RESUMEN
T cells are increasingly used for passive immunotherapy and bone marrow transplantation. Proper ex-vivo management of the cells is important for the desired therapeutic effects. For differentiation into effector cells of the Th1 and Th2 phenotypes, T-cells require signals from IFNγ and IL-4, respectively. Naïve cells have an extremely low expression of the specific receptors that recognize these cytokines, indicating that in order to differentiate, cells need to perceive other signals that will enable them to sense the cytokine milieu. CD43 has been proposed as one of the molecules that make the initial contacts with antigen presenting cells. We report here that in cord blood, adult naïve and total human T cells, CD43 signals induced the expression of both IFNγ and IL-4 receptors, mediate their capping, increased their signaling and augmented differentiation mediated by these receptors. CD43 signals also stimulated the expression of IFNγ and in neonatal cells that of IL-4 as well. These data demonstrate an important role for CD43 signals in T-cell preparedness for differentiation into effector cells.
Asunto(s)
Diferenciación Celular/fisiología , Citocinas/metabolismo , Leucosialina/metabolismo , Linfocitos T/metabolismo , Adulto , Citocinas/genética , Sangre Fetal , Regulación de la Expresión Génica , Humanos , Recién Nacido , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Transducción de Señal , Linfocitos T/citología , Receptor de Interferón gammaRESUMEN
OBJECTIVES: Systemic lupus erythematosus (SLE) is characterized by the production of multiple autoantibodies and also by T-cell dysfunction. CD43 is expressed by most immune cells, is involved in lymphocyte adhesion and activation, and interacts with galectin-1 (Gal-1). The aim of this work was to evaluate the plasma levels of autoantibodies against CD43 and Gal-1 as well as the levels of soluble Gal-1 in SLE Mexican mestizo patients, with the aim of establishing a correlation between these parameters and the clinical profile. METHODS: Serum levels of immunoglobulin (Ig)G autoantibodies against CD43 and Gal-1 and levels of soluble Gal-1 were measured by enzyme-linked immunosorbent assay (ELISA) in 55 patients with SLE and 71 healthy controls. RESULTS: We found significantly enhanced titres of anti-CD43 and anti-Gal-1 antibodies in sera from SLE patients compared to controls. In addition, the serum levels of Gal-1 were significantly higher in SLE patients than in healthy individuals. However, we could detect no correlation of these parameters with disease activity [using the Mexican Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI)], age, or a variety of different clinical or laboratory features. Similarly, no significant correlation with immunosuppressive or glucocorticoid therapy was observed. By contrast, a significant association was found between anti-CD43 titres and time of disease evolution, complement levels, and the presence of anti-Gal-1 antibodies. CONCLUSIONS: As CD43 and Gal-1 participate in modulating the immune system, we suggest that the presence of autoantibodies against these molecules may contribute to the immune deregulation observed in SLE.
Asunto(s)
Autoanticuerpos/sangre , Galectina 1/inmunología , Leucosialina/inmunología , Lupus Eritematoso Sistémico/inmunología , Adolescente , Adulto , Factores de Edad , Biomarcadores/sangre , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Galectina 1/sangre , Humanos , Leucosialina/sangre , Lupus Eritematoso Sistémico/diagnóstico , Masculino , Pronóstico , Valores de Referencia , Medición de Riesgo , Índice de Severidad de la Enfermedad , Factores Sexuales , Adulto JovenRESUMEN
OBJECTIVE: To assess the expression and function of the receptor for interleukin-10 (IL-10R) in immune cells from patients with systemic lupus erythematosus (SLE). METHODS: We assessed the expression and function of IL-10R in peripheral blood mononuclear cells (PBMCs) from 19 SLE patients and 15 healthy controls. The expression of IL-10R was assessed by flow cytometry, and the function of this receptor was determined by analysing both the activation of Jak-1, Tyk-2, Stat-1, and Stat-3 (Western blot) and the induction of gene expression (cDNA array test of 242 genes of cytokines, apoptosis and intracellular signalling) upon stimulation with IL-10. RESULTS: We found similar levels of IL-10R expression in SLE patients and controls. In addition, variable levels of Jak-1, Tyk-2, Stat-1, and Stat-3 activation were induced by IL-10 in PBMCs from SLE patients and controls, with no significant differences in protein phosphorylation or kinetics of activation. However, clear-cut differences in the gene expression induced through IL-10R were observed in SLE patients and controls, mainly in the genes involved in apoptosis and those encoding for cytokines and their receptors. CONCLUSIONS: Our data suggest that despite normal levels of IL-10R expression, and an apparent lack of abnormalities in the intracellular signals induced through this receptor, immune cells from SLE patients exhibit an aberrant pattern of gene expression induced through the IL-10R.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Receptores de Interleucina-10/metabolismo , Adolescente , Adulto , ADN/genética , Femenino , Regulación de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/patología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Interleucina-10/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , TYK2 Quinasa/genética , TYK2 Quinasa/metabolismoRESUMEN
Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll-like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)-293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co-expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of interleukin (IL)-10, IL-12p40, tumour necrosis factor (TNF)-alpha, and IL-8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2-/-) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild-type control and TLR2-/- animals succumbing to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.
