RESUMEN

Leu+ mutants from Salmonella typhimurium leu-500 strain MA412 arise at high frequencies and mutant colonies appear over a broad range of time on selective plates. This observation suggested that these Leu+ mutants might be induced or "directed."= If such a mechanism was responsible, mutants should originate on selective plates rather than in the preceding culture in nonselective conditions and should give rise to Poisson-like fluctuation curves upon plating of sister cultures on selective medium. Poisson-like distribution profiles were indeed observed for Leu+ mutants of S. typhimurium MA412. However, an explanation for the observed Poisson-like fluctuation patterns without a need for selection-induced mutations was found. Microscopical analysis and cell mass/viable count measurements showed that the size of Leu+ mutant cells was often much larger than those of nonmutants. This size difference was a stable characteristic of a large proportion of Leu+ mutants, was observed both in stationary and growing culture and did not measurably affect the division rates of the cells in nutrient broth. As the transition from normal-sized nonmutant to oversized mutant cells during the nonselective culture phase of the fluctuation experiment may have been accompanied by a period with no or few completed cell division cycles, the number of mutant offspring may have been smaller than that of sibling nonmutants. Such underrepresentation of mutants in the final culture is expected to give rise to Poisson-like fluctuation patterns without invoking "directed" mutations.

Asunto(s)

Leucina/metabolismo , Mutación , Salmonella typhimurium/genética , División Celular , Cinética , Distribución de Poisson , Salmonella typhimurium/citología , Salmonella typhimurium/crecimiento & desarrollo , Especificidad de la Especie , Factores de Tiempo

RESUMEN

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the gamma subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co, Zn, and Cd), Alcaligenes eutrophus (a member of the beta subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.

RESUMEN

Genetically modified microorganisms (GMMs) are frequently used as producers of mammalian immunomodulatory proteins, e.g. interferons and interleukins. Here we have examined the question of whether such GMMs interact in a way different from that of their non-modified parent micro-organisms with mammalian antimicrobial defence systems. As a typical GMM host micro-organism we used Escherichia coli K12, and as a typical immunomodulatory protein produced by a GMM we used mouse interferon-gamma (MuIFN-gamma). Two experimental systems are described in which synergistic "toxic" biological effects are induced by a combined treatment with E. coli and MuIFN-gamma but not, or less so, by the parental strain and the recombinant protein separately. First, it is shown that the IFN-gamma-producing GMM, or mixtures of E. coli cells and IFN-gamma, are cytolytic for mouse embryo fibroblastoid cells (MEF), whereas no cell killing occurs in MEF cultures treated with control E. coli cells or in those treated with bacteria-free recombinant IFN-gamma. Second, it is demonstrated that intraperitoneal injection in mice of high but not low numbers of control E. coli K12 cells induces a shock-like mortality, whereas co-injection with IFN-gamma induces killing at low numbers. IFN-gamma-producing E. coli cells cause a mortality rate that does not differ from that of control E. coli cells, probably because in these experimental conditions the level of recombinant MuIFN-gamma per cell is insufficiently high. Taken together, these data indicate that synergistic toxic effects induced by bacteria and their recombinant products can occur and may in certain situations enhance the intrinsic toxic capacity of the GMM. Synergistic toxic effects may thus be of relevance for identifying the safety level that should be employed when working with GMMs.

Asunto(s)

Toxinas Bacterianas/toxicidad , Escherichia coli/patogenicidad , Interferón gamma/toxicidad , Choque Séptico , Animales , Sinergismo Farmacológico , Escherichia coli/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes , Factores de Riesgo , Choque Séptico/mortalidad

RESUMEN

Monitoring of microbial DNA in soils by dot blot hybridization and PCR analysis is a useful technique for gaining insight into the survival and impact of genetically modified micro-organisms released in the environment. Most methods of DNA isolation from soils require a large number of purification steps rendering them unsuitable for quantitative analysis of multiple samples. Here we describe a very rapid method for the isolation and purification of multiple samples of soil DNA that can be used directly for dot blot hybridization and PCR analysis. Soil DNA extracts are prepared by lysozyme/SDS treatment at pH 9.0 and purified by ammonium acetate precipitation and Sephadex G50 gel filtration. In a practical application of this method, sandy soil samples were seeded with Alcaligenes eutrophus cells and exposed to high temperature (42 degrees C) or desiccation. As a result, the number of culturable A. eutrophus cells which could be recovered from the soil samples quickly declined. However, the concentration of a marker gene encoding resistance to cadmium, cobalt and zinc (czc) remained unaltered.

