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Kinetic analysis of the development of pancreatic lesions in mice infected with a murine retrovirus
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Clinical Immunology 109 (2003) 212–223
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Kinetic analysis of the development of pancreatic lesions in mice infected with a murine retrovirus Shiro Watanabe,a Kenji Suzuki,a,* Yusuke Kawauchi,a Satoshi Yamagiwa,a Hiroyuki Yoneyama,a Hiroshi Kawachi,b Yoshiaki Okada,c Fujio Shimizu,b Hitoshi Asakura,a and Yutaka Aoyagia a
b
Department of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan c Department of Bacterial and Blood Products, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan Received 28 April 2003; accepted with revision 8 July 2003
Abstract Sjo¨gren’s syndrome (SjS)-like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus, which induces a severe immunodeficiency termed murine AIDS (MAIDS). All mice with MAIDS showed advancing cellular infiltration around the pancreatic ducts as well as systemic exocrinopathy. The primary target tissue of the pancreas was acinar cells, and the pancreatic islets were well preserved until a late phase of the disease. Immunofluorescence and flow cytometry demonstrated that CD4⫹ T cells, Mac-1⫹ cells, and B220⫹ cells were major inflammatory components, and IFN-␥ and IL-10 were mainly detected on CD4⫹ T and Mac-1⫹ cells in the pancreas. Both Th1 and Th2 cells were found. TUNEL⫹ apoptotic cells were mostly detected among pancreasinfiltrating cells. Fas ligand and TNF-␣ were also detected among pancreas-infiltrating cells, whereas Fas was rarely expressed in the pancreatic acinar cells. Thus, MAIDS mice could be valuable for analyzing the pathogenesis of autoimmune-related pancreatitis associated with SjS. © 2003 Elsevier Inc. All rights reserved. Keywords: Autoimmune-related pancreatitis; Sjo¨gren’s syndrome; LP-BM5 murine leukemia virus; Murine AIDS; Interferon-␥; Interleukin-10; Apoptosis
Introduction In developed countries, most patients with chronic pancreatitis have a long history of alcohol abuse, which, therefore, is generally listed as the cause of the disease [1]. Nevertheless, approximately 30 to 40% of patients with chronic pancreatitis have no history of chronic alcoholism, so the etiology is regarded as idiopathic in these cases [1]. Since Sarles et al. reported a special form of chronic pancreatitis that may have been caused by “phenomena of self-immunization,” an autoimmune-related pathogenesis has been suggested in some cases of idiopathic chronic * Corresponding author. Department of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata City 951-8510, Japan. E-mail address: [email protected] (K. Suzuki). 1521-6616/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1521-6616(03)00197-9
pancreatitis [2,3]. Several authors have reported cases of chronic pancreatitis associated with Sjo¨gren’s syndrome (SjS) in which an autoimmune mechanism has been involved in the etiology and shown that steroid therapy was effective [4,5]. It has been suggested that there are two types of chronic pancreatitis with an autoimmune-related etiology [5–7]. One type is associated with another autoimmune disease such as SjS, primary biliary cirrhosis, primary sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis, Crohn’s disease), or systemic lupus erythematosus [4 –9]. Among them, SjS has the highest frequency of autoimmne-related pancreatitis [7]. Recently several reports have suggested another clinical type of chronic pancreatitis with autoimmune-related etiology that is not associated with another autoimmune disease [5,10]. In this type of chronic pancreatitis, high serum levels of IgG4 are characteristic and the values are closely associated with disease activity
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[10]. This disease has been referred to as sclerosing pancreatitis or autoimmune pancreatitis, and the clinicopathological features of this type of chronic pancreatitis mimic those of the disease associated with another autoimmune disease [10]. Thus, an autoimmune mechanism has been shown to play a rather large part in the pathogenesis of chronic pancreatitis [11,12]. However, the precise mechanism of the development of chronic pancreatitis with an autoimmune etiology is largely unknown. The LP-BM5 murine leukemia virus (MuLV) is a retrovirus that is known to induce profound immunodeficiency with splenomegaly and generalized lymphadenopathy in susceptible strains of mice such as C57BL/6(B6) and occasionally brings about lymphoid malignancy [13–15]. In the early phase of infection, hypergammaglobulinemia and polyclonal B and T cell activation were induced, and autoantibodies such as anti-nuclear and anti-ds-DNA antibodies were detected in the mice infected with the virus [13–15]. In the late phase of infection, the virus-infected B6 mice show symptoms similar to those of human acquired immunodeficiency syndrome (AIDS); therefore, they have been studied as a murine model of AIDS termed murine AIDS (MAIDS) [13–15]. We have reported previously that systemic exocrinopathy resembling SjS was induced in systemic exocrine glands such as salivary glands, lacrimal glands, and the pancreas of the virus-infected mice [16,17]. We have also shown that nude mice inoculated with lymph node cells and/or spleen cells from mice with MAIDS developed inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy [18 –21]. In MAIDS mice, the pancreatic lesions are characterized by inflammatory cell infiltration around the interlobular pancreatic ducts and acinar cell destruction [16,17]. Contrary to the injury to the exocrine system of the pancreas, endocrine pancreatic islets were well preserved until the late phase of the disease in the mice. Thus, the pancreatic lesions of the mice could be considered a model for chronic pancreatitis associated with SjS. To clarify the mechanism of the development of chronic pancreatitis with autoimmune-related etiology, especially in the form associated with SjS, we analyzed the kinetics of the development of chronic pancreatitis in mice with MAIDS.
Materials and methods
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Virus LP-BM5 MuLV was prepared from the supernatant of cloned G6 cells infected with the retrovirus. Twenty-fourhour culture supernatant of G6 cells contained approximately 5 ⫻ 104 plaque-forming units/ml of ecotropic virus, as determined by XC plaque assay. Aliquots containing the virus were stored at ⫺80°C until use. For the infection, 4-week-old B6 mice were inoculated intraperitoneally with 0.3 ml of the stock solution of LP-BM5 MuLV. Induction of MAIDS Four-week-old B6 female mice were injected intraperitoneally with 0.3 ml of LP-BM5 MuLV virus stock solution. Induction of MAIDS was confirmed when the mice developed splenomegaly and generalized lymphadenopathy. At 4, 8, 10, and 12 weeks after virus inoculation, mice with MAIDS were killed by cervical dislocation under ether anesthesia, and their pancreata were removed for further analysis. Age- and sex-matched B6 mice without virus inoculation were used as controls. Four mice were analyzed for each group and all experiments were repeated three or four times. Monoclonal antibodies For immunofluorescence and flow cytometric analyses, the following monoclonal antibodies were used: anti-CD4 (clone GK1.5, IgG2b), anti-CD8 (clone 53-6.7, IgG2a), anti-B220 (clone RA3-6B2, IgG2a), anti-Mac-1 (clone M-70.15, IgG2b), anti-mouse INF-␥ (clone XMG1.2), and anti-mouse IL-10 (clone JES5-16E3). Detection of LP-BM5 MuLV by PCR The PCR method used for detection of the virus was reported previously [16]. In brief, the oligonucleotides used as PCR primers were 5⬘-CCTCTTCCTTTATCGACACT-3⬘ and 5⬘-ATTAGGGGGGGAATAGCTCG-3⬘, which are present in the p15 and p12 regions of the gag gene, respectively. The template DNAs were extracted from the frozen blocks of pancreas and subjected to 30 cycles of amplification. The PCR products (237 bp) were separated and analyzed by electrophoresis on a 1.0% agarose gel and ethidium bromide staining.
Animals Quantitative RT-PCR to detect cytokine mRNAs Four-week-old female C57BL/6 (B6) mice were purchased from Charles River Japan (Atsugi, Kanagawa, Japan) and maintained at the Animal Center of Niigata University School of Medicine under specific-pathogen-free conditions. All animal experiments were performed according to the Guide for Animal Experiments of Niigata University School of Medicine.
