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Background and purpose of the study:Pseudomonas aeruginosa commonly colonizes the airway of patients with chronic obstructive pulmonary disease (COPD) and exacerbates their symptoms. P. aeruginosa carries flagellin that stimulates toll-like receptor (TLR)-5; however, the role of flagellin in the pathogenesis of COPD remains unclear. The aim of the study was to evaluate the mechanisms of the flagellin-induced innate immune response in bronchial epithelial cells, and to assess the effects of anti-inflammatory agents for treatment. Materials and methods: We stimulated BEAS-2B cells with P. aeruginosa-derived flagellin, and assessed mRNA expression and protein secretion of interleukin (IL)-6 and IL-8. We also used mitogen-activated protein kinases (MAPK) inhibitors to assess the signaling pathways involved in flagellin stimulation, and investigated the effect of clinically available anti-inflammatory agents against flagellin-induced inflammation. Results: Flagellin promoted protein and mRNA expression of IL-6 and IL-8 in BEAS-2B cells and induced phosphorylation of p38, ERK, and JNK; p38 phosphorylation-induced IL-6 production, while IL-8 production resulted from p38 and ERK phosphorylation. Fluticasone propionate (FP) and dexamethasone (DEX) suppressed IL-6 and IL-8 production in BEAS-2B cells, but clarithromycin (CAM) failed to do so. Conclusions:P. aeruginosa-derived flagellin-induced IL-6 and IL-8 production in bronchial epithelial cells, which partially explains the mechanisms of progression and exacerbation of COPD. Corticosteroids are the most effective treatment for the suppression of flagellin-induced IL-6 and IL-8 production in the bronchial epithelial cells. [ABSTRACT FROM AUTHOR]

Rationale: Neutrophilic airway inflammation plays a central role in chronic obstructive pulmonary disease (COPD). CXC chemokine ligand (CXCL)1 is a neutrophil chemokine involved in the pathogenesis of COPD. However, its clinical significance in COPD patients is poorly understood. Aim of the study: To assess the production of CXCL1 by bronchial epithelial cells in response to lipopolysaccharide (LPS) and tumor necrosis factor (TNF)α. Materials and Methods: We measured sputum CXCL1 and CXCL8 levels in patients with COPD, asthma, and asthma-COPD overlap (ACO), and compared them to those of patients with interstitial pneumonia (IP). Using primary human bronchial epithelial cells and BEAS-2B cells, CXCL1 protein release and mRNA expression were measured after LPS or TNFα stimulation. We evaluated signal transduction mechanisms for CXCL1 production using nuclear factor-κ B (NF-kB) and mitogen-activated protein kinase (MAPK) inhibitors, and examined the effects of anti-inflammatory agents on CXCL1 production in BEAS-2B cells. Results: Sputum CXCL1 levels in COPD and ACO patients were higher than in IP patients, whereas sputum CXCL8 levels were not. Sputum CXCL1 levels were not affected by inhaled corticosteroid usage, whereas sputum CXCL8 levels tended to be affected. LPS and TNFα stimulated CXCL1 production and mRNA expression in bronchial epithelial cells. NF-kB and MAPK p38 were involved in LPS-induced CXCL1 production. Therapeutic anti-inflammatory agents minimally attenuated CXCL1 production and considerably inhibited CXCL8 production in BEAS-2B cells. Conclusions: Sputum CXCL1 levels is a potentially better diagnostic marker for COPD than sputum CXCL8 levels, which is explained by that CXCL1 production in bronchial epithelial cells is less affected by therapeutic anti-inflammatory agents than CXCL8 production. [ABSTRACT FROM AUTHOR]

Background:Recent reports have suggested an involvement of neutrophilic inflammation driven by interleukin (IL)-17 from Th17 cells, especially in severe, refractory asthma. It remains unknown about the possible interactions of this cytokine and other proinflammatory cytokines to direct neutrophilic airway inflammation.Materials and Methods:We evaluated the effects of IL-17A, IL-17E, and IL-17F in combination with other stimuli such as tumor necrosis factor (TNF) –α on the production and expression of IL-8 in human bronchial epithelial cells. We also studied their effects on other cytokine production. The possible role of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways was evaluated by specific inhibitors. We examined the effects of anti-asthma drugs, such as steroids or salmeterol.Results:IL-17A alone induced only a minimal effect on IL-8 expression. IL-17A, but not IL-17E or IL-17F, in combination with TNF-α showed a synergistic effect on IL-8 expression. Similar findings were found when combination with IL-1β and IL-17A were used, but such was not the case with lipopolysaccharide (LPS). In addition, we further found such synergy on GM-CSF production. The synergy with TNF-α and IL-17A was significantly inhibited by MAPKs inhibitors. Corticosteroids such as fluticasone propionate and dexamethasone, but not salmeterol, partially suppressed the IL-17A and TNF-α-induced IL-8 production.Conclusions:IL-17A in the combination with TNF-α or IL-1β showed a synergistic augmenting effect on IL-8 and GM-CSF production in human airway epithelial cells. [ABSTRACT FROM PUBLISHER]

