Collagen-induced arthritis (CIA) is a widely used experimental model for human rheumatoid arthritis (RA) and has been instrumental in elucidating the cellular and molecular mechanisms that drive inflammatory joint pathology. Traditionally, CIA has been restricted to mouse strains expressing the major histocompatibility complex (MHC) class II haplotypes H-2q (DBA/1) or H-2r (B10.RIII), but not H-2b (C57BL/6: B6). Consequently, B6 mice were long regarded as relatively resistant to CIA due to their H-2b background.
However, subsequent studies demonstrated that CIA can be successfully induced in B6 mice using a modified immunization protocol utilizing chick type II collagen (Cat # 20011 or 20021) and Complete Freund’s Adjuvant (CFA) containing a high concentration (5 mg/ml) of Mycobacterium tuberculosis (Cat # 7023) for both the primary and booster injections.
This is especially relevant to B6 mice as their immune systems only recognize chick type II collagen as an antigen, and not bovine or mouse type II collagen. This modified protocol has been used to induce CIA in transgenic and gene knockout B6 background mice, enabling extensive investigations of RA pathogenesis (1–3).
Chondrex, Inc provides the following protocol for inducing CIA in B6 mice along with other important considerations for successful model establishment. As an alternative, Chondrex, Inc. also provides a collagen-antibody-induced arthritis model for B6 mice, which yields 100% arthritis incidence and greater severity.
Collagen-Induced Arthritis - Mouse
Collagen-Induced Arthritis - Mouse (Visual Guide)
Collagen Antibody Induced Arthritis
1. Materials
| Cat # | Products |
| 7023 | Complete Freund’s Adjuvant, 5 ml |
| 20011*1 | Chick type II collagen, 10 mg |
| 20012*1 | Chick type II collagen, 5 ml x 2 mg/ml |
| 9028 *2 | Lipopolysaccharide from E. coli O111:B4 0.5 mg/ml x 5 ml |
Note 1: Choose one of them as a collagen for immunization
Note 2: Optionally, use LPS Administration to synchronize and accelerate the induction of CIA
2. Immunization Schedule
Immunize mice with a collagen–CFA emulsion formulated with an equal volume of 2 mg/ml of chick type II collagen and 5 mg/ml of M. tuberculosis. A booster injection using the same collagen–CFA emulsion is administered on day 21. Arthritis typically appears on days 28–35 following the initial immunization, and the maximum incidence reaches approximately 50–70% between days 42–56.
3. Emulsion Preparation
The quality of the emulsion used for immunization is a major determinant of arthritis incidence, especially in low-responder strains such as B6 mice. Although various preparation methods exist, employing an electric homogenizer and verifying emulsion quality are strongly recommended to achieve consistent and reproducible CIA induction. For detailed procedures, please refer to our CIA protocols above.
4. Injection Site
Insert the needle bevel side up and parallel to the tail, 2 cm from the base of the tail until the needle tip is 0.5 cm from the base. Confirm that the full length of the needle remains subcutaneous and administer 0.1 ml of emulsion (100 μg collagen/mouse) at this site (Figure 1). For the booster injection, begin 3 cm from the tail base and insert the needle until the tip is 1.5 cm from the base. This injection must be given at a separate location from the primary inoculation. For complete procedural context, please refer to the CIA protocols above.
Note: Chondrex, Inc. does not recommend subcutaneous injections in the back or intraperitoneal (IP) injections, as such emulsions can elicit severe inflammatory responses within the peritoneal and thoracic cavities.
Figure 1. Subcutaneous Injection
5. LPS Administration: synchronizing and accelerating the induction of CIA
LPS exhibits a synergistic effect in inducing arthritis when combined with sub-arthritogenic doses of anti–type II collagen autoantibodies. In CIA, both the incidence and severity of disease can be enhanced through administration of LPS, a known B-cell mitogen. The bacterial toxins not only promote and amplify arthritic responses but also help synchronize the onset of arthritis. LPS (25–50 μg in saline) can be administered intraperitoneally on day 25–28, or 3–5 days prior to the desired onset of arthritis (4).
6. Recommendations for your IACUC
Because this model requires a high concentration of M. tuberculosis in CFA (5 mg/ml), your IACUC may hesitate to approve this protocol. You may clarify that a higher incidence and more robust arthritis scores are necessary to adequately compare the test and control groups. The references linked below demonstrate that increased M. tuberculosis concentration is essential for inducing CIA in B6 mice. However, as we are not familiar with your specific study design, we recommend providing your IACUC with references from similar studies that have successfully used our CIA protocol (3).
7. Two Factors to Consider When Using Mouse Disease Models
Mouse disease models have greatly advanced our understanding of many human diseases. Despite their value, these models are subject to several sources of variability that can influence experimental outcomes. For this reason, a pilot study is recommended for first-time users of any mouse model. The two primary sources of variability are summarized below.
7-1) Mouse Vendors
Genetic backgrounds and intestinal flora can differ among mice of the same strain when sourced from different vendors, which may alter their responses to experimental reagents and affect data quality (4). Therefore, it is advisable to test mice from multiple vendors using a standardized protocol before initiating a large-scale study.
7-2) Housing Conditions
The bacterial flora of laboratory animals plays an essential role in overall health and proper immune function (1). To minimize variability related to microbial exposure, we recommend maintaining mice under Specific Pathogen-Free (SPF) conditions rather than conventional housing (5).
For laboratories inducing CIA under conventional housing, the following practices are recommended:
A. Use sterilized food
B. Clean the animal room thoroughly; ensuring that floors, tables, and all work surfaces are properly sanitized
C. Autoclave cages prior to use
D. Use cage filters when possible (Figure 2)
Figure 2. Cage Filters
E. Handle animals with gloves and masks to reduce contamination.
F. Perform animal handling procedures under a hood when feasible.
Note: Careful attention to these major sources of variability will help reduce confounding factors and improve the reliability of experimental results.
References
- I. K. Campbell, J. A. Hamilton, I. P. Wicks, Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur. J. Immunol. 30, 1568–1575 (2000).
- H. Kai, K. Shibuya, Y. Wang, H. Kameta, T. Kameyama, S. Tahara-Hanaoka, A. Miyamoto, S.-I. Honda, I. Matsumoto, A. Koyama, T. Sumida, A. Shibuya, Critical role of M. tuberculosis for dendritic cell maturation to induce collagen-induced arthritis in H-2b background of C57BL/6 mice. Immunology. 118(2), 233-239 (2006).
- J. J. Inglis, G. Criado, M. Medghalchi, M. Andrews, A. Sandison, M. Feldmann, R. O. Williams, Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen. Arthritis Res. Ther. 9, R113 (2007).
- S. Tanaka, T. Toki, T. Akimoto, K. Morishita, Lipopolysaccharide accelerates collagen-induced arthritis in association with rapid and continuous production of inflammatory mediators and anti-type II collagen antibody. Microbiol Immunol. 57(6), 445-454 (2013).
- I. I. Ivanov, K. Atarashi, N. Manel, E. L. Brodie, T. Shima, U. Karaoz, D. Wei, K. C. Goldfarb, C. A. Santee, S. V. Lynch, T. Tanoue, A. Imaoka, K. Itoh, K. Takeda, Y. Umesaki, K. Honda, D. R. Littman, Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 139(3), 485-498 (2009).