INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 28: 993-999, 2011 Expression of interleukin-24 and its receptor in human pancreatic myofibroblasts HIROTSUGU IMAEDA1, ATSUSHI NISHIDA1, OSAMU INATOMI1, YOSHIHIDE FUJIYAMA1 and AKIRA ANDOH2 1Department of Medicine and 2Division of Mucosal Immunology, Graduate School of Medicine, Shiga University of Medical Science, Seta Tsukinowa, Otsu, Japan Received July 12, 2011; Accepted August 30, 2011 DOI: 10.3892/ijmm.2011.793 Abstract. Interleukin (IL)-24 is a member of the IL-10 family broblasts play a pivotal role in the progression of pancreatic of cytokines. In this study, we investigated IL-24 expression fibrosis (1-6). Pancreatic myofibroblasts actively proliferate, in chronic pancreatitis tissue and characterized the molecular migrate and produce large amounts of extracellular matrix mechanisms responsible for IL-24 expression in human (ECM) components such as type I collagen and fibronectin. pancreatic myofibroblasts. IL-24 expression in the tissues was In addition, these cells possess proinflammatory functions evaluated by immunohistochemical methods. IL-24 mRNA characterized by the expression of cytokines, chemokines and and protein expression in the pancreatic myofibroblasts was cell adhesion molecules (1-6). determined by real-time-PCR and ELISA, respectively. IL-24 Interleukin (IL)-24, a member of the IL-10 family of was expressed by α-smooth muscle actin-positive myofibro- cytokines (together with IL-10, -19, -20, -22, -26, -28 and -29), blasts in the chronic pancreatitis tissues. In isolated human was originally termed melanoma differentiation-associated pancreatic myofibroblasts, IL-1β significantly enhanced IL-24 protein 7 (MDA-7) (7), and was renamed IL-24 (8). The mRNA and protein expression. The p38 MAPK inhibitor human IL-24 gene is located on chromosome 1, within a SB203580 and the PI3K inhibitor LY294002 significantly 195-kb cytokine cluster containing the IL-10, IL-19, IL-20 and reduced the IL-β-induced IL-24 mRNA expression. An IL-24 genes (9). IL-24 shares a 20-30% amino acid homology adenovirus containing a dominant-negative mutant of c-Jun with IL-10, IL-20 and IL-22, and interacts with two different significantly inhibited the effects of IL-1β on IL-24 mRNA heterodimeric receptor complexes, IL-20R1/IL-20R2 and expression, indicating that the IL-1β-induced IL-24 mRNA IL-22R1/IL-20R2 (10-12). Binding to both receptors leads to expression was mediated by the activation of transcription the activation of signal transducer and activator of transcrip- factor AP-1. Pancreatic myofibroblasts expressed IL-22R1, tion 3 (STAT3) (10-13). Immune cells do not express IL-24 IL-20R1 and IL-20R2, and recombinant IL-24 induced the receptors (14), suggesting that IL-24 cannot stimulate the phosphorylation of p42/44, p38, JNK and STAT1/3. IL-24 is acquired immune response (15). In contrast, the restricted expressed in chronic pancreatitis tissue, and may play a role expression of the IL-24R components IL-20R1 and IL-22R1 to in the pathophysiology of chronic pancreatitis via autocrine non-immune tissues suggests that the innate immune response pathways. is the selective target of IL-24 (11,14). IL-24 expression has been identified in certain cell types Introduction (14,16), such as cultured melanocytes (7), dermal keratinocytes (15), lipopolysaccharide (LPS)-stimulated monocytes (16) and Fibrosis of the pancreas is one of the characteristic histo- Th2-polarized T cells (16). Furthermore, treatment with IFN-β pathologic findings in cases of chronic pancreatitis and the plus mezerein was found to induce a transient expression of desmoplastic reaction associated with pancreatic cancer. IL-24 mRNA in some cancer cell lines (9). However, the Previous studies have demonstrated that pancreatic myofi- precise molecular mechanisms of IL-24 induction still remain unclear. In the present study, we investigated IL-24 expression in chronic pancreatitis tissues. Furthermore, to investigate the Correspondence to: Dr Akira Andoh, Division of Mucosal Immu- molecular mechanisms responsible for IL-24 expression in the nology, Graduate School of Medicine, Shiga University of Medical pancreas, we analyzed IL-24 expression in human pancreatic Science, Seta Tsukinowa, Otsu 520-2192, Japan myofibroblasts. E-mail: [email protected] Materials and methods Key words: MAP-kinase, AP-1, chronic pancreatitis, interleukin-1β Reagents. Recombinant human cytokines were purchased from R&D Systems (Minneapolis, MN). Inhibitors for the p42/44 MAP-kinases (PD98059 and U0216), and an inhibitor for 994 IMAEDA et al: IL-24 AND THE PANCREAS Table I. Oligonucleotides used in this study. Gene name Primers Ref. IL-24 Sense 5'-GACTTTAGCCAGCAGACCCTT-3' 8 Antisense 5'-GGTTGCAGTTGTGACACGAT-3' IL-22R1 Sense 