Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells

Simon Read1, Fiona Powrie1

1 John Radcliffe Hospital, Oxford, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.13
DOI:  10.1002/0471142735.im1513s30
Online Posting Date:  May, 2001
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Abstract

The severe combined immunodeficiency (SCID) model of colitis shares many features of idiopathic inflammatory bowel disease (IBD) in humans. The SCID model is highly reproducible and easily manipulated, and as such provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. This unit describes a cell transfer system in which SCID mice are reconstituted with CD4+ CD45RBhigh cells to induce IBD. The CD4+ cells are isolated by immunomagnetic negative selection using monoclonal antibodies, and are then separated by fluorescent cell sorting into CD45RBhigh and CD45RBlow cells. The former population reproducibly induces disease in recipients. A support protocol describes methods to monitor disease progression, which is characterized by weight loss, loose stools, and histologically assessed lesions in the colon.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of IBD in SCID Mice by the Transfer of CD45RBhigh CD4+ T Cells
  • Support Protocol 1: Monitoring Development of IBD
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of IBD in SCID Mice by the Transfer of CD45RBhigh CD4+ T Cells

  Materials
  • Female BALB/c mice, 8 to 10 weeks old
  • Female C.B‐17 severe combined immunodeficiency (SCID) mice, 8 to 10 weeks old
  • recipeHBSS/BSA (see recipe)
  • PBS ( appendix 2A) and 2× PBS
  • 0.025% (w/v) trypan blue
  • recipeDepleting antibody mix (see recipe)
  • M450 anti‐rat‐IgG‐coated magnetic beads (Dynal, Advanced Magnetic)
  • recipePBS/BSA (see recipe)
  • Phycoerythrin‐conjugated anti‐CD4 antibody (CD4‐PE; Caltag)
  • Fluorescein isothiocyanate–conjugated anti‐CD45RB antibody (CD45RB‐FITC; Pharmingen)
  • FITC‐ and PE‐labeled isotype controls
  • Heat‐inactivated fetal bovine serum (FBS; appendix 2A)
  • 100‐mesh stainless steel screen (Fisher or equivalent)
  • 5‐ml disposable syringe
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Nylon cell strainer (Falcon)
  • MPC‐1 magnetic concentrator (Dynal or equivalent)
  • Rotary mixer (Dynal or equivalent)
  • 4‐ml polystyrene tubes for cell sorting (Falcon or equivalent)
  • Electronic cell sorter (Becton Dickinson Vantage or equivalent; unit 5.1)
  • Top‐pan balance
  • Additional equipment for removal of lymphoid organs (unit 1.9), preparing single‐cell suspensions (unit 3.1), negative selection of CD4+ cells using magnetic beads (unit 7.4), immunofluorescence staining of single‐cell suspensions (unit 5.3), trypan blue exclusion ( appendix 3B), managing immunocompromised animals (unit 1.2), cell sorting (units 5.1 & 5.3), intraperitoneal injection (unit 1.6), and unique identification of mice (unit 1.5)
NOTE: All manipulations should be carried out at 4°C.

Support Protocol 1: Monitoring Development of IBD

  Materials
  • Female C.B‐17 SCID mice with transferred T cells, weighed weekly (see protocol 1)
  • PBS ( appendix 2A), ice cold
  • 10% (v/v) buffered formalin (Baxter)
  • Hematoxylin counterstain (Mayer's; Sigma)
  • Dissecting instruments
  • 19‐G blunted‐ended needle
  • Tissue‐processing histology cassettes (Fisher)
  • Histology pencil (Fisher)
  • Additional reagents and equipment for CO 2 euthanasia (unit 1.8) and (optional) for paraffin embedding, sectioning, and hematoxylin staining (unit 5.8)
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Figures

Videos

Literature Cited

Literature Cited
   Aranda, R., Sydora, B.C., McAllister, P.L., Binder, S.W., Yang, H.Y., Targan, S.R., and Kronenberg, M. 1997. Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients. J. Immunol. 158:3464‐3473.
   Breese, E., Braegger, C.P., Corrigan, C.J., Walker‐Smith, J.A., and MacDonald, T.T. 1993. Interleukin‐2‐ and interferon‐gamma‐secreting T cells in normal and diseased human intestinal mucosa. Immunology 78:127‐131.
   Cahill, R.J., Foltz, C.J., Fox, J.G., Dangler, C.A., Powrie, F., and Schauer, D.B. 1997. Inflammatory bowel disease: An immunity‐mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infect. Immun. 65:3126‐3131.
   Claesson, M.H., Rudolphi, A., Kofoed, S., Poulsen, S.S., and Reinmann, J. 1996. CD4+ T lymphocytes injected into severe combined immunodeficient (SCID) mice lead to an inflammatory and lethal bowel disease. Clin. Exp. Immunol. 104:491‐500.
   Coffman, R.L. 1982. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre‐B cell development. Immunol. Rev. 69:5‐23.
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   Leach, M.W., Bean, A.G., Mauze, S., Coffman, R.L., and Powrie, F. 1996. Inflammatory bowel disease in C.B‐17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells. Am. J. Pathol. 148:1503‐1515.
   Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S., and Papaioannou, V.E. 1992. RAG‐1‐deficient mice have no mature B and T lymphocytes. Cell 68:869‐877.
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   Morrissey, P.J., Charrier, K., Braddy, S., Liggitt, D., and Watson, J.D. 1993. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178:237‐244.
   Powrie, F. 1995. T cells in inflammatory bowel disease: Protective and pathogenic roles. Immunity 3:171‐174.
   Powrie, F., Leach, M.W., Mauze, S., Caddle, L.B., and Coffman, R.L. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B‐17 scid mice. Int. Immunol. 5:1461‐1471.
   Powrie, F., Correa‐Oliveira, R., Mauze, S., and Coffman, R.L. 1994a. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell‐mediated immunity. J. Exp. Med. 179:589‐600.
   Powrie, F., Leach, M.W., Mauze, S., Menon, S., Caddle, L.B., and Coffman, R.L. 1994b. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1:553‐562.
   Powrie, F., Carlino, J., Leach, M.W., Mauze, S., and Coffman, R.L. 1996. A critical role for transforming growth factor‐beta but not interleukin 4 in the suppression of T helper type 1‐mediated colitis by CD45RB(low) CD4+ T cells. J. Exp. Med. 183:2669‐2674.
   Powrie, F., Mauze, S., and Coffman, R.L. 1998. CD4+ T cells in the regulation of inflammatory responses in the intestine. Res. Immunol. 148:576‐581.
   Roths, J.B. and Sidman, C.L. 1992. Both immunity and hyperresponsiveness to Pneumocystis carinii result from transfer of CD4+ but not CD8+ T cells into severe combined immunodeficiency mice. J. Clin. Invest. 90:673‐678.
   Rudolph, U., Finegold, M.J., Rich, S.S., Harriman, G.R., Srinivasan, Y., Brabet, P., Boulay, G., Bradley, A., and Birnbaumer, L. 1995. Ulcerative colitis and adenocarcinoma of the colon in G alpha i2‐deficient mice. Nature Genet. 10:143‐150.
   Sadlack, B., Merz, H., Schorle, H., Schimpl, A., Feller, A.C., and Horak, I. 1993. Ulcerative colitis‐like disease in mice with a disrupted interleukin‐2 gene. Cell 75:253‐261.
   Shimada, A., Rohane, P., Fathman, C.G., and Charlton, B. 1996. Pathogenic and protective roles of CD45RB(low) CD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse. Diabetes 45:71‐78.
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