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• W1978057084 abstract "Background & Aims: Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1u, lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1u) and iddm2 (lyp, Gimap5−/−) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels. Methods: Genetically lymphopenic rats and congenic wild-type partners were compared for gross pathologic, histologic, and immunologic parameters, the latter including cytokines and autoantibodies. Results: Genetic analysis demonstrated that homozygosity at the lyp locus was required for disease. All rats developed disease, and the median age at humane killing was ∼36 weeks. This panintestinal disease showed a conspicuous eosinophilic infiltrate in the submucosa and muscle layers, but the villi were unaffected. Diseased rats showed splenomegaly and massive enlargement of the mesenteric lymph nodes. This pathology resembles human eosinophilic gastroenteritis, and several further features indicate a Th2 basis. The rats developed high serum IgE and made IgG autoantibodies that detected a nonleukocytic cell present in the intestinal wall of all rats (including germ free). Conclusions: The T-lymphopenic state associated with GIMAP5 deficiency renders rats generally susceptible to T-cell-mediated autoimmunity, but the immunoregulatory bias (Th1/Th2) of any disease depends on other genetic (or environmental) factors. In the present model, we suggest that defective peripheral tolerance to an intestine-specific autoantigen leads to uncontrolled inflammation of the intestinal wall. Background & Aims: Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1u, lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1u) and iddm2 (lyp, Gimap5−/−) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels. Methods: Genetically lymphopenic rats and congenic wild-type partners were compared for gross pathologic, histologic, and immunologic parameters, the latter including cytokines and autoantibodies. Results: Genetic analysis demonstrated that homozygosity at the lyp locus was required for disease. All rats developed disease, and the median age at humane killing was ∼36 weeks. This panintestinal disease showed a conspicuous eosinophilic infiltrate in the submucosa and muscle layers, but the villi were unaffected. Diseased rats showed splenomegaly and massive enlargement of the mesenteric lymph nodes. This pathology resembles human eosinophilic gastroenteritis, and several further features indicate a Th2 basis. The rats developed high serum IgE and made IgG autoantibodies that detected a nonleukocytic cell present in the intestinal wall of all rats (including germ free). Conclusions: The T-lymphopenic state associated with GIMAP5 deficiency renders rats generally susceptible to T-cell-mediated autoimmunity, but the immunoregulatory bias (Th1/Th2) of any disease depends on other genetic (or environmental) factors. In the present model, we suggest that defective peripheral tolerance to an intestine-specific autoantigen leads to uncontrolled inflammation of the intestinal wall. See editorial on page 1629. See editorial on page 1629. Despite their clinical importance, the pathogenesis of many T-helper cell (Th)2-related diseases (eg, atopy, asthma, and food allergy) remains obscure. Elucidating the aberrant regulation underlying these diseases is crucial to developing rational therapeutic strategies, and relevant animal models are central to this development. The BioBreeding (BB) rat spontaneously develops insulin-dependent diabetes mellitus (IDDM) caused by a Th1-mediated destruction of pancreatic islet β cells and featuring high levels of interferon (IFN)-γ and interleukin (IL)-12.1Rabinovitch A. Suarez-Pinzon W. El-Sheikh A. Sorensen O. Power R.F. Cytokine gene expression in pancreatic islet-infiltrating leukocytes of BB rats: expression of Th1 cytokines correlates with β-cell destructive insulitis and IDDM.Diabetes. 