RESUMEN
Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.
Asunto(s)
Temperatura Corporal , Endotelio Vascular/fisiopatología , Fiebre/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/metabolismo , Humanos , Neutrófilos/fisiología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Temperatura , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
To study macrophage genes activated by lipopolysaccharide (LPS) we have constructed a cDNA library using the mouse macrophage cell line, RAW 264.7. By differential screening, a gene, designated LRG-21, was identified that showed nucleic acid sequence homology to rat liver regenerating factor-1 (LRF-1) and human activating transcription factor-3 (ATF3). Both LRG-21 and LRF-1 are transcribed within an hour following stimulation and in the absence of protein synthesis. The predicted protein sequence of LRG-21 consists of 181 amino acids with a molecular weight of 20.7 kDa. All three sequences contain basic and leucine zipper regions characteristic of the c-Fos and c-Jun family of transcription factors, but the remainder of the sequences are unrelated to this family. Recombinant LRG-21 has been shown to bind to a phorbol ester promoter element. Additional experiments have shown that LRG-21 is also induced by interferon-gamma (IFN-gamma) and by interleukin-4 (IL-4) in both RAW264.7 cells and murine peritoneal macrophages. Based on these observations, it is likely that LRG-21 plays an important role in macrophage activation.
Asunto(s)
Lipopolisacáridos/farmacología , Activación de Macrófagos/genética , Macrófagos/efectos de los fármacos , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor de Transcripción Activador 3 , Secuencia de Aminoácidos , Animales , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Línea Celular , Leucemia de Células B , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Factores de Transcripción/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
The response of macrophages to agents such as lipopolysaccharide (LPS) and interferon (IFN) includes the transcriptional activation of numerous genes. We have used the method of differential screening of a RAW 264.7 macrophage cell line cDNA library to isolate and characterize LPS-induced messages. One such message, LRG-47, is induced by LPS, IFN-gamma, and IFN-alpha/beta, but not by a panel of other cytokines or pharmacological activating agents. LRG-47 is homologous to two other IFN-gamma-induced genes, IRG-47 and Mg21. The LRG-47 sequence is approximately 33% identical and 52% similar to both these putative protein products. All three putative proteins, particularly Mg21, bear homology to a T cell product, Tgtp, induced by T cell receptor cross-linking. The three macrophage-derived proteins share areas of homology with GTP-binding proteins, are approximately 415 amino acids in length, and have similar kinetics of induction by IFN-gamma. This suggests that these genes may be members of a new family of IFN-inducible proteins.
Asunto(s)
ADN Complementario/análisis , ADN Complementario/biosíntesis , Interferones/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Secuencia de Bases , Células Cultivadas , Células Clonales , ADN Complementario/genética , Proteínas de Unión al GTP/química , Cinética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Estimulación QuímicaRESUMEN
Macrophages are stimulated by lipopolysaccharide (LPS) of gram-negative organisms. The changes in LPS-stimulated macrophages include transcriptional activation of multiple immediate-early genes, which may contribute to the natural immunity to microorganisms. We have defined by deletion and mutational analysis LPS-responsive elements (LREs) in two chemokine genes, MuRantes and crg-2, which are activated in an immediate-early manner. LRE consists of two motifs, TCAYR, which is an AP-1 half site with two flanking bases, and (A/T) (G/C)NTTYC(A/T)NTTY, which resembles in part the interferon-stimulated responsive element (ISRE). The orientation of these two motifs relative to each other in MuRantes differed from that in crg-2. These two motifs are separated by 10 and 6 nonconsensus nucleotides in the MuRantes and crg-2 LREs, respectively. Stimulation of macrophage-like RAW 264.7 cells with alpha/beta interferon did not activate MuRantes, indicating that the ISRE-like motif in MuRantes does not have ISRE activity. Upon stimulation of RAW 264.7 cells with LPS, proteins capable of binding to LRE accumulate in the nuclei as measured by electrophoretic mobility shift assay. These LRE-binding proteins include c-Jun and CREB.
