RESUMO
A functional hybrid receptor associating the common gamma chain (gammac) with the granulocyte/macrophage colony-stimulating factor receptor beta (GM-CSFRbeta) chain is found in mobilized human peripheral blood (MPB) CD34+ hematopoietic progenitors, SCF/Flt3-L primed cord blood (CB) precursors (CBPr CD34+/CD56-), and CD34+ myeloid cell lines, but not in normal natural killer (NK) cells, the cytolytic NK-L cell line or nonhematopoietic cells. We demonstrated, using CD34+ TF1beta cells, which express an interleukin (IL)-15Ralpha/beta/gammac receptor, that within the hybrid receptor, the GM-CSFRbeta chain inhibits the IL-15-triggered gammac/JAK3-specific signaling controlling TF1beta cell proliferation. However, the gammac chain is part of a functional GM-CSFR, activating GM-CSF-dependent STAT5 nuclear translocation and the proliferation of TF1beta cells. The hybrid receptor is functional in normal hematopoietic progenitors in which both subunits control STAT5 activation. Finally, the parental TF1 cell line, which lacks the IL-15Rbeta chain, nevertheless expresses both a functional hybrid receptor that controls JAK3 phosphorylation and a novel IL-15alpha/gammac/TRAF2 complex that triggers nuclear factor kappaB activation. The lineage-dependent distribution and function of these receptors suggest that they are involved in hematopoiesis because they modify transduction pathways that play a major role in the differentiation of hematopoietic progenitors.
Assuntos
Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos Monoclonais/metabolismo , Divisão Celular/fisiologia , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Interleucina-15 , Receptores de Interleucina-2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/fisiologiaRESUMO
Tumor-infiltrating p58+ T cells from a renal tumor were specifically expanded in response to tumor cell stimulation and cloned. These p58+ T cells were found to express a memory phenotype and corresponded to clonal TCRBV3 T-cell expansion. Functionally, p58(+) CTLs displayed a low lytic activity for HLA-A2 tumor and normal cells. However, this lytic activity was significantly increased after blockade of p58 with specific monoclonal antibodies. Interestingly, we demonstrated that stimulation by tumor cells was required to trigger the inhibitory effect of p58 on the lytic activity of antigen-specific CTLs and that stimulation of the inhibitory function of p58 by tumor cells correlated with an inhibition of nuclear factor-kappaB activation in p58+ tumor-specific CTLS.
Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Imunológicos/imunologia , Linfócitos T Citotóxicos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Complexo CD3/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores Imunológicos/antagonistas & inibidores , Receptores KIR , Receptores KIR2DL3RESUMO
There are two IL-15 isoforms and eight isoforms for the IL-15Ralpha chain whose biological role is poorly understood. Here, we have analysed the intracellular trafficking of IL-15 and IL-15Ralpha and tried to shed some light on their function(s). In IL-15/GFP CHO transfectants both IL-15 isoforms show nuclear localization. Two melanoma cell lines (MELP and MELREO) spontaneously expressing the IL-15 isoforms, display different intracellular trafficking of the IL-15/IL-15Ralpha complex. In MELP cells only IL-15Ralpha is detected inside the nucleus, whereas IL-15 and IL-15Ralpha assemble at the cell surface and are internalized. Moreover, the transducing molecule TRAF2 co-immunoprecipitates with IL-15Ralpha and may be deflected to TNFRI using anti-IL-15 blocking mAbs and TNF-alpha. By contrast, MELREO cells display IL-15Ralpha and IL-15 nuclear localization but only a partial co-localization of these molecules on the cell surface. In these cells, TRAF2 is strongly associated with IL-15Ralpha and cannot be deflected by any treatment. Since TRAF2 activates the transcription factor NF-kappaB, IL-15 through IL-15Ralpha, could have a role in the control of this pathway. Indeed, anti-IL-15 MaB inhibit the constitutive nuclear localization of NFkappaB and the phosphorylation of its inhibitor Ikappa-Balpha. Thus, IL-15Ralpha controls NF-kappaB activation, however differences in the intracellular trafficking of the IL-15 and/or IL-15Ralpha suggest a different biological role for this complex in MELP versus MELREO cells.