1996; 45: 749-754Crossref PubMed Scopus (85) Google Scholar, 2Zipris D. Greiner D.L. Malkani S. Whalen B. Mordes J.P. Rossini A.A. Cytokine gene expression in islets and thyroids of BB rats IFN-γ and IL-12p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats.J Immunol. 1996; 156: 1315-1321PubMed Google Scholar, 3Zipris D. Evidence that Th1 lymphocytes predominate in islet inflammation and thyroiditis in the BioBreeding (BB) rat.J Autoimmun. 1996; 9: 315-319Crossref PubMed Scopus (17) Google Scholar The lymphopenia (lyp) gene is essential for diabetes4MacMurray A.J. Moralejo D.H. Kwitek A.E. Rutledge E.A. Van Yserloo B. Gohlke P. Speros S.J. Snyder B. Schaefer J. Bieg S. Jiang J. Ettinger R.A. Fuller J. Daniels T.L. Pettersson A. Orlebeke K. Birren B. Jacob H.J. Lander E.S. Lernmark A. Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.Genome Res. 2002; 12: 1029-1039Crossref PubMed Scopus (187) Google Scholar, 5Guttmann R.D. Colle E. Michel F. Seemayer T. Spontaneous diabetes mellitus syndrome in the rat II. T lymphopenia and its association with clinical disease and pancreatic lymphocytic infiltration.J Immunol. 1983; 130: 1732-1735PubMed Google Scholar, 6Jacob H.J. Pettersson A. Wilson D. Mao Y. Lernmark A. Lander E.S. Genetic dissection of autoimmune type I diabetes in the BB rat.Nat Genet. 1992; 2: 56-60Crossref PubMed Scopus (217) Google Scholar: other susceptibility genes include the major histocompatibility complex (MHC) class II RT1u haplotype. The lyp gene encodes GTPase of the immunity associated protein (GIMAP5) (formerly known as Ian5 or Ian4), a member of the GTPase of the immunity-associated protein family,4MacMurray A.J. Moralejo D.H. Kwitek A.E. Rutledge E.A. Van Yserloo B. Gohlke P. Speros S.J. Snyder B. Schaefer J. Bieg S. Jiang J. Ettinger R.A. Fuller J. Daniels T.L. Pettersson A. Orlebeke K. Birren B. Jacob H.J. Lander E.S. Lernmark A. Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.Genome Res. 2002; 12: 1029-1039Crossref PubMed Scopus (187) Google Scholar, 7Hornum L. Romer J. Markholst H. The diabetes-prone BB rat carries a frameshift mutation in Ian4, a positional candidate of Iddm1.Diabetes. 2002; 51: 1972-1979Crossref PubMed Scopus (136) Google Scholar which may protect cells from apoptosis.8Sandal T. Aumo L. Hedin L. Gjertsen B.T. Doskeland S.O. Irod/Ian5: an inhibitor of γ-radiation- and okadaic acid-induced apoptosis.Mol Biol Cell. 2003; 14: 3292-3304Crossref PubMed Scopus (55) Google Scholar, 9Pandarpurkar M. Wilson-Fritch L. Corvera S. Markholst H. Hornum L. Greiner D.L. Mordes J.P. Rossini A.A. Bortell R. Ian4 is required for mitochondrial integrity and T-cell survival.Proc Natl Acad Sci U S A. 2003; 100: 10382-10387Crossref PubMed Scopus (71) Google Scholar The BB lyp gene contains a single nucleotide deletion, resulting in a frameshift and a significantly truncated predicted GIMAP5 protein.4MacMurray A.J. Moralejo D.H. Kwitek A.E. Rutledge E.A. Van Yserloo B. Gohlke P. Speros S.J. Snyder B. Schaefer J. Bieg S. Jiang J. Ettinger R.A. Fuller J. Daniels T.L. Pettersson A. Orlebeke K. Birren B. Jacob H.J. Lander E.S. Lernmark A. Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.Genome Res. 2002; 12: 1029-1039Crossref PubMed Scopus (187) Google Scholar, 7Hornum L. Romer J. Markholst H. The diabetes-prone BB rat carries a frameshift mutation in Ian4, a positional candidate of Iddm1.Diabetes. 2002; 51: 1972-1979Crossref PubMed Scopus (136) Google Scholar A pronounced T-cell lymphopenia results from defective thymic T-cell export10Zadeh H.H. Greiner D.L. Wu D.Y. Tausche F. Goldschneider I. Abnormalities in the export and fate of recent thymic emigrants in diabetes-prone BB/W rats.Autoimmunity. 1996; 24: 35-46Crossref PubMed Scopus (51) Google Scholar and increased T-cell apoptosis11Hernández-Hoyos G. Joseph S. Miller N.G. Butcher G.W. The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.Eur J Immunol. 1999; 29: 1832-1841Crossref PubMed Scopus (46) Google Scholar with a 5- to 10-fold reduction in peripheral CD4+ and negligible CD8+ T cells.12Poussier P. Nakhooda A.F. Falk J.A. Lee C. Marliss E.B. Lymphophenia and abnormal lymphocyte subsets in the “BB” rat: relationship to the diabetic syndrome.Endocrinology. 