Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis
- Takeshi Kobayashi†1, 2,
- Kensuke Tanaka†1, 2,
- Tetsuo Fujita†1, 2,
- Hiroki Umezawa†1, 2,
- Hiroyuki Amano1, 2,
- Kento Yoshioka1, 3,
- Yusuke Naito1, 2,
- Masahiko Hatano3,
- Sadao Kimura1,
- Koichiro Tatsumi2 and
- Yoshitoshi Kasuya1, 3Email author
© Kobayashi et al. 2016
Received: 10 March 2015
Accepted: 12 August 2015
Published: 20 August 2015
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.
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with an extremely poor prognosis . Likewise, epidemiological studies have demonstrated that the incidence and prevalence of IPF have been increasing in most western societies in recent years . Although there are many ongoing clinical trials of radical treatment for IPF, there is no effective pharmacological therapy to improve the survival of patients with IPF .
BLM-induced pulmonary fibrosis in mice is the most common experimental model of human IPF . Genetically modified mice subjected to bleomycin (BLM) instillation provide a useful target molecule for therapeutic intervention in IPF [5–8]. In these mice, fibrosis is closely linked to an inflammatory response in the lung. On the other hand, comprehensive gene expression analysis of BLM-induced fibrotic lung has revealed that two distinct groups of genes are involved in the inflammatory and fibrotic responses . A reciprocal relationship between lung inflammation and fibrosis has also been reported . Furthermore, most patients who present to clinicians with subjective symptoms show a reduction of forced vital capacity (FVC), indicating that fibrosis is already present . Hence, whether experimental evidence-based anti-inflammatory therapy is effective against lung fibrosis remains under debate . Recently, the concept that IPF results from alveolar epithelial cell injury with scant inflammation has been generally accepted . Many different molecular processes such as epithelial mesenchymal transition , apoptosis , endoplasmic reticulum stress , telomere shortening-associated senescence , and hypersecretion of MUC5B caused by a point mutation in the promoter region of the gene  are involved in the mechanisms of epithelial injury-based fibrosis. BLM administration can recapitulate epithelial injury-induced lung fibrosis in mice . Thus, to address the complex mechanisms of the pathophysiological events in the development of lung fibrosis, BLM is a useful tool.
IL-6 is a pleiotropic cytokine and functions as a proinflammatory factor as well as a profibrotic factor in BLM-induced lung fibrosis . Recently, besides TGF-β/Smad3 signaling, the signaling loop of IL-6/gp130/Stat3 has been shown to play a crucial role in the pathogenesis of lung fibrosis . Furthermore, blockade of the IL-6 signal during the chronic stages of lung injury shows a beneficial effect on lung fibrosis [21, 22]. In contrast, BLM-induced IL-6 has a cytoprotective effect on alveolar epithelial cells under stress with reactive oxygen species (ROS) . Likewise, the IL-6/Stat3/Akt signaling axis plays a protective role in type 2 pneumocytes by regulating surfactant homeostasis . These findings suggest the possibility that IL-6 also plays a protective role in epithelial injury-based fibrosis. However, this possibility has not been shown in vivo.
Here, we first elucidated which intracellular signal was predominantly activated at the early inflammatory stage of BLM-injured lung by a phospho-protein array and focused on the pathophysiological role of IL-6. Then, we demonstrated in vivo and in vitro that endogenous IL-6 shows a counter-effect on BLM-induced apoptosis of type 2 pneumocytes in an autocrine/paracrine manner through activation of the Stat3/Akt signaling axis. In the bronchoalveolar space, induction of IL-6 by BLM was characterized by a biphasic response. Neutralization of IL-6 at the early fibrotic stage of BLM-induced lung injury significantly ameliorated lung fibrosis. However, it is noteworthy that neutralization of IL-6 at the early inflammatory stage of BLM-induced lung injury accelerated the development of lung fibrosis.
Male C57BL/6 mice at 10 weeks of age were purchased from Clea Japan (Tokyo, Japan). Animals were housed in the Animal Resource Facility of Chiba University under pathogen-free conditions and cared for according to the animal care guidelines of Chiba University. The studies were performed according to an animal protocol approved by the Animal Welfare Committee of Chiba University.
BLM-induced lung injury model
Mice under anesthesia with isoflurane inhalation were given a single intratracheal injection of BLM hydrochloride (3 mg/kg; Nippon Kayaku, Tokyo, Japan) dissolved in phosphate-buffered saline (PBS), using a Microsprayer® atomizer (PennCentury, Philadelphia, PA). Control mice received sham treatment with PBS.
