Basic Protocol 1: Induction of Collagen-Induced Arthritis in ihe Mouse
The mouse model of collagen-induced arthritis (CIA) is particularly advantageous for study, given the vast number of inbred strains available and their corresponding immunologic reagents. Disease induction is more complex than that in other autoimmune disease models--e.g., experimental autoimmune encephalomyelitis (EAE; UNIT 15.1)--in that CIA requires both the activation of T cells and the production of antibodies specific for both the immunogen and the autoantigen, murine CII.
This protocol is for the DBA/1JLacJ strain of mouse; see Background Information for discussion of susceptibility of various mouse strains to CIA.
- Chick or bovine native CII (see Support Protocol 1 or purchase from MD Biosciences or Chondrex), a1(II) chains (see Support Protocol 2), or CB11 fragment of CII (see Support Protocol 3)
- 10 mM acetic acid, filter sterilized with 0.2-um filter
- Incomplete Freund's adjuvant (IFA; e.g., Difco)
- Mycobacterium tuberculosis (strain H37Ra; heat-killed; available by writing to Ministry of Agriculture, Fisheries, and Food, Central Veterinary Laboratory, Weybridge, Surrey, United Kingdom)
- DBA/1JLacJ mice (Jackson Labs)
- Mortar and pestle
- Virtis high-speed homogenizer (Virtis; optional)
- 1-ml glass syringes with locking hubs
- 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.
- Dissolve the CII in 10 mM acetic acid by stirring overnight at 4°C.
For 50-ul injection volumes containing 100 ug CII, dissolve CII at 4 mg/ml; for 100 ul injection volumes, dissolve CII at 2 mg/ml. Dosage is the same whether using native CII or fragments.
It is important that native CII be kept cold while being dissolved to prevent its denaturation. Once denatured, its efficacy in eliciting arthritis will drop by approximately one-half or more in comparison to native CII.
- Grind heat-killed Mycobacterium tuberculosis finely in a mortar and pestle and combine with IFA at a 4 mg/ml final concentration to prepare complete Freund's adjuvant (CFA).
Commercially prepared CFA may also be used, but may result in a lower incidence and severity of arthritis.
- Using a Virtis high-speed homogenizer or the emulsification technique described in UNIT 2.4, emulsify CII in an equal volume of CFA just prior to immunization.
A Virtis high-speed homogenizer is preferred for emulsifying CII and adjuvant, although its use is not critical as long as a stable emulsion is achieved (see UNIT 2.4). The collagen should be kept cold throughout the emulsification, especially if using a high-speed homogenizer.
Immunize mice and assess development of arthritis
- Using a glass syringe with a locking hub and a 26-G needle, inject mice with CII/CFA emulsion intradermally at the base of the tail (UNIT 1.6).
Typically, immunizations consist of a 100 ug of Mycobacterium tuberculosis and 100 ug of CII in a total volume of 50 ul; however, arthritis can be induced with as little as 10 ug of native CII.
If desired, a booster immunization of 50 ug of CII emulsified in incomplete Freund's adjuvant (IFA) can be given 2 to 3 weeks after the primary immunization. Booster immunizations are usually necessary only when using either a1(II) chains (see Support Protocol 2) or purified cyanogen bromide (CB) fragments of CII (see Support Protocol 3) to induce arthritis.
- Assess development of arthritis (Support Protocol 4, 5 or 6) and measure B cell (Support Protocol 7) and T cell responses to CII (see Support Protocol 8) if desired.
Arthritis typically appears in DBA/1LacJ mice 3 to 5 weeks after immunization with native CII, and usually peaks by week 6.
The arthritis is monophasic and eventually resolves as either a normal-appearing limb or, more commonly, a paw in which significant ankylosis has occurred. Arthritis can occur in any of the fore or hind paws or both, and at its peak involves the entire paw from the ankle to the digits. Although early signs of arthritis are difficult to detect, fully active disease is easily identified even by the untrained observer (see Support Protocols 4, 5, and 6).
Arnold Postlethwaite, Director
David Brand, PhD
Monica L. Brown, DO
Hongbo Chi, PhD
Weikuan Gu, PhD
Karen Hasty, PhD
Andrew H. Kang, MD
Linda K. Myers, MD
Eugene Pinkhassik, PhD
Arnold Postlethwaite, MD
Edward Rosloniec, PhD
Andrzej Slominski, MD, PhD
John Stuart, MD
Ae-Kyeung Yi, PhD