At an early stage of apoptosis cytochrome c (CYT) is released from mitochondria into the cytosol where it plays a role in activating the apoptotic proteolytic cascade (Cell 86:141). Since CYT is a minor antigen of systemic lupus erythematosus (SLE), we asked whether CYT may eventually appear in surface blebs like some other SLE antigens. Apoptosis was induced in DO11 T hybridoma cells by antibody (Ab) cross-linking of CD3, γ-irradiation, or dexamethasone treatment. Necrosis was also induced by heating the cells to 65° for 5 min. Using flow cytometry, in cells induced to apoptose by all three methods CYT was labeled by some but not all anti-native CYT monoclonal (m) Abs that were tested. The same mAbs that bound CYT in apoptotic cells also bound CYT in necrotic cells and in permeabilized live cells. To determine if CYT did appear at the cell surface of dead or dying cells, mAb binding to CYT was visualized by laser-scanning, confocal immunofluorescent microscopy. CYT assumed a punctate distribution in permeabilized live cells. In apoptotic cells CYT was often observed in blebs at the surface, while its distribution of was diffuse and polarized toward the surface of necrotic cells. However, in both apoptotic and necrotic cells CYT appears to be inaccessible at the surface, since 5 μ sulfate polystyrene latex microspheres coated with anti-CYT mAbs did not bind the cells. Thus, it seems unlikely that B lymphocytes become exposed to endogenous CYT on dying cells as has been suggested for other SLE antigens.
|Publication status||Published - Mar 20 1998|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology