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


CONCLUSIONS
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.
Wu
X
X
Loma Linda University School of Medicine, California, USA.
Molinaro
C
C
Johnson
N
N
Casiano
C A
CA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D001324:Autoantigens / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004338:Drug Combinations; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D001324:Autoantigens; D004338:Drug Combinations; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; C411023:anti-Fas monoclonal antibody; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D004264:DNA Topoisomerases, Type I; D019311:Staurosporine