1982; 110: 1825-1827Crossref PubMed Scopus (75) Google Scholar, 13Jackson R. Rassi N. Crump T. Haynes B. Eisenbarth G.S. The BB diabetic rat Profound T-cell lymphocytopenia.Diabetes. 1981; 30: 887-889Crossref PubMed Google Scholar, 14Elder M.E. Maclaren N.K. Identification of profound peripheral T-lymphocyte immunodeficiencies in the spontaneously diabetic BB rat.J Immunol. 1983; 130: 1723-1731PubMed Google Scholar Lymphopenia directly relates to disease as T-cell transfer prevents disease.15Whalen B.J. Greiner D.L. Mordes J.P. Rossini A.A. Adoptive transfer of autoimmune diabetes mellitus to athymic rats; synergy of CD4+ and CD8+ T cells and prevention by RT6+ T cells.J Autoimmun. 1994; 7: 819-831Crossref PubMed Scopus (48) Google Scholar Lymphopenia is seen in multiple human and animal autoimmune diseases (including systemic lupus erythematosus, rheumatoid arthritis, and Sjogren’s syndrome).16Schaller J.G. Immunodeficiency and autoimmunity.Birth Defects Orig Artic Ser. 1975; 11: 173-184PubMed Google Scholar, 17Sleasman J.W. The association between immunodeficiency and the development of autoimmune disease.Adv Dent Res. 1996; 10: 57-61Crossref PubMed Scopus (56) Google Scholar, 18Gleeson P.A. Toh B.H. van Driel I.R. Organ-specific autoimmunity induced by lymphopenia.Immunol Rev. 1996; 149: 97-125Crossref PubMed Google Scholar Indeed, several animal autoimmune models require the induction of lymphopenia via experimental or genetic means.19King C. Ilic A. Koelsch K. Sarvetnick N. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity.Cell. 2004; 117: 265-277Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 20Fowell D. McKnight A.J. Powrie F. Dyke R. Mason D. Subsets of CD4+ T cells and their roles in the induction and prevention of autoimmunity.Immunol Rev. 1991; 123: 37-64Crossref PubMed Scopus (184) Google Scholar To analyze the role of the lyp gene separately from other BB genetic factors, we created congenic rats in which the BB lyp gene was backcrossed onto the PVG-RT1u strain.11Hernández-Hoyos G. Joseph S. Miller N.G. Butcher G.W. The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.Eur J Immunol. 1999; 29: 1832-1841Crossref PubMed Scopus (46) Google Scholar The PVG-RT1u, lyp/lyp rat did not develop diabetes but unexpectedly developed intestinal inflammation with striking Th2-related characteristics. The pathologic features closely resemble those seen in human eosinophilic gastroenteritis (EG). PVG-RT1u, lyp/lyp; PVG-RT1u (used as wild-type [WT] controls); and PVG-RT1u, rnu/rnu rat strains were bred and maintained under specific pathogen-free conditions at either the Babraham Institute, Cambridge, United Kingdom, or the Sir William Dunn School of Pathology, Oxford, United Kingdom. Animals were used when between 1 and 12 months of age. All procedures were in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and with approval of the local ethical review committees. Inbred and backcross rats were genotyped by sequencing through the lyp-associated Gimap5 frameshift mutation (a single base deletion). Genomic DNA was prepared using Qiagen DNeasy tissue kit or QIAamp DNA blood mini kit (Qiagen, Crawley, United Kingdom). Thirty-cycle polymerase chain reaction (PCR) amplifications were carried out on 25 ng genomic DNA using the primers (forward) 5′-CCATGGCTTTGAGGAACTATCC-3′ and (reverse) 5′-TGTGGGTGAAGAGGACAATCAT-3′ to generate a 500-base pair (bp) fragment.4MacMurray A.J. Moralejo D.H. Kwitek A.E. Rutledge E.A. Van Yserloo B. Gohlke P. Speros S.J. Snyder B. Schaefer J. Bieg S. Jiang J. Ettinger R.A. Fuller J. Daniels T.L. Pettersson A. Orlebeke K. Birren B. Jacob H.J. Lander E.S. Lernmark A. Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.Genome Res. 2002; 12: 1029-1039Crossref PubMed Scopus (187) Google Scholar PCR products were purified using exonuclease and shrimp alkaline phosphatase (Amersham Biosciences, Piscataway, NJ), and DNA was sequenced commercially (Lark Technologies Inc., Saffron Walden, UK). Monoclonal antibodies (mAbs) against rat αβTCR (R73), CD45RC (OX-22), and CD172 (OX-41) were kindly provided by M. Puklavec, Oxford, and were conjugated to Alexa 647 or fluorescein isothiocyanate (FITC) according to the manufacturers’ instructions (Amersham Bioscience; Sigma-Aldrich, Poole, United Kingdom). Additional mAbs used were phycoerythrin (PE)- and FITC-conjugated OX-35 (anti-rat CD4), PerCP-OX-8 (anti-rat CD8α), PerCP-OX-6 (anti-rat MHC class II RT1-B), FITC-OX-33 (anti-rat CD45RA), PE-OX-39 (anti-rat CD25), PE-10/78 (anti-rat CD161) (all BD Pharmingen, Cowley, Oxford, UK), and PE-OX-40 (anti-rat CD134) (Serotec, Kidlington, Oxford, UK). Cells were stained by direct immunofluoresence and analyzed by flow cytometry on either a FACSort or a FACScalibur machine: at least 20,000 cells were gated and analyzed using CellQuest Software (Becton Dickinson, Franklin Lakes, NJ). An anti-mast cell protease mAb used is described in the Histology section. Mesenteric or cervical lymph nodes (MLN or CLN), or spleens, were harvested in ice-cold phosphate-buffered saline (PBS) and single-cell suspensions prepared by flushing through a 0.4-μm cell strainer followed by erythrocyte lysis with ammonium chloride-potassium bicarbonate (ACK) cell lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L Na2 EDTA, pH 7.2–7.4). For purification, cells were first enriched using the MACS CD4-specific beads (Miltenyi Biotech). Enriched cells were stained for CD4, αβTCR, and CD45RC prior to flow sorting for either CD4+ T cells or CD4+CD45RClo T cells. Cell purity was consistently >99%. Cells were cultured for 4 hours at 37°C in RPMI 1640 supplemented with 10% rat serum, 50 μmol/L 2-ME, 100 μg/mL penicillin, 50 U/mL streptomycin, and 100 μg/mL L-glutamine (all GIBCO) in the presence of phorbol myristic acetate (50 ng/mL) and ionomycin (500 ng/mL) (both Sigma-Aldrich). Supernatants were stored at −20°C prior to assay of IL-4 and IFN-γ, and cells were lysed in TriReagent (Sigma-Aldrich) and stored at −80°C. IL-4 and IFN-γ protein levels were determined by enzyme-linked immunosorbent assay (ELISA) using 96-well microtiter plates (Nalge Nunc International, Rochester, NY). For IFN-γ, biotinylated mAbs DB-1 and DB-12 (kind gifts of Dr Peter van der Meide, Biomedical Primate Research Centre, Rijswijk, The Netherlands) were used for antigen capture and detection, respectively. For IL-4, mAb OX-81 (capture) and biotinylated rabbit anti-IL-4 (Peprotech EC Ltd, London, UK) (detection) were used. Assays were developed with streptavidin-horseradish peroxidase (HRP) (BD Pharmingen) using the tetramethyl benzidine (TMB) enzyme substrate kit (BD Pharmingen). IFN-γ values are expressed as units/milliliter by reference to a standard curve constructed using rat recombinant IFN-γ. IL-4 measurements were calculated relative to a standard (BD Pharmingen). IL-13 and IL-5 messenger RNA (mRNA) were measured by quantitative PCR (QPCR) using SYBR green and normalized to β-actin mRNA. Cells were pelleted and RNA isolated using TriReagent (Sigma-Aldrich Ltd.) according to the manufacturer’s instructions. RNA was reverse transcribed using SuperscriptTM first-strand synthesis system (Invitrogen, Life Technologies Ltd., Strathclyde, UK) according to the manufacturer’s instructions. Genomic DNA contamination was assessed in reverse transcriptase (RT)-negative samples. QPCR was performed using SYBR green buffer (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) and 5 mmol/L of each primer. The primers used were taken from previously published RT-PCR analyses: IL-5: forward 5′-TGCTTCTGTGCTTGAACGTTCTAAC-3′; reverse 5′-TTCTCTTTTTGTCCGTCAATGTATTTC-3′.21Ide K. Hayakawa H. Yagi T. Sato A. Koide Y. Yoshida A. Uchijima M. Suda T. Chida K. Nakamura H. Decreased expression of Th2 type cytokine mRNA contributes to the lack of allergic bronchial inflammation in aged rats.J Immunol. 