RESUMO
We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citocinas/farmacologia , Expressão Gênica , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Peptídeos/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Citocinas/metabolismoRESUMO
BACKGROUND: Induction treatments with anti-thymocyte globulin (ATG) in solid organ transplantation may enhance the efficacy of maintenance immunosuppressive therapy. Since ATG can trigger Fas (CD95) mediated T cell apoptosis, a process antagonized in vitro by corticosteroids, an important issue is whether corticosteroids could interfere with T cell depleting and immunosuppressive activities of ATG. METHODS: MHC mismatched skin allografts were performed on cynomolgus and rhesus monkeys treated with ATG (20 mg/kg) associated or not with 6-methylprednisolone (10 mg/kg). RESULTS: There was no difference between the two immunosuppressive regimens as regards the intensity and duration of peripheral T lymphocyte depletion and the appearance of anti-ATG antibodies. Skin graft survival was increased in monkeys treated with 6-methylprednisolone as compared with ATG alone. CONCLUSIONS: In vivo, corticosteroids do not interfere with ATG ability to induce massive T cell depletion and to delay skin allograft rejection in non-human primates.
Assuntos
Soro Antilinfocitário/uso terapêutico , Imunossupressores/uso terapêutico , Depleção Linfocítica , Metilprednisolona/farmacologia , Transplante de Pele/imunologia , Linfócitos T , Animais , Soro Antilinfocitário/efeitos adversos , Apoptose , Calafrios/etiologia , Calafrios/prevenção & controle , Cólica/etiologia , Cólica/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Feminino , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Isoanticorpos/biossíntese , Macaca fascicularis , Macaca mulatta , Masculino , Metilprednisolona/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transplante Homólogo/imunologia , Receptor fas/imunologiaAssuntos
Imunossupressores/farmacologia , Interferon gama/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Linfócitos T/imunologia , Animais , Soro Antilinfocitário/farmacologia , Células Cultivadas , Humanos , Interferon gama/sangue , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Muromonab-CD3/farmacologia , Ácido Micofenólico/análogos & derivados , Fito-Hemaglutininas , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The mechanisms of action of polyclonal antithymocyte globulins (ATGs) are still poorly understood and the selection of doses used in different clinical applications (prevention or treatment of acute rejection in organ allografts, treatment of graft-versus-host disease, or conditioning for allogeneic stem cell transplantation) remains empirical. Low T-cell counts are usually achieved in peripheral blood during ATG treatment but the extent of T-cell depletion in lymphoid tissues is unknown. METHODS: Experiments were conducted in cynomolgus monkeys using Thymoglobuline at low (1 mg/kg), high (5 mg/kg), and very high (20 mg/kg) doses. RESULTS: ATG treatment induced a dose-dependent lymphocytopenia in the blood and a dose-dependent T-cell depletion in spleen and lymph nodes but not in the thymus, indicating a limited access of ATG to this organ. T-cell apoptosis in peripheral lymphoid tissues was the main mechanism of depletion. Remaining T cells in peripheral lymphoid organs were coated by antibodies and had down-modulated surface expression of CD2, CD3, CD4, and CD8 molecules, whereas their responsiveness in mixed leukocyte reaction was impaired. The survival of MHC-mismatched skin and heart allografts was prolonged in a dose-dependent fashion, despite the occurrence of a strong anti-ATG antibody response resulting in the rapid clearance of circulating ATGs. CONCLUSION: The results indicate that T-cell depletion is achieved rapidly and primarily in peripheral lymphoid tissues at high ATG dosage. Short ATG treatments could therefore be clinically evaluated when major peripheral T-cell depletion is required.
