Tohoku J. Exp. Med., 2009, 217, 175-184Fas/FasL-Mediated Cytotoxicity in Pancreatitis 175

Toll-Like Receptor 3 Signaling Induces Chronic Pancreatitis through the Fas/-Mediated Cytotoxicity

1 1 1 1 YOSHIKO SOGA, HIROAKI KOMORI, TATSUHIKO MIYAZAKI, NORIMASA ARITA, 1 1 2 2 3 MIHO TERADA, KAZUO KAMADA, YUKI TANAKA, TAKAHIRO FUJINO, YOICHI HIASA, 3 3 1 BUNZO MATSUURA, MORIKAZU ONJI and MASATO NOSE

1Department of Pathogenomics, Ehime University Graduate School of Medicine, Ehime, Japan 2Integrated Center for Sciences, Ehime University, Ehime, Japan 3Department of Gastroenteology and Metabology, Ehime University Graduate School of Medicine, Ehime, Japan

Innate immunity plays important roles in host defense against pathogens, but may also contribute to the development of autoimmune diseases under certain conditions. Toll-like receptors (TLRs) recognize various pathogens and induce innate immunity. We herein present a mouse model for chronic pancreatitis, which was induced by TLR3 signaling that generated the Fas/Fas ligand (FasL)-mediated cytotoxicity. An analogue of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C), which is recognized by TLR3, was injected into autoimmune-prone strains: MRL/Mp mice (MRL/+), MRL/Mp mice with a deficit of Fas (MRL/lpr) and MRL/Mp mice with a deficit of functional FasL (MRL/gld). The pancreatitis in MRL/+ mice was initiated by the destruction of pancreatic ductules, and its severity was significantly higher than that in MRL/lpr mice or MRL/gld mice. Using a pancreatic duct epithelial cell line MRL/S-1 newly established from the MRL/gld mouse that lacks FasL, we showed that treatment with poly I:C significantly induced the expression of Fas on the cultured cells. MRL/S-1 cells were destructed when co-cultured with splenocytes bearing intact FasL prepared from MRL/+ or MRL/lpr mice, but the magnitude of cytotoxicity was smaller with splenocytes of MRL/gld mice. Likewise, synthetic FasL showed cytotoxicity on MRL/S-1 cells. Furthermore, MRL/S-1 cells expressed higher levels of after the treatment with poly I:C, suggesting that the poly I:C-mediated induction of chemokines may be responsible for recruitment of lymphoid cells to the pancreatic periductular regions. These findings indicate that TLR3 signaling generates the Fas/FasL-mediated cytotoxicity, thereby leading to the development of chronic pancreatitis. ──── poly I:C; MRL/lpr; MRL/gld; innate immunity; pancreatic duct epithelial cell. Tohoku J. Exp. Med., 2009, 217 (3), 175-184. © 2009 Tohoku University Medical Press

Genetic and environmental factors are responsible for such as / and dendritic cells, fol- the development of autoimmune diseases. Environmental lowed by inducing immunomodulation, such as the activa- factors have a potential to induce loss of tolerance during tion of macrophages, NK and B cells and the production of central and peripheral differentiation of the adaptive and chemokines (Alexopoulou et al. 2001; immune response, followed by uncontrolled activation of Matsukura et al. 2006). In the previous study, poly I:C self-reactive B and T cells inducing autoimmunity and tis- injection to MRL/Mp mice generated chronic pancreatitis sue injury, which may be assisted by the cells of the innate (Qu et al. 2002). It has been shown that an RNA virus, immune system (Wen and Wong 2005). Toll-like receptors Coxackievirus B4, which is a prevalent human pathogen (TLRs) play important roles in innate immunity, thus lead- that is associated with pancreatits, autoimmune diabetes and ing to intracellular signaling to macrophages and dendritic myocarditis, induces TLR3 signaling (Richer et al. 2009). cells after the stimulation with various pathogens (Takeda Thus, TLR3 signaling contributes to the development of and Akira 2005). The inappropriate activation of TLR path- progressive inflammatory diseases. ways by these ligands may lead to the initiation and/or pro- In this study, we focused on analyzing the pathogenesis gression of autoimmune responses and tissue injury. of chronic pancreatitis induced by TLR3 signaling using an TLR3 recognizes viral double-stranded RNA (dsRNA) MRL/Mp mouse model. MRL/Mp mice spontaneously (Alexopoulou et al. 2001). Polyinosinic:polycytidylic acid develop chronic pancreatitis at an older age, possibly due to (poly I:C) is a synthetic dsRNA (Field et al. 1967) and rec- an autoimmune mechanism, since the pancreatitis was suc- ognized by TLR3 expressed on antigen-presenting cells cessfully transferred to younger mice by the injection of Received December 30, 2008; revision accepted for publication January 28, 2009. Correspondence: Masato Nose, MD, PhD, Department of Pathogenomics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan. e-mail: [email protected]