Asunto(s)

Alcaligenes/genética , ADN Bacteriano/aislamiento & purificación , Monitoreo del Ambiente/métodos , Microbiología del Suelo , Secuencia de Bases , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa

RESUMEN

Normal mouse embryo fibroblasts (MEF) are killed by treatment with low doses of interferon gamma (IFN-gamma) in combination with lipopolysaccharide (LPS). This cytotoxicity has previously been shown to represent an active suicidal reaction. Here we show that the time period between first contact with IFN-gamma/LPS (t = 0 h) and cell death (t = 48 h) can be separated into two distinct periods, during which glycolytic metabolism of glucose either has a positive (8-24 h) or a negative (30-48 h) effect on cytotoxicity. During the first period (8-24 h), withdrawal of glucose from the culture medium, or inclusion in the medium of the glycolytic inhibitors deoxy-D-glucose, NaF or iodoacetate, prevented later cell death. During the second period (30-48 h), withdrawal of glucose or supplementation of the culture medium with glycolytic inhibitors was no longer protective; instead it was a requirement for cell suicide to occur. Glycolytic activity during the first period was found to be increased twofold in LPS-treated MEF and almost threefold in IFN-gamma/LPS-treated MEF. A variety of agents were found both to protect cells against IFN-gamma/LPS-induced cytotoxicity and to inhibit increased glycolysis in these cells: glucocorticoids, the serine-type protease inhibitor N-acetyl-DL-phenylalanine-beta-naphthyl ester, the ADP-ribosylation inhibitors 3-aminobenzamide and nicotinamide, and the transcription and translation inhibitors actinomycin and cycloheximide. Mitochondrial function, although normal in LPS-treated cells, was markedly depressed in IFN-gamma/LPS-treated MEF. Specifically, malate- and succinate-driven respiration was found to be impaired. Furthermore, IFN-gamma/LPS-treated MEF contained one-third of the ATP level of LPS-treated MEF. Withdrawal of L-arginine from the culture medium prevented cell death in IFN-gamma/LPS-treated MEF. N-Methyl-L-arginine, which is an inhibitor of nitric oxide (NO.) biosynthesis from L-arginine, also inhibited cell death. In conclusion, we propose that cell death in our experiments is due to an L-arginine/glycolysis-dependent impairment of mitochondrial respiration.

Asunto(s)

Arginina/farmacología , Muerte Celular , Fibroblastos/citología , Glucólisis , Interferón gamma/farmacología , Lipopolisacáridos , Adenosina Trifosfato/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Embrión de Mamíferos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Cinética , Ratones , Mitocondrias/metabolismo , Consumo de Oxígeno

RESUMEN

Interferon-gamma (IFN-gamma) is coded for by a single gene containing three introns, localized within the coding region. We have previously cloned the IFN-gamma gene from a pig genomic DNA lambda library and have determined its nucleotide sequence. In order to construct the porcine IFN-gamma DNA without intervening sequence, the four exons were separately amplified by the polymerase chain reaction (PCR) using primers matching the exon-termini. From the amplified exon-fragments the complete intron-free DNA was obtained by a strategy consisting of alternate rounds of PCR and ligation. The sequence so-obtained was used for expression in E. coli. The recombinant protein appeared as inclusion bodies which were solubilized and refolded in order to obtain biologically active IFN-gamma.

Asunto(s)

ADN/genética , Escherichia coli/genética , Exones , Interferón gamma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular/métodos , Biblioteca Genómica , Interferón gamma/aislamiento & purificación , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Porcinos

RESUMEN

Nude mice were inoculated with CHO/IFN-gamma cells, a line of Chinese hamster ovary tumor cells, that had been genetically engineered to produce murine IFN-gamma. Severe cachexia, as evident from body weight loss and reduced food intake, occurred in these mice, but not in those injected with CHO/control cells, i.e. the original, non-IFN-gamma-producing line. The essential role of IFN-gamma in the pathogenesis of cachexia was confirmed by the demonstration that monoclonal antibodies (MAbs) against IFN-gamma, given prior to injection of the tumor cells, prevented cachexia. In addition to IFN-gamma, the presence of the tumor cells was also required for cachexia to develop. As evident from pair-feeding experiments, reduced food intake could only partially account for the rapid and extensive body-weight loss. Cachexia was characterized by a marked reduction in the amount of interscapular fat tissue. Injected tumor cells exclusively invaded intraperitoneal adipose tissue and elicited an inflammatory cell infiltrate, indicating that interscapular fat loss was due to humoral factors. Our data suggest that, among the humoral factors responsible for cancer-associated cachexia, IFN-gamma plays a prominent role.

