RESUMEN
The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.
Asunto(s)
Integrasas/genética , Lentivirus/enzimología , Proteínas Virales/genética , Animales , Células de la Médula Ósea/enzimología , Encéfalo/enzimología , Ciclo Celular , División Celular , Línea Celular , Supervivencia Celular , Células Cultivadas , Chlorocebus aethiops , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Ratones , Ratones EndogámicosRESUMEN
The major regulator of lipolysis in white adipocytes and brown adipocytes is cAMP and the actions of cAMP are mediated by protein kinase A (PKA). Multiple subunits of PKA, including RII beta, R1 alpha, C alpha, and C beta 1, are expressed in fat cells but the major holoenzyme assembled under normal conditions contains RII beta and C alpha. Targeted disruption of the RII beta gene in mice revealed that both white and brown adipocytes are capable of compensating by increasing the level of RI alpha. Nevertheless, the mice display a lean phenotype, have an elevated metabolic rate due to activation and induction of uncoupling protein in brown fat, and are resistant to diet-induced obesity and insulin resistance. Although the metabolic disturbances in white and brown fat tissue may explain most of the phenotypic changes, the loss of neuronal expression of RII beta may also contribute to the alterations in energy balance. Specific neuronal defects have been characterized that prevent the normal changes in gene expression seen with drugs that act through the dopaminergic pathway. The RII beta mutant mouse provides an interesting model of obesity resistance and demonstrates that chronic changes in the PKA signaling system can lead to stable alterations in energy storage and utilization.
Asunto(s)
Tejido Adiposo/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Animales , RatonesRESUMEN
New neurons are continuously born in the dentate gyrus of the adult mouse hippocampus, and regulation of adult neurogenesis is influenced by both genetic and environmental determinants. Mice of the 129/SvJ strain have significantly less hippocampal neurogenesis than other inbred mouse strains [1] and do not perform well in learning tasks. Here, the impact of environmental stimuli on brain plasticity during adulthood of 129/SvJ mice was studied using 'enriched environments' where mice receive complex inanimate and social stimulation [2,3]. In contrast to our earlier reports on mice of the C57BL/6 strain - which are competent in learning tasks and in which environmental stimulation did not influence cell proliferation [4,5] - environmentally stimulated 129/SvJ mice were found to have twice as many proliferating cells in the dentate gyrus compared with mice in standard housing. Environmental stimulation fostered the survival of newborn cells in 129/SvJ mice; this effect had also been seen in C57BL/6 mice. Phenotypic analysis of the surviving cells revealed that environmental stimulation resulted in 67% more new neurons. In combination with our earlier results, these data indicate a differential impact of inheritable traits on the environmental regulation of adult hippocampal neurogenesis. In addition, we observed behavioral changes in environmentally stimulated 129/SvJ mice.
Asunto(s)
Giro Dentado/citología , Giro Dentado/fisiología , Aprendizaje por Laberinto , Plasticidad Neuronal , Neuronas/citología , Neuronas/fisiología , Medio Social , Animales , División Celular , Femenino , Vivienda para Animales , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Especificidad de la EspecieRESUMEN
Motor behavior is modulated by dopamine-responsive neurons in the striatum, where dopaminergic signaling uses G-protein-coupled pathways, including those that result in the activation of cAMP-dependent protein kinase (PKA). The RIIbeta isoform of PKA is highly enriched in the striatum, and targeted disruption of the RIIbeta gene in mice leads to a dramatic reduction in total PKA activity in this region. Although the mutant mice show typical locomotor responses after acute administration of dopaminergic drugs, they display abnormalities in two experience-dependent locomotor behaviors: training on the rotarod task and locomotor sensitization to amphetamine. In addition, amphetamine induction of fos is absent, and the basal expression of dynorphin mRNA is reduced in the striatum. These results demonstrate that motor learning and the regulation of neuronal gene expression require RIIbeta PKA, whereas the acute locomotor effects of dopaminergic drugs are relatively unaffected by this PKA deficiency.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Actividad Motora/fisiología , Anfetamina/farmacología , Animales , Conducta Animal/fisiología , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dopamina/fisiología , Dopaminérgicos/farmacología , Relación Dosis-Respuesta a Droga , Dinorfinas/genética , Fertilidad/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Hibridación in Situ , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Locomoción/efectos de los fármacos , Locomoción/fisiología , Longevidad/genética , Masculino , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Neuronas/enzimología , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismoRESUMEN
Developing sensory systems are sculpted by an activity-dependent strengthening and weakening of connections. Long-term potentiation (LTP) and depression (LTD) in vitro have been proposed to model this experience-dependent circuit refinement. We directly compared LTP and LTD induction in vitro with plasticity in vivo in the developing visual cortex of a mouse mutant of protein kinase A (PKA), a key enzyme implicated in the plasticity of a diverse array of systems. In mice lacking the RIbeta regulatory subunit of PKA, we observed three abnormalities of synaptic plasticity in layer II/III of visual cortex in vitro. These included an absence of (1) extracellularly recorded LTP, (2) depotentiation or LTD, and (3) paired-pulse facilitation. Potentiation was induced, however, by pairing low-frequency stimulation with direct depolarization of individual mutant pyramidal cells. Together these findings suggest that the LTP defect in slices lacking PKA RIbeta lies in the transmission of sufficient net excitation through the cortical circuit. Nonetheless, functional development and plasticity of visual cortical responses in vivo after monocular deprivation did not differ from normal. Moreover, the loss of all responsiveness to stimulation of the originally deprived eye in most cortical cells could be restored by reverse suture of eyelids during the critical period in both wild-type and mutant mice. Such an activity-dependent increase in response would seem to require a mechanism like potentiation in vivo. Thus, the RIbeta isoform of PKA is not essential for ocular dominance plasticity, which can proceed despite defects in several common in vitro models of neural plasticity.
