Asunto(s)
Dependencia de Morfina/inmunología , Receptores Opioides mu/inmunología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Corticosterona/sangre , Humanos , Inmunidad/efectos de los fármacos , Inmunosupresores/toxicidad , Ratones , Ratones Noqueados , Morfina/toxicidad , Tamaño de los Órganos/efectos de los fármacos , ARN/genética , ARN/metabolismo , Receptores Opioides delta/inmunología , Receptores Opioides kappa/inmunología , Receptores Opioides mu/deficiencia , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/genética , Timo/efectos de los fármacos , Timo/patologíaRESUMEN
Vascular endothelial growth factor (VEGF) is an angiogenic mitogen, specific for endothelial cells. Hypoxia-induced VEGF in endothelial cells and cardiomyocytes leads to autocrine and paracrine stimulation, respectively. During myocardial ischemia, VEGF is upregulated in the endothelium and myocardium, and may mediate angiogenesis. Morphine sulfate is commonly used in pain relief for patients with acute myocardial infarction. We investigated the effect of morphine sulfate on VEGF expression in cultured endothelial cells and cardiac myocytes subjected to hypoxia. Enzyme-linked immunosorbent assays showed that morphine sulfate significantly inhibited hypoxia-induced VEGF expression in mouse heart microvascular endothelial cells (SMHEC4), primary cultures of human umbilical vein endothelial cells (HUVECs) and in primary cultures of rat cardiac myocytes (P<0.05). Real time reverse transcriptase polymerase chain reaction showed that morphine treatment (100 ng/ml) of hypoxic HUVECs resulted in a significant reduction in mRNA levels of VEGF(121) and VEGF(165) isoforms. Transfection of HUVECs with a human VEGF promoter-luciferase construct showed that hypoxia-induced transcriptional activation of VEGF was markedly inhibited by morphine sulfate (P<0.05). Phosphatidyl inositol-3 kinase and protein kinase C-mediated activation of the VEGF promoter was also inhibited by morphine. The opioid antagonist naloxone significantly reversed the inhibitory effects of morphine in endothelial cells suggesting the involvement of opioid receptors. Our results show that the inhibitory effects of morphine on hypoxia-induced VEGF expression in endothelial cells and cardiac myocytes can lead to a decrease in the autocrine and paracrine stimulation and hence limit neovascularization of the ischemic myocardium.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/metabolismo , Linfocinas/genética , Morfina/farmacología , Miocardio/citología , Narcóticos/farmacología , Animales , Células Cultivadas , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Corazón/efectos de los fármacos , Linfocinas/efectos de los fármacos , Linfocinas/metabolismo , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , ARN Mensajero/efectos de los fármacos , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The major objective of this paper is to characterize the mechanism by which morphine modulates lymphocyte function and if these effects are mediated through the mu-opioid receptor. We evaluated the in vitro effects of morphine on lymphocytes that were freshly isolated from lymph nodes from wild type (WT) and mu-opioid receptor knock-out (MORKO) mice. Results show that morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-gamma synthesis in a dose-dependent manner. This effect was abolished in lymph node cells isolated from MORKO mice. The inhibition of T-cell function with low-dose morphine was associated with an increase in caspase-3- and caspase-8-mediated apoptosis. The inhibition of T-cell function with high-dose morphine was associated with an increase in the inducible NO synthase mRNA expression. N(G)-nitro-L-arginine methyl ester (L-NAME) antagonized the apoptosis induced by high-dose morphine. Our results suggest that low-dose morphine, through the mu-opioid receptor, can induce lymph node lymphocyte apoptosis through the cleavage activity of caspase-3 and caspase-8. Morphine at high doses induces NO release. This effect of morphine is also mediated through the mu-opioid receptor present on the surface of macrophages.
