Tumor necrosis factor-alpha enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-beta1.

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.

Procaterol inhibits lung fibroblast migration.

Fibroblasts are important cells that are involved in modulation of fibrosis after injuries. In some uncontrollable inflammatory processes, excess fibroblasts migrate around the small airway. The pathogenesis of chronic obstructive pulmonary disease is related to fibrosis around the small airways. The aim of the current study was to investigate the effect of procaterol, a second-generation beta (2)-agonist, on migration of human fetal lung fibroblasts (HFL-1) induced by human plasma fibronectin (HFn). Using the blindwell chamber technique, 10(-8) M procaterol inhibited migration of HFL-1 (control, 100%; 10(-8) M, 73.2 +/- 4.9%; n = 6, p < 0.05). The inhibitory effect of procaterol was concentration-dependent. Although a beta 2-receptor inhibitor, ICI 181551, blocked the inhibitory effect of procaterol, a beta 1-receptor inhibitor, atenolol, did not. Because a cyclic AMP-dependent protein kinase (PKA) inhibitor, KT5720, blocked the effect of procaterol, the cyclic AMP-PKA pathway may be involved in the migration inhibitory process. Procaterol, which is prescribed mainly for treatment of bronchial asthma, might be a useful drug for inhibiting lung fibrosis following injuries to the lung.

C-reactive protein modulates human lung fibroblast migration.

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 microg/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 microM) and SB203580 (25 microM) 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.

Leukotriene D4 potentiates fibronectin-induced migration of human lung fibroblasts.

Fibroblasts play an important role in the repair and remodeling processes following injury. Leukotriene D4 (LTD4) is a potent mediator in inflammatory processes, but the direct effect of cysteinyl leukotrienes on fibroblast migration remains unelucidated. In this study, the effect of the LTD4 on normal human lung fibroblasts (NHLF) chemotaxis induced by human plasma fibronectin (HFn) was investigated using the modified Boyden's chamber technique. LTD4 potentiated NHLF chemotaxis to HFn in concentration-dependent manner. A specific cysteinyl leukotriene receptor type 1 antagonist, pranlukast inhibited this effect, indicating that LTD4 affected cell migration via its specific receptor. The potentiating effect of LTD4 on fibroblast chemotaxis was completely abolished by pertussis toxin (PTX), suggesting that LTD4-induced effect was dependent on PTX-sensitive Gi/o signaling. These findings suggest that LTD4 has a potential to augment fibroblast chemotaxis, and to contribute to regulation of the wound healing and following remodeling in fibrotic processes of the lung.

Molecular mechanisms of anti-inflammatory action of erythromycin in human bronchial epithelial cells: possible role in the signaling pathway that regulates nuclear factor-kappaB activation.

Long-term macrolide therapy has been proven to improve survival in patients with diffuse panbronchiolitis. Although its mechanisms remain unknown, previous studies have suggested the effects of macrolide might be anti-inflammatory rather than antibacterial. To elucidate the molecular mechanisms of its action, we studied here the effects of erythromycin (EM) and its new derivative, EM703, which shows no antibacterial action, on the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in human bronchial epithelial cells. Western blotting analysis showed that EM did not inhibit the degradation of IkappaBalpha, suggesting the molecular target for EM was not the dissociation of NF-kappaB from IkappaB. An electrophoretic mobility shift assay showed that EM did not interrupt the NF-kappaB DNA-binding activity in the nucleus under the conditions tested. Moreover, not only EM but also EM703 suppressed the activation of NF-kappaB and the production of interleukin-8, demonstrating that the anti-inflammatory action of the macrolide is independent of its antibacterial activity. Taken together, these data suggest EM has an anti-inflammatory action, presumably via an interaction with the NF-kappaB signaling pathway in the downstream of the dissociation from IkappaB, resulting in the inhibition of NF-kappaB.

Methotrexate induces interleukin-8 production by human bronchial and alveolar epithelial cells.

