IL-13 promotes the proliferation of rat pancreatic stellate cells through the suppression of NF-κB/TGF-β1 pathway

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Abstract

In chronic pancreatitis, pancreatic stellate cells (PSCs) play a central role in tissue fibrogenesis. Transforming growth factor β1 (TGF-β1) and the Th2 lymphokines such as interleukin (IL)-13 are major profibrogenic cytokines in many organs. Activated PSCs produce various inflammatory cytokines including TGF-β1. In this study, we investigated whether IL-13 affects pancreatic fibrogenesis by modulating the functions of PSCs. IL-13 promoted PSCs proliferation without activation through the suppression of autocrine TGF-β1. IL-13 enhanced Stat6 phosphorylation in PSCs but Stat6 was not involved in the suppression of TGF-β1. IL-13 inhibited the transcriptional activity of NF-κB, and the expression of mutant I-κB reproduced the suppression of autocrine TGF-β1 and promoted PSCs proliferation. Taken together, we demonstrated that IL-13 promotes PSCs proliferation through the suppression of the transcriptional activity of NF-κB, resulting in the decrease of autocrine TGF-β1. This finding provides an unequivocal evidence of IL-13 participation in pancreatic fibrosis, illustrating a new strategy for chronic pancreatitis.

Introduction

Tissue fibrosis is a well-documented consequence of Th2 cytokine-dominated inflammatory responses. Th2 inflammation plays a central role in the pathogenesis of a variety of other fibrotic disorders including systemic sclerosis, idiopathic pulmonary fibrosis, asthma, hepatic fibrosis and ulcerative colitis [1], [2], [3], [4], [5]. This is probably because Th2 cytokines induce monocytes and macrophages to produce TGF-β1, a potent regulator of extracellular matrix formation and tissue remodeling in vivo[6]. IL-13, secreted predominantly by activated Th2 cells, has been proposed as one of the central mediators in Th2 cytokine pathologies [7]. IL-13 was also reported to promote organ fibrosis by increasing the TGF-β1 expression levels [8], [9] and has been recognized as a profibrotic cytokine in liver fibrosis induced by schistosomiasis as well as in pulmonary fibrosis caused by asthma via allergic mechanisms [7]. Furthermore, IL-13 induces B lymphocytes to produce IgE, and this mechanism (termed class switching) causes the onset of asthma and allergic inflammatory changes.

The receptor system for IL-13 is composed of IL-4Rα and IL-13Rα1. IL-13 binds to IL-13Rα1, heterodimerizing with IL-4Rα, thus leading to the activation of the signal transducer and activator of transcription protein 6 (Stat6) signaling pathway. Both receptors are expressed in fibroblasts [7], [10]. IL-4 shares the same receptor system and intracellular signal transduction pathway with IL-13 [10].

The main pathological feature of chronic pancreatitis is tissue fibrosis caused by sustained pancreatic inflammation. Recent studies have identified, isolated and characterized pancreatic stellate cells (PSCs) [11]. These cells play a central role in pancreatic fibrogenesis. In a stable, non-inflammatory state of the pancreas, PSCs are quiescent and are located in peri-acinar region. After the onset of inflammation, PSCs are rapidly activated and transform into myofibroblast-like cells. Rapidly proliferating activated PSCs produce various inflammatory cytokines including IL-1β, IL-6, and TGF-β1 and migrate to the injured area of pancreas, resulting in the expansion of pancreatic fibrosis [12], [13], [14]. TGF-β1 is one of the major profibrogenic cytokines associated with organ fibrosis, and it is well known to promote pancreatic fibrogenesis [15].

As a model system to study the involvement of IL-13 in pancreatic fibrosis, we used rat cultured PSCs and examined whether IL-13 affects pancreatic fibrogenesis by modulating the function of PSCs, which play a central role in the pathogenesis of chronic pancreatitis. In the present study, we examined the expressions of IL-13 in the pancreas and the various effects of IL-13 on culture-activated PSCs in order to elucidate its function. Clarifying the molecular mechanisms of the lymphokine that regulates PSCs will therefore enable us to develop a new therapeutic strategy for the treatment of pancreatic fibrosis.

Section snippets

Materials and methods

Materials. Recombinant rat IL-13 and anti-Stat6 antibody were purchased from R&D Systems (Abington, UK). Pronase, Nycodenz, and anti-α-SMA antibody were from Sigma (St. Louis, MO). DNase-1 was from Roche (Basel, Switzerland). Collagenase P was from Boehringer Mannheim (Mannheim, Germany). Anti-α-tubulin antibody was from Santa Cruz (Santa Cruz, CA) and anti-phospho-Stat6 (Y641) antibody was from Upstate (Billerica, MA). Horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG,

IL-13 promotes the proliferation of PSCs without activation

We first tried to elucidate the expressions of IL-13 in human and rat pancreas. Immunohistochemistry study was carried out using anti-IL-13 antibody on normal pancreas and chronic pancreatitis tissues. Although normal human and rat pancreas did not show any IL-13 signals, a high degree of IL-13 signals were detected in human and rat chronic pancreatitis tissues, corresponding to the fibrotic region indicated by azan staining (Supplementary Fig. S1). We presumed that PSCs might secrete IL-13

Discussion

In this study, we demonstrated that IL-13 promotes PSCs proliferation through the reduction of NF-κB activity and the suppression of autocrine TGF-β1.

In 1989, IL-13 was first reported as “p600” produced by Th2 lymphocytes. Inflammation is primarily modulated by Th1 cytokines, such as interferon-γ or IL-12, and fibrosis is mainly controlled by Th2 cytokines, such as IL-13 or IL-4 [7]. IL-13 shares many functional activities with IL-4 because both cytokines use the same receptor component

Conflicts of Interest

All of the authors disclose no conflicts.

Acknowledgments

The authors thank Ms. Naoko Sugaya and Ms. Keiko Sasaki for their excellent technical assistances. Contract grant sponsor: Grants-in-Aid from Ministry of Education, Culture, Sports, Science and Technology of Japan.

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