RESUMEN

Macrophages are fundamental for initiation, maintenance, and resolution of inflammation. They can be activated by 'Toll-like receptor' (TLR) engagement, which initiates critical pathways to fight infections. 'Interleukin receptor-associated kinase 2' (IRAK2) is part of the membrane-proximal Myddosome formed at IL-1R/TLRs, but utility and regulation of IRAK2 within is not completely understood. In this study, we addressed the importance of the evolutionary conserved extreme C-terminus of IRAK2 in TLR signaling. The last 55 amino acids lack any known functional domain. The C-terminus deletion mutant IRAK2Δ55 was hypofunctional and disabled to conduct TLR4-inducible NF-κB and ERK2 activation. Accordingly, it could neither fully support subsequent CD40 cell surface expression nor IL-6 and nitric oxide release. Interestingly, IRAK2Δ55 was still capable to bind to 'tumor necrosis factor receptor-associated factor 6' (TRAF6), which is requisite to activate TRAF6 as an E3-ubiquitin ligase for further downstream signaling. However, IRAK-dependent auto-ubiquitination of TRAF6 was impaired, when IRAK2Δ55 was bound. Thus, the conserved last 55 amino acids enable IRAK2 to sustain an optimal TLR response. This knowledge might spark ideas how overshooting inflammatory responses could be modified without blocking the entire immune response.

Asunto(s)

Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Animales , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Células RAW 264.7 , Factor 6 Asociado a Receptor de TNF/inmunología , Receptores Toll-Like/inmunología , Ubiquitinación

RESUMEN

CD4+CXCR5+Foxp3+ T-follicular regulatory (TFR) cells control the germinal center responses. Like T-follicular helper cells, they express high levels of Nuclear Factor of Activated T-cells c1, predominantly its short isoform NFATc1/αA. Ablation of NFATc1 in Tregs prevents upregulation of CXCR5 and migration of TFR cells into B-cell follicles. By contrast, constitutive active NFATc1/αA defines the surface density of CXCR5, whose level determines how deep a TFR migrates into the GC and how effectively it controls antibody production. As one type of effector Treg, TFR cells express B lymphocyte-induced maturation protein-1 (Blimp-1). Blimp-1 can directly repress Cxcr5 and NFATc1/αA is necessary to overcome this Blimp-1-mediated repression. Interestingly, Blimp-1 even reinforces the recruitment of NFATc1 to Cxcr5 by protein-protein interaction and by those means cooperates with NFATc1 for Cxcr5 transactivation. On the contrary, Blimp-1 is necessary to counterbalance NFATc1/αA and preserve the Treg identity. This is because although NFATc1/αA strengthens the follicular development of Tregs, it bears the inherent risk of causing an ex-Treg phenotype.

Asunto(s)

Movimiento Celular/inmunología , Centro Germinal/inmunología , Factores de Transcripción NFATC/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Animales , Movimiento Celular/genética , Ratones , Ratones Transgénicos , Factores de Transcripción NFATC/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética

RESUMEN

Posttranslational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is largely unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is unclear. To this end, we generated a novel transgenic mouse in which SUMO modification of NFATc1 is prevented. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. Elevated IL-2 production in T cells promoted T reg expansion and suppressed autoreactive or alloreactive immune responses. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself, as well as the induced, proliferation-associated survival factor Bcl2A1. Collectively, these data demonstrate that prevention of NFATc1 SUMOylation fine-tunes T cell responses toward lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.

Asunto(s)

Autoinmunidad , Encefalomielitis Autoinmune Experimental/inmunología , Factores de Transcripción NFATC/inmunología , Sumoilación/inmunología , Linfocitos T Reguladores/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/genética , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Factores de Transcripción NFATC/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Sumoilación/genética

RESUMEN

Drug resistance is a significant obstacle in cancer treatment and therefore a frequent subject of research. Developed or primary resistance limits the treatment success of inhibitors of the B cell receptor (BCR) pathway in mantle cell lymphoma (MCL) patients. Recent research has highlighted the role of the nuclear factor-kappa B (NFκB) pathway in the context of resistance to BCR inhibitors in MCL. In this study, we analyzed the dependency of MCL cell lines on NFκB signaling and illustrated the ability of CD40L to activate the alternative NFκB pathway in MCL. This activation leads to independency of classical NFκB signaling and results in resistance to BCR inhibitors. Therefore, ligands (such as CD40L) and their activation of the alternative NFκB pathway have a major impact on the drug response in MCL. Furthermore, this study indicates a protective role for cells expressing specific ligands as microenvironmental niches for MCL cells and underlines the significance of therapeutically targeting alternative NFκB signaling in MCL.

