Collagen‐Induced Arthritis

Edward F. Rosloniec1, Michael Cremer1, Andrew H. Kang1, Linda K. Myers1, David D. Brand1

1 Veterans Affairs Medical Center, and University of Tennessee Health Science Center, Memphis, Tennessee
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.5
DOI:  10.1002/0471142735.im1505s89
Online Posting Date:  April, 2010
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Abstract

The mouse model collagen‐induced arthritis (CIA) is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization with type II collagen (CII) emulsified in complete Freund's adjuvant. This unit describes the steps necessary for the acquisition, handling, and preparation of CII, in addition to the selection of mouse strains, proper immunization technique, and methods for evaluation of the incidence and severity of arthritis. In this model, the first signs of arthritis appear approximately 21 to 28 days after immunization. The protocols in this unit should provide the investigator with all the necessary information required to reproducibly induce a high incidence of CIA in genetically susceptible strains of mice, and to critically evaluate the pathology of the disease. Curr. Protoc. Immunol. 89:15.5.1‐15.5.25. © 2010 by John Wiley & Sons, Inc.

Keywords: collagen; arthritis; mouse; model; method

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Collagen‐Induced Arthritis in the Mouse
  • Basic Protocol 2: Induction of Collagen‐Induced Arthritis in the Rat
  • Support Protocol 1: Purification of Type II Collagen
  • Support Protocol 2: Purification of Collagen α1(II) Chains
  • Support Protocol 3: Purification of Cyanogen Bromide Fragments from Collagen α1(II) Chains
  • Assessment of Arthritis in the Mouse and Rat
  • Support Protocol 4: Visual Assessment of Arthritis Severity
  • Support Protocol 5: Determining Arthritis Severity by Plethysmography
  • Support Protocol 6: Determining Arthritis Severity by Caliper Measurement of Paw Swelling
  • Support Protocol 7: Measurement of B Cell Responses to CII in CIA
  • Support Protocol 8: Measurement of T Cell Responses to CII in CIA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Collagen‐Induced Arthritis in the Mouse

  Materials
  • Chick or bovine native CII (see protocol 3 or purchase from Sigma, Chondrex, or MD Biosciences), α1(II) chains (see protocol 4), or CB11 fragment of CII (see protocol 5)
  • 10 mM acetic acid, filter sterilized with 0.2‐µm filter
  • Incomplete Freund's adjuvant (IFA; BD Biosciences)
  • Mycobacterium tuberculosis (strain H37Ra; heat‐killed; BD Biosciences)
  • DBA/1JLacJ mice (Jackson Labs) or other genetically susceptible strain (see )
  • Mortar and pestle
  • High‐speed homogenizer (Virtis; optional)
  • 1‐ ml Luer‐lok syringes (BD Biosciences)
  • 26‐G needles
  • Additional reagents and equipment for preparing antigen/CFA emulsions (unit 2.4) and intradermal injection of mice (unit 1.6)
CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced intradermally or into the eyes, and may cause profound sloughing of skin or loss of sight. Self‐injection can cause a positive TB test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

Basic Protocol 2: Induction of Collagen‐Induced Arthritis in the Rat

  Materials
  • Chick, porcine, rat, or bovine native CII (see protocol 3)
  • 10 mM acetic acid, filter sterilized with 0.2‐µm filter
  • Incomplete Freund's adjuvant (IFA; BD Biosciences)
  • Rats (see )
  • High‐speed homogenizer (Virtis, optional)
  • 1‐ml Luer‐lok syringes (BD Biosciences)
  • 26‐G needles
  • Additional reagents and equipment for preparing antigen/CFA emulsions (unit 2.4) and intradermal injection of rat (unit 1.6)