Asunto(s)
Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Peptidoglicano/inmunología , Fosfolípidos/inmunología , Receptores de Superficie Celular/inmunología , Receptores Inmunológicos/inmunología , Animales , Línea Celular , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12 , Interleucina-8/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Monocitos/inmunología , FN-kappa B/metabolismo , Subunidades de Proteína/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
When activated, CD4(+) T cells differentiate into two major sub-populations differing in their profiles of secreted cytokines. Type One, or TH1, cells secrete IL-2, IFNgamma, and TNFbeta and mediate a cellular immune response. Type Two, or TH2, cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13 and potentiate a humoral response. The nature of any specific immune response depends on the interaction of antigen-presenting cells and T cells. The role of antigen-presenting cells is to respond to the nature of the immune challenge and signal differentiation of CD4(+) T cells. A number of factors are involved in the effector phenotype of T cells-nature and affinity of antigen, co-receptors signals, and cytokine environment. T-cell differentiation is a complex process comprising four defined developmental stages: activation of particular cytokine genes, commitment of the cells, silencing of the opposing cytokine genes, and stabilization of the phenotype. In each of these stages, the cells respond to the products of many signaling cascades from many membrane-bound receptors. The stages in development are mediated by different molecular mechanisms, involving control of gene expression and chromatin remodeling. This review centers on the factors, cellular interactions, and molecular mechanisms involved in the maturation of naïve CD4(+) T lymphocytes into fully effector cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD/fisiología , Diferenciación Celular , Citocinas/fisiología , Activación de Linfocitos , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.
Asunto(s)
Antígenos CD , Proteínas de Ciclo Celular , Proteínas Oncogénicas de Retroviridae/metabolismo , Sialoglicoproteínas/metabolismo , Linfocitos T/metabolismo , Proteínas 14-3-3 , Anticuerpos Monoclonales , Activación Enzimática , Genes Reporteros , Humanos , Células Jurkat , Leucosialina , Activación de Linfocitos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Oncogénica v-cbl , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteína Quinasa C/metabolismo , Subunidades de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Agregación de Receptores , Proteínas Oncogénicas de Retroviridae/inmunología , Serina/genética , Serina/metabolismo , Sialoglicoproteínas/inmunología , Transducción de Señal , Linfocitos T/inmunología , Transfección , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Most dramatic examples of actin reorganization have been described during host-microbe interactions. Plasticity of actin is, in part, due to posttranslational modifications such as phosphorylation or ubiquitylation. Here, we show for the first time that actins found in root nodules of Phaseolus vulgaris are modified transiently during nodule development by monoubiquitylation. This finding was extended to root nodules of other legumes and to other plants infected with mycorrhiza or plant pathogens such as members of the genera Pseudomonas and Phytophthora. However, neither viral infections nor diverse stressful conditions (heat shock, wounding, or osmotic stress) induced this response. Additionally, this phenomenon was mimicked by the addition of a yeast elicitor or H2O2 to Phaseolus vulgaris suspension culture cells. This modification seems to provide increased stability of the microfilaments to proteolytic degradation and seems to be found in fractions in which the actin cytoskeleton is associated with membranes. All together, these data suggest that actin monoubiquitylation may be considered an effector mechanism of a general plant response against microbes.
