RESUMEN
Osteopetrosis is a rare and heterogeneous genetic disorder characterized by dense bone mass that is a consequence of defective osteoclast function and/or development. Autosomal recessive osteopetrosis (ARO) is the most severe form and is often fatal within the first years of life; early hematopoietic stem cell transplant (HSCT) remains the only curative treatment for ARO. The majority of the ARO-causing mutations are located in the TCIRG1 gene. We report here the identification and characterization of an A to T transversion in the fourth base of the intron 2 donor splice site (c.117+4AâT) in TCIRG1, a mutation not previously seen in the Ashkenazi Jewish (AJ) population. Analysis of a random sample of individuals of AJ descent revealed a carrier frequency of approximately 1 in 350. Genotyping of five loci adjacent to the c.117+4AâT-containing TCIRG1 allele revealed that the presence of this mutation in the AJ population is due to a single founder. The identification of this mutation will enable population carrier testing and will facilitate the identification and treatment of individuals homozygous for this mutation.
Asunto(s)
Efecto Fundador , Osteopetrosis/genética , Mutación Puntual , Sitios de Empalme de ARN/genética , ATPasas de Translocación de Protón Vacuolares/genética , Análisis Mutacional de ADN , Frecuencia de los Genes , Humanos , Lactante , Intrones , Judíos/genéticaRESUMEN
Leigh syndrome is a neurodegenerative disorder of infancy or childhood generally due to mutations in nuclear or mitochondrial genes involved in mitochondrial energy metabolism. We performed linkage analysis in an Ashkenazi Jewish (AJ) family without consanguinity with three affected children. Linkage to microsatellite markers D5S1969 and D5S407 led to evaluation of the complex I gene NDUFS4, in which we identified a novel homozygous c.462delA mutation that disrupts the reading frame. The resulting protein lacks a cAMP-dependent protein kinase phosphorylation site required for activation of mitochondrial respiratory chain complex I. In a random sample of 5000 healthy AJ individuals, the carrier frequency of the NDUFS4 mutation c.462delA was 1 in 1000, suggesting that it should be considered in all AJ patients with Leigh syndrome.
Asunto(s)
Judíos/genética , Enfermedad de Leigh/genética , Mutación , NADH Deshidrogenasa/genética , Adulto , Secuencia de Bases , Preescolar , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/metabolismo , Resultado Fatal , Femenino , Frecuencia de los Genes , Ligamiento Genético , Predisposición Genética a la Enfermedad , Haplotipos , Herencia , Homocigoto , Humanos , Lactante , Enfermedad de Leigh/complicaciones , Enfermedad de Leigh/diagnóstico , Enfermedad de Leigh/enzimología , Enfermedad de Leigh/etnología , Masculino , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Linaje , Fenotipo , Fosforilación , Embarazo , Procesamiento Proteico-PostraduccionalRESUMEN
The autosomal recessive disorder familial dysautonomia (FD) has recently been demonstrated to be caused by mutations in the IKBKAP gene, so named because an initial report suggested that it encoded an IkappaB kinase complex associated protein (IKAP). Two mutations in IKBKAP have been reported to cause FD. The major mutation is a T-->C transition in the donor splice site of intron 20 and the minor mutation is a missense mutation in exon 19 that disrupts a consensus serine/threonine kinase phosphorylation site. We have characterized the cDNA sequences of the mouse, rat and rabbit IKBKAP-encoded mRNAs and determined the genomic organization and chromosomal location of mouse IKBKAP. There is significant homology in the amino acid sequence of IKAP across species and the serine/threonine kinase phosphorylation site altered in the minor FD mutation of IKAP is conserved. The mouse and human IKBKAP genes exhibit significant conservation of their genomic organization and the intron 20 donor splice site sequence, altered in the major FD mutation, is conserved in the human and mouse genes. Mouse IKBKAP is located on the central portion of chromosome 4 and maps to a region in which there is conserved linkage homology between the human and mouse genomes. The homologies observed in the human and mouse sequences should allow, through the process of homologous recombination, for the generation of mice that bear the IKBKAP mutations present in individuals with FD. The characterization of such mice should provide significant information regarding the pathophysiology of FD.
Asunto(s)
Proteínas Portadoras/genética , Genes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Exones , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Muridae , Proteínas de Unión al ARN , Conejos , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Elongación TranscripcionalRESUMEN
The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IkappaB kinase complex-associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T-->C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.