Asunto(s)

Caquexia/etiología , Interferón gamma/fisiología , Neoplasias Experimentales/fisiopatología , Tejido Adiposo/patología , Animales , Anorexia/fisiopatología , Peso Corporal , Cricetinae , Cricetulus , Conducta Alimentaria , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Análisis de Supervivencia

RESUMEN

In several biological systems interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) act synergistically. We therefore examined whether it would be possible to construct IFN-gamma/IL-1 hybrid proteins that would be more active than the individual components. Hybrid proteins were examined that consisted of the amino-terminal 118 residues of mouse IFN-gamma and the 156 or 152 carboxyl-terminal residues of mouse IL-1 alpha or IL-1 beta, respectively. They were obtained by ligation of the respective coding sequences and expression of the fused genes under control of the PL promotor in Escherichia coli. Both the IFN-gamma/IL-1 alpha and the IFN-gamma/IL-1 beta fusion proteins were purified by affinity chromatography on an anti-IFN-gamma monoclonal antibody column. Analysis of biological activities showed that these fusion proteins were less active than the individual cytokines. Specific antiviral activity of the IFN-gamma/IL-1 beta hybrids was less than 0.1% that of IFN-gamma and D10.G4.1 T-cell proliferative (IL-1) activity amounted to 0.1% that of mouse IL-1. Affinity-purified preparations of the IFN-gamma/IL-1 alpha hybrid were found to contain variable proportions of a Mr 14,000 degradation product possessing IFN-gamma activity, whereas the undegraded Mr 30,000 fusion protein, while being devoid of detectable IFN-gamma activity, did possess IL-1 activity (1%). Serum from rats immunized with the IFN-gamma/IL-1 alpha hybrid contained high levels of IL-1 alpha-binding and -neutralizing antibodies and IFN-gamma-binding antibodies, but no detectable levels of IFN-gamma-neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Anticuerpos Monoclonales/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-1/genética , Proteínas Recombinantes de Fusión/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Bioensayo , Línea Celular , Escherichia coli/genética , Femenino , Hibridomas/inmunología , Inmunoensayo , Interferón gamma/aislamiento & purificación , Interferón gamma/farmacología , Interleucina-1/inmunología , Interleucina-1/farmacología , Activación de Linfocitos , Ratones , Plásmidos , Ratas , Ratas Endogámicas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Transfección

RESUMEN

In mammalian species, interferon-gamma (IFN-gamma) is a lymphokine with a wide range of biological effects, of which the antiviral and macrophage-activating capacities are those best characterized. In birds, no equivalent with a similar range of actions has as yet been isolated. Chicken splenocytes were stimulated by mitogens in conditions that were similar to those used for the induction of mammalian IFN-gamma. Culture fluids were assayed for antiviral and macrophage-activating capacities. As much as 1000 units/ml of an interferon-like antiviral activity was found in the culture fluid of Staphylococcus aureus lysate-induced spleen cells. Seroneutralization assays with a polyclonal antiserum against purified interferon and physicochemical studies revealed that the antiviral activity is identical to or closely related to type I interferon (interferon-alpha/beta). The presence of macrophage activating factors (MAF) in the splenocyte medium was demonstrated by measuring increased production of H2O2 by chicken peritoneal macrophage cultures and a chicken macrophage cell line (HD11). The heat stability of this MAF activity was similar to that of the antiviral factor, and was completely neutralized by the anti-IFN-alpha/beta antiserum. These results show that when the classical procedure used for the production of mammalian IFN-gamma is applied to chicken splenocytes, it does not yield an equivalent for IFN-gamma/MAF. This suggests that the classification of interferons into types (alpha, beta and gamma), while generally applicable in mammals, may not be applicable in birds.

Asunto(s)

Pollos/inmunología , Interferón gamma/biosíntesis , Macrófagos/inmunología , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Factores Activadores de Macrófagos/biosíntesis , Factores Activadores de Macrófagos/inmunología , Ratones , Mitógenos , Virus de los Bosques Semliki/inmunología , Bazo/inmunología , Temperatura

Asunto(s)

Infecciones/inmunología , Inflamación/inmunología , Interferón gamma/inmunología , Macrófagos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Diseño de Fármacos , Retroalimentación/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Inmunización , Interferón gamma/antagonistas & inhibidores , Interferón gamma/uso terapéutico , Macrófagos/efectos de los fármacos , Modelos Biológicos

Asunto(s)

Interferón gamma/genética , Porcinos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico

RESUMEN

Preincubation of rat anterior pituitary (AP) cells with homologous interferon-gamma (IFN-gamma) caused a dose-dependent inhibition of ACTH secretion stimulated by CRF. The effect was seen in both monolayer and aggregate AP cell cultures and was not due to cytotoxicity. In monolayer cultures IFN-gamma also inhibited PRL and GH release stimulated by various hypothalamic releasing factors. IFN-gamma did not affect the time kinetics of the ACTH response to CRF. The dose needed for half-maximal inhibition amounted to approximately 1 (antiviral) U/ml. The effect of IFN-gamma was abrogated by an IFN-gamma-neutralizing monoclonal antibody. Furthermore, ACTH secretion by the AP cells was not affected by the anti-IFN-gamma antibody added alone, indicating that in the culture system no endogenous IFN-gamma is operational in regulating the ACTH response studied. Of the other cytokines tested [interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-alpha/beta (IFN-alpha/beta)] only TNF-alpha and IL-6 were found to inhibit CRF-stimulated ACTH release, although this inhibition was less pronounced than that caused by IFN-gamma. Lipopolysaccharide, even at high doses, did not significantly inhibit the ACTH response to CRF. These results identify IFN-gamma as one of the inflammatory cytokines that, like IL-1, TNF-alpha, and IL-6, have the potential to regulate pituitary function.

Asunto(s)

Hormona Adrenocorticotrópica/metabolismo , Hormona del Crecimiento/metabolismo , Interferón gamma/farmacología , Adenohipófisis/metabolismo , Prolactina/metabolismo , Animales , Células Cultivadas , Hormona Liberadora de Corticotropina/farmacología , Femenino , Interferón Tipo I/farmacología , Interleucina-1/farmacología , Interleucina-6/farmacología , Cinética , Adenohipófisis/efectos de los fármacos , Ratas , Ratas Endogámicas , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.

Asunto(s)

Interferón gamma/fisiología , Lipopolisacáridos/farmacología , Fenómeno de Shwartzman/etiología , Animales , Anticuerpos Monoclonales , Factores Biológicos/sangre , Citocinas , Relación Dosis-Respuesta Inmunológica , Femenino , Interferón gamma/inmunología , Interferón gamma/farmacología , Ratones , Ratones Endogámicos , Premedicación , Proteínas Recombinantes , Fenómeno de Shwartzman/sangre

RESUMEN

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.

Asunto(s)

Antígenos Ly/biosíntesis , Diterpenos , Interferones/farmacología , Proteína Quinasa C/fisiología , Animales , Antígenos Ly/genética , Antígenos de Superficie/genética , Calmodulina/antagonistas & inhibidores , Calmodulina/fisiología , Línea Celular , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Ratones , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/genética , Terpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Antígenos Thy-1 , Factores de Tiempo

RESUMEN

Gamma interferon (IFN-gamma) can be cytolytic for normal mouse fibroblasts isolated from embryonic or adult tissue (R. Dijkmas, B. Decock, H. Heremans, J. Van Damme, and A. Billiau, Lymphokine Res. 8:25-34, 1989). This cytotoxicity has been shown to be transcription and translation dependent, thereby suggesting involvement of a suicidelike mechanism. The dose of IFN-gamma required for cytotoxicity is higher than that needed for antiviral and macrophage activation but can be reduced 10- to 100-fold by cotreatment of the cells with tumor necrosis factor or interleukin-1 (IL-1) or both, two cytokines that by themselves are not toxic for these cells. Here, we show that bacterial lipopolysaccharide (LPS), which alone has no effect on the viability of mouse fibroblasts, stimulates cell suicide induced by IFN-gamma. The effect was observed in cultures that were virtually free of nonfibroblastoid cells. LPS showed its toxicity-enhancing effect only if applied on the cells simultaneously with or immediately after treatment with IFN-gamma. Pretreatment of the cells with LPS was ineffective. Inclusion of antibodies directed against tumor necrosis factor alpha or IL-1 alpha in the culture medium did not block the cytotoxic effect of combined IFN-gamma plus LPS treatment. The time courses of cell toxicity appearance in fibroblasts treated with combined IFN-gamma plus LPS or IFN-gamma plus IL-1 were similar. In addition to LPS, heat-killed gram-negative (Escherichia coli) but also gram-positive (Staphylococcus aureus, Listeria monocytogenes) bacteria were found to enhance IFN-gamma-induced cell death. These findings suggest that IFN-gamma formed in vivo during infectious processes directly aggravates tissue destruction.

