Purpose: Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM.
Methods: EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry.
Results: Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells.
Conclusions: Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.