175 176 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 177 splenocytes from the older mice (Kanno et al. 1992), and gation and/or infiltration in the periductular regions without any poly I:C injection into younger MRL/Mp mice accelerates parenchymal destruction; grade 2+, focal parenchymal destruction the development of pancreatitis (Qu et al. 2002). Pancreatitis with mononuclear cell infiltration into ductular epithelium; grade 3+, in these mice is histopathologically characterized by the diffuse parenchymal destruction with granulomatous lesions and/or severe infiltration of lymphocytes, plasma cells and macro- the replacement of acinar tissues with fibrosis or adipose tissues (Fig. phages into the pancreatic parenchyma. Such lesions are 1). More than 10 lesions were examined in each individual. The average score was used as the pancreatitis index of each individual. thought to be initiated by the accumulation of lymphoid cells into the periductular regions and then the ductular epi- Immunohistochemistry thelial cells, followed by their destruction (Kanno et al. Paraffin-embedded tissues were used for identification of Fas. 1992; Qu et al. 2002), similar to autoimmune pancreatitis in After sectioning they were mounted of coated multi spot slides. human (Finkelberg et al. 2006; Zandieh and Byrne 2007). Immunohistochemical procedures were performed using the This initial event in the pancreas leads to the destruction of EnvisionTM Systems (Dako, Glostrup, Denmark) method, according to pancreatic acini, associated with the formation of granulo- the manufacturer’s instructions. Briefly, the sections were micro- matous lesions, with a partial regeneration of the ductules, waved for 10 min and washed with Tris-buffered saline (TBS). The thus showing so called “epithelial islands” (Okazaki et al. sections were incubated in PBS with 3% bovine serum albumin (BSA) 2008). Interestingly, the incidence of poly I:C-induced pan- to prevent nonspecific binding for 30 min. After washing twice in creatitis in MRL/Mp mice was higher than that in MRL/Mp TBS, the sections were incubated with rabbit anti-Fas (Wako, Osaka, mice bearing a Fas with the lpr mutation, MRL/Mp- Japan) for 1 overnight at 4°C followed by horseradish peroxidase lpr/lpr (Qu et al. 2002), which induces a deficit in Fas- labeled polymer-conjugated to goat anti-rabbit immunoglobulin for 1 mediated cytotoxicity (Watanabe-Fukunaga et al. 1992). hr. Antibody localization was effected using a peroxidase reaction The present study therefore demonstrates a critical role of with 3,3’-diaminobenzidine tetrahydrochloride (DAB) (Dako) as TLR3 signaling to generate the Fas/Fas ligand (FasL)- chromogen. The section was counterstained with hematoxylin. mediated cytotoxicity, leading to the development of pan- creatitis, associated with the effect of poly I:C directly act- Cell culture The pancreatic duct epithelial cell line MRL/S-1 was established ing on pancreatic epithelial cells. from the pancreas of an MRL/gld mouse to exclude the influence of functional FasL in subsequent studies. First, we prepared an MRL/gld MATERIALS AND METHODS mouse congenic with the SV40T-antigen transgene in the immortal- Mice ized strain of mice (Immortmice®) previously reported (Jat et al. lpr/lpr MRL/Mp-+/+ (MRL/+) and MRL/Mp- (MRL/lpr) mice 1991). In brief, an immortalized strain of mice was backcrossed for (Murphy and Roths 