Masashi Desaki

Molecular Mechanisms of Anti-Inflammatory Action of Erythromycin in Human Bronchial Epithelial Cells: Possible Role in the Signaling Pathway That Regulates Nuclear Factor-κB Activation

ABSTRACT 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-κB (NF-κB) in human bronchial epithelial cells. Western blotting analysis showed that EM did not inhibit the degradation of IκBα, suggesting the molecular target for EM was not the dissociation of NF-κB from IκB. An electrophoretic mobility shift assay showed that EM did not interrupt the NF-κB DNA-binding activity in the nucleus under the conditions tested. Moreover, not only EM but also EM703 suppressed the activation of NF-κB 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-κB signaling pathway in the downstream of the dissociation from IκB, resulting in the inhibition of NF-κB.

Erythromycin and clarithromycin attenuate cytokine-induced endothelin-1 expression in human bronchial epithelial cells

Erythromycin and its fourteen-member macrolide analogues have attracted attention for their efficacy in bronchial asthma. However, their mechanisms of action remain unclear. We evaluated the effects of the macrolide antibiotics on endothelin-1 (ET-1) expression in normal and transformed human bronchial epithelial cells, one of the sources of this potent bronchoconstrictor important in the pathogenesis of asthma. Human bronchial epithelial cells were obtained from the resected bronchi, and the effect of several antimicrobial and antiasthmatic drugs on the production and messenger ribonucleic acid (mRNA) levels of ET-1 was evaluated. Bronchoepithelial cells were also isolated from the mucosa of asthmatic patients under fibreoptic bronchoscopy, and the modulating effects of the drugs were studied. Erythromycin and clarithromycin uniquely suppressed mRNA levels as well as the release of ET- at therapeutic and non-cytotoxic concentrations (percentage inhibition of ET-1 protein release: 26.4+/-5.22% and 31.2+/-7.45%, respectively, at 10(-6) M). Furthermore, erythromycin and clarithromycin inhibited ET-1 expression in bronchoepithelial cells from patients with chronic, stable asthma. A glucocorticosteroid, dexamethasone, also inhibited ET-1 expression. In contrast, theophylline, salbutamol and FK506 had no effect on ET-1 production. Our findings demonstrated that these fourteen-member macrolide antibiotics had an inhibitory effect on endothelin-1 expression in human bronchial epithelial cells. Moreover, this new mode of action may have some relevance to their clinical efficacy in bronchial asthma.

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 invivo. BALB/c and C57BL/6 mice were exposed to DE with low (100 μ g/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]-1 β, 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-1 β, 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-1 β 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.

Tumor necrosis factor-α enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-β1

ABSTRACT 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.

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.

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 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.