Asunto(s)

Ligando de CD40/metabolismo , Resistencia a Antineoplásicos , Linfoma de Células del Manto/metabolismo , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/metabolismo , Proteínas Recombinantes/farmacología

Asunto(s)

Técnicas de Inactivación de Genes , Antígenos de Histocompatibilidad Menor/genética , Familia de Multigenes , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Muerte Celular , Humanos , Ratones , Mitocondrias/metabolismo , Modelos Biológicos

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls proliferation and survival of myeloid cells including monocytes. Here, we describe a time-dependent licensing process driven by GM-CSF in murine Ly6Chigh and human CD14+ monocytes that disables their inflammatory functions and promotes their conversion into suppressor cells. This 2-step licensing of monocytes requires activation of the AKT/mTOR/mTORC1 signaling cascade by GM-CSF followed by signaling through the interferon-γ receptor (IFN-γR)/interferon regulatory factor-1 (IRF-1) pathway. Only licensing-dependent adaptations in Toll-like receptor/inflammasome, IFN-γR, and phosphatidylinositol 3-kinase/AKT/mTOR signaling lead to stabilized expression of inducible nitric oxide synthase by mouse and indoleamine 2,3-dioxygenase (IDO) by human monocytes, which accounts for their suppressor activity. This study suggests various myeloid cells with characteristics similar to those described for monocytic myeloid-derived suppressor cells, Mreg, or suppressor macrophages may arise from licensed monocytes. Markers of GM-CSF-driven monocyte licensing, including p-Akt, p-mTOR, and p-S6, distinguish inflammatory monocytes from potentially suppressive monocytes in peripheral blood of patients with high-grade glioma.

RESUMEN

In this article, we report the complete coding sequence and to our knowledge, the first functional analysis of two homologous nonclassical MHC class II genes: RT1-Db2 of rat and H2-Eb2 of mouse. They differ in important aspects compared with the classical class II ß1 molecules: their mRNA expression by APCs is much lower, they show minimal polymorphism in the Ag-binding domain, and they lack N-glycosylation and the highly conserved histidine 81. Also, their cytoplasmic region is completely different and longer. To study and compare them with their classical counterparts, we transduced them in different cell lines. These studies show that they can pair with the classical α-chains (RT1-Da and H2-Ea) and are expressed at the cell surface where they can present superantigens. Interestingly, compared with the classical molecules, they have an extraordinary capacity to present the superantigen Yersinia pseudotuberculosis mitogen. Taken together, our findings suggest that the b2 genes, together with the respective α-chain genes, encode for H2-E2 or RT1-D2 molecules, which could function as Ag-presenting molecules for a particular class of Ags, as modulators of Ag presentation like nonclassical nonpolymorphic class II molecules DM and DO do, or even as players outside the immune system.

Asunto(s)

Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Secuencia de Bases , Western Blotting , Separación Celular , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción Genética

RESUMEN

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

Asunto(s)

Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/genética , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Animales , Apoptosis/genética , Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/ultraestructura , Diferenciación Celular/inmunología , Regulación de la Expresión Génica , Inmunidad Humoral , Inmunomodulación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Células Plasmáticas/inmunología , Células Plasmáticas/ultraestructura , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Maturation of high-affinity B lymphocytes is precisely controlled during the germinal center reaction. This is dependent on CD4(+)CXCR5(+) follicular helper T cells (TFH) and inhibited by CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (TFR). Because NFAT2 was found to be highly expressed and activated in follicular T cells, we addressed its function herein. Unexpectedly, ablation of NFAT2 in T cells caused an augmented GC reaction upon immunization. Consistently, however, TFR cells were clearly reduced in the follicular T cell population due to impaired homing to B cell follicles. This was TFR-intrinsic because only in these cells NFAT2 was essential to up-regulate CXCR5. The physiological relevance for humoral (auto-)immunity was corroborated by exacerbated lupuslike disease in the presence of NFAT2-deficient TFR cells.

