RESUMEN
As it has been shown for Mcl-1, Bcl-xl and Bcl-2, proteins of the Bcl-2 family play a crucial role during T-cell development in the thymus. We here show that the expression of the antiapoptotic gene A1 is specifically enhanced at the DN3/DN4 transition and in DP thymocytes that have been positively selected suggesting that A1 expression might be considered as a transcriptional signature of thymocytes that have received pre-TCR or TCR survival signal. Furthermore, we observed that A1-a overexpression in recombination activation gene 1-deficient mice transgenic for the major histocompatibillity complex class I-restricted F5 TCR enhances cell survival of DP thymocytes and permits accumulation of DP cells awaiting positive selection. However, A1-a overexpression has no effect on negative selection. Therefore, our results suggest that A1 plays a specialized role in allowing survival of DP thymocytes and that its role can be distinguished from that of Mcl-1, Bcl-xl and Bcl-2.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T/metabolismo , Timo/metabolismo , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Citometría de Flujo , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Timo/citología , Proteína bcl-X/metabolismoRESUMEN
Following stimulation, primary B cells either directly undergo terminal differentiation to IgM-secreting plasma cells or enter the memory pathway characterized by affinity maturation and isotype switching. Which of the various fates is adopted by B cells is determined by the strength and duration of the antigenic signal, the availability and quality of T cell help and additional signals derived from the germinal center milieu. High rate secretion is correlated with endogenous Blimp-1 levels and can be caused by ectopic expression of Blimp-1. Using cultures of resting primary mouse B cells stimulated in vitro in various combinations with IL-4, anti-mu F(ab')2 or anti-CD40 in the absence or presence of lipopolysaccharide, we show that IgM secretion and the expression of Blimp-1 is either not induced or even suppressed by B cell receptors (BCR) or CD40 ligation and by IL-4. Additional treatment with IL-2 and IL-5 induces Blimp-1 expression and facilitates IgM and IgG1 secretion, which can also be achieved by retroviral transduction of Blimp-1. On the other hand, the drastic increase in membrane IgG1(+) cells with time in cultures treated with IL-4 is greatly diminished in cells forced to express Blimp-1. We conclude that suppression of Blimp-1 by antigen-BCR interaction and T helper cell-dependent CD40 and IL-4 signaling are necessary to facilitate entrance into the memory pathway and to prevent terminal differentiation.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/fisiología , Cambio de Clase de Inmunoglobulina , Interleucina-4/farmacología , Proteínas Represoras , Factores de Transcripción/biosíntesis , Factores de Transcripción/fisiología , Animales , Linfocitos B/efectos de los fármacos , Antígenos CD40/inmunología , Diferenciación Celular , Línea Celular , Células Cultivadas , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Interleucina-2/farmacología , Interleucina-5/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , ARN Mensajero/biosíntesis , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Activation of the transcription factor NF-kappaB is necessary for full expression of tumor necrosis factor alpha (TNF-alpha)-inducible endothelial chemokines and adhesion molecules. However, a detailed analysis regarding contribution of the different NF-kappaB upstream components to endothelial activation has not been performed yet. We employed a retroviral infection approach to stably express transdominant (TD) mutants of IkappaBalpha, IkappaBbeta, or IkappaBepsilon and dominant negative (dn) versions of IkappaB kinases (IKK) 1 or 2 as well as a constitutively active version of IKK2 in human endothelial cells. TD IkappaBalpha, IkappaBbeta, and IkappaBepsilon were not degraded upon TNF-alpha exposure, and each prevented NF-kappaB activation. These TD IkappaB mutants almost completely inhibited the induction of monocyte chemoattractant protein-1, interleukin-8, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression by TNF-alpha, whereas interferon-gamma-mediated up-regulation of intercellular adhesion molecule-1 and HLA-DR was not affected. Expression of dn IKK2 completely blocked TNF-alpha-induced up-regulation, whereas dn IKK1 showed a partial inhibition of expression of these molecules. Importantly, expression of constitutively active IKK2 was sufficient to drive full expression of all chemokines and adhesion molecules in the absence of cytokine. We conclude that the IKK/IkappaB/NF-kappaB pathway is crucial and sufficient for proinflammatory activation of endothelium.
