RESUMO
Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met alpha-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential.
Assuntos
Fígado Gorduroso/fisiopatologia , Hepatócitos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Receptor fas/fisiologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Colágeno/metabolismo , Fígado Gorduroso/patologia , Humanos , Imuno-Histoquímica , Células Jurkat , Cinética , Neoplasias Hepáticas , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-met , Receptor fas/isolamento & purificaçãoRESUMO
To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.
Assuntos
Baculoviridae/genética , Bombyx/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Receptor fas/genética , Receptor fas/isolamento & purificação , Animais , Bovinos , Clonagem Molecular , Expressão Gênica , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Trombina/metabolismo , Receptor fas/metabolismoRESUMO
Immunohistochemical methods were used to search for Fas receptor/Fas ligand system involvement in multiple sclerosis (MS) white matter brain lesions. We found large numbers of Fas ligand (Fas-L)-bearing cells present in two acute lesions and 12 of 16 chronic MS lesions, and very few positive cells in non-inflammatory controls. Four of six brains from non-MS neuropathologic conditions associated with inflammation and white matter disease were, however, also positive for Fas-L. Double staining with cell-specific markers revealed that the pattern of ligand-positive cells in chronic MS lesions was complex and composed of several different cell types which were primarily resident glial cells with a small overlay of macrophages. Fas/APO 1 (CD95) receptor expression in MS tissue was also evaluated and marked upregulation of the receptor was found. In addition, Fas receptor was induced, but to a lesser extent, in numerous control brains. The observations that TUNEL-positive dying cells were present in MS lesions and showed excellent co-localization with Fas-L, indicate that the Fas death system may contribute to plaque pathogenesis and could lead to the development of a new category of therapeutic agents for MS.
Assuntos
Encéfalo/patologia , Morte Celular , Glicoproteínas de Membrana/isolamento & purificação , Esclerose Múltipla/etiologia , Receptor fas/isolamento & purificação , Bancos de Espécimes Biológicos , Proteína Ligante Fas , Histocitoquímica/métodos , Humanos , Imuno-Histoquímica , Neuroglia/química , Neuroglia/patologia , Distribuição Tecidual , Regulação para CimaRESUMO
Apoptosis has been shown to be associated with altered glycosylation patterns and biosynthesis of glycoproteins. A major cell surface receptor involved in the induction of apoptosis is Fas that is activated by binding Fas ligand but can also be activated by binding anti-Fas antibody. In order to determine whether the Fas receptor is glycosylated, the extracellular domain of human Fas (shFas) was expressed as a cleavable fusion protein (shFas-Fc) in HeLa cells. These cells were shown to express activities of glycosyltransferases involved in N- and O-glycan biosynthesis. The secreted shFas-Fc was shown to be a glycoprotein with heterogeneous glycan chains. MALDI mass spectrometry revealed a disperse molecular weight of shFas with an average of 23.4kDa. Western blots of shFas-Fc secreted from tunicamycin treated transfected HeLa cells showed that only N-glycosylated glycoforms were secreted, while the unglycosylated shFas-Fc remained intracellular. The results suggest that both N-glycosylation sites of the extracellular domain of Fas are occupied with large N-glycans that play a role in the expression of the glycoprotein.
Assuntos
Receptor fas/química , Receptor fas/metabolismo , Sequência de Aminoácidos , Apoptose , Glicosídeo Hidrolases/metabolismo , Glicosilação , Células HeLa , Humanos , Lectinas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Polissacarídeos/análise , Polissacarídeos/biossíntese , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Receptor fas/isolamento & purificaçãoRESUMO
The protein BigH3 is a cell-adhesion molecule induced by transforming growth factor-beta (TGF-beta). It consists of four homologous repeat domains known as FAS1 domains; mutations in these domains have been linked to corneal dystrophy. The fourth FAS1 domain was expressed in Escherichia coli B834 (DE3) (a methionine auxotroph) and purified by DEAE anion-exchange and gel-filtration chromatography. The FAS1 domain was crystallized using the vapour-diffusion method. A SAD diffraction data set was collected to a resolution of 2.5 A at 100 K. The crystal belonged to space group P6(1) or P6(5) and had two molecules per asymmetric unit, with unit-cell parameters a = b = 62.93, c = 143.27 A, alpha = beta = 90.0, gamma = 120.0 degrees.