1999; 163: 396-402PubMed Google Scholar IL-13: forward 5′-CAGGGAGCTTATCGAGGAGC-3′; reverse 5′-AAGTTGCTTGGAGTAATTGAGC-3′.22Seddon B. The role of thymic and peripheral T cell subsets in the control of autoimmune disease. D. Phil. Thesis, University of Oxford, UK1996Google Scholar β-actin: forward 5′-TCCTGTGGCATCCATGAAACT-3′ reverse 5′-GAAGCATTTGCGGTGCACGAT-3′.21Ide K. Hayakawa H. Yagi T. Sato A. Koide Y. Yoshida A. Uchijima M. Suda T. Chida K. Nakamura H. Decreased expression of Th2 type cytokine mRNA contributes to the lack of allergic bronchial inflammation in aged rats.J Immunol. 1999; 163: 396-402PubMed Google Scholar QPCR was performed using the Taqman Sequence detector 1.6.3 ABI software (Applied Biosystems, Warrington, UK). All QPCR reactions were as follows: 94°C for 2 minutes, followed by 45 cycles of 96°C 10 seconds, 58°C 15 seconds, 72°C 20 seconds, and 82°C 1 second. Specificity was assessed using melting curve analysis. Data were first normalized to β-actin expression and then expressed as values relative to that of WT CD4+ T cells. Tissues were fixed in 10% formalin and paraffin sections cut at 5 μm. For eosinophils, sections were stained using Chromotrope 2R. Mast cell staining was by anti-RMCPII (69F522, a kind gift from Prof. Hugh Miller, Edinburgh, United Kingdom), detected using HRP-donkey anti-mouse Ig (Jackson Immunoresearch, Newmarket, UK) and diaminobenzidine (Polysciences, Warrington, PA). Counterstaining used methyl green. For general histology, sections were stained with H&E, Alcian Blue, or Masson’s Trichrome. Serum IgG1, IgG2, and IgE were measured by ELISA. Plates were coated overnight with either 3 μg/mL anti-rat IgG1 (MARG1-2), 3 μg/mL anti-rat IgG2b (MARG2b-2), or 5 μg/mL anti-IgE (MARE-1) (all Serotec) and blocked for 30 minutes with 1% (wt/vol) bovine serum albumin (BSA). Serial dilutions of serum were incubated for 2 hours and plates washed, and bound Ig was detected with either HRP-rat anti-Igκ+λ (MARK-1/MARL-15, Serotec) or 2 μg/mL biotin-anti-IgE (B41-3, Pharmingen) and subsequently with streptavidin-HRP (1/1000, Pharmingen). The assay was developed with TMB substrate, as above. Antibody levels were determined by comparison with IgG1 (IR27), IgG2b (IR863), or IgE (IR162) standards (all Serotec). Intestinal sections prepared as above were blocked with 10% donkey serum (Sigma-Aldrich, Poole, United Kingdom). Endogenous peroxidase was quenched with 0.7 U/mL glucose oxidase (Sigma) in prewarmed PBS containing 25 mmol/L D-glucose, 1 mmol/L sodium azide (30 minutes at 37°C). Sections were washed and stained for IgE (MARE-1; Serotec) followed by donkey anti-mouse HRP (Jackson Immunoresearch). Staining was amplified with Cy5 tyramide (NEN Life Science, Boston, MA) and counterstaining was with 4’,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR). Fresh tissues from PVG-RT1u, rnu/rnu rats or germ-free Wistar (RT1a) rats (a generous gift from Dr R. Stepankova, Prague, Czech Republic) were frozen in Tissue-tek OCT compound (Sakura Fineteck Europe B.V., Zoeterwoude, The Netherlands) over isopentane on dry ice. Five- to six-micrometer sections were stored at −20°C until use. Sections were fixed in 100% ethanol at 4°C for 15 minutes. Autoantibodies were detected by incubating sections overnight with serum from PVG-RT1u, lyp/lyp or WT rats. Binding was detected by FITC-donkey anti-rat Ig (Jackson Immunoresearch). Counterstaining was with DAPI. Double staining for leukocytes and myofibroblasts was by anti-rat CD45 (OX-1) and anti-human smooth muscle α-actin (mAb Clone 1A4; Serotec). Secondary antibody was Cy5-conjugated donkey anti-mouse IgG (Jackson Immunoresearch). Values are expressed as means ± 1 SD. Significance of differences between means was by an unpaired t test. Differences were taken as significant at P < .05. PVG-RT1u, lyp/lyp rats were clinically normal until ∼20 weeks of age. Subsequently, they developed symptoms eventually necessitating killing on welfare grounds, all animals being affected by ∼50 weeks. The decline in health of the animals was generally consistent with loss of proper gut function. Symptoms varied from animal to animal but included weight loss (general wasting), listlessness, and diarrhea, and postmortem analyses often revealed gut blockages and ascites. Generally, there