At 3 days post-instillation (dpi), mice under anesthesia were intracardially perfused with ice-cold PBS to thoroughly wash out blood cells in the lungs and sacrificed. Lung lobes separated from the trachea and the main bronchi were homogenized in ice-cold lysis buffer  and centrifuged at 9000 × g for 15 min at 4 °C. The resulting supernatant (400 μg protein) was analyzed using a Pathscan Antibody Array kit (Cell Signaling Technology, Danvers, MA). Detection of phosphorylation of 39 proteins in the lung sample was performed according to the manufacturer’s protocol. Using a densitometer, each signal was normalized to the positive internal control included in the array membrane and expressed in arbitrary units.
Mice were sacrificed at 3 or 8 dpi, and the lung lobes were fixed, dehydrated and frozen. Freshly cut lung sections (5 μm thickness) placed on poly-L-lysine-coated slides were pretreated with 1:10 FcR blocking agent (Miltenyi Biotech, Gladbach, Germany) for 10 min and reacted with various antibodies as follows: goat anti-prosurfactant protein (proSP)-C antibody (Santa Cruz Biotech., Santa Cruz, CA), rabbit anti-phospho-Stat3 (Cell Signaling Technology), rabbit anti-phospho-Akt antibody (Cell Signaling Technology), rabbit anti-Iba 1 antibody (WAKO, Osaka, Japan), rat anti-mouse IL-6 antibody (Biolegend, San Diego, CA), rabbit anti-S100A4 antibody (Abcam, Cambridge, UK) or mouse anti-smooth muscle α-actin/SMA (Sigma-Aldrich, St. Louis, MO). After staining with each appropriate fluorescein-conjugated second antibody, the sections were observed under a fluorescence microscope (Axio Imager A2, Zeiss, Oberkochen, Germany). Nuclei were stained with 4’6’-diamino-2-phenylindole (DAPI).
Effect of IL-6 signal on BLM-induced apoptosis in A549 cells and primary cultured alveolar epithelial cells
A human lung adenocarcinoma epithelial cell line, A549, was purchased from European Collection of Cell Cultures (Salisbury, UK). Primary cultured alveolar epithelial cells were prepared from mice according to a method previously described with modifications . The primary cultured cells were characterized by immunofluorescence study with rabbit anti-proSP-C antibody  and hamster anti-podoplanin/gp36 antibody (Abcam) in combination with Alexa Fluor 488-chicken anti-rabbit IgG and Alexa Fluor 594-goat anti-hamster IgG (Life Technologies, Carlsbad, CA). Then, SP-C+gp36− cells (purity > 90 %) were used as alveolar epithelial cells for the following experiments.
After 24 h of serum deprivation in Dulbecco's Modified Eagle's Medium (DMEM) (WAKO) containing 0.2 % fatty acid-free BSA (Sigma-Aldrich), A549 cells and primary cultured alveolar epithelial cells on 24-well plates were stimulated with BLM (100 μg/ml) for 12 h. The cell lysates from A549 and primary cultured alveolar epithelial cells were subjected to western blot (WB) with anti-human cleaved caspase-8 (Asp391) antibody (Cell Signaling Technology) and anti-cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology), respectively. To investigate the effects of human IL-6 (3 and 10 ng/ml, Peprotech, Rocky Hill, NJ), human IL-6-neutralizing antibody (2 μg/ml, clone MQ2-13A5, Biolegend), mouse IL-6-neutralizing antibody (2 μg/ml, clone MP5-20 F3, Biolegend), rat IgG1κ isotype control for IL-6-neutralizing antibodies (2 μg/ml, Biolegend), LY294002 (a PI3K inhibitor, 1 μM, Cell Signaling Technology) and S3I-201 (a Stat3 inhibitor, 1 μM, Sigma-Aldrich), each agent was added to the culture medium 30 min prior to BLM stimulation.
Effect of IL-6 signal on BLM-induced cytokine expression in primary cultured alveolar epithelial cells
Primary cultured alveolar epithelial cells on 12-well plates were stimulated with BLM (100 μg/ml) for 12 h in the presence of mouse IL-6-neutralizing antibody (2 μg/ml) or isotype control. Then, supernatants were collected and centrifuged at 400 × g for 15 min at 4 °C. The resulting supernatants were subjected to Mouse Cytokine Antibody Array C1 (RayBiotech, Inc., Norcross, GA) and changes in expression levels of 22 inflammation-related proteins in the supernatant sample were evaluated. The array was performed according to the manufacturer’s instructions.
