Citrullinated mouse collagen administered to DBA/1J mice in the absence of adjuvant initiates arthritis
Highlights
► Induction of arthritis in mice in the absence of an adjuvant was evaluated. ► Injection of citrullinated mouse type II collagen increases the severity of arthritis. ► Injection of citrullinated mouse type II collagen increases T-cell proliferation against citrullinated peptides. ► Increased bone erosion is evident by mirco-CT following injection of citrullinated mouse type II collagen. ► A potential new T-cell mediated model of arthritis is proposed.
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.
Section snippets
Animals
Male DBA/1 (H-2q) mice were purchased from Jackson Laboratory (Bar Harbor, Maine, U.S.A.) and maintained on a Purina breeder's diet [11]. Animals were allowed free access to their food and/or water up to 1 h prior to sacrifice. All procedures were approved by the Animal Subcommittee of the Omaha VA Medical Center in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals.
Antigen preparation
Mouse type II collagen (Col), purchased from Chondrex (Redmond, WA, U.S.A.), was
Results
To determine if Cit-Col would induce an RA like autoimmune disease (Citrullinated Collagen‐induced arthritis = CCIA), mice were immunized with Col (control), Cit-Col, and FCA-Col. CCIA was first assessed for inflammation using the caliper method at weeks 0, 3, and 5 as described in Materials and methods. As shown in Fig. 1, prior to injection of antigens, the average baseline ankle thickness was recorded and used to calculate percent change in the right ankle joint of the immunized animals. At
Discussion
Citrullination of self‐proteins has been suggested to play a significant role in the pathogenesis of rheumatoid arthritis (RA). Recent studies have used animal models to study these citrullinated self‐proteins and how they interact to induce an RA like disease [4], [5], [13], [14]. These studies have systematically used robust inflammatory adjuvants (i.e. Freund’s complete adjuvant) as the catalyst for induction of the disease. In contrast, it was the purpose of this study to inject
Abbreviations
- RA
Rheumatoid Arthritis
- FCA
Freund's Complete Adjuvant
- IFA
Incomplete Freund's Adjuvant
- PAD
Peptidylargine Deiminase
- CIA
Collagen‐Induced Arthritis
- CCIA
Citrullinated Collagen‐induced Arthritis
- RSA
Rat Serum Albumin
- BSA
Bovine Serum Albumin
- HPMA
N-(2-hydroxypropyl)methacrylamide
- VOI
Volume of Interest
- MTP
Metatarsophalangeal
- CCP
Cyclic Citrullinated Peptide
- SEM
Standard Error of the Mean
- BV
Bone Volume
- SI
Stimulation Index
- ACPA
Anti Citrullinated Protein Antigens
Competing interests
The authors have no competing interest to disclose for this manuscript.
Author contributions
GMT contributed to the conception, design, interpretation of data, and preparation of the manuscript. MJD contributed to the conception, design, interpretation of data, and preparation of the manuscript. AD carried out the procedures, contributed interpretation of data and preparation of the manuscript. CDH carried out the procedures, contributed interpretation of data and preparation of the manuscript. JPL CDH carried out the procedures, contributed interpretation of data and preparation of
Acknowledgments
The authors would like to thank the members of the Experimental Immunology Laboratory for their valuable support of this work. The Department of Internal Medicine and the College of Medicine at UNMC, the American College of Rheumatology Research and Education Foundation within Our Reach award. Also, the Omaha VA Medical Center Research Services.
References (32)
Collagen-induced arthritis in the mouse
Methods Enzymol
(1988)- et al.
Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis
Autoimmun Rev
(2007) - et al.
Adjuvant oils induce arthritis in the DA rat. I. Characterization of the disease and evidence for an immunological involvement
J Autoimmun
(1991) - et al.
Anti-CCP antibodies: the past, the present and the future
Nat Rev Rheumatol
(2011) - et al.
Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value
Arthritis Res
(2002) - et al.
Citrullination of fibronectin in rheumatoid arthritis synovial tissue
Rheumatology (Oxford)
(2005) - et al.
Autoimmunity against fibrinogen mediates inflammatory arthritis in mice
J Immunol
(2010) - et al.
Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis
J Clin Invest
(2006) - et al.
Citrullination of self-proteins and autoimmunity
Scand J Immunol
(2004) - et al.
Citrullination of synovial proteins in murine models of rheumatoid arthritis
Arthritis Rheum
(2003)
N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis
J Immunol
Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis
Immunol Rev
Autoimmunity to type II collagen an experimental model of arthritis
J Exp Med
Oral administration of lipopolysaccharide exacerbates collagen-induced arthritis in mice
J Immunol
Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity
Arthritis Res Ther
In the rat, citrullinated autologous fibrinogen is immunogenic but the induced autoimmune response is not arthritogenic
Clin Exp Immunol
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