RESUMO
Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-alpha/TNFR interactions because although type 1 cells secreted more TNF-alpha than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.
Assuntos
Ativação Linfocitária , Macrolídeos , Glicoproteínas de Membrana/fisiologia , Serina Endopeptidases/fisiologia , Subpopulações de Linfócitos T/citologia , Receptor fas/fisiologia , Adulto , Clorometilcetonas de Aminoácidos/farmacologia , Antibacterianos/farmacologia , Cálcio , Caspase 8 , Caspase 9 , Inibidores de Caspase , Quelantes/farmacologia , Grânulos Citoplasmáticos/metabolismo , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Etanercepte , Exocitose , Proteína Ligante Fas , Granzimas , Humanos , Imunoglobulina G/farmacologia , Interferon gama/metabolismo , Interleucina-12/farmacologia , Interleucina-2/farmacologia , Interleucina-4/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Muromonab-CD3/farmacologia , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The class II human leucocyte antigens (HLA class II) are principally peptide presentation molecules. Signal transduction by these molecules has also been shown to transmit activation signals in both B and T lymphocytes by a pathway including protein tyrosine kinase activation, an intracellular calcium flux, and both the activation and transcriptional regulation of protein kinase C (PKC) isoforms. Apoptosis can also result from human leukocyte antigen class II stimulation. Inhibitors of gene transcription were used to inhibit activation and, therefore, to distinguish the signal transduction pathways important for apoptosis. This approach provided evidence that cellular activation and apoptosis undertook separate signaling pathways, and that PKC and intracellular calcium were shared between the two pathways, while tyrosine kinase activity was essential for cell activation. Further studies using cord blood B cells showed that these cells were incapable of generating a calcium flux after HLA class II ligation and were not subject to cell death. The importance of sustained levels of calcium for programmed cell death (PCD) was underlined since the restoration of a calcium flux enabled PCD of cord blood B cells via HLA class II. These results demonstrate that HLA class II stimulation initiates two distinct signal transduction pathways.
Assuntos
Apoptose/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Adulto , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Transplante de Células/fisiologia , Cesárea , Dactinomicina/farmacologia , Feminino , Sangue Fetal/citologia , Feto/citologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Gravidez , Proteína Quinase C/farmacologia , Sistemas do Segundo Mensageiro , Baço/citologiaRESUMO
Cord blood is increasingly used for hematopoietic stem cell transplantation since less severe graft-versus-host disease has been reported leading to the notion that cord blood is "naive." Human leucocyte antigen (HLA) class II molecules are expressed throughout B lymphocyte ontogeny (except the plasmocytes), are responsible for antigen presentation, and can also transmit signals. Cord blood B stimulate an allogeneic response, and this property is believed to indicate the presence of a class II-associated peptide. In this study we examined the capacity of cord blood B to transmit signals via HLA-DR. Activation and relocalization of protein kinase C (PKC) isoenzymes alpha and betaII was detected along with tyrosine kinase activation and proliferation. However, in contrast to resting adult B, generation of an intracellular calcium ([Ca++]i) flux and rapid aggregation were not detected. To address the question of whether or not HLA-DR signals throughout B lymphocyte ontogeny, we extended this study to include malignant adult B (B chronic lymphocytic leukemia [B-CLL], B mantle cell lymphoma, and B large cell leukemia). Tyrosine kinase activation and proliferation were observed in all these cell populations, albeit in the absence of [Ca++]i flux or an increase in PKC. HLA-DR therefore transmits signals throughout B lymphocyte ontogeny, although different signaling pathways are initiated in adult vs. fetal vs. malignant B. The lack of intracellular [Ca++]i flux in both cord blood and malignant B lymphocytes may represent a feature of HLA class II signaling at a particular stage of differentiation, although the downregulation of PKC clearly distinguishes between cord blood B and B-CLL.
Assuntos
Apresentação de Antígeno/fisiologia , Linfócitos B/imunologia , Sangue Fetal/imunologia , Antígenos HLA-DR/imunologia , Células-Tronco Neoplásicas/imunologia , Transdução de Sinais , Adulto , Fatores Etários , Agregação Celular , Diferenciação Celular , Divisão Celular , Ativação Enzimática , Sangue Fetal/citologia , Humanos , Recém-Nascido , Isoenzimas/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Fosforilação , Fosfotirosina/análise , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-alfa , Processamento de Proteína Pós-TraducionalRESUMO
The class II human leukocyte antigens (HLA class II) are constitutively expressed on antigen presenting cells (APC) and are essential for peptide presentation to helper T lymphocytes. Signal transduction by HLA class II molecules on B lymphocytes has been described and has been shown in many cases to induce cellular proliferation. However, since signalling via HLA class II can also lead to apoptosis, it has not been clear how the outcome of the signals is determined. We have distinguished two separate HLA class II-initiated pathways leading to either proliferation or apoptosis of primary human B lymphocytes. Proliferation requires new gene transcription and activation of src family tyrosine kinases. In contrast, apoptosis is significantly increased in the absence of transcription/translation. It is dependent on serine/threonine phosphatases and cytoskeletal mobility. An extracellular source of calcium was essential for apoptosis, suggesting the need for sustained high level of intracellular calcium. Activation of iso-enzymes of the protein kinase C family was needed for both pathways. We therefore conclude that HLA class II molecules can initiate two distinct signalling pathways leading to either proliferation or apoptosis of APC.