Total RNA was extracted from pancreas specimens with Trizol (Gibco BRL) according to the standard protocol and reverse transcribed. Thereafter, cDNA was amplified using the ABI 7700 sequence-detector system (Applied Biosystems, Foster City, CA) with a set of primers and probes corresponding to IFN-␥ and IL-10 and glyceraldehyde-3-
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phosphate dehydrogenase (GAPDH) as previously described. Histopathological examination Tissue samples were taken from the pancreas, fixed in 10% buffered formalin, and then embedded in paraffin wax blocks. Sections 4 m thick were made in the usual way and stained with hematoxylin and eosin and the aldehyde–fuchsin Masson–Goldner staining method. The stained sections were then examined by light microscopy. To assess the degree of pancreatitis, the number of inflammatory cells in a high-power field (⫻400) were counted under a microscope. Cell numbers at three different points in pancreatic inflammatory focuses of each mouse were counted and the data of three mice of each group were compared statistically. The severity of each lesion in at least one section of pancreas was scored on a 0 – 4⫹ scale based on the histopathological changes described by Kanno et al. [22,23]. Briefly, 0, pancreas without mononuclear cell infiltration, indicating that it is almost normal; 1⫹, mononuclear cell aggregation and/or infiltration within the interstitium without any parenchymal destruction; 2⫹, focal parenchymal destruction with mononuclear cell infiltration; 3⫹, diffuse parenchymal destruction but some intact parenchymal residue is retained; 4⫹, almost all pancreatic tissue except pancreatic islets destroyed or replaced with fibrotic or adipose tissue. The maximal score was used as the pancreatitis grade of each individual. IF staining procedure Frozen sections of the pancreas were prepared in a cryostat and stained with several fluorescent-dye-conjugated anti-mouse antibodies as described above. The sections were observed by fluorescence microscopy. Double IF staining procedure For the simultaneous demonstration of cell surface antigens and cytokines, acetone-fixed frozen sections were incubated sequentially with biotinylated anti-cell surface antigen antibody and then with Alexa 594 (Molecular Probes, Inc.)-conjugated avidin as the first step. As the second step, they were incubated with fluorescein conjugated anti-cytokine antibody. The sections were observed by fluorescence microscopy. Controls for the double staining were prepared by omitting the primary antibodies in the first or second step. To assess the distinct cytokine expression, the number of CD4⫹ and Mac-1⫹ cells that were double-positive for IFN-␥ or IL-10 was counted under a microscope in a highpower field (⫻400). Cell numbers at three different points in the pancreas of each mouse were counted and the data of three mice of each group were compared statistically.
Terminal deoxynucleotide transferase labeling Apoptotic cells were identified using an in situ apoptosisdetection kit (Chemicon International, USA) according to the manufacturer’s instruction. In brief, acetone-fixed 5-m fresh-frozen pancreas sections were permeabilized on ice and incubated with the terminal deoxynucleotide transferase mixture for 1 h at 37°C. Biotin-labeled dNTP was treated with streptavidin HRP for 30 min, visualized with DAB, and counterstained with methyl green. Preparation and flow cytometric analysis of cells that infiltrated the pancreas Mice were anesthetized with ether and killed by cervical vertebral dislocation. The pancreas was taken and then pressed through a 200-gauge stainless-steel mesh and cells were suspended in Eagle’s minimum essential medium (MEM). After being washed twice in MEM, the cell suspension was used for flow cytometric analysis. The surface phenotypes of pancreas-infiltrating cells were analyzed using the dye-labeled monoclonal antibodies described above with a FACScan (Becton–Dickinson, CA). Statistical analysis Data are expressed as means ⫾ SD. Statistical analyses were performed using the unpaired Student t test or the nonparametric Mann–Whitney test. Differences were considered significant at P ⬍ 0.05.
Results Exocrine pancreatitis developed in mice with MAIDS All of the mice infected with LP-BM5 MuLV developed generalized lymphadenopathy and hepatosplenomegaly, and died with changes characteristic of MAIDS at around 12–16 weeks after the virus infection (n ⫽ 20). As we have previously reported, periductal mononuclear cellular infiltration resembling SjS was detected in salivary glands and lacrimal glands beginning at around 4 weeks after infection and continuing thereafter. In the pancreas of the mice, mononuclear cellular infiltration was observed at around 4 weeks after infection and the number of infiltrating cells then increased gradually (Fig. 1A–D and H). The inflammatory cells were initially detected around the pancreatic ducts, from where they progressively expanded, pressing the acinar architecture outward. At the interface lesions between the infiltrating cells and the pancreatic parenchyma, destructed acinar cells were detected occasionally beginning at about 4 weeks after infection, and their number then increased gradually and reached a peak at 8 and 12 weeks after infection (Fig. 1D and I). However, the degree of acinar cell destruction by infiltrating cells was rather mild
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Fig. 1. Development of pancreatic lesions in mice with MAIDS. The number of inflammatory cells infiltrating into the pancreas of MAIDS mice increased and reached a peak at 8 weeks after infection. Pancreas from mice infected with LP-BM5 at 4 weeks (A), 8 weeks (B, D, G), and 12 weeks (C) after infection, and pancreas from normal mice (E, F). (A–E); hematoxylin and eosin staining. Original magnification, ⫻200. (F,G); Aldehyde–fuchsin Masson–Goldner staining. Original magnification, ⫻400. The number of cells infiltrating the pancreas after infection (H), and histological score of acinar cell destruction of the pancreas (I). The data were counted under a microscope at high-power magnification for three different areas per mouse and the data of each time point were collected from three mice and compared with each other. Fig. 2. Detection of LP-BM5 MuLV in the pancreas of MAIDS mice. (A) The template DNAs were extracted from frozen sections of the pancreas and then analyzed by PCR. Lane M, molecular size marker; lane 1, MAIDS spleen cells (positive control); lane 2, the pancreas of untreated B6 mice (negative control); lane 3, the pancreas of MAIDS mice 4 weeks after infection; lane 4, the pancreas of MAIDS mice 8 weeks after infection; lane 5, the pancreas of MAIDS mice 12 weeks after infection. (B) immunostaining for the p12 antigen of LP-BM5 MuLV in the pancreas of MAIDS mice.
and parenchymal fibrosis, edema, and hemorrhage were rarely observed (Fig. 1D and I). Peri-islet cellular infiltration was observed frequently, but Langerhans islets were well preserved until a late phase and no marked insulitis was detected (Fig. 1D–G). Detection of LP-BM5 MuLV in the pancreas of mice with MAIDS A defective LP-BM5 virus genome was detected by PCR in the same frozen sections of the pancreas of mice with MAIDS as those used for immunohistochemical staining to detect the virus (Fig. 2A). Immunohistochemistry using anti-gag p12 antibody revealed that the viral antigen was
present on some of the pancreas-infiltrating cells, but not on acinar cells or duct epithelial cells of the pancreas (Fig. 2B). Pancreas-infiltrating cells were composed of CD4⫹T, Mac-1⫹, and B220⫹ cells in mice with MAIDS We further characterized the phenotype of inflammatory cells in the pancreas in situ using immunofluorescence. Infiltrating cells were composed of CD4⫹, CD8⫹T cells, Mac-1⫹ macropahges, and B220⫹ B cells but not NK cells or granulocytes (Fig. 3A–D). The ratio of CD4⫹T cells, Mac-1⫹, and B220⫹ cells was nearly equal except for a minor population of CD8⫹T cells. The number of infiltrating CD4⫹T cells, Mac-1⫹, and B220⫹ cells reached a peak
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Fig. 3. Kinetic appearance of lymphocyte subpopulations in the pancreas of MAIDS mice. Immunofluorescenc for CD4 (A), CD8 (B), Mac-1 (C), and B220 (D). Quantitative analysis of immune cells infiltrating the pancreas (E). Each focus of cellular infiltration was examined for the presence of CD4, CD8, B220, and Mac-1 cells. Final results are presented as the number per high-powered microscope field. Representative findings (means ⫾ standard deviation, n ⫽ 10) from three independent experiments are shown. *Data are significantly different from the number of CD8⫹ cells at each time point as determined by Student’s t test (P ⬍ 0.05). Fig. 6. Double-color immunofluorescence staining for CD4 and IFN-␥ (A-C), CD4 and IL-10 (D-F), Mac-1 and IFN-␥ (G-I), and Mac-1 and IL-10 (J-L) of the pancreas of MAIDS mice 8 weeks after infection. CD4 (A, D, C, F; red) and Mac-1 (G, I, J, L; red); IFN-␥ (B, C, H, I; green), and IL-10 (E, F, K, L; green).
at 8 weeks and stayed at that level throughout the infection (Fig. 3E). We also confirmed the number of each phenotype of infiltrating cells with flow cytometry, and it was revealed that the major populations of pancreas-infiltrating cells comprised CD4⫹T cells with ␣T cell receptors, Mac-1⫹, and B220⫹ cells (Fig. 4). The lower number of Mac-1⫹ cells, compared with that found by IF, was probably caused by loss through their tendency to adhesion during the preparation of pancreas-infiltrating cells for flow cytometry. Only negligible numbers of NK1.1⫹ NK cells and Gr1⫹ granulocytes were found. IFN-␥ and IL-10 were produced by CD4⫹T and Mac-1⫹ cells in the pancreatic lesions of mice with MAIDS To reveal the immune response in the pancreas of mice with MAIDS, we analyzed the cytokine mRNA expression of IFN-␥ and IL-10 by RT-PCR. We chose IFN-␥ as a
representative for a proinflammatory cytokine (or Th1 response) and IL-10 as a representative of an anti-inflammatory cytokine (or Th2). The levels of expression of the mRNAs of both cytokines were significantly increased after the infection (Fig. 5A and B). Immunofluorescence showed that IFN-␥- and IL-10-positive cells were detected through 4 to 12 weeks after infection, and throughout the course the number of IL-10-positive cells was larger, but not statistically significantly, than that of IFN-␥-positive ones (Fig. 5C). Next we characterized the phenotypes of cells producing these cytokines by a double-color immunofluorescence method. IFN-␥ and IL-10 were mainly present on CD4⫹ T cells (Fig. 6A–F) and Mac-1⫹ cells (Fig. 6G–L), but not on B220⫹ or CD8⫹ T cells (data not shown). We could detect neither mRNA nor protein expression for IFN-␥ or IL-10 in the presence of normal B6 mice. Some CD4⫹T cells expressing either IFN-␥ or IL-10
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were detected at the 4th week in the pancreas of MAIDS mice, and their number significantly increased and reached a plateau at 8 weeks after infection (Fig. 6A–F and 5D). The number of IL-10-positive CD4⫹ T cells was significantly greater than that of IFN-␥ -positive CD4⫹T cells through 4 to 12 weeks after infection (Fig. 6A–F and 5D). Some Mac-1⫹ cells also expressed either IFN-␥ or IL-10 in the pancreas of MAIDS mice at 4 weeks after infection. These cells increased remarkably at 8 and 12 weeks after infection, and the numbers of IFN-␥-positive Mac-1⫹ cells and IL-10-positive Mac-1⫹ cells were almost equal through the course (Fig. 6G–L and 5E). Apoptotic cells were mostly detected among pancreasinfiltrating cells in mice with MAIDS Histological analysis revealed that advancing pancreatic lesions were composed of inflammatory cellular infiltration around the interlobular pancreatic ducts and scattered destruction of acinar cells. We further analyzed the role of apoptosis in tissue destruction of the pancreas using terminal deoxynucleotide transferase labeling (TUNEL) staining. Few TUNEL⫹ acinar cells were detected in the interface lesions of each inflammatory focus in the pancreas of MAIDS mice (Fig. 7A–C and E). The results suggest that apoptosis might not be the primary type of cell death of acinar cells in the pancreatic lesions of these mice. In contrast to the paucity of apoptotic acinar cells, TUNEL⫹ mononuclear cells (MNCs) were detected, and their number increased gradually in each focus of the pancreas of MAIDS mice (Fig. 7A and D). Flow cytometric analysis for the pancreas-infiltrating MNCs also revealed that annexin-Vpositive cells were detected and increased in number after infection (Fig. 7F). Fas ligand and TNF-␣ were detected among pancreasinfiltrating inflammatory cells, whereas Fas was rarely expressed in the pancreas of mice with MAIDS As reported in cases with chronic pancreatitis, the Fas/ Fas-ligand system and TNF-␣ are supposed to be important mechanisms involved in the apoptosis of pancreatic acinar cells in autoimmune-related pancreatitis [24,25]. Therefore, we next analyzed the Fas/FasL and TNF-␣ in the pancareas of the MAIDS mice. The expression levels of the mRNAs of FasL and TNF-␣ were increased at 8 weeks after infection in the pancreas of mice with MAIDS (Fig. 8A and B). Immunohistochemistry revealed that FasL and TNF-␣ were expressed on some of the MNCs in the inflammatory focus of the pancreas of MAIDS mice (Fig. 8C and D) and that
Fas was positive on a few MNCs but negative on acinar cells of the pancreas (Fig. 8E). In the pancreas of untreated control mice, FasL, TNF-␣, and Fas were not detected by immunohistochemisry.
Discussion In this report, we demonstrated that advancing cellular infiltration in the pancreas was induced in mice with MAIDS, and the lesions have characteristics similar to those in the exocrine glands in mice with MAIDS that we have reported previously [16,17]. The pancreas is composed of the exocrine tissue of acinar cells and the endocrine tissue of islet cells. The present histopathological study revealed that the lesions in the pancreas of mice with MAIDS are more dominant in the exocrine than the endocrine system. The common pathological characteristics of most cases of autoimmune-related pancreatitis are fibrotic changes with lymphocytic infiltration, absence of pancreatic calcificaiton, and absence of pancreatic cysts [7,8]. The endocrine tissue of pancreatic islets might be involved in the disease, but the primary arena of inflammation is the pancreatic parenchyma, that is, the exocrine system [7,8], as it is in mice with MAIDS. Some unknown target antigens, adhesion molecules, and chemokines might regulate the organ specificity of the lesions of mice with MAIDS, as is the case with several autoimmune diseases, including SjS and autoimmune-related pancreatitis [11]. We have reported that CD4⫹ T cells from MAIDS were able to induce lesions in exocrine glands, such as the salivary glands and pancreas, but could not induce colitis when transfered to B6 nude mice [21]. We speculate that the most important population of MAIDS cells to induce exocrinopathy is T, especially CD4⫹ T cell population (manuscript in preparation). We have observed oligoclonal expansion of T cell recepter ␣ CD4⫹ T cell clones in salivary glands and spleen in mice with MAIDS (unpublished data), and these CD4⫹ T cell clones might recognize some common target antigens in the pancreas and systemic exocrine glands of mice with MAIDS. We reported previously that the virus was integrated in Ly-1 B cells but not in T cells [26], although several reports showed that B cells [27], and macrophages [28], as well as T cells [29], can serve as targets for infection of the virus. A double color immunofluorescence study revealed that the viral antigen was detected on some of B220⫹ and Mac-1⫹ cells, but not on CD4⫹ or CD8⫹ cells (unpublished observation). Using electron microscopy and IF with anti-p 12 antibody, we were unable to detect the virus on acinar cells of the pan-
Fig. 4. Phenotypic characterization of mononuclear cells in the pancreas of MAIDS mice. Two-color staining for B220 and CD3, CD4 and CD8, and TCR-␣ and TCR-␥␦. Single-color staining for Mac-1. Pancreas-infiltrating mononuclear cells in MAIDS mice infected after 4 and 10 weeks were analyzed by flow cytometry; pancreas-residing mononuclear cells of untreated B6 mice were used as controls. Numbers represent the percentages of fluorescence-positive cells in corresponding areas. Representative results of three experiments are depicted.
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Fig. 5. Kinetic analyses of cytokine production by MNCs infiltrating the pancreas in mice with MAIDS. (A, B) Real-time quantitative PCR of IFN-␥ (A) and IL-10 (B) mRNA expression in the pancreas of MAIDS mice. Each amount was normalized to the level of GAPDH and the final relative values were expressed relative to calibrators on day 0. Kinetic quantitative analysis of IFN-␥ and IL-10 expression patterns of cells that infiltrated the pancreas at 4, 8, and 12 weeks after infection (C). Stained cryostat sections of three focuses of cellular infiltration of the individual pancreas were examined by counting the number of cells expressing IFN-␥ and cells expressing IL-10. Three mice were analyzed for each time point. In control normal B6 mice, a negligible number of cells expressed IFN-␥ or IL-10, so the data are not shown. Kinetic quantitative analysis of IFN-␥ and IL-10 expression patterns of CD4⫹ and Mac-1⫹ cells that infiltrated the pancreas at 4, 8, and 12 weeks after infection (D, E). Stained cryostat sections of three focuses of cellular infiltration of the individual pancreas were examined by counting the number of CD4⫹ cells expressing IFN-␥ or IL-10 and Mac-1⫹ cells expressing IFN-␥ or IL-10. Three mice were analyzed for each time point. *P ⬍ 0.01. In control normal B6 mice, a negligible number of cells expressed IFN-␥ or IL-10, so the data are not shown.
