Mercuric Chloride–Induced Autoimmunity

A. Badou1, A. Saoudi1, G. Dietrich1, E. Druet1, P. Druet1, L. Pelletier1

1 Institut Fédératif de Recherche, Toulouse, France
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.15
DOI:  10.1002/0471142735.im1515s32
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl2 injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti‐laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4+ TH2 anti‐self MHC class II molecules and preparation of T cell lines. IL‐4 is produced very early after the first HgCl2 injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL‐4 mRNA expression may be detected in spleen and lymph nodes from HgCl2‐injected BN rats. The fact that HgCl2 induces in vitro mRNA IL‐4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic TH2 cells; a test for this condition is therefore also included.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of Mercury Disease in BN Rats
  • Basic Protocol 2: Adoptive Transfer of Autoimmunity by Autoreactive CD4+ Anti–Self MHC Class II T Cell Lines
  • Support Protocol 1: Measurement of Serum IgE Concentration in Rats by ELISA
  • Support Protocol 2: Measurement of Anti‐Laminin Antibody Titer in Rats
  • Support Protocol 3: Detection of Renal Immunoglobulin Deposits in HgCl2‐Treated Rats
  • Support Protocol 4: Generation of Autoreactive Anti–Self MHC Class II T Cell Lines from Gold Salt–Injected Rats
  • Support Protocol 5: Measurement of IL‐4 mRNA Expression in BN Rat Spleen Cells Exposed In Vitro or In Vivo to HgCl2
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Mercury Disease in BN Rats

  Materials
  • Female or male BN rats 8 to 12 week old (e.g., Charles River Labs)
  • HgCl 2 (e.g., Sigma)
  • Distilled water, PBS, or 0.9% (w/v) NaCl, sterile
  • Protein assay kit (Bio‐Rad) or equivalent
  • Metabolic cages for housing rats
  • 1‐ml syringes with 26‐G needles
  • 50‐ml conical polypropylene tubes, sterile
  • Additional reagents and equipment for rat identification (unit 1.5); anesthesia (unit 1.4); and euthanasia (unit 1.8); blood collection (unit 1.7); serum preparation (unit 2.4); determination of IgE (see protocol 3) and autoantibody titers (see protocol 4Support Protocols 2 and protocol 53); and evaluation of arthritis symptoms (unit 15.4)

Basic Protocol 2: Adoptive Transfer of Autoimmunity by Autoreactive CD4+ Anti–Self MHC Class II T Cell Lines

  Materials
  • Anti–self MHC class II T cell line derived from gold salt–injected BN rats (see protocol 6)
  • Irradiated (2000 rad) or mitomycin C–treated syngeneic thymocytes (unit 3.12)
  • Complete RPMI‐10 ( appendix 2A)
  • RPMI 1640
  • Male or female BN recipient rats (e.g., Charles River Labs)
  • 1 mg/ml purified recipeanti–rat CD8+ (OX8) MAb (see recipe) in sterile PBS
  • 1 mg/ml isotype‐matched control MAb (e.g., MOPC‐21; purchased from Pharmingen or prepared from B cell hybridomas) in sterile PBS
  • FITC‐labeled OX8 MAb (Pharmingen)
  • FITC‐labeled RAM (rabbit anti‐mouse Ig; Sigma)
  • PBS ( appendix 2A) containing 10% normal rat serum (heat inactivated 30 min at 56°C min and filtered through 0.22‐µm‐pore‐size Millipore filters)
  • Additional reagents and equipment for T cell culture (unit 3.12), removal of dead cells by one‐step Ficoll‐Paque gradient centrifugation (unit 3.1; optional), counting of viable cells with trypan blue ( appendix 3B), euthanasia (unit 1.8), preparation of single‐cell spleen suspension and depletion of red blood cells (unit 3.1), and flow cytometry (unit 5.3)

Support Protocol 1: Measurement of Serum IgE Concentration in Rats by ELISA

  Materials
  • recipe0.05 M sodium carbonate buffer, pH 9.6 (see recipe), containing 0.01% NaN 3
  • IgG mouse anti–rat IgE (MARE‐1; LO‐IMEX, University of Louvain, Brussels)
  • PBS ( appendix 2A) containing 0.05% Tween 20 (PBS/Tween)
  • Milk powder
  • Gelatin (Sigma)
  • Test samples: serum samples obtained before and at weekly intervals after the first HgCl 2 injection (see protocol 1)
  • Serum samples from control animals (see protocol 1)
  • recipeIgE standard (see recipe)
  • Biotinylated mouse anti–rat κ (MARK1) and λ chain (MARL15) antibody mixture (mixture available from LO‐IMEX, University of Louvain, Brussels)
  • Peroxidase‐labeled streptavidin (Amersham)
  • recipeortho‐Phenylenediamine (OPD) substrate solution (see recipe)
  • 2 M HCl
  • 96‐well microtiter plates (Nunc)
  • Microtiter plate reader (Molecular Devices)

Support Protocol 2: Measurement of Anti‐Laminin Antibody Titer in Rats

  Materials
  • Engelbreth‐Holm‐Swarm (EHS) mouse sarcoma laminin (Sigma), dissolved at 10 µg/ml in PBS containing 0.01% NaN 3
  • PBS/Tween: PBS containing 0.05% Tween 20
  • Test samples: serum samples obtained before and at days 7, 14, 21, 28, and 35 after the first HgCl 2 injection (see protocol 1)
  • Serum samples from control animals (see protocol 1)
  • Anti‐laminin standard: pool of sera (unit 2.4) obtained from BN rats injected with HgCl 2 for 15 days (see protocol 1)
  • Biotin‐labeled sheep anti‐Fcγ rat Ig antibodies (Jackson Immunoresearch)
  • Gelatin (Sigma)
  • 96‐well microtiter plates (Nunc)
  • Microtiter plate reader (Molecular Devices)
  • Additional reagents and equipment for serum collection and preparation (units 1.7 & 2.4) and streptavidin/OPD detection of antibodies (see 15.15, steps 15.15 to 15.15)

Support Protocol 3: Detection of Renal Immunoglobulin Deposits in HgCl2‐Treated Rats

  Materials
  • Kidneys from rats treated 2 weeks to 1 month with HgCl 2 (see protocol 1)
  • Kidneys from sex and age‐matched normal or NaCl‐injected BN rats (controls)
  • PBS ( appendix 2A)
  • Fluoresceinated anti–rat IgG (Sigma or other source)
  • recipeSlide‐mounting solution (see recipe)
  • UV‐light microscope
  • Additional reagents and equipment for immunohistochemistry (unit 21.4)

Support Protocol 4: Generation of Autoreactive Anti–Self MHC Class II T Cell Lines from Gold Salt–Injected Rats

  Materials
  • BN rats (e.g., Charles River Labs)
  • Allochrysine (aurothiopropanolsulfonate sodium salt; Laboratoire Sarbach): 1 vial (containing 100 mg) dissolved in 5 ml of 0.9% (w/v) NaCl
  • Complete RPMI ( appendix 2A) supplemented with 1% normal rat serum (heat inactivated 30 min at 56°C min and filtered through 0.22‐µm‐pore‐size Millipore filters)
  • Concanavalin A (Con A)–activated supernatant (see unit 3.13) or human rIL‐2 (e.g., Boehringer Mannheim)
  • Complete RPMI‐10 medium ( appendix 2A)
  • 75‐cm2 tissue flasks, sterile
  • Additional reagents and equipment for treatment of rats with HgCl 2 (see protocol 1), euthanasia (unit 1.8), removal of rat lymphoid organs (unit 1.9), preparation of cell suspensions (unit 3.1), and irradiation of thymocytes (unit 3.12)

Support Protocol 5: Measurement of IL‐4 mRNA Expression in BN Rat Spleen Cells Exposed In Vitro or In Vivo to HgCl2

  Materials
  • BN rats (e.g., Charles River Labs), untreated or treated with HgCl 2 (see protocol 1, steps and )
  • 1.35 mg/ml (5 mM) HgCl 2 in 0.9% (w/v) NaCl, prepared fresh (see protocol 1, step )
  • 0.9% (w/v) NaCl
  • 1 mg/ml ionomycin in dimethyl sulfoxide (DMSO; store in aliquots at −80°C and discard unused thawed solution)
  • Rat‐IL‐4‐specific PCR primers (Life Technologies):
    • Sense: 5′‐TGA TGG GTC TCA GCC CCC ACC TTG C‐3′
    • Antisense: 5′‐CTT TCA GTG TTG TGA GCG TGG ACT C‐3′
  • β‐actin‐specific PCR primers:
    • Sense: 5′‐TGG AAT CCT GTG GCA TCC ATG AAA C‐3′
    • Antisense: 5′‐TAA AAC GCA GCT CAG TAA CAG TCC G‐3′
  • Thermal cycler (Perkin Elmer)
  • High‐performance CCD camera coupled to a computer for densitometric analysis
  • Gel Analyst program (Iconix) or other program for quantitation of band intensity
  • Additional reagents and equipment for removal of rat lymphoid organs (unit 1.9), preparation of single‐cell suspensions (unit 3.1), RNA extraction (unit 10.11), and reverse transcription, PCR, and gel electrophoresis (units 10.20 & 10.23)
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Figures

