RESUMEN
In this study, we showed that the transforming growth factor beta (TGFbeta)-mediated apoptosis of Burkitt's lymphoma BL41 cells is dependent on the BH3-only protein Bim. In contrast to what has been observed with other cell types, TGFbeta activation did not promote Bim upregulation in BL41 cells, but instead resulted in Bim release from the mitochondria. Indeed, Bim levels were high in healthy BL41 cells, in which they dimerized with the Bcl-2-like protein Mcl-1 at the mitochondrial surface. In healthy and TGFbeta-activated BL41 cells, unlike in epithelial cells or hepatocytes, Bim did not associate with Bcl-2 or Bcl-xL. TGFbeta activation of BL41 cells triggered the p38-dependent activation of caspase-8, causing the cleavage of Mcl-1 and the transfer of Bim from the mitochondria to the cytoskeleton. In addition to mitochondrial activation, this relocation of Bim may facilitate the complete demise of a cell death that is beyond the commitment point to apoptosis and may represent a hallmark of the TGFbeta-mediated apoptosis of human lymphoma B cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Proteínas de la Membrana/metabolismo , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Western Blotting , Caspasa 8/metabolismo , Proliferación Celular , Células Cultivadas , Citoesqueleto , Dimerización , Activación Enzimática , Células Epiteliales/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Inmunoprecipitación , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Factor de Crecimiento Transformador beta/genética , Regulación hacia Arriba , Proteína bcl-X/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/inmunología , Proteínas Portadoras/fisiología , Caspasas/fisiología , Proteínas de Unión al ADN/fisiología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptor fas/fisiología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD40/inmunología , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Células Clonales , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Activación Enzimática/inmunología , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Sueros Inmunes/farmacología , Membranas Intracelulares/enzimología , Membranas Intracelulares/inmunología , Péptidos y Proteínas de Señalización Intracelular , Potenciales de la Membrana/inmunología , Mitocondrias/enzimología , Mitocondrias/inmunología , Mitocondrias/metabolismo , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos B/fisiología , Solubilidad , Células Tumorales CultivadasRESUMEN
Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.
Asunto(s)
Apoptosis/fisiología , Linfoma de Burkitt/enzimología , Caspasas/metabolismo , Activación Enzimática/fisiología , Zinc/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Células Cultivadas/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Manganeso/metabolismo , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Cell cycle progression is under the control of cyclin-dependent kinases (cdks), the activity of which is dependent on the expression of specific cdk inhibitors. In this paper we report that the two cdk inhibitors, p27(Kip1) and p18(INK4c), are differently expressed and control different steps of human B lymphocyte activation. Resting B cells contain large amounts of p27(Kip1) and no p18(INK4c). In vitro stimulation by Staphylococcus aureus Cowan 1 strain or CD40 ligand associated with IL-10 and IL-2 induces a rapid decrease in p27(Kip1) expression combined with cell cycle entry and progression. In contrast, in vitro Ig production correlates with specific expression of p18(INK4c) and early G(1) arrest. This G(1) arrest is associated with inhibition of cyclin D3/cdk6-mediated retinoblastoma protein phosphorylation by p18(INK4c). A similar contrasting pattern of p18(INK4c) and p27(Kip1) expression is observed both in B cells activated in vivo and in various leukemic cells. Expression of p18(INK4c) was also detected in various Ig-secreting cell lines in which both maximum Ig secretion and specific p18(INK4c) expression were observed during the G(1) phase. Our study shows that p27(Kip1) and p18(INK4c) have different roles in B cell activation; p27(Kip1) is involved in the control of cell cycle entry, and p18(INK4c) is involved in the subsequent early G(1) arrest necessary for terminal B lymphocyte differentiation.
