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• W2055575075 abstract "Background & Aims: It has been unclear which chemokine network is involved in migration of T-cell subsets to chronically inflamed lesions of the intestine. SAMP1/YP mice develop a spontaneous chronic transmural intestinal lesion specifically in the ileum. Using these mice, we investigated the gut chemokine network involved in specific migration of T-cell subsets to the inflamed lesion of the intestine. Methods: We performed expression analyses of chemokines and their receptors, chemokine receptor blocking studies, and migration studies in vitro and in vivo to identify the gut chemokine network induced in intestinal inflammation and to determine its role in migration of conventional and FoxP3+ suppressor T cells to the inflamed intestine. Results: The expression of homeostatic chemokines was largely unchanged in the inflamed lesion of SAMP1/YP mice compared with control mice. However, an additional chemokine axis (CCL5-CCR5) was up-regulated in the inflamed intestine of SAMP1/YP mice compared with control mice. Activated T cells of SAMP1/YP mice compared with control mice were hyperresponsive to CCL5 in chemotaxis. CCR5+ T cells preferentially migrated to the inflamed lesion, which can be blocked by a CCR5 antagonist. Importantly, the FoxP3+ regulatory T cells of the inflamed lesion of SAMP1/YP mice highly expressed CCR5. CCR5 blockade suppressed the migration of FoxP3+ T cells into the inflamed intestine and significantly exacerbated the intestinal inflammation. Conclusions: The CCL5-CCR5 chemokine axis is involved in preferential recruitment of FoxP3+ regulatory T cells, which prevents further exacerbation of chronic inflammation in the intestine. Background & Aims: It has been unclear which chemokine network is involved in migration of T-cell subsets to chronically inflamed lesions of the intestine. SAMP1/YP mice develop a spontaneous chronic transmural intestinal lesion specifically in the ileum. Using these mice, we investigated the gut chemokine network involved in specific migration of T-cell subsets to the inflamed lesion of the intestine. Methods: We performed expression analyses of chemokines and their receptors, chemokine receptor blocking studies, and migration studies in vitro and in vivo to identify the gut chemokine network induced in intestinal inflammation and to determine its role in migration of conventional and FoxP3+ suppressor T cells to the inflamed intestine. Results: The expression of homeostatic chemokines was largely unchanged in the inflamed lesion of SAMP1/YP mice compared with control mice. However, an additional chemokine axis (CCL5-CCR5) was up-regulated in the inflamed intestine of SAMP1/YP mice compared with control mice. Activated T cells of SAMP1/YP mice compared with control mice were hyperresponsive to CCL5 in chemotaxis. CCR5+ T cells preferentially migrated to the inflamed lesion, which can be blocked by a CCR5 antagonist. Importantly, the FoxP3+ regulatory T cells of the inflamed lesion of SAMP1/YP mice highly expressed CCR5. CCR5 blockade suppressed the migration of FoxP3+ T cells into the inflamed intestine and significantly exacerbated the intestinal inflammation. Conclusions: The CCL5-CCR5 chemokine axis is involved in preferential recruitment of FoxP3+ regulatory T cells, which prevents further exacerbation of chronic inflammation in the intestine. Homeostatic chemokines are expressed often in a tissue-specific manner to guide lymphocyte migration for development and effector function.1Rot A. von Andrian U.H. Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells.Annu Rev Immunol. 2004; 22: 891-928Crossref PubMed Scopus (1005) Google Scholar, 2Kim C.H. The greater chemotactic network for lymphocyte trafficking: chemokines and beyond.Curr Opin Hematol. 