Assuntos
Apoptose/imunologia , Antígenos HLA-D/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/imunologia , Apoptose/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Cálcio/fisiologia , Ciclo Celular/imunologia , Citoesqueleto/imunologia , Humanos , Interfase/imunologia , Proteína Quinase C/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Baço , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
The usual features of non-Hodgkin lymphomas (NHL) of the mucosa-associated lymphoid tissue (MALT) include monocytoid B-cell proliferation, and chronic inflammatory precursor lesions. Despite a reputation of being indolent, NHL of the MALT may disseminate to other MALT areas, and raise difficult therapeutic questions. We report a case of gastric NHL of the MALT whose evolution, despite an initial surgical treatment considered radical, was noticeable for a well documented breast relapse.
Assuntos
Neoplasias da Mama/secundário , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma não Hodgkin/patologia , Neoplasias Gástricas/patologia , Adulto , Feminino , HumanosRESUMO
HLA class II molecules, expressed on the surface of antigen-presenting cells, are responsible for the presentation of antigen-derived peptides to CD4+ helper T lymphocytes. Signaling via these molecules initiates the generation of second messengers leading to programed cell death (PCD) of activated B lymphocytes. The present study examined the mechanism of HLA class II-mediated apoptosis and describes the essential role of the molecule Fas and its ligand (FasL). FasL was expressed in B lymphocytes after stimulation via HLA class II or with phorbol esters. Expression of FasL protein was significantly increased in 50% of B lymphocytes after stimulation via HLA class II, and the level of FasL mRNA was also increased either by activation with phorbol esters and ionomycin or by signaling via HLA class II. Although HLA class II signaling did not change the expression of the Fas molecule, it did lead to increased sensitivity to Fas-mediated apoptosis. The crucial role of Fas/FasL interactions was confirmed by the absence of cell death via HLA class II in B cells lacking Fas expression, and by the significant inhibition of HLA class II-mediated apoptosis in the presence of either an antagonistic anti-Fas or anti-FasL antibody. These data demonstrate FasL expression on activated human B lymphocytes and support the idea that antigen presentation could contribute to the regulation of lymphocyte populations via Fas and FasL interactions.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/fisiologia , Glicoproteínas de Membrana/metabolismo , Receptor fas/metabolismo , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Proteína Ligante Fas , Humanos , Ligantes , Ativação Linfocitária , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , RNA Mensageiro/biossíntese , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Receptor fas/imunologiaRESUMO
HLA class II molecules are constitutively expressed on human B lymphocytes and are induced on human T lymphocytes after activation, through which signal transduction via these molecules has been extensively described. We have observed cell death of as many as 60% after stimulation of lymphocytes via HLA class II DR molecules, but not via DP or DQ. Propidium iodide fluorescence, DNA fragmentation and morphology of the dead cells were examined. The reported cell death was very rapid, and independent of Fc receptors and of complement. A morphologically distinct sub-population of activated B cells was sensitive to HLA-DR mediated death, while resting B lymphocytes from the same donor did not die as a result of HLA class II mediated stimulation. Death via HLA-DR was distinguishable from necrosis. Cytoskeletal integrity, serine/threonine phosphatase activity and endonucleases were required for the pathway leading to HLA-DR mediated death. The 'ladder' pattern of DNA fragmentation which typically characterizes apoptosis was not observed, despite the observation of cell and nuclear shrinkage normally associated with apoptosis. These data suggest that HLA class II mediated death is a means of rapidly removing either T or B lymphocytes which have already served their role in the immune response, thereby avoiding the inflammatory responses associated with necrosis and concentrating the ligands for new TCR and/or CD4 interactions.
Assuntos
Apoptose/imunologia , Linfócitos B/fisiologia , Antígenos HLA-DR/fisiologia , Linfócitos T/fisiologia , Anticorpos Monoclonais , Apoptose/genética , Linfócitos B/ultraestrutura , Linhagem Celular , DNA/metabolismo , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Linfócitos T/ultraestruturaRESUMO
Development of naive T cells into type 1 (Th1, Tc1) or type 2 (Th2, Tc2) effector cells is thought to be under the control of cytokines. In this study, we show that when both IL-12 and IL-4 are present, murine and human T cell differentiation is regulated by the balance of protein kinase C (PKC) and calcium signaling within T cells. Although both biochemical signals were required for T cell activation via the TCR, altering the balance between them redirected type 1 cells to type 2 and vice versa. Stimulation of calcium signaling or inhibition of PKC favored type 1 differentiation, whereas stimulation of PKC or inhibition of calcineurin resulted in type 2 effectors. Altered peptide ligands induced distinct balances of PKC/calcium signaling and altered Tc1/Tc2 development in TCR-transgenic CD8 T cells. The data suggest novel strategies for manipulation of the immune response in vivo.