Asunto(s)
Actinas/metabolismo , Plantas/metabolismo , Plantas/microbiología , Ubiquitina/metabolismo , Actinas/química , Fabaceae/metabolismo , Fabaceae/microbiología , Fijación del Nitrógeno , Phaseolus/metabolismo , Phaseolus/microbiología , Phytophthora/patogenicidad , Enfermedades de las Plantas/microbiología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Pseudomonas/patogenicidad , SimbiosisRESUMEN
Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal peripheral T cells was sufficient to induce interleukin-2, CD69, and CD40-L gene expression, without requiring signals provided by additional receptor molecules. This response was partially inhibited by cyclosporin A and staurosporine, suggesting the participation of both the Ca(2+) and the protein kinase C pathways in CD43 signaling. Consistent with the transient CD43-dependent intracellular Ca(2+) peaks reported by others, signals generated through the CD43 molecule resulted in the induction of NF-AT DNA binding activity. CD43-dependent signals resulted also in AP-1 and NFkappaB activation, probably as a result of protein kinase C involvement. AP-1 complexes bound to the AP-1 sequence contained c-Jun, and those bound to the NF-AT-AP-1 composite site contained c-Jun and Fos. NFkappaB complexes containing p65 could be found as early as 1 h after CD43 cross-linking, suggesting that CD43 participates in early events of T cell activation. The induction of the interleukin-2, CD69, and CD-40L genes and the participation of AP-1, NF-AT, and NFkappaB in the CD43-mediated signaling cascade implicate an important role for this molecule in the regulation of gene expression and cell function.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares , Sialoglicoproteínas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Ligando de CD40/metabolismo , Inhibidores de la Calcineurina , Calcio/metabolismo , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Interleucina-2/metabolismo , Iones , Células Jurkat , Lectinas Tipo C , Leucosialina , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , FN-kappa B/genética , Factores de Transcripción NFATC , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Estaurosporina/farmacología , Sinaptotagmina I , Sinaptotagminas , Linfocitos T/metabolismo , Factores de Tiempo , Factor de Transcripción AP-1/genética , Factores de Transcripción/genéticaRESUMEN
To initiate a specific immune response, lymphoid cells integrate a variety of signals generated through the orchestrated interaction of multiple cell surface molecules with their counter-receptors. As a result of the specific recognition of the antigen through antigen-specific receptors, and of the monitoring of their particular environment through the so-called coreceptor molecules, lymphoid cells go through elaborate processes of maturation and activation, contributing to the plasticity and sensitivity of immune response. CD43 is the major sialic acid rich protein on the surface of lymphocytes. However, the specific roles of this protein in different lymphoid cells under normal physiological conditions remain largely unknown. In this review we will mainly focus on the recent advances concerning the functions of this molecule as a coreceptor of different lymphoid cells as well as on the participation of this molecule in different pathologies.
Asunto(s)
Antígenos CD , Sialoglicoproteínas/fisiología , Animales , Adhesión Celular , Diferenciación Celular , Polaridad Celular , Humanos , Leucosialina , Linfocitos/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Transducción de Señal , Síndrome de Wiskott-Aldrich/inmunologíaRESUMEN
CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Antígenos CD , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Sialoglicoproteínas/fisiología , Linfocitos T/fisiología , Tirosina/metabolismo , Células 3T3 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Proteína Adaptadora GRB2 , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Genes fos/genética , Humanos , Interleucina-2/genética , Leucosialina , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transfección/genéticaRESUMEN
Apoptosis has an important role in several key immunological phenomena such as regulation of the immune response, and deletion of auto-reactive cells. This phenomenon is induced following the interaction of several cell membrane receptors with their respective ligands or after cell activation. We have studied the possible effect of signaling through CD50/ICAM-3 and CD69/AIM on apoptosis of peripheral blood lymphocytes. Apoptosis was assessed by both flow cytometry analysis (content of cell DNA and binding to annexin V), and detection of DNA fragmentation by agarose gel electrophoresis. We found that a stimulatory anti-CD50 mAb was able to induce a small but significant degree of apoptosis in resting peripheral blood mononuclear cells from most donors; this effect was dose-dependent and was evident as early as at 12 h, with a maximal induction at 48 h. Studies with T and non-T cells showed that only the former cell population was sensitive to the induction of apoptosis through CD50. Further experiments revealed that the anti-ICAM-3 mAb preferentially induced apoptosis of TCR gamma delta-bearing cells. In addition, we found a significant increase in Cai2+ in PBMC stimulated with an anti-CD50 mAb, suggesting the involvement of this signaling pathway in the induction of apoptosis through this adhesion receptor. In contrast, under our experimental conditions, stimulation through CD69 did not have any effect on the induction of apoptosis on either cultured T lymphoblasts or PMA-stimulated PBMC. Our findings suggest that the interaction of CD50 with its natural ligand LFA-1 results in the induction of apoptosis in a significant fraction of resting PBMC. This phenomenon may be involved in immune regulation, lymphocyte turnover and peripheral deletion of auto-reactive cells.
