Oxidative stress is thought to play a role in the pathogenesis of asthma. Clusterin is a sensitive cellular biosensor of oxidative stress and has antioxidant properties. The function and expression of clusterin in patients with asthma have not been fully investigated. OBJECTIVE: To investigate whether the expression of clusterin in patients with asthma is regulated by increased oxidative burden and whether clusterin expression could be used to assess the response to inhaled corticosteroids. METHODS: Clusterin levels in serum, induced sputum, and peripheral blood mononuclear cells of patients with asthma were measured by enzyme-linked immunosorbent assay and western blotting and compared with pulmonary function and levels of expression of hyperoxidized peroxiredoxins. Serum concentrations of clusterin in treatment-naive patients were compared before and after inhaled corticosteroid use. RESULTS: Serum clusterin concentration was significantly elevated in patients with severe asthma and was inversely correlated with pulmonary function. The expression of hyperoxidized peroxiredoxins was greatly increased in peripheral blood mononuclear cells of patients with asthma and was strongly correlated with clusterin expression. Serum clusterin concentrations in treatment-naive patients with asthma were decreased significantly after initial treatment with inhaled corticosteroids. CONCLUSION: Clusterin may be a biomarker of asthma severity and the burden of oxidative stress in patients with asthma. Moreover, clusterin may be useful for the prompt assessment of airway inflammation.

Metformin reduces airway inflammation and remodeling via activation of AMP-activated protein kinase.

Recent reports have suggested that metformin has anti-inflammatory and anti-tissue remodeling properties. We investigated the potential effect of metformin on airway inflammation and remodeling in asthma. The effect of metformin treatment on airway inflammation and pivotal characteristics of airway remodeling were examined in a murine model of chronic asthma generated by repetitive challenges with ovalbumin and fungal-associated allergenic protease. To investigate the underlying mechanism of metformin, oxidative stress levels and AMP-activated protein kinase (AMPK) activation were assessed. To further elucidate the role of AMPK, we examined the effect of 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) as a specific activator of AMPK and employed AMPKα1-deficient mice as an asthma model. The role of metformin and AMPK in tissue fibrosis was evaluated using a bleomycin-induced acute lung injury model and in vitro experiments with cultured fibroblasts. Metformin suppressed eosinophilic inflammation and significantly reduced peribronchial fibrosis, smooth muscle layer thickness, and mucin secretion. Enhanced AMPK activation and decreased oxidative stress in lungs was found in metformin-treated asthmatic mice. Similar results were observed in the AICAR-treated group. In addition, the enhanced airway inflammation and fibrosis in heterozygous AMPKα1-deficient mice were induced by both allergen and bleomycin challenges. Fibronectin and collagen expression was diminished by metformin through AMPKα1 activation in cultured fibroblasts. Therefore metformin reduced both airway inflammation and remodeling at least partially through the induction of AMPK activation and decreased oxidative stress. These data provide insight into the beneficial role of metformin as a novel therapeutic drug for chronic asthma.

Grape seed proanthocyanidin extract attenuates allergic inflammation in murine models of asthma.

BACKGROUND: Antioxidants have been suggested to alleviate the pathophysiological features of asthma, and grape seed proanthocyanidin extract (GSPE) has been reported to have powerful antioxidant activity. PURPOSE: This study was performed to determine whether GSPE has a therapeutic effect on allergic airway inflammation in both acute and chronic murine model of asthma. METHODS: Acute asthma model was generated by intraperitoneal sensitization of ovalbumin (OVA) with alum followed by aerosolized OVA challenges, whereas chronic asthma model was induced by repeated intranasal challenges of OVA with fungal protease twice a week for 8 weeks. GSPE was administered by either intraperitoneal injection or oral gavage before OVA challenges. Airway hyperresponsiveness (AHR) was measured, and airway inflammation was evaluated by bronchoalveolar lavage (BAL) fluid analysis and histopathological examination of lung tissue. Lung tissue levels of various cytokines, chemokines, and growth factors were analyzed by quantitative polymerase chain reaction and ELISA. Glutathione assay was done to measure oxidative burden in lung tissue. RESULTS: Compared to untreated asthmatic mice, mice treated with GSPE showed significantly reduced AHR, decreased inflammatory cells in the BAL fluid, reduced lung inflammation, and decreased IL-4, IL-5, IL-13, and eotaxin-1 expression in both acute and chronic asthma models. Moreover, airway subepithelial fibrosis was reduced in the lung tissue of GSPE-treated chronic asthmatic mice compared to untreated asthmatic mice. Reduced to oxidized glutathione (GSH/GSSG) ratio was increased after GSPE treatment in acute asthmatic lung tissue. CONCLUSION: GSPE effectively suppressed inflammation in both acute and chronic mouse models of asthma, suggesting a potential role of GSPE as a therapeutic agent for asthma.

The role of nitrosative stress in the pathogenesis of unexplained chronic cough with cough hypersensitivity.

