Background/Purpose:

Antibodies to proteins/peptides containing citrulline (commonly measured using cyclic citrullinated peptide, CCP2) are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE), a major risk factor for RA. RA patients also generate antibodies to homocitrulline-containing proteins/peptides. The latter antibodies are often referred to as anti-carbamylated protein antibodies (Anti-CarP). Citrulline and homocitrulline are structurally similar. There is evidence supporting an important role for immune responses to citrulline in the pathogenesis of RA. We hypothesize that immune responses to homocitrulline are related to citrulline (cross-reactive) and are also arthritogenic. We aimed to study: (1) the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express human SE, (2) whether homocitrulline immune responses are dependent on the SE and (3) induce arthritis in this animal model.

Methods:

We used DR4tg mice on a C57BL/6 (B6) background; these mice express the human SE, lack endogenous murine MHC class II and are known to develop immune responses to citrulline and arthritis when immunized with citrullinated fibrinogen. DR4tg and B6 mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). We examined immune responses to HomoCitJED and CitJED, which have an identical number of modified amino acids on the same peptide backbone. Splenic T cell proliferation was evaluated by ³H-thymidine incorporation assay. IgG anti-HomoCitJED, anti-CitJED, and anti-CCP2 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with CitJED and HomoCitJED. Some HomoCitJED immunized mice also received intra-articular injection (IA) of HomoCitJED and joint swelling was measured using calipers.

Results:

HomoCitJED immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to CitJED or anti-CCP2 antibodies. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. DR4tg mice immunized with HomoCitJED developed knee swelling following IA injections of HomoCitJED.

Conclusion: DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice. HomoCitJED is immunogenic and its intra-articular presence is required for knee swelling in DR4tg mice.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-responses-to-peptides-containing-homocitrulline-or-citrulline-in-the-dr4-transgenic-mouse-model-of-rheumatoid-arthritis/