Asunto(s)
Linfocitos B/citología , Linfocitos B/enzimología , Proteínas Portadoras/biosíntesis , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Proteínas Supresoras de Tumor , Antígenos CD40/fisiología , Proteínas Portadoras/metabolismo , Diferenciación Celular/inmunología , Células Cultivadas , Ciclina D3 , Quinasa 6 Dependiente de la Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/antagonistas & inhibidores , Ciclinas/biosíntesis , Regulación hacia Abajo/inmunología , Fase G1/inmunología , Humanos , Leucemia de Células B/inmunología , Leucemia de Células B/metabolismo , Activación de Linfocitos , Sustancias Macromoleculares , Proteínas Asociadas a Microtúbulos/biosíntesis , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/inmunologíaRESUMEN
Cell proliferation is controlled by cdk2 which in association with cyclin E and A regulates G1/S transition and S phase progression. cdk2 activation is dependent on its localization in the nucleus where regulatory mediators are found. We report that activation of cdk2 is associated with the formation of cdk2/MAP Kinase complexes. cdk2 associates with both inactive and activated MAP Kinase. Prevention of MAP Kinase activation by the MEK inhibitor PD98059 inhibits both activation and nuclear localization of cdk2 and S phase entry. These findings indicate that the nuclear translocation of cdk2 is associated with the formation of molecular complexes containing active MAP Kinase and is dependent on MAP Kinase activation. Oncogene (2000) 19, 4184 - 4189
Asunto(s)
Quinasas CDC2-CDC28 , Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Interleucina-2/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ciclo Celular/fisiología , Fraccionamiento Celular , Línea Celular , Núcleo Celular/enzimología , Quinasa 2 Dependiente de la Ciclina , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Immunoblotting , Microscopía Confocal , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Señales de Localización Nuclear/fisiología , Fosforilación , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis. Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.
Asunto(s)
Apoptosis , Linfocitos B/metabolismo , Caspasa 1/metabolismo , Caspasas/metabolismo , Manganeso/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Caspasa 3 , Muerte Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Humanos , Manganeso/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales CultivadasRESUMEN
In this study, we investigated the involvement of caspases in TGFbeta-induced apoptosis in human B cells. Our results show that TGFbeta-mediated nuclear fragmentation, observed in the Epstein-Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by zVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Specific activation of caspase-3 during TGFbeta-induced apoptosis was demonstrated by the DEVD-fmk-sensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFbeta treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFbeta. Our data thus demonstrate that TGFbeta-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein.
Asunto(s)
Apoptosis/fisiología , Linfocitos B/fisiología , Caspasas/fisiología , Proteína de Retinoblastoma/fisiología , Factor de Crecimiento Transformador beta/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Linfoma de Burkitt/patología , Linfoma de Burkitt/fisiopatología , Caspasa 2 , Caspasa 3 , Inhibidores de Caspasas , División Celular/fisiología , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Activación Enzimática , Humanos , Oligopéptidos/farmacología , Proteínas , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
We previously reported that p56lck expression is upregulated in human B lymphocytes upon mitogenic stimulation. In this report, we characterized the molecules associated with p56lck in vivo in leukemic B cells costimulated with anti-mu Ab and IL-2 for 72 h. In vitro phosphorylation after p56lck immunoprecipitation indicated that p56lck is associated in vivo with the beta chain of the IL-2 receptor and p42 MAP kinase as well as a number of other proteins. Moreover, p56lck-associated MAP kinase is tyrosine and threonine phosphorylated, suggesting that it is activated. Prevention of DNA synthesis with aphidicolin abrogated this molecular association, and furthermore, cell cycle analysis with IL-2-dependent T cells showed that in cells in G1, MAP kinase was not associated to p56lck, whereas this p56lck-MAP kinase association was observed when cells are in S phase. Thus, p56lck and MAP kinase are only associated during S phase. These data suggest that MAP kinase in association with p56lck is directly involved in the control of IL-2-mediated DNA synthesis of both B and T lymphocytes.
Asunto(s)
Linfocitos B/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Interleucina-2/farmacología , Proteínas Tirosina Quinasas/análisis , Familia-src Quinasas/análisis , Afidicolina/farmacología , Linfocitos B/inmunología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Activación de Linfocitos/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Proteína Quinasa 1 Activada por Mitógenos , Fosforilación , Pruebas de Precipitina , Fase S , Células Tumorales CultivadasRESUMEN
Transforming growth factor beta 1 (TGF beta 1) has been shown to inhibit growth stimulation in normal human B cells as well as in Epstein Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines. The mechanisms for this potent growth inhibition are not completely defined. Here we show that a number of EBV-negative lymphoma B cell lines (BL-41, Ramos and CAPA-2), when exposed in vitro to TGF beta 1, undergo apoptosis. Maximum apoptosis was observed at 48 h following TGF beta 1 treatment, with no apparent effect on the expression of c-myc and bcl-2 proteins. Similar induction of apoptosis was observed when these lymphoma cell lines were treated with aphidicolin, a DNA synthesis inhibitor. In contrast, various preparations (14 out of 17) of normal human tonsilar B cells showed no significant apoptosis, although both TGF beta 1 and aphidicolin inhibited anti-mu/IL-4 induced DNA synthesis in all preparations. Furthermore, another TGF beta 1 sensitive EBV-negative BL cell line, CA46, exhibited no apoptosis in response to TGF beta 1 and aphidicolin, corroborating the findings in normal human B cells. Taken together, these data support the hypothesis that exposure to TGF beta 1, which results in cell cycle arrest and DNA synthesis inhibition, may not be obligatory or sufficient for the induction of apoptosis. Rather, induction of apoptosis or lack of it may be intrinsically determined by an interplay between extracellular and intracellular regulators of cellular growth.
