ovalbumin and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

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.. 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.. 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.. 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.

Topics: Adjuvants, Immunologic; Amino Acid Chloromethyl Ketones; Animals; Antigens; Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Dendritic Cells; DNA; Flow Cytometry; L-Lactate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Necrosis; Ovalbumin; Phagocytosis; Plant Bark; Saponaria; Saponins

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.

Topics: Aerosols; Allergens; Amino Acid Chloromethyl Ketones; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Caspase Inhibitors; Cell Movement; Cysteine Proteinase Inhibitors; Disease Models, Animal; Inflammation; Interleukin-4; Interleukin-5; Intubation, Intratracheal; Leukocytes; Lung; Lymphocyte Activation; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; T-Lymphocytes

Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1-/- thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Differentiation; Cell Survival; Clonal Deletion; Crosses, Genetic; Cysteine Proteinase Inhibitors; Enzyme Activation; Female; H-Y Antigen; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Transgenic; Nucleopolyhedroviruses; Ovalbumin; Peptides; Receptors, Antigen, T-Cell; T-Lymphocytes; Thymus Gland; Viral Proteins