Overexpression of Interleukin-1β in the Murine Pancreas Results in Chronic Pancreatitis
2008; Elsevier BV; Volume: 135; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2008.06.078
ISSN1528-0012
AutoresFrédéric Marrache, Shui Ping Tu, Govind Bhagat, Swaroop Pendyala, Christoph H. Österreicher, Shanisha Gordon, Vigneshwaran Ramanathan, Melitta Penz-Österreicher, Kelly S. Betz, Zhigang Song, Timothy C. Wang,
Tópico(s)Phagocytosis and Immune Regulation
ResumoBackground & Aims: Chronic pancreatitis is a significant cause of morbidity and a known risk factor for pancreatic adenocarcinoma. Interleukin-1β is a proinflammatory cytokine involved in pancreatic inflammation. We sought to determine whether targeted overexpression of interleukin-1β in the pancreas could elicit localized inflammatory responses and chronic pancreatitis. Methods: We created a transgenic mouse model (elastase sshIL-1β) in which the rat elastase promoter drives the expression of human interleukin-1β. Mice were followed up for up to 2 years. Pancreata of elastase sshIL-1β mice were analyzed for chronic pancreatitis-associated histologic and molecular changes. To study the potential effect of p53 mutation in chronic pancreatitis, elastase sshIL-1β mice were crossed with p53R172H mice. Results: Three transgenic lines were generated, and in each line the pancreas was atrophic and occasionally showed dilation of pancreatic and biliary ducts secondary to proximal fibrotic stenosis. Pancreatic histology showed typical features of chronic pancreatitis. There was evidence for increased acinar proliferation and apoptosis, along with prominent expression of tumor necrosis factor-α; chemokine (C-X-C motif) ligand 1; stromal cell-derived factor 1; transforming growth factor-β1; matrix metallopeptidase 2, 7, and 9; inhibitor of metalloproteinase 1; and cyclooxygenase 2. The severity of the lesions correlated well with the level of human interleukin-1β expression. Older mice displayed acinar-ductal metaplasia but did not develop mouse pancreatic intraepithelial neoplasia or tumors. Elastase sshIL-1β*p53R172H/+ mice had increased frequency of tubular complexes, some of which were acinar-ductal metaplasia. Conclusions: Overexpression of interleukin-1β in the murine pancreas induces chronic pancreatitis. Elastase sshIL-1β mice consistently develop severe chronic pancreatitis and constitute a promising model for studying chronic pancreatitis and its relationship with pancreatic adenocarcinoma. Background & Aims: Chronic pancreatitis is a significant cause of morbidity and a known risk factor for pancreatic adenocarcinoma. Interleukin-1β is a proinflammatory cytokine involved in pancreatic inflammation. We sought to determine whether targeted overexpression of interleukin-1β in the pancreas could elicit localized inflammatory responses and chronic pancreatitis. Methods: We created a transgenic mouse model (elastase sshIL-1β) in which the rat elastase promoter drives the expression of human interleukin-1β. Mice were followed up for up to 2 years. Pancreata of elastase sshIL-1β mice were analyzed for chronic pancreatitis-associated histologic and molecular changes. To study the potential effect of p53 mutation in chronic pancreatitis, elastase sshIL-1β mice were crossed with p53R172H mice. Results: Three transgenic lines were generated, and in each line the pancreas was atrophic and occasionally showed dilation of pancreatic and biliary ducts secondary to proximal fibrotic stenosis. Pancreatic histology showed typical features of chronic pancreatitis. There was evidence for increased acinar proliferation and apoptosis, along with prominent expression of tumor necrosis factor-α; chemokine (C-X-C motif) ligand 1; stromal cell-derived factor 1; transforming growth factor-β1; matrix metallopeptidase 2, 7, and 9; inhibitor of metalloproteinase 1; and cyclooxygenase 2. The severity of the lesions correlated well with the level of human interleukin-1β expression. Older mice displayed acinar-ductal metaplasia but did not develop mouse pancreatic intraepithelial neoplasia or tumors. Elastase sshIL-1β*p53R172H/+ mice had increased frequency of tubular complexes, some of which were acinar-ductal metaplasia. Conclusions: Overexpression of interleukin-1β in the murine pancreas induces chronic pancreatitis. Elastase sshIL-1β mice consistently develop severe chronic pancreatitis and constitute a promising model for studying chronic pancreatitis and its relationship with pancreatic adenocarcinoma. Chronic pancreatitis leads to a progressive and irreversible destruction of the pancreatic gland.1Witt H. Apte M.V. Keim V. et al.Chronic pancreatitis: challenges and advances in pathogenesis, genetics, diagnosis, and therapy.Gastroenterology. 