Measurement of IL-6 level in bronchoalveolar lavage fluid
Mice with instillation of BLM were anesthetized and sacrificed on days 0, 0.5, 1, 2, 3, 5, 7, 8, 9, 10 and 11. Then, the lungs of each mouse were lavaged with 1 ml ice-cold PBS twice via the exposed trachea cannulated with a 20-gauge catheter. Collected bronchoalveolar lavage fluid (BALF) was centrifuged at 400 x g for 10 min. The resulting supernatant was subjected to IL-6 measurement using a mouse IL-6 ELISA MAX™ Standard (Biolegend), according to the manufacturer’s protocol.
Effects of IL-6-neutralizing antibody on BLM-induced lung injury
Mouse IL-6-neutralizing antibody was intratracheally injected in BLM-instilled mice using a Microsprayer® atomizer. IL-6-neutralizing antibody was administered at 15 μg/body, and isotype control at 15 μg/body, for each injection.
For neutralization of IL-6 at the early inflammatory stage of BLM-induced lung injury, IL-6-neutralizing antibody was administered at 6, 30 and 54 h (0, 1 and 2 dpi) after instillation of BLM. Control mice received treatment with isotype control. Then, examination of apoptosis of type 2 pneumocytes, analysis of cell populations in BALF and histopathological study were performed. For examination of apoptosis, mice were anesthetized, sacrificed and intracardially perfused with PBS at 3 dpi, and the lungs were lavaged twice with 1 ml PBS via the exposed trachea cannulated with a 20-gauge catheter. Then, 1 ml PBS containing YO-PRO-1 (1:100, Life Technologies, Carlsbad, CA) and propidium iodide (0.5 μM, Life Technologies) was injected into the bronchoalveolar space through the catheter. After 15 min, the lungs were lavaged four times with 1 ml ice-cold PBS, and the bronchoalveolar space was filled with 4 % PFA. The lungs were dissected out, further fixed, frozen and sectioned. The population of apoptotic and necrotic cells was examined under a fluorescence microscope, and type 2 pneumocytes were confirmed by observing the phase-contrast of the same visual field. Then, the apoptotic or necrotic signal, corresponding to type 2 pneumocytes, was estimated. For evaluation of infiltrated cells, mice under anesthesia were sacrificed at 7 dpi, and the lungs of each mouse were lavaged with 1 ml ice-cold PBS twice via the exposed trachea cannulated with a 20-gauge catheter. After centrifugation of BALF, collected total cell count was measured using a hemocytometer. The differential cell count was determined by manually counting 200 cells/mouse after staining with Diff-Quick (Sysmex Co., Kobe, Japan). For histopathological studies, mice under anesthesia were sacrificed at 7 and 14 dpi. The lungs were perfused, dissected out, fixed, sectioned and stained with Masson’s trichrome to visualize fibrotic lesions. Semi-quantitative elucidation of lung fibrotic changes was performed according to the previously described method with a slight modification . In brief, longitudinal sections along the central axis of the right lung (apical, azygous and diaphragmatic lobes) and the left lung were prepared, and one section from each lung was randomly selected (2 sections/mouse). Under 200x magnification, 10 fields in each section were randomly chosen, scored, and the average score was calculated.
For neutralization of IL-6 at the early fibrotic stage of BLM-induced lung injury, IL-6-neutralizing antibody was administered at 8, 9 and 10 days after instillation of BLM. Control mice received treatment with isotype control. Then, changes in body weight and survival rate were monitored until 14 dpi. At 14 dpi, mice were sacrificed under anesthesia, and the fibrotic changes in lung sections were visualized with Masson’s trichrome staining and scored according to the method described above.
Data are expressed as mean ± S.E.M. Statistical analysis was conducted using Graphpad Prism Version 6 (GraphPad Software Inc., San Diego, CA). Statistical significance was determined by Student’s t test or analysis of variance (ANOVA) followed by the Tukey’s test, and p values < 0.05 were considered significant.
