RESUMEN
Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.
Asunto(s)
Calcineurina/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Ciclosporina/farmacología , Humanos , Inmunosupresores/farmacología , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Ratones , Ratones Transgénicos , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Unión Proteica , Transducción de Señal , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
Glucocorticoid (GC) signaling in thymocytes shapes the TCR repertoire by antagonizing thymocyte negative selection. The transcription factors Nur77 and Helios, which are upregulated in TCR-signaled thymocytes, have been implicated in negative selection. In this study, we found that GCs inhibited Helios and, to a lesser extent, Nur77 upregulation in TCR-stimulated mouse thymocytes. Inhibition was increased by GC preincubation, and reductions in mRNA were prevented by a protein synthesis inhibitor, suggesting that GCs suppress indirectly via an intermediary factor. Upregulation of Helios in TCR-stimulated thymocytes was unaffected by deletion of Nur77, indicating Nur77 and Helios are regulated independently. Whereas CD4+ thymocytes are positively selected in wild-type AND TCR-transgenic B6 mice, loss of GC receptor expression resulted in increased negative selection. Correspondingly, Helios and Nur77 levels were elevated in TCRhiCD4+CD8+ (TCR-signaled) thymocytes. Notably, deletion of Helios fully reversed this negative selection, whereas deletion of Nur77 had no effect on CD4+CD8+ cell numbers but reversed the loss of mature CD4+ thymocytes. Thus, Nur77 and Helios are GC targets that play nonredundant roles in setting the signaling threshold for thymocyte negative selection.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Glucocorticoides/farmacología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/antagonistas & inhibidores , Timocitos/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Animales , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficiencia , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Timocitos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.
Asunto(s)
Genes Codificadores de los Receptores de Linfocitos T/fisiología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencia de Aminoácidos , Regulación de la Expresión Génica , Humanos , Células Jurkat , Fosforilación , Transducción de Señal , Proteína Tirosina Quinasa ZAP-70/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Glucocorticoid (GC) signaling in thymocytes counters negative selection and promotes the generation of a self-tolerant yet Ag-responsive T cell repertoire. Whereas circulating GC are derived from the adrenals, GC are also synthesized de novo in the thymus. The significance of this local production is unknown. In this study we deleted 11ß-hydroxylase, the enzyme that catalyzes the last step of GC biosynthesis, in thymic epithelial cells (TEC) or thymocytes. Like GC receptor-deficient T cells, T cells from mice lacking TEC-derived but not thymocyte-derived GC proliferated poorly to alloantigen, had a reduced antiviral response, and exhibited enhanced negative selection. Strikingly, basal expression of GC-responsive genes in thymocytes from mice lacking TEC-derived GC was reduced to the same degree as in GC receptor-deficient thymocytes, indicating that at steady-state the majority of biologically active GC are paracrine in origin. These findings demonstrate the importance of extra-adrenal GC even in the presence of circulating adrenal-derived GC.
Asunto(s)
Antígenos/metabolismo , Células Epiteliales/metabolismo , Glucocorticoides/metabolismo , Timocitos/metabolismo , Animales , Células Cultivadas , Activación de Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxigenasas de Función Mixta/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfocitos T/metabolismoRESUMEN
Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR(CD11c-cre)) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR(CD11c-cre) mice, CD8(+) DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c(+) cells rescued LPS-induced loss of CD8(+) DCs but not other DC subsets. Unlike wild-type animals, exposure of GR(CD11c-cre) mice to low-dose LPS did not induce CD8(+) DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Células Dendríticas/efectos de los fármacos , Glucocorticoides/agonistas , Interleucina-12/antagonistas & inhibidores , Receptores de Glucocorticoides/agonistas , Choque Séptico/prevención & control , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Células Cultivadas , Cruzamientos Genéticos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Relación Dosis-Respuesta a Droga , Femenino , Glucocorticoides/antagonistas & inhibidores , Glucocorticoides/sangre , Glucocorticoides/metabolismo , Inmunidad Innata/efectos de los fármacos , Interleucina-12/sangre , Interleucina-12/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Choque Séptico/inmunología , Choque Séptico/metabolismo , Choque Séptico/patología , Transducción de Señal/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Bazo/patologíaRESUMEN
Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.