Support Protocol 1: Purification of Type II Collagen

  Materials
  • Fetal bovine joints or chick sternums
  • 70% (v/v) isopropyl alcohol
  • Tris‐buffered guanidine (see recipe)
  • 10 mM, 100 mM, and 500 mM acetic acid
  • 70% (v/v) formic acid
  • Pepsin (3× crystallized; Sigma)
  • 5 M NaCl ( appendix 2A)
  • 10 mM Na 2HPO 4
  • Tris‐buffered saline (TBS): 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A)/200 mM NaCl
  • DE‐52 anion‐exchange resin (Whatman)
  • Scalpel or single‐edged razor blade
  • Food processor or blender with sharp blades
  • Sorvall RC‐5B or equivalent centrifuge with 250‐ml and 1‐liter centrifuge bottles
  • Dialysis tubing (MWCO 10,000)
  • 5 × 20–cm chromatography column
  • 5% SDS‐PAGE gel (unit 8.4)
  • Additional reagents and equipment for dialysis ( appendix 3H), DE‐52 ion‐exchange chromatography (as for IgG purification; unit 2.7), and SDS‐PAGE (unit 8.4)
CAUTION: Special precautions need to be taken when working with fetal bovine and chicken tissue. Bovine tissue is a potential source of infection with Brucella abortus, and chicken tissue may be contaminated with Salmonella or Campylobacter. Gloves, mask, and eye protection should be worn and the tissue rinsed liberally with 70% isopropyl alcohol before dissection. Residual tissues should be handled as potentially biohazardous material and disposed of accordingly (see Chapter 7 introduction).NOTE: All steps in this procedure must be performed at ≤4°C unless otherwise indicated. Although it is nearly impossible to perform this procedure under totally sterile conditions, maintaining the collagen preparation at such a low temperature will not only help to maintain its native form, but will also help reduce bacterial or mycobacterial growth.

Support Protocol 2: Purification of Collagen α1(II) Chains

  Materials
  • Lyophilized type II collagen (CII; see protocol 3)
  • 0.06 M and 0.5 M sodium acetate, pH 4.8, filter sterilized with 0.2‐µm filter
  • Gradient solutions:
    • 0.06 M sodium acetate, pH 4.8, filter sterilized
    • 0.06 M sodium acetate, pH 4.8/0.1 M NaCl, filter sterilized
  • Dialysis tubing (MWCO 10,000) or Sephadex G‐10 resin (Pharmacia Biotech) equilibrated with 0.1 M acetic acid in 5 × 10–cm chromatography column
  • 45°C water bath
  • 2.5 × 15–cm chromatography column packed with CM‐52 resin (Whatman) and equilibrated with 0.06 M sodium acetate, pH 4.8 (see appendix 3I for packing and equilibration)
  • Chromatography apparatus including:
    • Water jacket attached to temperature‐controlled water bath via circulating pump
    • Gradient mixer
    • Fraction collector
    • Spectrophotometer for monitoring column effluent
  • Additional reagents and equipment for cation‐exchange chromatography ( appendix 3I) and dialysis ( appendix 3H) or size‐exclusion chromatography ( appendix 3I)
NOTE: Keep buffers sterile prior to use. Enzymes produced by bacteria can digest the α1(II) chains.

Support Protocol 3: Purification of Cyanogen Bromide Fragments from Collagen α1(II) Chains

  Materials
  • Purified collagen α(II) chains (see protocol 5)
  • 70% (v/v) formic acid
  • Cyanogen bromide (CNBr)
  • 0.02 M sodium citrate/0.02 M NaCl, pH 3.6 (filter sterilized with 0.2‐µm filter and preheated to 42°C)
  • Gradient solutions:
    • 0.02 M citrate/0.02 M NaCl, pH 3.6 (filter sterilized)
    • 0.02 M citrate/0.15 M NaCl, pH 3.6 (filter sterilized)
  • Bio‐Gel A‐1.5 m agarose gel (200 to 400 mesh; Bio‐Rad)
  • 10% SDS‐PAGE gel (unit 8.4)
  • Round‐bottom flask fitted with dual‐valve bubbler stopcock and N 2 source
  • 2.5 × 20–cm chromatography column packed with CM‐52 resin (Whatman) and equilibrated with 0.02 M sodium citrate/0.02 M NaCl, pH 3.6 (see appendix 3I for packing and equilibration)
  • Chromatography apparatus including:
    • Water jacket attached to temperature‐controlled water bath via circulating pump
    • Gradient mixer
    • Fraction collector
    • Spectrophotometer for monitoring column effluent
  • 2 × 110–cm chromatography column
  • Additional reagents and equipment for cation‐exchange chromatography and size‐exclusion chromatography ( appendix 3I) and SDS‐PAGE (unit 8.4)
NOTE: Keep chromatography buffers free of bacterial growth to prevent proteolytic digestion of the CB3 fragment.

