Research paper
Collagen antibody-induced arthritis in mice: Development of a new arthritogenic 5-clone cocktail of monoclonal anti-type II collagen antibodies

https://doi.org/10.1016/j.jim.2009.01.009Get rights and content

Abstract

A cocktail of 4 monoclonal anti-type II collagen antibodies recognizing conserved epitopes located within the CB11 fragment (CII 124-402) of type II collagen is currently used as an arthritogenic antibody preparation for inducing collagen antibody induced arthritis (CAIA). In order to increase the arthritogenicity of this cocktail, we have developed 7 new monoclonal antibodies to anti-type II collagen from spleen cells of DBA/1J mice immunized with bovine type II collagen, and tested for their additional effect on the arthritogenicity over that of the current 4-clone cocktail. Three of the clones (CII-3, -5 and -6) bind to the LyC1 (CII 124-290) peptide of CB11 and 1 (CII-7) of the clones binds to CB9.7 (CII 898-1020), and highly cross-reacted with other species of type II collagen. This indicates that these clones recognize conserved epitopes within type II collagen, including mouse type II collagen. On the other hand, 2 other clones (CII-1 and -4) directed against CB9.7 and 1 clone (CII-2) against CB8 (CII 403-551) were less reactive with other species of type II collagen.

The arthritogenicity of the current 4-clone cocktail was significantly increased by addition of a fifth clone, CII-3. No effects were observed with other clones. The arthritogenicity of this new 5-clone cocktail was 2-fold greater than the current 4-clone cocktail in all strains of mice tested: the CIA-responder strain DBA/1J, the CIA-resistant BALB/c (H-2d), the T-cell deficient C.B-17/l scid/scid and the CAIA-low responder C57BL/6 (H-2b) strain. These results clearly indicated the importance of epitope specificity of arthritogenicity of autoantibodies to type II collagen. Due to its enhanced arthritogenicity, this 5-clone cocktail is capable of inducing a more consistent and severe arthritis with lower doses compared to the current 4-clone cocktail, and will provide an effective new reagent for inducing arthritis in various strains of CAIA low responder mice.

Introduction

Collagen-induced arthritis (CIA) is mediated by autoantibodies against type II collagen. A similar arthritis can be induced in naïve mice by transferring sera from arthritic mice (Stuart and Dixon, 1983) and partially purified antibodies from a patient with rheumatoid arthritis (Wooley et al., 1984). However, not all autoantibodies to type II collagen are capable of inducing arthritis because the activation of complement on the surfaces of cartilage requires that multiple antibodies sequestered within cartilage layers are available for this activation as the first step in the inflammatory cascade (Watson and Townes, 1985, Reife et al., 1991). The importance of complement activation in CIA pathology has been further confirmed in detail by many studies (Hietala et al., 2004). Importantly, the dominant arthritogenic epitopes are all clustered within the CNBr-digest fragment number 11 (CB11) of type II collagen in DBA/1J (H-2q) mice (Terato et al., 1985) and CB8 in B10.RIII (H-2r) mice (Myers et al., 1995). Therefore, several combinations of complement-fixing IgG subtype monoclonal antibodies recognizing these clustered epitopes within CB11 is capable of inducing arthritis (Terato et al., 1992). Furthermore, Terato et al. (1995) found that bacteria toxins such as LPS has a strong synergistic effect with autoantibodies to type II collagen, and can be used to reduce the threshold level and numbers of monoclonal antibody required for induction of arthritis. This collagen antibody-induced arthritis (CAIA) model which employs a cocktail of monoclonal antibodies and LPS has been widely used for studying the pathogenesis of autoimmune arthritis and evaluating therapeutics (Kagari et al., 2002, Kagari et al., 2003, Tanaka et al., 2006, Banda et al., 2006, Banda et al., 2007). It is an exceedingly valuable tool because consistent and severe arthritis can be induced within several days instead of the 4 weeks required to induce CIA in various strains of mice including CIA resistant strains, such as BALB/c (H-2d), C57BL/6 (H-2b) and even T-cell deficient C.B-17/l scid/scid mice (Kagari et al., 2002, Tanaka et al., 2006), which do not carry CIA-susceptible MHC-haplotypes, H-2q and H-2r (Wooley et al., 1981, Wooley et al., 1985).

Although the current arthritogenic monoclonal antibody cocktail consisting of 4 monoclonal antibodies is cable of effectively inducing consistent arthritis in mice, its arthritogenicity is limited, and relatively large doses are required for inducing arthritis in CAIA-low responder strains of mice such as C57BL/6 mice.

In the present study, we developed IgG2a and IgG2b subtype anti-type II collagen monoclonal antibodies, and tested these clones for their additional effect on arthritogenicity over that of the current 4-clone cocktail. The arthritogenicity of the current 4-clone cocktail was significantly increased through the addition of clone CII-3 in all strains of mice tested such as DBA/1J, BALB/c, C.B-17/l, and C57BL/6 mice, but the other clones did not provide any additional effect. This indicated the importance of epitope specificity of autoantibodies to type II collagen for induction of inflammatory arthritis. This newly developed 5-clone cocktail provides a new useful tool for inducing arthritis in CAIA-low responder strains and variety of gene knockout and transgenic mice whose susceptibility to CAIA is unknown.

Section snippets

Animals

Male DBA/1J mice (8 weeks of age) were bred in the animal breeding unit of Kobe Pharmaceutical University, Kobe, Japan. BALB/c and C3H/He mice were purchased from Charles River Laboratory (Kanagawa, Japan). C.B-17/l mice (6–7 week of age) and C57BL/6 mice were obtained from Charles River (Wilmington, MA) and Harlan (Indianapolis, IL), respectively. Mice were housed in specific pathogen-free environment and fed standard rodent chow and water ad libitum. All procedures were performed with the

Epitope analysis of newly developed 7 mAbs

The species specificity and species cross-reactivity of newly developed 7 mAbs were analyzed by ELISA using chick, bovine, rat, porcine, human, and mouse type II collagen as antigen (Table 1). All clones were specific to bovine type II collagen used for immunization, and all clones cross-reacted with other species of type II collagen.

To localize epitopes, all clones were tested for the reactivity with renatured CB-peptide fragments of bovine type II collagen (Table 2). Importantly, 3 of 3

Discussion

The present study demonstrates that arthritogenicity of the current 4-clone cocktail of monoclonal anti-type II collagen antibodies can be significantly increased through the addition of a single monoclonal antibody (CII-3) recognizing the LyC1 fragment of type II collagen. Importantly, other clones which recognize epitopes located even within LyC1 fragment and other CB-peptide fragments were not capable of increasing the arthritogenicity of 4-clone cocktail, indicating the importance of

Acknowledgments

This work is supported by a grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture of Japan.

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