Activation of IL-6 signal in type 2 pneumocytes at early inflammatory stage of BLM-induced lung injury
IL-6 has counter effect on BLM-induced cell death of type 2 pneumocytes in vitro
Endogenous IL-6 modulates BLM-induced cytokine production by type 2 pneumocytes
Time-dependent induction of IL-6 in BLM-instilled lung
Blockade of IL-6 at early inflammatory stage of BLM-induced lung injury accelerates lung fibrosis
Blockade of IL-6 at early fibrotic stage of BLM-induced lung injury ameliorates lung fibrosis
Comprehensive profiling of mRNA and protein expression using microarray and WB array technology is useful for evaluating the complex mechanisms of pathophysiological events and to define a novel therapeutic target in a certain disease [5, 9, 34, 35]. In addition to these analytical options, WB array for phospho-proteins is a useful tool to determine which intracellular signal is predominantly activated under a certain condition . Based on the results of phospho-protein array, we focused on Akt and Stat3, downstream signaling molecules of the IL-6 receptor, for the following reasons: 1) IL-6 induced in a variety of acute and chronic inflammatory diseases plays a major role as a trigger for acute-phase protein synthesis . 2) In pulmonary inflammatory diseases, bipotential functions of IL-6 in inflammation were shown using IL-6−/− mice. IL-6 can mediate persistent inflammation and subsequent fibrotic changes in the lung . On the other hand, IL-6 contributes to host defense against pneumococcal pneumonia through downregulating activation of the cytokine network in the lung . 3) During the fibrotic stage of BLM-induced lung injury, the IL-6/Stat3 signaling axis promotes lung fibrosis [21, 22]. However, radical oxygen species (ROS) mimicking cytotoxicity of BLM induce apoptosis of several types of cells including type II alveolar epithelial cells in organotypic lung slices, which was marked in IL-6−/− slices and wild-type (WT) slices treated with IL-6-neutralizing antibody compared with the case of WT slices treated with isotype control . This finding tempts us to consider that IL-6 may also play an antifibrotic role in epithelial injury-based fibrosis. Hence, temporal and spatial differences in IL-6-acting site might affect the pathophysiological outcome in the BLM-injured lung. Indeed, the concept that the role of IL-6 signaling may differ between acute and chronic stages of lung disease is suggested . However, whether IL-6 modulates epithelial injury-based fibrosis in the BLM-instilled mouse model has still not been clarified.
At the early inflammatory stage of BLM-induced lung injury, immunofluorescence studies revealed the predominantly IL-6-acting site and IL-6-producing cell. The phosphorylated forms of Akt and Stat3 were mostly restricted to type 2 pneumocytes, indicating that type 2 pneumocytes respond as the predominantly IL-6-acting site. The phosphorylated form of Stat3 was detected in SP-C+ cells even in the PBS-treated lung, which is similar to the result of phospho-protein array showing a clear signal of phospho-Stat3 in the homogenate of PBS-treated lung, suggesting that intrinsic activity of Stat3 may be relatively high in type 2 pneumocytes. On the other hand, type 2 pneumocytes also function as the predominant IL-6- producing cells when activation of Stat3 and Akt was detected in type 2 pneumocytes. Although macrophages could also produce IL-6, their contribution was less than that of type 2 pneumocytes. This finding is supported by a previous report that IL-6 secretion from type 2 pneumocytes is markedly higher than that from alveolar macrophages . These results suggest the possibility that IL-6 induced by BLM affects the cell fate and function of type 2 pneumocytes in an autocrine/paracrine manner, at least in the early inflammatory phase. Then, we examined this possibility using cultured cells.