Assuntos
Interleucina-15/metabolismo , Melanoma/metabolismo , Receptores de Interleucina-2/metabolismo , Animais , Células CHO , Compartimento Celular , Núcleo Celular , Cricetinae , Proteínas de Fluorescência Verde , Humanos , Interleucina-15/genética , Interleucina-15/isolamento & purificação , Proteínas Luminescentes/genética , Proteínas Luminescentes/isolamento & purificação , Microscopia Confocal , NF-kappa B/metabolismo , Ligação Proteica , Sinais Direcionadores de Proteínas , Subunidades Proteicas , Transporte Proteico , Proteínas/isolamento & purificação , Receptores de Interleucina-15 , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNFRESUMO
IL-15 and SCF fail to induce NK differentiation and proliferation of CD34+ hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although, both normal and leukemic CD34+ cells display comparable expression of c-kit or IL-15 receptor subunits. Interestingly, confocal microscopy analysis revealed that leukemic and most normal CD34+ cells produce and secrete IL-15, as shown by its trafficking through the Golgi apparatus and early endosomes. However, only leukemic progenitors express the membrane bound IL-15. Colocalization and internalization of IL-15Rbeta/gammac and IL-15Ralpha/gammac complexes indicated that IL-15 was specifically uptaken by leukemic progenitors. We also demonstrated that in both normal and leukemic progenitors, the signaling kinase Jak3 is constitutively pre-associated with the gammac chain. Anti-IL-15 neutralizing mAb treatment resulted in down-regulation of gammac chain and disruption of gammac/Jak3 interaction in normal but had no effect in leukemic progenitors. Our results suggest the existence in both normal and leukemic CD34+ cells of a constitutive production of a bioactive IL-15 that does not lead to NK differentiation and further indicate that membrane bound IL-15 and constitutive activation of gammac are hallmarks of leukemic progenitors. Oncogene (2000).
Assuntos
Antígenos CD34/metabolismo , Células Matadoras Naturais/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Subpopulações de Linfócitos/patologia , Diferenciação Celular , Divisão Celular , Linhagem Celular , Humanos , Interleucina-15/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Subpopulações de Linfócitos/fisiologia , Microscopia Confocal , Receptores de Interleucina-15 , Receptores de Interleucina-2/metabolismo , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Células Tumorais CultivadasRESUMO
Affinity purified, freshly isolated CD34+ progenitors were shown to express low levels of type I interferon (IFN) receptors (740 +/- 60 binding sites/cell, K(d) 0.7 +/- 0.04 nM) determined by Scatchard's analysis using a radiolabelled, neutralizing, monoclonal antibody directed against the IFNAR1 chain of the human type I IFN receptor. Treatment of freshly isolated (day 0), highly purified (>95% pure) CD34+ cells with recombinant IFN-alpha resulted in rapid tyrosine phosphorylation and activation of STAT1, Tyk2 and JAK1 as shown by Western immunoblotting. Similarly, IFN treatment was shown by confocal microscopy to result in rapid nuclear localization of the transcription factors IRF1 and STAT2, demonstrating the presence of functional IFN receptors on freshly isolated (day 0) CD34+ cells. The number of specific type I IFN receptor binding sites expressed on hematopoietic progenitor cells increased to some 1440 +/- 40 per cell after 11 days of cultivation of CD34+ cells in vitrosuggesting that receptor expression increases with cell differentiation. IFN-mediated signal transduction and the inhibitory effect of IFN-alpha on 7 or 14 days CFU-GM and BFU-E colony formation was abrogated in the presence of the anti-IFNAR1 mAb, indicating that IFN-alpha acts directly on the proliferation of human hematopoietic progenitor cells via receptor activated signal transduction without excluding the induction of other cytokines or growth factors by residual accessory cells.
Assuntos
Antígenos CD34/análise , Células-Tronco Hematopoéticas/metabolismo , Receptores de Interferon/fisiologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Anticorpos Monoclonais/farmacologia , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/química , Humanos , Fator Regulador 1 de Interferon , Interferon-alfa/antagonistas & inibidores , Janus Quinase 1 , Cinética , Proteínas de Membrana , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/antagonistas & inibidores , Receptores de Interferon/imunologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , TYK2 Quinase , Transativadores/metabolismoRESUMO
BACKGROUND: Lung fibroblasts may have a pivotal role in airway inflammation as they are involved in continuous cycles of mediator secretion, proliferation, activation and cross-talk with recruited inflammatory cells. The role of fibroblasts as intermediate participants in the inflammatory network suggests that they could represent an important target for drugs commonly used in asthma; thus, we investigated the effects of triamcinolone acetonide (TAA) on primary human lung fibroblasts. METHODS: The in vitro activity of increasing concentrations (10(-9) to 10(-7) M) of TAA in fibroblast cultures was evaluated as regards the following parameters: proliferation, extracellular matrix (ECM) release, cytokine/chemokine secretion and surface antigen expression. RESULTS: All concentrations of TAA decreased fetal calf serum (FCS)-induced fibroblast proliferation, whereas in the presence of FCS plus basic fibroblast growth factor TAA was only effective at 10(-8) and 10(-7) M. TAA failed to decrease ECM, whereas at 10(-8) and 10(-7) M it decreased IL-6 and IL-8 secretion to different extents. In the presence of IFN-gamma the drug was able to reduce VCAM-1 expression at all of the tested concentrations; on the other hand, in TGF-beta 1-driven cultures a decrease in CD54 expression was detected with TAA at 10(-8) and 10(-7) M. CONCLUSIONS: TAA acts on some functional properties of human lung fibroblasts that make these cells active participants in the inflammatory network. The ability of TAA to inhibit lung fibroblast proliferation may prevent or even reverse some of the histological changes that characterize airway remodeling in chronic inflammatory diseases; moreover, IL-6, IL-8 and surface molecule decreases by TAA may suggest a direct anti-inflammatory effect of the drug by suppression of resident lung cell function.