Asunto(s)
Linfocitos B/citología , Inhibidores de Crecimiento/farmacología , Linfoma/patología , Factor de Crecimiento Transformador beta/farmacología , Afidicolina/farmacología , Apoptosis , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Cromatina/ultraestructura , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Genes myc , Humanos , Inhibidores de la Síntesis del Ácido Nucleico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
Chronic B lymphocytic leukemia cells (B-CLL), characterized by the accumulation in vivo of long-life span B cells, exhibit spontaneous programmed cell death or apoptosis when cultured in vitro. We show that interferon-alpha (IFN-alpha), although able to decrease in vivo the number of leukemic cells, protects chronic B lymphocytic leukemia cells from in vitro programmed cell death or apoptosis. This inhibition of spontaneous in vitro apoptosis of leukemic B cells was observed after 24-48 hr of culture with 100-1000 U of either Interferon-alpha 2a or 2b. The protective activity was observed in the majority of the patients tested (6 out of 8) independent of the amount of apoptosis observed. Furthermore, in contrast to IL-4, IFN-alpha did not up-regulate the expression of Bcl-2. This suggests that B-CLL cells can be prevented from undergoing apoptosis in vitro by at least two different mechanisms: one, triggered for instance by IL-4, is associated with Bcl-2 production and the second triggered by Interferon-alpha is Bcl-2 independent. To elucidate the pathways mobilized by Interferon-alpha we also studied the regulation of c-myc expression in our experimental system. We found that (i) induction of in vitro B-CLL apoptosis was not associated with up-regulation of c-myc, (ii) c-myc expression as assessed by mRNA and protein determinations was increased after in vitro or in vivo Interferon-alpha stimulation. Additional experiments using c-myc specific oligonucleotides demonstrated that when Interferon-alpha-mediated c-myc expression was decreased by 60%, the in vitro protective effect of Interferon-alpha was not modified. Thus our data show that in contrast to the situation in vivo, Interferon-alpha prevents spontaneous in vitro B-CLL cells apoptosis through a Bcl-2-independent pathway which is probably not related to c-myc up-regulation.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/citología , Interferón-alfa/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Proto-Oncogénicas/fisiología , Anciano , Secuencia de Bases , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2 , Regulación hacia Arriba/fisiologíaRESUMEN
In this study, we investigated the role of B-T cell contacts in interleukin Ia-mediated inhibition of human B lymphocyte proliferation induced by mitogenic doses of soluble anti-mu monoclonal antibody (mAb). We show that additional cross-linking of B cell antigens, using Sepharose beads coated with anti-mu, anti-(IL)-4 mAb (but not soluble mAb) or anti-CD40 antigen counteracted the inhibitory activity of IL-4. More importantly, cell contacts between B cells and activated T cells (but not unstimulated T cells) were sufficient to counteract IL-4-mediated inhibition of DNA synthesis. In addition, the inhibitory activity of IL-4 on chronic lymphocytic leukemia B cells stimulated with anti-mu and IL-2 was itself reduced by the presence of fixed activated T cells. Our data suggest that a major role for IL-4 would be to prepare B cells to receive additional mitogenic signals through cell contact interactions with activated T lymphocytes. When such interactions do not occur IL-4 may block DNA synthesis, preventing uncontrolled B cell proliferation.
Asunto(s)
Linfocitos B/inmunología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos CD40 , Humanos , Técnicas In Vitro , Interleucina-2/farmacología , Cooperación Linfocítica , Receptores de Antígenos de Linfocitos B/fisiologíaRESUMEN
In this study, we analyzed the expression and regulation of src kinase p56lck expression during human B lymphocyte activation. We show that upon mitogenic stimulation with anti-IgM antibodies and interleukin-2, specific mRNA for p56lck becomes detectable in B cells after 24 h of activation and is followed by an increase in p56lck protein expression on days 2 and 3. This up-regulation is specific for p56lck since expression of other src kinases such as fyn, lyn, or yes was not modified. Furthermore, immune complex kinase assays show that p56lck protein expressed on day 2 is associated with kinase activity. Experiments using lck-specific antisense oligonucleotides show that the G0-G1 transition does not require p56lck, whereas DNA synthesis is dependent upon its expression. Thus, our data demonstrate that p56lck expression is up-regulated during human B cell stimulation and this kinase plays an important role during the control of late steps of B lymphocyte activation such as S phase entry.