In both A549 cells and primary cultured type 2 pneumocytes, application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor augmented BLM-induced production of cleaved caspases. These results clearly suggest that BLM mobilizes an apoptotic signal in type 2 pneumocytes and simultaneously induces IL-6 production as a compensatory mechanism. Likewise, the IL-6/PI3K/Akt and IL-6/ Stat3 signaling axes can protect alveolar epithelial cells from BLM-induced cell death. This notion is supported by previous reports as follows: blockade of the PI3K/Akt pathway potentiates apoptosis induced by a cyclin-dependent kinase inhibitor in A549 cells ; keratinocyte growth factor can inhibit Fas-mediated apoptosis of A549 cells through activation of the PI3K/Akt pathway ; Stat3 in type 2 pneumocytes possibly contributes to alveolar epithelial cell survival and surfactant/lipid synthesis, which are necessary for protection of the lung during injury ; and the IL-6/Stat3/Akt signaling axis plays a protective role in type 2 pneumocytes . Hence, Akt activated by IL-6 may play a cytoprotective role in concert with Stat3 in type 2 pneumocytes of the lung instilled with BLM. We have confirmed that activation of Akt and Stat3 is induced by BLM in alveolar type II cells within a few hours, which is relatively faster than the time course of BLM-induced IL-6 synthesis/release (data not shown). This finding indicates that IL-6-independent and BLM-induced PI3K/Akt and Stat3 activation also exist in alveolar type II cells and can explain the finding that the apoptotic signal augmented by a PI3K inhibitor or a Stat3 inhibitor was stronger than that augmented by IL-6-neutralizing antibody. However, the effect of IL-6-neutralizing antibody suggests that at least IL-6-dependent Akt and Stat3 activation can function as a survival signal in BLM-treated type 2 pneumocytes in an autocrine/paracrine manner. Moreover, application of IL-6-neutralizing antibody to type 2 pneumocytes also affected their cytokine-producing activity under stimulation with BLM. The BLM-induced production of several cytokines such as GM-CSF, IL-2, IL-9, IL-13, MCP-1 and THPO was modulated by endogenous IL-6 in type 2 pneumocytes. GM-CSF and IL-9 play a protective role against BLM-induced lung fibrosis through a prostaglandin-dependent mechanism [43, 44]. In contrast, IL-13 and MCP-1 contribute to the development of BLM-induced lung fibrosis [45, 46]. On the other hand, IL-2 is shown to be involved not in fibrosis but in lymphocytic infiltration in the lung instilled with BLM . THPO has not been shown to be related to lung injury, but plays a protective role in liver fibrosis . Hence, the stimulatory effect of IL-6 on expression of antifibrotic cytokines, IL-9 and THPO, and the inhibitory effect of IL-6 on expression of proinflammatory and profibrotic cytokines, IL-2 and IL-13, may improve the microenvironment in the lung exposed to BLM. In conjunction with the cytoprotective activity of IL-6, at least IL-6 was upregulated predominantly in type 2 pneumocytes at the early inflammatory stage, and may exert counter effects on the development of BLM-induced lung injury. In the BLM-instilled lung, IL-6 was induced at both the early inflammatory stage and early fibrotic stage. Then, we further investigated how blockade of IL-6 at each stage affects the pathophysiological outcome of BLM-induced lung injury.
According to our expectation, blockade of IL-6 at the early inflammatory stage of BLM-induced lung injury enhanced apoptosis of type 2 pneumocytes. In addition, an increase in population of neutrophils in BALF from BLM-instilled mice was also enhanced by IL-6-neutralizing antibody but not isotype control. Although a precise evaluation of the inflammatory cell populations in the lung parenchyma contributing to the severity of fibrosis is needed , the increase in neutrophils in BALF suggests that blockade of IL-6 at the early inflammatory stage of BLM-induced lung injury at least enhances lung inflammation. Furthremore, even at the transitional period from the inflammatory phase to the fibrotic phase, the application of IL-6-neutralizing antibody to the lung instilled with BLM resulted in obvious fibrosis. The exact reason why blockade of IL-6 at the early inflammatory stage of BLM-induced lung injury enhanced fibrotic formation at 7 dpi but not 14 dpi is unclear in the present study. At 14 dpi, alveoli nearly obliterated with fibrous masses that are Grade 7 by Ashcroft score were observed more frequently in BLM + anti-IL-6 group than both BLM and BLM + control IgG groups. Hence, histopathological observation or biochemical analysis more than 14 days may be needed. In the normal alveoli of most patients with IPF, numerous type 2 pneumocytes actively undergo programmed cell death, suggesting epithelial injury-based mechanisms of lung fibrosis . This concept has been clearly validated by the finding that induction of type 2 pneumocyte-specific cell death by a genetically engineered method leads to pulmonary fibrosis . Taking these findings together, IL-6 at the early inflammatory stage of BLM-induced lung injury functions as an inhibitory factor in the epithelial injury-based mechanisms of lung fibrosis. The question arose as to whether blockade of the protective role of IL-6 could affect BLM-induced TGF-β1 expression because a correlation between IL-6 signal and TGF-β1 expression has been shown in BLM-challenged mice . It is well known that TGF-β/smad3 signaling can stimulate fibroblast differentiation and epithelial mesenchymal transition and that mouse with Smad3-deficiency shows resistance to BLM-induced lung fibrosis . Moreover, a genetic inhibition of TGF-β/TβRII signaling axis in alveolar type II cells limits BLM-induced fibrogenesis by increasing fibroblast apoptosis . Thus, TGF-β1 is closely associated with epithelial cell fate and subsequent fibrotic formation. Blockade of IL-6 at the early inflammatory stage of BLM-induced lung injury significantly enhanced BLM-induced TGF-β1 mRNA expression at 7 dpi (Additional file 2: Figure S2). Although the precise mechanism of this finding is still unclear, the upregulation of BLM-induced TGF-β1 may partly contribute to obvious fibrotic formation manifested by blocking IL-6 at the early inflammatory stage of BLM-induced lung injury.
