Abstract
BackgroundVarious 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.MethodsMice 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.ResultsPhospho-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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0261-z) contains supplementary material, which is available to authorized users.
Highlights
Various signals are known to participate in the pathogenesis of lung fibrosis
Many different molecular processes such as epithelial mesenchymal transition [14], apoptosis [15], endoplasmic reticulum stress [16], telomere shortening-associated senescence [17], and hypersecretion of MUC5B caused by a point mutation in the promoter region of the gene [18] are involved in the mechanisms of epithelial injury-based fibrosis
Activation of IL-6 signal in type 2 pneumocytes at early inflammatory stage of BLM-induced lung injury It is well known that BLM-induced lung injury is composed of two phases, an inflammatory phase characterized by
Summary
Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis. BLM-induced pulmonary fibrosis in mice is the most common experimental model of human IPF [4]. Modified mice subjected to bleomycin (BLM) instillation provide a useful target molecule for therapeutic intervention in IPF [5,6,7,8]. In these mice, fibrosis is closely linked to an inflammatory response in the lung. BLM administration can recapitulate epithelial injury-induced lung fibrosis in mice [4].
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