2007; 132: 1557-1573Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar In advanced cases, this may result in pancreatic exocrine insufficiency and/or diabetes. Significant morbidity is also associated with occasional biliary or pancreatic ductal obstruction secondary to fibrotic stenosis or ductal obliteration. Furthermore, chronic pancreatitis is a well-described risk factor for pancreatic adenocarcinoma,2Malka D. Hammel P. Maire F. et al.Risk of pancreatic adenocarcinoma in chronic pancreatitis.Gut. 2002; 51: 849-852Crossref PubMed Scopus (390) Google Scholar, 3Lowenfels A.B. Maisonneuve P. Cavallini G. et al.Pancreatitis and the risk of pancreatic cancer International Pancreatitis Study Group.N Engl J Med. 1993; 328: 1433-1437Crossref PubMed Scopus (1575) Google Scholar especially in cases of hereditary chronic pancreatitis.4Lowenfels A.B. Maisonneuve P. DiMagno E.P. et al.Hereditary pancreatitis and the risk of pancreatic cancer International Hereditary Pancreatitis Study Group.J Natl Cancer Inst. 1997; 89: 442-446Crossref PubMed Scopus (857) Google Scholar Although the pathogenesis of the initial pancreatic lesion depends on the etiology of the disease, it is believed that subsequent progression to chronic pancreatitis implies a common mechanism. Two main hypotheses have been proposed. The first one is called the necrosis-fibrosis sequence hypothesis and is based on clinical and experimental data, which suggest that chronic pancreatitis could result from repetitive episodes of acute pancreatitis. However, chronic pancreatitis is sometimes observed without evidence of associated necrosis, suggesting a different pathogenic mechanism, also referred to as the sentinel acute pancreatitis episode hypothesis. According to the latter, an initial episode of acute pancreatitis elicits chronic inflammation by recruiting immune cells and activating pancreatic stellate cells. Finally, it is possible that both mechanisms occur simultaneously and that their respective contributions vary depending on the etiology of the disease and the presence of additional host or environmental factors. Several mouse models of chronic pancreatitis have been reported to date, supporting the different aforementioned hypotheses. The most commonly employed model consists of repeated injections of cerulein, an analogue of cholecystokinin, which induces chronic pancreatitis if injected repeatedly over a sufficient period of time, in agreement with the necrosis-fibrosis sequence hypothesis.5Neuschwander-Tetri B.A. Burton F.R. Presti M.E. et al.Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.Dig Dis Sci. 2000; 45: 665-674Crossref PubMed Scopus (99) Google Scholar Pancreatic duct ligation is another model that can induce inflammation and pancreatic atrophy.6Watanabe S. Abe K. Anbo Y. et al.Changes in the mouse exocrine pancreas after pancreatic duct ligation: a qualitative and quantitative histological study.Arch Histol Cytol. 1995; 58: 365-374Crossref PubMed Scopus (74) Google Scholar Recently, transgenic mouse models have been developed, some of which are based on genetic mutations associated with hereditary pancreatitis. Archer et al reported a model relying on the acinar expression of a mutated version of cationic trypsinogen PRSS1,7Archer H. Jura N. Keller J. et al.A mouse model of hereditary pancreatitis generated by transgenic expression of R122H trypsinogen.Gastroenterology. 2006; 131: 1844-1855Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and Durie et al reported cystic fibrosis transmembrane conductance regulator knockout mice.8Durie P.R. Kent G. Phillips M.J. et al.Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model.Am J Pathol. 