RESUMEN
The molecular repertoire of porcine alveolar macrophages (PAMs) is greatly affected by the microenvironment they are exposed to, and specifically by inflammatory cytokines, such as interferon gamma (IFN-γ) released by activated lymphocytes, and microbial products, such as lipopolysaccharide (LPS). In our previous study, we found that IFN-γ- and LPS-activated PAMs (M1) could inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication. In this study, comprehensive analysis of the expression profiles of the genes associated with the polarization of M0-type PAMs (resting) toward M1 phenotypes (activated by IFN-γ and LPS) led to the following main results: 1) 1551 and 1823 genes were upregulated or downregulated in M1-type PAMs, respectively, compared with M0-type PAMs; 2) Among these, genes encoding ASS1 and CRTAM were the most upregulated and downregulated, respectively; 3) Genes involved in cytokine-cytokine receptor interaction and the JAK/STAT signaling pathway were significantly upregulated, suggesting their critical role in cellular activation; and 4) Genes involved in antigen proteolysis and presentation (immunoproteasome subunits), and inhibition of virus replication (host restriction factors) were significantly upregulated, emphasizing the critical role of these cytokines in immunity. Thus, our results provide important information for future studies on the role of PAM polarization in modulation of infection.
Asunto(s)
Argininosuccinato Sintasa/genética , Inmunoglobulinas/genética , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Transcriptoma/inmunología , Animales , Argininosuccinato Sintasa/inmunología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Ontología de Genes , Inmunoglobulinas/inmunología , Janus Quinasa 1/genética , Janus Quinasa 1/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/virología , Anotación de Secuencia Molecular , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Cultivo Primario de Células , Receptores de Citocinas/genética , Receptores de Citocinas/inmunología , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/inmunología , Porcinos , Replicación Viral/efectos de los fármacosRESUMEN
AIM: In this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-α-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved. MAIN METHODS: The TNF-α-stimulated inflammatory HUVECs were treated with acetylcholine (ACh) and/or FA. NO productions were measured by monitoring nitrite and nitrate using a 2,3-diaminonaphthalene Kit. Expressions of mRNA and proteins were evaluated by RT-PCR and Western blotting, respectively. KEY FINDINGS: FA treatment resulted in a dose-dependent (10-200µM) restoration of ACh-mediated NO production in TNF-α-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O2(-) production in TNF-α-stimulated HUVECs. N(G)-monomethyl-l-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and α-methyl-dl-aspartic acid (an argininosuccinate synthase (ASS) inhibitor) counteracted the effects of FA on the NO production. While FA treatment did not significantly affect the protein expression of p-eNOS or eNOS, the protein expression of ASS as well as mRNA expression was restored to normal levels upon exposure to FA in TNF-α-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-κB) expression by TNF-α. SIGNIFICANCE: FA treatment rescues the defect in ACh-induced NO production resulting from TNF-α-stimulation in inflammatory HUVECs. This effect was likely due, in part, to the FA-mediated up-regulation of ASS expression via the suppression of NF-κB inflammatory signaling cascade.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Argininosuccinato Sintasa/inmunología , Ácidos Cumáricos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Óxido Nítrico/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Argininosuccinato Sintasa/genética , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , FN-kappa B/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Lipopolysaccharide (LPS), a structural component of Gram-negative bacteria, is implicated in the pathogenesis of endotoxemia/sepsis and multi-organ injury, including liver damage. We have shown that argininosuccinate synthase (ASS), a hepatic enzyme of the urea cycle, accumulates in circulation within 1h after treatment with both LPS alone and hepatotoxic combination of LPS and D-Galactosamine. These findings indicate ASS as a sensitive biomarker of liver responses to bacterial endotoxin. Furthermore, we suggest that the ASS release represents a potential counteracting liver reaction to LPS, and demonstrates anti-LPS activity of recombinant ASS (rASS) in vitro and in rodent models of endotoxemia in vivo. rASS physically bound to LPS, as indicated by a gel shift assay, and suppressed Escherichia coli growth in cultures consistent with direct antimicrobial properties of ASS. rASS reduced LPS cytotoxicity, TNF-α production, and increased cell viability in cultured mouse macrophages, even when added one hour following LPS challenge. Intraperitoneal injection of rASS (5 mg/kg) after treatment with a high dose of LPS remarkably increased survival of rodents, with a concomitant decrease of sepsis markers TNF-α, C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels in blood. These results suggest that the endogenous ASS constitutes a novel liver-derived component of the innate immune response to bacterial LPS, and that recombinant ASS could mitigate the lethal effects of bacterial endotoxins during sepsis.