2005; 12: 298-304Crossref PubMed Scopus (57) Google Scholar, 3Kunkel E.J. Campbell D.J. Butcher E.C. Chemokines in lymphocyte trafficking and intestinal immunity.Microcirculation. 2003; 10: 313-323Crossref PubMed Google Scholar In the intestine, CCL25 and CCL28 are expressed to regulate the migration of lymphocytes. CCL25 is expressed in the crypts of the small intestine4Kunkel E.J. Campbell J.J. Haraldsen G. Pan J. Boisvert J. Roberts A.I. Ebert E.C. Vierra M.A. Goodman S.B. Genovese M.C. Wardlaw A.J. Greenberg H.B. Parker C.M. Butcher E.C. Andrew D.P. Agace W.W. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (538) Google Scholar, 5Papadakis K.A. Prehn J. Nelson V. Cheng L. Binder S.W. Ponath P.D. Andrew D.P. Targan S.R. The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system.J Immunol. 2000; 165: 5069-5076Crossref PubMed Scopus (278) Google Scholar and attracts immunoglobulin A+ plasma cells6Bowman E.P. Kuklin N.A. Youngman K.R. Lazarus N.H. Kunkel E.J. Pan J. Greenberg H.B. Butcher E.C. The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells.J Exp Med. 2002; 195: 269-275Crossref PubMed Scopus (207) Google Scholar and T cells.7Mora J.R. Bono M.R. Manjunath N. Weninger W. Cavanagh L.L. Rosemblatt M. Von Andrian U.H. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells.Nature. 2003; 424: 88-93Crossref PubMed Scopus (894) Google Scholar CCL28 is expressed by epithelial cells in various mucosal tissues8Pan J. Kunkel E.J. Gosslar U. Lazarus N. Langdon P. Broadwell K. Vierra M.A. Genovese M.C. Butcher E.C. Soler D. A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues.J Immunol. 2000; 165: 2943-2949Crossref PubMed Scopus (247) Google Scholar, 9Wang W. Soto H. Oldham E.R. Buchanan M.E. Homey B. Catron D. Jenkins N. Copeland N.G. Gilbert D.J. Nguyen N. Abrams J. Kershenovich D. Smith K. McClanahan T. Vicari A.P. Zlotnik A. Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2).J Biol Chem. 2000; 275: 22313-22323Crossref PubMed Scopus (227) Google Scholar and attracts immunoglobulin A+ lymphocytes.10Kunkel E.J. Kim C.H. Lazarus N.H. Vierra M.A. Soler D. Bowman E.P. Butcher E.C. CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells.J Clin Invest. 2003; 111: 1001-1010Crossref PubMed Scopus (270) Google Scholar, 11Lazarus N.H. Kunkel E.J. Johnston B. Wilson E. Youngman K.R. Butcher E.C. A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts.J Immunol. 2003; 170: 3799-3805Crossref PubMed Scopus (204) Google Scholar, 12Hieshima K. Ohtani H. Shibano M. Izawa D. Nakayama T. Kawasaki Y. Shiba F. Shiota M. Katou F. Saito T. Yoshie O. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.J Immunol. 2003; 170: 1452-1461Crossref PubMed Scopus (196) Google Scholar CCL11 is implicated in the migration of eosinophils into the intestine.13Rothenberg M.E. Eotaxin An essential mediator of eosinophil trafficking into mucosal tissues.Am J Respir Cell Mol Biol. 1999; 21: 291-295Crossref PubMed Scopus (147) Google ScholarIt has been reported that other chemokines are up-regulated in intestinal tissues with inflammatory bowel diseases (IBD). Chemokines such as CCL2, CCL4, CCL5, CCL9, CCL17, CCL19, CCL20, CXCL1, CXCL5, and CL1 are up-regulated in chronically inflamed colon tissues of interleukin (IL)-10 (−/−) mice.14Scheerens H. Hessel E. de Waal-Malefyt R. Leach M.W. Rennick D. Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10−/− mice and Rag-2−/− mice reconstituted with CD4+CD45RBhigh T cells.Eur J Immunol. 2001; 31: 1465-1474Crossref PubMed Scopus (99) Google Scholar In Rag2 (−/−) mice reconstituted with CD4+CD45RBhigh T cells, CCL2, CCL5, CCL9, CCL17, CCL22, and CXCL10 are up-regulated in the mouse intestine.14Scheerens H. Hessel E. de Waal-Malefyt R. Leach M.W. Rennick D. Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10−/− mice and Rag-2−/− mice reconstituted with CD4+CD45RBhigh T cells.Eur J Immunol. 