ARTHRITIS & RHEUMATISM Vol. 44, No. 11, November 2001, pp 2642–2652 © 2001, American College of Rheumatology Published by Wiley-Liss, Inc. Secondary Necrosis Is a Source of Proteolytically Modified Forms of Specific Intracellular Autoantigens Implications for Systemic Autoimmunity Xiwei Wu, Christine Molinaro, Neal Johnson, and Carlos A. Casiano Objective. Specific autoantigens targeted in sys- temic autoimmunity undergo posttranslational modifi- cations, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunologic conse- quences of defective clearance of apoptotic cells, we sought to determine whether autoantigens cleaved dur- ing apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. Methods. Apoptosis was induced in Jurkat cells with etoposide, anti-Fas antibody, or staurosporine (STS), and in HeLa cells with STS. Progression to secondary necrosis was assessed morphologically and quantified by trypan blue uptake. Autoantigen proteol- ysis during cell death was examined by immunoblotting of cell lysates using highly specific human autoantibod- ies as detecting probes. Results. Cells treated with the different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several autoantigens, including poly(ADP-ribose) polymerase, topoisomerase I (or Scl- 70), SSB/La, and U1–70 kd, were cleaved into their signature apoptotic fragments. Progression of apoptosis to secondary necrosis was associated with additional proteolysis of these and other autoantigens in a caspase- independent manner. Some autoantigens (e.g., ribo- somal RNP, Ku, and SSA/Ro) appeared to be resistant to proteolysis during cell death. Conclusion. In the absence of phagocytosis, apo- ptotic cells may undergo secondary necrosis, a process associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflamma- tory conditions. The origin of antinuclear autoantibodies (ANA) in rheumatic diseases remains elusive. While there is strong evidence that these antibodies are driven by proteins and nucleic acids associated with intracellular particles (1,2), it is unclear how these normally seques- tered particles are exposed to the immune system and rendered capable of sustaining a prolonged autoimmune response. An emerging view is that this process could be facilitated by abnormalities in cell death, such as in- creased rates of apoptosis or defective clearance of apoptotic cells, in combination with cell death– associated posttranslational modifications that might enhance autoantigen immunogenicity (3–5). Many intra- cellular antigens targeted by autoantibodies in rheu- matic diseases undergo dramatic modifications during apoptosis, such as relocalization to the cell surface and clustering in apoptotic blebs (6,7), caspase- and nuclease-mediated cleavage (8–10), and hyperphospho- rylation (11). It has been suggested that these modifica- tions may expose to the immune system novel or cryptic Presented in part at the 63rd Annual Scientific Meeting of the American College of Rheumatology, Boston, MA, November 1999. Supported by NIH grant AI-44088. Support for microscopy and imaging facilities was provided by the Hedco Foundation. Xiwei Wu, MD, Christine Molinaro, MS, Neal Johnson, BS, Carlos A. Casiano, PhD: Loma Linda University School of Medicine, Loma Linda, California. Address correspondence and reprint requests to Carlos A. Casiano, PhD, Department of Microbiology and Molecular Genetics, Center for Molecular Biology and Gene Therapy, Loma Linda Uni- versity School of Medicine, Loma Linda, CA 92354. E-mail: ccasiano@som.llu.edu. Submitted for publication January 3, 2001; accepted in re- vised form June 18, 2001. 2642