RESUMO
In this paper, we sought to identify the CD4(+) T-cell dynamics in the course of HIV infection in response to continuous and intermittent intravenous courses of interleukin-2 (IL-2), the principal cytokine responsible for progression of CD4(+) T-lymphocytes from the G1 to the S phase of the cell cycle. Based on multivariate regression models, previous literature has concluded that the increase in survival of CD4(+) T-cell appears to be the critical mechanism leading to sustained CD4(+) T-cell levels in HIV-infected patients receiving intermittent IL-2 therapy. Underscored by comprehensive mathematical modeling, a major finding of the present work is related to the fact that, rather than due to any increase in survival of CD4(+) T-cells, the expressive, selective and sustained CD4(+) T-cell expansions following IL-2 administration may be related to the role of IL-2 in modulating the dynamics of Fas-dependent apoptotic pathways, such as activation-induced cell death (AICD) or HIV-specific apoptotic routes triggered by viral proteins.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/terapia , Linfócitos T CD4-Positivos/imunologia , HIV-1/imunologia , Imunoterapia , Interleucina-2 , Síndrome da Imunodeficiência Adquirida/patologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos T CD4-Positivos/patologia , Feminino , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Humanos , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Masculino , Fase S/efeitos dos fármacos , Fase S/imunologia , Proteínas Virais/imunologia , Receptor fas/imunologiaRESUMO
Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.
Assuntos
Apoptose/imunologia , Membrana Celular/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Quinase CDC2 , Caspase 8/imunologia , Caspase 8/metabolismo , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Ciclina B/imunologia , Ciclina B/metabolismo , Quinase 2 Dependente de Ciclina/imunologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Fase G1/imunologia , Humanos , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/imunologia , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase S/imunologia , Proteína de Morte Celular Associada a bcl/imunologia , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
PPARalpha ligands are medications used clinically to prevent cardiovascular events, however studies have shown that these agents are also anti-inflammatory. Our previous studies have shown that PPARalpha ligands induce lymphocyte depletion. PPARalpha ligands also potently upregulate TRB3, a protein that has been associated with cell cycle arrest. Therefore the following studies were undertaken to determine the mechanisms associated with lymphocyte depletion. Our studies demonstrate that WY14,643, a PPARalpha ligand, decreases the amount of lymphocytes recovered after stimulation and reduces cellular divisions. Cells treated with WY14,643 also accumulate in the G2/S phase of the cell cycle. TRB3 has been shown to inhibit the phosphorylation of AKT/Protein Kinase B, and reduced activation of AKT has been associated with decreased cellular divisions and survival. However in lymphocytes, TRB3 did not reduce the phosphorylation of AKT, and WY14,643 treatment was associated with enhanced activation of AKT. Drosophila tribbles (TRB3 homolog) causes G2 arrest by decreasing the expression of a Cdc25c homolog. Lymphocytes stimulated and treated with WY14,643 have reduced expression of Cdc25c, however this is not associated with enhanced expression of phosphorylated-Cdc2 which induces G2 arrest. Instead we observed that WY14,643 consistently reduces the protein and mRNA expression of Cyclin B1. Moreover, TRB3 inhibits activation of a Cyclin B1 promoter construct. In summary, we propose that PPARalpha ligands may reduce cellular number by augmenting TRB3 expression, which in turn induces cell cycle arrest by reducing the expression of Cyclin B1. Reduced cellular divisions and cell cycle arrest may be responsible for some of the immunomodulatory effects of these agents that have been consistently observed in human trials.
Assuntos
Proteínas de Ciclo Celular/imunologia , Fase G2/efeitos dos fármacos , Linfócitos/imunologia , PPAR alfa/agonistas , PPAR alfa/imunologia , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Fase S/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Ciclina B/biossíntese , Ciclina B/imunologia , Ciclina B1 , Fase G2/imunologia , Humanos , Ligantes , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , PPAR alfa/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/imunologia , Fase S/imunologia , Regulação para Cima/imunologia , Fosfatases cdc25/imunologiaRESUMO
The canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus-encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle-associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC-expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active.