Asunto(s)
Envejecimiento/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Plasticidad Neuronal/fisiología , Corteza Visual/fisiología , Potenciales de Acción/fisiología , Animales , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Espacio Extracelular/fisiología , Lateralidad Funcional/fisiología , Potenciación a Largo Plazo/fisiología , Ratones , Fenómenos Fisiológicos Oculares , Técnicas de Placa-Clamp , Valores de Referencia , Privación Sensorial/fisiología , Sinapsis/fisiología , Visión Monocular/fisiología , Corteza Visual/crecimiento & desarrolloRESUMEN
The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-fos mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIbeta subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIbeta mutant mice fail to induce either c-fos or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.
Asunto(s)
Antipsicóticos/farmacología , Catalepsia , Cuerpo Estriado/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Regulación de la Expresión Génica/efectos de los fármacos , Haloperidol/farmacología , Animales , Conducta Animal/efectos de los fármacos , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ratones , Ratones Mutantes , Neurotensina/biosíntesis , Neurotensina/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , ARN Mensajero/aislamiento & purificación , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Sulpirida/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
To assess the contribution of PKA to injury-induced inflammation and pain, we evaluated nociceptive responses in mice that carry a null mutation in the gene that encodes the neuronal-specific isoform of the type I regulatory subunit (RIbeta) of PKA. Acute pain indices did not differ in the RIbeta PKA mutant mice compared with wild-type controls. However, tissue injury-evoked persistent pain behavior, inflammation of the hindpaw, and ipsilateral dorsal horn Fos immunoreactivity was significantly reduced in the mutant mice, as was plasma extravasation induced by intradermal injection of capsaicin into the paw. The enhanced thermal sensitivity observed in wild-type mice after intraplantar or intrathecal (spinal) administration of prostaglandin E2 was also reduced in mutant mice. In contrast, indices of pain behavior produced by nerve injury were not altered in the mutant mice. Thus, RIbeta PKA is necessary for the full expression of tissue injury-evoked (nociceptive) pain but is not required for nerve injury-evoked (neuropathic) pain. Because the RIbeta subunit is only present in the nervous system, including small diameter trkA receptor-positive dorsal root ganglion cells, we suggest that in inflammatory conditions, RIbeta PKA is specifically required for nociceptive processing in the terminals of small-diameter primary afferent fibers.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Isoenzimas/genética , Mutación , Nociceptores/fisiología , Nervio Ciático/lesiones , Heridas no Penetrantes/patología , Animales , Capsaicina/farmacología , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Umbral Diferencial , Dinoprostona/farmacología , Expresión Génica , Marcación de Gen , Calor , Ratones , Ratones Mutantes , Neuritis/etiología , Neuritis/fisiopatología , Dolor , Umbral Sensorial/efectos de los fármacos , Transgenes/genética , Heridas no Penetrantes/fisiopatologíaRESUMEN
In mammals, the cAMP-dependent protein kinase (PKA) family of enzymes is assembled from the products of four regulatory and two catalytic subunit genes, all of which are expressed in neurons. Specific isoforms of PKA display differences in biochemical properties and subcellular localization, but it has been difficult to ascribe specific physiological functions to any given isoform. The recent development of gene knockout and transgenic mouse models has allowed for a more integrated examination of the in vivo roles of specific PKA isoforms in gene expression, synaptic plasticity, and behaviour.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Vías Nerviosas/fisiología , Animales , Hipocampo/metabolismo , Ratones , Modelos BiológicosRESUMEN
The cAMP-dependent protein kinase holoenzyme is assembled from regulatory (R) and catalytic (C) subunits that are expressed in tissue-specific patterns. Despite the dispersion of the R and C subunit genes to different chromosomal loci, mechanisms exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved. We have created null mutations in the RIbeta and RIIbeta regulatory subunit genes in mice, and find that both result in an increase in the level of RIalpha protein in tissues that normally express the beta isoforms. Examination of RIalpha mRNA levels and the rates of RIalpha protein synthesis in wild type and RIIbeta mutant mice reveals that the mechanism of this biochemical compensation by RIalpha does not involve transcriptional or translational control. These in vivo findings are consistent with observations made in cell culture, where we demonstrate that the overexpression of Calpha in NIH 3T3 cells results in increased RIalpha protein without increases in the rate of RIalpha synthesis or the level of RIalpha mRNA. Pulse-chase experiments reveal a 4-5-fold increase in the half-life of RIalpha protein as it becomes incorporated into the holoenzyme. Compensation by RIalpha stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células 3T3 , Tejido Adiposo/enzimología , Animales , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ratones , Ratones Mutantes , Biosíntesis de ProteínasRESUMEN