2004; 164: 1481-1493Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Both mouse models showed somewhat mild pancreatic lesions. Other models have also been described with a variable degree of similarity with human chronic pancreatitis. These are based on defective ciliary function,9Cano D.A. Sekine S. Hebrok M. Primary cilia deletion in pancreatic epithelial cells results in cyst formation and pancreatitis.Gastroenterology. 2006; 131: 1856-1869Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar overexpression of keratin 8,10Casanova M.L. Bravo A. Ramirez A. et al.Exocrine pancreatic disorders in transgenic mice expressing human keratin 8.J Clin Invest. 1999; 103: 1587-1595Crossref PubMed Scopus (69) Google Scholar disruption of genes encoding factors associated with the transforming growth factor (TGF)-β signaling pathway,11Bottinger E.P. Jakubczak J.L. Roberts I.S. et al.Expression of a dominant-negative mutant TGF-β type II receptor in transgenic mice reveals essential roles for TGF-β in regulation of growth and differentiation in the exocrine pancreas.EMBO J. 1997; 16: 2621-2633Crossref PubMed Scopus (221) Google Scholar and knockout of PARK,12Harding H.P. Zeng H. Zhang Y. et al.Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival.Mol Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar or EIF1 and 2.13Iglesias A. Murga M. Laresgoiti U. et al.Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.J Clin Invest. 2004; 113: 1398-1407Crossref PubMed Scopus (84) Google Scholar An interesting observation in human patients is the variability in individual susceptibility to chronic pancreatitis.14Uomo G. Manes G. Risk factors of chronic pancreatitis.Dig Dis. 2007; 25: 282-284Crossref PubMed Scopus (10) Google Scholar This could be related to variability in the immune response, which has been demonstrated to influence the penetrance and severity of chronic inflammatory diseases. Interleukin (IL)-1β is an inflammatory cytokine,15Bjorkdahl O. Akerblad P. Gjorloff-Wingren A. et al.Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1β gene product.Immunology. 1999; 96: 128-137Crossref PubMed Scopus (8) Google Scholar, 16Lappalainen U. Whitsett J.A. Wert S.E. et al.Interleukin-1β causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung.Am J Respir Cell Mol Biol. 2005; 32: 311-318Crossref PubMed Scopus (373) Google Scholar and polymorphisms in this gene have been shown to affect the immune response.17Macarthur M. Hold G.L. El-Omar E.M. Inflammation and cancer II Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G515-G520Crossref PubMed Scopus (342) Google Scholar This cytokine has also been shown to have an important role in the pathogenesis of pancreatitis.18Norman J.G. Fink G.W. Sexton C. et al.Transgenic animals demonstrate a role for the IL-1 receptor in regulating IL-1β gene expression at steady-state and during the systemic stress induced by acute pancreatitis.J Surg Res. 1996; 63: 231-236Abstract Full Text PDF PubMed Scopus (42) Google Scholar We sought to determine whether local activation of the immune system within the pancreas could elicit chronic pancreatitis. We hypothesized that expression of a single proinflammatory cytokine in the pancreas could induce chronic pancreatitis, and, consequently, we created a transgenic mouse, elastase sshIL-1β, in which overexpression of IL-1β is targeted to the pancreas using the elastase promoter.19Heller R.S. Stoffers D.A. Bock T. et al.Improved glucose tolerance and acinar dysmorphogenesis by targeted expression of transcription factor PDX-1 to the exocrine pancreas.Diabetes. 2001; 50: 1553-1561Crossref PubMed Scopus (50) Google Scholar Elastase sshIL-1β mice consistently developed severe chronic pancreatitis that exhibited an early onset and phenocopied well the human disease. All mice procedures were performed according to the protocol (AC-AAAA5113) approved by the Institutional Animal Use and Care Committee of Columbia University. The sshIL-1β gene15Bjorkdahl O. Akerblad P. Gjorloff-Wingren A. et al.Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1β gene product.Immunology. 