Asunto(s)
Caspasas/fisiología , Ganglios Linfáticos/inmunología , Morfina/farmacología , Óxido Nítrico/fisiología , Linfocitos T/inmunología , Animales , Apoptosis , Caspasa 3 , Caspasa 8 , Caspasa 9 , Células Cultivadas , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Morfina/administración & dosificación , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/biosíntesis , Receptores Opioides mu/genética , Receptores Opioides mu/fisiologíaRESUMEN
BACKGROUND: Failure of cell-mediated immunity is thought to increase the morbidity and mortality rates after trauma and major surgical procedures and to be the result, in part, of a redirection of CD4(+) T cells toward T(H2) differentiation. We tested the hypothesis that morphine treatment after injury promotes T(H2) differentiation of precursor T cells through the mu-opioid receptor. METHODS: Human peripheral blood mononuclear cells (PBMCs) or splenocytes from either wild type or mu-opioid receptor knock-out mice were treated in vitro with either vehicle or morphine and then stimulated with anti-CD3/anti-CD28. The supernatant was assayed for T(H1) (interleukin-2 [IL-2], interferon gamma [IFN gamma]) and T(H2) (IL-4, IL-5) cytokines (enzyme-linked immunosorbent assay). Morphine regulation of IL-4 transcription was investigated in PBMCs (IL-4 messenger RNA, nuclear factor of activated T-cells) and Jurkat T cells transfected with a murine IL-4 promoter-luciferase construct. Morphine-induced nuclear factor of activated T-cell (NFAT) binding was assayed with the electromobility shift assay in Jurkat T cells. RESULTS: Morphine treatment of PBMCs decreases IL-2 and IFN gamma and increases IL-4 and IL-5 as a function of morphine concentration. Morphine treatment in wild type splenocytes inhibited IFN gamma and stimulated IL-4 protein synthesis. Changes in cytokine synthesis were abolished in mu-opioid receptor knockout mice. Morphine treatment increases IL-4 messenger RNA accumulation in PBMCs and increases IL-4 promoter activity in Jurkat T cells. Morphine increases NFAT nuclear protein binding to an NFAT DNA response element. CONCLUSIONS: We conclude that morphine treatment promotes T(H2) differentiation through a mu-opioid receptor mechanism and that morphine treatment increases IL-4 transcription, in part, through an NFAT mechanism.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Células Th2/citología , Células Th2/efectos de los fármacos , Animales , Antígenos CD28 , Complejo CD3/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Células Jurkat , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/inmunología , Receptores Opioides mu/genética , Receptores Opioides mu/inmunología , Bazo/citología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , TransfecciónRESUMEN
Mu-opioid receptor knockout mice (MORKO), were used to address two questions: (1) if morphine induced decrease in thymic weight and cell distribution is mediated by the mu-opioid receptor and (2) the role of corticosteroids in morphine mediated alteration in thymic cell distribution. Our result show that morphine mediated increase in plasma corticosterone is mediated by the mu-opioid receptor since morphine at doses as high as 25 mg/kg-body weight does not increase plasma corticosterone levels in the MORKO. In addition, we have also shown that morphine treatment results in the differentiation of CD4+CD8+ (double positive cells) to single positive CD4+ cells while dexamethasone treatment results in the deletion of CD4+CD8+ (double positive) cells.
Asunto(s)
Analgésicos Opioides/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Morfina/farmacología , Hipófisis/efectos de los fármacos , Receptores Opioides mu/genética , Timo/citología , Hormona Adrenocorticotrópica/sangre , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Corticosterona/sangre , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Glucocorticoides/farmacología , Sistema Hipotálamo-Hipofisario/inmunología , Ratones , Ratones Noqueados , Tamaño de los Órganos , Hipófisis/inmunología , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides mu/inmunología , Timo/efectos de los fármacosRESUMEN
BACKGROUND: Interleukin-4 (IL-4) treatment after lipopolysaccharide (LPS) induction inhibits macrophage (Mphi) IL-12 synthesis; however, IL-4 pretreatment (PreTx) primes the Mphi for increased LPS-induced IL-12 production. In this study we study the role of c-fos in the IL-4 priming of Mphi IL-12 synthesis. METHODS: With a murine in vitro peritoneal M phi model, we studied the effect of either c-fos deficiency (wild type, WT; homozygous c-fos knockout, Homo KO) or c-fos overexpression to study the role of c-fos in IL-4 priming of LPS-induced M phi IL-12 synthesis. RESULTS: (1) We first show that IL-4 PreTx results in a 72% decrease in Mphi c-fos mRNA compared with vehicle PreTx. (2) With respect to IL-12 p70 protein, IL-4 PreTx in the WT group increased LPS-induced Mphi IL-12 p70 2.2-fold compared with vehicle PreTx. Compared with vehicle PreTx in the WT group, vehicle PreTx in the Homo KO group followed by LPS stimulation resulted in a 2.8-fold increase in IL-12 p70 in the Homo KO group. IL-4 PreTx did not significantly increase IL-12 p70 over vehicle PreTx in the Homo KO group. (3) We studied the effect of c-fos overexpression on LPS-induced Mphi IL-12 production when primed with IL-4. Overexpression of c-fos completely inhibited IL-4 primed LPS-induced IL-12 p70 protein synthesis. CONCLUSIONS: These data demonstrated that down-regulation of c-fos is an integral part of the IL-4 priming process for Mphi IL-12 production.
