RESUMEN

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPS(d), although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-kappa B, which is shown by an NF-kappa B-driven reporter gene construct. In the presence of an inhibitor of NF-kappa B activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-kappa B drives the expression of IL-9 via three NF-kappa B binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Interleucina-13/biosíntesis , Interleucina-9/biosíntesis , Lipopolisacáridos/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , FN-kappa B/fisiología , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Interleucina-9/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Congénicos , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , FN-kappa B/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología

RESUMEN

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Proteínas Nucleares , Linfocitos T/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Calcineurina/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Activación de Linfocitos , Mutación , Factores de Transcripción NFATC , Conformación Proteica , Transducción de Señal , Células TH1/fisiología , Células Th2/fisiología , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética

RESUMEN

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/metabolismo

RESUMEN

Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis without further requirement for TCR coengagement. This observation prompted us to postulate that signal 2 (costimulatory signal) alone without signal 1 (TCR signal) can activate T cells. To test whether this putative function of CD28 is mediated via a particular signaling pathway, we compared early signaling events initiated in resting T cells by the stimulatory mAb BW 828 with signals triggered by the nonstimulating CD28 mAb 9.3. Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did not lead to detectable activation of the protein kinases p56(lck) and c-Raf-1. This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but was unable to induce the binding of transcription factors to the IL-2 promoter. In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), indicating that the signaling capacity of CD28 changes with activation. These data support the view that stimulation through CD28 can induce some effector functions in T cells and suggest that this capacity is associated with a particular pattern of early signaling events.

Asunto(s)

Antígenos CD28/fisiología , Citocinas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Transcripción Genética/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD28/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Interfase/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Factores de Transcripción/biosíntesis

RESUMEN

HMG I(Y) proteins bind to double-stranded A + T oligonucleotides longer than three base pairs. Such motifs form part of numerous NF-AT-binding sites of lymphokine promoters, including the interleukin 4 (IL-4) promoter. NF-AT factors share short homologous peptide sequences in their DNA-binding domain with NF-kappa B factors and bind to certain NF-kappa B sites. It has been shown that HMG I(Y) proteins enhance NF-kappa B binding to the interferon beta promoter and virus-mediated interferon beta promoter induction. We show that HMG I(Y) proteins exert an opposite effect on the DNA binding of NF-AT factors and the induction of the IL-4 promoter in T lymphocytes. Introduction of mutations into a high-affinity HMG I(Y)-binding site of the IL-4 promoter, which decreased HMG I(Y)-binding to a NF-AT-binding sequence, the Pu-bB (or P) site, distinctly increased the induction of the IL-4 promoter in Jurkat T leukemia cells. High concentrations of HMG I(Y) proteins are able to displace NF-ATp from its binding to the Pu-bB site. High HMG I(Y) concentrations are typical for Jurkat cells and peripheral blood T lymphocytes, whereas E14 T lymphoma cells and certain T helper type 2 cell clones contain relatively low HMG I(Y) concentrations. Our results indicate that HMG I(Y) proteins do not cooperate, but instead compete with NF-AT factors for the binding to DNA even though NF-AT factors share some DNA-binding to DNA even though NF-AT factors share some DNA-binding properties with NF-kB factors. This competition between HMG I(Y) and NF-AT proteins for DNA binding might be due to common contacts with minor groove nucleotides of DNA and may be one mechanism contributing to the selective IL-4 expression in certain T lymphocyte populations, such as T helper type 2 cells.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Interleucina-4/biosíntesis , Interleucina-4/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Clonación Molecular , Glutatión Transferasa/biosíntesis , Proteína HMGA1a , Células HeLa , Humanos , Leucemia de Células T , Activación de Linfocitos , Linfoma de Células T , Ratones , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Factores de Transcripción NFATC , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología

Asunto(s)

Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/inmunología , Interleucina-4/genética , Proteínas Nucleares , Regiones Promotoras Genéticas/inmunología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Regiones Promotoras Genéticas/efectos de los fármacos , Factores de Transcripción/biosíntesis

RESUMEN

The promoters of IL-2 and IL-4 genes contain multiple binding sites for octamer factors. In peripheral T lymphocytes and several T cell lines, both the ubiquitous Oct factor Oct-1 and the lymphocyte-specific factor Oct-2 are expressed and bind to the IL-2 and IL-4 promoters. Prominent octamer binding sites of IL-2 and IL-4 promoters are their upstream promoter sites (UPS) which share 14 identical nucleotides. Multiple copies of the IL-2 and IL-4 UPS act as inducible enhancers in T cells, and their induction is inhibited by the immunosuppressant cyclosporin A (CsA). Closely linked to the octamer site, the IL-2 UPS contains a non-canonical AP-1 binding (TRE) site, and mutation in either site to a non-functional factor binding site impairs the induction of the IL-2 promoter. The binding of AP-1 and octamer factors to the IL-2 UPS DNA overlaps, and the tight association and functional cooperation of octamer with AP-1 factors is of crucial importance for the inducible IL-2 UPS activity. Introduction of five or ten spacer nucleotides between both IL-2 UPS sites results in a drastic reduction of inducible UPS activity, both in the loss of suppression by CsA and stimulation by the Ca(2+)-dependent phosphatase calcineurin. Within the IL-4 UPS the Oct and TRE-like motifs are separated by a binding site of nuclear factor of activated T cells (NF-AT). This site shares nine out of ten bp with an IL-2 NF-AT site. The strong binding of NF-ATp to the IL-4 UPS site suppresses the simultaneous binding of Oct factors to the IL-4 UPS. Because the two other Oct binding sites of IL-4 promoter show a similar sequence configuration, the binding of NF-AT seems to prevent the simultaneous binding of Oct factors to the IL-4 promoter. By contrast, both classes of factors bind simultaneously to the IL-2 promoter, and their tight association with AP-1 enhances the IL-2 promoter activity.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Interleucina-2/genética , Interleucina-4/genética , Proteínas Nucleares , Oligodesoxirribonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Factor de Transcripción AP-1/fisiología , Transfección , Células Tumorales Cultivadas