5'-CTGTCCGAGATCACCTACTTAGG-3' 14 Antisense 5'-GCACATTTGGGTCAGATGTTCTGTC-3' IL-20R1 Sense 5'-GCTCAGCCTTCTGAGAAGCAGTG-3' 20 Antisense 5'-CGCACAAATGTCAGTGGTTCTGAC-3' IL-20R2 Sense 5'-GCTGGTGCTCACTCACTGAAGGT-3' 20 Antisense 5'-TCTGTCTGGCTGAAGGCGCTGTA-3' Figure 1. Representative immunohistochemical staining for IL-24 and α-smooth muscle actin (α-SMA) in chronic pancreatitis tissues. IL-24 protein was clearly detected in the spindle-shaped cells (green fluorescence; left panel). Myofibroblasts were detected as α-SMA-immunopositive cells (red fluorescence; middle panel). α-SMA/IL-24 double-immunopositive cells were detected as yellow, and the α-SMA-immunopositive cells coincided with some of the IL-24- immunopositive cells (right panel). Magnification, x200. p38 MAPK (SB203580) were purchased from Cell Signaling cence using a digital confocal laser scanning microscope Technology (Beverly, MA). The procollagen type I C-peptide (LSM510 ver. 3.0; Carl Zeiss Japan, Tokyo, Japan). Standard EIA kit was purchased from Takara Bio (Otsu, Japan). immunohistochemical analyses were performed according to the methods described in our previous report (17). Antibodies. Goat anti-human IL-24 antibodies (R&D System), mouse anti-α-SMA antibodies (Sigma Chemical Co., Human pancreatic myofibroblast cultures.Primary cultures of St. Louis, MO), goat anti-JAK1 antibodies, anti-MAP kinase, pancreatic myofibroblasts were isolated from normal pancreatic and anti-STAT1 and 3 antibodies (Cell Signaling Technology), tissues obtained from patients who underwent total gastropan- goat anti-human c-Jun and c-Fos antibodies (Santa Cruz createctomies due to gastric cancer, according to methods Biotechnology, Santa Cruz, CA, USA) were purchased from previously described (4). The cells were cultured in DMEM commercial suppliers. containing 10% FBS. All culture media were supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. Over 98% Tissue samples and immunochemistry. Human pancreatic of the cells were immunopositive for α-SMA, a marker for tissues were obtained from patients with clinically diagnosed myofibroblasts. The studies were performed on passages 2-6 of chronic pancreatitis undergoing surgery for intractable back myofibroblasts isolated from 4 different resection specimens. pain, or from patients with significant suspicion of pancre- atic cancer. Normal pancreatic tissues were obtained from Quantification of human IL-24 and IL-24R.Antigenic IL-24 in patients who underwent total gastropancreatectomies due to all samples was quantified by sandwich enzyme-linked immu- gastric cancer. The study design was approved by the Ethics nosorbent assay (ELISA) kits purchased from R&D systems. Committee of the Shiga University of Medical Science. The expression of mRNA in the samples was assessed by real- Informed consent was obtained from all patients prior to time PCR analysis. The oligonucleotide primers used in this sample collection. study were described in our previous report (18). Real-time Paraffin-embedded tissues were cut into 3 µm sections. PCR was performed using a LightCycler 2.0 system (Roche Goat anti-human IL-24 antibodies and anti-human α-SMA Applied Science, Tokyo, Japan). The PCR was performed using antibodies were then applied. Cy2-labeled anti-goat rabbit a SYBR-Green PCR master mix (Applied Biosystems, Foster IgG (Chemicon, Temecula, CA) and Cy3-labeled anti-mouse City, CA). The oligonucleotide primers used in this study are donkey IgG (Millipore, Billerica, MA) were used as the shown in Table I (7,13,19). The data were normalized versus secondary antibodies. The slides were analyzed for fluores- β-actin for human IL-24. The expression of IL-24R mRNA INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 28: 993-999, 2011 995 bacterial β-galactosidase cDNA (Ad-LacZ). The dominant- negative mutant c-Jun (TAM67) lacks the transactivational domain of amino acids 3 to 122 of the wild type c-Jun (21), but retains the DNA-binding domain. The detailed procedures have been described in our previous report (22). Statistical analysis. The statistical significance of the differ- ences was determined by the Mann-Whitney U test (Statview version 4.5). Differences resulting in P-values <0.05 were considered to be statistically significant. Results IL-24 expression in chronic pancreatitis. To evaluate the expression of IL-24 mRNA in the pancreas, the tissues of chronic pancreatitis patients were double immunostained with anti-human IL-24 antibodies and anti-α-smooth muscle actin (α-SMA; a marker for myofibroblasts) antibodies (Fig. 1). IL-24 protein was clearly detected in the spindle-shaped cells (green fluorescence; Fig. 1, left panel). Myofibroblasts were detected as α-SMA-positive cells (red fluorescence; Fig. 1, middle panel). As shown in Fig. 1 (right