Assuntos
Linfócitos B/fisiologia , Proliferação de Células , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Receptor Notch1/fisiologia , Receptor Notch2/fisiologia , Proteínas Virais/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Transformação Celular Viral/imunologia , Células Cultivadas , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Modelos Biológicos , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Proteínas Recombinantes/farmacologia , Fase S/efeitos dos fármacos , Fase S/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas Virais/metabolismo , Proteínas Virais/farmacologiaRESUMO
The effect of mild hypothermic (32 degrees C) conditions on cell growth, cell-cycle progress, and antibody production of hybridoma C2E7 cells was investigated in the present study. The growth of hybridoma cells was slower during the mild hypothermic condition compared to that at 37 degrees C; this led to about 10% decrease in maximum viable cell density and volumetric antibody productivity. However, under mild hypothermic growth conditions, the culture viability was substantially improved and the specific antibody productivity was enhanced compared to that at 37 degrees C. The average specific productivity for the entire batch culture at 32 degrees C is about 5% higher than that at 37 degrees C. Cell-cycle analysis data showed that there was no growth arrestment during the mild hypothermic growth of hybridoma cells. The G1-phase cells were increased, while the S-phase cells were decreased gradually as the culture time progressed. Further analysis showed that the specific antibody productivity of hybridoma cells was correlated to the fraction of S-phase cells.
Assuntos
Anticorpos Monoclonais/biossíntese , Ciclo Celular/imunologia , Proliferação de Células , Temperatura Baixa , Hibridomas/imunologia , Hibridomas/metabolismo , Imunoglobulina M/biossíntese , Animais , Especificidade de Anticorpos , Células CHO , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fase S/imunologiaRESUMO
Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity.
Assuntos
Caspase 8/fisiologia , Proteína de Domínio de Morte Associada a Fas/fisiologia , Interleucina-2/fisiologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Fase S/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/enzimologia , Síndrome de Alstrom , Animais , Caspase 8/genética , Células Cultivadas , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/fisiologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Proteínas Quinases S6 Ribossômicas/deficiência , Fase S/genética , Transdução de Sinais/genética , Subpopulações de Linfócitos T/imunologiaRESUMO
CD74 is an integral membrane protein that was thought to function mainly as an MHC class II chaperone. However, CD74 was recently shown to have a role as an accessory-signaling molecule. Our studies demonstrated that CD74 regulates B-cell differentiation by inducing a pathway leading to the activation of transcription mediated by the NF-kappaB p65/RelA homodimer and its coactivator, TAF(II)105. Here, we show that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-kappaB activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-X(L). These studies therefore demonstrate that surface CD74 functions as a survival receptor.
Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária/imunologia , Fase S/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos B/citologia , Diferenciação Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , DNA/genética , DNA/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/imunologia , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Células Tumorais Cultivadas , Proteína bcl-X/genética , Proteína bcl-X/imunologiaRESUMO
The pleiotropic effects of statin, including its anti-inflammatory effects, via chemokines may be independent of statin-induced cholesterol reduction. Therefore, we examined the effect of pitavastatin on cell proliferation and the association between chemokine receptors (CCR2 and CCR5) and their ligands, RANTES (regulated upon activation, normal T cell-expressed and secreted) and monocyte chemotactic protein-1 (MCP-1), in monocytes. Pitavastatin but not pravastatin inhibited cell proliferation in a dose-dependent manner and showed S-phase arrest associated with the downregulation of CCR2 and CCR5 expression in human monocytic tumor cells (U937 cells). Although the anti-proliferative effects of pitavastatin were not inhibited by lower concentrations of RANTES and MCP-1, overexpression of CCR2/CCR5 significantly blocked the anti-proliferation with a low concentration of RANTES or MCP-1. Pitavastatin upregulated p21(waf1) but not p27(kip1), and did not change the expression levels of cyclin D1 or cdk4. In addition, RANTES and MCP-1 upregulated cyclin D1 in the presence of pitavastatin. In conclusion, the anti-proliferative effect of pitavastatin, but not pravastatin, through the downregulation of CCR2/CCR5 may be a pleiotropic effect. This effect may be anti-atherogenic in monocytes.