Asunto(s)
Linfocitos B/fisiología , Replicación del ADN , Regulación Enzimológica de la Expresión Génica , Activación de Linfocitos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , ADN/biosíntesis , Humanos , Interleucina-2/farmacología , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Tonsila Palatina , Fitohemaglutininas , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Timidina/metabolismo , Factores de TiempoRESUMEN
Surface immunoglobulin (Ig) cross-linking by anti-IgM (mu) antibodies leads to B cell activation resulting in numerous early biochemical events including an increase in intracellular [Ca2+]. Furthermore, anti-mu-activated B cells become able to proliferate in response to interleukin (IL)2 and IL4. These studies examined the effect of the calcium ionophore ionomycin, an enhancer of cytoplasmic [Ca2+] levels, on IL2 and IL4-mediated proliferation of anti-mu-stimulated normal human B cells. Ionomycin inhibited the proliferative response of anti-mu-activated B cells to IL4. In contrast, IL2 and phorbol 12,13 dibutyrate (PBu2)-mediated B cell proliferation was refractory to the growth inhibitory effects of ionomycin. In an attempt to delineate a possible mechanism(s) for this differential growth effect of ionomycin, we first studied direct effects of ionomycin on activated B cells. Our data suggested that ionomycin induced DNA fragmentation in anti-mu-costimulated B cells. Interestingly, in contrast to PBu2, IL4 did not prevent ionomycin-dependent DNA fragmentation. Importantly, H7, an inhibitor of protein kinase C activation, down-regulated only the IL2 and PBu2-driven B cell proliferation but not B cell proliferative response to IL4. These results suggest that putative protein kinase C activation, either by direct treatment with phorbol ester or during IL2 signaling, counteracts the inhibitory effects of ionomycin. In contrast, IL4 signaling does not exhibit the same protective properties.
Asunto(s)
Linfocitos B/fisiología , Calcio/fisiología , Muerte Celular/efectos de los fármacos , Interleucina-2/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Linfocitos B/citología , Daño del ADN , Humanos , Técnicas In Vitro , Interleucina-6/biosíntesis , Isoquinolinas/farmacología , Forbol 12,13-Dibutirato/farmacología , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidoresRESUMEN
In this report we show that IL-4 inhibits DNA synthesis induced by stimulation of human B cells with mitogenic doses of either soluble anti-mu mAb DA44 or phorbol ester. In contrast, earlier steps of anti-mu-induced B cell stimulation, such as RNA synthesis, CD23 expression and IL-6 production, were not inhibited but rather increased in the presence of IL-4. From these results, IL-4 appears therefore to exert two opposite effects on DA44 anti-mu mAb-induced human B cell activation: early steps are stimulated, and later steps inhibited. The results of kinetic analysis were consistent with this model. The inhibitory activity of IL-4 required an active cAMP-dependent pathway since IL-4-mediated inhibition of anti-mu-induced B cell proliferation was abolished in the presence of two specific inhibitors of the cAMP pathway (H8 and 2',5'-dideoxyadenosine which are specific for cAMP-dependent protein kinase and adenylate cyclase respectively). Furthermore, IL-4 induced a delayed and prolonged increase in intracellular cAMP concentrations (observed between 4 and 48 hours of culture), and this strongly suggests that the late inhibitory effects of IL-4 is cAMP-dependent. Moreover, this delayed IL-4-mediated cAMP production is probably sufficient to prevent anti-mu induced DNA synthesis since addition of the cAMP agonist forskolin on day 1 or 2 of culture also suppresses the anti-mu-mediated B cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Monoclonales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Linfocitos B/inmunología , AMP Cíclico/fisiología , Interleucina-4/farmacología , Interleucina-6/biosíntesis , Activación de Linfocitos , Receptores Fc/fisiología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Células Cultivadas , Niño , Colforsina/farmacología , Replicación del ADN/efectos de los fármacos , Humanos , Interleucina-6/análisis , Cinética , Activación de Linfocitos/efectos de los fármacos , Tonsila Palatina/inmunología , Forbol 12,13-Dibutirato/farmacología , ARN/biosíntesis , ARN/efectos de los fármacos , Receptores Fc/inmunología , Receptores de IgE , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Timidina/metabolismoRESUMEN
The second messenger cAMP is a modulator of cellular growth possessing both inhibitory and stimulatory properties. In this report, we show that IL-2- and IL-4-dependent DNA synthesis of anti-mu-activated human B cells is modulated in opposite ways by agents increasing intracellular levels of cAMP. Forskolin and 2'-O-dibutyriladenosine-3',5'-cyclic monophosphate had no proliferative effect by themselves. Nevertheless they decreased IL-2-driven proliferation and increased IL-4-mediated DNA synthesis. IL-4 and cAMP each inhibited the IL-2-dependent proliferation with similar patterns of reactivity. Both IL-4 and forskolin needed to be present during the first 48 h of culture to display inhibitory activity, and preactivation of B cells for 16 h with forskolin and IL-4 did not prevent further B cell response to IL-2. This suggests that cAMP and IL-4 directly interact with IL-2 signaling. In addition, we show that the cAMP-dependent protein kinase inhibitor N-(2-methylamino-ethyl)-5-iso-quinoline-sulfamide reversed the IL-4-inhibitory effect on IL-2-driven proliferation. Our data suggest that the IL-4-inhibitory signal to IL-2-driven human B cell proliferation involves cAMP-dependent protein kinase activation.