1978) were purchased from The Jackson MRL/gld more than 10 generations to establish MRL/Mp-gld/gld-TAg gld/gld Laboratory (Bar Harbor, ME, USA) and MRL/Mp- (MRL/gld) (MRL/gld-TAg). Thereafter, pancreatic tissue was prepared to per- mice which are deficit of functional FasL (Ito et al. 1997) were pur- form the culture in William’s E medium (Invitrogen, Carlsbad, CA, chased from Japan SLC, Inc. (Hamamatsu, Japan). They were main- USA) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen), tained in an animal center under specific pathogen-free conditions. supplemented with insulin (5 mg/l) (Sigma-Aldrich), IFNγ (200 The housing and care of the animals and all the procedures used in U/ml) (Biosource, Camarillo,CA, USA), penicillin (100 U/ml) (Banyu, these studies were performed in accordance with the guidelines and Tokyo, Japan), streptomycin (100 μg/ml) (Meiji, Tokyo, Japan) and regulations of the Ehime University Graduate School of Medicine. EGF (10 μg/l) (Sigma) at 33ºC. Mice were divided into two groups; poly I:C (5 mg/kg) (Sigma- MRL/S-1 cells were characterized using light microscopy, elec- n Aldrich, St. Louis, MO, USA) ( = 10, each of strains) or phosphate tron microscopy and immunocytochemistry. For electron microscopy, n bufferd saline (PBS) as control ( = 10, each of strains) was injected the cultured cells in plastic dishes were fixed with 5% glutaraldehyde intraperitoneally once every three days, starting when the animals and 2% paraformaldehyde in 0.1 M PB. The cells were post fixed in were 6 wk old and continuing until they were 18 wk old, according to 2% osmium tetroxide and embedded in epoxy resin. For morphologi- the method previously described (Qu et al. 2002). cal examinations, the cells were cultured on glass chamber slides (BD Bioscieneces, Franklin Lakes, NJ, USA) and fixed with methanol for Histopathological evaluation of pancreatitis 20 min at -20ºC. The cells were stained with HE for the examination At 18 wk of age, each strain of mice were anaesthetized with by light microscopy. In addition, the cells were reacted with fluores- ether and sacrificed by cervical dislocation. Half of the pancreases cein isothiocyanate (FITC)-conjugated mouse anti-mouse E-cadherin and spleens were removed from each mouse for histopathological antibody (BD Biosciences), sheep anti-human carbonic anhydrase II examinations. The tissue specimens were fixed in 10% formalin in antibody (The Binding Site, Birmingham, AL, USA), goat anti-mouse 0.01 M phosphate buffer (PB) (pH 7.2) and then were embedded in Fas antibody (R&D systems, Minneapolis, MN, USA) and rabbit anti- paraffin. After sectioning they were stained with hematoxylin and mouse TLR3 antibody (Imgenex, San Diego, CA, USA). As the sec- eosin (HE). ond antibodies, FITC-conjugated goat anti-rabbit IgG (H+L) (Jackson The histopathological evaluation of pancreatic lesions was per- ImmunoResearch, Baltimore Pike, PA, USA), FITC-conjugated rabbit formed under light microscopy. The severity grade of each lesion in anti-sheep IgG (KPL, Gaithersburg, MD, USA) and FITC-conjugated at least one section of the pancreas was scored on a 0-3+ scale as fol- rabbit anti-goat IgG (MP Biomedicals Inc, Solon, OH, USA). lows. Grade 0, pancreas without mononuclear cell infiltration, thus indicating it to be almost normal; grade 1+, mononuclear cell aggre- 176 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 177

