Abstract
Deimination, a posttranslational modification of arginine to citrulline carried out by peptidylarginine deiminases, may compromise tolerance of self-antigens. Patients with connective tissue autoimmunity, particularly rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or Felty’s syndrome, present with autoantibodies to deiminated histones (dH), which thus form a category of antibodies to citrullinated protein antigens (ACPA). In general, ACPA are a sensitive diagnostic for RA and may form in response to the release of nuclear chromatin (DNA plus dH) from granulocytes, usually referred to as neutrophil extracellular traps. The aim of this study was to examine spontaneously autoimmune mice for autoantibodies and T cell responses to dH. We compared IgG binding to deiminated and non-deiminated histones (nH) by ELISA and Western blotting in spontaneously autoimmune strains of (NZB × NZW) F1 and NZM2410 together with their derivative congenic strains, C57BL/6.Sle1 and C57BL/6.Sle1.Sle3, which display profound autoreactivity against nuclear self-antigens. The splenocyte proliferation against the two antigens was determined in the spontaneously autoimmune (NZB × NZW) F1 strain from which other autoimmune strains used in the study were derived. Immunizations with dH and nH were attempted in BALB/c mice to assess their splenocyte response. Splenocytes from BALB/c mice and from autoimmune mice at the time of conversion to autoimmunity proliferated strongly in response to dH, yet serum IgG from autoimmune (NZB × NZW) F1, NZM2410, and C57BL/6.Sle1.Sle3 mice displayed a remarkable bias against binding to dH. At the time of seroconversion, the antibodies already exhibited preference for nH, and only nH were recovered from circulating immune complexes. Analysis of histone deimination showed constitutive deimination in thymic extracts from C57BL/6 and C57BL/6.Sle1.Sle2.Sle3 triply congenic mice and in spleens of autoimmune triply congenic mice. Our study demonstrates that tolerance mechanisms against dH are intact in BALB/c and C57BL/6 mice and continue to be effective in mice with overt autoimmunity to nH. We conclude that, in contrast to human RA and SLE patients, where we frequently observe autoantibodies against dH, autoimmune mice maintain strong tolerance mechanisms to prevent the development of autoantibodies to dH.
Highlights
Antibodies to citrullinated protein antigens (ACPA) are diagnostic markers for rheumatoid arthritis (RA) (1) and arise in other human autoimmune disorders such as systemic lupus erythematosus (SLE) and Felty’s syndrome (2, 3)
We observed that autoimmune mice exhibit a striking B cell bias toward binding non-deiminated histones (nH) over deiminated histones (dH), a bias that is present at the earliest stages of anti-histone autoimmunity (Figure 2G)
To exclude the possibility that antidH antibodies are only transiently expressed, or ensconced in immune complexes, we carried out longitudinal antibody-binding assays that consistently revealed preferential or exclusive binding to nH (Figure 2G)
Summary
Antibodies to citrullinated protein antigens (ACPA) are diagnostic markers for rheumatoid arthritis (RA) (1) and arise in other human autoimmune disorders such as systemic lupus erythematosus (SLE) and Felty’s syndrome (2, 3). Citrullines are introduced into proteins by peptidylarginine deiminase (PAD) family of enzymes (4), and much effort has been devoted to learning the circumstances that activate PADs and lead to the PADmediated conversion of arginine residues into citrulline residues (5, 6). Several citrullinated antigens have been identified in RA, and a common mechanism has been proposed to account for the generation of citrullinated autoantigens (7–9). The proposed mechanism places particular importance on PAD2 and PAD4, enzymes that are expressed in cells of the innate and adaptive immune system (10, 11). These calcium-dependent enzymes are activated under inflammatory conditions (5). Direct stimuli of PADs include microbial pathogens and pro-inflammatory chemokines and cytokines (5). It has been argued that any perforation to the plasma membrane could lead to the activation of PADs (12)
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