Asunto(s)
Linfocitos B/efectos de los fármacos , AMP Cíclico/fisiología , Interleucina-2/farmacología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos B/inmunología , Bucladesina/farmacología , Células Cultivadas , Colforsina/farmacología , Humanos , Forbol 12,13-Dibutirato/farmacologíaRESUMEN
Cystic fibrosis is associated with an cAMP-regulated channel defect, which has been evidenced in many cell types including B lymphocytes. To document a B-cell dysfunction potentially related to this defect, we studied the in vitro IgG production by lymphocytes from 11 cystic fibrosis patients. B lymphocytes were co-cultured with autologous monocytes and stimulated with Staphylococcus aureus Cowan or with Nocardia-delipidated cell mitogen in the presence of low concentrations of IL2. Cystic fibrosis patients' cells produced amounts of IgG comparable with that of normal and control patients' cells. However, dexamethasone (10(-7) mol l-1) had no effect on the response of cystic fibrosis patients' cells, whereas it enhanced that of the latter two groups. This resistance of cystic fibrosis cells was true with concentrations of dexamethasone up to 10(-6) mol l-1, whereas this agent induced a dose-related enhancement from 10(-8) to 10(-6) mol l-1 in cultures of normal cells. Co-culture experiments showed that cystic fibrosis B lymphocytes themselves are resistant to the effect of dexamethasone. In contrast dexamethasone normally suppressed the anti-CD3 antibody-induced response of cystic fibrosis T cells in the presence of IL2 and the IL1 alpha- or beta-induced collagenase production of cystic fibrosis fibroblast cell lines. Thus cystic fibrosis B lymphocytes exhibit a selective defect which may interfere with the normal interactions between the hormonal and immune systems and may participate in the sensitivity of cystic fibrosis patients to bacterial bronchopulmonary infections.
Asunto(s)
Linfocitos B/efectos de los fármacos , Fibrosis Quística/inmunología , Dexametasona/farmacología , Adolescente , Adulto , Linfocitos B/inmunología , Niño , Preescolar , Dexametasona/administración & dosificación , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Inmunoglobulina G/biosíntesis , Técnicas In Vitro , Activación de Linfocitos/efectos de los fármacos , Masculino , Colagenasa Microbiana/biosíntesis , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
In order to analyze the effects of interleukin (IL) 6 on human in vitro Ig production B lymphocytes were activated by Staphylococcus aureus Cowan strain I (SAC) in the presence of low concentrations of IL2 (1 U/ml) and dexamethasone (10(-7) M). Previously we showed that this model of B cell response is completely monocyte dependent. We here demonstrate that, under these experimental conditions, IL6 is able to replace monocytes and stimulate Ig production provided IL1 is also present. Dose-effect curves show that these two monokines act synergistically. This synergy is demonstrable only in the presence of dexamethasone, when B lymphocytes are activated (by SAC) and when T cell help (provided by IL2) is present. It results in the production of both IgM and IgG. Both IL1 and IL6 have to be present during the first 48 h of culture to exert an optimal effect. These results show that IL6 may act on early (as well as on late) stages of normal B lymphocyte differentiation. Moreover, glucocorticosteroids potentiate the synergistic effect of IL1 and IL6 on their B lymphocyte target, an effect comparable to that exerted on hepatocytes.
Asunto(s)
Linfocitos B/citología , Diferenciación Celular/efectos de los fármacos , Glucocorticoides/farmacología , Interleucina-1/farmacología , Interleucinas/farmacología , Factores Biológicos/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Técnicas In Vitro , Interleucina-6 , MonocinasRESUMEN
A high m.w. B cell growth factor (50,000 BCGF) prepared from lectin-activated human peripheral blood lymphocyte culture supernatants acts only on B cells preactivated by a first signal. This first signal can be delivered in vitro (with anti-mu antibody (Ab)) or in vivo. Upon costimulation with anti-mu Ab the 50,000 BCGF induces an early and transient proliferative response, whereas the response to interleukin 2 (IL-2) develops more progressively. To determine the respective targets of the 50,000 BCGF and of IL-2, B cells were activated with anti-mu Ab and separated according to the expression of the IL-2 receptor (cluster designation (CD)25 antigen). CD25+ B cells do not respond to the 50,000 BCGF and do not acquire this responsiveness after an additional culture with IL-2. CD25- B cells respond to the 50,000 BCGF and not to IL-2. However, when CD25- B cells are cultured for 3 days with the 50,000 BCGF they become responsive to IL-2. These results demonstrate a pathway of B cell activation based on the ordered and sequential action of anti-mu Ab, the 50,000 BCGF, and IL-2.
Asunto(s)
Linfocitos B/efectos de los fármacos , Interleucinas/farmacología , Anticuerpos Antiidiotipos/inmunología , Linfocitos B/inmunología , División Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Inmunoglobulina M/inmunología , Interleucina-2/farmacología , Interleucina-4 , Cinética , Activación de Linfocitos , Peso Molecular , Receptores Inmunológicos/análisis , Receptores de Interleucina-2RESUMEN
After activation by anti-mu antibody human B cells acquire the ability to proliferate in the presence of recombinant interleukin 2 (IL 2), a 20-kDa mol. mass B cell growth factor (BCGF) and a high mol. mass BCGF (50-kDa BCGF). An anti-IL 2 receptor (IL 2R) monoclonal antibody inhibits the IL 2-dependent proliferation without affecting that induced by BCGF. B cells expressing the IL 2R after anti-mu antibody activation (IL 2R+ cells) were separated from those not expressing IL 2R (IL 2R- cells). IL 2 stimulated the proliferation of only IL 2R+ cells whereas the 20-kDa BCGF acted on both IL 2R+ and IL 2R- cells. Importantly, the 50-kDa BCGF supported the proliferation of IL 2R- cells whereas it was inactive on IL 2R+ cells. Thus, the B cell subset responding to the 50-kDa BCGF after anti-mu antibody activation is distinct from that responding to IL 2.
Asunto(s)
Linfocitos B/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Interleucina-2 , Linfocinas/farmacología , Anticuerpos Monoclonales/inmunología , Linfocitos B/clasificación , Linfocitos B/inmunología , Sustancias de Crecimiento/aislamiento & purificación , Humanos , Interleucina-4 , Activación de Linfocitos , Linfocinas/aislamiento & purificación , Peso Molecular , Receptores Inmunológicos/análisis , Receptores Inmunológicos/fisiología , Receptores de Interleucina-2RESUMEN
Nonspecific T cell factors produced by lectin-activated human peripheral blood lymphocytes (PBL) were used to restore the T-dependent B cell response to trinitrophenyl-polyacrylamide (TNP-PAA). Preincubation experiments with the particulate antigen TNP-PAA and/or a soluble TNP-protein conjugate show that a first specific signal provided by the antigen and nonspecific lymphokines sequentially acts on B cells. By gel filtration the T cell-replacing factor (TRF) activity is present in the 30-15-kDa fraction of T cell supernatants and is associated to interleukin 2 (IL2). However, absorption of IL2 does not abolish the TRF activity. Moreover, chromatofocusing of this 30-15-kDa material allows the obtaining of an IL2-free fraction containing a differentiation factor (with an isoelectric point of 5.7 +/- 0.2). The ability of this fraction to restore the anti-TNP response is manifest in the presence of a 50-kDa B cell growth factor. This latter, prepared by a combination of absorption on concanavalin A-Sepharose and gel filtration, was IL2 free and unable to support the anti-TNP response. We thus directly demonstrate that in the absence of IL2 three separate signals (the antigen, T cell-derived growth and differentiation factors) are involved in human-specific B cell response.