Asunto(s)
Lipopolisacáridos/farmacología , Linfocinas/genética , Macrófagos/metabolismo , Monocinas/genética , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Quimiocina CCL5 , Quimiocina CXCL10 , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , Análisis Mutacional de ADN , Expresión Génica , Genes Inmediatos-Precoces/efectos de los fármacos , Humanos , Interferón Tipo I/farmacología , Linfocinas/biosíntesis , Macrófagos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Monocinas/biosíntesis , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Eliminación de Secuencia , Transcripción Genética , TransfecciónRESUMEN
The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Supervivencia Celular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad , Adenosina Difosfato Ribosa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Benzamidas/farmacología , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Líquido Intracelular/metabolismo , Cinética , Ratones , NAD/metabolismo , Niacinamida/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/toxicidad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Stimulation of rat astrocytes in vitro by calcium ionophore A23187 and/or lipopolysaccharide results in the generation of a cytotoxic factor that is functionally similar to the previously described macrophage-derived cytotoxic factor, tumor necrosis factor. Like the macrophage product, the astrocyte cytotoxic factor kills murine L 929 cell targets. In addition, it kills rat oligodendrocytes, the myelin-producing cells of the central nervous system. Human recombinant tumor necrosis factor also has cytotoxic activity directed against rat oligodendrocytes.
Asunto(s)
Astrocitos/fisiología , Citotoxinas/fisiología , Enfermedades Desmielinizantes/fisiopatología , Neuroglía/fisiología , Oligodendroglía/fisiología , Animales , Calcimicina/farmacología , Inflamación/fisiopatología , Lipopolisacáridos/farmacología , Conejos , Ratas , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.
Asunto(s)
Calcio/fisiología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Neoplasias/inmunología , Aminoquinolinas , Animales , Calcimicina/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxinas/biosíntesis , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Macrófagos/inmunología , Ratones , Ratones EndogámicosAsunto(s)
Citotoxicidad Inmunológica , Citotoxinas/análisis , Activación de Macrófagos , Macrófagos/inmunología , Proteínas , Animales , Células Cultivadas , Citotoxinas/fisiología , Escherichia coli , Femenino , Cobayas , Células L/inmunología , Lipopolisacáridos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C3H , ConejosRESUMEN
Peritoneal macrophages from C3HeB/FeJ mice became cytotoxic for 6C3HED lymphosarcoma cells, P815 mastocytoma cells, and L-929 fibroblasts when treated with the calcium ionophore, A23187, at concentrations ranging from 1.0 to 20 microM. The effect of A23187 on other activation processes was also tested. It was found that A23187 and lipopolysaccharide (LPS) acted synergistically, but no consistent synergy with macrophage-activating factor (MAF) was observed. Cytotoxic activity (M phi-CF) was found in cellfree supernatants from M phi activated by A23187 or LPS. Furthermore, these two activating agents synergize in the production of M phi-CF. The cytotoxic activity of the crude material was not blocked by catalase or protease inhibitors. Fractionation of supernatants by high pressure liquid chromatography has shown that there was a peak of cytotoxic activity with a m.w. of approximately 45,000. Interestingly, L-929 cells were 30-fold more sensitive to M phi-CF than a lymphotoxin-resistant subline of L-929.