Fig. 1. Histopathological grading of pancreatic lesions. (A) Pancreas without mononuclear cell infiltration, indicating almost normal; grade 0. (× 100, bar; 200 μm) (B) Mononuclear cell aggregation and/or infiltration into the periductular regions without any parenchymal destruction; grade 1+. (× 100, bar; 200 μm) (C) Focal parenchymal destruction with mononu- clear cell infiltration into ductular epithelium; grade 2+. (× 100, bar; 200μ m). The inset shows mononuclear cell infiltra- tion into the ductular epithelium, which is the characteristic finding of grade 2+. (× 400) (D) Diffuse parenchymal destruction with granulomatous lesions but retaining some intact parenchymal residue (left) or with almost all pancreatic tissue, except the pancreatic islets, destroyed or replaced with fibrosis or adipose tissue (right), grade 3+. (× 100, bar; 200 μm). Hematoxylin and eosin (HE) staining.

Fig. 2. Representative histopathological features of poly I:C-induced pancreatitis in MRL/+, MRL/lpr and MRL/gld mice. (A) The destruction of the pancreatic parenchyma followed by the formation of granulomatous lesions with the regenerative ductules and diffuse infiltration of mononuclear cells was observed in MRL/+ mice. (B) The replacement of parenchyma with adipose tissue was also observed in MRL/+ mice. (C) In MRL/lpr mice, the lesions were almost limited in the accumulation of mononuclear cells in the periductular regions. (D) Lymphoid cell infiltration to the ductular epithelial cells and parenchyma was observed, but the severity of such a lesion was low in MRL/gld mice. HE staining, × 100, bar; 200 μm. 178 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 179

Real-time RT-PCR The protein solution was loaded onto the column and thereafter the

Total RNA was isolated from and MRL/S-1 cells using TRIzol column was washed with wash buffer (20 mM PB, 300 mM NaCl, 50 Regent (Invitrogen). High capacity cDNA Reverse Transcription Kit mM Imidazole, pH 7.5). His-fusion FasL was eluted with elution buf- (Applied Biosystems, Foster City, CA, USA) was used following the fer (20 mM PB, 300 mM NaCl, 500 mM Imidazole, pH 7.5) and dia- manufacturer’s protocol for reverse transcription. To validate gene lyzed against PBS using the Mini Dialysis kit (GE Healthcare Bio- expression changes, a quantitative real-time RT-PCR analysis was Sciences). performed with an Applied Biosystems 7500 Sequence Detection sys- tem using TaqMan universal PCR master mix according to the manu- Cytotoxicity assay facturer’s specifications (Applied Biosystems). The TaqMan probes The measurement of cytotoxicity of MRL/S-1 cells in vitro was and primers for Fas (assay identification number Mm_01204974_m1) performed using the MTT assay based on measuring the cell viability and TLR3 (assay identification number Mm01207404_m1) were (Roche, Basel, Switzerland) (Hu et al. 2004; Shiraishi et al. 2004). In assay-on-demand products. The murine hypoxan- brief, MRL/S-1 cells were plated in 96-well tissue culture plates at 1 thine guanine phosphoribosyl transferase 1 (HPRT1) gene was × 105 cells/well and incubated in William’s E medium with 10% FCS used as endogenous control (assay identification number and with poly I:C (100 μg/ml) for 24 hr. To enrich the expression of Mm03024075_m1) (Applied Biosystems). The reactions were incu- Fas, the splenocytes of MRL/+, MRL/lpr or MRL/gld mice at 4 bated in a 96-well optical plate at 50ºC for 2 min, at 95ºC for 10 min, months of age were cultured in the presence of IL-2 (0.4 ng/ml) (R&D following by 40 cycles of 95ºC for 15 sec and 60ºC for 1 min. The Systems) and concanavalin A (2 μg/ml) (Miles Laboratories, Elkhart, real-time PCRs for each assay were run in triplicate. Amplification IN, USA) for 24 hr. Then, MRL/S-1 cells were reacted with these data were analyzed with an Applied Biosystems Sequence Detection splenocytes at 5 × 104 cells/well for 24 hr. Following treatment, the Soft ware (Applied Biosystems). cells were incubated with MTT labeling reagent 10 μl (5 mg/ml) for 4 hr at 37ºC. Precipitated formazan salt was solubilized with 100 μl PCR microarray analysis 10% SDS in 0.01 M HCl. The cell viability was estimated by the RNA was