Assuntos
Proteínas da Matriz Extracelular/isolamento & purificação , Fator de Crescimento Transformador beta/isolamento & purificação , Receptor fas/isolamento & purificação , Animais , Cristalização , Cristalografia por Raios X , Drosophila melanogaster , Proteínas da Matriz Extracelular/química , Humanos , Fator de Crescimento Transformador beta/química , Receptor fas/químicaRESUMO
Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
Assuntos
Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Fosfoproteínas/isolamento & purificação , Fosfotirosina/metabolismo , Receptor fas/isolamento & purificação , Receptor fas/metabolismo , Linhagem Celular , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Immunoblotting , Fosfoproteínas/química , Fosforilação , Fosfotirosina/química , Receptor fas/químicaRESUMO
This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.
Assuntos
Apoptose , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/análise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/isolamento & purificação , Transdução de Sinais , Receptor fas/química , Receptor fas/isolamento & purificação , Autoanticorpos/metabolismo , Biotina/metabolismo , Western Blotting , Cromatografia de Afinidade , Humanos , Células Jurkat , Microesferas , Complexos Multienzimáticos/metabolismo , Coloração e Rotulagem , Estreptavidina/metabolismo , Receptor fas/metabolismoRESUMO
An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purification of GST::CD95IC from 32P-labelled L929 cells and cleavage with thrombin revealed that CD95IC was phosphorylated in vivo when produced as a GST fusion protein. Therefore, pLEF may facilitate the mapping of in vivo-modified sites of eukaryotic proteins.
Assuntos
Expressão Gênica/genética , Vetores Genéticos/genética , Glutationa Transferase/genética , Receptor fas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/metabolismo , Receptor fas/química , Receptor fas/isolamento & purificaçãoRESUMO
The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.
Assuntos
Apoptose , Eosinófilos/fisiologia , Citometria de Fluxo/métodos , Anexina A5/isolamento & purificação , Antígenos de Diferenciação , Células Cultivadas , Fragmentação do DNA , Eosinófilos/efeitos dos fármacos , Eosinófilos/ultraestrutura , Humanos , Luz , Necrose , Fosfatidilserinas/isolamento & purificação , Propídio , Espalhamento de Radiação , Azida Sódica/farmacologia , Coloração e Rotulagem , Receptor fas/isolamento & purificaçãoRESUMO
CD95/Fas/Apo-1 is a cell surface receptor that, upon contact with its ligand, induces cells to die by apoptosis. In view of the importance of Fas receptor (FasR) in immunologic tolerance, an immunohistochemical analysis of FasR expression was performed in the lymphoid and certain parenchymal tissues of normal and mutant MRL/lpr mice using a rabbit polyclonal anti-Fas receptor antibody. FasR was expressed by immunoperoxidase (IP) in the cortex and at the corticomedullary junction of the thymus of normal mice. By immunoelectron microscopy FasR was detected on the cell membrane of normal thymocytes. In MRL/lpr mice, FasR protein expression could not be clearly detected. FasR protein expression was not detected in the heart, liver or ovary by IP, presumably reflecting the low number of receptors in these tissues.
Assuntos
Receptores de Superfície Celular/isolamento & purificação , Timo/ultraestrutura , Receptor fas/isolamento & purificação , Animais , Apoptose , Feminino , Tolerância Imunológica , Técnicas Imunoenzimáticas , Linfócitos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/genética , Distribuição Tecidual , Receptor fas/genéticaRESUMO
PROBLEM: Apoptosis has been proposed as a mechanism for maintaining homeostasis in the immune system. Activated lymphocytes are removed by a Fas/FasL-mediated programmed cell death process called activation induced cell death (AICD). The aim of the study was to investigate surface Fas antigen (APO-1, CD95) expression on T lymphocytes and NK cells and also soluble Fas antigen concentrations in pre-eclamptic patients and healthy pregnant women. MATERIALS AND METHODS: Sixteen pre-eclamptic patients and 18 healthy pregnant women were studied. Peripheral blood lymphocytes were isolated, labeled by direct staining with anti-CD95 monoclonal antibodies and analyzed using the flow cytometric method. Furthermore, the concentrations of soluble CD95 molecule in serum of patients with severe pre-eclampsia and women with uncomplicated pregnancy were measured using ELISA method. RESULTS: We found that Fas antigen expression and fluorescence intensity on T CD8+ lymphocytes were higher in patients with severe pre-eclampsia in comparison with healthy pregnant women (P<0.05). Furthermore, the concentrations of soluble CD95 molecule were higher in the group of pre-eclamptic patients when compared to controls (P<0.001). CONCLUSIONS: These findings suggest that T CD8+ lymphocytes in patients with severe pre-eclampsia can be activated. Moreover, higher concentrations of soluble CD95 antigen can suggest altered possibilities to undergo Fas/FasL-mediated activation induced cell death process of lymphocytes in severe pre-eclampsia.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Pré-Eclâmpsia/imunologia , Receptor fas/isolamento & purificação , Adulto , Morte Celular , Proteína Ligante Fas , Feminino , Humanos , Glicoproteínas de Membrana/metabolismo , GravidezRESUMO
Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft-associated proteins are detected. In this report, we demonstrate that by incorporating 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) crosslinking and freezing at -80°C into the membrane raft isolation procedure of HaCaT cells, both membrane raft-associated proteins caveolin-1 and Fas receptor are able to be reproducibly isolated into a single fraction containing the membrane rafts of the cells.