Assuntos
Inibidores Enzimáticos/farmacologia , Monócitos/efeitos dos fármacos , Quinolinas/farmacologia , Receptores CCR5/metabolismo , Receptores de Quimiocinas/metabolismo , Anticolesterolemiantes/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacologia , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Monócitos/citologia , Monócitos/metabolismo , Pravastatina/farmacologia , Receptores CCR2 , Fase S/efeitos dos fármacos , Fase S/imunologia , Células U937 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Interleukin-3 (IL-3) mediates hematopoietic cell survival and proliferation via several signaling pathways such as the Janus kinase/signal transducer and activator of transcription pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide-3 kinase (PI-3K) pathway. Mammalian target of rapamycin (mTOR) is one of the downstream targets of the PI-3K pathway, and it plays an important role in hematopoiesis and immune cell function. To better elucidate how mTOR mediates proliferation signals from IL-3, we assessed the role of S6 kinase 2 (S6K2), one of the downstream targets of mTOR, in IL-3 signaling. We show that S6K2 is activated by IL-3 in the IL-3-dependent Ba/F3 cell line and that this is mediated by mTOR and its upstream activator PI-3K but not by the MAPK kinase/extracellular signal-regulated kinase pathway. S6K2 is also activated in primary mouse bone marrow-derived mast cells upon IL-3 stimulation. Expression of a rapamycin-resistant form of S6K2, T388E, in Ba/F3 cells provides a proliferation advantage in the absence or presence of rapamycin, indicating that S6K2 can potentiate IL-3-mediated mitogenic signals. In cells expressing T388E, rapamycin still reduces proliferation at all doses of rapamycin, showing that mTOR targets other than S6K2 play an important role in IL-3-dependent proliferation. Cell-cycle analysis shows that T388E-expressing Ba/F3 cells enter S phase earlier than the control cells, indicating that the proliferation advantage may be mediated by a shortened G1 phase. This is the first indication that S6K2 plays a role in IL-3-dependent cell proliferation.
Assuntos
Células da Medula Óssea/enzimologia , Proliferação de Células/efeitos dos fármacos , Interleucina-3/metabolismo , Mastócitos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Humanos , Interleucina-3/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Fase S/efeitos dos fármacos , Fase S/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Homeostasis-driven T cell proliferation occurs in response to a lymphopenic environment and is mediated by TCR and IL-7 signaling. In this report, we demonstrate a defect in the proliferation of murine naive and memory T cells lacking both E2F1 and E2F2 in response to lymphopenic conditions, suggesting that E2F1 and E2F2 function redundantly downstream of TCR and/or IL-7 signaling during homeostasis-driven proliferation. In contrast, T cell proliferation in response to antigenic stimulation is either unaffected (in vivo) or potentiated (ex vivo) by loss of E2F1 and E2F2, indicating divergent requirements for these E2F factors in T cell proliferation mediated by distinct stimuli. E2F1/E2F2 double knockout (DKO) T cells enter S phase in response to homeostatic signaling, but fail to divide, suggesting that S phase progression is either incomplete or defective. In addition, E2F1/E2F2 DKO mice do not recover normal T cell numbers following exposure to a sublethal dose of radiation, indicating that this defect in homeostasis-driven proliferation is physiologically relevant. Consistent with their failure in cell cycle progression, the differentiation of DKO T cells into memory T cells in response to homeostatic signals is significantly reduced. These observations support the idea that proliferation is required for memory T cell formation and also have implications for the development of clinical strategies to minimize the occurrence of lymphopenia-induced autoimmunity.
Assuntos
Antígenos/administração & dosagem , Antígenos/imunologia , Proteínas de Ciclo Celular/fisiologia , Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Homeostase/imunologia , Linfócitos T/imunologia , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Homeostase/genética , Memória Imunológica/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fase S/genética , Fase S/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Phagocytosis of microorganisms by macrophages is an important host defense mechanism. While studying the phagocytosis of the human pathogenic fungus Cryptococcus neoformans, we noted that macrophage-like J774 cells with ingested fungal cells had frequent mitotic figures. By analyzing the relative proportion of phagocytic cells as a function of cell cycle phase, we observed an increase in S phase cells after Fc-mediated phagocytosis of polystyrene beads, live or heat-killed C. neoformans. This result was confirmed by increased nuclear BrdU incorporation after Fc-mediated phagocytosis. The induced progression to S phase was observed after both Fc- and complement-mediated phagocytosis of live yeasts. Fc-mediated stimulation of cell division did not require ingestion, because it could be triggered by incubating cells in IgG1-coated plates. Phagocytosis-mediated stimulation of replication was confirmed in vitro using primary bone marrow macrophages and in vivo for peritoneal macrophages. We conclude that phagocytosis of microbes or inert particles can stimulate macrophages to enter S phase and commence cell division. This observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also promote the spread of infection.