was progressive, and eventually severe, enlargement of the gut itself, gut-associated lymphoid tissue, spleen, and liver. This often brought about obvious distension of the body wall. Apart from the infiltration with eosinophils and mast cells (see below), the gut-associated lymphoid tissue, spleen, and liver also showed the involvement of plasma cells and Mott cells and evidence of extramedullary hematopoiesis, all of which increased with time. The lungs also contained infiltrates. Clinically affected rats displayed severe intestinal inflammation from duodenum to rectum, accompanied by splenomegaly and massive MLN enlargement (Figure 1A). The stomach and esophagus were usually uninvolved. H&E staining of the large intestine (LI) (Figure 1B and C) revealed marked cellular infiltration of the submucosa and muscular layers, whereas the inner mucosa was less affected. Similar inflammation was present in the caecum (Figure 1D), large intestine (Figure 1E), and small intestine (Figure 1G). Importantly, villous atrophy and crypt hyperplasia, conspicuous in many Th1 inflammatory bowel disease (IBD) models,23Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1870) Google Scholar were absent, and goblet cells were present in at least normal numbers (Figure 1J). Collagen deposition in the submucosa signified fibrosis (Figure 1K). Most infiltrating cells were eosinophils (Figure 1H), confirmed by Chromotrope 2R24Roque A.L. Chromotrope aniline blue method of staining Mallory bodies of Laennec’s cirrhosis.Lab Invest. 1953; 2: 15-21PubMed Google Scholar staining (Figure 2A). Infiltration was confined to the submucosal and muscular layers, villi being unaffected. The pathology strongly suggests that intestinal inflammation results from a Th2 response. Disease progression was accompanied by blood eosinophilia. Eosinophils were identified using flow cytometry (high side scatter [SSC] and expression of CD172). Sorted SSChi, CD172+ cells were confirmed as eosinophils microscopically by bilobed nuclei and dark granular cytoplasm (unpublished data). The percentage of eosinophils in blood increased from ∼1% to ∼10% of total blood leukocytes as the disease progressed, and this represented an absolute numerical increase (Figure 2C). Staining for the rat mast cell-specific protease RMCPII revealed a marked panintestinal mast cell infiltration in lyp/lyp rats (Figure 2B).Figure 2Eosinophils and mast cells in PVG-RT1u, lyp/lyp rats. (A) Large intestine stained for eosinophils by Chromotrope 2R (original magnification, 630×) (Left panel) WT, (Right panel) lyp/lyp. (B) Large intestine stained for the mast cell-specific protease RCMPII (brown) (L panel) WT, (R panel) lyp/lyp. Counterstain was Methyl Green (original magnification, 100×). (C) Eosinophil numbers in peripheral blood. Eosinophils were identified in flow cytometry by expression of CD172 and high side scatter signal. Data are presented as means ± SEM.View Large Image Figure ViewerDownload (PPT) Intestinal infiltration with eosinophils and mast cells was only seen in clinically affected rats, although a moderate blood eosinophilia was detectable prior to disease onset (unpublished data). This suggests that these cells were acting as effector cells rather than in the induction of the response. To test formally the genetic involvement of the lyp region, the WT PVG-RT1u, RT7b strain was backcrossed onto PVG-RT1u, lyp/lyp. Offspring were genotyped for the Gimap5 frameshift mutation (the basis of the lyp defect). Results accorded with genotyping using polymorphic microsatellites (data not shown). All homozygous lyp/lyp progeny (n = 28) from the backcross became ill within a 50-week period while none of their lyp/+ littermates (n = 16) did so. The median age at killing on welfare grounds (∼36 weeks) was not significantly different between the lyp/lyp progeny from the backcross and the established PVG-RT1u, lyp/lyp strain (P = .223). No sex bias was detected. These results confirmed the involvement of the lyp region in the disease. The role of the MHC haplotype was