Asunto(s)
Antígenos de Diferenciación , Apoptosis , Moléculas de Adhesión Celular/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Humanos , Células Jurkat , Lectinas Tipo C , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signal transduction pathways that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in CD43's signal transduction pathway are poorly understood. In the present report we show that activation of both purified T lymphocytes and Jurkat cells, through CD43 cross-linking with the anti-CD43 L10 monoclonal antibody, induced CD43 association to Fyn kinase. This association is mediated by the Src homology 3 (SH3) domain of Fyn, since a glutathione S-transferase-Fyn SH3 fusion protein was able to precipitate CD43 from lysates of CD43-activated T cells. A synthetic peptide containing the SH3 binding sites of p85, located within the amino acid sequence 300ERQPAPALPPKPPKP314, was able to inhibit binding of CD43 to Fyn as well as to the glutathione S-transferase-Fyn SH3 fusion protein. We also provide evidence that upon CD43 cross-linking, Fyn is tyrosine-phosphorylated in a time-dependent manner. Our results suggest that CD43 cross-linking on the T cell surface induces the interaction between CD43 and Fyn, presumably through the Fyn SH3 domain and a putative SH3 binding site in CD43, leading to Fyn tyrosine phosphorylation and signal propagation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Activación de Linfocitos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Linfocitos T/enzimología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Sitios de Unión , Proteínas de Ciclo Celular/química , Membrana Celular/metabolismo , Secuencia de Consenso , Reactivos de Enlaces Cruzados , Citometría de Flujo , Glutatión Transferasa/metabolismo , Humanos , Células Jurkat , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes de Fusión/metabolismo , Dominios Homologos srcRESUMEN
A convenient--simple, sensitive, rapid and reproducible--enzyme immunoassay to measure H-2 particulated and solubilized cellular antigens is described. Cellular antigens were measured by ELISA through the binding of specific biotinylated antibodies and streptoavidin-peroxidase conjugate to cells in suspension. Endogenous peroxidase activity of activated cells was inhibited by addition of sodium azide and H2O2 in acid conditions. The assay proved capable of distinguishing between two cell lines (EL-4/H-2b and P815/H-2d) and even between the cells of three congenic mouse strains (BALB/B, H-2b, BALB/c, H-2d, and BALB/K, H-2k) and was sensitive to as few as 2.5 x 10(4) cells/well. Results were comparable to those obtained with FACS. An inhibition version of this assay was found to be very useful for the detection of H-2 antigens present in whole antigen cells extracts.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Antígenos H-2/análisis , Animales , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Estudios de Evaluación como Asunto , Haplotipos , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Tumorales Cultivadas/inmunologíaRESUMEN
The T-cell glycoprotein CD2 not only contributes to intercellular adhesion but also plays a direct role in T-cell activation. Here we demonstrate that the interaction of CD2 with its ligand lymphocyte function-associated antigen 3 (CD58) is regulated by T-cell receptor-CD3 signaling. T-cell receptor-CD3 crosslinking by specific antigen or monoclonal antibodies rapidly increases the avidity with which cell-surface CD2 binds immunoaffinity-purified CD58. Mutational analysis of the CD2 cytoplasmic domain demonstrates that the carboxyl-terminal asparagine is essential for T-cell receptor-induced changes in CD2 avidity but is not essential for CD2-mediated signaling, establishing that the cytoplasmic portion of CD2 consists of distinct functional domains. Furthermore, cell lines expressing CD2 molecules incapable of avidity regulation exhibit a marked deficiency in an antigen-specific response. Thus, the regulation of CD2 adhesiveness has a profound effect on the ability of CD2 to enhance antigen responsiveness. These observations demonstrate that adhesion strengthening resulting from increased CD2 avidity contributes directly to T-cell responsiveness independently of CD2-mediated signal transduction.