Assuntos
Soro Antilinfocitário/imunologia , Imunossupressores/farmacologia , Macaca fascicularis/imunologia , Animais , Antígenos de Superfície/fisiologia , Soro Antilinfocitário/uso terapêutico , Contagem de Células Sanguíneas , Relação Dose-Resposta Imunológica , Regulação para Baixo , Feminino , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Transplante de Coração/imunologia , Masculino , Coelhos , Transplante de Pele/imunologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA), is a potent immunosuppressive agent used in clinical organ transplantation. MPA preferentially inhibits the type II isoform of inosine monophosphate dehydrogenase, depletes GTP, suppresses transfer of mannose and fucose to glycoproteins, and prevents lymphocyte proliferation in vivo. Whether MMF can also delete activated T cells in vivo by triggering an apoptotic signal was addressed in this study. To this end we analyzed the activity of MMF in mice injected with the bacterial superantigen staphylococcal enterotoxin B (SEB). Superantigens bind to MHC class II molecules without requirement for processing, and activate subsets of CD4+ and CD8+ T cells whose T cell receptor beta chains express Vbeta family-specific homologous sequences. This model that shares several features with direct allorecognition has the unique advantage of allowing a precise monitoring of activated T cells. METHODS: BALB/c mice treated with MMF (100 mg/kg/ day) or vehicle were injected with SEB. Serum cytokines, CD4+ and CD8+ Vbeta8+ cells were monitored in blood and lymphoid tissues, and apoptosis was determined by externalization of membrane phosphatidyl serine, double strand DNA breaks, and expression of B220 antigen by Vbeta8+ cells. RESULTS: MMF treatment decreased tumor necrosis factor alpha, interferon gamma, and interleukin-10 secretion induced by SEB. It did not modify other early activation events (blast transformation, CD69 and CD25 expression) but completely inhibited SEB-induced expansion of Vbeta8+ cells by inducing apoptosis of SEB-reactive T cells. A similar effect was observed in CD95-ligand-deficient mice. Repeated SEB injections associated with MMF resulted in a marked decrease of CD8+ Vbeta8+ T cells. SEB-induced increase of Vbeta8+ thymocytes was not prevented by MMF treatment. CONCLUSION: Results obtained in this in vivo model suggest that MMF treatment may induce deletion of activated peripheral T cells and decrease early cytokine responses.
Assuntos
Apoptose/efeitos dos fármacos , Enterotoxinas/farmacologia , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapêutico , Superantígenos/farmacologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Proteína Ligante Fas , Ativação Linfocitária/imunologia , Depleção Linfocítica , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Methotrexate (MTX), a folate antagonist with multiple enzymatic targets, is used in the treatment of malignancies as well as in autoimmune and chronic inflammatory diseases, and ZD1694 (tomudex), a water-soluble quinazoline specific inhibitor of thymidylate synthase (TS), is used in the treatment of adenocarcinomas. In this study, we investigated the effects of these folate analogues on superantigen (SAg)-reactive peripheral T cells in vivo. In BALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokine secretion, IL-2R (CD25) expression and early deletion of a fraction of SEB-reactive V(beta)8(+) T cells were not impaired by either MTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTX and tomudex prevented V(beta)8-selective T cell expansion and accelerated their peripheral elimination. Administration of thymidine (500 mg/kg/12 h) completely abrogated this effect, indicating that inhibition of TS but not that of other folate-dependent enzymes was the main mechanism involved. Furthermore, a marked increase of apoptotic cells restricted to the V(beta)8(+) T cell subset indicated that proliferation inhibition was associated with apoptosis. In contrast with peripheral V(beta)8(+) T cell deletion, MTX and tomudex did not prevent the increase of V(beta)8(+) thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lpr mice further demonstrated that deletion of V(beta)8(+) T cells induced by folate analogues was independent of Fas-Fas ligand interaction. Our results provide evidence that folate analogues may selectively delete dividing peripheral T cells through TS inhibition, but do not interfere with other events triggered by SAg.
Assuntos
Deleção Clonal/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Metotrexato/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Superantígenos/imunologia , Subpopulações de Linfócitos T/imunologia , Timidina/antagonistas & inibidores , Receptor fas/fisiologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Enterotoxinas/administração & dosagem , Proteína Ligante Fas , Antagonistas do Ácido Fólico/farmacologia , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/farmacologia , Injeções Intraperitoneais , Injeções Intravenosas , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Depleção Linfocítica , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Quinazolinas/farmacologia , Staphylococcus aureus/imunologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/metabolismo , Tiofenos/farmacologia , Timidina/biossíntese , Timidilato Sintase/antagonistas & inibidoresRESUMO
Autoimmune diseases include systemic or tissue-specific disorders induced by antibodies or CD8+ cytotoxic T lymphocytes and(or) CD4+ T lymphocytes that produce pro-inflammatory type-1 cytokines (gamma interferon and tumour necrosis factor). Some may be triggered by infectious agents. Search for predisposition genes is rapidly progressing. Disease-associated autoantibodies production requires T-B cell co-operation. Current research is focused on the characterisation of peptides processed from autoantigens and that of T lymphocytes involved in the initiation of the process. Those studies might eventually lead to immuno-intervention based on antigen administration aiming at the restoration of natural tolerance.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/fisiopatologia , Linfócitos T/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/fisiologia , Humanos , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The utilization of accurate and sensitive methods for the measurement of cytokines in body fluids is prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokine quantitation. Bioassays historically preceded immunoassays, which are now very popular, but there is a need for standardization. Nevertheless, due to the local effects of cytokines, the study of their blood levels is of limited value for an understanding of the pathophysiology of these mediators. This explains the development of alternative approaches to assess the ability of cells to produce cytokines. These include the Enzyme-Linked Immuno Spot Assay (ELISPOT), the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction, appear to be complementary tools for a better understanding of the biology of cytokines. Selected examples of possible clinical applications related to infectious diseases, cancer, autoimmune diseases, allergy, transplantation and preclinical evaluation of drugs and biotechnology products are given.
