Combination of TWEAK and TGF-ß1 induces the production of TSLP, RANTES, and TARC in BEAS-2B human bronchial epithelial cells during epithelial-mesenchymal transition.

AIM OF THE STUDY: In patients with asthma, chronic inflammatory processes and the subsequent remodeling of the airways contribute to the symptoms and the pathophysiological changes. Epithelial-mesenchymal transition (EMT) is thought to play an important role in tissue remodeling. Previous reports show that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF superfamily, exerts pro-inflammatory effects, and enhances transforming growth factor (TGF)-ß-induced EMT in bronchial epithelial cells. In this study, we investigated the TWEAK-induced cytokine and chemokine production in the human bronchial epithelial cell line BEAS-2B during EMT. MATERIALS AND METHODS: Quantitative real-time RT-PCR, enzyme-linked immunosorbent assays, western blotting, and immunohistochemistry were used to define the production of cytokines and chemokines. RESULTS: We found that TWEAK increases mRNA and protein levels of thymic stromal lymphopoietin (TSLP), monocyte chemoattractant protein -1 (MCP-1), regulated upon activation normal T cell express sequence (RANTES), and IL-8 in BEAS-2B bronchial epithelial cells. Moreover, co-treatment with TWEAK and TGF-ß1 induces not only features of EMT but also enhances the production of TSLP and RANTES. Thymus- and activation-regulated chemokines (TARC) production is induced by the co-treatment of TWEAK and TGF-ß1 but not by TWEAK or TGF-ß1 stimulation alone. Furthermore, the increased mRNA expression of TSLP and RANTES after co-treatment with TWEAK and TGF-ß1 is prevented by inhibitors of Smad-independent signaling pathways. CONCLUSIONS: In the present study, we have revealed a novel mechanism for the production of asthma-related cytokines and chemokines in EMT driven by the co-stimulation with TWEAK and TGF-ß1. We conclude that cellular EMT processes caused by TWEAK and TGF-ß1 may contribute to chronic airway inflammation and remodeling.

Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease.

Alterations in microRNA (miRNA) expression may contribute to COPD pathogenesis. In COPD, lung fibroblast repair functions are altered in multiple ways, including extracellular mediator release. Our prior study revealed miR-503 expression is decreased in COPD lung fibroblasts, although the exact role played by miR-503 is undetermined. The current study examined a role of miR-503 in cytokine, growth factor and fibronectin production by lung fibroblasts from patients with and without COPD. Primary adult lung fibroblasts were isolated from patients with or without COPD. MiR-503 expression and interleukin (IL)-6, -8, PGE2, HGF, KGF, VEGF and fibronectin release were examined with or without inflammatory cytokines, IL-1ß and tumor necrosis factor (TNF)-α. MiR-503 expression was decreased in COPD lung fibroblasts. The expression of miR-503 was positively correlated with %FVC, %FEV1, and %DLco as well as IL-6, -8, PGE2, HGF, KGF, and VEGF in the absence or presence of IL-1ß/TNF-α. In addition, IL-8 and VEGF release from COPD lung fibroblasts were increased compared to those from control. Exogenous miR-503 inhibited VEGF release from primary adult and fetal lung fibroblasts but not IL-8 release. As expected, COPD fibroblasts proliferated more slowly than control fibroblasts. MiR-503 did not affect proliferation of either control or COPD lung fibroblasts. MiR-503 inhibition of VEGF protein production and mRNA was mediated by direct binding to the 3' untranslated region of VEGF mRNA. Endogenous miR-503 was differently regulated by exogenous stimulants associated with COPD pathogenesis, including IL-1ß/TNF-α, TGF-ß1 and PGE2. Endogenous miR-503 inhibition augmented VEGF release by IL-1ß/TNF-α and TGF-ß1 but not by PGE2, demonstrating selectivity of miR-503 regulation of VEGF. In conclusions, reduced miR-503 augments VEGF release from lung fibroblasts from patients with COPD. Since VEGF contributes to disturbed vasculature in COPD, altered miR-503 production might play a role in modulating fibroblast-mediated vascular homeostasis in COPD.

TWEAK enhances TGF-ß-induced epithelial-mesenchymal transition in human bronchial epithelial cells.