Asunto(s)
Afinidad de Anticuerpos , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Ácido Aspártico , Antígenos CD2 , Complejo CD3 , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , Citoplasma/inmunología , Humanos , Hibridomas/inmunología , Interleucina-2/biosíntesis , Cinética , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
THE protein CD43 (also known as sialophorin, leukosialin, large sialoglycoprotein or gp115) is expressed on the surface of T lymphocytes, monocytes, neutrophils, platelets and some B lymphocytes. Expression of CD43 is deficient and/or defective in the X-chromosome-linked immunodeficiency disorder Wiscott-Aldrich syndrome, suggesting that CD43 might have a role in T-cell activation. We have shown that expression of human CD43 in an HLA-DR-specific murine T-cell hybridoma enhances the antigen-specific response to stimulation by the human lymphoblastoid cell line Daudi, and that Daudi cells bind specifically to purified immobilized CD43. These data indicate that the specific interaction of CD43 with a ligand on the surface of Daudi cells might contribute to T-cell activation. Here we report evidence that intercellular adhesion molecule-1 (ICAM-1, or CD54), is a ligand for CD43.
Asunto(s)
Antígenos CD , Moléculas de Adhesión Celular/metabolismo , Sialoglicoproteínas/genética , Síndrome de Wiskott-Aldrich/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Antígenos HLA-DR/biosíntesis , Humanos , Hibridomas/metabolismo , Molécula 1 de Adhesión Intercelular , Leucosialina , Ligandos , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Ratones , Receptores Inmunológicos/fisiología , Sialoglicoproteínas/deficienciaRESUMEN
CD4 functions to enhance T cell activation by increasing intercellular adhesion and/or by transduction of an intracellular signal. To study the role of human CD4 in T cell activation we have used a murine T cell hybridoma, By 155.16, which produces IL-2 when stimulated by HLA-DR-bearing cells. Previously, we have shown that expression of human CD4 by this hybridoma enhances its ability to produce IL-2 in response to HLA-DR-bearing cells. Furthermore, deletion of the majority of the cytoplasmic domain renders CD4 less efficient at enhancing IL-2 production. We describe studies of a glycolipid-anchored mutant of the CD4 molecule, CD4PI. This mutant is composed of the entire extracellular domain of CD4 anchored to the outlet leaflet of the membrane via a covalent bond to glycosylphosphatidylinositol and, therefore, has no transmembrane or cytoplasmic domains. When expressed in By155.16, CD4PI shows no defect in its ability to increase intercellular adhesion but is unable to augment IL-2 production. These results clearly demonstrate that CD4 enhances T cell activation by mechanisms other than increasing intercellular adhesion.