Asunto(s)
Receptores de Glucocorticoides/química , Animales , Células Cultivadas , ADN/metabolismo , Ratones , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/metabolismoRESUMEN
OBJECTIVE: Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. APPROACH AND RESULTS: Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)-null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI(+/+) mice, but no thymocyte apoptosis in SR-BI(-/-) mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI(-/-) mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI(+/+) HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI(-/-) HDL had no such activity. Further study revealed that SR-BI(+/+) HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI(-/-) HDL loses such regulatory activity. To understand why SR-BI(-/-) HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI(-/-) HDL compared with SR-BI(+/+) HDL. Normalization of unesterified cholesterol in SR-BI(-/-) HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI(+/+) HDL rendered SR-BI(+/+) HDL dysfunctional. Using lckCre-GR(fl/fl) mice in which thymocytes lack cecal ligation and puncture-induced thymocyte apoptosis, we showed that lckCre-GR(fl/fl) mice were significantly more susceptible to cecal ligation and puncture-induced septic death than GR(fl/fl) control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis. CONCLUSIONS: The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.
Asunto(s)
Apoptosis , HDL-Colesterol/sangre , Receptores Depuradores de Clase B/metabolismo , Sepsis/metabolismo , Timocitos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciego/microbiología , Ciego/cirugía , Células Cultivadas , Corticosterona/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ligadura , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Probucol/farmacología , Transporte de Proteínas , Punciones , Receptores de Glucocorticoides/metabolismo , Receptores Depuradores de Clase B/deficiencia , Receptores Depuradores de Clase B/genética , Sepsis/sangre , Sepsis/microbiología , Sepsis/patología , Transducción de Señal , Timocitos/efectos de los fármacos , Timocitos/patologíaRESUMEN
Stimulation via the T-cell receptor (TCR) activates p38α and p38ß by phosphorylation of p38 Tyr-323 (p38(Y323)). Here we characterize knockin mice in which p38α and/or ß Tyr-323 has been replaced with Phe. We find that p38α accounts for two-thirds and p38ß the remainder of TCR-induced p38 activation. T cells from double knockin mice (p38αß(Y323F)) had defects in TCR-mediated proliferation and Th1 and Th17 skewing, the former corresponding with an inability to sustain T-bet expression. Introduction of p38α(Y323F) into Gadd45α-deficient mice, in which the alternative p38 pathway is constitutively active, reversed T-cell hyperproliferation and autoimmunity. Furthermore, p38αß(Y323F) mice had delayed onset and reduced severity of the inflammatory autoimmune diseases collagen-induced arthritis and experimental autoimmune encephalomyelitis. Thus, T cell-specific alternative activation of p38 is an important pathway in T-cell proliferation, Th skewing, and inflammatory autoimmunity, and may be an attractive tissue-specific target for intervention in these processes.
Asunto(s)
Artritis Experimental/metabolismo , Autoinmunidad , Proteínas de Ciclo Celular/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación/metabolismo , Proteínas Nucleares/genética , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/patología , Western Blotting , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/inmunología , Proliferación Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Activación Enzimática/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnicas de Sustitución del Gen , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/inmunología , Fenilalanina/inmunología , Fenilalanina/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Tirosina/inmunología , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
T cells possess a p38 activation alternative pathway in which stimulation via the antigen receptor (T-cell receptor [TCR]) induces phosphorylation of p38alpha and beta on Tyr323. To assess the contribution of this pathway to normal T-cell function, we generated p38alpha knockin mice in which Tyr323 was replaced with Phe (p38alpha(Y323F)). TCR-mediated stimulation failed to activate p38alpha(Y323F) as measured by phosphorylation of the Thr-Glu-Tyr activation motif and p38alpha catalytic activity. Cell-cycle entry was delayed in TCR-stimulated p38alpha(Y323F) T cells, which also produced less interferon (IFN)-gamma than wild-type T cells in response to TCR-mediated but not TCR-independent stimuli. p38alpha(Y323F) mice immunized with T-helper 1 (Th1)-inducing antigens generated normal Th1 effector cells, but these cells produced less IFN-gamma than wild-type cells when stimulated through the TCR. Thus, the Tyr323-dependent pathway and not the classic mitogen-activated protein (MAP) kinase cascade is the physiologic means of p38alpha activation through the TCR and is necessary for normal Th1 function but not Th1 generation.
