RESUMO
IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.
Assuntos
Citotoxicidade Imunológica , Granzimas/fisiologia , Imunofenotipagem , Interferon gama/fisiologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Neoplasias Uveais/imunologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/fisiologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/imunologia , Células Clonais , Regulação para Baixo/imunologia , Granzimas/antagonistas & inibidores , Humanos , Tolerância Imunológica , Imunidade Inata/imunologia , Interferon gama/sangue , Melanoma/metabolismo , Melanoma/patologia , Melanoma/secundário , Perforina/fisiologia , Prognóstico , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/metabolismo , Regulação para Cima/imunologia , Neoplasias Uveais/enzimologia , Neoplasias Uveais/patologiaRESUMO
OBJECTIVE: T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death. METHODS AND RESULTS: The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation. CONCLUSIONS: These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin.
Assuntos
Apoptose/fisiologia , Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/deficiência , Miócitos de Músculo Liso/fisiologia , Serina Endopeptidases/fisiologia , Animais , Caspase 3 , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Matriz Extracelular/enzimologia , Proteínas da Matriz Extracelular/metabolismo , Granzimas , Células HeLa/química , Células HeLa/metabolismo , Humanos , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/enzimologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Ratos , Serina Endopeptidases/metabolismo , Baço/citologia , Baço/metabolismoRESUMO
Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake.