Secondary necrosis is a source of proteolytically modified forms of specific intracellular autoantigens: Implications for systemic autoimmunity

Abstract

Objective Specific autoantigens targeted in systemic autoimmunity undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunologic consequences of defective clearance of apoptotic cells, we sought to determine whether autoantigens cleaved during 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 proteolysis during cell death was examined by immunoblotting of cell lysates using highly specific human autoantibodies 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., ribosomal RNP, Ku, and SSA/Ro) appeared to be resistant to proteolysis during cell death. Conclusion In the absence of phagocytosis, apoptotic 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 proinflammatory conditions.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose

“…In the case of Topo I, a signature necrotic fragment of 45 kDa was detected, consistent with previous reports demonstrating that the generation of this fragment is a distinctive feature of necrotic cell death. 42,43 The pattern of Figure 3 Transfected HA-E6 retains biological activity in both sensitive and resistant clones. (a) ELISA measurement of baseline and induced levels of p53 in E6-expressing clones.…”

Cooperation between C1q and DNase I in the clearance of necrotic cell–derived chromatin

“…A disturbed clearance of apoptotic cells, as has been observed in some patients with SLE, facilitates the release of heat-shock proteins (33), nucleosomes (34), calreticulin (35,36), and other intracellular contents. In addition, modifications of these autoantigens during programmed cell death can render them more immunogenic (34,37,38). It has also been shown that in mice genetically predisposed to autoimmune disease, bacterial DNA can elicit anti-DNA autoantibodies under conditions in which mammalian DNA is inactive (39).…”

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…In some experiments, cells were pretreated with the cathepsin L inhibitor Z-FY-CHO (150 M) or with individual caspase inhibitors 1 hour prior to addition of TNF␣. Quantification of cytoplasmic membrane rupture, indicative of necrosis, was performed by trypan blue exclusion (11). Morphologic analysis of cells was performed using an Olympus IX70 inverted microscope (Olympus, Melville, NY) equipped with Hoffman modulation contrast.…”

“…Topo I fragments of 72-75 kd are also produced by granzyme B both in vitro and during cell death induced by cytotoxic T lymphocytes (14). Our group demonstrated previously that topo I is also cleaved into fragments of approximately 70 kd and 45 kd in cells undergoing primary or secondary necrosis (10,11).…”

“…There is compelling evidence indicating that dying cells, both apoptotic and necrotic, are reservoirs of fragmented or cleaved forms of intracellular autoantigens (8)(9)(10)(11)(12). The excessive accumulation of these cleavage products, which could potentially expose cryptic epitopes, might break immune tolerance in a proinflammatory microenvironment and elicit specific humoral and cellular immune responses in patients with systemic autoimmune diseases.…”