Asunto(s)
Interleucina-12/genética , Interleucina-4/farmacología , Macrófagos Peritoneales/inmunología , Proteínas Proto-Oncogénicas c-fos/fisiología , Animales , Células Cultivadas , Genes fos , Homocigoto , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-fos/deficiencia , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , TransfecciónRESUMEN
Gram-negative sepsis syndrome is an increasingly common complication in medical and surgical patients. The molecular and cellular mechanisms underlying this dreaded complication are yielding to investigation. These studies have led to a multiplicity of targets for novel therapies. Despite highly promising results in many animal studies, clinical studies have been disappointing.
Asunto(s)
Infecciones por Bacterias Gramnegativas , Síndrome de Respuesta Inflamatoria Sistémica/microbiología , Síndrome de Respuesta Inflamatoria Sistémica/terapia , Infecciones Urinarias/microbiología , Infecciones Urinarias/terapia , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Gramnegativas/fisiopatología , Infecciones por Bacterias Gramnegativas/terapia , Humanos , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/fisiopatología , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/metabolismo , Infecciones Urinarias/fisiopatologíaRESUMEN
BACKGROUND: Interleukin 12 (IL-12) p70 is a heterodimeric protein (p35, p40 subunits) that promotes T-helper TH1-type cytokine response. In critically ill patients, after severe trauma or sepsis, IL-12 production is markedly impaired. We tested the hypothesis that deficiency of the transcription factor c-fos will increase macrophage IL-12 production. METHODS: We harvested adherent peritoneal macrophages harvested from wild-type (WT), heterozygous c-fos knockout (Hetero KO), or homozygous c-fos knockout (Homo KO) mice and investigated lipopolysaccharide (LPS)-induced IL-12 p70 protein synthesis (by enzyme-linked immunosorbent assay), IL-12 p35 and IL-12 p40 messenger RNA accumulation (mRNA) (by reverse transcriptase-polymerase chain reaction), and the transcription rate (by nuclear runoff). RESULTS: (1) LPS treatment compared with vehicle increases c-fos mRNA accumulation 5-fold and AP-1 DNA protein binding (electrophoretic mobility shift assay), which precedes either IL-12 p35 or IL-12 p40 mRNA accumulation. (2) LPS induces a significant increase in IL-12 p70 protein, IL-12 p40 mRNA, and the transcription rate in the Homo KO group compared with either the Hetero KO or WT groups. (3) Compared with vehicle control, we demonstrate that interferon gamma priming increases LPS-stimulated macrophage IL-12 p70 protein in the Hetero KO or WT groups to the level of the Homo KO group but has no significant effect on the Homo KO group. CONCLUSIONS: These data suggest that deficiency of the transcription factor c-fos increases LPS-induced macrophage IL-12 production, possibly by simulating the effect of interferon gamma priming.
