Antibodies against citrullinated proteins are specific and predictive markers for rheumatoid arthritis although the pathologic relevance of these antibodies remains unclear. To investigate the significance of these autoantibodies, collagen-induced arthritis (CIA) in mice was used to establish an animal model of antibody reactivity to citrullinated proteins. DBA/1J mice were immunized with bovine type II collagen (CII) at days 0 and 21, and serum was collected every 7 days for analysis. Antibodies against both CII and cyclic citrullinated peptide, one such citrullinated antigen, appeared early after immunization, before joint swelling was observed. Further, these antibodies demonstrated specific binding to citrullinated filaggrin in rat esophagus by indirect immunofluorescence and citrullinated fibrinogen by Western blot. To evaluate the role of immune responses to citrullinated proteins in CIA, mice were tolerized with a citrulline-containing peptide, followed by antigen challenge with CII. Tolerized mice demonstrated significantly reduced disease severity and incidence compared with controls. We also identified novel murine monoclonal antibodies specific to citrullinated fibrinogen that enhanced arthritis when coadministered with a submaximal dose of anti-CII antibodies and bound targets within the inflamed synovium of mice with CIA. These results demonstrate that antibodies against citrullinated proteins are centrally involved in the pathogenesis of autoimmune arthritis.

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.

Intestinal ischemia-reperfusion (IR) injury is initiated when natural Abs recognize neoantigens that are revealed on ischemic cells. Cr2−/− mice, deficient in complement receptors (CR)1 and CR2, demonstrate defects in T-dependent B-2 B cell responses to foreign Ags and have also been suggested to manifest abnormalities of the B-1 subset of B lymphocytes. To determine whether these CRs might play a role in the generation of the natural Abs that initiate intestinal IR injury, we performed experiments in Cr2−/− and control Cr2+/+ mice. We found that Cr2−/− mice did not demonstrate severe intestinal injury that was readily observed in control Cr2+/+ mice following IR, despite having identical serum levels of IgM and IgG. Pretreatment of Cr2−/− mice before the ischemic phase with IgM and IgG purified from the serum of wild-type C57BL/6 mice reconstituted all key features of IR injury, demonstrating that the defect involves the failure to develop this subset of natural Abs. Pretreatment with IgM and IgG individually demonstrates that each contributes to unique features of IR injury. In sum, CR2/CR1 play an unanticipated but critical role in the development of a subset of the natural Ab repertoire that has particular importance in the pathogenesis of IR injury.

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.