BACKGROUND: Chronic airway inflammatory disorders, such as asthma, are characterized by airway inflammation and remodeling. Chronic inflammation and damage to the airway epithelium cause airway remodeling, which is associated with improper epithelial repair, and is characterized by elevated expression of transforming growth factor-ß (TGF-ß). Epithelial-mesenchymal transition (EMT) is an important mechanism during embryonic development and tissue remodeling whereby epithelial cells gain the capacity to increase motility by down-regulation of epithelial markers and up-regulation of mesenchymal markers. TGF-ß is a central inducer of EMT, and TGF-ß-induced EMT is enhanced by pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1ß. We investigated whether the pro-inflammatory cytokine TWEAK (TNF-like weak inducer of apoptosis) enhanced TGF-ß1-induced EMT in the human bronchial epithelial cell line BEAS-2B. METHODS: Quantitative RT-PCR and western blotting were used to define alterations in epithelial and mesenchymal marker expression in BEAS-2B cells. The cells were assessed for 48 h after stimulation with TGF-ß1 alone or in combination with TWEAK. RESULTS: TGF-ß1 induced spindle-like morphology and loss of cell contact, and reduced the expression of epithelial marker E-cadherin and increased the expression of mesenchymal markers N-cadherin and vimentin. Our data, for the first time, show that TWEAK reduced the expression of E-cadherin, and that co-treatment with TGF-ß1 and TWEAK enhanced the TGF-ß1-induced features of EMT. Moreover, hyaluronan synthase 2 expression was up-regulated by a combination with TGF-ß1 and TWEAK, but not TNF-α. We also demonstrated that the Smad, p38 MAPK, and NF-κB signaling pathways, and the transcriptional repressor ZEB2 might mediate N-cadherin up-regulation by TGF-ß1 in combination with TWEAK. CONCLUSIONS: These findings suggest that the pro-inflammatory cytokine TWEAK and TGF-ß1 have synergistic effects in EMT and may contribute to chronic airway changes and remodeling.

TIM-4 has dual function in the induction and effector phases of murine arthritis.

T cell Ig and mucin domain (TIM)-4 is involved in immune regulation. However, the pathological function of TIM-4 has not been understood and remains to be clarified in various disease models. In this study, DBA/1 mice were treated with anti-TIM-4 mAb during the induction or effector phase of collagen-induced arthritis (CIA). Anti-TIM-4 treatment in the induction phase exacerbated the development of CIA. In vitro experiments suggest that CD4 T cells bind to TIM-4 on APCs, which induces inhibitory effect to CD4 T cells. In contrast, therapeutic treatment with anti-TIM-4 mAb just before or after the onset or even at later stage of CIA significantly suppressed the development and progression by reducing proinflammatory cytokines in the ankle joints without affecting T or B cell responses. Consistently, clinical arthritis scores of collagen Ab-induced arthritis, which is not mediated by T or B cells, were significantly reduced in anti-TIM-4-treated mice with a concomitant decrease of proinflammatory cytokines in the joints. In vitro, macrophages secreted proinflammatory cytokines in response to TIM-4-Ig protein and LPS, which were reduced by the anti-TIM-4 mAb. The anti-TIM-4 mAb also inhibited the differentiation and bone-resorbing activity of osteoclasts. These results indicate that TIM-4 has two distinct functions depending on the stage of arthritis. The therapeutic effect of anti-TIM-4 mAb on arthritis is mediated by the inhibition of proinflammatory cytokine production by inflammatory cells, osteoclast differentiation, and bone resorption, suggesting that TIM-4 might be an appropriate target for the therapeutic treatment of arthritis.

Blockade of CD70-CD27 interaction inhibits induction of allergic lung inflammation in mice.

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T-cell activation. In this study, we investigated the effects of neutralizing anti-CD70 monoclonal antibody (mAb) in a murine model of allergic lung inflammation to determine whether CD27 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. BALB/c mice were immunized by an injection of ovalbumin (OVA) with alum adjuvant and challenged with aerosolized OVA in PBS. Some groups of mice were treated with anti-CD70 mAb or control rat IgG during the induction or effector phase. The administration of anti-CD70 mAb during the induction phase, but not the effector phase, reduced eosinophil infiltration in lung tissue compared with control IgG-treated mice. Treatment with anti-CD70 mAb also resulted in the decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the bronchoalveolar lavage fluid and draining lymph node cell cultures. We further revealed that antigen-specific CD4 T cells were separated into CD27(+) and CD27(-) populations in the lymph nodes of OVA-immunized DO11.10/Rag-2(-/-) mice. The CD27(+) CD4 T cells produced a high concentration of IFN-γ, representing Th1 cells. In contrast, CD27(-) CD4 T cells produced high concentrations of IL-4, IL-5, and IL-13, representing Th2 cells. Moreover, the population of CD27(-) Th2 cells was significantly reduced by the anti-CD70 mAb treatment. These results indicate an important role for CD27 in the development of pathogenic Th2 cells in a murine model of allergic lung inflammation.