isolated from the whole spleen and MRL/S-1 cells MTT absorbance which was determined at 590 nm on an ELISA read- using TRIzol RNA isolation reagent (Invitrogen). Two μg of total er with a reference wavelength of 700 nm. The absorbance of wells RNA was reverse transcribed to cDNA using RT2 First Strand Kit with medium alone was determined as control values. All assays were (SABioscience, Frederick, MD, USA). Samples were analyzed performed in triplicate for each experiment. Cytotoxicity inducing according to the manufacturer’s recommendations using the “Mouse activity of synthetic His-fusion FasL (10 ng/ml) were also measured inflammatory Cytokines and Receptors” array in conjunction with the by the MTT assay in the presence of mouse for RT2 Profiler PCR Array System (SABioscience). All target the detection of His6-tagged recombinant (Anti-His6) (Roche), were normalized to the reference housekeeping genes; HPRT1, glyc- which makes a aggregate form of His-fusion FasL to be efficient in eraldehyde-3-phosphate dehydrogenase and β-actin. the induction of the Fas/FasL-mediated cytotoxicity as indicated (Dupont and Warrens 2007). Preparation of Fas ligands Statistical analysis FasL were prepared according to a cell-free protein synthesis One-way analysis of variance and Bonferroni/Dunn’s post hoc system using wheat germ ribosomal RNA, in which there is no risk of test and Student’s t-test were applied to analyze the statistical signifi- a lipopolysaccharide (LPS) contamination (Madin et al. 2000). The cance of our results. P < 0.05 was considered to be significant. extracellular domain of FasL cDNA obtained from an MRL/lpr mouse, which expresses a higher amount of intact FasL, was inserted RESULTS into pEUE01-His-N2 expression vector containing a His tag region (Suda et al. 1996). The FasL protein was automatically synthesized Reduced severity of poly I:C-induced pancreatitis in by the Robotic Protein Synthesizer Protemist® DT (CellFree MRL/lpr and MRL/gld mice Sciences, Matsuyama, Japan) according to the manufacturer’s instruc- MRL/+ mice treated with poly I:C developed severe tion manual as previously reported (Sawasaki et al. 2002). In brief, pancreatitis; namely, almost all of the treated mice showed the transcription mixture (250 μl), containing 25 μg of the plasmid the destruction of the pancreatic parenchyma, associated DNA, 80 mM HEPES-KOH, pH 7.8, 16 mM magnesium acetate, 2 with invasive infiltration of lymphoid cells into the ductular mM spermidine, 10 mM dithiothreitol, 2.5 mM each of nucleoside tri- epithelial cells and acini, followed by the formation of gran- phosphates, 250 U of SP6 RNA polymerase (Promega, Madison, WI, ulomatous lesions with regenerative ductules and finally the USA) and 250 U of RNasin (Promega), was incubated for 6 hr at 37°C. replacement of acini with adipose tissue (Fig. 2A, B). After the incubation, the transcription solution containing transcribed However, most of MRL/lpr mice, which are deficit of Fas μ mRNA was mixed with 250 l of wheat germ extract WEPRO1240H (Watanabe-Fukunaga et al. 1992), retained the pancreatic (CellFree Sciences) supplemented with 2 μl of 20 mg/ml creatine parenchyma limited with the accumulation of mononuclear kinase in a single well of a six-well plate. The 5.5-ml substrate mix (30 cells in the periductular regions (Fig. 2C). In MRL/gld mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.7 mM mag- nesium acetate, 0.4 mM spermidine, 2.5 mM dithiothreitol, 0.3 mM mice, which lack the functional FasL due to a point muta- amino acid mix, 1.2 mM ATP, 0.25 mM GTP and 16 mM creatine tion in the FasL gene (Takahashi et al. 1994), the severity of phosphate) (CellFree Sciences) was then added on top of the transla- the pancreatitis lesions was lower than that of MRL/+ mice, tion mix and incubated at 17°C for 20 hr. thus showing mainly the lymphoid cell infiltration into the Synthetic His-fusion FasL was purified with Ni Sepharose High ductular epithelial cells, but only a slight association with Performance (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). the destruction of ductules and acini (Fig. 2D). 178 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 179