Assuntos
Citocinas/análise , Diagnóstico , Líquidos Corporais/química , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: On September 1998, the first human hand allograft was successfully performed in Lyon. METHODS: A 48-year-old white man who had suffered accidental amputation of the arm in 1984, received a forearm and hand allograft from a 42-year-old white male cadaveric heart-beating donor. Immunosuppressive therapy included prednisone, mycophenolate mofetil, FK506, and antithymocyte globulins. Sequential skin biopsies were taken from the grafted limb and examined (immuno)histologically to detect a possible graft rejection and to evaluate the structural integrity of the skin of the allograft. RESULTS: The skin showed histologically a normal appearance, except on days 57 and 63, when a mononuclear perivascular cell infiltrate was observed in the dermis; this appeared concomitantly with erythematous lesions of the skin that developed after a slight decrease of the immunosuppressive treatment. These changes were considered as signs of graft rejection, and were reversed by an increase of the immunosuppressive treatment. No skin necrosis was seen at any time. Immunohistochemically, the main cell types of the skin were present throughout the study. From day 77 onward the epidermis of the grafted hand harbored some epidermal Langerhans cells of recipient's origin. CONCLUSION: This study shows that the skin of the hand allograft maintains overall a normal histological structure and contains most essential cell types, including cells of recipient origin, such as Langerhans cells. Furthermore, it shows that in this system of composite tissue transplantation, skin biopsies may reveal a starting graft rejection, before the appearance of clinically obvious lesions.
Assuntos
Transplante de Mão , Mãos/patologia , Pele/patologia , Adulto , Biópsia , Relação Dose-Resposta a Droga , Eritema/etiologia , Rejeição de Enxerto/complicações , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/patologia , Humanos , Imuno-Histoquímica , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Células de Langerhans/patologia , Masculino , Pessoa de Meia-Idade , Cuidados Pós-OperatóriosRESUMO
The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Daunorrubicina/farmacologia , Doxorrubicina/farmacologia , Depleção Linfocítica , Linfócitos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Linfócitos B/citologia , Camptotecina/farmacologia , Células Cultivadas , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Fase G1 , Humanos , Linfonodos/imunologia , Linfócitos/citologia , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fase de Repouso do Ciclo Celular , Baço/imunologia , Linfócitos T/citologia , Timo/imunologiaRESUMO
The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.
Assuntos
Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Linfócitos/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Células Cultivadas , Humanos , Ativação Linfocitária , Transdução de Sinais/efeitos dos fármacos , Inibidores da Topoisomerase I , Receptor fasAssuntos
Apoptose , Imunossupressores/farmacologia , Ativação Linfocitária , Linfócitos T/imunologia , Receptor fas/imunologia , Animais , Apoptose/efeitos dos fármacos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Receptor fas/efeitos dos fármacosAssuntos
Soro Antilinfocitário/farmacologia , Apoptose/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Glicoproteínas de Membrana/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologia , Animais , Células Cultivadas , Proteína Ligante Fas , Humanos , Imunossupressores/farmacologia , Interleucina-2/farmacologia , Ionomicina/farmacologia , Linfócitos/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.
Assuntos
Apoptose , Deleção Clonal/imunologia , Imunossupressores/farmacologia , Metotrexato/farmacologia , Linfócitos T/efeitos dos fármacos , Adenosina/farmacologia , Artrite Reumatoide/sangue , Ciclo Celular , Células Cultivadas , Meios de Cultura , Antagonistas do Ácido Fólico/farmacologia , Humanos , Leucócitos Mononucleares , Ativação Linfocitária , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologia , Fase S , Linfócitos T/citologia , Linfócitos T/imunologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidoresRESUMO
Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells.