Asunto(s)

Autoinmunidad/inmunología , Centro Germinal/inmunología , Inmunidad Humoral/inmunología , Factores de Transcripción NFATC/inmunología , Receptores CXCR5/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Análisis de Varianza , Animales , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR5/metabolismo

RESUMEN

Blimp-1 is a master regulator of terminal B cell differentiation and plays a pivotal role in various developmental processes. In addition to full length Blimp-1, a Blimp-1 mRNA lacking exon 7 (Blimp-1Delta7) has been described to occur in murine B cells. The activity and function of the mutant mRNA-encoded protein (Blimp-1Delta7), lacking three crucial zinc fingers necessary for DNA interaction, is completely unknown. Since isoforms of other prdm family proteins affect each other's functions, we wondered whether Blimp-1Delta7 still plays a role in B cells, independent of direct DNA binding. In this study, we found that Blimp-1Delta7 is preferentially expressed in naïve CD19(+) B cells. A fraction of Blimp-1Delta7 migrates to the nucleus, colocalizes with HDAC2 and is found at sites of repressed chromatin, although it does not bind to the Blimp-1 DNA consensus site. Unexpectedly, Blimp-1 and Blimp-1Delta7 homodimerize as well as heterodimerize with each other. Ectopic expression of Blimp-1Delta7 in WEHI 231 cells, a Blimp-1-negative murine lymphoma line, leads to cessation of proliferation and enhancement of apoptosis. Importantly, LPS-induced differentiation is suppressed in the presence of Blimp-1Delta7. This is in agreement with our finding that Blimp-1Delta7 interferes with endogenous Blimp-1 expression. Thus, our data suggest an auto-regulatory mechanism of Blimp-1 activation.

Asunto(s)

Eliminación de Gen , Regulación de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Antígenos CD19/metabolismo , Apoptosis/genética , Comunicación Autocrina/genética , Linfocitos B/metabolismo , Ciclo Celular/genética , Células Cultivadas , Exones , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transfección

RESUMEN

Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin. Recently, we have shown that MCC cells in situ are characterized by a complete absence of mitogen-activated protein kinase (MAPK) pathway signaling, which is preserved in the MCC cell line UISO. Here we present data suggesting that silencing of the MAPK pathway is essential for the survival of MCC cells. Activation of the MAPK pathway could be achieved by inducing a regulatable form of the c-Raf-1 kinase domain in UISO cells. Consequently, MAPK signaling led to morphological changes, loss of actin stress fibers, and induction of apoptosis, which could be prevented by the MAP kinase kinase-specific inhibitor U0126. Hence, despite the fact that activation of the MAPK pathway contributes to oncogenesis in many cancers, it seems to be a negative selection factor for MCC cells. Since ERK phosphorylation was also inducible by the Raf-activating pharmacological agent ZM336372, these results provide new perspectives for potential therapeutics for this highly aggressive tumor.

Asunto(s)

Apoptosis , Carcinoma de Células de Merkel/enzimología , Carcinoma de Células de Merkel/patología , Sistema de Señalización de MAP Quinasas , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Actinas/metabolismo , Línea Celular Tumoral , ADN/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal

RESUMEN

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.

Asunto(s)

Presentación de Antígeno , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Antígenos CD1/genética , Antígenos CD1/inmunología , Antígenos CD1/metabolismo , Antígenos CD1d , Línea Celular , Células Cultivadas , Galactosilceramidas/administración & dosificación , Galactosilceramidas/inmunología , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Inmunofenotipificación , Hígado/citología , Hígado/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Ratas , Ratas Endogámicas F344 , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Especificidad de la Especie

RESUMEN

The transcription factor C/EBPbeta transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPbeta also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPbeta represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPbeta-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPbeta. Whereas sumoylation does not affect the C/EBPbeta-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPbeta and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPbeta.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Genes myc , Interleucina-4/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Proliferación Celular , ADN/genética , ADN/metabolismo , Fase G1 , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Inmunológicos , Regiones Promotoras Genéticas , Supresión Genética , Linfocitos T/citología , Activación Transcripcional

RESUMEN

Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transferred could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis.

Asunto(s)

Apoptosis/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Supervivencia Celular , Células Cultivadas , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo

RESUMEN

Terminal B-cell differentiation is a multi-step process, from short-lived plasmablasts to mature long-lived plasma cells (PC). The anti-apoptotic Bcl-2 family member Bfl-1/A1 plays a critical role in the survival of mature B cells. However, its potential involvement at the later stages of B-cell development remains highly controversial. Our aim was thus to clarify the place of Bfl-1/A1 in the biology of normal PC and in the pathogenesis of multiple myeloma (MM), the major PC dyscrasia. Using gene expression profiling and quantifiable reverse transcription polymerase chain reaction experiments, we found a similar down-regulation of Bfl-1/A1 in both normal immature plasmablasts and mature PC when compared with B cells. In myeloma cells, the level of Bfl-1/A1 was low and Bfl-1/A1 was not a nuclear factor kappaB-inducible gene. Collectively, these data demonstrate that Bfl-1/A1 is not involved in the prolonged survival of normal mature PC, and that Bfl-1/A1 deregulation is not a common oncogenic event in MM. However, overexpression of Bfl-1/A1 by retroviral transduction promoted autonomous survival of an interleukin-6-dependent myeloma cell line and rendered it less sensitive to dexamethasone. Thus, Bfl-1/A1 transduction could be an interesting tool to obtain myeloma cell lines from primary samples and to favour the in vitro generation of antibody-secreting, long-lived normal PC.