In contrast to the case of blocking the first peak of IL-6, blockade of IL-6 at the early fibrotic stage of BLM-induced lung injury improved both the body weight loss and the survival rate of mice. Likewise, BLM-induced lung fibrosis was significantly inhibited by IL-6-neutralizing antibody at 14 dpi. The possibility that blockade of IL-6 at the early fibrotic stage of BLM-induced lung injury simply delayed the onset of BLM-induced fibrotic formation remains. In a preliminary study, however, severe fibrosis induced by BLM was not observed up to 40 dpi in mice administered with IL-6-neutralizing antibody. These results suggest that IL-6 positively contributes to lung fibrosis under a fibrosis-establishing state. This finding showed good agreement with previous reports that lung fibrosis is ameliorated by genetic or pharmacologic blockade of IL-6 [19, 21]. In addition, the abundant localization of phospho-Stat3 in fibrotic areas at the early fibrotic stage of BLM-induced lung injury was in accord with previous reports on lung sections from both mice with lung fibrosis and patients with IPF [20, 22]. It has been clearly shown that IL-6/gp130/Stat3 signaling axis in lung fibroblasts derived from IPF patients enhances the resistance to apoptosis by upregulating Bcl-2 expression . Likewise, viral delivery of oncostatin M, one of IL-6 family members sharing gp130-signaling subunit, to the lung induced severe fibrosis associated with Stat3 activation in a TGF-β/smad3-independent manner [20, 53]. Hence, localization of activated Stat3 in fibrotic area may be one of pathophysiological indices in IL-6 family-mediated fibrogenesis. At the early fibrotic stage of BLM-induced lung injury, the IL-6-producing cells were mainly macrophages and fibroblasts but not type 2 pneumocytes, and phospho-Stat3 was not observed in type 2 pneumocyte. Thus, an autocrine/paracrine loop of IL-6 signaling was not observed in type 2 pneumocytes. The changes in IL-6-producing and -acting cells between different injury stages may at least support a bidirectional role of IL-6 in BLM-induced lung fibrosis.
Targeting IL-6 is a rational approach to various autoimmune and chronic inflammatory diseases . Likewise, the IL-6/gp130/Stat3 signaling axis is expected to be a new therapeutic target in IPF [20, 22]. Furthermore, neutralization of IL-6, especially at the fibrotic stage of lung injury, significantly inhibits the progression of lung fibrosis . Thus, an anti-IL-6 strategy may be beneficial for IPF patients. However, recent clinical case reports presented patients with established rheumatoid arthritis (RA) with RA-associated interstitial lung disease (ILD) treated with tocilizumab, an anti-IL-6 receptor monoclonal antibody, who had an acute exacerbation of interstitial infiltrates or pulmonary fibrosis [55, 56]. Based on the accumulating information on clinical cases, the effects of an anti-IL-6 strategy on ILD should be observed carefully.
In the present study, we clearly demonstrated that the role of IL-6 signaling could differ between the inflammatory and fibrotic stages of BLM-induced lung injury. In particular, blockade of IL-6 at the early inflammatory stage of BLM-induced lung injury can lead to apoptosis and functional change of type 2 pneumocytes and accelerate lung fibrotic formation partly by regulating TGF-β1 expression. BLM eventually effectively induces lung fibrosis, suggesting that the protective role of IL-6 as a compensatory mechanism cannot intrinsically overcome BLM-induced lung fibrosis and is masked in the final pathological outcome. Considering an anti-IL-6 strategy against lung inflammatory disease, however, this compensatory mechanism could be a crucial element in management of the disease.
This work was supported in part by Grants-in-Aid for Scientific Research ((B), 24390137 to Y.K.) and for Challenging Exploratory Research (25670256 to Y.K.), and by the Takeda Science Foundation for Visionary Research (to Y.K.). We thank Dr. Wendy Gray for editing our manuscript.
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