Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. ihsekat_job@yahoo.co.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. ihsekat_job@yahoo.co.jp.
Takeshi
T
Kobayashi
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. kensuke_piglet@ybb.ne.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. kensuke_piglet@ybb.ne.jp.
Kensuke
K
Tanaka
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. phajita0318@yahoo.co.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. phajita0318@yahoo.co.jp.
Tetsuo
T
Fujita
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. h-umepan@hotmail.co.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. h-umepan@hotmail.co.jp.
Hiroki
H
Umezawa
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. purapi71202000@yahoo.co.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. purapi71202000@yahoo.co.jp.
Hiroyuki
H
Amano
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. kent.y.mb@gmail.com.|Department of Biomedical Science, Graduate School of Medicine, Chiba University, Chiba, Japan. kent.y.mb@gmail.com.
Kento
K
Yoshioka
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. yusukenaito1980@yahoo.co.jp.|Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. yusukenaito1980@yahoo.co.jp.
Yusuke
Y
Naito
Department of Biomedical Science, Graduate School of Medicine, Chiba University, Chiba, Japan. hatanom@faculty.chiba-u.jp.
Masahiko
M
Hatano
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. s-kimura@faculty.chiba-u.jp.
Sadao
S
Kimura
Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. tatsumi@faculty.chiba-u.jp.
Koichiro
K
Tatsumi
Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. kasuya@faculty.chiba-u.jp.|Department of Biomedical Science, Graduate School of Medicine, Chiba University, Chiba, Japan. kasuya@faculty.chiba-u.jp.
Yoshitoshi
Y
Kasuya
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001992:Bronchoalveolar Lavage Fluid; D045744:Cell Line, Tumor; D006801:Humans; D015850:Interleukin-6 / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011658:Pulmonary Fibrosis / Q000378:metabolism* / Q000473:pathology*
D015850:Interleukin-6