Asunto(s)
Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/deficiencia , Animales , Interferón gamma/farmacología , Macrófagos/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/análisis , Factor de Transcripción AP-1/metabolismoRESUMEN
Emergent or elective surgical procedures may be complicated by sepsis, resulting in critical illness that can lead to organ failure and death. The opioid drug, morphine is widely used to alleviate pain in post-surgical patients; however, it is well documented that chronic treatment of mice with morphine affects the proliferation, differentiation and function of immune cells. Thus, morphine might be expected to exacerbate the effects of sepsis, which also compromises the immune system. To test this notion, we investigated the effect on several immune functions of a clinical dose of morphine (4 mg/kg) superimposed upon a lipopolysaccharide (LPS)-induced infection model. Our results show that this relatively low dose of morphine, though generally having no effects on immune parameters by itself, significantly augmented LPS responses. A clinical dose of morphine (4 mg/kg body weight) superimposed upon an animal model of sepsis resulted in a significant increase in mortality at 48 h. In the absence of the drug, most septic animals died after 96 h. Phenotypic responses such as, decreased thymic cellularity, compromised mitogenic response and inhibition of IL-2 synthesis that are evident at 48-72 h after LPS injection appear as early as 24 h in animals that receive morphine in addition to LPS. In addition, our results show that in T cells there is a shift from TH1 type cytokine elaboration to a TH2 type cytokine elaboration in animals that receive both LPS and morphine.
Asunto(s)
Endotoxemia/inducido químicamente , Lipopolisacáridos/farmacología , Morfina/farmacología , Narcóticos/farmacología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Endotoxemia/mortalidad , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/inmunología , Inmunofenotipificación , Interferón gamma/análisis , Interferón gamma/inmunología , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-12/análisis , Interleucina-12/inmunología , Interleucina-2/análisis , Interleucina-2/inmunología , Interleucina-4/análisis , Interleucina-4/inmunología , Interleucina-6/análisis , Interleucina-6/genética , Interleucina-6/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos ICR , Tamaño de los Órganos , Hipófisis/química , Hipófisis/inmunología , ARN Mensajero/análisis , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Timo/citología , Timo/inmunología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The role of the mu-opioid receptor in immune function was investigated using mu-opioid receptor knockout mice (MOR-KO). Morphine modulation of several immune functions, including macrophage phagocytosis and macrophage secretion of TNF-alpha, was not observed in the MOR-KO animals, suggesting that these functions are mediated by the classical mu-opioid receptor. In contrast, morphine reduction of splenic and thymic cell number and mitogen-induced proliferation were unaffected in MOR-KO mice, as was morphine inhibition of IL-1 and IL-6 secretion by macrophages. These latter results are consistent with morphine action on a naloxone insensitive morphine receptor, a conclusion supported by previous studies characterizing a nonopioid morphine binding site on immune cells. Alternatively, morphine may act either directly or indirectly on these cells, by a mechanism mediated by either delta or kappa opioid receptors.
Asunto(s)
Sistema Inmunológico/efectos de los fármacos , Ratones Noqueados/inmunología , Morfina/farmacología , Receptores Opioides mu/inmunología , Receptores Opioides mu/fisiología , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Modelos Animales de Enfermedad , Implantes de Medicamentos , Exones/genética , Marcación de Gen , Terapia de Inmunosupresión , Interleucina-2/análisis , Interleucina-2/metabolismo , Ligandos , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Noqueados/genética , Mitógenos/farmacología , Morfina/administración & dosificación , Naltrexona/farmacología , Tamaño de los Órganos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Receptores Opioides mu/genética , Bazo/efectos de los fármacos , Trastornos Relacionados con Sustancias , Timidina/metabolismo , Timo/efectos de los fármacos , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Inmunosupresores/farmacología , Morfina/farmacología , Sepsis/inmunología , Animales , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Sistema Inmunológico/efectos de los fármacos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-2/biosíntesis , Lipopolisacáridos , Ratones , Ratones Endogámicos ICR , Tamaño de los Órganos/efectos de los fármacos , Fenotipo , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sepsis/inducido químicamente , Análisis de Supervivencia , Timo/citología , Timo/efectos de los fármacosRESUMEN