Fig. 3. The severity of poly I:C-induced pancreatitis in MRL/+, MRL/lpr and MRL/gld mice. Each strain was divided to two groups (n = 10 each) treated with poly I:C or PBS as control, and the severity of the lesions as indicated by pancreatitis index was histopathologically evaluated under light microscopy (see the Methods). The pancreatitis index was signifi- cantly higher in MRL/+ mice than in MRL/lpr mice and that in MRL/gld mice was intermediate. **P < 0.01 by one- way analysis of variance and Bonferroni/Dunn’s post hoc test.

A quantitative study was conducted by scoring the Induction of FasL-mediated cytotoxicity of MRL/S-1 pancreatitis lesions in MRL/+, MRL/lpr and MRL/gld mice cells treated with poly I:C, showing a remarkable difference in To examine whether splenocytes with functional FasL the severity of pancreatitis among these strains. The pan- can induces apoptosis of MRL/S-1 cells, splenocytes creatitis index of MRL/+ mice was significantly higher than derived from MRL/+, MRL/lpr or MRL/gld mice were pre- that in MRL/lpr mice, while the index of MRL/gld mice pared, and pre-treated with IL-2 and concanavalin A in vitro was intermediate (Fig. 3). to enrich in FasL expression and then reacted with MRL/S-1 cells. There was lower number of viable cells among those Expression of Fas in the pancreatic ductular cells treated with splenocytes from MRL/+ and MRL/lpr mice These results indicate that poly I:C-induced pancreati- than those from MRL/gld mice (Fig. 7A). Moreover, syn- tis is associated with the Fas/FasL-mediated cytotoxicity, thetic His-fusion FasL derived MRL/lpr mice induced cyto- which leads to the destruction of pancreatic duct epithelial toxicity of MRL/S-1 cells in the presence of Anti-His6 (Fig. cells. In immunohistological study, Fas was found to be 7B). significantly expressed in the ductular epithelial cells and also infiltrating lymphoid cells in MRL/+ mice treated with Expression of chemokines in MRL/S-1 cells and poly I:C (Fig. 4). The lymphoid cells in these lesions were receptors in splenocytes after the treatment composed of various cells positive for CD3, CD4, CD8α, with poly I:C CD11b, CD11c or CD49b (data not shown). These results may explain why poly I:C-induced pan- The expression of Fas in the pancreatic ductular cells creatitis was more severe in MRL/+ mice than MRL/lpr was confirmed in the cultured pancreatic duct cell line mice and in a lesser extent in the MRL/gld mice. The Fas/ MRL/S-1, which was newly established from MRL/gld-TAg FasL-mediated cytotoxicity plays an important role on the mice as described in Materials and Methods. These cells effector phase of the development of pancreatitis. However, retained the characteristics of duct epithelial cells, showing the mechanisms of cell infiltration into periductular regions a cobblestone appearance under permissive culture condi- were not examined in the mice treated with poly I:C. To tions and positive for desmosomes observed by electron investigate whether poly I:C directly induced chemokine microscopy and E-cadherin and carbonic anhydrase II, both expression in pancreatic duct epithelial cells, which may of which are expressed in duct epithelial cells (Blouin et al. lead to the recruitment of effector cells to the periductular 1997; Koisumi et al. 2004) observed by immunocytochem- regions, a cultured pancreatic duct epithelial cell line istry (Figs. 5A-D). Moreover, after the treatment with poly MRL/S-1 was used. When MRL/S-1 cells were treated with I:C, MRL/S-1 cells became significantly positive for Fas poly I:C in vitro, these cells expressed higher mRNA levels and TLR3 (Figs. 5E, F). The expression of Fas mRNA and of chemokines including CCL2 (MCP-1), CCL5 TLR3 mRNA in the MRL/S-1 cells was enhanced by 12 hr (RANTES), CCL7, CCL17 and CXCL10 (IP-10) (Fig. 8A). after the treatment with poly I:C (Figs. 6A, B) . Thereafter, the association of chemokine expression in pancreatic ductular epithelial cells with the expression of chemokine receptors was examined on the splenocytes that 180 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 181

Fig. 4. The Fas expression in pancreatic ductular epithelial cells of MRL/+ mice treated with poly I:C. (A) The destruction of the pancreatic parenchyma followed by the formation of granulomatous lesions was observed in MRL/+ mice treated with poly I:C. (HE staining, × 200) (B) Pancreatic ductules showed positive for Fas (arrowheads) (Immu- nostaining, × 200). The inset shows cytoplasmic staining for Fas in pancreatic ductular epithelial cells and infiltrating lymphoid cells. bar; 50 μm.

Fig. 5. The characterization of a cultured pancreatic duct cell line MRL/S-1. (A) Light microscopy showed cobblestone appearance under permissive culture conditions. (HE staining, × 200, bar; 100 μm) (B) Electron microscopy revealed intercellular junctions characteristic of desmosomes (arrowheads) and a lack of zymogen granules (× 9000, bar; 1 μm) (C) Immunofluorescence staining with antibody to E-cadherin shows that almost half of the MRL/S-1 cells were posi- tive on the cell surface (× 400). (D) Immunofluorescence staining with antibody to carbonic anhydrase II shows positive in the cytoplasm of MRL/S-1 cells. (× 400). Cells were stained at variable levels in the cytoplasm. (E,F) MRL/S-1 cells treated with poly I:C (100 μg/ml) for 24 hr showed positive for Fas in the cytoplasm and cell membrane (E) and TLR3 in the cytoplasm (F). (× 400 each) 180 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 181