2001; 31: 1465-1474Crossref PubMed Scopus (99) Google Scholar Expression of CCL17 and CCL22, along with others, has been reported in Crohn’s disease (CD) in humans.15Jugde F. Alizadeh M. Boissier C. Chantry D. Siproudhis L. Corbinais S. Quelvennec E. Dyard F. Campion J.P. Gosselin M. Bretagne J.F. Semana G. Heresbach D. Quantitation of chemokines (MDC, TARC) expression in mucosa from Crohn’s disease and ulcerative colitis.Eur Cytokine Netw. 2001; 12: 468-477PubMed Google Scholar The functions of these proinflammatory chemokines in T-cell migration to the intestine have yet to be elucidated.SAMP1/Yit mice spontaneously develop intestinal inflammation similar to that of human CD in many aspects: a discontinuous lesion localized in the terminal ileum and heavily infiltrated with T cells, neutrophils, and macrophages.16Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar, 17Strober W. Nakamura K. Kitani A. The SAMP1/Yit mouse: another step closer to modeling human inflammatory bowel disease.J Clin Invest. 2001; 107: 667-670Crossref PubMed Google Scholar The localized transmural ileitis is a unique feature not shared with other mouse models of IBD.18Pizarro T.T. Arseneau K.O. Bamias G. Cominelli F. Mouse models for the study of Crohn’s disease.Trends Mol Med. 2003; 9: 218-222Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Mizoguchi A. Mizoguchi E. Bhan A.K. Immune networks in animal models of inflammatory bowel disease.Inflamm Bowel Dis. 2003; 9: 246-259Crossref PubMed Scopus (64) Google Scholar The intestinal lesion of SAMP1/Yit mice has increased crypts and decreased numbers of intraepithelial lymphocytes.16Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar, 20Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn’s disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (216) Google Scholar, 21Makita S. Kanai T. Matsumoto S. Iiyama R. Uraushihara K. Totsuka T. Yamazaki M. Nakamura T. Ishikawa H. Watanabe M. The role of cryptopatch-derived intraepithelial lymphocytes in the development of chronic ileocecitis.Scand J Immunol. 2003; 58: 428-435Crossref PubMed Scopus (4) Google Scholar There is an increase in Th1 cell numbers, expression of IL-5, and infiltration of eosinophils.20Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn’s disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (216) Google Scholar, 22Takedatsu H. Mitsuyama K. Matsumoto S. Handa K. Suzuki A. Funabashi H. Okabe Y. Hara T. Toyonaga A. Sata M. Interleukin-5 participates in the pathogenesis of ileitis in SAMP1/Yit mice.Eur J Immunol. 2004; 34: 1561-1569Crossref PubMed Scopus (39) Google Scholar A substrain called SAMP1/YitFc derived after more than 20 generations of inbreeding displays more accelerated ileitis, muscularis hypertrophy, ulceration, and fistulae formation.23Rivera-Nieves J. Bamias G. Vidrich A. Marini M. Pizarro T.T. McDuffie M.J. Moskaluk C.A. Cohn S.M. Cominelli F. Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis.Gastroenterology. 2003; 124: 972-982Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar An initial genetic study has identified peroxisome proliferator-activated receptor γ as one of the potential susceptibility genes in SAMP1/YitFc mice.24Sugawara K. Olson T.S. Moskaluk C.A. Stevens B.K. Hoang S. Kozaiwa K. Cominelli F. Ley K.F. McDuffie M. Linkage to peroxisome proliferator-activated receptor-gamma in SAMP1/YitFc mice and in human Crohn’s disease.Gastroenterology. 