Assuntos
Caveolina 1/isolamento & purificação , Reagentes de Ligações Cruzadas , Congelamento , Microdomínios da Membrana/química , Receptor fas/isolamento & purificação , Fracionamento Celular/métodos , Linhagem Celular , Centrifugação com Gradiente de Concentração , SuccinimidasAssuntos
Apoptose , Transdução de Sinais , Receptor fas/fisiologia , Linhagem Celular , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Focalização Isoelétrica/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , Receptor fas/química , Receptor fas/isolamento & purificaçãoRESUMO
The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during spermatogenesis. The presence of other molecular markers of apoptosis has, however, not been shown. More importantly, differences in these markers have not been investigated in men with normal and abnormal sperm parameters. In this study we examine for the presence of the cell surface protein Fas in ejaculated human spermatozoa. Ejaculated spermatozoa (55 samples) were labeled with anti-human Fas antibody and the number of spermatozoa displaying Fas were counted using a fluorescence-activated cell sorter (FACS). In 30/31 (96.8%) normal males (>20 million sperm per milliliter), less than 10% of the spermatozoa were Fas positive. In contrast, 14/24 (58.3%) oligozoospermic samples (<20 million sperm per milliliter) contained more than 10% Fas-positive spermatozoa. Similar differences were observed in men whose spermatozoa had poor motility and morphology. These results indicate that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are clear differences in molecular markers of apoptosis between males with normal and abnormal sperm parameters. We propose that the presence of Fas-labeled spermatozoa in the ejaculate of these men is indicative of an "abortive apoptosis" having taken place, whereby the normal apoptotic mechanisms have misfunctioned, have been overridden, or have not been completed.
Assuntos
Apoptose , Infertilidade Masculina/etiologia , Espermatozoides/patologia , Receptor fas/isolamento & purificação , Citometria de Fluxo , Humanos , Masculino , Oligospermia/etiologia , Sêmen/citologia , Transdução de Sinais , Contagem de EspermatozoidesRESUMO
OBJECTIVE: The aim of the study was to compare accepted surrogate markers of HIV disease progression with markers of lymphocyte apoptosis in their ability to predict short-term disease progression. METHODS: In all, 40 HIV-positive patients were studied prospectively and observed during follow-up for HIV-related adverse clinical events. Ex vivo apoptosis was measured with the markers CD95 expression, annexin V binding, and Apostain dye uptake by flow cytometry at baseline. Established markers of disease progression (CD4 count, HIV-RNA level, and CD8/38 count), CD8, B-cell, and natural killer (NK) cell counts were determined by standard procedures at baseline and after 6 months. RESULTS: In HIV-infected patients, CD95 expression and annexin V binding showed significantly elevated apoptosis in peripheral blood lymphocytes and all lymphocyte subsets at baseline compared with HIV-negative, healthy controls. Apostain failed to differentiate between HIV-infected patients and healthy controls. HIV-related complications could be predicted by CD4 and CD8/38 counts, but not HIV viral load as assessed by relative operating characteristic (ROC) analysis (CD4, p = .003; CD8/38, p = .031). A similar or even better diagnostic accuracy was found for CD95 expression in total lymphocytes (p<.001), the CD4+ (p = .003) and CD8+ (p = .005) T-cell subsets and for annexin V binding in CD4+ T cells (p = .005). When patients with CD4 counts <200 cells/microl were analyzed separately, only annexin V binding in CD4+ T cells, but none of the other prognostic markers could predict complications (p = .001). CONCLUSION: Determination of annexin V binding on CD4+ T cells may be a useful tool to monitor HIV-infected patients with low (<200 cells/microl) CD4 counts, as it can reliably assess the risk for imminent complications in such patients.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/etiologia , Antígenos de Diferenciação/isolamento & purificação , Apoptose , Infecções por HIV/complicações , HIV-1 , Adulto , Anexina A5/metabolismo , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/isolamento & purificação , Antígenos de Diferenciação de Linfócitos T/isolamento & purificação , Feminino , Previsões , Infecções por HIV/etiologia , Humanos , Células Matadoras Naturais , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Fatores de Risco , Receptor fas/isolamento & purificaçãoRESUMO
BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.