Asunto(s)
Proteínas Portadoras/genética , Cromosomas Humanos Par 9 , Disautonomía Familiar/genética , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Secuencia de Consenso , Exones , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Factores de Elongación TranscripcionalRESUMEN
An RNA species has been identified whose nucleotide sequence is closely related to the mRNA encoding the murine interferon (IFN)-induced guanylate-binding protein-1 (mGBP1) and an mRNA encoding an isoprenylated protein that is constitutively expressed in various organs in the rat. Sequence analysis of the gene encoding this newly identified RNA reveals that in its 5'-region it is identical to a DNA fragment reported to represent the 5'-region of a gene termed mGBP2. In light of this homology, we term this newly identified gene product mGBP2. mGBP2 is inducible following IFN treatment in animals bearing Gbp1a alleles, in which mGBP1 is transcriptionally upregulated by IFN treatment, as well as in animals bearing Gbp1b alleles, in which mGBP1 is not induced in response to IFN treatment. The genomic organizations of the genes encoding mGBP1 and mGBP2 are similar, and the nucleotide sequences of their IFN-responsive-like elements and their relative locations are conserved. Gbp1 and Gbp2 map to a genetically indistinguishable site on the distal arm of chromosome 3.
Asunto(s)
Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Proteínas de Unión al GTP , Genoma , Interferones/farmacología , Animales , Secuencia de Bases , Proteínas de Unión al ADN/biosíntesis , Datos de Secuencia Molecular , Prenilación de Proteína , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
A cDNA encoding the human guanylate binding protein-1 (hGBP-1) was expressed in HeLa cells using a constitutive expression vector. Stably transfected clones expressing hGBP-1 exhibited resistance to the cytopathic effect mediated by both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) and produced less viral progeny than control cells following infection with these viruses. To study the role hGBP-1 plays in the IFN-mediated antiviral effect, cells were stably transfected with a construct expressing antisense RNA for hGBP-1. VSV infection of IFN-alpha-treated antisense RNA-expressing cells produced an amount of virus comparable to that produced in the parental cell line, while EMCV infection of the IFN-alpha-treated transfected cells and VSV and EMCV infection of the IFN-gamma-treated transfected cells produced far more virus than was produced in the parental cell line. These results demonstrate that GBP-1 mediates an antiviral effect against VSV and EMCV and plays a role in the IFN-mediated antiviral response against these viruses.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Virus de la Encefalomiocarditis/fisiología , Proteínas de Unión al GTP , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación Viral/fisiología , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Células HeLa , Humanos , ARN sin Sentido/genética , Proteínas Recombinantes/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
Daily parenteral administration of exogenous interferon-gamma (IFN-gamma) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed. The complete coding region of IFN-gamma was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-gamma minigene (dAv-IFN-gamma) was isolated from 293 cells co-transfected with the linearized plasmid and an E1 region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-gamma, dose-dependent and time-dependent expression of IFN-gamma, mRNA and production of soluble protein were demonstrated in human diploid fibroblat and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for > = 4 weeks following initial transfection, and secreted IFN-gamma induced both antiviral activity (8000-25,000 U/ml) and macrophage activation with killing of intracellular Toxoplasma gondii and leishmania donovani. These results establish that dAv-IFN-gamma generates long-term secretion of biologically active IFN-gamma in vitro and suggest that this vector may be a useful delivery system for cytokine therapy.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Interferón gamma/genética , Activación de Macrófagos , Animales , Northern Blotting , Clonación Molecular , Citomegalovirus/genética , Expresión Génica , Células HeLa , Humanos , Interferón gamma/biosíntesis , Leishmania donovani/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Toxoplasma/inmunologíaRESUMEN
The interferon-mediated induction of the gene encoding the human tryptophanyl tRNA synthetase (WRS) results in the production of two mRNA species differing in size by approximately 800 base pairs (bp). Two distinctly sized cDNAs differing by approximately 800 bp were isolated from a cDNA library generated from mRNA prepared from IFN-gamma-treated cells. Northern blot analysis using cDNA probes recognizing different regions of the WRS mRNA reveals distinctly sized mRNAs differing in the length of their 3' untranslated regions. Differential display analysis using oligo dT primers demonstrates that the different sized WRS mRNAs result from alternative polyadenylation of this transcript.