1999; 96: 128-137Crossref PubMed Scopus (8) Google Scholar, 20Wingren A.G. Bjorkdahl O. Labuda T. et al.Fusion of a signal sequence to the interleukin-1 β gene directs the protein from cytoplasmic accumulation to extracellular release.Cell Immunol. 1996; 169: 226-237Crossref PubMed Scopus (34) Google Scholar was amplified by polymerase chain reaction (PCR), adding EcoRI restriction sites at 5′ and 3′ ends. After EcoRI digestion, sshIL-1β was inserted downstream the rat elastase promoter (kind gift from Doris Stoffer19Heller R.S. Stoffers D.A. Bock T. et al.Improved glucose tolerance and acinar dysmorphogenesis by targeted expression of transcription factor PDX-1 to the exocrine pancreas.Diabetes. 2001; 50: 1553-1561Crossref PubMed Scopus (50) Google Scholar) and the rabbit β-globin enhancer, and upstream a rabbit β-globin polyA sequence (Figure 1A). Orientation was checked by HindIII digestion, and the plasmid was sequenced. Elastase sshIL-1β transgene was then extracted from pBluescript by Xho and Xba digestion and microinjected in the pronucleus of fertilized CBA × C57BL/6J oocytes. Pups were genotyped using 3 different pairs of primers spanning the entire transgene (see supplementary Table 1, genotyping primers, online at www.gastrojournal.org). Positive founders were mated with C57BL/6J mice, and F1 or F2 generations were killed for analysis. Two hours before death, mice were injected with 50 mg/kg BrdU (Sigma–Aldrich, St. Louis, MO) intraperitoneally. To generate elastase sshIL-1β mice heterozygous for the p53R172H germ-line mutation (elastase sshIL-1β*p53R172H/+), elastase sshIL-1β mice (L124) were crossed with mice homozygous for p53R172H mutation (kind gift from Tyler Jacks, Massachusetts Institute of Technology, Cambridge, MA).21Olive K.P. Tuveson D.A. Ruhe Z.C. et al.Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome.Cell. 2004; 119: 847-860Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar For the cerulein-based model of chronic pancreatitis, C57BL/6 mice were injected intraperitoneally with 50 μg/kg cerulein (American Peptide Company, Inc, Sunnyvale, CA; dissolved in sterile water) on 7 consecutive hours, twice a week for 10 weeks, and once a week for 10 additional weeks. Mice were killed 1 week after the last cerulein injection. For tissue preparations, see Supplementary materials (see Supplementary materials online at www.gastrojournal.org). For histology, immunohistochemistry, immunofluorescence, and microscopy, see Supplementary materials. H&E-stained sections were analyzed, and lesions were graded using a modification of the scoring system previously described by Demolset al,22Demols A. Van Laethem J.L. Quertinmont E. et al.Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G1105-G1112Crossref PubMed Scopus (100) Google Scholar taking into account the extent of inflammation, glandular atrophy, and tubular complexes and the percentage of area involved (see supplementary Table 2 online at www.gastrojournal.org). For fibrosis quantification, 6 nonoverlapping ×100 magnification fields of Sirius red stained slides were captured. Morphometric analysis was performed using the IPLab 3.6 software (BD Biosciences, Franklin Lakes, NJ). Preneoplastic lesions were defined according to the criteria of Hruban et al.23Hruban R.H. Adsay N.V. Albores-Saavedra J. et al.Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations.Cancer Res. 2006; 66: 95-106Crossref PubMed Scopus (327) Google Scholar For proliferation and apoptosis quantification, see Supplementary materials. For complementary DNA (cDNA) synthesis and PCR, see Supplementary materials. For IL quantification, see Supplementary materials. For flow cytometry, see Supplementary materials. For magnetic resonance imaging, see Supplementary materials. For glucose tolerance test, see Supplementary materials. For research of fat malabsorption, see Supplementary materials. Statistical analysis was performed using t test or Wilcoxon signed-rank test when appropriate, and P values <.05 were considered significant. Data are expressed as means ± SEM. Three founder mice were generated and subsequently bred to generate 3 separate lines (L122, L123, L124). The litter size was normal for all 3 lines. For lines L122 and L123, approximately 