Assuntos
Antígenos de Diferenciação de Linfócitos T/isolamento & purificação , Terapia Antirretroviral de Alta Atividade , Apoptose , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Linfócitos T/imunologia , Adolescente , Fatores Etários , Antígenos CD28/isolamento & purificação , Criança , Pré-Escolar , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Antígenos Comuns de Leucócito/isolamento & purificação , Masculino , Potenciais da Membrana , Mitocôndrias/metabolismo , Fenótipo , Receptor fas/isolamento & purificaçãoRESUMO
Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.
Assuntos
Apoptose , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos , Técnicas de Cocultura , Antígenos de Histocompatibilidade Classe II/genética , Homeostase , Interleucina-2/biossíntese , Linfonodos/imunologia , Camundongos , Camundongos Transgênicos , Mutação , Receptores de Interleucina-2/isolamento & purificação , Baço/imunologia , Receptor fas/isolamento & purificaçãoRESUMO
CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.
Assuntos
Antígenos CD , Apoptose , Linfócitos B/imunologia , Imunoglobulinas/biossíntese , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anexina A5/isolamento & purificação , Antígenos CD20/isolamento & purificação , Antígenos de Diferenciação/isolamento & purificação , Subpopulações de Linfócitos B/imunologia , Separação Celular , Sobrevivência Celular , Regulação para Baixo , Proteína Ligante Fas , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , NAD+ Nucleosidase/isolamento & purificação , Proteínas Recombinantes de Fusão , Solubilidade , Receptor fas/isolamento & purificaçãoRESUMO
Although the identity of the T cells that protect against bacteria in humans remains unknown, it is clear that patients with bacterial infection have reduced numbers of T cells in their blood. Here we have determined whether this T cell loss is a consequence of bacterial antigen-mediated activation-induced cell death (AICD). By flowcytometric analysis, less than 0.3% of freshly isolated T cells from healthy volunteers and patients with severe pneumonia were identified as apoptotic. However, during culture the rate of apoptosis in peripheral blood T cells from patients was 3.0 +/- 0.9%; and increased further in the presence of anti-CD3 (7.4 +/- 2.1%) and decreased when IL-2 was added (4.4 +/- 1.3%). In contrast, no changes were observed in healthy volunteers on addition of anti-CD3. Further, anti-CD3 significantly increased the susceptibility to apoptosis of CD45RO+ T cells, but not CD45RA+ T cells from patients, and the percentage of CD45RO+ T cells in patients was significantly higher than that in healthy volunteers. Flowcytometric analysis revealed the expression level of Fas to be higher in the patients than healthy volunteers. Collectively, these findings demonstrated that bacteria-reactive T cells were more susceptible to AICD and that Fas-FasL pathways of apoptosis were involved. AICD of CD45RO+ T cells, therefore, provides an explanation for the loss of bacteria-reactive T cells during bacterial infection.
Assuntos
Apoptose/imunologia , Antígenos Comuns de Leucócito/imunologia , Infecções Respiratórias/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Interleucina-2/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-2/sangue , Receptor fas/isolamento & purificaçãoRESUMO
Human Fas/Apo-1 is a cell-surface protein that mediates apoptosis upon ligation with Fas ligand. The gene lies on the long arm of chromosome 10, consists of nine exons, and spans more than 26 kb of DNA. We previously reported the presence of a Fas variant mRNA, designated as Fas delta TM, in human peripheral blood mononuclear cells. Fas delta TM is generated by alternative splicing of the intact exon 6, which encodes the Fas transmembrane domain. In the present study, we describe three novel forms of Fas mRNA that are generated by alternative splicing of exons 3, 4, 6 and 7. These three mRNA variants undergo a frameshift and produce truncated polypeptides because of the appearance of a stop codon in the altered open reading frame. On activation of the peripheral blood mononuclear cells, a decreased expression of alternatively spliced Fas mRNA species correlated with increased cell-surface expression of Fas. These results suggest that differential expression of alternatively spliced Fas mRNAs may play a role in regulation of Fas function via regulation of the production of the membrane-bound and the soluble, secreted Fas protein products.