Kobayashi Takeshi, Tanaka Kensuke, Fujita Tetsuo, Umezawa Hiroki, Amano Hiroyuki, Yoshioka Kento, Naito Yusuke, Hatano Masahiko, Kimura Sadao, Tatsumi Koichiro, Kasuya Yoshitoshi
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.
Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Respir Res. 2015 Aug 20;16(1):99. doi: 10.1186/s12931-015-0261-z.
Various signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis.
Mice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated.
Phospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis.
The present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.
已知多种信号参与肺纤维化的发病机制。我们的目的是确定在早期炎症阶段主要被激活且随后调节肺纤维化发展的信号。
小鼠接受单次剂量3毫克/千克体重的博来霉素（BLM），并在滴注后指定天数（dpi）处死。分别对处于早期炎症阶段的小鼠的肺匀浆和切片进行磷酸化蛋白阵列分析和免疫荧光研究。对小鼠的支气管肺泡灌洗液（BALF）进行白细胞介素（IL）-6的酶联免疫吸附测定（EIA）以及浸润细胞群体的评估。阐明内源性和外源性IL-6对BLM诱导的A549细胞和Ⅱ型肺细胞凋亡信号的影响。此外，评估IL-6中和抗体对BLM诱导的肺损伤的作用。
磷酸化蛋白阵列显示，在3 dpi时，BLM诱导肺中IL-6受体下游分子如Stat3和Akt的磷酸化。在3 dpi时，免疫荧光研究表明磷酸化Stat3和-Akt信号定位于Ⅱ型肺细胞，并且BLM诱导的IL-6样免疫反应主要在Ⅱ型肺细胞中观察到。应用IL-6中和抗体、PI3K抑制剂或Stat3抑制剂可增强BLM处理的A549细胞和Ⅱ型肺细胞中半胱天冬酶的激活。EIA显示，BLM诱导的BALF中的IL-6呈双相性，第一次升高发生在0.5至3 dpi，随后第二次升高发生在8至10 dpi。用IL-6中和抗体阻断IL-6的第一次升高可增强Ⅱ型肺细胞的凋亡和中性粒细胞浸润，并显著加速肺纤维化。相反，用IL-6中和抗体阻断IL-6的第二次升高可改善肺纤维化。
本研究表明IL-6在肺纤维化发病机制中可发挥双向作用。特别是，在BLM损伤肺的早期炎症阶段，IL-6的上调通过以自分泌/旁分泌方式调节Ⅱ型肺细胞的细胞命运而具有抗纤维化活性。
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