BACKGROUND: Animal models of stress and sepsis demonstrate increased hypophyseal gene expression of the transcription factor c-fos and the cytokines interleukin-1 and interleukin-6. Chronic central nervous system exposure to interleukin-1 results in hypermetabolism, accelerated nitrogen loss, anorexia, and cachexia. We test the hypothesis that the host response to ovarian carcinoma recapitulates the host response to sepsis regarding the elaboration of the transcription factors and cytokines in the central nervous system, liver, and lung. MATERIALS AND METHODS: Nude mice were seeded intraperitoneally with either ovarian carcinoma (MA-148) or vehicle. The animal subjects were observed for 5 weeks and sacrificed for brain, pituitary, lung, and liver mRNA. We studied the mRNA accumulation of the transcription factors c-fos, c-jun, and C/EBP alpha and the cytokines interleukin-1 and interleukin-6 using reverse-transcriptase polymerase chain reaction. RESULTS: Compared with the control, ovarian carcinoma in the mouse model resulted in the following: (1) Pituitary c-fos and c-jun mRNA increased 3-fold (P = 0.012) and 6-fold (P < 0.001), respectively; (2) pituitary IL-1 and IL-6 mRNA increased 4-fold (P < 0.001) and 8-fold (P = 0.037), respectively; (3) liver c-fos mRNA increased > 8-fold (P < 0.001); and (4) lung C/EBP alpha mRNA decreased greater than 10-fold (P < 0.001). CONCLUSIONS: We conclude that the host response to ovarian carcinoma in this animal model recapitulates many aspects of the host response to bacterial sepsis especially concerning pituitary gene expression. These data suggest that, as in sepsis, a hypothalamic-hypophyseal-mediated cytokine response in ovarian carcinoma may result in hypermetabolism, accelerated nitrogen loss, anorexia, and cachexia.
Asunto(s)
Carcinoma/metabolismo , Infecciones/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Neoplasias Ováricas/metabolismo , Hipófisis/fisiopatología , Animales , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/genética , Femenino , Interleucina-1/genética , Interleucina-6/genética , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Chronic use of morphine affects the immune system and predisposes an individual to opportunistic infections. Macrophages play an important role in conferring a first line of defense against invading pathogens. Understanding the mechanisms by which morphine affects the functioning of macrophages would have significant therapeutic benefit in treatment against infections such as HIV and AIDS related syndromes. Two of the major cytokines secreted by activated macrophages are Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Our studies show that morphine differentially modulates lipopolysaccharide (LPS) induced expression of IL-6 and TNF-alpha. Nanomolar concentrations of morphine synergize with LPS and augment the secretion of both IL-6 and TNF-alpha. However, at micromolar concentrations morphine inhibits LPS induced synthesis of IL-6 and TNF-alpha. Expression of both these cytokine genes is dependent on the activation of a transcription factor, NF kappa B. Interestingly, morphine treatment also modulated the activation of NF kappa B by LPS. Pretreatment with a low dose of morphine (nanomolar) resulted in an increase in NF kappa B activation. In contrast pretreatment with a high dose of morphine (micromolar) led to a significant decrease in NF kappa B activation. Furthermore unlike the augmentation which was naloxone reversible, the inhibition of NF kappa B by morphine was not reversed by naloxone, suggesting the involvement of a nonclassical opioid receptor.
Asunto(s)
Proteínas I-kappa B , Macrófagos Peritoneales/efectos de los fármacos , Morfina/farmacología , FN-kappa B/metabolismo , Activación Transcripcional/efectos de los fármacos , Animales , Proteínas de Unión al ADN/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Ratones , Naloxona/farmacología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Chronic treatment of mice with morphine affects the proliferation, differentiation, and function of immune cells. In the present study, we investigated the mechanism by which morphine inhibits phytohemagglutinin (PHA)/interleukin-1 (IL-1)-induced thymocyte proliferation. When compared to control cultures, morphine-treated thymocytes showed decreased steady-state levels of bioactive IL-2 and IL-2 mRNA. The reduced IL-2 concentration and reduced transcript levels correlated well with a decreased rate of synthesis of IL-2 mRNA as determined by nuclear runoff assays. Subsequent studies showed that morphine treatment affected transcriptional control elements of the IL-2 promoter by inhibiting the synthesis of a specific trans-activating nuclear factor, c-Fos. c-Fos mRNA levels measured by semiquantitative RT-PCR were significantly decreased in thymocytes following treatment with morphine and activation with PHA and IL-1. Under identical conditions, c-Jun mRNA levels were not altered. Electrophoretic mobility shift studies with the AP-1 consensus oligonucleotide showed significantly decreased levels of AP-1-protein complex formation in nuclear extracts prepared from morphine-treated cells. These studies demonstrate for the first time that opioid alkaloids such as morphine can impair mitogen-lymphokine-activated thymocyte proliferation by interfering with transcriptional activation of the IL-2 gene.