Asunto(s)
Interferones/farmacología , Poli A/metabolismo , ARN Mensajero/biosíntesis , Triptófano-ARNt Ligasa/genética , Empalme Alternativo , Secuencia de Bases , ADN Complementario , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A cDNA clone complementary to an interferon (IFN)-induced mRNA approximately 3 kb in length was identified and sequenced revealing homology with the endoplasmic reticular heat shock protein/ATPase gp96. Both IFN-alpha and -gamma transcriptionally upregulate expression of this gene. gp96 transcripts, protein, and ATPase activity are shown to be enhanced as a result of IFN treatment in two human cell lines and this effect requires de novo protein synthesis. gp96 molecules have recently been implicated in the presentation of endogenous antigens. A number of the key elements in this pathway, the transporter proteins, the major histocompatibility complex (MHC)-linked units of the proteasomes and the MHC class I molecules are known to be IFN inducible. Our results show that yet another molecule suggested to play an accessory role in the endogenous presentation pathway is IFN inducible. Further, our studies represent the first demonstration of modulation of expression of a heat shock protein by a cytokine and identify a new enzymatic activity upregulated in IFN-treated cells.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/biosíntesis , Interferones/farmacología , Adenosina Trifosfatasas/biosíntesis , Secuencia de Bases , Línea Celular , ADN Complementario/aislamiento & purificación , Proteínas de Choque Térmico/genética , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Regulación hacia ArribaRESUMEN
A lambda cDNA library prepared from polyadenylated RNA isolated from Daudi cells was differentially screened to isolate cDNAs that recognize mRNA whose levels are reduced following interferon (IFN) treatment. Southern blot and DNA sequence analysis of 20 cDNA clones that were isolated revealed that they represented mitochondrially encoded mRNAs for the following proteins: cytochrome c oxidase subunits II and III, ATPase 6, cytochrome b, and subunit 1 of the NADH dehydrogenase. Northern blot analysis employing these cDNAs and oligonucleotides generated to the remaining mitochondrially encoded mRNAs demonstrated that IFN-alpha treatment of Daudi cells mediates a time-dependent suppression of the level of all of the mitochondrially encoded mRNAs. Study of this IFN-mediated effect reveals that: (i) the suppression of the level of these mRNAs is dependent on protein synthesis, (ii) it can be observed to occur prior to any detectable effect on thymidine incorporation, (iii) the degree of suppression correlates with the sensitivity of the cells to the anticellular action of IFN, and (iv) the suppression of the level of these RNAs appears to result from an effect on the level of transcription rather than on the stability of these mRNAs. A study of the level of cellular respiration in IFN-treated Daudi cells reveals a clear suppression 3 h following IFN treatment.
Asunto(s)
Interferón-alfa/farmacología , Mitocondrias/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Secuencia de Bases , Línea Celular , Humanos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Consumo de Oxígeno/efectos de los fármacos , ARN/efectos de los fármacos , ARN/metabolismo , ARN Mensajero/metabolismo , ARN Mitocondrial , Timidina/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The promyelocytic leukemia line HL-60 can be terminally differentiated in vitro to either monocyte/macrophages or granulocytes. We used this cell line to test whether the state of differentiation of a cell changes its response to interferon (IFN). The characteristics of expression of several IFN-alpha- and IFN-gamma-inducible genes in undifferentiated and differentiated HL-60 cells were examined. p67, an IFN-gamma-inducible protein, was induced similarly in three cell types, whereas another IFN-gamma-inducible protein, p56, was induced strongly only in undifferentiated cells. In contrast, two isozymes of 2,5(A)-synthetase were induced better in differentiated cells in response to either IFN. Several IFN-alpha-inducible mRNAs, e.g., 561, 6-16, 1-8, and 2A, were induced much more strongly in granulocytes than in macrophages or in undifferentiated cells. Electrophoretic mobility shift assays using the IFN-stimulated response element of gene 561 and nuclear extracts of IFN-alpha-treated cells revealed the appearance of one complex and the disappearance of another one, concomitant with differentiation of the cells to granulocytes. These observations suggest that expression of IFN-inducible genes in HL-60 cells is regulated by trans-acting factors whose activity changes with the state of differentiation of the cells. Our study may have implications in the optimal clinical use of IFNs. Inducing cellular differentiation may augment the efficacy of IFNs as antitumor agents.
Asunto(s)
Diferenciación Celular , Interferón-alfa/farmacología , Interferón gamma/farmacología , Biosíntesis de Proteínas , Secuencia de Bases , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Granulocitos/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Sondas de Oligonucleótidos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.