Asunto(s)

Apoptosis , Mieloma Múltiple/metabolismo , Células Plasmáticas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Regulación hacia Abajo , Humanos , Interleucina-6/farmacología , Antígenos de Histocompatibilidad Menor , Mieloma Múltiple/patología , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Células Plasmáticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , Células Tumorales Cultivadas

RESUMEN

Human CD23 (the low affinity IgE receptor) is a B cell differentiation marker involved in inflammatory responses. Two isoforms (CD23a and CD23b) are known, which differ only in their cytoplasmic domain. Whereas CD23b expression is specifically induced by IL-4 on B cells and cells of the myeloid lineage, CD23a expression is restricted to B cells. Each isoform is regulated by its own promoter. Pax-5 is a B-cell-restricted transcription factor with an essential role in early and late B cell development. Analyses of the CD23a promoter revealed a Pax-5-binding site, which can compete a high affinity Pax-5-binding site or directly bind Pax-5 protein in electrophoretic mobility shift assays. Introducing mutations into this site abrogates the binding. Expression of Pax-5 in 293 cells resulted in a seven- to tenfold activation of a CD23a core promoter construct. Most importantly, ectopic expression of Pax-5 in the monocytic cell line U-937, which regularly expresses only the CD23b isoform, led to CD23a expression after stimulation with IL-4 and PMA. Our results suggest that Pax-5 is a key regulator of the B-cell-restricted expression of the CD23a isoform.

Asunto(s)

Linfocitos B/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Receptores de IgE/genética , Factores de Transcripción/fisiología , Secuencia de Bases , Sitios de Unión , Humanos , Interleucina-4/farmacología , Datos de Secuencia Molecular , Factor de Transcripción PAX5 , Regiones Promotoras Genéticas , Isoformas de Proteínas , Acetato de Tetradecanoilforbol/farmacología , Células U937

RESUMEN

RelB is an unusual member of the NF-kappaB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-kappaB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-kappaB1 or p52/NF-kappaB2, the precise molecular mechanisms through which it represses NF-kappaB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-alpha-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA.RelB heterodimers that were unable to bind to kappaB sites in vitro strongly suggesting that these newly described NF-kappaB dimers cannot bind DNA. Expression pattern analysis revealed that RelA.RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKbeta. Functional characterization of RelA.RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IkappaB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA.RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-kappaB-dependent gene expression.

Asunto(s)

FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Dimerización , Fibroblastos/metabolismo , Ratones , Factor de Transcripción ReIB , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

A decade after the first description of IL-2-deficient mice, the redundancy of IL-2 as a T cell growth factor is well accepted and the focus of research has shifted to the unexpected multiorgan autoimmunity and inflammation observed in mice lacking components of the IL-2/IL-2R system. So far, a set of defects at the levels of repertoire selection, the generation of suppressive regulatory T cells, T cell homing and clonal contraction via activation induced cell death (AICD) have been documented. We propose that these individual defects jointly contribute to the severe disturbance of T cell homeostasis and self-tolerance underlying the immunopathology of the IL-2 deficiency syndrome.

Asunto(s)

Enfermedades Autoinmunes/fisiopatología , Interleucina-2/fisiología , Animales , Apoptosis , Enfermedades Autoinmunes/genética , Movimiento Celular , Supresión Clonal , Células Dendríticas/inmunología , Homeostasis , Humanos , Tolerancia Inmunológica/fisiología , Interleucina-2/deficiencia , Interleucina-2/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos , Ratones Noqueados , Receptores de Interleucina-2/deficiencia , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/fisiología , Síndrome , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor fas/fisiología

RESUMEN

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.

Asunto(s)

Apoptosis/inmunología , Caspasas/metabolismo , Linfoma de Células B/enzimología , Linfoma de Células B/inmunología , Mitocondrias/enzimología , Receptores de Antígenos de Linfocitos B/fisiología , Animales , Apoptosis/genética , Caspasa 7 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/genética , Caspasas/fisiología , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Fragmentación del ADN/genética , Fragmentación del ADN/inmunología , Activación Enzimática/genética , Activación Enzimática/inmunología , Humanos , Ligandos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Mitocondrias/metabolismo , Mitocondrias/fisiología , Fragmentos de Péptidos/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción Genética , Células Tumorales Cultivadas