Support Protocol 4: Visual Assessment of Arthritis Severity

  Materials
  • Plethysmometer for measuring paw volume of rats or mice, (Ugo Basile,
  • product no.7140)
NOTE: A number of investigators have devised similar alternative instruments and approaches for measuring paw volume (Goodfellow et al., ; Lee et al., ; Wang et al., ; Zhang et al., ).

Support Protocol 5: Determining Arthritis Severity by Plethysmography

  Materials
  • Constant‐tension or Vernier calipers

Support Protocol 6: Determining Arthritis Severity by Caliper Measurement of Paw Swelling

  Materials
  • Purified CII (see protocol 3)
  • Potassium phosphate buffer, pH 7.4 (see recipe), 4°C
  • Blocking buffer (see recipe)
  • 0.15 M NaCl/0.05% (v/v) Tween 20
  • Serum (unit 1.7) from CII‐sensitized mouse (see protocol 1) or rat (see protocol 2)
  • ELISA buffer (see recipe)
  • 2 µg/ml goat anti‐mouse or anti‐rat IgG Fc–specific horseradish peroxidase conjugate (Sigma)
  • ELISA substrate buffer (see recipe)
  • Stopping solution: 2.5 N H 2SO 4
  • 96‐well microtiter plates (Serocluster from Costar)
  • 12‐channel pipettor
  • ELISA plate reader equipped with a 490 nm and a 650 nm (optional) filter
  • Additional reagents and equipment for ELISA (unit 2.1)

Support Protocol 7: Measurement of B Cell Responses to CII in CIA

  Materials
  • CII‐immunized mice (see protocol 1) or rats (see protocol 2)
  • HL‐1 serum‐free medium (Lonza, cat. no. 77201; use for mouse assay) or DMEM medium (see recipe in appendix 2A, omit serum; use for rat assay)
  • Antigen‐presenting cell suspension (see recipe in Reagents and Solutions; for rat assay)
  • Antigen: native CII (see protocol 3), α1(II) (see protocol 4), or CNBr fragment of CII (see protocol 5)
  • Pseudo‐complete HL‐1 (pcHL‐1) serum‐free medium with additional reagents including 0.3% (w/v) BSA (see recipe in Reagents and Solutions) (for mouse assay) or DMEM medium ( appendix 2A) containing 3% normal rat serum (for rat assay)
  • Bovine serum albumin (BSA), Fraction V, low endotoxin, IgG free (Sigma, cat. no. A‐2058)
  • [3H]thymidine
  • 96‐well flat‐bottom microtiter plate
  • 0.2‐µm syringe filters
  • Semi‐automated microtiter plate cell harvester and glass microfiber strips (e.g., Cambridge Technology)
  • Additional reagents and equipment for preparing suspensions of lymph node or spleen cells (unit 3.1), T cell enrichment by nylon wool (for rat cells only; unit 3.2) or MACS beads (for rat cells only; unit 3.5), and counting viable cells ( appendix 3B)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: Where HL‐1 medium is used (mouse cells) all culture incubations are performed in a humidified 37° C, 5% CO 2 incubator; where DMEM is used (for rat cells), all culture incubations are performed in a humidified 37° C, 10% CO 2 incubator.NOTE: Cell washes can be performed with any sterile, isotonic tissue culture medium.
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Figures

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Literature Cited

Literature Cited
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