Systemic autoimmune diseases are a genetically complex, heterogeneous group of disorders in which the immune system targets a diverse but highly specific group of intracellular autoantigens. The molecules targeted are not unified by common structure, function, or distribution in control cells but become clustered and concentrated in surface blebs when cells undergo apoptosis. We show here that the majority of autoantigens targeted across the spectrum of human systemic autoimmune diseases are efficiently cleaved by granzyme B in vitro and during cytotoxic lymphocyte granule–induced death, generating unique fragments not observed during any other form of apoptosis. These molecules are not cleaved by caspase-8, although this protease has a very similar specificity to granzyme B. The granzyme B cleavage sites in autoantigens contain amino acids in the P2 and P3 positions that are preferred by granzyme B but are not tolerated by caspase-8. In contrast to autoantigens, nonautoantigens are either not cleaved by granzyme B or are cleaved to generate fragments identical to those formed in other forms of apoptosis. The striking ability of granzyme B to generate unique fragments is therefore an exclusive property of autoantigens and unifies the majority of molecules targeted in this spectrum of diseases. These results focus attention on the role of the cytotoxic lymphocyte granule–induced death pathway in the initiation and propagation of systemic autoimmunity.

Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.

Peptidylarginine deiminases (PADs) play a critical role in generating autoantigens in rheumatoid arthritis (RA), but the mechanisms underlying their dysregulation in this disease remain unknown. Although PADs require supraphysiologic concentrations of calcium for activity in vitro, the enzymes are clearly active in vivo (e.g. in RA synovial fluid) where calcium concentrations are much lower. In this study, we have discovered a novel subset of anti-PAD4 autoantibodies (identified by their cross-reactivity with PAD3) which strikingly increase the catalytic efficiency of PAD4 by decreasing the enzyme’s requirement for calcium into the physiologic range. Patients with these novel PAD3/PAD4 cross-reactive autoantibodies had higher baseline radiographic damage scores and a higher likelihood of radiographic progression compared to individuals negative for these antibodies. The ability of autoantibodies to activate an enzyme that itself generates citrullinated autoantigens identifies an important feed-forward loop which may drive the erosive outcome observed in RA patients with these autoantibodies. PAD3 autoantibodies may therefore identify RA patients who would benefit from early aggressive treatment or addition of PAD-inhibitor therapy.