15% of the pups showed significant growth retardation and needed to be killed. The remainder of the elastase sshIL-1β mice displayed normal behavior and growth, although the average weight was significantly lower in L122 mice at 7 weeks and in L122 and L123 male mice at 20 weeks (Figure 1B). For line L124, mice lived up to 2 years, whereas the oldest mice from line 123 are currently 22 months old and appear healthy. Although a detailed survival analysis was not carried out, the elastase sshIL-1β mice do not appear to show any significant decreased longevity compared with wild-type control mice. We confirmed the expression of the sshIL-1β transgene in the pancreas by reverse-transcription (RT)-PCR and by enzyme-linked immunosorbent assay (ELISA) measurement of human IL-1β peptide in pancreatic protein extracts (Figure 1C and D). Expression of sshIL-1β could not be detected in any of the other tissues we analyzed, including liver, submaxillary glands, kidney, lung, and spleen (data not shown). The level of human IL-1β expression was different in the 3 transgenic lines, and these were classified as high (L122), medium (L123), and low (L124) expressers. No human IL-1β was detectable in control mice, which were age matched wild-type mice or mice with cerulein-induced chronic pancreatitis. It is noteworthy that the level of human IL-1β expression decreased with time in all 3 lines, probably related to progressive acinar atrophy as also described in other acinar promoter based transgenic mice.7Archer H. Jura N. Keller J. et al.A mouse model of hereditary pancreatitis generated by transgenic expression of R122H trypsinogen.Gastroenterology. 2006; 131: 1844-1855Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar IL-1β itself is a known downstream target of the nuclear factor (NF)-κB pathway. Consistent with the notion that sshIL-1β activates the NF-κB pathway, we could detect strong expression of endogenous murine IL-1β in the pancreata of elastase sshIL-1β mice at the age of 7 weeks, whereas there was only very weak expression in the majority of age-matched, wild-type control mice (Figure 1E). In addition, to demonstrate that human IL-1β can directly activate the murine IL-1 receptor, we isolated myeloid cells from the bone marrow of mice and stimulated these cells with human IL-1β. Treatment of marrow-derived myeloid cells with standard doses of human IL-1β resulted in a >3-fold increase in IL-6 messenger RNA (mRNA) and a >7-fold increase in IL-6 protein secretion (not shown). Because IL-6 is the most commonly used downstream surrogate for IL-1 receptor signaling and NF-κB pathway activation, these findings indicate that human IL-1β can directly stimulate the murine IL-1 receptor. Pancreata of elastase sshIL-1β mice were atrophic (Figure 2A). In addition, as early as 7 weeks, L122 and L123 mice displayed dilatation of the main pancreatic duct alone, or, in some cases, associated with dilatation of the biliary duct (Figure 2B) because of fibrotic stenosis at the level of the head of the pancreas, mimicking human chronic pancreatitis. Histologic analysis showed other features typical of chronic pancreatitis, including acinar atrophy, tubular complexes, mixed inflammatory infiltrate, and fibrosis, the latter confirmed by trichrome staining (Figure 3). These lesions appeared as early as 1 week after birth, starting with a mixed inflammatory infiltrate and initially mild fibrosis. Interestingly, we did not observe pancreatic necrosis at any time point. Complete autopsy did not reveal abnormality in any other organ.Figure 3H&E (A, C, E, and F) and Masson's trichrome staining (B and D). Wild-type pancreas (original magnification, ×40; A and B) and pancreas of 7- to 8-week-old transgenic mice: (C) elastase sshIL-1β L124, acinar atrophy, inflammatory infiltrate, and fibrosis surrounding a major duct (original magnification, ×40); (D) L122, extensive fibrosis evidenced by Masson's trichrome staining (original magnification, ×40); (E) L124, loss of acinar cells, extensive fibrosis, tubular complexes, mixed cellular infiltrate, and increased fat (original magnification, ×100); (F) higher magnification (original magnification, ×400): acini and tubular complexes amid mixed inflammatory infiltrate composed of lymphocytes, macrophages, and granulocytes (original magnification, ×400).