Aim of the Study: Interleukin (IL)-10 is an anti-inflammatory cytokine, but its role in cigarette smoke (CS)-induced inflammation and chronic obstructive pulmonary disease (COPD) has not been fully elucidated. The purpose of this study was to investigate the effect of IL-10 deficiency on CS-induced pulmonary inflammation in mice in vivo and in vitro.Materials and Methods: IL-10-deficient and wild-type control mice with a C57BL6/J genetic background were exposed to CS, and inflammatory cells in bronchoalveolar lavage fluid (BALF) and mRNA of cytokines in lung were evaluated with enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR).Results: During 12 days of daily CS exposure to wild-type mice, neutrophil counts in BAL fluid and tumor necrosis factor (TNF)-α mRNA expression were increased, peaked at day 8, and then declined on day 12 when the level of IL-10 reached its peak. In IL-10-deficient mice, neutrophil recruitment and TNF-α mRNA levels induced by CS exposure were significantly greater than those in wild-type mice. Keratinocyte-derived chemokine (KC; murine ortholog of human CXCL8) and granulocyte macrophage colony-stimulating factor (GM-CSF) mRNA levels or matrix metalloproteinase(MMP)-9 protein levels were not correlated with neutrophil count.Conclusions: IL-10 had a modulatory effect on CS-induced pulmonary neutrophilic inflammation and TNF-α expression in mice in vivo and therefore appears to be an important endogenous suppressor of airway neutrophilic inflammation. [ABSTRACT FROM PUBLISHER]

Background: Airway remodeling is implicated in irreversible airflow limitation of refractory asthma, which includes increased smooth muscle mass and subepithelial fibrosis. Activated fibroblasts acquire contractile phenotype to participate in tissue contraction and structural alteration of extracellular matrices. Histamine is a potent mediator of allergic inflammation, substantially involved in asthmatic pathophysiology. Objective: We hypothesized that histamine might play a role in airway remodeling, and investigated its effect on fibroblast-mediated collagen gel contraction. Methods: Fibroblast-mediated collagen gel contraction was studied. Histamine's regulation of collagen gel contraction was characterized by using specific histamine-receptor antagonists, an IP3 receptor antagonist and a PKC inhibitor. Results: Histamine induced contraction of collagen gels embedded with human lung fibroblasts, in a time-dependent manner, and at the concentration more than 10−6 M, both in four primary cultured adult lung fibroblasts and three fetal lung fibroblast cell lines. This effect was attenuated by H1 receptor antagonist, whereas those for H2 to H4 receptors failed to show an inhibitory effect. Furthermore, IP3 receptor-mediated Ca2+ mobilization was implicated in histamine's action on collagen gel contraction. Conclusions: Our results suggest that histamine is involved in airway remodeling through its action on lung fibroblasts, and antihistamine drugs, especially H1 receptor antagonists, might be potentially beneficial for a subset of asthmatic patients. [ABSTRACT FROM AUTHOR]

Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-β1 signaling. Tumor necrosis factor (TNF)-α has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-α affected TGF-β1-induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-β1 (5 ng/mL), with/without TNF-α (10 ng/mL). TGF-β1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-α with TGF-β1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-β1 induced cell contraction, as did TNF-α. Moreover, costimulation with TGF-β1 and TNF-α enhanced the cell contraction. Although IFN-γ suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-β1. In conclusion, TNF-α enhances not only EMT but also cell contraction induced by TGF-β1. EMT might contribute to tissue fibrosis through induction of cell contraction. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) has been classically used as a marker of inflammation. The aim of this study was to investigate the effect of CRP on migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn). Using the blindwell chamber technique, CRP inhibited HFL-1 migration in a dose-dependent fashion (at 1 μ g/mL, inhibition: 32.5% ± 7.1%; P < .05). Western blot analysis showed that CRP inhibited the p38 mitogen-activated protein kinase (MAPK) activity in the presence of HFn. Moreover, the MAPK inhibitors SB202190 (25 μ M) and SB203580 (25 μ M) inhibited HFn-induced cell migration, suggesting an important role of p38 MAPK in HFn-induced migration. Taken together, these results suggest that the inhibitory effect of CRP is mediated by blocking MAPK. In summary, this study demonstrates that CRP directly modulates human lung fibroblasts migration. Thus, CRP may contribute to regulation of wound healing and may be endogenous antifibrotic factor acting on lung fibrosis. [ABSTRACT FROM AUTHOR]