2005; 128: 351-360Abstract Full Text Full Text PDF PubMed Scopus (74) Google ScholarIn this study, we characterized the altered gut chemokine network in the SAMP1/Yit mice maintained at Purdue University (referred to as “SAMP1/YP mice” to differentiate them from other mouse colonies) and investigated the role of this additional chemokine network in migration of T-cell subsets to the intestinal lesion and in intestinal inflammation. While the 2 housekeeping chemokines CCL25 and CCL28 were expressed unchanged in a segment-specific manner in the SAMP1/YP mice compared with control AKR/J mice, we found a new gut chemokine axis (CCL5-CCR5) induced in the chronically inflamed intestine. Our results suggest that this newly induced gut chemokine axis plays an important role in migration of T cells, particularly FoxP3+ regulatory T cells, to the chronically inflamed intestine.Materials and MethodsAnimalsSAMP1/YP (H-2k) mice, originally derived as previously described,16Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar and AKR/J mice, purchased from Jackson Laboratories (Bar Harbor, ME), were maintained for up to ∼18 generations at Purdue University in conventional cages. Severe combined immunodeficient (SCID; C3HSmn.C-Prkdcscid/J) mice were purchased from Jackson Laboratories and maintained in barrier cages under a specific pathogen-free condition. All the experiments with animals in this study were approved by the Purdue Animal Care and Use Committee.Assessment of Spontaneous Inflammation in SAMP1/YP MiceSAMP1/YP mice and AKR/J control mice were killed at indicated ages, and the intestine was histologically evaluated. In a subset of experiments, AKR/J and SAMP1/YP mice were treated once every 2 days for 11 weeks (from 4 to 15 weeks of age) with intraperitoneal injections of 100 μg of TAK-779 (NIH AIDS Research & Reference Reagent Program) in 100 μL of 5% d-mannitol (control medium) or the control medium only. Mice were killed, and the small intestine was fixed in neutral-buffered formalin, embedded in paraffin, and stained with H&E. For histologic assessment, the severity of tissue infiltration by mononuclear cells and polymorphonuclear cells, numbers of goblet cells, and thickness of crypts and muscularis propria in affected lesions of SAMP1/YP mice and control tissues of AKR/J mice were scored in 10 randomly selected high-power fields to calculate the average scores for each lesion.Assessment of Induced Intestinal Inflammation in SCID MiceFor the adoptive transfer model, adult female major histocompatibility complex–matched SCID mice were used as recipients in the following experiments. Mesenteric lymph node (MLN) cells (1 × 106 cells/mouse) of SAMP1/YP mice (∼30 weeks old) were injected intraperitoneally into 6-week-old SCID mice to induce intestinal inflammation. Some mice were injected with control medium. TAK-779 was injected as described previously. Mice were monitored for weight change, and all mice were killed 6 weeks after cell transfer or when mice with severe inflammation became moribund and/or lost >20% of their initial weight. The intestines were removed and evaluated histologically. The intestinal inflammation in SCID mice was scored by combining the severity scores (0–4; 0, no inflammation; 1, slight infiltration of inflammatory cells in the lamina propria; 2, moderate infiltration with inflammatory cells with mild mucosa hyperplasia; 3, marked infiltration with inflammatory cells with disturbed mucosal architecture, crypt abscesses, and marked hyperplasia; and 4, massive infiltration with inflammatory cells with severe mucosa hyperplasia) and the area scores (0–4; 0, no area affected; 1, 1%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%).RNA Expression Analyses of Chemokines and Chemokine ReceptorsTotal RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) from the terminal ileum, Peyer’s patches, and MLNs of 30-week-old AKR/J or SAMP1/YP mice. The RNA was reverse transcribed using SuperScript First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. Serially diluted complementary DNA samples were analyzed for expression of a panel of chemokines by real-time polymerase chain reaction (PCR) using SYBR Green (BMA, Rockland, ME) on an ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA). The expression levels of chemokines in each sample were assessed after normalization for β-actin. For conventional