concanamycin-a and 3-4-dichloroisocoumarin

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis.

Topics: Animals; Apoptosis; beta-Galactosidase; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cellular Senescence; Coumarins; Cytoplasmic Granules; Enzyme Inhibitors; Exocytosis; Extracellular Matrix Proteins; Hepatic Stellate Cells; Humans; Immunologic Surveillance; Isocoumarins; Killer Cells, Natural; Liver; Liver Cirrhosis; Macrolides; Mice; Mice, Inbred C57BL; Mice, Knockout; Perforin; RNA Interference; RNA, Small Interfering; Serine Proteinase Inhibitors; Tumor Necrosis Factor Decoy Receptors

Elimination of tumor cells by cytotoxic T lymphocytes (CTL) is mediated by two major pathways: the granule exocytosis and the death receptor pathway, transduced by Fas, TNF and TRAIL. The usage of these distinct pathways in the alloreactive setting across major and minor HLA barriers still remains controversial. We generated CTLs against allogeneic Epstein-Barr virus (EBV)-transformed cell lines (LCL) from HLA-unmatched healthy donors and assessed their cytotoxicity by flow cytometrically measuring mitochondrial membrane permeability (MMP) of target cells. Mitochondrial apoptosis induced by CTL was abrogated by selectively inhibiting the granule exocytosis-mediated pathway with Concanamycin A (CMA). Conversely, apoptosis was not decreased in the presence of the caspase 8 inhibitor IETD, which is specific for all death receptor pathways. In general, caspases were not involved in MMP as shown using the pan-caspase inhibitor zVAD. This effector mechanism was preserved when using purified CD4 + and CD8 + T-lymphocyte subsets to generate CTL. We further showed, that death receptor signalling was not used as a salvage mechanism when granule exocytosis was inhibited even at longer incubation times sufficient for slow kinetic death receptor caspase signalling. Our results clearly demonstrate that mitochondrial apoptosis induced by human alloreactive CTLs is mainly mediated by granule exocytosis but not by death receptor caspase dependent pathways. Furthermore, the granule exocytosis pathway does not require caspases to induce MMP.

Topics: Apoptosis; Caspase Inhibitors; Cell Line, Transformed; Coumarins; Cysteine Proteinase Inhibitors; Cytotoxicity Tests, Immunologic; fas Receptor; Humans; Intracellular Membranes; Isocoumarins; Macrolides; Mitochondria; Oligopeptides; Permeability; Receptors, Tumor Necrosis Factor; Signal Transduction; T-Lymphocytes, Cytotoxic