Wound-induced TGF-ß1 and TGF-ß2 enhance airway epithelial repair via HB-EGF and TGF-α.

The abundance of transforming growth factor-beta (TGF-ß) in normal airway epithelium suggests its participation in physiological processes to maintain airway homeostasis. The current study was designed to address the hypothesis that TGF-ß1 and TGF-ß2 might contribute to normal reparative response of airway epithelial cells (AECs). Treatments with exogenous TGF-ß1 or TGF-ß2 significantly enhanced wound repair of confluent AEC monolayers. Mechanical injury of AEC monolayers induced production of both TGF-ß1 and TGF-ß2. Wound repair of AECs was significantly reduced by a specific inhibitor of TGF-ß type I receptor kinase activity. We investigated whether the TGF-ß-enhanced repair required epidermal growth factor receptor (EGFR) transactivation and secretion of EGFR ligands. Both TGF-ß1 and TGF-ß2 enhanced EGFR phosphorylation and induced production of heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-α) in AECs. Moreover, treatment with a broad-spectrum metalloproteinase inhibitor or anti-HB-EGF and anti-TGF-α antibodies inhibited the wound repair and the EGFR phosphorylation by TGF-ß1 and TGF-ß2, indicating that the TGF-ß1 and TGF-ß2 effects on wound repair required the release of HB-EGF and TGF-α. Our data, for the first time, have shown that both TGF-ß1 and TGF-ß2 play a stimulatory role in airway epithelial repair through EGFR phosphorylation following autocrine production of HB-EGF and TGF-α. These findings highlight an important collaborative mechanism between TGF-ß and EGFR in maintaining airway epithelial homeostasis.

Patients with mild-to-moderate asthma may develop clinically significant chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Little is known about the aetiology and clinical characteristics of COPD among people who have never smoked. METHODS: A case-control study was conducted to describe the potential risk factors for clinically significant COPD among smokers and people who had never smoked. Medical record reviews and scoring of high-resolution computed tomography (HRCT) findings were performed in patients with clinically significant COPD (defined as having post-bronchodilator FEV(1)/FVC < 0.7 and FEV(1) < 60% of the predicted values). Pathological analyses were performed in some patients following autopsy. RESULTS: Among the 9493 subjects screened, 424 (4.5%) were diagnosed with clinically significant COPD. Forty-nine (11.6% of clinically significant COPD) were never smokers (NSCOPD), and a comparative group of 98 smoking patients with COPD was randomly selected (SMCOPD). NSCOPD was characterized by predominantly female sex (87.8%) and a high prevalence of physician-diagnosed asthma (61.2%). Similar levels of reduction in %FEV(1) and %FEF(25-75%) were found in NSCOPD and SMCOPD, but there were higher %DL(CO) values and fewer low attenuation areas on HRCT in NSCOPD. More than half of the NSCOPD patients without a history of asthma had features of asthma. More than one-third of NSCOPD patients with an asthma history had never had a severe attack. At autopsy, both NSCOPD and SMCOPD showed wall thickening and inflammatory cell infiltration in small airways, and the number of CD4(+)-T cells was increased in NSCOPD. CONCLUSION: In elderly Japanese, COPD among never smokers is largely attributable to asthma. Airflow limitation in NSCOPD results from small airway disease (airway predominant phenotype) rather than parenchymal destruction (emphysematous phenotype).

A case with life-threatening interstitial pneumonia associated with bucillamine treatment.

We report a case of bucillamine-induced interstitial pneumonia accompanied by severe hypoxemia in an 83-year-old woman who had rheumatoid arthritis. Respiratory failure worsened even after withdrawal of bucillamine and administration of high-dose corticosteroids, and mechanical ventilation was required. A review of 15 cases with bucillamine-induced pulmonary injury suggests that advanced age may be associated with the development of severe interstitial pneumonia. Bucillamine can cause corticosteroid-resistant and life-threatening lung injury, especially in the elderly.