Assuntos
Anticorpos Monoclonais/imunologia , Apoptose/imunologia , Antígenos CD2/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Receptor fas/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Proteína Ligante Fas , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/patologiaRESUMO
Polyclonal horse antilymphocyte and rabbit antithymocyte globulins (ATGs) are currently used in severe aplastic anemia and for the treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs activate the human classic complement pathway and induce lysis of both resting and phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells. At low, submitogenic, concentration ATGs induce antibody-dependent cell cytotoxicity of PHA-activated cells, but not resting cells. They also trigger surface Fas (Apo-1, CD95) expression in naive T cells and Fas-ligand gene and protein expression in both naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and rapamycin. The data suggest that low doses of ATGs could be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.
Assuntos
Anticorpos/imunologia , Apoptose/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Receptor fas/imunologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Coelhos , Linfócitos T/patologiaRESUMO
We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.
Assuntos
Apoptose , Caspases , Ceramidas/biossíntese , Cisteína Endopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos T/imunologia , Receptor fas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Anticorpos Monoclonais/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Caspase 1 , Caspase 3 , Inibidores de Cisteína Proteinase/farmacologia , Citocalasinas/farmacologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Norbornanos , Ácido Okadáico/farmacologia , Oligopeptídeos/farmacologia , Força Próton-Motriz/efeitos dos fármacos , Transdução de Sinais , Tiocarbamatos , Tionas/farmacologiaRESUMO
In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the alpha1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the alpha1 (B9.12.1), alpha2 (W6/32), or alpha3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas- and HLA class I-mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab' fragments. The data indicate that MoAb90 and YTH862 directed against the alpha1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.
Assuntos
Apoptose/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/patologia , Receptor fas/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Humanos , Ratos , Transdução de Sinais/imunologiaRESUMO
We analyzed herein whether antibodies to HLA class I alpha1 domain, which trigger apoptosis of activated T cells, may also control the growth/survival of human B lymphocytes. Addition of monoclonal antibody (MoAb) 90 (mouse IgG1) or YTH862 (rat IgG2b) was found to strongly inhibit the proliferation of CD40-activated total tonsil B cells as well as that of purified naive, germinal center, and memory B-cell subsets. This inhibitory effect was not prevented by addition of B-cell tropic factors, such as interleukin-2 (IL-2), IL-4, and IL-10, and was a result of induced B-cell apoptosis as shown by using a TUNEL assay and DNA electrophoresis. In contrast, engagement of another epitope of the alpha1 domain, as well as that of the alpha2 and alpha3 domains by specific anti-HLA class I MoAbs, failed to inhibit DNA synthesis and to induce apoptosis of CD40-activated B cells. As recently reported for acquisition of sensitivity to Fas (APO-1/CD95) -dependent apoptosis, susceptibility to MoAb90-and YTH862-induced death was restricted to CD40-activated B cells, because resting and anti-IgM-activated B cells did not undergo apoptosis after HLA class I engagement. Moreover, ligation of the B-cell receptor protected CD40-activated B cells from both HLA class I- and Fas-mediated growth inhibition and apoptosis. Taken together, these results show that engagement of the alpha1 domain of HLA class I induces apoptotic cell death of CD40-activated, but not of antigen-activated B cells, and would, therefore, suggest a possible role for HLA class I molecules in the control of B-cell homeostasis.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/imunologia , Linfócitos B/imunologia , Antígenos CD40/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Animais , Apoptose/efeitos dos fármacos , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/citologia , Linfócitos B/fisiologia , Células Cultivadas , Humanos , Imunoglobulina G , Células L , Camundongos , Tonsila Palatina/imunologia , Ratos , Transfecção , Receptor fas/biossínteseRESUMO
Antibody interaction with a specific epitope of the HLA class I alpha1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor-alpha. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I-mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I alpha1 domain (mAb9O, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)-activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin-2 (rIL-2). Blocking the IL-2 pathway by cyclosporin A, FK506, rapamycin, anti-IL-2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL-2 and, to a lesser extent, IL-4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL-7 and recombinant interferon-gamma restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I-mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL-2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I-mediated apoptosis depends on both activation signals and IL-2 or IL-4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen-activated peripheral T cells in vivo, provided that the IL-2 pathway is not blocked by other immunosuppressive agents.