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Fig. 7. Immunostaining for TUNEL⫹ apoptotic cells in the pancreas of MAIDS mice. TUNEL⫹ cells are colored brown. Positive cells are scattered within a focus of cellular infiltration around the pancreatic duct (A), and there are few positive cells among destructed acinar cells at the interface lesions of a cellular focus of the pancreas (B, C) of MAIDS mice 8 weeks after infection. The arrow indicates TUNEL⫹ apoptotic acinar cells, and the arrowhead indicates TUNEL-negative necrotic acinar cells. Quantitative analysis of TUNEL⫹ mononuclear and acinar cells in the pancreas (D, E). Numbers of TUNEL⫹ mononuclear (D) and acinar cells (E) at three different points in pancreatic inflammatory focuses of each mouse were counted, and the data of three mice with MAIDS are shown. The pancreas-infiltrating cells were analyzed with flow cytometry for expression of annexin-V at 8 weeks after infection (F). Cells of the pancreas from untreated normal B6 mice were served as control. Fig. 8. Fas-ligand and TNF-␣ expression in MNC infiltrating the pancreas of MAIDS mice. Real-time quantitative PCR of Fas-ligand (A) and TNF-␣ (B) mRNA expression in the pancreas of MAIDS mice. Each amount was normalized to the level of GAPDH, and the final relative values were expressed relative to calibrators on day 0. Results of three independent experiments are shown. Immunofluorescence of Fas-ligand (C), TNF-␣ (D), and Fas (E).
creas or in salivary and lacrimal glands [16]. Therefore, we cannot assume that the viral antigen of the p12 molecule is a target molecule on the pancreatic acinar cells for the pancreas-infiltrating T cells. However, the molecular mimicry mechanism cannot be excluded as an explanation of the organ specificity of the pancreatitis, and we are now attempting to determine potential target molecules. Recently, Th1 and Th2 imbalance has been considered one of the important mechanisms in the development of some autoimmune diseases [30]. Using double color IF study, we demonstrated a coexistence of Th1 and Th2 cells in the pancreatic lesions of mice with MAIDS, although there were more Th2 than Th1 cells. IL-10 is known to be an anti-inflammatory cytokine that inhibits a wide variety of leukocyte functions, including the production of inflammatory cytokines and the expression of costimulatory molecules [31]. In murine experimental acute pancreatitis, Van Laethem et al. reported that intraperitoneal recombinant IL-10 injection prevented necrosis of acinar cells, at least in part, by inhibition of TNF-␣ [32,33]. In addition, IL-10 neutralization by anti-IL-10 Ab worsened the acute pancre-
atitis of mice [34]. In patients with acute pancreatitis, the level of IL-10 is reported to be correlated with the disease severity [35]. Therefore, IL-10 produced by CD4⫹ T cells in the pancreas of mice with MAIDS might play a protective role in suppressing the further progression of pancreatitis. In contrast, IL-10 is considered a key cytokine in the pathogenesis of exocrinopathy in patients with SjS. Recently, Saito et al. reported that mice transgenic for IL-10 developed SjS-like exocrinopathy, suggesting an inflammatory role of this cytokine in the inflammatory lesions [36]. In addition, Wynn et al. have shown that IL-10 is an important endogenous down-regulator of type 2 as well as type 1 cytokine synthesis by their studies of granuloma formation in double cytokine-deficient mice [37]. Thus, IL-10 not only is a Th2 cytokine but also plays a critical role in multiple biologic events. The role of IL-10 in pancreatic lesions in MAIDS remains to be elucidated precisely. Unlike IL-10, IFN-␥ is an inflammatory cytokine and a Th1 type immune-response has been shown to be involved in the pathogenesis of autoimmune-related chronic pancreatitis. Okazaki et al. have reported that CD4⫹ T cells pro-
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duce interferon-␥ and that the secreted levels were significantly increased in patients with the disease [8]. IFN-␥ mainly produced by Th1 cells is an important factor responsible for macrophage activation, an event known to be pivotal in the development of inflammation and necrosis during acute pancreatitis [38]. The chemokine interferon-␥inducible protein 10 (IP-10) is known as a ligand of CXCR3 on Th1 cells [24]. We showed that neutralization of IP-10 by administration of anti-IP-mAb ameliorated the pancreatic lesions of mice with MAIDS (manuscript in preparation). These data suggest that Th1CD4⫹T cells attack and destroy the exocrine system of pancreas. Thus, because several distinct types of CD4⫹ T cells infiltrate and form the pancreatic lesions in mice with MAIDS, it is important to identify these CD4⫹ T cells and clarify how they initiate the first step of pancreatitis. In the mechanism of tissue destruction in autoimmunerelated pancreatitis, not only T cells but also macrophages are considered effecter cells by producing several cytokines and inflammatory mediators. Emmrich et al. reported that the immune cell infiltrates in chronic pancreatitis consist of 50% T lymphocytes and 30% macrophages [25]. Goecke et al. also reported similar data for patients with chronic pancreatitis of alcoholic and idiopathic origin [39]. In the pancreatic lesions of mice with MAIDS, we showed that Mac-1⫹ cells as well as CD4⫹ T cells predominated in the pancreas-infiltrating cells. We have also shown that adoptive transfer of lymph node cells of MAIDS mice induces inflammatory bowel disease-like colitis, termed MAIDS colitis, as well as SjS-like exocrinopathy and pancreatitis [18 –20]. The proportion of phenotypes and cytokine production pattern of inflammatory cells in the pancreas are the same as in the colitis lesions of MAIDS colitis [19 –21]. We considered that Mac-1⫹ cells and CD4⫹ T cells, producing IFN-␥ and IL-10, cooperatively form the pancreatitis, exocrinopathy, and colitis lesions in mice with MAIDS. Goecke et al. reported that the majority of CCR5-positive cells in chronic pancreatitis are macrophages and suggested that the concomitant upregulation of the CCR5 ligands RANTES and MIP-1␣ indicates that CCR5 is most likely involved in the attraction and activation of these macrophages [39]. To resolve this question it must be clarified whether Mac-1⫹ macrophages express CCR5 in the pancreas of mice with MAIDS. This study revealed that the severity of exocrine pancreatitis of MAIDS mice was relatively mild to moderate. The levels of serum amylase, lipase, and blood glucose were stable and showed no difference between mice with MAIDS and normal mice (unpublished observation). Glucose tolerance was also well preserved until later stages. To elucidate the inhibitory mechanism against progression of pancreatic lesions in MAIDS, we analyzed the role of apoptosis by the TUNEL method. There were some TUNEL⫹ apoptotic cells in the pancreas of mice with MAIDS, although most of them were among pancreas-infiltrating mononuclear cells; these cells are considered activated T lymphocytes to be elimi-
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nated through activation-induced cell death, in other words, Fas/FasL system-mediated apoptosis [40]. In contrast, TUNEL⫹ apoptotic acinar cells in MAIDS were fewer than in another experimental pancreatitis [41]. Activation of Fas by certain anti-Fas antibodies or by FasL results in apoptotic cell death in susceptible cells. Recently, Kornmann et al. reported that Fas mRNA is overexpressed in CP and that Fas and FasL colocalization was accompanied by marked activation of the apoptotic process in CP [42]. In WBN/Kob rats which spontaneously develop chronic pancreatitis, apoptosis was shown to be involved in the development of pancreatitis, and the Fas/FasL system was suggested as one of the mediators of apoptosis [41]. In the pancreas of MAIDS mice, we showed the moderate expression of mRNA and proteins of TNF-␣ and the weak expression of those of Fas and FasL. We suggest that the weak activation of Fas/FasL system might be attributed partially to the mild tissue destruction in the pancreatitis of MAIDS mice. In minor salivary glands of SjS, Xanthou et al. [43] reported that CD4⫹ cytotoxic cell populations exist that express perforin mRNA. They suggest that CD4⫹ cytotoxic cells utilizing perforin play a critical role in the immunopathological lesions of SjS. In MAIDS, the functions of both CD4⫹ T and CD8⫹ T cells are suppressed [13–15]; therefore, CD4⫹ cytotoxic cells might not participate in the pathogenesis of pancreatic lesions. In autoimmune-related chronic pancreatitis as well as MAIDS, the role of CD4⫹ cytotoxic cells must be elucidated more precisely. Additionally, the potential roles of cytokine and FasL could be established by infecting IFN-deficient, IL10-deficient, and gld mice on a B6 background with the virus. In conclusion, we have shown that chronic exocrine pancreatitis was induced in mice with MAIDS. The results suggest that the lesions, accompanied by systemic exocrinopathy resembling SjS, could be considered a model for autoimmune-related chronic pancreatitis associated with SjS. This model should provide valuable insights in the development of autoimmune-related pancreatitis.
Acknowledgments We thank Dr. Xiu-Hua Yang and Mr. Norio Honda for technical assistance and Dr. Minoru Nomoto and Dr. Terasu Honma for helpful discussions. This work was supported by grants from the Ministry of Education and Science and Technology and the Ministry of Health, Welfare, and Labor of the Government of Japan.
