Citrullinated mouse collagen administered to DBA/1J mice in the absence of adjuvant initiates arthritis

Abstract

Introduction

Citrullinated self‐proteins are thought to be involved in the onset/progression of rheumatoid arthritis (RA). Numerous studies have been performed to look for the self‐antigen that becomes citrullinated and induces RA. Importantly, these studies have been performed using citrullinated self‐antigens injected into an animal model in the presence of a strong adjuvant in order to derive the response. However, to date no studies have been performed to determine if these phenotypes can be induced in the absence of an adjuvant.

Methods

To investigate this possibility, mice were immunized with citrullinated or non-citrullinated mouse Type II collagen (Cit-Col or Col) in the presence or absence of Freund's Complete Adjuvant (FCA).

Results

An autoimmune-like RA response was observed in mice immunized with Cit-Col in the absence of FCA; by the increase in caliper score, visual observation, and micro-CT analysis of bone erosions. Antibody and T-cell responses were increased in the Cit-Col injected mice to Cit-Col as well as antibody to Anti-Citrullinated Peptide Antigens (ACPA) as determined by a commercially available test kit.

Conclusions

Therefore, the use of citrullinated mouse collagen induces an autoimmune‐like RA in the absence of an adjuvant. These data also suggest that citrullinate self‐proteins may be potential molecular adjuvants that assist in driving an inflammatory response, that increases the production of PAD in joint tissue, resulting in the citrullination of other self‐proteins to exacerbate the disease.

Introduction

Rheumatoid arthritis (RA) is a severe and potentially debilitating disease characterized by inflammation and the progressive destruction of joint tissues. While numerous studies have implicated the involvement of various antigens in the disease, the pathogenesis and cause of RA remain uncertain. In the last decade autoantibodies to citrullinated protein antigens (ACPA) and/or peptides have been used as diagnostic tools and have been implicated in the disease process [1]. A number of citrullinated proteins have been implicated as playing a central role in disease pathogenesis; including, filaggrin, fibrin, fibrinogen, vimentin, alpha enolase, collagen, and even peptidylagrinine deiminase (PAD), the enzyme responsible for the post-translational modification of arginine into citrulline [2], [3], [4], [5], [6], [7], [8], [9].

Animal models in RA have traditionally used type II or IV collagen from a variety of animal sources in the presence of Freund's complete adjuvant (FCA) to initiate the disease [10], [11], [12]. With an increased understanding of their potential role in disease pathogenesis, citrullinated proteins have been evaluated as immunogens in the presence of FCA [4], [5], [6], [13]. These studies showed that there was a slight increase in immune responses towards the injected immunogen, and a slight increase in joint damage reflected in the ankle. For example, citrullinated human and rat fibrinogen were injected into rats and mice with FCA, resulting in increased joint damage compared to that observed in animals immunized with FCA plus unmodified collagen [4], [6], [14]. When citrullinated rat fibrinogen was injected into rats in the presence of FCA, tolerance was broken and an autoimmune response was initiated and characterized by the production of IgG antibodies to both citrullinated fibrinogen and native fibrinogen [4]. These studies have also been performed using autologous rat serum albumin (RSA) that had been citrullinated [13]. In this study RSA was citrullinated and injected into animals in the presence of FCA which produced autoantibodies and T-cell proliferations directed against citrullinated RSA. These data indicate that tolerance can be broken by citrullinating a self‐protein.

Previous studies using the collagen‐induced arthritis model in rats have shown that antibodies could be detected against citrullinated fibrinogen following immunization, again underscoring the role of citrullinated antigens in RA pathogenesis [5]. As with the aforementioned studies, this study also involved the immunization of the antigen type II collagen from calf articular cartilage in the presence of FCA. The studies described above all used FCA to induce arthritis. To date, there have been no animal studies that have used a citrullinated self‐protein to induce arthritis in the absence of an adjuvant. This is important since a disease model that does not require an adjuvant could lend added physiologic relevance to human disease. Therefore, it was the purpose of this study to evaluate whether citrullinated mouse type II collagen (Cit-Col) initiated an autoimmune collagen-induced arthritis in the absence of an adjuvant and, if so, to characterize the resulting immune responses utilizing a number of complimentary scientific approaches.