Asunto(s)
Activación Enzimática/inmunología , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células TH1/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Animales , Western Blotting , Ciclo Celular , Activación Enzimática/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Sustitución del Gen , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Ratones , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Glucocorticoids are lipid-soluble hormones that signal via the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Circulating glucocorticoids derive from the adrenals, but it is now apparent that paracrine glucocorticoid signaling occurs in multiple tissues. Effective local glucocorticoid concentrations and whether glucocorticoid delivery can be targeted to specific cell subsets are unknown. We use fluorescence detection of chromatin-associated GRs as biosensors of ligand binding and observe signals corresponding to steroid concentrations over physiological ranges in vitro and in vivo. In the thymus, where thymic epithelial cell (TEC)-synthesized glucocorticoids antagonize negative selection, we find that CD4+CD8+TCRhi cells, a small subset responding to self-antigens and undergoing selection, are specific targets of TEC-derived glucocorticoids and are exposed to 3-fold higher levels than other cells. These results demonstrate and quantitate targeted delivery of paracrine glucocorticoids. This approach may be used to assess in situ nuclear receptor signaling in a variety of physiological and pathological contexts.
Asunto(s)
Glucocorticoides/metabolismo , Timo/metabolismo , Animales , Técnicas Biosensibles , Línea Celular , Cromatina/metabolismo , Sistemas de Liberación de Medicamentos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Timo/citologíaRESUMEN
Endogenous levels of glucocorticoids rise during pregnancy to warrant development and maturation of the fetal organs close to birth. However, during most of the gestation, the fetus is protected from excessive biologically active endogenous glucocorticoids by placental and fetal expression of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2). Maternal stress, which may overwhelm placental 11ß-HSD2 activity with high glucocorticoid levels, or administration of synthetic glucocorticoids to improve the survival chances of the premature newborn, are associated to postnatal increased risk for immune diseases. Fetal exposure to excessive glucocorticoids may underlie this altered postnatal immunity. Here, we revise the role that placental and fetal 11ß-HSD2, fetal glucocorticoid exposure, and programming of the offspring's the hypothalamic-pituitary-adrenal (HPA) axis play on concerted steps in immune fetal development. We could identify gaps in knowledge about glucocorticoid-induced programming of immune diseases. Finally, based on current evidence about glucocorticoid and HPA axis-mediated immune regulation, we hypothesize on mechanisms that could drive the enhanced risk for atopies, infections, and type I diabetes in offspring that were prenatally exposed to glucocorticoids.
Asunto(s)
Glucocorticoides/administración & dosificación , Glucocorticoides/metabolismo , Inmunidad/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Femenino , Feto/efectos de los fármacos , Feto/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/efectos adversos , Humanos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , Placenta/metabolismo , Embarazo , Atención Prenatal , Efectos Tardíos de la Exposición Prenatal , Fenómenos Fisiológicos ReproductivosRESUMEN
Pathogen-associated molecular pattern (PAMP) recognition leads to TANK-binding kinase (TBK1) polyubiquitination and activation by transautophosphorylation, resulting in IFN-ß production. Here, we describe a mouse model of optineurin insufficiency (OptnΔ(157) ) in which the TBK1-interacting N-terminus of optineurin was deleted. PAMP-stimulated cells from OptnΔ(157) mice had reduced TBK1 activity, no phosphorylation of optineurin Ser(187) , and mounted low IFN-ß responses. In contrast to pull-down assays where the presence of N-terminus was sufficient for TBK1 binding, both the N-terminal and the ubiquitin-binding regions of optineurin were needed for PAMP-induced binding. This report establishes optineurin as a positive regulator TBK1 via a bipartite interaction between these molecules.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/genética , Interferón beta/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Proteínas de Transporte de Membrana , Ratones , Fosforilación/efectos de los fármacos , Unión Proteica , Eliminación de SecuenciaRESUMEN
Because antigen-stimulated naive T cells either die as effectors or enter the activated/memory pool, continuous egress of new T lymphocytes from thymus is essential for maintenance of peripheral immune homeostasis. Unexpectedly, we found that systemic infection with the protozoan Toxoplasma gondii triggers not only a transient increase in activated CD4+ Th1 cells but also a persistent decrease in the size of the naive CD4+ T lymphocyte pool. This immune defect is associated with decreased thymic output and parasite-induced destruction of the thymic epithelium, as well as disruption of the overall architecture of that primary lymphoid organ. Importantly, the resulting quantitative and qualitative deficiency in naive CD4+ T cells leads to an immunocompromised state that both promotes chronic toxoplasma infection and leads to decreased resistance to challenge with an unrelated pathogen. These findings reveal that systemic infectious agents, such as T. gondii, can induce long-term immune alterations associated with impaired thymic function. When accumulated during the lifetime of the host, such events, even when occurring at low magnitude, could be a contributing factor in immunological senescence.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Timo/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Timo/patología , Toxoplasmosis/genética , Toxoplasmosis/patologíaRESUMEN