Videos

Literature Cited

Literature Cited
   Aten, J., Bosman, C.B., Rozing, J., Stjnen, T., Hoedemaeker, P.J., and Weening, J.J. 1988. Mercuric chloride–induced autoimmunity in the Brown Norway rat. Cellular kinetics and major histocompatibility complex antigen expression. Am. J. Pathol. 133:127‐138.
   Badou, A., Savignac, M., Moreau, M., Leclerc, C., Pasquier, R., Druet, P., and Pelletier, L. 1997. HgCl2‐induced interleukin‐4 gene expression in T cells involves a protein kinase C–dependent calcium influx through L‐type calcium channels. J. Biol. Chem. 272:32411‐32418.
   Berra, E., Diaz‐Meco, M., Dominguez, I., Municio, M.M., Sanz, L., Lozano, J., Chapkin, R.S., and Moscat, J. 1993. Protein kinase C ζ isoform is critical for mitogenic signal transduction. Cell 74:555‐563.
   Cantrell, D. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:259‐274.
   Castedo, M., Pelletier, L., Pasquier, R., and Druet, P. 1994. Improvement of TH1 functions during the regulation phase of mercury disease in Brown Norway rats. Scand. J. Immunol. 39:144‐150.
   Cornacchia, E., Golbus, J., Maybaum, J., Strahler, J., Hanash, S., and Richardson, B. 1988. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J. Immunol. 140:2197‐2200.
   Druet, P., Sapin, C., Druet, E., and Hirsch, F. 1982. Genetic control of mercury‐induced immune response in the rat. In Nephrotoxic Mechanisms of Drugs and Environmental Toxins (G. Porter, ed.) pp. 425‐435. Plenum, New York.
   Dubey, C., Bellon, B., Hirsch, F., Kuhn, J., Vial, M.‐C., Goldman, M., and Druet, P. 1991. Increased expression of class II major histocompatibility complex molecules on B cells in rats susceptible or resistant to HgCl2‐induced autoimmunity. Clin. Exp. Immunol. 86:118‐125.
   Dubey, C., Kuhn, J., Vial, M.C., Druet, P., and Bellon, B. 1993. Anti‐interleukin‐2 receptor monoclonal antibody therapy supports a role for TH1‐like cells in HgCl2‐induced autoimmunity in rats. Scand. J. Immunol. 37:406‐412.
   Fillon, J., Baccala, R., Kuhn, J., Druet, P., and Bellon, B. 1997. Evidence for heterogenous TCRVβ expression in mercury‐induced autoimmune disorders in rats. Int. Immunol. 9:263‐271.
   Fraser, J.D., Straus, D., and Weiss, A. 1993. Signal transduction events leading to T‐cell lymphokine gene expression. Immunol. Today 14:357‐362.
   Gillespie, K.M., Qasim, F.J., Tibbats, L.M., Thiru, S., Oliveira, D.B.G., and Mathieson, P.W. 1995. Interleukin‐4 gene expression in mercury‐induced autoimmunity. Scand. J. Immunol. 41:268‐272.
   Gillespie, K.M., Saoudi, A., Kuhn, J., Whittle, C.J., Druet, P., Bellon, B., and Mathieson, P.W. 1996. TH1/TH2 cytokine gene expression after mercuric chloride in susceptible and resistant rat strains. Eur. J. Immunol. 10:2388‐2392.
   Hirsch, F., Couderc, J., Sapin, C., Fournie, G., and Druet, P. 1982. Polyclonal effect of HgCl2 in the rat, its possible role in an experimental autoimmune disease. Eur. J. Immunol. 12:620‐625.
   Kermarrec, N., Blanpied, C., Pelletier, L., Feingold, N., Mandet, C., Druet, P., and Hirsch, F. 1995. Genetic study of gold salt–induced immune disorders in the rat. Nephrol. Dial. Transplant. 10:2187‐2191.
   Kermarrec, N., Dubay, C., De Gouyon, B., Blanpied, C., Gauguier, D., Gillespie, K., Druet, P., Lathrop, M., and Hirsch, F. 1996. Serum IgE concentration and other immune manifestations of treatment with gold salts are linked to MHC and IL‐4 regions in the rat. Genomics 31:111‐114.
   Mathieson, P.W., Stapleton, K.J., Oliveira, D.B.G., and Lockwood, C.M. 1991. Immunoregulation of mercuric chloride–induced autoimmunity in Brown Norway rats: A role for CD8+ T cells revealed by in vivo depletion studies. Eur. J. Immunol. 21:2105‐2109.
   Mathieson, P.W., Thiru, S., and Oliveira, D.B.G. 1992. Mercuric chloride–treated Brown Norway rats develop widespread tissue injury including necrotizing vasculitis. Lab. Invest. 67:121‐129.
   Mathieson, P.W., Thiru, S., and Oliveira, D.B.G. 1993. Regulatory role of OX22high T cells in mercury‐induced autoimmunity in the Brown Norway rat. J. Exp. Med. 177:1309‐1316.