Cyclic AMP is an important second messenger in the coordinated regulation of cellular metabolism. Its effects are mediated by cAMP-dependent protein kinase (PKA), which is assembled from two regulatory (R) and two catalytic (C) subunits. In mice there are four R genes (encoding RI alpha, RI beta, RII alpha, and RII beta) and two C gene (encoding C alpha and C beta), expressed in tissue-specific patterns. The RII beta isoform is abundant in brown and white adipose tissue and brain, with limited expression elsewhere. To elucidate its functions, we generated RII beta knockout mice. Here we report that mutants appear healthy but have markedly diminished white adipose tissue despite normal food intake. They are protected against developing diet-induced obesity and fatty livers. Mutant brown adipose tissue exhibits a compensatory increase in RI alpha, which almost entirely replaces lost RII beta, generating an isoform switch. The holoenzyme from mutant adipose tissue binds cAMP more avidly and is more easily activated than wild-type enzyme. This causes induction of uncoupling protein and elevations of metabolic rate and body temperature, contributing to the lean phenotype. Our results demonstrate a role for the RII beta holoenzyme in regulating energy balance and adiposity.
Asunto(s)
Proteínas Portadoras , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Delgadez/enzimología , Tejido Adiposo/enzimología , Tejido Adiposo Pardo/enzimología , Animales , Peso Corporal/fisiología , AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Grasas de la Dieta/farmacología , Ingestión de Alimentos , Metabolismo Energético , Femenino , Canales Iónicos , Isoenzimas/genética , Isoenzimas/metabolismo , Leptina , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales , Unión Proteica , Proteínas/metabolismo , Delgadez/genética , Triglicéridos/metabolismo , Proteína Desacopladora 1RESUMEN
Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hipocampo/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Animales , Conducta Animal/fisiología , Sitios de Unión , Encéfalo/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Genes , Hipocampo/enzimología , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Transducción de SeñalRESUMEN
Using a genetic approach, we assessed the effects of mutations in protein kinase A (PKA) on long-term potentiation (LTP) in the mossy fiber pathway and its relationship to spatial and contextual learning. Ablation by gene targeting of the C beta 1 or the RI beta isoform of PKA produces a selective defect in mossy fiber LTP, providing genetic evidence for the role of these isoforms in the mossy fiber pathway. Despite the elimination of mossy fiber LTP, the behavioral responses to novelty, spatial learning, and conditioning to context are unaffected. Thus, contrary to current theories about hippocampal function, mossy fiber LTP does not appear to be required for spatial or contextual learning. In the absence of mossy fiber LTP, adequate spatial and contextual information might reach the CA1 region via other pathways from the entorhinal cortex.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Hipocampo/citología , Potenciación a Largo Plazo/genética , Aprendizaje por Laberinto/fisiología , Conducta Espacial/fisiología , Animales , Colforsina/farmacología , Condicionamiento Psicológico/fisiología , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electrofisiología , Femenino , Hipocampo/fisiología , Isoenzimas , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MutaciónRESUMEN
The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Animales , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Electrofisiología , Hipocampo/anatomía & histología , Hipocampo/enzimología , Homocigoto , Técnicas In Vitro , Ratones , Ratones Mutantes , Ratones Transgénicos , ARN Mensajero/análisisAsunto(s)
Genoma , Ratones Noqueados , Ratones/genética , Animales , Desarrollo Embrionario y Fetal/genética , Predicción , Expresión Génica , MutaciónRESUMEN