Asunto(s)
Citotoxicidad Inmunológica , Activación de Macrófagos , Macrófagos/inmunología , Animales , Calcimicina/farmacología , Línea Celular , Cinética , Células L/inmunología , Lipopolisacáridos/farmacología , Linfocinas/fisiología , Factores Activadores de Macrófagos , Macrófagos/enzimología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Peso Molecular , Inhibidores de Proteasas/farmacologíaRESUMEN
Guinea pig peritoneal macrophages, when activated for cytotoxicity by the calcium ionophore A23187 or lipopolysaccharide, produce a cytotoxic factor [macrophage cytotoxic factor (M phi-CF)] that is not blocked by catalase or protease inhibitors. Fractionation of culture supernates containing M phi-CF by gel filtration revealed one peak of cytotoxic activity of Mr approximately 45,000, the same as guinea pig lymphotoxin (LT). Antiserum prepared against purified guinea pig LT completely neutralized the cytotoxic activity of M phi-CF. In addition, the cytotoxic factor in guinea pig tumor necrosis serum was found to have a Mr of 45,000 and was neutralized by anti-LT. Thus, M phi-CF is physicochemically and immunochemically similar to LT and tumor necrosis factor, if not identical. To investigate the role of M phi-CF in macrophage-mediated cytotoxicity, anti-LT was added to A23187- or lipopolysaccharide-activated macrophages before addition of L-929 target cells. In 10 of 16 experiments, the inhibition of macrophage-mediated cytotoxicity was 100%. In the others, cytotoxicity was blocked partially, the lowest inhibition being 49%. The effectiveness of inhibition appeared to be inversely related to the intensity of macrophage activation. These results indicate that M phi-CF plays a significant role in macrophage-mediated cytotoxicity but involvement of another mechanism cannot be excluded.
Asunto(s)
Citotoxicidad Inmunológica , Macrófagos/inmunología , Animales , Complejo Antígeno-Anticuerpo , Glicoproteínas/inmunología , Cobayas , Linfotoxina-alfa/inmunología , Activación de Macrófagos , Peso Molecular , Factor de Necrosis Tumoral alfaRESUMEN
Macrophages can be activated for tumor cell cytotoxicity by endotoxin- or lymphokine-containing solutions, and in both cases activation can be blocked by the addition of indomethacin. In contrast, the activation of macrophages by nonadherent or inflammatory peritoneal cells or antibody-coated tumor cells is not affected by indomethacin. These results demonstrate that there are at least 2 distinct pathways of macrophage activation, only 1 of which is affected by the addition of the drug. Activation by endotoxin that is indomethacin sensitive requires 2 steps. At present, the first is undefined and not blocked by indomethacin, but the second is inhibited by the drug and appears to require the production of E series prostaglandins. Our results also suggest that neither step alone is sufficient to activate macrophages for cytotoxicity.
Asunto(s)
Citotoxicidad Inmunológica , Linfoma no Hodgkin/inmunología , Macrófagos/inmunología , Sarcoma de Mastocitos/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Aspirina/farmacología , Bovinos , Adhesión Celular , Indometacina/farmacología , Lipopolisacáridos/farmacología , Linfocinas/farmacología , Ratones , Ratones Endogámicos C3H , Prostaglandinas E/farmacologíaRESUMEN
A radioimmunoassay was used to quantitate the number of tumor cell-bound IgG1 anti-tumor antibody molecules required to cause suppression of tumor growth in C3H mice. Radiolabeled anti-IgG1 was used to detect cell-bound IgG1 antibody. The assay was calibrated by using TNP-coupled tumor cells that had a known number of bound IgG1 anti DNP molecules. Between 70,000 and 130,000 IgG1 anti-tumor antibody molecules per tumor cell were sufficient to cause 50% suppression of tumor growth in mice inoculated with appriximately 50,000 tumor cells.
Asunto(s)
Anticuerpos Antineoplásicos/análisis , Especificidad de Anticuerpos , Transformación Celular Neoplásica , Inmunoglobulina G/análisis , Inmunosupresores , Isoanticuerpos/análisis , Animales , Sitios de Unión de Anticuerpos , Femenino , Linfoma/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmacitoma/inmunología , Conejos , RadioinmunoensayoRESUMEN
Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.
Asunto(s)
Ácido Aminolevulínico/metabolismo , Citocromos/metabolismo , Genes , Ácidos Levulínicos/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Ácido Aminolevulínico/farmacología , Genotipo , Mitocondrias/efectos de los fármacos , Mutación , Saccharomyces cerevisiae/efectos de los fármacos , EspectrofotometríaRESUMEN
The need for the provision of primary experience in the course of residential treatatment of certain deeply emotionally deprived children has been considered, in the context of therapeutic work in a boarding school for maladjusted children. The nature of primary experience and means of providing this have been discussed. A distinction has been made between the needs of such children before and after integration as individuals. Some of the special problems faced by the therapeutic providers of primary experience have been noted.