Assuntos
Ciclo Celular/imunologia , Fase G1/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Receptores de Complemento/metabolismo , Receptores Fc/metabolismo , Fase S/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Células Cultivadas , Ativação do Complemento/imunologia , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/metabolismo , Imunoglobulina G/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/imunologia , Receptores de Complemento/fisiologia , Receptores Fc/fisiologiaRESUMO
Recurrent chromosomal rearrangements are associated with the development of acute myeloid leukemia (AML). The frequent inversion of chromosome 16 creates the CBFB-MYH11 fusion gene that encodes the fusion protein CBFbeta-SMMHC. This fusion protein inhibits the core-binding factor (CBF), resulting in a block of hematopoietic differentiation, and induces leukemia upon the acquisition of additional mutations. A recent genetic screen identified Plag1 and Plagl2 as CBF beta-SMMHC candidate cooperating proteins. In this study, we demonstrate that Plag1 and Plagl2 independently cooperate with CBF beta-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and Plagl2 increased proliferation by inducing G1 to S transition that resulted in the expansion of hematopoietic progenitors and increased cell renewal in vitro. Finally, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples. Interestingly, PLAGL2 was preferentially increased in samples with chromosome 16 inversion, suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Overall, this study shows that Plag1 and Plagl2 are novel leukemia oncogenes that act by expanding hematopoietic progenitors expressing CbF beta-SMMHC.
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Doença Aguda , Adolescente , Adulto , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fase G1/imunologia , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Mieloide/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Pessoa de Meia-Idade , Mutagênese Insercional , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Retroviridae/genética , Fase S/imunologia , Fatores de Transcrição/metabolismoRESUMO
High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.
Assuntos
Apoptose/imunologia , Catepsina B/fisiologia , Catepsinas/fisiologia , Ativação Linfocitária/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Antígenos CD28/farmacologia , Inibidores de Caspase , Catálise , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina L , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Morte Celular/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Citocromos c/metabolismo , Citosol/enzimologia , Citosol/imunologia , Citosol/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/imunologia , Relação Dose-Resposta Imunológica , Fase G1/imunologia , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/imunologia , Lisossomos/enzimologia , Muromonab-CD3/farmacologia , Permeabilidade , Fase S/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.
Assuntos
Ciclo Celular/imunologia , Nucleotídeos de Purina/farmacologia , Nucleotídeos de Pirimidina/farmacologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Agregação Celular/efeitos dos fármacos , Agregação Celular/imunologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Inibidores do Crescimento/farmacologia , Humanos , Cinética , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotídeos de Purina/antagonistas & inibidores , Nucleotídeos de Purina/biossíntese , Nucleotídeos de Pirimidina/antagonistas & inibidores , Nucleotídeos de Pirimidina/biossíntese , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/imunologia , Fase S/efeitos dos fármacos , Fase S/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
Cells expressing indoleamine 2,3-dioxygenase (IDO), an enzyme which catabolizes tryptophan, prevent T-cell proliferation in vitro, suppress maternal antifetal immunity during pregnancy and inhibit T-cell-mediated responses to tumour-associated antigens. To examine the mechanistic basis of these phenomena we activated naïve murine T cells in chemically defined tryptophan-free media. Under these conditions T cells expressed CD25 and CD69 and progressed through the first 12 hr of G0/G1 phase but did not express CD71, cyclin D3, cdk4, begin DNA synthesis, or differentiate into cytotoxic effector cells. In addition, activated T cells with their growth arrested by tryptophan deprivation exhibited enhanced tendencies to die via apoptosis when exposed to anti-Fas antibodies. Apoptosis was inhibited by caspase inhibitor and was not observed when T cells originated from Fas-deficient mice. These findings suggest that T cells activated in the absence of free tryptophan entered the cell cycle but cell cycle progression ceased in mid-G1 phase and T cells became susceptible to death via apoptosis, in part though Fas-mediated signalling. Thus, mature antigen-presenting cells expressing IDO and Fas-ligand may induce antigen-specific T-cell tolerance by blocking T-cell cycle progression and by rapid induction of T-cell activation induced cell death in local tissue microenvironments.
Assuntos
Apoptose/imunologia , Linfócitos T/imunologia , Triptofano/deficiência , Animais , Antígenos CD/imunologia , Western Blotting/métodos , Divisão Celular/imunologia , Linhagem Celular/imunologia , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo/métodos , Indolamina-Pirrol 2,3,-Dioxigenase , Interfase/imunologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fase S/imunologia , Baço/imunologia , Triptofano Oxigenase/imunologiaRESUMO
CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti-CD26 mAb 1F7 appears to be induced through activation of extracellular signal-regulated kinase (ERK) pathway. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft-vs.-host disease.