Mice in which exon 2 of the glucocorticoid receptor (GR) has been disrupted [GR exon 2 knockout (GR2KO)] have been used as a model to study the requirement for this receptor in a number of biological systems. A recent report showed that these mice actually express a truncated ligand-binding GR fragment, prompting us to ask whether this mutation truly results in a glucocorticoid-insensitive phenotype. Based on cDNA microarray analysis of fetal thymocytes, we found that glucocorticoids were able to enhance or repress activation-induced gene expression in GR2KO and wild-type thymocytes to a similar degree. Moreover, although changes in gene expression induced by glucocorticoids alone were blunted, the expression of a substantial number of genes in GR2KO thymocytes was modulated by stimulation with glucocorticoids. Among these genes, as confirmed by quantitative real-time PCR, was the classic glucocorticoid-responsive gene glutamine synthetase as well as genes implicated in T cell development and function such as IL-7 receptor alpha-chain and glucocorticoid-induced leucine zipper (GIL2). Thus, the truncated C-terminal GR2KO product, which lacks the major transactivation domain, retains, to a large extent, the ability to regulate gene expression both positively and negatively in a ligand-responsive manner when expressed in vivo.
Asunto(s)
Exones , Regulación de la Expresión Génica , Ligandos , Receptores de Glucocorticoides/genética , Animales , Células Cultivadas , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Glutamato-Amoníaco Ligasa/genética , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Interleucina-7/genética , Elementos de Respuesta , Linfocitos T/fisiología , Timo/citología , Timo/efectos de los fármacos , Timo/embriología , Factores de Transcripción/genéticaRESUMEN
Synthetic glucocorticoids (GCs) are commonly used in the treatment of inflammatory diseases, but the role of endogenous GCs in the regulation of host-protective immune responses is poorly understood. Here we show that GCs are induced during acute Toxoplasma gondii infection and directly control the T cell response to the parasite. When infected with toxoplasma, mice that selectively lack GC receptor (GR) expression in T cells (GR(lck-Cre)) rapidly succumb to infection despite displaying parasite burdens indistinguishable from control animals and unaltered levels of the innate cytokines IL-12 and IL-27. Mortality in the GR(lck-Cre) mice was associated with immunopathology and hyperactive Th1 cell function as revealed by enhanced IFN-γ and TNF production in vivo. Unexpectedly, these CD4(+) T lymphocytes also overexpressed IL-10. Importantly, CD4(+) T cell depletion in wild-type or GR(lck-Cre) mice led to ablation of the GC response to infection. Moreover, in toxoplasma-infected RAG(-/-) animals, adoptive transfer of CD4(+) T lymphocytes was required for GC induction. These findings establish a novel IL-10-independent immunomodulatory circuit in which CD4(+) T cells trigger a GC response that in turn dampens their own effector function. In the case of T. gondii infection, this self-regulatory pathway is critical for preventing collateral tissue damage and promoting host survival.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Glucocorticoides/metabolismo , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/metabolismo , Animales , Citocinas/biosíntesis , Citocinas/inmunología , Ratones , Ratones Noqueados , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Células TH1/inmunología , Toxoplasmosis/genéticaRESUMEN
Generation of a self-tolerant but antigen-responsive T cell repertoire occurs in the thymus. Although glucocorticoids are usually considered immunosuppressive, there is also evidence that they play a positive role in thymocyte selection. To address the question of how endogenous glucocorticoids might influence the adaptive immune response, we generated GRlck-Cre mice, in which the glucocorticoid receptor gene (GR) is deleted in thymocytes prior to selection. These mice were immunocompromised, with reduced polyclonal T cell proliferative responses to alloantigen, defined peptide antigens, and viral infection. This was not due to an intrinsic proliferation defect, because GR-deficient T cells responded normally when the TCR was cross-linked with antibodies or when the T cell repertoire was "fixed" with αß TCR transgenes. Varying the affinity of self ligands in αß TCR transgenic mice showed that affinities that would normally lead to thymocyte-positive selection caused negative selection, and alterations in the TCR repertoire of polyclonal T cells were confirmed by analysis of TCR Vß CDR3 regions. Thus, endogenous glucocorticoids are required for a robust adaptive immune response because of their promotion of the selection of T cells that have sufficient affinity for self, and the absence of thymocyte glucocorticoid signaling results in an immunocompromised state.