BACKGROUND: Unexplained chronic cough is a common condition without specific causes. A hyperreactivity of the cough reflex has been suggested as a mechanism for inducing chronic cough. We hypothesized that nitrosative stress in the upper airway might play a role in cough hypersensitivity by causing neurochemical abnormalities. METHODS: Fifty-one patients with unexplained chronic cough and 27 controls were enrolled. A capsaicin cough provocation test was performed to determine cough sensitivity. Nitrosative stress in the upper airway was assessed by quantifying 3-nitrotyrosine (3-NT) immunostaining in nasal epithelial cells (NECs) and measuring nasal nitric oxide (nNO). The effect of NO on airway epithelium was investigated by measuring the levels of substance P (SP) in nasal lavage fluid and evaluating SP expression in airway epithelial cells. RESULTS: Based on the results of the capsaicin test, patients were divided into two groups: a cough hypersensitivity (CHS) group and a normal cough sensitivity (NCS) group. The levels of 3-NT immunoreactivity in NECs were significantly higher in CHS (49 ± 2.9%) than in NCS (27 ± 3.3%) and controls (12 ± 1.6%), a pattern that was also reflected in the values of nNO (350 ± 43, 215 ± 23, and 138 ± 23 ppb in CHS, NCS, and controls, respectively). SP concentration was also elevated in nasal lavage fluids from CHS (746 ± 28 pg/mL) compared with that from NCS (624 ± 40 pg/mL) and controls (526 ± 41 pg/mL). SP expression in airway epithelial cells was greatly enhanced by exposure to NO donor, which was attenuated by pretreatment with either NO scavenger or NO synthase inhibitor. CONCLUSION: Increased nitrosative stress in the upper airway may play a role in the pathogenesis of unexplained chronic cough with CHS through enhanced secretion of SP.

Chlamydophila pneumoniae inhibits corticosteroid-induced suppression of metalloproteinase-9 and tissue inhibitor metalloproteinase-1 secretion by human peripheral blood mononuclear cells.

Chlamydophila pneumoniae infection has been suggested to be associated with severe asthma characterized by persistent airway limitation, which may be related to airway remodelling. We investigated whether C. pneumoniae infection affected the secretion of metalloproteinase-9 (MMP9) and tissue inhibitor metalloproteinase-1 (TIMP1), and altered the responsiveness of inflammatory cells to corticosteroids. Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence or absence of C. pneumoniae. Secretion of both MMP9 and TIMP1 was strongly suppressed by dexamethasone treatment in uninfected cells. MMP9 secretion was also significantly inhibited by dexamethasone in C. pneumoniae-infected cells, but TIMP1 secretion was not; hence the MMP9 to TIMP1 ratio decreased. Interestingly, expression of human glucocorticoid receptor ß, which is believed to confer resistance to corticosteroids, was enhanced by dexamethasone treatment in C. pneumoniae-infected PBMCs. We conclude that C. pneumoniae infection may promote airway remodelling by decreasing the ratio of MMP9 to TIMP1 secreted by inflammatory cells, and by altering cellular responsiveness to corticosteroids.

Hyperoxidized peroxiredoxins in peripheral blood mononuclear cells of asthma patients is associated with asthma severity.

AIMS: Oxidative stress is involved in the pathogenesis of asthma, and peroxiredoxins (PRDX) may be critical in controlling intracellular oxidative stress. The aim of this study was to evaluate expressions of PRDX and their hyperoxidized forms in asthmatic individuals. MAIN METHODS: The levels of expression of PRDX1, PRDX2, PRDX3, and PRDX6 and their hyperoxidized forms (PRDX-SO(3)) were measured in PBMCs from asthma patients and control subjects. In addition, cells from these subjects were treated with hydrogen peroxide (H(2)O(2)) and their intracellular concentrations of reactive oxygen species (ROS) were measured. KEY FINDINGS: The ratios of hyperoxidized to total PRDX (PRDX-SO(3/)PRDX) in PBMCs were significantly higher in asthma patients than in normal subjects and were correlated with disease severity, with the highest ratio seen in patients with severe asthma. Furthermore, H(2)O(2) treatment of PBMCs, particularly lymphocytes, increased intracellular ROS concentrations with greater and more persistent increases observed in cells from asthmatic than from control subjects. SIGNIFICANCE: Hyperoxidation of PRDX may serve as a biomarker of asthma severity and may predict enhanced susceptibility to oxidative stress load in PBMCs of asthmatics.

Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation.

Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-κB and AP-1, induced by pro-inflammatory stimuli, such as TNF-α, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-α. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity.

Increased oxidative stress in the airway and development of allergic inflammation in a mouse model of asthma.