Fig. 6. Real-time RT-PCR of Fas mRNA and TLR3 mRNA in a cultured pancreatic duct cell line MRL/S-1. The cells were treated with poly I:C for 12, 24 and 48 hr. After harvest, cDNA from these cells were prepared for real-time PCR (see the Methods). (A) At 12 hr, Fas was highly expressed in response to the treatment with poly I:C and at 48 hr, the expres- sion levels of Fas decreased to the level without poly I:C treatment. (B) Similar results were found in the TLR3 expres- sion. The study was repeated three times and similar results were obtained in all experiments. **P < 0.01, *P < 0.05; one-way analysis of variance and Bonferroni/Dunn’s post hoc test.

may infiltrate periductular regions in the poly I:C-treated tant to elucidate the pathogenesis of autoimmune diseases. mice. The expression of chemokine receptors in spleno- In past studies, the major research works on this subject has cytes derived from poly I:C-treated MRL/+ and MRL/lpr focused on the antigen specificity of immune cells bearing mice were examined by real-time PCR array (Fig. 8B). antigen receptors according to the concept of adaptive Several chemokine receptors corresponding to the chemo- immunity. Even recent studies of the roles of innate immu- kines expressed in MRL/S-1 cells were highly expressed in nity relating to this subject have focused on the immune a mRNA level on splenocytes, including CCR1, the receptor cells engaged in adaptive immunity. In general, the patho- for CCL5 and CCL7, CCR2, the receptor for CCL2 and logical phenotypes of autoimmune diseases with progres- CCL7, CCR3, the receptor for CCL5 and CCL7, CCR4, the sive inflammatory lesions can be considered as a result of receptor for CCL17, CCR5, the receptor for CCL5 and the cumulative effect of multiple genes that lead to a cas- CXCR3, the receptor for CXCL10. These receptor-bearing cade reaction. Some of these genes are involved in the tar- cells may accumulate in the periductular regions of the pan- geted organs in situ and susceptible to environmental factors creas in response to chemokines produced by the pancreatic possibly involving viral infection. The poly I:C-induced ductular cells corresponding to the histopathological mani- pancreatitis model presented in this study may support this festations as shown in Fig. 1. However, there was no dis- idea and pointed out an important role of the Fas/FasL- tinct difference between the splenocytes of MRL/+ and mediated cytotoxicity in local innate immunity in the target MRL/lpr mice treated with poly I:C, suggesting that the dif- organs. ferent severity of poly I:C-induced pancreatitis between Several reports have shown the expression of Fas and MRL/+ and MRL/lpr mice did not depend on the difference FasL in the duct epithelium of human chronic pancreatitis in the expression level of chemokine receptors on spleno- immunohistologically, thus suggesting an important role of cytes of these strains. Fas-mediated cytotoxicity in the development of chronic pancreatitis (Kornmann et al. 2000). However, there is no DISCUSSION direct evidence indicating the Fas-mediated cytotoxicity and The organ specificity in autoimmune diseases is impor- the developmental mechanism has remained unclear. In this 182 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 183

Fig. 7. The FasL-mediated cytotoxicity of MRL/S-1 cells in an MTT assay. (A) MRL/S-1 cells were pre-treated with poly I:C, then incubated with FasL-rich splenocytes derived from MRL/+, MRL/lpr or MRL/gld mice (see the Methods). The splenocytes from MRL/+ and MRL/lpr mice induced more cytotoxicity than those from MRL/gld mice. **P < 0.01 by one-way analysis of variance and Bonferroni/Dunn’s post hoc test. (B) Synthetic His-fusion FasL from the cDNA of MRL/lpr mice (see the Methods) induced cytotoxicity of MRL/S-1 cells in the presence of Anti-His6. *P < 0.05 by Stu- dent’s t-test. Each of the studies was repeated three times and similar results were obtained in all experiments. Cytotox- icity was represented as cell viability; a relative value was estimated by the MTT absorbance as described in Materials and Methods. study, taking advantage of a newly established pancreatic produced by pancreatic duct epithelial cells, a pancreatic duct epithelial cell line MRL/S-1, we were able to show that duct epithelial cell line was established from a FasL defi- poly I:C directly induced those cells to express Fas signifi- cient mouse, MRL/gld. Finally, the results showed that the cantly (Fig. 6A) and then clarified that the Fas/FasL- FasL protein induced cytotoxicity in pancreatic duct epithe- mediated cytotoxicity led to their destruction by splenocytes lial cells. derived from MRL/+ and MRL/lpr mice (Fig. 7A). Finally, The observation that poly I:C directly induced the synthetic extracellular domain of FasL induced cytotox- MRL/S-1 cells to express TLR3 yielded further evidence of icity in MRL/S-1 cells (Fig. 7B). the significance of innate immunity in the development of Soluble FasL can induce apoptosis in a hexameric form chronic pancreatitis (Fig. 6B); thus indicating the presence (Holler et al. 2003). In the present study, the soluble FasL- of a positive feed back mechanism in the expression of mediated cytotoxicity in MRL/S-1 cells treated with poly I:C TLR3 of these cells. This suggests the presence of a self was examined using synthetic His-fusion FasL in the pres- perpetuation mechanism in pancreatic duct epithelial cells ence of Anti-His6 to make a aggregate form according to the to express TLR3 in response to the treatment with poly I:C method previously described (Dupont and Warrens 2007). in vivo. Moreover, MRL/S-1 cells expressed higher mRNA His-fusion FasL was prepared in a cell-free protein synthe- levels of chemokines including CCL2 (MCP-1), CCL5, sis system using wheat germ ribosomal RNA to avoid a risk CCL7, CCL17 and CXCL10 (IP-10) in vitro after the treat- of contamination with LPS, which induces TLR4-mediated ment with poly I:C (Fig. 8A). These results may indicate that signaling. Moreover, to avoid an autocrine effect of FasL several chemokines produced by pancreatic duct epithelial 182 Y. Soga et al. Fas/FasL-Mediated Cytotoxicity in Pancreatitis 183

