RESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer in children around the globe. Mexico City has one of the highest incidence rates of childhood leukemia worldwide with 49.5 cases per million children under the age of 15 which is similar to that reported for Hispanic populations living in the United States. In addition, it has been noted a dismal prognosis in Mexican and Hispanic ALL pediatric population. Although ALL, like cancer in general, has its origins in endogenous, exogenous, and genetic factors, several studies have shown that the immune system also plays a deterministic role in cancer development. Among various elements of the immune system, T lymphocytes and NK cells seem to dominate the immune response against leukemia. The aim of the present study was to perform a phenotypic and functional characterization of NK cells in ALL Mexican children at the moment of diagnosis and before treatment initiation. A case-control study was conducted by the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia (MIGICCL). 41 cases were incident ALL children younger than 17 years old and residents of Mexico City. 14 controls were children without leukemia, matched by age and sex with cases. NK cell function was evaluated by degranulation assays towards K562 cells and SLAM-associated protein (SAP) expression was measured by intracellular staining. All assays were performed using peripheral blood mononuclear cells from controls and patients. The results indicate that NK mediated cytotoxicity, measured by CD107a degranulation assays in response to K562 cells, was reduced in ALL patients compared to controls. Interestingly, an impaired NK cell killing of target cells was not equally distributed among ALL patients. In contrast to patients classified as high-risk, standard-risk patients did not display a significant reduction in NK cell-mediated cytotoxicity. Moreover, patients presenting a leukocyte count ≥ 50,000xmm3 displayed a reduction in NK-cell mediated cytotoxicity and a reduction in SAP expression, indicating a positive correlation between a reduced SAP expression and an impaired NK cell-mediated citotoxicity. In the present study it was observed that unlike patients with standard-risk, NK cells from children presenting high-risk ALL, harbor an impaired cytotoxicity towards K562 at diagnosis. In addition, NK cell function was observed to be compromised in patients with a leukocyte count ≥50,000xmm3, where also it was noticed a decreased expression of SAP compared to patients with a leukocyte count <50,000xmm3. These data indicate NK cell-mediated cytotoxicity is not equally affected in ALL patients, nevertheless a positive correlation between low SAP expression and decreased NK cell-mediated cytotoxicity was observed in ALL patients with a leukocyte count ≥50,000xmm3. Finally, an abnormal NK cell-mediated cytotoxicity may represent a prognostic factor for high-risk acute lymphoblastic leukemia.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Linfócitos T Citotóxicos/metabolismo , Adolescente , Estudos de Casos e Controles , Degranulação Celular/genética , Degranulação Celular/imunologia , Criança , Pré-Escolar , Citotoxicidade Imunológica/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células K562 , Células Matadoras Naturais/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Masculino , México , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Linfócitos T Citotóxicos/patologiaRESUMO
Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of ß-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Degranulação Celular , Mastócitos/metabolismo , Complexo 3 de Proteínas Adaptadoras/genética , Animais , Biomarcadores , Degranulação Celular/genética , Linhagem Celular , Citocinas/metabolismo , Endocitose , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Mediadores da Inflamação/metabolismo , Masculino , Mastócitos/imunologia , Estabilidade Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Ratos , Receptores de IgE/genética , Receptores de IgE/metabolismoRESUMO
Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nphi) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nphi culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nphi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nphi number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nphi toxicity as well as Nphi degranulation regardless of infection. Furthermore, Gal-3 expression by Nphi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nphi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nphi life span and activation during early T. gondii infection.