Asunto(s)
Interferón-alfa/farmacología , Triptófano-ARNt Ligasa/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/genética , Electroforesis en Gel de Agar , Inducción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Cinética , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Triptófano-ARNt Ligasa/genéticaRESUMEN
A partial cDNA clone for the interferon (IFN)-induced 67,000-dalton (67K) protein was isolated by immunological screening and used as a probe to study the expression of mRNAs encoding this protein. Northern blot analyses of RNA from IFN-treated GM2767 cells revealed the presence of two major 67K-specific RNA species, 2.7 and 4.3 kb in length, and two minor RNA species, 5.7 and 7.2 kb long. All of these 67K-specific RNAs were polyadenylated. Multiple 67K-specific mRNAs were observed to be induced in several cell lines. IFN-gamma was more effective at inducing these mRNAs than was IFN-alpha. In IFN-alpha-treated GM2767 cells, the 67K-specific mRNAs were detectable 6 h following IFN treatment, but not 12, 18, or 24 h following treatment. In IFN-gamma-treated cells, these mRNAs were detectable 6 h after treatment and continued to be present 24 h after treatment. The induction of the 67K-specific mRNAs in GM2767 cells did not require protein synthesis as the RNAs were induced by IFN-alpha or IFN-gamma in the presence of cycloheximide (CHX, 50 micrograms/ml). Treatment of cells with the combination of CHX and IFN-alpha mediated an enhanced accumulation of the 67K-specific mRNAs, suggesting that ongoing protein synthesis may downregulate the induction or accumulation of the IFN-alpha-induced 67K-specific mRNAs. Western blot analysis employing a monoclonal antibody to the 67K protein revealed that several distinctly sized but immunologically related proteins were induced in IFN-treated cells.
Asunto(s)
ADN/genética , Interferones/farmacología , Proteínas/genética , ARN Mensajero/genética , Línea Celular , Cicloheximida/farmacología , Humanos , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Cinética , Peso Molecular , Biosíntesis de Proteínas , ARN Mensajero/biosíntesisRESUMEN
The capacity of BALB/c mice to acquire resistance to and eliminate intracellular visceral Leishmania donovani is T cell dependent, associated with a granulomatous tissue reaction, and correlates with the ability to secrete the macrophage-activating lymphokine, IFN-gamma. These responses appear by 4 wk after infection and are fully established by 8 wk. To examine the role of endogenous IFN-gamma, BALB/c mice were injected with anti-IFN-gamma mAb before and for 8 wk after infection. At 4 wk, mAb treatment inhibited the acquisition of resistance to L. donovani and abolished mature granuloma formation. Although liver parasite burdens in mAb-treated mice were fivefold higher than in controls at 8 wk, continually treated mice nevertheless began for form tissue granulomas and decreased their parasite loads by 50% from peak values. The levels of anti-IFN-gamma antibody in the serum of mice injected for 8 wk were appreciably reduced, thus raising the possibilities of either insufficient neutralization of endogenous IFN-gamma at this time point or a pathway independent of IFN-gamma. Although the role of IFN-gamma and the potential effect of an IFN-gamma-independent mechanism in the resolution of visceral infection remain to be defined, these results indicate that IFN-gamma plays a critical role in the early immune response that both optimally controls L. donovani infection and induces the tissue granuloma.
Asunto(s)
Granuloma/inmunología , Interferón gamma/fisiología , Leishmaniasis Visceral/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Anticuerpos Antiprotozoarios/administración & dosificación , Anticuerpos Antiprotozoarios/sangre , Cricetinae , Femenino , Granuloma/patología , Inmunidad Innata , Interferón gamma/biosíntesis , Interferón gamma/sangre , Leishmaniasis Visceral/patología , Hígado/inmunología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , BazoRESUMEN
Tumor necrosis factor (TNF) or lymphotoxin (LT) treatment of cells sensitive to the anticellular action of TNF results in the degradation of their cellular DNA into fragments that are multiples of about 200 base pairs. The specificity of this DNA fragmenting effect was examined. The DNA of cells dying either as a result of exposure to interferon-gamma (IFN-gamma) or as a result of having exhausted their culture media was observed to be fragmented into multiples of 200 base pairs. Antibody to TNF or LT failed to block the IFN-gamma-mediated DNA fragmentation and antibodies to IFN-gamma, TNF, and LT failed to block the DNA fragmentation observed in the cells dying as a result of having exhausted their culture media. Thus the fragmentation of cellular DNA appears to be nonspecific effect of cell death that can be induced by a variety of treatments.