Fig. 8. The expression of chemokines in MRL/S-1 cells and chemokine receptors in splenocytes after the treatment with poly I:C in PCR array analysis. (A) The chemokine expression of MRL/S-1 cells treated with poly I:C (100 μg/ml) for 12 hr, revealed a significantly higher levels of chemokine gene expression involving Ccl2, Ccl5, Ccl7, Ccl17 and Cxcl10. (B) The expression of splenocytes obtained from MRL/+ and MRL/lpr mice was shown. These mice were prepared by intraperitoneal injection with poly I:C at a dose of 5 mg/kg twice a week, starting when the animals were 6 wk old and continuing until they were 10 wk old. The results showed the gene expression of Ccr1, Ccr2, Ccr3, Ccr4, Ccr5 and Cxcr3. But, no significant difference was observed between the splenocytes of MRL/+ and MRL/lpr mice. Each of the studies was repeated three times and similar results were obtained in all experiments.

cells can effectively induce the recruitment of inflammatory may support this idea (Axelsson et al. 2008; Moreno et al. cells from the circulation even if they might have no anti- 2006). The question of whether antigen-specific T cells are gen-specific receptors. Furthermore, no synergistic effect fundamentally required in poly I:C-induced pancreatitis was observed with the expression of chemokine receptors should be investigated further; although a previous study corresponding to these chemokines in the splenocytes treat- reported that splenic cell transfer of pancreatitis is success- ed with poly I:C (Fig. 8B). ful in MRL/+ mice (Qu et al. 2002). It is important to consider which chemokines are In conclusion, this study demonstrated that Fas/FasL- responsible for the recruitment of FasL-bearing lymphoid mediated cytotoxicity plays an important role in poly I:C- cells and subsequent Fas/FasL-mediated cytotoxicity of induced pancreatitis in MRL/Mp mice with the effect of pancreatic duct epithelial cells. FasL is strongly expressed signal transduction of TLR3 directly acting on pancreatic on NK cells following the administration of poly I:C (Wang duct epithelial cells. The above findings suggest that the et al. 2006). Considering this in conjunction with the results TLR3 signal-mediated innate immunity may be an impor- of the current study that the lymphoid cells infiltrating into tant factor determining the target organ specificity in auto- the pancreatitis lesions were composed of various cells posi- immunity. tive for CD3, CD4, CD8α, CD11b, CD11c and CD49b (data not shown), among which CD8α, CD11b, CD11c and/or Acknowledgments CD49b are expressed on NK cells, at least CCL2, CCL5 We wish to thank Prof. H. Hasegawa for helpful discussions and CXCL10 produced by MRL/S-1 cells treated with poly in the study of chemokines. This work was supported in part by I:C may be candidate chemokines to recruit NK cells to the a Grant-in Aid for Scientific Research (B) of the Ministry of pancreatic periductular regions. The previous reports that Education, Science and Culture of Japan (#18390123 to M.N). CCL2 contributes to the development of heparan sulfate- induced pancreatitis and also cerulein-induced pancreatitis 184 Y. Soga et al.

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