   McMenamin, C. and Holt, P.G. 1993. The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class I–restricted CD8+ T cell–mediated but MHC class II–restricted CD4+ T cell–dependent immune deviation resulting in selective suppression of immunoglobulin E production. J. Exp. Med. 178:889‐899.
   Oliveira, D.B.G., Gillespie, K., Wolfreys, K., Mathieson, P.W., Qasim, F., and Coleman, J.W. 1995. Compounds that induce autoimmunity in the Brown Norway rat sensitize mast cells for mediator release and interleukin‐4 expression. Eur. J. Immunol. 25:2259‐2264.
   Pelletier, L., Pasquier, R., Rossert, J., Vial, M.‐C., Mandet, C., and Druet, P. 1988. Autoreactive T cells in mercury‐induced autoimmunity. Ability to induce the autoimmune disease. J. Immunol. 140:750‐754.
   Prigent, P., Saoudi, A., Pannetier, C., Graber, P., Bonnefoy, Y., Druet, P., and Hirsch, F. 1995. Mercuric chloride, a chemical responsible for TH2‐mediated autoimmunity in Brown‐Norway rats, directly triggers T cells to produce IL‐4. J. Clin. Invest. 96:1484‐1489.
   Prouvost‐Danon, A., Abadie, A., Sapin, C., Bazin, H., and Druet, P. 1981. Induction of IgE synthesis and potentiation of anti‐ovalbumin antibody IgE response by HgCl2 in the rat. J. Immunol. 126:699‐702.
   Pusey, C.D., Bowman, C., Morgan, A., Weetman, A.P., Hartley, B., and Lockwood, C.M. 1990. Kinetics and pathogenicity of autoantibodies induced by mercuric chloride in the Brown Norway rat. Clin. Exp. Immunol. 81:76‐82.
   Qasim, F.J., Thiru, S., Mathieson, P.W., and Oliveira, D.B. 1995. The time course and characterization of mercuric chloride–induced immunopathology in the brown Norway rat. J. Autoimmun. 8:193‐208.
   Qasim, F.J., Mathieson, P.W., Sendo, F., Thiru, S., and Oliveira, D.B. 1996. Role of neutrophils in the pathogenesis of experimental vasculitis. Am. J. Pathol. 149:81‐89.
   Quddus, J., Johnson, K., Gavalchin, J., Amento, E., Chrisp, C., Yung, R., and Richardson, B. 1993. Treating activated CD4+ T cells with either of two distinct methyltransferase inhibitors, 5‐azacytidine or procainamide, is sufficient to cause a lupus‐like disease in syngeneic mice. J. Clin. Invest. 92:38‐46.
   Rossert, J., Pelletier, L., Pasquier, R., and Druet, P. 1988. Autoreactive T cells in mercury‐induced autoimmunity. Demonstration by limiting dilution analysis. Eur. J. Immunol. 18:1761‐1766.
   Saoudi, A., Kuhn, J., Huygen, K., de Kozak, Y., Velu, T., Goldman, M., Druet, P., and Bellon, B. 1993. TH2 activated cells prevent experimental autoimmune uveoretinitis, a TH1‐dependent autoimmune disease. Eur. J. Immunol. 23:3096‐3103.
   Saoudi, A., Castedo, M., Nochy, D., Mandet, C., Pasquier, R., Druet, P., and Pelletier, L. 1995. Self reactive anti–class II TH2 cell lines derived from gold salt–injected rats trigger B cell polyclonal activation and transfer autoimmunity in CD8+ depleted normal syngeneic recipients. Eur. J. Immunol. 25:1972‐1979.
   Sapin, C., Druet, E., and Druet, P. 1977. Induction of anti–glomerular basement antibodies in the Brown‐Norway rat by mercuric chloride. Clin. Exp. Immunol. 28:173‐178.
   Sapin, C., Hirsch, F., Delaporte, J.‐P., and Druet, P. 1984. Polyclonal IgE increase after HgCl2 injections in BN and LEW rats: A genetic analysis. Immunogenetics 20:227‐236.
   Spickett, G.P., Brandon, M.R., Mason, D.W., Williams, A.F., and Woollett, G.R. 1983. MRC OX‐22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte‐common antigen. J. Exp. Med. 158:795‐810.
   Woolfson, R.G., Qasim, F.J., Thiru, S., Oliveira, D.B., Neild, G.H., and Mathieson, P.W. 1995. Nitric oxide contributes to tissue injury in mercuric chloride–induced autoimmunity. Biochem. Biophys. Res. Commun. 217:515‐521.
   Yung, R.L., Quddus, J., Chrisp, C.E., Johnson, K.J., and Richardson, B.C. 1995. Mechanisms of drug‐induced lupus I. Clones TH2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo. J. Immunol. 154:3025‐3035.
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