The authors used BALB/c and C57BL/6 mouse strains to search for genetically based differences in response to prolonged (6 months) low-dose (100 μg/m3) diesel exhaust particle (DEP) exposure from birth in terms of airway inflammatory responses. Histopathological assessment showed that inflammatory cells infiltrated the perivascular areas only in C57BL/6 mice. The count of DEP-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid was significantly greater in BALB/c mice (P < .05) than in C57BL/6 mice. The lymphocyte and eosinophil count in BAL fluid was significantly greater in C57BL/6 mice (P < .05) than in BALB/c mice. Immunoglobulin (Ig) IgG1 and IgG2 levels in serum, and the monocyte chemoattractant protein (MCP)-1 level in BAL fluid were significantly greater in BALB/c mice than in C57BL/6 mice. The interleukin (IL)-12 level in BAL fluid was significantly greater in C57BL/6 mice than in BALB/c mice, but the IL-13 level in BAL fluid was significantly less in BALB/c mice than in C57BL/6 mice. Glutathione S-transferase (GST) mRNA expression and protein production in lung tissues were significantly lower in C57BL/6 mice than in BALB/c mice, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) level in the lung tissues were significantly greater in C57BL/6 mice than in BALB/c mice. In conclusion, prolonged low-dose DEP exposure induces airway inflammatory responses that differ remarkably among mouse strains; these differences are caused by differences in the host defense response to the oxidative stress induced by DEP exposure and may be useful in the development of biomarkers. [ABSTRACT FROM AUTHOR]

Low-dose diesel exhaust particle (DEP) exposure induces airway inflammation and exaggerates asthmatic responses in mice, but it is unclear whether strains differ in their susceptibility to adverse effects from low-dose DEP exposure. The authors used BALB/c and C57BL/6 mouse strains to search for genetically based differences in response to low-dose DEP (100 µg/m3) exposure in terms of airway inflammatory response. The macrophage count in bronchoalveolar lavage (BAL) fluid soon after DE exposure began was significantly greater in C57BL/6 mice (P < .05) than that in BALB/c mice. The count did not increase significantly in BALB/c mice until later. Heme oxygenase-1 (HO-1) mRNA expression and protein production in lung tissues soon after exposure began were more marked in BALB/c mice than in C57BL/6 mice, but the reverse was true later on. The increases in interleukin (IL)-1β and interferon (IFN)-γ levels in BAL fluid after DE exposure were significant only in BALB/c mice; there were significantly increases in monocyte chemoattractant protein (MCP)-1, IL-12, IL-10, IL-4, and IL-13 in both strains, but these were more marked in C57BL/6 mice. These interstrain differences in airway inflammatory response after DE exposure were significantly attenuated by antioxidant N-acetylcysteine (NAC) treatment. Changes in airway hyperresponsiveness were independent of the airway inflammation induced by low-dose DEP. Thus, in BALB/c mice, innate immunity may play a central role in DE exposure response, whereas in C57BL/6 mice Th2-dominant responses play a central role. Low-dose DEP exposure induces airway inflammatory responses that differ among strains, and these differences may be caused by differences in sensitivity to oxidative stress. [ABSTRACT FROM AUTHOR]

The authors investigated the effects of inhalation of diesel exhaust (DE) on murine mycobacterial infection in vivo. Eight-week-old female BALB/c mice were exposed to DE (3 mg/m 3 of diesel exhaust particles [DEPs]) for 1 month, 2 months, or 6 months (for 7 hours a day, 5 days a week). Control mice were housed in a clean room for the same periods. On the day following the last DE exposure, control mice and DE-exposed mice were aerially infected with Mycobacterium tuberculosis (1?×?10 6 colony-forming units (CFU), Kurono strain). At 7 weeks after mycobacterial infection, the authors examined the lung tissues for histopathological changes and performed reverse transcriptase–polymerase chain reaction (RT-PCR) to measure the messenger RNA (mRNA) expression of several proinflammatory cytokines and inducible nitric oxide synthase (iNOS). Then, the homogenates of lungs and spleens were cultured on 1% ( v / v ) Ogawa's egg slant medium, and after a 4-week incubation period at 37°C, colonies on the medium were counted. After 1 month of DE exposure, the mycobacterial infection had slightly ameliorated. After 2 months of DE exposure, the expression levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1ß, IL-12p40, interferon (IFN)-?, and iNOS mRNAs were slightly increased. However, after 6 months of DE exposure, the expression levels of IL-1ß, IL-12p40, IFN-?, and iNOS mRNAs were decreased, and the infection as measured by increased lung burden (CFU) actually increased. These results indicate that long-term DE exposure may increase pulmonary mycobacterial burden. [ABSTRACT FROM AUTHOR]

The authors investigated the effects of exposure to diesel exhaust (DE) on murine lung tissues invivo. BALB/c and C57BL/6 mice were exposed to DE with low (100 μg/m 3 ) and high (3 mg/m 3 ) DE particle levels for 3 months. The authors then examined morphological changes and the expression of mRNAs for various cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-4, IL-6, IL-10, IL-12p40, and interferon [IFN]-γ) and inducible nitric oxide synthase (iNOS) in the lungs, as well as TNF-α, IL-1β, IL-10, IL-12p40, and Mac-1 mRNA expression in alveolar macrophages (AMs). TNF-α, IL-12p40, IL-4, and IL-10 mRNA expression were mildly increased, whereas IL-1β mRNA and iNOS expression were slightly decreased, in the low- and high-level exposure groups. Flow cytometry of bronchoalveolar lavage fluid revealed a significant increase in Mac-1-positive cells in the high-level exposure group. On histological examination, bronchus-associated lymphoid tissue (BALT), containing B and T lymphocytes, had developed only in the high-level exposure group. Chronic inhalation of DE influences cytokine expression in the murine lung, and induces phagocytosis and BALT development. These findings suggest that DE may provoke immunological responses by acting as a foreign body in the lung, and that even low-level exposure may induce allergic reactions. [ABSTRACT FROM AUTHOR]

The authors investigated the effect of diesel exhaust (DE) on cytokine expression in murine lung tissues. BALB/c mice were exposed to DE for 1 month at different dose levels of DE (low dose: diesel exhaust particles [DEP] 100 µg/m[sup 3]; high dose: 3mg/m[sup 3]). After exposure, the authors examined mRNA expression of cytokines (tumor nocrosis factor alpha [TFN-α], Interleukin [IL]-1β, IL-4, IL-6, IL-10, IL-12p40, and interferon gamma [IFN-γ] and inducible nitric oxide synthase (iNOS) in the lung, and also measured the secretion of TNF-α and IL-10 protein by alveolar macrophages (AM). The mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-12p40, IFN-γ) and iNOS, which are important for host defense, were suppressed significantly. However, the IL-10 mRNA level was increased by DE exposure. The IL-4 mRNA level was increased by low-dose DE exposure but suppressed by high-dose DE exposure. TNF-α and IL-10 secretion by AM paralleled mRNA expression. Chronic inhalation of DE affects cytokine expression in murine lung. These results suggest that DE alters immunological responses in the lung and may increase susceptibility to pathogens, and that increased IL-4 expression by low-dose DE exposure may induce allergic reaction such as asthma. [ABSTRACT FROM AUTHOR]

To investigatethe effects of diesel exhaust particles (DEP) and mycobacterial infection on macrophages, we examined protein and mRNA expression levels of various cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-12, and IL-18 in BALB/c mouse alveolar macrophages (AM)and a macrophagecell line (RAW264.7) after in vitro stimulation with diesel exhaust (DE), with and without Mycobacterium bovis bacillus Calmette-Guerin (BCG). The cells were exposed to DE freshly generated in an in vitro system. When AM were exposed to DE (mean DEP exposure level, 300 µ g/m[sup 3]), the levels of TNF-α and IL-12 decreased significantly (by 51% to 61% for TNF-α and by 69% to 78% for IL-12), whereas those of IL-1β and IL-18 remained unchanged. When AM were exposed to DE and then treated with BCG, the level of TNF-α decreased by 45% to 71%, whereas the level of IL-1β increased by 154%, compared with AM treated with BCG alone. Similarly, RAW264.7 cells were stimulated with DE with and without M. bovis BCG and cytokine mRNA levels examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Longer exposure to DE decreased TNF-α and IL-12 mRNA levels in the RAW264.7 cells. When the cells were exposed to DE and subsequently treated with BCG, levels of TNF-α, IL-1β, and IL-12 mRNAs decreased compared with those of cells treated with BCG alone. These results show that DE exposure has complex and diverse effects on cytokine production by AM, and that longer exposure (> 8 hours) may suppress cytokine production by AM in vitro. Longer exposure of DE may therefore suppress the host defense in the lung and may increase susceptibility to lung infections such as mycobacterial infection. [ABSTRACT FROM AUTHOR]

Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-beta1 signaling. Tumor necrosis factor (TNF)-alpha has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-alpha affected TGF-beta1-induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-beta1 (5 ng/mL), with/without TNF-alpha (10 ng/mL). TGF-beta1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-alpha with TGF-beta1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-beta1 induced cell contraction, as did TNF-alpha. Moreover, costimulation with TGF-beta1 and TNF-alpha enhanced the cell contraction. Although IFN-gamma suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-beta1. In conclusion, TNF-alpha enhances not only EMT but also cell contraction induced by TGF-beta1. EMT might contribute to tissue fibrosis through induction of cell contraction.