Asunto(s)
ADN/efectos de los fármacos , Linfotoxina-alfa/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Supervivencia Celular/efectos de los fármacos , ADN/aislamiento & purificación , Células HeLa , Humanos , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
In response to cytomegalovirus (CMV) and Toxoplasma gondii antigens, T4+ cells from seropositive donors produce interferon-gamma (IFN-gamma) by different mechanisms; one (T. gondii) dependent upon and the other (CMV) largely independent of interleukin-2 (IL-2) and its receptor. To determine whether IFN-gamma-generating mechanisms unrelated to IL-2 also differ, we examined the requirement for accessory cells and their expressed or secreted products. In response to both specific antigens, IFN-gamma secretion was strictly dependent upon the presence of accessory cells (monocytes), and was largely inhibited by monoclonal antibodies to class II (HLA-DR and -DQ) but not class I MHC antigens. Both CMV and T. gondii antigens stimulated monocytes to release interleukin-1 (IL-1), and IFN-gamma production in response to both antigens was abolished by pretreatment with anti-IL-1 antibody. In contrast, the secretion of tumour necrosis factor (TNF) was not stimulated by either antigen, and IFN-gamma production was not diminished by antisera directed at TNF-alpha or TNF-beta. We conclude that CMV and T. gondii antigen-induced IFN-gamma production requires a similar accessory cell mechanism, and that soluble antigen-stimulated IFN-gamma secretion by human T4+ cells is dependent on monocytes, expression of class II MHC antigens, and the presence of IL-1.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Antígenos de Protozoos/inmunología , Antígenos Virales/inmunología , Interferón gamma/biosíntesis , Monocitos/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Células Presentadoras de Antígenos/inmunología , Citomegalovirus/inmunología , Antígenos HLA-D/inmunología , Humanos , Interleucina-1/biosíntesis , Monocitos/inmunología , Toxoplasma/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Interferon-gamma (IFN-gamma) treatment of polymorphonuclear leukocytes (PMNs) results in an activation of their functions. Studying the IFN responsiveness of PMNs and using antibodies to the IFN-induced proteins, we have observed the ability of IFN-alpha to stimulate the production of the IFN-induced 67,000 and 56,000 dalton proteins and the ability of IFN-gamma to induce the synthesis of the 67,000, 56,000, and 42,000 dalton proteins. The induction of these proteins is dependent on de novo RNA synthesis, as its induction is inhibited if the IFNs and actinomycin D are added to the cells simultaneously. The results of this study confirm the ability of PMNs to carry out gene activation and demonstrate the ability of PMNs to respond to both IFN-alpha and IFN-gamma.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Neutrófilos/efectos de los fármacos , Proteínas , Proteínas Adaptadoras Transductoras de Señales , Humanos , Técnicas In Vitro , Peso Molecular , Neutrófilos/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas , Proteínas de Unión al ARN , Proteínas RecombinantesRESUMEN
To determine whether extracellular tryptophan degradation represents an oxygen-independent antimicrobial mechanism, we examined the effect of exogenous tryptophan on the intracellular antimicrobial activity of gamma interferon (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,3-dioxygenase (IDO) activity and stimulated MDM, alveolar macrophages, and oxidatively deficient chronic granulomatous disease MDM to degrade tryptophan. All IFN-gamma-activated, tryptophan-degrading macrophages killed or inhibited Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani. Although exogenous tryptophan partially reversed this activity, the increases in intracellular replication were variable for normal MDM (T. gondii [5-fold], C. psittaci [3-fold], L. donovani [2-fold]), chronic granulomatous disease MDM (T. gondii [2.5-fold], C. psittaci [5-fold]), and alveolar macrophages (T. gondii [1.5-fold], C. psittaci [1.5-fold]). In addition, IFN-alpha and IFN-beta also stimulated normal MDM to express IDO and degrade tryptophan but failed to induce antimicrobial activity, and IFN-gamma-treated mouse macrophages showed neither IDO activity nor tryptophan degradation but killed T. gondii and L. donovani. These results suggest that while tryptophan depletion contributes to the oxygen-independent antimicrobial effects of the activated human macrophage, in certain cytokine-stimulated cells, tryptophan degradation may be neither sufficient nor required for antimicrobial activity.
Asunto(s)
Actividad Bactericida de la Sangre , Interferón gamma/farmacología , Macrófagos/fisiología , Triptófano/fisiología , Animales , Humanos , Técnicas In Vitro , Indolamina-Pirrol 2,3,-Dioxigenasa , Macrófagos/efectos de los fármacos , Ratones , Monocitos/fisiología , Consumo de Oxígeno , Oxigenasas/metabolismo , Proteínas Recombinantes , Triptófano OxigenasaRESUMEN
Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.