BACKGROUND: The exact pathogenic role of oxidative stress in the development of allergic airway inflammation is still largely unknown. OBJECTIVE: To investigate a possible link between increased pulmonary oxidative stress and the pivotal features of asthma during the mounting of an allergic inflammatory response. METHODS: To determine the relationship between oxidative stress and allergic inflammatory responses, we evaluated the sequential kinetics of oxidative stress in the lung, the development of airway inflammation, mucin hypersecretion, and airway hyperresponsiveness (AHR) in an ovalbumin (OVA)-sensitized and challenged mouse with and without antioxidant. Parameters were measured at 9 points for more than 28 days, starting from the first day of OVA challenge with or without antioxidant treatment. The ratio of reduced to oxidized glutathione in the lungs and levels of intracellular reactive oxygen species (ROS) in the bronchial epithelium were serially measured. Bronchoalveolar lavage fluid cells, histopathologic features, and AHR were analyzed at the same time points. RESULTS: The reduced to oxidized glutathione ratio was reduced from immediately after OVA challenge to day 1, remained at this level until day 1, and rapidly recovered to the normal level after more than 2 days. Intracellular ROS levels in the bronchial epithelium followed similar kinetics. The inflammatory cells in bronchoalveolar lavage fluid reached a maximum of 3 days and decreased progressively thereafter. Histopathologic examination revealed that substantial airway inflammation persisted through day 28. The proportion of mucin-producing epithelial cells significantly increased after day 1, reached a maximum at day 3, and remained at this level until day 5. The AHR peaked on day 1 and normalized within 5 days. The pretreatment of antioxidant significantly reduced not only the increased ROS levels but also development of other phenotypes of asthma. CONCLUSION: These results indicate that increased oxidative stress in the lung precedes other pivotal phenotypes of allergic airway disease, suggesting a critical role for increased oxidative stress in the induction of allergic airway inflammation.

Vascular endothelial growth factor levels in induced sputum and emphysematous changes in smoking asthmatic patients.

BACKGROUND: Although asthmatic patients frequently smoke cigarettes, the effect of smoking on asthmatic lungs has not been extensively studied. Vascular endothelial growth factor (VEGF) has been suggested to play a role in the pathogenesis of asthma and emphysema. OBJECTIVE: To determine whether the level of airway VEGF might be related to emphysematous lung changes in asthmatic patients with a history of smoking. METHODS: Pulmonary function tests and volumetric computed tomography (CT) of the lungs were performed in 10 ex-smoking and 15 nonsmoking asthmatic patients. Levels of VEGF were evaluated in the supernatants of induced sputum using an enzyme-linked immunosorbent assay method in these 25 asthmatic patients and in 10 healthy nonsmoking controls. Associations among sputum VEGF levels, emphysema indices on CT, and clinical variables were analyzed. RESULTS: Ex-smoking asthmatic patients showed a higher emphysema index on volumetric CT and a decreased mean forced expiratory volume in 1 second to forced vital capacity ratio compared with nonsmoking asthmatic patients. Sputum VEGF levels in asthmatic patients with a history of smoking were significantly lower than those in nonsmoking asthmatic patients but remained higher than those in control subjects. Furthermore, the emphysema index was significantly correlated with sputum VEGF levels in asthmatic patients. CONCLUSION: Lower VEGF levels in the airway might bea surrogate marker for emphysematous changes in the lungs, especially in asthmatic patients with a history of smoking.

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo.

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.

Allergen-induced CD11b+ CD11c(int) CCR3+ macrophages in the lung promote eosinophilic airway inflammation in a mouse asthma model.

Although the recruitment of macrophages to the lung is a central feature of airway inflammation, its function in ongoing T(h)2 cell-mediated eosinophilic airway inflammation remains controversial. Here, we have demonstrated that the allergen-induced CD11b(+) CD11c(int) macrophage expressing CC chemokine receptor 3 (CCR3) in the lung performs a crucial function in the induction of eosinophilic asthma in a murine model. In the lungs of normal mice, residential cells evidencing high granularity phenotypically evidenced CD11b(int) CD11c(+) or CD11b(+) CD11c(int) cells, appearing at a 2:1 ratio. After allergen challenge, however, this reverses dramatically, up to a ratio of one to six. Approximately 91% of increased CD11b(+) CD11c(int) cells evidenced the expression of the CCR3 eotaxin receptor, but not other chemokine receptors, such as CCR5 and CXCR4. Interestingly, the CD11b(+) CD11c(int) cells purified from the lungs of OVA (ovalbumin)-sensitized and challenged mice evidenced higher antigen-presenting activity than was observed in CD11b(int) CD11c(+) cells. In order to investigate the in vivo function of CD11b(+) CD11c(int) cells, the cells were isolated from the lungs of OVA-sensitized and challenged mice and then adoptively transferred prior to the allergen challenge of normal mice. In the CD11b(+) CD11c(int)-transferred mice airway hyperresponsiveness, eosinophilic inflammation in the lung and T(h)2 cytokine secretion in the bronchoalveolar lavage fluids were significantly enhanced as the result of OVA challenge, as compared with the mice that received OVA-primed CD90(+) T cells or CD11b(int) CD11c(+) cells. These findings show that CD11b(+) CD11c(int) macrophages expressing CCR3 as key pro-inflammatory cells are both necessary and sufficient for allergen-specific T cell stimulation during ongoing eosinophilic airway inflammation.