Asunto(s)

Fibroblastos/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Animales , Bacterias/inmunología , Supervivencia Celular/efectos de los fármacos , Técnicas In Vitro , Interleucina-1/farmacología , Ratones , Ratas , Proteínas Recombinantes , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Interferon-gamma (IFN-gamma) is a cytokine produced by T lymphocytes and Natural Killer cells which has a key function in resistance against infections. Baboon (Papio anubis) IFN-gamma was produced by stimulation of baboon splenocytes with a lysate of Staphylococcus aureus. This interferon was active on human cells and could be seroneutralized with a polyclonal antiserum against human IFN-gamma, but not with antisera against human interferon-alpha and interferon-beta. Poly(A)(+)-RNA was isolated from baboon splenocytes and fractionated according to its sedimentation coefficient by sucrose density centrifugation. BaIFN-gamma mRNA was present in the 15 S fraction as was shown by hybridization with a human IFN-gamma cDNA probe. A cDNA library was constructed and a clone containing the complete BaIFN-gamma cDNA was isolated. The cDNA codes for a polypeptide of 165 amino acids of which the 23 N-terminal may serve as signal peptide. BaIFN-gamma differs at 11 residues from human IFN-gamma. Southern analysis of chromosomal DNA confirmed some of the nucleotide sequence differences between baboon and human IFN-gamma. The baboon IFN-gamma cDNA was placed under control of a trc promoter and brought to expression in Escherichia coli cells. Recombinant baboon IFN-gamma could be seroneutralized with certain monoclonal anti-human IFN-gamma antibodies. The presented work leads to the availability of recombinant baboon IFN-gamma for animal experiments but also yields new insight in the structure-function relationship of IFN-gamma.

Asunto(s)

ADN/genética , Interferón gamma/genética , Papio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Inmunoquímica , Interferón gamma/inmunología , Datos de Secuencia Molecular , Papio/inmunología , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Especificidad de la Especie

RESUMEN

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are cytotoxic for certain tumor cells but have a proliferative effect on normal cells. Here we show that interferon-gamma (IFN-gamma) can be cytotoxic for normal cells, in particular mouse embryonic fibroblasts. The cytotoxicity effect is observed with immuno-purified recombinant mouse IFN-gamma (MuIFN-gamma) at concentrations of 1,000 I.U./ml and can be neutralized by anti-MuIFN-gamma monoclonal antibodies. The effect appears 48 h after initial contact with IFN-gamma and is not influenced by infection of the target cells with mengovirus. Although TNF and IL-1 are not toxic for mouse fibroblasts, they can strongly enhance the IFN-gamma-induced cytotoxicity. Interferon-alpha (IFN-alpha), interferon-beta (IFN-beta) and interleukin-6 (IL-6) neither are cytotoxic themselves nor have any influence on the IFN-gamma-induced cytotoxicity. The cytotoxicity of IFN-gamma, in contrast to that of TNF is inhibited by actinomycin or cycloheximide. These data suggest that the cytotoxic effect of IFN-gamma requires active cooperation of target cells and that the mechanism of action is different from that of the TNF-induced cytotoxicity.

Asunto(s)

Interferón gamma/efectos adversos , Interleucina-1/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Fibroblastos/efectos de los fármacos , Interferón Tipo I/efectos adversos , Interleucina-1/efectos adversos , Interleucina-6 , Interleucinas/efectos adversos , Macrófagos , Ratones , Ratones Endogámicos , Proteínas Recombinantes , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Studies are reviewed in which the role of IFN-gamma in different models of inflammation in mice is examined: LPS-induced generalized Shwartzman reaction, experimental allergic encephalomyelitis (EAE) and experimental cerebral malaria (ECM). The particular role of the cytokine was studied by systemic administration and by blocking the endogenously produced cytokine by the use of neutralizing antibodies. IFN-gamma was found, depending on the model and circumstances, to exert an anti- or a pro-inflammatory effect. In the generalized Shwartzman reaction and ECM this cytokine has a disease promoting role. In EAE, on the contrary, endogenous as well as exogenous IFN-gamma exert a down-regulating effect.

Asunto(s)

Inflamación/etiología , Interferón gamma/fisiología , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/etiología , Malaria/etiología , Fenómeno de Shwartzman/etiología

Asunto(s)

Infecciones Bacterianas/inmunología , Interferón gamma/farmacología , Linfocitos T/inmunología , Animales , Ratones

Asunto(s)

Interferón gamma/fisiología , Animales , Formación de Anticuerpos/fisiología , Humanos , Inmunidad Celular/fisiología , Inmunidad Innata/fisiología , Inflamación/fisiopatología