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www.elsevier.com/locate/yclim
Kinetic analysis of the development of pancreatic lesions in mice infected with a murine retrovirus Shiro Watanabe,a Kenji Suzuki,a,* Yusuke Kawauchi,a Satoshi Yamagiwa,a Hiroyuki Yoneyama,a Hiroshi Kawachi,b Yoshiaki Okada,c Fujio Shimizu,b Hitoshi Asakura,a and Yutaka Aoyagia a
b
Department of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan c Department of Bacterial and Blood Products, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan Received 28 April 2003; accepted with revision 8 July 2003
Abstract Sjo¨gren’s syndrome (SjS)-like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus, which induces a severe immunodeficiency termed murine AIDS (MAIDS). All mice with MAIDS showed advancing cellular infiltration around the pancreatic ducts as well as systemic exocrinopathy. The primary target tissue of the pancreas was acinar cells, and the pancreatic islets were well preserved until a late phase of the disease. Immunofluorescence and flow cytometry demonstrated that CD4⫹ T cells, Mac-1⫹ cells, and B220⫹ cells were major inflammatory components, and IFN-␥ and IL-10 were mainly detected on CD4⫹ T and Mac-1⫹ cells in the pancreas. Both Th1 and Th2 cells were found. TUNEL⫹ apoptotic cells were mostly detected among pancreasinfiltrating cells. Fas ligand and TNF-␣ were also detected among pancreas-infiltrating cells, whereas Fas was rarely expressed in the pancreatic acinar cells. Thus, MAIDS mice could be valuable for analyzing the pathogenesis of autoimmune-related pancreatitis associated with SjS. © 2003 Elsevier Inc. All rights reserved. Keywords: Autoimmune-related pancreatitis; Sjo¨gren’s syndrome; LP-BM5 murine leukemia virus; Murine AIDS; Interferon-␥; Interleukin-10; Apoptosis
Introduction In developed countries, most patients with chronic pancreatitis have a long history of alcohol abuse, which, therefore, is generally listed as the cause of the disease [1]. Nevertheless, approximately 30 to 40% of patients with chronic pancreatitis have no history of chronic alcoholism, so the etiology is regarded as idiopathic in these cases [1]. Since Sarles et al. reported a special form of chronic pancreatitis that may have been caused by “phenomena of self-immunization,” an autoimmune-related pathogenesis has been suggested in some cases of idiopathic chronic * Corresponding author. Department of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata City 951-8510, Japan. E-mail address: [email protected] (K. Suzuki). 1521-6616/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1521-6616(03)00197-9
pancreatitis [2,3]. Several authors have reported cases of chronic pancreatitis associated with Sjo¨gren’s syndrome (SjS) in which an autoimmune mechanism has been involved in the etiology and shown that steroid therapy was effective [4,5]. It has been suggested that there are two types of chronic pancreatitis with an autoimmune-related etiology [5–7]. One type is associated with another autoimmune disease such as SjS, primary biliary cirrhosis, primary sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis, Crohn’s disease), or systemic lupus erythematosus [4 –9]. Among them, SjS has the highest frequency of autoimmne-related pancreatitis [7]. Recently several reports have suggested another clinical type of chronic pancreatitis with autoimmune-related etiology that is not associated with another autoimmune disease [5,10]. In this type of chronic pancreatitis, high serum levels of IgG4 are characteristic and the values are closely associated with disease activity
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[10]. This disease has been referred to as sclerosing pancreatitis or autoimmune pancreatitis, and the clinicopathological features of this type of chronic pancreatitis mimic those of the disease associated with another autoimmune disease [10]. Thus, an autoimmune mechanism has been shown to play a rather large part in the pathogenesis of chronic pancreatitis [11,12]. However, the precise mechanism of the development of chronic pancreatitis with an autoimmune etiology is largely unknown. The LP-BM5 murine leukemia virus (MuLV) is a retrovirus that is known to induce profound immunodeficiency with splenomegaly and generalized lymphadenopathy in susceptible strains of mice such as C57BL/6(B6) and occasionally brings about lymphoid malignancy [13–15]. In the early phase of infection, hypergammaglobulinemia and polyclonal B and T cell activation were induced, and autoantibodies such as anti-nuclear and anti-ds-DNA antibodies were detected in the mice infected with the virus [13–15]. In the late phase of infection, the virus-infected B6 mice show symptoms similar to those of human acquired immunodeficiency syndrome (AIDS); therefore, they have been studied as a murine model of AIDS termed murine AIDS (MAIDS) [13–15]. We have reported previously that systemic exocrinopathy resembling SjS was induced in systemic exocrine glands such as salivary glands, lacrimal glands, and the pancreas of the virus-infected mice [16,17]. We have also shown that nude mice inoculated with lymph node cells and/or spleen cells from mice with MAIDS developed inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy [18 –21]. In MAIDS mice, the pancreatic lesions are characterized by inflammatory cell infiltration around the interlobular pancreatic ducts and acinar cell destruction [16,17]. Contrary to the injury to the exocrine system of the pancreas, endocrine pancreatic islets were well preserved until the late phase of the disease in the mice. Thus, the pancreatic lesions of the mice could be considered a model for chronic pancreatitis associated with SjS. To clarify the mechanism of the development of chronic pancreatitis with autoimmune-related etiology, especially in the form associated with SjS, we analyzed the kinetics of the development of chronic pancreatitis in mice with MAIDS.
Materials and methods
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Virus LP-BM5 MuLV was prepared from the supernatant of cloned G6 cells infected with the retrovirus. Twenty-fourhour culture supernatant of G6 cells contained approximately 5 ⫻ 104 plaque-forming units/ml of ecotropic virus, as determined by XC plaque assay. Aliquots containing the virus were stored at ⫺80°C until use. For the infection, 4-week-old B6 mice were inoculated intraperitoneally with 0.3 ml of the stock solution of LP-BM5 MuLV. Induction of MAIDS Four-week-old B6 female mice were injected intraperitoneally with 0.3 ml of LP-BM5 MuLV virus stock solution. Induction of MAIDS was confirmed when the mice developed splenomegaly and generalized lymphadenopathy. At 4, 8, 10, and 12 weeks after virus inoculation, mice with MAIDS were killed by cervical dislocation under ether anesthesia, and their pancreata were removed for further analysis. Age- and sex-matched B6 mice without virus inoculation were used as controls. Four mice were analyzed for each group and all experiments were repeated three or four times. Monoclonal antibodies For immunofluorescence and flow cytometric analyses, the following monoclonal antibodies were used: anti-CD4 (clone GK1.5, IgG2b), anti-CD8 (clone 53-6.7, IgG2a), anti-B220 (clone RA3-6B2, IgG2a), anti-Mac-1 (clone M-70.15, IgG2b), anti-mouse INF-␥ (clone XMG1.2), and anti-mouse IL-10 (clone JES5-16E3). Detection of LP-BM5 MuLV by PCR The PCR method used for detection of the virus was reported previously [16]. In brief, the oligonucleotides used as PCR primers were 5⬘-CCTCTTCCTTTATCGACACT-3⬘ and 5⬘-ATTAGGGGGGGAATAGCTCG-3⬘, which are present in the p15 and p12 regions of the gag gene, respectively. The template DNAs were extracted from the frozen blocks of pancreas and subjected to 30 cycles of amplification. The PCR products (237 bp) were separated and analyzed by electrophoresis on a 1.0% agarose gel and ethidium bromide staining.
Animals Quantitative RT-PCR to detect cytokine mRNAs Four-week-old female C57BL/6 (B6) mice were purchased from Charles River Japan (Atsugi, Kanagawa, Japan) and maintained at the Animal Center of Niigata University School of Medicine under specific-pathogen-free conditions. All animal experiments were performed according to the Guide for Animal Experiments of Niigata University School of Medicine.