Asunto(s)
Endotelio Vascular/citología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Western Blotting , Células Cultivadas , Quimiocina CCL2/metabolismo , Selectina E/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Genes Dominantes , Humanos , Quinasa I-kappa B , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/metabolismo , Interleucina-8/metabolismo , Mutación , FN-kappa B/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Retroviridae/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales/citología , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
RelB is an unusual member of the Rel/NF-kappaB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkappaBs, the cytosolic NF-kappaB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkappaBs. However, RelB degradation differs from that of IkappaBs since it is not induced by TNFalpha but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB(DM), site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-kappaB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kappaB activity.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Animales , Línea Celular , Células Cultivadas , Cisteína Endopeptidasas/fisiología , Humanos , Células Jurkat , Cinética , Ratones , Complejos Multienzimáticos/fisiología , FN-kappa B/antagonistas & inhibidores , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Eliminación de Secuencia , Transducción de Señal , Linfocitos T/efectos de los fármacos , Factor de Transcripción ReIB , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
C/EBP transcription factors have been described to control the activity of the human IL-4 promoter. The C/EBP binding sites within the IL-4 promoter overlap with composite NF-AT and AP-1 binding motifs. We show here that similar binding sites are part of the murine IL-4 promoter. Retroviral overexpression of C/EBPbeta in murine EL-4 thymoma cells led to a strong induction of endogenous IL-4 and a reduction in IL-2 and IFN-gamma expression. Similarily, in primary murine T cells C/EBPbeta induction resulted in an increase in IL-4 levels, whereas in human Jurkat T cells a decrease in IL-2 RNA was detected. Like AP-1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP-1, C/EBPbeta does not act in synergy with NF-AT in the induction of the murine IL-4 promoter. Instead, both factors compete in their binding to the P4/Pu-bD site, one of the most important sequence elements of the IL-4 promoter. Whereas NF-AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL-4 promoter activity at specific developmental stages of T cells.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Humanos , Interleucina-4/genética , Ratones , Factores de Transcripción NFATC , Factor de Transcripción AP-1/metabolismoRESUMEN
Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. We show here that cross-linking CD40 stimulates the expression of A1, a member of the anti-apoptotic Bcl-2 family, in primary murine B lymphocytes. CD40-dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line. We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein. A1-transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor. In contrast, constitutive expression of A1 did not abrogate anti-IgM-induced c-myc down-regulation. Consistent with this, A1 did not release anti-IgM-induced cell cycle arrest. Our data indicate that CD40-stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti-IgM antibodies and suggests a protective role for A1 in antigen receptor-mediated apoptosis in B cells.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos CD40/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Homeodominio , Inmunoglobulina M/inmunología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Animales , Anticuerpos Antiidiotipos/inmunología , Linfocitos B/metabolismo , Ciclo Celular/inmunología , División Celular/inmunología , Supervivencia Celular/inmunología , Fragmentación del ADN/inmunología , Proteínas de Unión al ADN/fisiología , Linfoma de Células B , Ratones , Antígenos de Histocompatibilidad Menor , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , ARN Mensajero/biosíntesis , Proteína de Replicación C , Células Tumorales CultivadasRESUMEN
Blimp-1 (B lymphocyte-induced maturation protein 1) is strongly expressed during the late stages of B cell differentiation to immunoglobulin-secreting plasma cells. Overexpression of Blimp-1 in B lymphoma cells has been reported to induce either growth arrest and cell death or Ig secretion and terminal differentiation, depending on the developmental stage of the recipient lymphomas. By using a retroviral expression system we show that Blimp-1-transduced immature WEHI 231 murine B lymphoma cells produce J chain, increased levels of the secretory form of micro heavy chain mRNA and secrete IgM for a short period of time. Concomitantly, they exhibit altered ratios of c-myc/mad4 mRNA levels, a reduction in the expression of the anti-apoptotic bcl-2 family member A1 and a distinct growth disadvantage, followed by cell death. Reintroduction of A1 by retroviral transduction greatly extends the life span of Blimp-1-expressing WEHI 231 cells which continue to secrete IgM. These data suggest that levels of A1 may determine the checkpoint between death and survival of Blimp-1-expressing B cells at different stages of differentiation.