High concentrations of tumor necrosis factor (TNF) alpha have been detected in the plasma of patients undergoing immunotherapy with interleukin 2 (IL-2), suggesting that this cytokine may play a role in the fever and shocklike state induced by the administration of high-dose IL-2. Dexamethasone has been shown to inhibit the synthesis of TNF by monocytes activated in vitro by endotoxin. To determine if dexamethasone can exert a similar suppressive effect on IL-2-induced TNF synthesis in vivo, the concentration of TNF alpha was measured in plasma samples serially obtained (a) from cancer patients participating in a phase I dose escalation clinical trial with high-dose IL-2 administered in conjunction with dexamethasone (IL-2/Dex) and (b) from patients participating in concurrent studies with IL-2 alone. In contrast to the high plasma levels of TNF alpha detected in patients receiving IL-2 alone, TNF levels in most of the IL-2/Dex patients remained below the threshold of detectability of our TNF radioimmunoassay. The concurrent administration of dexamethasone also prevented the IL-2-induced increase in serum levels of C-reactive protein, a hepatic acute phase reactant whose synthesis is regulated by proinflammatory cytokines such as TNF. The steroid-treated patients also failed to develop the neutrophil chemotactic defect characteristic of IL-2 recipients. The concomitant administration of dexamethasone increased the maximum tolerated dose of IL-2 approximately threefold and markedly reduced the hypotension and organ dysfunction ordinarily observed in these patients. These results demonstrate that dexamethasone inhibits the release of TNF into the circulation of patients undergoing immunotherapy with IL-2. They further suggest that the altered spectrum and reduced severity of IL-2 side effects observed in patients receiving dexamethasone may be attributable in part to the suppressive effect of steroids on IL-2-induced TNF synthesis.
Asunto(s)
Dexametasona/farmacología , Interleucina-2/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína C-Reactiva/análisis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Relación Dosis-Respuesta a Droga , Evaluación de Medicamentos , Humanos , Interleucina-2/efectos adversos , Interleucina-2/toxicidad , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , FenotipoRESUMEN
We previously demonstrated that IL-2 promotes the adhesion of NK cells to endothelial cells (EC) and that EC are readily lysed by lymphokine-activated killer (LAK) cells in vitro, suggesting that cell mediated endothelial injury may contribute to the capillary leak syndrome observed in patients treated with IL-2. In this investigation, we sought to determine the effects of EC activation on the in vitro susceptibility of EC to LAK cell-mediated cytolysis. Despite increased binding of CD16+ lymphocytes to TNF-activated EC monolayers, prior exposure of EC to any of several IL-2-inducible cytokines including TNF-alpha, IL-1 beta, and IFN-gamma not only failed to render the EC more vulnerable to cytolysis but increased their resistance to LAK cells in 111Indium release cytolysis assays. This decrement in susceptibility to cytolysis resulting from prior exposure to cytokines preceded any detectable increase in HLA class I or II Ag expression. In cold target competition experiments with LAK cell effectors and radiolabeled K562 target cells, TNF-primed EC were no more competitive than unstimulated EC, and in assays with unstimulated PBMC effectors, the addition of unlabeled TNF-activated EC actually increased the cytolysis of the radiolabeled tumor cells. The effects of various cytokines and lymphocyte preparations on EC permeability were also evaluated. In these experiments, saphenous vein EC were cultured on porous filter disks, exposed to cytokines or lymphocytes, and the diffusion of 125I-BSA through the filters was then measured. Exposure to IL-2, IFN-gamma, or TNF-alpha did not increase the diffusion of the BSA through the EC-coated filters, whereas LAK cells markedly increased their permeability. Consistent with the results of the cytolysis assays, pretreatment of the EC with TNF, IL-1, or IFN-gamma diminished the LAK cell-induced increase in BSA diffusion. These results suggest that although circulating IL-2-inducible cytokines such as TNF and IFN-gamma may activate EC in vivo and contribute to lymphocyte margination and lymphopenia, they may not be directly responsible for the IL-2-induced capillary leak syndrome and may actually protect EC from LAK cell-mediated injury.
Asunto(s)
Factores Biológicos/fisiología , Permeabilidad Capilar , Citotoxicidad Inmunológica , Endotelio Vascular/inmunología , Interleucina-2/uso terapéutico , Células Asesinas Naturales/inmunología , Antígenos de Diferenciación , Factores Biológicos/biosíntesis , Adhesión Celular , Citocinas , Endotelio Vascular/patología , Filtración/instrumentación , Antígenos HLA/metabolismo , Humanos , Inmunidad Celular , Inmunidad Innata , Interferón gamma/farmacología , Linfocitos/fisiología , Fenotipo , Receptores Fc , Receptores de IgG , Vena Safena , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.