investigated by comparing disease in PVG-lyp/lyp rats carrying either the RT1u or the RT1c haplotype (the latter derived from PVG). Disease was seen in both strains (>90% incidence in both strains over a 63-week inspection period), but the time at which killing on welfare grounds became necessary differed significantly: 32 ± 8.4 (SD) weeks for PVG-RT1u, lyp/lyp (n = 24) and 48 ± 9.2 (SD) weeks for PVG-RT1c, lyp/lyp (n = 37) (difference significant at P < .0001). As PVG-RT1u, lyp/lyp rats became clinically affected, they developed substantial enlargement of the spleen and MLN (Figures 1A and 3A). Splenocyte numbers increased ∼3-fold, whereas MLN numbers increased to ∼10 times normal (Figure 3A). This enlargement was not seen in other peripheral lymph nodes (eg, cervical) (Figure 3A). The T-cell lymphopenia seen in the diabetes-prone BB rat is also present in PVG-RT1u, lyp/lyp rats.11Hernández-Hoyos G. Joseph S. Miller N.G. Butcher G.W. The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.Eur J Immunol. 1999; 29: 1832-1841Crossref PubMed Scopus (46) Google Scholar In the present study, nondiseased PVG-RT1u, lyp/lyp rat CD4+ T cells were ∼40%–50% (Figure 3B) and CD8+ T cells <10% of controls (Figure 3C). The lyp gene mutation does not affect B cells,11Hernández-Hoyos G. Joseph S. Miller N.G. Butcher G.W. The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.Eur J Immunol. 1999; 29: 1832-1841Crossref PubMed Scopus (46) Google Scholar, 25Ramanathan S. Norwich K. Poussier P. Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat.J Immunol. 1998; 160: 5757-5764PubMed Google Scholar and B-cell numbers were not decreased in PVG-RT1u, lyp/lyp animals (Figure 3D). PVG-RT1u, lyp/lyp rats also possessed negligible natural killer T cells in all immune compartments tested, including spleen (Figure 3E) and MLN. As for BB rats,25Ramanathan S. Norwich K. Poussier P. Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat.J Immunol. 1998; 160: 5757-5764PubMed Google Scholar, 26Moore J.K. Scheinman R.I. Bellgrau D. The identification of a novel T cell activation state controlled by a diabetogenic gene.J Immunol. 2001; 166: 241-248PubMed Google Scholar, 27Lang J.A. Kominski D. Bellgrau D. Scheinman R.I. Partial activation precedes apoptotic death in T cells harbouring an IAN gene mutation.Eur J Immunol. 2004; 34: 2396-2406Crossref PubMed Scopus (22) Google Scholar PVG-RT1u, lyp/lyp CD4+ T cells display activation markers. Compared with controls, more lyp/lyp CD4+ T cells express OX-40 and CD25 and are CD45RClo (Figure 4A). These changes appeared by 1 month, and the proportion of activated CD4+ T cells increased with age (unpublished data). In contrast to the Th1 bias seen in diabetic BB rats, the pathology in PVG-RT1u, lyp/lyp intestines suggested a Th2 bias. Therefore, purified lyp/lyp CD4+ T cells were activated in vitro and secreted cytokines analyzed. These cells produced increased amounts of IL-4, whereas IFN-γ levels were similar to controls (Figure 4B). In the absence of ELISA assays for rat IL-5 and IL-13, we investigated gene expression in lyp/lyp T cells by QPCR. In vitro-activated lyp/lyp CD4+ T cells expressed ∼10 times more IL-5 and IL-13 than WT cells (Figure 4B). To ensure that these differences did not represent differences in the proportions of activated/memory T cells in control and lyp/lyp rats, we assessed cytokine production by purified CD4+ CD45RClo cells isolated from WT and lyp/lyp rats. Results showed a similar striking increase in Th2 cytokine expression in lyp/lyp CD4+CD45RClo cells compared with controls, whereas IFN-γ production was similar. Thus, in contrast to BB rats, the lyp/lyp gene on a PVG-RT1u background is associated with a strong Th2 bias. A Th2 bias was seen in splenic and MLN T cells between 1 and 2 months and increased with age (unpublished data). Total serum IgG (unpublished data) and levels of IgG isotypes in lyp/lyp rats were normal (Figure 5A). In control rats, serum IgE was undetectable, but, in lyp/l" @default.
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