Asunto(s)
Interleucina-2/genética , Morfina/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-2/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero , Timo/citología , Factor de Transcripción AP-1/metabolismoRESUMEN
Chronic treatment of mice with morphine selectively abrogates the terminal differentiation of committed bone marrow progenitor cells to form macrophage colony forming units. To understand the molecular mechanisms involved in morphine-mediated suppression of myeloid cell differentiation, we investigated the use of a macrophage cell line, Bac 1.2 F5. In vitro proliferation of this cell line is dependent on the exogenous supply of macrophage colony stimulating factor. Treatment of Bac 1.2F5 cells in vitro with morphine showed a dose-dependent inhibition of proliferation which was associated with morphological changes. Characterization of the binding site revealed that the binding site for morphine on these cells is different from the classical opioid receptors described in the brain. In addition to the putative novel class of morphine receptors, Bac 1.2F5 cells also expressed the delta opioid receptors as determined by RT-PCR analyses. These studies show that Bac 1.2F5 cells are suitable for the molecular characterization of opioid effects on the proliferation and differentiation of myeloid progenitor cells.
Asunto(s)
Macrófagos/efectos de los fármacos , Morfina/toxicidad , Animales , Secuencia de Bases , Unión Competitiva/efectos de los fármacos , Unión Competitiva/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Línea Celular , ADN Complementario/aislamiento & purificación , Leucina Encefalina-2-Alanina/farmacología , Macrófagos/citología , Ratones , Datos de Secuencia Molecular , Receptores Opioides/química , Receptores Opioides/efectos de los fármacos , Receptores Opioides/genéticaRESUMEN
Peritoneal sepsis results in downregulation of the gene that codes for the hepatic mitochondrial enzyme carnitine palmitoyltransferase (CPT). The inhibition of hepatic CPT transcription by sepsis is thought to be mediated, in part, by increased expression of the leucine-zipper DNA transcription factor c-fos. In a cecal ligation and puncture (CLP) model, we examined the temporal effect of surgical treatment (cecal excision) on sepsis-induced inhibition of CPT gene expression. We investigated the hypothesis that Fos protein level will inversely correlate with the regulation of CPT gene expression. Specifically, we studied hepatic Fos nucleoprotein accumulation and CPT gene expression as measured by total mitochondrial CPT activity, CPT protein, and CPT mRNA. We investigated the following groups: (i) CLP followed by cecal excision 6, 12, or 24 hr following initial insult, (ii) concurrent CLP control group, and (iii) concurrent sham CLP reference group. When measured 48 hr following initial surgical insult, we conclude that: (i) in the absence of surgical treatment, peritoneal contamination results in a decrease in hepatic CPT gene expression and an increase in Fos nucleoprotein accumulation; (ii) surgical treatment at 6 or 12 hr following initial insult prevents the downregulation in hepatic CPT gene expression and does not result in Fos nucleoprotein accumulation; and (iii) surgical treatment at 24 hr following insult did not prevent the downregulation of hepatic CPT gene expression and results in an increase in hepatic Fos nucleoprotein accumulation. These data are consistent with the hypothesis that sepsis-induced regulation of hepatic c-fos gene expression, in part, is responsible for the downregulation of CPT gene expression.
Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Expresión Génica , Hígado/fisiopatología , Enfermedades Peritoneales/enzimología , Enfermedades Peritoneales/cirugía , Proteínas Proto-Oncogénicas c-fos/metabolismo , Sepsis/enzimología , Sepsis/cirugía , Animales , Carnitina O-Palmitoiltransferasa/genética , Masculino , Mitocondrias Hepáticas/enzimología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de Transcripción AP-1/genéticaRESUMEN
Amiodarone, a widely used antiarrhythmic drug, is associated with pulmonary toxicity, with an estimated mortality of 1% to 33%. Standard treatment for amiodarone pulmonary toxicity (APT) has been discontinuance of the drug and steroid therapy. We report a case of APT that recurred after withdrawal of steroids and failed to respond to reinstatement of steroid therapy. Recurrent APT is a rare clinical entity that has been reported only twice in recent literature.
Asunto(s)
Amiodarona/efectos adversos , Antiarrítmicos/efectos adversos , Pulmón/efectos de los fármacos , Síndrome de Dificultad Respiratoria/inducido químicamente , Anciano , Humanos , Masculino , Recurrencia , Taquicardia Ventricular/tratamiento farmacológicoRESUMEN
OBJECTIVE: To investigate the hypothesis that a central dopaminergic mechanism may regulate hepatic c-fos and c-jun gene expression following peritoneal sepsis. METHODS: First, dopamine or vehicle was instilled into a stereotaxically placed intracerebral-ventricular (ICV) cannula with or without D1 (SCH 23390) or D2 (haloperidol) antagonist pretreatment in a rat model, and the effect on hepatic c-fos or c-jun protein expression was investigated. Second, we investigated the effect of haloperidol and vehicle treatment following cecal ligation and puncture (CLP)-induced sepsis with respect to hepatic c-fos protein expression, c-jun protein expression, and survival. RESULTS: Intracerebral-ventricular dopamine treatment increased hepatic c-fos immunoreactive protein but had no effect on hepatic c-jun immunoreactive protein expression. Pretreatment with SCH 23390 inhibited ICV dopamine treatment-induced hepatic c-fos immunoreactive protein expression. Haloperidol pretreatment synergized with ICV dopamine treatment to overexpress hepatic c-fos protein. Haloperidol treatment significantly increased CLP-induced hepatic c-fos and c-jun protein expression and improved survival following CLP. CONCLUSIONS: Hepatic c-fos protein expression may be regulated, in part, by a central nervous system-mediated dopaminergic D1 receptor mechanism. Treatment with the D2 receptor antagonist, haloperidol, increases sepsis-induced hepatic c-fos and c-jun protein expression and improves survival following peritoneal contamination.
Asunto(s)
Dopamina/fisiología , Genes fos/genética , Genes jun/genética , Hígado/metabolismo , Receptores Dopaminérgicos/fisiología , Sepsis/metabolismo , Animales , Encéfalo/fisiología , Antagonistas de Dopamina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The transcription factors C/EBP alpha and C/EBP beta belong to the leucine-zipper C/EBP (CCAAT/enhancer binding protein) family of DNA-binding proteins. C/EBP alpha and C/EBP beta are expressed in the liver and are implicated in the control of transcriptional events following following sepsis. It is hypothesized that inhibition of C/EBP alpha gene expression following sepsis may lead to some of the phenotypic features we recognize as sepsis syndrome such as decreased visceral protein (albumin) synthesis. In this study we demonstrate that C/EBP alpha mRNA accumulation is transiently inhibited 12 hr following peritoneal insult, consistent with previous data. However, we demonstrate that (1) there is increased binding of hepatic nuclear protein to the C/EBP alpha DNA response element 48 hr following insult, (2) a marked increase in C/EBP alpha protein is observed 48 hr following CLP insult compared with no increase in hepatic C/EBP alpha protein at 12 hr postinsult, (3) the increase in hepatic C/EBP alpha protein at 48 hr following cecal ligation and puncture is not associated with an increase in C/EBP alpha mRNA accumulation, (4) the increase in hepatic C/EBP alpha protein is associated with an increase in C/EBP beta protein, and (5) hepatic albumin mRNA accumulation is decreased at 12 and 48 hr following insult and does not correlate with the C/EBP alpha protein synthesis. We conclude that the possible role of the transcription factor C/EBP alpha with respect to decreased albumin gene expression following sepsis must be reevaluated.