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The inflammatory infiltrate consisted mainly of lymphocytes, but variable numbers of macrophages and granulocytes were seen admixed. Characterization of the infiltrate by flow cytometry analysis (Figure 4A) demonstrated a predominance of T cells (CD3+) and confirmed the presence of macrophages (CD11b+), granulocytes (gr1+), and B cells (CD19+). The predominance of T lymphocytes was in agreement with the chronic nature of the inflammation. Increased proliferation and apoptosis in the exocrine pancreas are hallmarks of chronic pancreatitis in humans. We quantified proliferation using immunohistochemical staining for BrdU and apoptosis by the TUNEL assay (Figure 4B). Both proliferation and apoptosis were significantly elevated compared with age-matched, wild-type controls. Chronic pancreatitis is associated with a stromal reaction involving inflammatory cell infiltration as well as proliferation and activation of a specific type of mesenchymal cells called pancreatic stellate cells.24Omary M.B. Lugea A. Lowe A.W. et al.The pancreatic stellate cell: a star on the rise in pancreatic diseases.J Clin Invest. 2007; 117: 50-59Crossref PubMed Scopus (558) Google Scholar These cells can be detected by staining for desmin. Figure 5A shows increased number of pancreatic stellate cells in elastase sshIL-1β mice, interspersed between acini, located around ducts or regenerative ductules, or scattered amongst the inflammatory infiltrate. Some of these cells, located in the periacinar or periductal area were activated pancreatic stellate cells, as demonstrated by expression of α-smooth muscle actin. By RT-PCR (Figure 5B), we detected the expression of genes associated with chronic pancreatitis, including TNF-α, which is also a downstream target of IL-1β; chemokines, like stromal cell-derived factor 1 (SDF1) and chemokine (C-X-C motif) ligand 1 (CXCL1); TGF-β1, a growth factor that is involved in pancreatic fibrosis and the activation of pancreatic stellate cells; metalloproteases involved in the remodeling of the extracellular matrix matrix metallopeptidase [MMP] 2, 7, and 9; tissue inhibitor of metalloproteinase [TIMP] 1); and cyclooxygenase 2. These genes all showed minimal expression in the pancreas of wild-type control mice. To correlate the extent and severity of the lesions with the level of human IL-1β expression, we graded the lesions and quantified fibrosis by morphometric analysis after Sirius red staining (Figure 6A and B). Mice from lines 122 and 123 (with high and moderate level of sshIL-1β expression, respectively) had more severe lesions and higher levels of fibrosis than mice from the low expressing line (line 124). We also compared the histologic findings in 20-week-old transgenic mice with those of mice treated for 20 weeks with repeated injections of cerulein because the latter has been the most commonly employed model of murine chronic pancreatitis. Despite the protracted protocol, mice that received cerulein had milder lesions and less fibrosis than elastase sshIL-1β mice from any of the 3 lines. Severe cases of pancreatitis can be associated with pancreatic endocrine and/or exocrine insufficiency. To assess glucose tolerance in these mice, we performed a glucose tolerance test, which did not demonstrate significant abnormalities of glucose tolerance in transgenic mice compared with wild-type controls (see supplementary Figure 1 online at www.gastrojournal.org). We also evaluated pancreatic exocrine function by assessing for fat malabsorption in mice fed a high fat diet. We performed Oil Red O staining on stool smears (see supplementary Figure 2 online at www.gastrojournal.org) but did not detect any fat in the stools of elastase sshIL-1β mice. It is noteworthy that these tests were performed in mice 8–10 months old to study mice with advanced disease. Chronic pancreatitis is a well-described risk factor for pancreatic adenocarcinoma. We followed a cohort of transgenic elastase sshIL-1β up to 2 years to determine whether they might develop