Total RNA was extracted from pancreas specimens with Trizol (Gibco BRL) according to the standard protocol and reverse transcribed. Thereafter, cDNA was amplified using the ABI 7700 sequence-detector system (Applied Biosystems, Foster City, CA) with a set of primers and probes corresponding to IFN-␥ and IL-10 and glyceraldehyde-3-
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phosphate dehydrogenase (GAPDH) as previously described. Histopathological examination Tissue samples were taken from the pancreas, fixed in 10% buffered formalin, and then embedded in paraffin wax blocks. Sections 4 m thick were made in the usual way and stained with hematoxylin and eosin and the aldehyde–fuchsin Masson–Goldner staining method. The stained sections were then examined by light microscopy. To assess the degree of pancreatitis, the number of inflammatory cells in a high-power field (⫻400) were counted under a microscope. Cell numbers at three different points in pancreatic inflammatory focuses of each mouse were counted and the data of three mice of each group were compared statistically. The severity of each lesion in at least one section of pancreas was scored on a 0 – 4⫹ scale based on the histopathological changes described by Kanno et al. [22,23]. Briefly, 0, pancreas without mononuclear cell infiltration, indicating that it is almost normal; 1⫹, mononuclear cell aggregation and/or infiltration within the interstitium without any parenchymal destruction; 2⫹, focal parenchymal destruction with mononuclear cell infiltration; 3⫹, diffuse parenchymal destruction but some intact parenchymal residue is retained; 4⫹, almost all pancreatic tissue except pancreatic islets destroyed or replaced with fibrotic or adipose tissue. The maximal score was used as the pancreatitis grade of each individual. IF staining procedure Frozen sections of the pancreas were prepared in a cryostat and stained with several fluorescent-dye-conjugated anti-mouse antibodies as described above. The sections were observed by fluorescence microscopy. Double IF staining procedure For the simultaneous demonstration of cell surface antigens and cytokines, acetone-fixed frozen sections were incubated sequentially with biotinylated anti-cell surface antigen antibody and then with Alexa 594 (Molecular Probes, Inc.)-conjugated avidin as the first step. As the second step, they were incubated with fluorescein conjugated anti-cytokine antibody. The sections were observed by fluorescence microscopy. Controls for the double staining were prepared by omitting the primary antibodies in the first or second step. To assess the distinct cytokine expression, the number of CD4⫹ and Mac-1⫹ cells that were double-positive for IFN-␥ or IL-10 was counted under a microscope in a highpower field (⫻400). Cell numbers at three different points in the pancreas of each mouse were counted and the data of three mice of each group were compared statistically.
Terminal deoxynucleotide transferase labeling Apoptotic cells were identified using an in situ apoptosisdetection kit (Chemicon International, USA) according to the manufacturer’s instruction. In brief, acetone-fixed 5-m fresh-frozen pancreas sections were permeabilized on ice and incubated with the terminal deoxynucleotide transferase mixture for 1 h at 37°C. Biotin-labeled dNTP was treated with streptavidin HRP for 30 min, visualized with DAB, and counterstained with methyl green. Preparation and flow cytometric analysis of cells that infiltrated the pancreas Mice were anesthetized with ether and killed by cervical vertebral dislocation. The pancreas was taken and then pressed through a 200-gauge stainless-steel mesh and cells were suspended in Eagle’s minimum essential medium (MEM). After being washed twice in MEM, the cell suspension was used for flow cytometric analysis. The surface phenotypes of pancreas-infiltrating cells were analyzed using the dye-labeled monoclonal antibodies described above with a FACScan (Becton–Dickinson, CA). Statistical analysis Data are expressed as means ⫾ SD. Statistical analyses were performed using the unpaired Student t test or the nonparametric Mann–Whitney test. Differences were considered significant at P ⬍ 0.05.
Results Exocrine pancreatitis developed in mice with MAIDS All of the mice infected with LP-BM5 MuLV developed generalized lymphadenopathy and hepatosplenomegaly, and died with changes characteristic of MAIDS at around 12–16 weeks after the virus infection (n ⫽ 20). As we have previously reported, periductal mononuclear cellular infiltration resembling SjS was detected in salivary glands and lacrimal glands beginning at around 4 weeks after infection and continuing thereafter. In the pancreas of the mice, mononuclear cellular infiltration was observed at around 4 weeks after infection and the number of infiltrating cells then increased gradually (Fig. 1A–D and H). The inflammatory cells were initially detected around the pancreatic ducts, from where they progressively expanded, pressing the acinar architecture outward. At the interface lesions between the infiltrating cells and the pancreatic parenchyma, destructed acinar cells were detected occasionally beginning at about 4 weeks after infection, and their number then increased gradually and reached a peak at 8 and 12 weeks after infection (Fig. 1D and I). However, the degree of acinar cell destruction by infiltrating cells was rather mild
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Fig. 1. Development of pancreatic lesions in mice with MAIDS. The number of inflammatory cells infiltrating into the pancreas of MAIDS mice increased and reached a peak at 8 weeks after infection. Pancreas from mice infected with LP-BM5 at 4 weeks (A), 8 weeks (B, D, G), and 12 weeks (C) after infection, and pancreas from normal mice (E, F). (A–E); hematoxylin and eosin staining. Original magnification, ⫻200. (F,G); Aldehyde–fuchsin Masson–Goldner staining. Original magnification, ⫻400. The number of cells infiltrating the pancreas after infection (H), and histological score of acinar cell destruction of the pancreas (I). The data were counted under a microscope at high-power magnification for three different areas per mouse and the data of each time point were collected from three mice and compared with each other. Fig. 2. Detection of LP-BM5 MuLV in the pancreas of MAIDS mice. (A) The template DNAs were extracted from frozen sections of the pancreas and then analyzed by PCR. Lane M, molecular size marker; lane 1, MAIDS spleen cells (positive control); lane 2, the pancreas of untreated B6 mice (negative control); lane 3, the pancreas of MAIDS mice 4 weeks after infection; lane 4, the pancreas of MAIDS mice 8 weeks after infection; lane 5, the pancreas of MAIDS mice 12 weeks after infection. (B) immunostaining for the p12 antigen of LP-BM5 MuLV in the pancreas of MAIDS mice.
and parenchymal fibrosis, edema, and hemorrhage were rarely observed (Fig. 1D and I). Peri-islet cellular infiltration was observed frequently, but Langerhans islets were well preserved until a late phase and no marked insulitis was detected (Fig. 1D–G). Detection of LP-BM5 MuLV in the pancreas of mice with MAIDS A defective LP-BM5 virus genome was detected by PCR in the same frozen sections of the pancreas of mice with MAIDS as those used for immunohistochemical staining to detect the virus (Fig. 2A). Immunohistochemistry using anti-gag p12 antibody revealed that the viral antigen was
present on some of the pancreas-infiltrating cells, but not on acinar cells or duct epithelial cells of the pancreas (Fig. 2B). Pancreas-infiltrating cells were composed of CD4⫹T, Mac-1⫹, and B220⫹ cells in mice with MAIDS We further characterized the phenotype of inflammatory cells in the pancreas in situ using immunofluorescence. Infiltrating cells were composed of CD4⫹, CD8⫹T cells, Mac-1⫹ macropahges, and B220⫹ B cells but not NK cells or granulocytes (Fig. 3A–D). The ratio of CD4⫹T cells, Mac-1⫹, and B220⫹ cells was nearly equal except for a minor population of CD8⫹T cells. The number of infiltrating CD4⫹T cells, Mac-1⫹, and B220⫹ cells reached a peak
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Fig. 3. Kinetic appearance of lymphocyte subpopulations in the pancreas of MAIDS mice. Immunofluorescenc for CD4 (A), CD8 (B), Mac-1 (C), and B220 (D). Quantitative analysis of immune cells infiltrating the pancreas (E). Each focus of cellular infiltration was examined for the presence of CD4, CD8, B220, and Mac-1 cells. Final results are presented as the number per high-powered microscope field. Representative findings (means ⫾ standard deviation, n ⫽ 10) from three independent experiments are shown. *Data are significantly different from the number of CD8⫹ cells at each time point as determined by Student’s t test (P ⬍ 0.05). Fig. 6. Double-color immunofluorescence staining for CD4 and IFN-␥ (A-C), CD4 and IL-10 (D-F), Mac-1 and IFN-␥ (G-I), and Mac-1 and IL-10 (J-L) of the pancreas of MAIDS mice 8 weeks after infection. CD4 (A, D, C, F; red) and Mac-1 (G, I, J, L; red); IFN-␥ (B, C, H, I; green), and IL-10 (E, F, K, L; green).
at 8 weeks and stayed at that level throughout the infection (Fig. 3E). We also confirmed the number of each phenotype of infiltrating cells with flow cytometry, and it was revealed that the major populations of pancreas-infiltrating cells comprised CD4⫹T cells with ␣T cell receptors, Mac-1⫹, and B220⫹ cells (Fig. 4). The lower number of Mac-1⫹ cells, compared with that found by IF, was probably caused by loss through their tendency to adhesion during the preparation of pancreas-infiltrating cells for flow cytometry. Only negligible numbers of NK1.1⫹ NK cells and Gr1⫹ granulocytes were found. IFN-␥ and IL-10 were produced by CD4⫹T and Mac-1⫹ cells in the pancreatic lesions of mice with MAIDS To reveal the immune response in the pancreas of mice with MAIDS, we analyzed the cytokine mRNA expression of IFN-␥ and IL-10 by RT-PCR. We chose IFN-␥ as a
representative for a proinflammatory cytokine (or Th1 response) and IL-10 as a representative of an anti-inflammatory cytokine (or Th2). The levels of expression of the mRNAs of both cytokines were significantly increased after the infection (Fig. 5A and B). Immunofluorescence showed that IFN-␥- and IL-10-positive cells were detected through 4 to 12 weeks after infection, and throughout the course the number of IL-10-positive cells was larger, but not statistically significantly, than that of IFN-␥-positive ones (Fig. 5C). Next we characterized the phenotypes of cells producing these cytokines by a double-color immunofluorescence method. IFN-␥ and IL-10 were mainly present on CD4⫹ T cells (Fig. 6A–F) and Mac-1⫹ cells (Fig. 6G–L), but not on B220⫹ or CD8⫹ T cells (data not shown). We could detect neither mRNA nor protein expression for IFN-␥ or IL-10 in the presence of normal B6 mice. Some CD4⫹T cells expressing either IFN-␥ or IL-10
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were detected at the 4th week in the pancreas of MAIDS mice, and their number significantly increased and reached a plateau at 8 weeks after infection (Fig. 6A–F and 5D). The number of IL-10-positive CD4⫹ T cells was significantly greater than that of IFN-␥ -positive CD4⫹T cells through 4 to 12 weeks after infection (Fig. 6A–F and 5D). Some Mac-1⫹ cells also expressed either IFN-␥ or IL-10 in the pancreas of MAIDS mice at 4 weeks after infection. These cells increased remarkably at 8 and 12 weeks after infection, and the numbers of IFN-␥-positive Mac-1⫹ cells and IL-10-positive Mac-1⫹ cells were almost equal through the course (Fig. 6G–L and 5E). Apoptotic cells were mostly detected among pancreasinfiltrating cells in mice with MAIDS Histological analysis revealed that advancing pancreatic lesions were composed of inflammatory cellular infiltration around the interlobular pancreatic ducts and scattered destruction of acinar cells. We further analyzed the role of apoptosis in tissue destruction of the pancreas using terminal deoxynucleotide transferase labeling (TUNEL) staining. Few TUNEL⫹ acinar cells were detected in the interface lesions of each inflammatory focus in the pancreas of MAIDS mice (Fig. 7A–C and E). The results suggest that apoptosis might not be the primary type of cell death of acinar cells in the pancreatic lesions of these mice. In contrast to the paucity of apoptotic acinar cells, TUNEL⫹ mononuclear cells (MNCs) were detected, and their number increased gradually in each focus of the pancreas of MAIDS mice (Fig. 7A and D). Flow cytometric analysis for the pancreas-infiltrating MNCs also revealed that annexin-Vpositive cells were detected and increased in number after infection (Fig. 7F). Fas ligand and TNF-␣ were detected among pancreasinfiltrating inflammatory cells, whereas Fas was rarely expressed in the pancreas of mice with MAIDS As reported in cases with chronic pancreatitis, the Fas/ Fas-ligand system and TNF-␣ are supposed to be important mechanisms involved in the apoptosis of pancreatic acinar cells in autoimmune-related pancreatitis [24,25]. Therefore, we next analyzed the Fas/FasL and TNF-␣ in the pancareas of the MAIDS mice. The expression levels of the mRNAs of FasL and TNF-␣ were increased at 8 weeks after infection in the pancreas of mice with MAIDS (Fig. 8A and B). Immunohistochemistry revealed that FasL and TNF-␣ were expressed on some of the MNCs in the inflammatory focus of the pancreas of MAIDS mice (Fig. 8C and D) and that
Fas was positive on a few MNCs but negative on acinar cells of the pancreas (Fig. 8E). In the pancreas of untreated control mice, FasL, TNF-␣, and Fas were not detected by immunohistochemisry.
Discussion In this report, we demonstrated that advancing cellular infiltration in the pancreas was induced in mice with MAIDS, and the lesions have characteristics similar to those in the exocrine glands in mice with MAIDS that we have reported previously [16,17]. The pancreas is composed of the exocrine tissue of acinar cells and the endocrine tissue of islet cells. The present histopathological study revealed that the lesions in the pancreas of mice with MAIDS are more dominant in the exocrine than the endocrine system. The common pathological characteristics of most cases of autoimmune-related pancreatitis are fibrotic changes with lymphocytic infiltration, absence of pancreatic calcificaiton, and absence of pancreatic cysts [7,8]. The endocrine tissue of pancreatic islets might be involved in the disease, but the primary arena of inflammation is the pancreatic parenchyma, that is, the exocrine system [7,8], as it is in mice with MAIDS. Some unknown target antigens, adhesion molecules, and chemokines might regulate the organ specificity of the lesions of mice with MAIDS, as is the case with several autoimmune diseases, including SjS and autoimmune-related pancreatitis [11]. We have reported that CD4⫹ T cells from MAIDS were able to induce lesions in exocrine glands, such as the salivary glands and pancreas, but could not induce colitis when transfered to B6 nude mice [21]. We speculate that the most important population of MAIDS cells to induce exocrinopathy is T, especially CD4⫹ T cell population (manuscript in preparation). We have observed oligoclonal expansion of T cell recepter ␣ CD4⫹ T cell clones in salivary glands and spleen in mice with MAIDS (unpublished data), and these CD4⫹ T cell clones might recognize some common target antigens in the pancreas and systemic exocrine glands of mice with MAIDS. We reported previously that the virus was integrated in Ly-1 B cells but not in T cells [26], although several reports showed that B cells [27], and macrophages [28], as well as T cells [29], can serve as targets for infection of the virus. A double color immunofluorescence study revealed that the viral antigen was detected on some of B220⫹ and Mac-1⫹ cells, but not on CD4⫹ or CD8⫹ cells (unpublished observation). Using electron microscopy and IF with anti-p 12 antibody, we were unable to detect the virus on acinar cells of the pan-
Fig. 4. Phenotypic characterization of mononuclear cells in the pancreas of MAIDS mice. Two-color staining for B220 and CD3, CD4 and CD8, and TCR-␣ and TCR-␥␦. Single-color staining for Mac-1. Pancreas-infiltrating mononuclear cells in MAIDS mice infected after 4 and 10 weeks were analyzed by flow cytometry; pancreas-residing mononuclear cells of untreated B6 mice were used as controls. Numbers represent the percentages of fluorescence-positive cells in corresponding areas. Representative results of three experiments are depicted.
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Fig. 5. Kinetic analyses of cytokine production by MNCs infiltrating the pancreas in mice with MAIDS. (A, B) Real-time quantitative PCR of IFN-␥ (A) and IL-10 (B) mRNA expression in the pancreas of MAIDS mice. Each amount was normalized to the level of GAPDH and the final relative values were expressed relative to calibrators on day 0. Kinetic quantitative analysis of IFN-␥ and IL-10 expression patterns of cells that infiltrated the pancreas at 4, 8, and 12 weeks after infection (C). Stained cryostat sections of three focuses of cellular infiltration of the individual pancreas were examined by counting the number of cells expressing IFN-␥ and cells expressing IL-10. Three mice were analyzed for each time point. In control normal B6 mice, a negligible number of cells expressed IFN-␥ or IL-10, so the data are not shown. Kinetic quantitative analysis of IFN-␥ and IL-10 expression patterns of CD4⫹ and Mac-1⫹ cells that infiltrated the pancreas at 4, 8, and 12 weeks after infection (D, E). Stained cryostat sections of three focuses of cellular infiltration of the individual pancreas were examined by counting the number of CD4⫹ cells expressing IFN-␥ or IL-10 and Mac-1⫹ cells expressing IFN-␥ or IL-10. Three mice were analyzed for each time point. *P ⬍ 0.01. In control normal B6 mice, a negligible number of cells expressed IFN-␥ or IL-10, so the data are not shown.