PCR, complementary DNA samples were serially diluted and first analyzed by PCR (20 cycles) for β-actin to determine appropriate dilution factors for each sample and then PCR analyzed for genes of interest. A total of 5–20 μg of total RNA was assayed by the RiboQuant Multi-Probe RNase Protection Assay System (BD Biosciences, San Diego, CA).In Situ Fluorescent ImmunohistochemistryTerminal ileum tissues from 20- to 30-week-old AKR/J or SAMP1/YP mice frozen in Tissue-Tek freezing medium (Miles, Elkhart, IN) were made into 5-μm sections. Frozen sections were fixed in cold acetone and blocked with 10% rabbit serum for 20 minutes at room temperature. Sections were stained with goat anti-mCCL5 antibody (10 μg/mL, AF478; R&D Systems, Minneapolis, MD) or goat control immunoglobulin G antibody (Sigma-Aldrich, St Louis, MO) for 1 hour at 4°C. The sections were further incubated with biotinylated anti-goat immunoglobulin G (Vector Laboratories, Burlingame, CA). Sections were blocked with 5% goat serum and further stained with streptavidin-phycoerythrin, anti–CD4/fluorescein isothiocyanate (L3T4), or anti–CD8/fluorescein isothiocyanate (53-6.7). The sections were further stained with Hoechst 33342 and examined with a Nikon E400 microscope (Tokyo, Japan) equipped with epifluorescence.Assessment of CCL5 Expression by Enzyme-Linked Immunosorbent AssayCD4+ or CD8+ T cells were isolated from MLNs by magnetic beads (Miltenyi Biotec, Auburn, CA; ∼90% pure). The MLN CD4+ or CD8+ T cells (0.4 × 106 cells/well) were cultured in 96-well plates (BD Biosciences, San Jose, CA) coated with 10 μg/mL of anti-CD3 (17A2; BD Biosciences) for 2 days in a co2 incubator. When indicated, anti-CD28 (37.51; BD Biosciences; 2 μg/mL) was added to provide costimulation signals. The conditioned culture medium was examined by CCL5 enzyme-linked immunosorbent assay (ELISA). Additionally, tissue proteins were extracted from the terminal ileum tissues of 30-week-old SAMP1/YP and age-matched control AKR/J mice by grinding the tissues in liquid nitrogen using a mortar and a pestle followed by homogenization using zirconium beads in phosphate-buffered saline buffer. A protease inhibitor cocktail (Sigma-Aldrich) was added to prevent degradation of proteins during and after homogenization. Serially diluted homogenates were examined for CCL5 protein levels by a 3-step sandwich ELISA (R&D Systems). The total protein concentrations were determined by a bicinchoninic acid method (Pierce, Rockford, IL).Chemotaxis AssayThe chemotaxis assay was performed by a 2-chamber Transwell migration assay system (pore size, 5.0 μm; Corning, Corning, NY) as previously described.25Kim C.H. Kunkel E.J. Boisvert J. Johnston B. Campbell J.J. Genovese M.C. Greenberg H.B. Butcher E.C. Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential.J Clin Invest. 2001; 107: 595-601Crossref PubMed Scopus (277) Google Scholar, 26Kim C.H. Qu C.K. Hangoc G. Cooper S. Anzai N. Feng G.S. Broxmeyer H.E. Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.J Exp Med. 1999; 190: 681-690Crossref PubMed Scopus (81) Google Scholar, 27Kim C.H. Rott L. Kunkel E.J. Genovese M.C. Andrew D.P. Wu L. Butcher E.C. Rules of chemokine receptor association with T cell polarization in vivo.J Clin Invest. 2001; 108: 1331-1339Crossref PubMed Scopus (423) Google Scholar MLN cells were isolated from 30-week-old AKR/J or SAMP1/YP mice and activated with concanavalin A (2.5 μg/mL; Sigma Chemical Co) for 2 days and then cultured in murine IL-2 (5 ng/mL) for an additional 2 days. A total of 0.5 × 106 cells/well was added to the upper chambers. In some experiments, TAK-779 (1.0 μg/mL) was added to both chambers. After 3 hours, the cells that migrated to the lower chambers were harvested, stained with anti–CD4/APC and anti–CD8/cychrome, and counted by FACScalibur (BD Biosciences).Retroviral Expression of Chemokine Receptors and Chemokine–Chemokine Receptor InteractionChemokine receptor–overexpressing T cells were generated by a retroviral transfer method.28Ouyang W. Ranganath S.H. Weindel K. Bhattacharya D. Murphy T.L. Sha W.C. Murphy K.M. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism.Immunity. 1998; 9: 745-755Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar CD4+ T cells were activated for 36 hours with concanavalin A and IL-2 (5 ng/mL; R&D Systems). CD4+ T cells were then transduced with retrovirus made in Phoenix-ecotropic retrovirus packaging cells (a gift from Dr Gary Nolan, Stanford University). Retroviral gene transfer rates were assessed based on green fluorescent protein expression by fluorescence-activated cell sorter analysis. A pCDNA3.1 vector for expression of CCL5-Fc fusion protein was constructed12Hieshima K. Ohtani H. Shibano M. Izawa D. Nakayama T. Kawasaki Y. Shiba F. Shiota M. Katou F. Saito T. Yoshie O. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.J Immunol. 2003; 170: 1452-1461Crossref PubMed Scopus (196) Google Scholar and transfected into HEK-293 cells for production of CCL5-Fc fusion protein in 3-day-old culture supernatant. Biotinylated anti-human immunoglobulin G and streptavidin-APC were used to detect the binding of CCL5-Fc proteins to cell surface chemokine receptors.Expression of FoxP3 and CCR5 by Intestinal T CellsThe lamina propria lymphocytes were isolated after removing the epithelial cells with 5 mmol/L EDTA (5 times), digestion (3 times, 45 minutes each) with 300 U/mL collagenase (type 3; Worthington, Lakewood, NJ) and 100 μg/mL deoxyribonuclease I (Worthington), filtration through a nylon mesh, and centrifugation in a 40/75% Percoll gradient. The cells, isolated from the intestine, Peyer’s patches, and MLNs, were stained with antibodies to CCR5 (C34-3448; BD Biosciences, San Jose, CA), CD4, and FoxP3 (FJK-16s; eBioscience) for flow cytometric determination of CCR5 expression and frequency of FoxP3+ cells.T-Cell Homing Into Inflamed IntestineTest CCR5+ CD4+ T cells were generated by retroviral gene transfer as described previously from the MLN cells of SAMP1/YP mice. The test T cells and control T cells (untransduced) were cultured in the presence of retinoic acid (10 nmol/L; Sigma-Aldrich) to induce the gut-homing phenotype.29Iwata M. Hirakiyama A. Eshima Y. Kagechika H. Kato C. Song S.Y. Retinoic acid imprints gut-homing specificity on T cells.Immunity. 2004; 21: 527-538Abstract Full Text Full Text PDF PubMed Scopus (1194) Google Scholar Control T cells were labeled with the red fluorescent dye tetramethylrhodamine-5-(and-6)-isothiocyanate, while CCR5+ CD4+ T cells were labeled with the green dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). The 2 T-cell subsets (107 cells per mouse) were coinjected into SAMP1/YP mice via a tail vein. T cells were isolated from various tissues of the SAMP1/YP or AKR/J mice 20–24 hours after injection and analyzed by flow analysis. We also generated gut-homing FoxP3+ (and FoxP3−) CD4+ T cells by retroviral transduction of murine FoxP3 gene into spleen T cells in the presence of retinoic acid. For this, spleen T cells from AKR/J mice were activated for 36 hours as described previously and infected with FoxP3 retrovirus. Cells were further cultured for 4 days in the presence of retinoic acid and cytokines (5 ng/mL of IL-2 alone or IL-2 and 2 ng/mL of IL-12). Naive CD4+ T cells become regulatory T cells when they are transduced with the FoxP3 gene.30Hori S. Nomura T. Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (47) Google Scholar, 31Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (6022) Google Scholar The cells (∼2 × 107 cells per mouse) were labeled with CFSE and injected intravenously into SAMP1/YP mice for 20- to 24-hour homing. Lamia propria cells were isolated and stained for CD4, FoxP3, and CCR5 as described previously. Normalized homing indexes were calculated by the formula HI = a ÷ b, where a represents [Test Cells Migrated Into the Tissue Site] ÷ [Control Cells Migrated Into the Tissue Site] and b represents [Test Cells in Input] ÷ [Control Cells in Input].In Vitro Suppression of Target T Cells by CD4+CD25+ CellsCD4+CD25− T cells (responders) and CD4+CD25+ T cells (suppressors) were isolated from MLNs of AKR/J and SAMP1/YP mice and cocultured for 72 hours at indicated ratios in the presence of anti-CD3 (5 μg/mL; 145-2C11; eBioscience) and 1 × 105 irradiated splenocytes as antigen-presenting cells in U-bottomed 96-well plates. Cells were further incubated with 1 μCi/well of 3H-thymidine for 8 hours, and 3H-thymidine incorporation was measured by a beta scintillation counter (Packard Instruments, Meriden, CT).Statistical AnalysesStudent t test (paired or unpaired; 2-tailed) was used to determine the significance of the differences between 2 sets of related data. Statistical significance was set at P values less than .05.ResultsThe Gut Chemokine Network of SAMP1/YP MiceAs previously reported,16Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar, 20Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn’s disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (216) Google Scholar the SAMP1/YP mice develop a CD-like lesion with the thickening of the lamina propria infiltrated with immune cells in the ileum segment (Figure 1A and B). The ileitis started early at 4–5 weeks of age and became more severe with age. Most mice had advanced ileitis at 10 weeks of age. The ileitis was characterized by severe discontinuous transmural inflammation, localized in the terminal ileum (the last 7–8 cm before the ileocecal junction), with mild inflammation extending into the proximal ileum in some mice. Bowel wall thickening and constriction of the lumen in the terminal ileum, caused by inflammation and hyperplasia of the mucosa, submucosa, and muscularis externa, were readily detected grossly. The lesions were more opaque than uninflamed segments and devoid of digested food content due to bowel thickening and constriction. Inflamed lamina propria of SAMP1/YP ileum was heavily infiltrated with mononuclear and polymorphonuclear leukocyte cells. Infiltration of polymorphonuclear leukocytes and mononuclear cells in the submucosa and muscularis externa was relatively mild compared with that in the lamina propria. Hyperplasia of the crypts and fusion of villi were observed in the lesions, and goblet cells were greatly increased in number (Figure 1B). Mice with intestinal fistulae were rare. Histologic analysis of intestinal segments other than ileum revealed no apparent inflammation (Figure 1C). MLNs of SAMP1/YP mice were enlarged and fused together. The results indicate that the inflammation was more advanced than in the mice described in the original report16Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar and similar or slightly less advanced than in the SAMP1/YitFc mice described by Rivera-Nieves et al.23Rivera-Nieves J. Bamias G. Vidrich A. Marini M. Pizarro T.T. McDuffie M.J. Moskaluk C.A. Cohn S.M. Cominelli F. Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis.Gastroenterology. 2003; 124: 972-982Abstract Full Text Full Text PDF PubMed Scopus (124) Google ScholarSAMP1/YP mice were originally derived from AKR/J mice. Therefore, AKR/J mice have commonly been used as normal controls for SAMP1/YP mice. We assessed the basal expression levels of gut chemokines in AKR/J mice and SAMP1/YP mice. Intestinal tissues were divided into 4 parts: jejunum, ileum, cecum, and colon. A quantitative reverse-transcription polymerase chain reaction technique was used to examine the message levels of 19 major CC and CXC chemokines (Figure 2A). Conventional PCR was also performed for selected chemokines highly expressed in the" @default.
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