Assuntos
Dipeptidil Peptidase 4/imunologia , Fase G1/imunologia , Fase S/imunologia , Linfócitos T/imunologia , Anticorpos Monoclonais/imunologia , Técnicas de Cultura de Células , Divisão Celular/imunologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases/imunologiaRESUMO
The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.
Assuntos
Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas de Membrana , Animais , Linfócitos B/efeitos dos fármacos , Proteínas de Transporte/genética , Linhagem da Célula , Núcleo Celular/genética , Replicação do DNA , Eletroforese em Gel Bidimensional , Hibridização in Situ Fluorescente , Proteína Jagged-2 , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos , Fase S/genética , Fase S/imunologia , Células Tumorais CultivadasRESUMO
TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in susceptible cells, which can be both malignant and nontransformed. Despite homologies among the death ligands, there are great differences between the TRAIL system on the one hand and the TNF and CD95 systems on the other hand. In particular, TRAIL-induced apoptosis differs between rodents and man. Studies on animal models of autoimmune diseases suggested an influence of TRAIL on T cell growth and effector functions. Because we previously demonstrated that TRAIL does not induce apoptosis in human (auto)antigen-specific T cells, we now asked whether TRAIL exhibits other immunoregulatory properties in these cells. Active TRAIL inhibited calcium influx through store-operated calcium release-activated calcium channels, IFN-gamma/IL-4 production, and proliferation. These effects were independent of APC, Ag specificity, and Th differentiation, and no differences were detected between healthy donors and multiple sclerosis patients. TRAIL affected neither the expression of the cell cycling inhibitor p27(Kip1) nor the capacity of T cells to produce IL-2 upon Ag rechallenge, indicating that signaling via TRAIL receptor does not induce T cell anergy. Instead, the TRAIL-induced hypoproliferation could be attributed to the down-regulation of the cyclin-dependent kinase 4, indicating a G(1) arrest of the cell cycle. Thus, although it does not contribute to mechanisms of peripheral T cell tolerance such as clonal anergy or deletion by apoptosis, TRAIL can directly inhibit activation of human T cells via blockade of calcium influx.
Assuntos
Apoptose/imunologia , Autoantígenos/imunologia , Regulação para Baixo/imunologia , Epitopos de Linfócito T/imunologia , Inibidores do Crescimento/fisiologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/fisiologia , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Células Apresentadoras de Antígenos/imunologia , Proteínas Reguladoras de Apoptose , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Divisão Celular/imunologia , Linhagem Celular , Anergia Clonal/imunologia , Fase G1/imunologia , Inibidores do Crescimento/farmacologia , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-4/antagonistas & inibidores , Interleucina-4/biossíntese , Células Jurkat , Ligantes , Glicoproteínas de Membrana/farmacologia , Fase S/imunologia , Solubilidade , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Engagement of CTLA-4 is critical for inhibiting T cell immune responses. Recent studies have shown that CTLA-4 plays a key role in regulating peripheral T cell tolerance. It has been suggested that one mechanism by which CTLA-4 performs this function is by regulating cell cycle progression. Here, we investigate in depth the role of CTLA-4 in regulating cell cycle progression in naive T cells by comparing the immune responses in the absence or presence of CTLA-4. In the absence of CLTA-4, T cells exhibit marked increases in T cell proliferation, IL-2 mRNA and protein secretion, and cells cycling in the S and G2-M phase. Analyses of cyclins, cyclin-dependent kinases, and cell cycle inhibitors involved in the transition from the G1 to S phase reveal that cell cycle progression is prolonged in the absence of CTLA-4. This is due to the early exit from the G1 phase, entry into the S phase, and prolonged S phase period. Re-expression of the cell cycle inhibitor p27(kip1) is delayed in the absence of CTLA-4. These studies demonstrate that the B7 : CTLA-4 pathway exerts its major effects on T cell immune responses via regulation of the cell cycle.
Assuntos
Antígenos de Diferenciação/fisiologia , Ciclo Celular/imunologia , Imunoconjugados , Linfócitos T/citologia , Linfócitos T/imunologia , Abatacepte , Animais , Antígenos CD/genética , Antígenos CD/fisiologia , Antígenos de Diferenciação/genética , Antígeno B7-1/genética , Antígeno B7-1/fisiologia , Antígeno B7-2 , Antígeno CTLA-4 , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/biossíntese , Feminino , Fase G1/imunologia , Técnicas In Vitro , Interleucina-2/biossíntese , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Fase S/imunologia , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.