Assuntos
Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Triancinolona Acetonida/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Colágeno/imunologia , Colágeno/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibronectinas/imunologia , Fibronectinas/metabolismo , Glucocorticoides/imunologia , Glucocorticoides/uso terapêutico , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/imunologia , Interferon gama/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Interleucina-8/biossíntese , Interleucina-8/imunologia , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Triancinolona Acetonida/imunologia , Triancinolona Acetonida/uso terapêutico , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
NK cells express receptors characterized by opposite functions that finely regulate their activities. Among inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are still orphan receptors. On the contrary, various activating receptors are involved in the triggering of NK-mediated natural cytotoxicity. In general, their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Thus, the process of NK cell triggering mediated by Natural Cytotoxicity Receptors can be mainly considered as a non MHC-restricted mechanism. Here, a brief description of the molecular nature of these receptors, as well as, of their 3D-structures and of the implications for ligand recognition, is given.
Assuntos
Células Matadoras Naturais/imunologia , Receptores Imunológicos/fisiologia , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/química , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/fisiologia , Cromossomos Humanos Par 17/genética , Citotoxicidade Imunológica , Humanos , Lectinas/química , Lectinas/genética , Lectinas/fisiologia , Modelos Imunológicos , Modelos Moleculares , Família Multigênica , Receptor 1 Desencadeador da Citotoxicidade Natural , Receptor 2 Desencadeador da Citotoxicidade Natural , Psoríase/genética , Psoríase/imunologia , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores KIR , Receptores de Células Matadoras NaturaisRESUMO
Asthma is characterized by an irreversible subepithelial fibrosis with the appearance of myofibroblasts, which can be now considered important early participants in inflammatory responses as well as potential targets for anti-inflammatory drugs. In this study, we show that fluticasone propionate (FP), a powerful inhaled corticosteroid (ICS), displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation. Indeed, FP inhibits in lung myofibroblasts, at a very early stage of differentiation, the activation of Janus kinase/STAT pathways induced by IL-13 (tyrosine kinase 2, STAT1, STAT3, STAT6, mitogen-activated protein kinase). Contrarily, in mildly or fully differentiated myofibroblastic cultures, FP still displays a potential anti-inflammatory activity even if it only inhibits tyrosine kinase 2 phosphorylation. Moreover, FP inhibits constitutive and TGF-beta-induced expression of alpha-smooth muscle actin, the main marker of myofibroblastic differentiation, both in very early and in mild differentiated myofibroblasts. Finally, FP displays an additional powerful anti-inflammatory effect, decreasing nuclear translocation of NF-kappaB independent of the degree of myofibroblastic differentiation. These data 1) suggest that myofibroblasts are priority targets for ICS, which is able to revert them to a normal phenotype even if they appear to be already engaged in their differentiation, and 2) may help to explain why asthma is improved by an early ICS treatment, whereas advanced asthma is more resistant to these drugs.
Assuntos
Androstadienos/farmacologia , Anti-Inflamatórios/farmacologia , Pulmão/efeitos dos fármacos , Proteínas Tirosina Quinases , Actinas/análise , Administração por Inalação , Adulto , Androstadienos/administração & dosagem , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Fluticasona , Humanos , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Pulmão/citologia , Microscopia Confocal , NF-kappa B/metabolismo , Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3 , TYK2 Quinase , Transativadores/fisiologiaRESUMO
Microglial cells (MC) are IL-12 producers in the central nervous system. Here, we found that IL-12 receptor subunits beta1 and beta2 were both constitutively expressed, and up-regulated by IFN-gamma, in human primary MC. IL-12p70, after binding to its receptor, is internalized into vesicles that qualify as early endosomes as indicated by intracellular colocalization with transferrin. IL-12 induced tyrosine phosphorylation and nuclear translocation of STAT4. IL-12 signaling in human MC also involved members of the NFkappaB family. IL-12p70 and, more effectively, the combination of IL-12p70 and IFN-gamma, induced IL-12p40 mRNA expression and bioactive IL-12p70 production. Human MC, thus, express a functional IL-12 receptor and produce bioactive IL-12 following IL-12 stimulation.