Asunto(s)
Inmunidad Adaptativa , Glucocorticoides/fisiología , Timocitos/inmunología , Animales , Antígenos Virales/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Glicoproteínas/inmunología , Isoantígenos/inmunología , Ganglios Linfáticos/citología , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Timocitos/metabolismo , Timocitos/fisiología , Timo/citología , Proteínas Virales/inmunologíaRESUMEN
p38 MAPKs are typically activated by upstream MAPK kinases that phosphorylate a Thr-X-Tyr motif in the activation loop. An exception is the T cell antigen receptor signaling pathway, which bypasses the MAPK cascade and activates p38alpha and p38beta by phosphorylation of Tyr-323 and subsequent autophosphorylation of the activation loop. Here we show that, unlike the classic MAPK cascade, the alternative pathway results primarily in mono-phosphorylation of the activation loop residue Thr-180. Recombinant mono-phosphorylated and dual phosphorylated p38alpha differed widely with regard to activity and substrate preference. Altered substrate specificity was reproduced in T cells in which p38 was activated by the alternative or classical MAPK pathways. These findings suggest that T cells have evolved a mechanism to utilize p38 in a specialized manner independent of and distinct from the classical p38 MAPK signaling cascade.
Asunto(s)
Receptores de Antígenos de Linfocitos T/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Activación Enzimática , Humanos , Cinética , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , Especificidad por Sustrato , Linfocitos T/enzimología , Acetato de Tetradecanoilforbol/farmacología , Treonina/metabolismo , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Mitogen-activated protein kinases (MAPKs) participate in signaling initiated by a wide variety of extracellular stimuli. MAPKs are most commonly activated by a series of phosphorylation events in which one kinase phosphorylates another, the "MAPK cascade". The cascade concludes with the dual phosphorylation of MAPKs on a conserved Thr-X-Tyr motif. In the case of the p38 MAPK, an exception to this paradigm has been found when signaling via the T cell antigen receptor (TCR). Rather than trigger the MAPK cascade, TCR-mediated stimulation activates proximal tyrosine kinases, which results in the phosphorylation of p38 on a noncanonical activating residue, Tyr-323. This phosphorylation activates p38 to phosphorylate third party substrates as well as its own Thr-Gyl-Tyr motif. Here we discuss the structural and functional implications of this alternative p38 activation pathway, which may provide a new target for tissue-specific pharmacologic inhibition.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencias de Aminoácidos , Animales , Activación Enzimática , Humanos , Modelos Biológicos , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Quinasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Treonina/química , Tirosina/químicaRESUMEN
The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor-activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45alpha, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a(-/-) mice had spontaneously increased p38 activity in the absence of 'upstream' MAPK kinase activation. The p38 from resting Gadd45a(-/-) T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45alpha in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45alpha, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.
Asunto(s)
Proteínas de Ciclo Celular/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Nucleares/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular/metabolismo , Activación Enzimática , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Tirosina/inmunología , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70 , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.