Methotrexate (MTX) has been widely used for the treatment of a variety of tumours as well as for inflammatory diseases. MTX-induced pneumonitis has been a serious unpredictable side effect of the treatment and an important clinical problem. However, its mechanism remains largely unclear. Possible causes include allergic, cytotoxic or immunologic reactions to this agent. To elucidate the proinflammatory mechanism of MTX-induced pneumonitis, we evaluated the effect of MTX on the production of IL (interleukin)-8 by human bronchial and alveolar epithelial cells in vitro and the role of p38 MAPK (mitogen-activated protein kinase) in order to clarify the intracellular signal regulating IL-8 expression. MTX induced IL-8 secretion by human bronchial and alveolar epithelial cells in a dose- and time-dependent manner within the range of the clinically observed serum concentrations. Although addition of LPS (lipopolysaccharide) and glucose showed no significant enhancing effect, addition of IL-1beta or TNF-alpha (tumour necrosis factor-alpha) with MTX to bronchial epithelial cells showed a significant augmenting effect. SB203580, the specific inhibitor of p38 MAPK, inhibited MTX-induced IL-8 production. MTX induced the phosphorylation of Thr(180) and Tyr(182) on p38 MAPK. These results suggest that MTX activates bronchial and alveolar epithelial cells to induce IL-8 production through p38 MAPK, which might play an important role as one of the mechanisms of MTX-induced lung inflammation.

Inhalation of diesel exhaust for three months affects major cytokine expression and induces bronchus-associated lymphoid tissue formation in murine lungs.

The authors investigated the effects of exposure to diesel exhaust (DE) on murine lung tissues in vivo. BALB/c and C57BL/6 mice were exposed to DE with low (100 microg/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]-alpha, interleukin [IL]-1beta, IL-4, IL-6, IL-10, IL-12p40, and interferon [IFN]-gamma) and inducible nitric oxide synthase (iNOS) in the lungs, as well as TNF-alpha, IL-1beta, IL-10, IL-12p40, and Mac-1 mRNA expression in alveolar macrophages (AMs). TNF-alpha, IL-12p40, IL-4, and IL-10 mRNA expression were mildly increased, whereas IL-1beta 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.

Diesel exhaust particles upregulate eotaxin gene expression in human bronchial epithelial cells via nuclear factor-kappa B-dependent pathway.

Fine particles derived from diesel engines, diesel exhaust particles (DEP), have been shown to augment gene expression of several inflammatory cytokines in human airway epithelial cells in vitro. However, it remains unclear whether or not DEP have any effect on the expression and production of eotaxin, an important chemokine involved in eosinophil recruitment into the airways. We studied the effects of DEP by using a conventional suspended DEP and by a recently established in vitro cell exposure system to diesel exhaust (Abe S, Takizawa H, Sugawara I, and Kudoh S, Am J Respir Cell Mol Biol 22: 296-303, 2000). DEP showed a dose-dependent stimulatory effect on eotaxin production by normal human peripheral airway epithelial cells as well as by bronchial epithelial cell line BET-1A as assessed by specific ELISA. mRNA levels increased by DEP were shown by RT-PCR. DEP showed an additive effect on IL-13-stimulated eotaxin expression. DEP induced NF-kappaB activation by EMSA as previously reported but did not induce signal transducer and activator of transcription (STAT) 6 activation according to Western blot analysis. Finally, antioxidant agents (N-acetyl cysteine and pyrrolidine dithiocarbamate), which inhibited NF-kappaB activation but failed to affect STAT6 activation, almost completely attenuated DEP-induced eotaxin production, whereas these agents failed to attenuate IL-13-induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-kappaB-dependent, but STAT6-independent, pathways.

Role of interferon-gamma in the development of murine bronchus-associated lymphoid tissues induced by silica in vivo.

Exposure to silica is associated with the development of chronic airflow obstruction as well as pulmonary fibrosis, probably mediated in part by silica-induced small airway disease. To elucidate the mechanism of mucosal immune responses in the small airways, we analyzed the roles of interferon-gamma (IFN-gamma) using mice deficient of this cytokine in silicotic lung. IFN-gamma knockout mice (-/-) and wild-type C57BL/6 mice were treated with either a single fibrogenic dose of silica or an equivalent volume of saline and euthanized 21 days after intratracheal instillation. Total cell counts in bronchoalveolar lavage fluids increased in silica-instilled mice compared to saline-instilled mice, but there were no significant differences between IFN-gamma knockout mice and wild-type mice treated with silica. Morphometric estimation for fibrotic lesions within the lung did not show any differences between these mice. However, bronchus-associated lymphoid tissues (BALT), which are known to be involved in the mucosal immune responses, were significantly larger in the lungs of IFN-gamma knockout mice than in those of wild-type mice treated with silica. In addition, we evaluated the development of BALT in interleukin 4 (IL-4) knockout mice in order to clarify the effect of Th2 cytokine. Morphometric estimation for BALT did not show any differences between IL-4 knockout mice and wild-type mice in silicotic lung. These results suggest that IFN-gamma has an inhibitory effect on the development of BALT and may be involved in small airway disease in silicotic lung.