Asunto(s)
Apoptosis/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas de Homeodominio , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/inmunología , Animales , Diferenciación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Cadenas J de Inmunoglobulina/metabolismo , Linfoma de Células B , Ratones , Antígenos de Histocompatibilidad Menor , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína de Replicación C , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Engagement of the antigen receptor on WEHI 231 murine B lymphoma cells leads to growth arrest and induction of apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. At the molecular level, CD40 has been shown to activate nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK). The aim of our present study was to define the stretch of the CD40 cytoplasmic tail responsible for mediating these effects in WEHI 231 cells. Using recombinant retroviruses with the enhanced green fluorescent protein as selection marker we transduced WEHI 231 cells with chimeric molecules consisting of the extracellular and transmembrane region of human CD40 or rat CD4 and selected portions of the murine CD40 tail. Chimeric molecules with cytoplasmic fragments encompassing the "CD40 tumor necrosis factor-associated factor family member interacting motif" (TIM) were able to sustain growth and to uphold NF-kappaB activity as efficiently as the whole intracellular region of CD40. While the potential of the motif relative to the whole cytoplasmic tail was independent of the heterologous part of the chimeras it was strongly influenced by its distance to the membrane. Placing the 17-amino acid stretch of the motif too close to the membrane, i. e. only two or four amino acids apart, destroyed its capacity to mitigate the anti-IgM effect. Activation of SAPK through the chimeric molecules always correlated with their ability to activate NF-kappaB activity and to rescue the cells from apoptosis induced by antigen receptor ligation. Our data indicate that CD40-TIM carries most if not all of the information needed to deliver the signals responsible for sustaining growth in anti-IgM-stimulated WEHI 231 cells.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Antígenos CD40/fisiología , Linfoma de Células B/patología , Proteínas Quinasas Activadas por Mitógenos , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , División Celular , Activación Enzimática , Humanos , Ratones , Proteínas Recombinantes de Fusión/fisiología , Células Tumorales Cultivadas , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
CD40 crosslinking on B cells activates NF-kappaB and stress-activated protein kinase (SAPK) pathways. Since CD40 crosslinking rescues WEHI 231 B cells from anti-IgM-induced apoptosis, those pathways were likely candidates to be involved. Indeed, both signaling cascades predominated in anti-IgM-treated WEHI 231 cells, treated concurrently with anti-CD40 to rescue them from apoptosis. Crosslinking of CD40 activated the NF-kappaB proteins c-Rel and p50, but had no influence on their cytoplasmic steady state level. However, in contrast to-and even in the presence of-anti-IgM-mediated signals, engagement of CD40 resulted in a prolonged nuclear translocation of c-Rel, thereby allowing the formation of active NF-kappaB complexes. Consistent with this, the upstream regulatory element of the c-myc promoter, known to be regulated by NF-kappaB, was differently regulated after BCR ligation vs BCR plus CD40 crosslinking. The level of c-myc RNA was rapidly downregulated after BCR engagement, but persistent in the presence of CD40 signaling.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/inmunología , Inmunoglobulina M/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Proto-Oncogénicas/inmunología , Transducción de Señal/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/citología , División Celular/inmunología , Línea Celular , Humanos , Activación de Linfocitos , Proteínas Proto-Oncogénicas c-rel , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
The B cell-associated surface molecule CD40 plays a key role in T cell-dependent B cell maturation, as individuals with defects in either CD40 or its ligand are impaired in immunoglobulin isotype class switching and germinal center formation. CD40 signaling activates downstream effectors, including the tyrosine protein kinase, Lyn, the phosphatidylinositol-3-kinase (PI-3 kinase), and the transcription factor, NF-kappa B. In this study, we demonstrate that stress-activated protein kinases (SAPK) are activated after CD40 cross-linking on various B cell lines or human tonsillar B cells. The activation is rapid and transient and is mediated through a cyclosporin A-insensitive pathway. Furthermore, this signaling pathway appears not to rely on protein kinase C. While CD40 ligation strongly activates the SAPKs (up to 25-fold), it does not affect members of the mitogen-activated protein kinase family (MAPK; ERK1 and ERK2). Consistent with these data, CD40 signals up-regulate c-jun but not c-fos mRNA and alter the transcription factor ATF2 but not the Raf-1 protein. In summary, CD40 signaling preferentially induces SAPK but not MAPK.