Asunto(s)
Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/fisiología , Antígenos CD4/química , Adhesión Celular , Línea Celular , Clonación Molecular , Glucolípidos/fisiología , Glicosilfosfatidilinositoles , Humanos , Técnicas In Vitro , Activación de Linfocitos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Fosfatidilinositoles/fisiología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
CD2 (T11, the T cell E receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, is a T cell-specific surface protein that plays an important role in T lymphocyte adhesion, signal transduction, and differentiation. A natural ligand of CD2 is lymphocyte function associated Ag-3 (LFA-3 (CD58)), a widely expressed glycoprotein of 50 to 70 kDa. The physiologic interaction of CD2 with LFA-3 functions to increase intercellular adhesion and plays a role in T cell activation. This interaction, however, in the absence of other stimuli, has not previously been shown to induce intracellular signals such as Ca2+ mobilization or IL-2 production. To investigate whether cAMP may play a role in ligand-triggered CD2-mediated signal transduction, we have studied the ability of purified LFA-3 and anti-CD2 mAb to induce changes in intracellular cAMP content in murine Ag-specific T cell hybridomas that stably express wild-type and mutated human CD2 molecules. By using a RIA sensitive to the femtomolar range and specific for cAMP, we demonstrate that purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3 alone to CD2-expressing hybridoma cells, however, did not stimulate phosphatidylinositol turnover nor IL-2 production. The cytoplasmic domain of CD2 is necessary for these ligand-induced cAMP changes, demonstrating that LFA-3 binding to CD2 transduces a signal to the cell. Experiments using the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine showed that CD2-mediated regulation of cAMP levels occurs primarily by the stimulation of cAMP production rather than by the inhibition of cAMP degradation. These results demonstrate that the interaction of LFA-3 with CD2, in the absence of other stimuli, is capable of initiating intracellular biochemical changes and suggest that CD2/LFA-3 interactions may regulate T cell function at least in part through the generation of intracellular cAMP.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos de Superficie/fisiología , AMP Cíclico/biosíntesis , Glicoproteínas de Membrana/fisiología , Receptores Inmunológicos/fisiología , Linfocitos T/fisiología , Animales , Anticuerpos Monoclonales , Antígenos CD/fisiología , Antígenos CD2 , Antígenos CD58 , Humanos , Hibridomas , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Interleucina-2/biosíntesis , Ratones , Transducción de Señal , TransfecciónRESUMEN
CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.
Asunto(s)
Antígenos CD4/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Animales , Antivirales/farmacología , Sitios de Unión , Infecciones por VIH/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Mutación , Fosfatidilinositoles/análisisRESUMEN
CD43 (sialophorin, leukosialin, leukocyte large sialoglycoprotein), a heavily sialylated molecule found on most leukocytes and platelets, was initially identified as a major glycoprotein of mouse, rat and human T cells. CD43 expression is defective on the T cells of males with the Wiskott-Aldrich syndrome, an X chromosome-linked recessive immunodeficiency disorder. Affected males are susceptible to opportunistic infections and do not respond to polysaccharide antigens, reflecting defects in cytotoxic and helper T-cell functions. Anti-CD43 monoclonal antibodies have a modest costimulatory effect on T cells, natural killer cells, B cells and monocytes, and one such antibody has been shown to activate T cells directly. To investigate a possible physiological role for CD43, a complementary DNA encoding the human protein was introduced into an antigen-responsive murine T-cell hybridoma. We observed that CD43 enhances the antigen-specific activation of T cells and that the intracellular domain of CD43, which is hyperphosphorylated during T-cell activation, is required for this function. We also found that antigen-presenting cells can bind specifically to immobilized purified CD43 and that the binding can be inhibited by liposomes containing CD43 as well as by anti-CD43 monoclonal antibodies.
Asunto(s)
Antígenos CD/inmunología , Activación de Linfocitos , Sialoglicoproteínas/inmunología , Linfocitos T/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Animales , Anticuerpos Monoclonales , Linfocitos B/inmunología , Línea Celular , Membrana Celular/inmunología , Clonación Molecular , Humanos , Leucosialina , Masculino , Ratones , Plásmidos , Sialoglicoproteínas/genética , Transfección , Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Infection by human immunodeficiency virus type-1 (HIV-1) is initiated when its envelope protein, gp120, binds to its receptor, the cell surface glycoprotein CD4. Small molecules, termed N-carbomethoxycarbonyl-prolyl-phenylalanyl benzyl esters (CPFs), blocked this binding. CPFs interacted with gp120 and did not interfere with the binding of CD4 to class II major histocompatibility complex molecules. One CPF isomer, CPF(DD), preserved CD4-dependent T cell function while inhibiting HIV-1 infection of H9 tumor cells and human T cells. Although the production of viral proteins in infected T cells is unaltered by CPF(DD), this compound prevents the spread of infection in an in vitro model system.