spontaneous pancreatic neoplasia. Acinar atrophy and fibrosis were progressive, and the majority of the pancreatic gland was replaced over time by adipose tissue. None of the mice developed pancreatic tumors or mouse pancreatic intraepithelial neoplasia (mPanIN). Eosinophilic metaplasia was seen in the large dilated ducts in a few mice, but this lesion is typically considered a reactive change (Figure 7A). We also observed acinar-ductal metaplasia starting between 20 (L122 and L123) and 40 weeks of age (L124) (Figure 7B). This lesion was confirmed by performing immunofluorescence staining for amylase and cytokeratin 19 (Figure 7C): in some tubular complexes, cells expressing both acinar and ductal markers were observed. On staining for BrdU (Figure 7D), these lesions had a low proliferation index suggesting acinar transdifferentiation25Means A.L. Meszoely I.M. Suzuki K. et al.Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates.Development. 2005; 132: 3767-3776Crossref PubMed Scopus (278) Google Scholar rather than expansion of centroacinar cells as suggested by some studies.26Stanger B.Z. Stiles B. Lauwers G.Y. et al.Pten constrains centroacinar cell expansion and malignant transformation in the pancreas.Cancer Cell. 2005; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar In patients with hereditary chronic pancreatitis, exposure to tobacco, a known mutagen, significantly increases the risk of pancreatic adenocarcinoma.27Lowenfels A.B. Maisonneuve P. Whitcomb D.C. et al.Cigarette smoking as a risk factor for pancreatic cancer in patients with hereditary pancreatitis.JAMA. 2001; 286: 169-170Crossref PubMed Scopus (268) Google Scholar We thus hypothesized that, in addition to chronic inflammation, an additional genetic event might be required to initiate carcinogenesis. Consequently, we crossed elastase sshIL-1β (L124) with mice harboring a R172H mutation in p5321 and generated elastase sshIL-1β mice heterozygous for p53R172H mutation (elastase sshIL-1β*p53R172H/+). These mice had normal growth and development compared with elastase sshIL-1β mice. On comparing the severity of lesions in elastase sshIL-1β mice with or without p53R172H/+ mutation, no significant difference in the extent of inflammation or acinar atrophy was observed, but a significantly increased number of tubular complexes, including some showing evidence of acinar-ductal metaplasia, was noted in 40-week-old elastase sshIL-1β*p53R172H/+ mice (Figure 8A). No difference was observed at 20 weeks old (data not shown). Because p53 is involved in apoptosis regulation,21Olive K.P. Tuveson D.A. Ruhe Z.C. et al.Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome.Cell. 2004; 119: 847-860Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar we compared acinar apoptotic indexes according to p53 status and found that elastase sshIL-1β*p53R172H/+ mice had a lower apoptotic index, although the difference was not statistically significant (Figure 8B). We did not observe any mPanIN lesion in elastase sshIL-1β*p53R172H/+ mice, even in mice up to 15 months of age. Follow-up of this cohort was shorter than that of elastase sshIL-1β mice because, starting at 12 months, mice began to die from p53 germ-line mutation- associated tumors (lymphomas and sarcomas essentially). These tumors occurred with the same frequency as in the single transgenic p53R172H/+ control group. Finally, we observed 1 single case of pancreatic tumor (out of 15 mice), an adenocarcinoma with sarcomatoid and giant cell features in a 43-week-old mouse (Figure 7E and F). The pancreas was enlarged and nodular, and the tumor showed extensive invasion with peritoneal carcinomatosis. No liver or lung metastases were identified. In areas of nontumoral pancreas, chronic pancreatitis was evident with acinar-ductal metaplasia, but no mPanIN was observed. Elastase sshIL-1β mice have a phenotype of early onset chronic pancreatitis, with chronic inflammatory infiltrate consisting predominantly of T lymphocytes, progressive glandular atrophy with acinar cell loss, and severe fibrosis, particularly in murine lines with moderate to high levels of human IL-1β expression,
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