Asunto(s)
Linfocitos B/enzimología , Antígenos CD40/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclosporina/farmacología , Activación Enzimática , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ésteres del Forbol/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Transducción de SeñalRESUMEN
The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/inmunología , FN-kappa B/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Antígenos CD40 , Línea Celular , Proteínas de Unión al ADN/fisiología , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Oxidación-Reducción , Tonsila Palatina/citología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/inmunología , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiología , TransfecciónRESUMEN
T cells provide help for B cell antibody production by cell-cell contact and by soluble factors. CD40L is the predominant protein induced on activated T cells which constitutes contact-dependent help, and lack of CD40L or blocking CD40-CD40L interactions leads to severely impaired antibody production. In addition to CD40-CD40L, B and T cells express costimulatory, accessory molecules which amplify T and B cell function and allow for reciprocal dialogues during antigen presentation. Interactions between costimulatory counter-receptors can determine lymphocyte activation or nonresponsiveness, and provides a means for regulating self-tolerance.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/inmunología , Glicoproteínas de Membrana/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Antígenos CD40 , Ligando de CD40 , Comunicación Celular/inmunología , Humanos , Cooperación Linfocítica/inmunología , Transducción de Señal/inmunologíaRESUMEN
When B cells are stimulated with lipopolysaccharide (LPS) they start to proliferate and mature into immunoglobulin (Ig)-secreting cells. Co-stimulation with F(ab')2 fragments of antibodies directed against the B cell antigen receptor leads to an inhibition of Ig secretion but not of proliferation. This effect can be mimicked by phorbol esters alone or by a combination of phorbol esters and the Ca2+ ionophore ionomycin, which activate protein kinase C. Here we report that co-stimulation with phorbol esters and ionomycin differentially affects a group of genes normally up-regulated during the course of LPS-dependent B cell activation. Thus, the mRNA coding for the membrane-bound form of IgM and the interleukin 2 receptor (55-kDa protein) continue to be expressed at the levels typical of LPS-stimulated cells, while the mRNA coding for the secreted form of IgM (mu S) and for the J chain are reduced. The loss of mu S mRNA is attributable to an altered processing behavior with respect to the mu precursor and/or a decreased stability of the mRNA itself.
Asunto(s)
Linfocitos B/fisiología , Genes de Inmunoglobulinas , Receptores de Antígenos de Linfocitos B/fisiología , Animales , Diferenciación Celular , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Cadenas J de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/metabolismo , Ionomicina/farmacología , Activación de Linfocitos , Ratones , Ésteres del Forbol/farmacología , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Receptores de Interleucina-2/genéticaRESUMEN
Resting and activated B lymphocytes were used to study the stability of mu-specific precursor and mature mRNA. Resting cells which predominantly process the mu precursor towards micron do so rather slowly as reflected in a precursor half-life (T1/2) of 1-2 h. The small amount of secreted mu chain mRNA is fairly stable (T1/2 approximately 8 h) compared to membrane mu chain (T1/2 approximately 4 h). After activation the precursor processing is very fast (T1/2 approximately 10 min) and the stability of mu2, which now predominates, increases (T1/2 approximately 16 h) while the half-life of micron remains at about 4 h. The data indicate that normal B cells regulate mu-specific mRNA stability differently from tumor cells of the B cell lineage.