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Fig. 7. Immunostaining for TUNEL⫹ apoptotic cells in the pancreas of MAIDS mice. TUNEL⫹ cells are colored brown. Positive cells are scattered within a focus of cellular infiltration around the pancreatic duct (A), and there are few positive cells among destructed acinar cells at the interface lesions of a cellular focus of the pancreas (B, C) of MAIDS mice 8 weeks after infection. The arrow indicates TUNEL⫹ apoptotic acinar cells, and the arrowhead indicates TUNEL-negative necrotic acinar cells. Quantitative analysis of TUNEL⫹ mononuclear and acinar cells in the pancreas (D, E). Numbers of TUNEL⫹ mononuclear (D) and acinar cells (E) at three different points in pancreatic inflammatory focuses of each mouse were counted, and the data of three mice with MAIDS are shown. The pancreas-infiltrating cells were analyzed with flow cytometry for expression of annexin-V at 8 weeks after infection (F). Cells of the pancreas from untreated normal B6 mice were served as control. Fig. 8. Fas-ligand and TNF-␣ expression in MNC infiltrating the pancreas of MAIDS mice. Real-time quantitative PCR of Fas-ligand (A) and TNF-␣ (B) mRNA expression in the pancreas of MAIDS mice. Each amount was normalized to the level of GAPDH, and the final relative values were expressed relative to calibrators on day 0. Results of three independent experiments are shown. Immunofluorescence of Fas-ligand (C), TNF-␣ (D), and Fas (E).
creas or in salivary and lacrimal glands [16]. Therefore, we cannot assume that the viral antigen of the p12 molecule is a target molecule on the pancreatic acinar cells for the pancreas-infiltrating T cells. However, the molecular mimicry mechanism cannot be excluded as an explanation of the organ specificity of the pancreatitis, and we are now attempting to determine potential target molecules. Recently, Th1 and Th2 imbalance has been considered one of the important mechanisms in the development of some autoimmune diseases [30]. Using double color IF study, we demonstrated a coexistence of Th1 and Th2 cells in the pancreatic lesions of mice with MAIDS, although there were more Th2 than Th1 cells. IL-10 is known to be an anti-inflammatory cytokine that inhibits a wide variety of leukocyte functions, including the production of inflammatory cytokines and the expression of costimulatory molecules [31]. In murine experimental acute pancreatitis, Van Laethem et al. reported that intraperitoneal recombinant IL-10 injection prevented necrosis of acinar cells, at least in part, by inhibition of TNF-␣ [32,33]. In addition, IL-10 neutralization by anti-IL-10 Ab worsened the acute pancre-
atitis of mice [34]. In patients with acute pancreatitis, the level of IL-10 is reported to be correlated with the disease severity [35]. Therefore, IL-10 produced by CD4⫹ T cells in the pancreas of mice with MAIDS might play a protective role in suppressing the further progression of pancreatitis. In contrast, IL-10 is considered a key cytokine in the pathogenesis of exocrinopathy in patients with SjS. Recently, Saito et al. reported that mice transgenic for IL-10 developed SjS-like exocrinopathy, suggesting an inflammatory role of this cytokine in the inflammatory lesions [36]. In addition, Wynn et al. have shown that IL-10 is an important endogenous down-regulator of type 2 as well as type 1 cytokine synthesis by their studies of granuloma formation in double cytokine-deficient mice [37]. Thus, IL-10 not only is a Th2 cytokine but also plays a critical role in multiple biologic events. The role of IL-10 in pancreatic lesions in MAIDS remains to be elucidated precisely. Unlike IL-10, IFN-␥ is an inflammatory cytokine and a Th1 type immune-response has been shown to be involved in the pathogenesis of autoimmune-related chronic pancreatitis. Okazaki et al. have reported that CD4⫹ T cells pro-
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duce interferon-␥ and that the secreted levels were significantly increased in patients with the disease [8]. IFN-␥ mainly produced by Th1 cells is an important factor responsible for macrophage activation, an event known to be pivotal in the development of inflammation and necrosis during acute pancreatitis [38]. The chemokine interferon-␥inducible protein 10 (IP-10) is known as a ligand of CXCR3 on Th1 cells [24]. We showed that neutralization of IP-10 by administration of anti-IP-mAb ameliorated the pancreatic lesions of mice with MAIDS (manuscript in preparation). These data suggest that Th1CD4⫹T cells attack and destroy the exocrine system of pancreas. Thus, because several distinct types of CD4⫹ T cells infiltrate and form the pancreatic lesions in mice with MAIDS, it is important to identify these CD4⫹ T cells and clarify how they initiate the first step of pancreatitis. In the mechanism of tissue destruction in autoimmunerelated pancreatitis, not only T cells but also macrophages are considered effecter cells by producing several cytokines and inflammatory mediators. Emmrich et al. reported that the immune cell infiltrates in chronic pancreatitis consist of 50% T lymphocytes and 30% macrophages [25]. Goecke et al. also reported similar data for patients with chronic pancreatitis of alcoholic and idiopathic origin [39]. In the pancreatic lesions of mice with MAIDS, we showed that Mac-1⫹ cells as well as CD4⫹ T cells predominated in the pancreas-infiltrating cells. We have also shown that adoptive transfer of lymph node cells of MAIDS mice induces inflammatory bowel disease-like colitis, termed MAIDS colitis, as well as SjS-like exocrinopathy and pancreatitis [18 –20]. The proportion of phenotypes and cytokine production pattern of inflammatory cells in the pancreas are the same as in the colitis lesions of MAIDS colitis [19 –21]. We considered that Mac-1⫹ cells and CD4⫹ T cells, producing IFN-␥ and IL-10, cooperatively form the pancreatitis, exocrinopathy, and colitis lesions in mice with MAIDS. Goecke et al. reported that the majority of CCR5-positive cells in chronic pancreatitis are macrophages and suggested that the concomitant upregulation of the CCR5 ligands RANTES and MIP-1␣ indicates that CCR5 is most likely involved in the attraction and activation of these macrophages [39]. To resolve this question it must be clarified whether Mac-1⫹ macrophages express CCR5 in the pancreas of mice with MAIDS. This study revealed that the severity of exocrine pancreatitis of MAIDS mice was relatively mild to moderate. The levels of serum amylase, lipase, and blood glucose were stable and showed no difference between mice with MAIDS and normal mice (unpublished observation). Glucose tolerance was also well preserved until later stages. To elucidate the inhibitory mechanism against progression of pancreatic lesions in MAIDS, we analyzed the role of apoptosis by the TUNEL method. There were some TUNEL⫹ apoptotic cells in the pancreas of mice with MAIDS, although most of them were among pancreas-infiltrating mononuclear cells; these cells are considered activated T lymphocytes to be elimi-
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nated through activation-induced cell death, in other words, Fas/FasL system-mediated apoptosis [40]. In contrast, TUNEL⫹ apoptotic acinar cells in MAIDS were fewer than in another experimental pancreatitis [41]. Activation of Fas by certain anti-Fas antibodies or by FasL results in apoptotic cell death in susceptible cells. Recently, Kornmann et al. reported that Fas mRNA is overexpressed in CP and that Fas and FasL colocalization was accompanied by marked activation of the apoptotic process in CP [42]. In WBN/Kob rats which spontaneously develop chronic pancreatitis, apoptosis was shown to be involved in the development of pancreatitis, and the Fas/FasL system was suggested as one of the mediators of apoptosis [41]. In the pancreas of MAIDS mice, we showed the moderate expression of mRNA and proteins of TNF-␣ and the weak expression of those of Fas and FasL. We suggest that the weak activation of Fas/FasL system might be attributed partially to the mild tissue destruction in the pancreatitis of MAIDS mice. In minor salivary glands of SjS, Xanthou et al. [43] reported that CD4⫹ cytotoxic cell populations exist that express perforin mRNA. They suggest that CD4⫹ cytotoxic cells utilizing perforin play a critical role in the immunopathological lesions of SjS. In MAIDS, the functions of both CD4⫹ T and CD8⫹ T cells are suppressed [13–15]; therefore, CD4⫹ cytotoxic cells might not participate in the pathogenesis of pancreatic lesions. In autoimmune-related chronic pancreatitis as well as MAIDS, the role of CD4⫹ cytotoxic cells must be elucidated more precisely. Additionally, the potential roles of cytokine and FasL could be established by infecting IFN-deficient, IL10-deficient, and gld mice on a B6 background with the virus. In conclusion, we have shown that chronic exocrine pancreatitis was induced in mice with MAIDS. The results suggest that the lesions, accompanied by systemic exocrinopathy resembling SjS, could be considered a model for autoimmune-related chronic pancreatitis associated with SjS. This model should provide valuable insights in the development of autoimmune-related pancreatitis.
Acknowledgments We thank Dr. Xiu-Hua Yang and Mr. Norio Honda for technical assistance and Dr. Minoru Nomoto and Dr. Terasu Honma for helpful discussions. This work was supported by grants from the Ministry of Education and Science and Technology and the Ministry of Health, Welfare, and Labor of the Government of Japan.
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