Asunto(s)
Argininosuccinato Sintasa/inmunología , Infecciones Bacterianas/inmunología , Inmunidad Innata/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Hígado/inmunología , Animales , Argininosuccinato Sintasa/sangre , Argininosuccinato Sintasa/metabolismo , Infecciones Bacterianas/metabolismo , Proteína C-Reactiva/antagonistas & inhibidores , Supervivencia Celular/inmunología , Células Cultivadas , Endotoxemia/sangre , Endotoxemia/inmunología , Endotoxemia/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Galactosamina/inmunología , Humanos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/sangre , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, tissue regrowth and remodelling. Wound healing follows tissue destruction so we hypothesized that antibodies might bind to wounded tissues, which would facilitate the engulfment of damaged tissues by macrophages. Here, we show that B cells, which produce antibodies to damaged tissues, are engaged in the process of wound healing. Splenectomy delayed wound healing, and transfer of spleen cells into splenectomized mice recovered the delay in wound healing. Furthermore, wound healing in splenectomized nude mice was also delayed. Transfer of enriched B220(+) cells by magnetic beads accelerated wound healing in splenectomized mice. We detected immunoglobulin G1 (IgG1) binding to wounded tissues by using fluorescein isothiocyanate-labelled anti-IgG1 6-24 hr after wounding. Splenectomy reduced the amount of IgG1 binding to wounded tissues. Immunoblotting studies revealed several bands, which were reduced by splenectomy. Using immunoprecipitation with anti-IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham-operated mice. These bands were matched to myosin IIA, carbamoyl-phosphate synthase, argininosuccinate synthase, actin and alpha-actinin-4 by liquid chromatography tandem mass spectrometry analysis.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos B/metabolismo , Inmunoglobulina G/metabolismo , Piel/metabolismo , Cicatrización de Heridas/inmunología , Actinina/inmunología , Actinina/metabolismo , Actinas/inmunología , Actinas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo/inmunología , Argininosuccinato Sintasa/inmunología , Argininosuccinato Sintasa/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/trasplante , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Células Cultivadas , Femenino , Inmunoglobulina G/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Miosina Tipo IIA no Muscular/metabolismo , Piel/inmunología , Piel/lesiones , Bazo/patología , Bazo/cirugía , EsplenectomíaRESUMEN
The distribution of the urea cycle enzyme, argininosuccinate synthetase, in the rat brain was determined using immunohistochemistry. This enzyme participates in the only known metabolic pathway for citrulline, its condensation with aspartate to form argininosuccinate, which can then be cleaved to fumarate and arginine. It may thus provide a mechanism to recycle citrulline, formed in the nervous system via nitric oxide synthase activity, back to the nitric oxide precursor, L-arginine. Argininosuccinate synthetase immunoreactivity was detected in discrete populations of neurons throughout the brain. Double-staining with nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry for the localization of nitric oxide synthase demonstrated that argininosuccinate synthetase coexists with nitric oxide synthase in some brain regions. However, many neurons were found that contained one of these two enzymes, but not the other. Thus some nitric oxide synthase-containing neurons appear able to recycle citrulline via argininosuccinate, while others do not. Additional roles for argininosuccinate synthetase in the brain are discussed.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Argininosuccinato Sintasa/metabolismo , Encéfalo/enzimología , Neuronas/enzimología , Animales , Argininosuccinato Sintasa/inmunología , Encéfalo/citología , Cerebelo/enzimología , Cerebelo/inmunología , Colina O-Acetiltransferasa/inmunología , Colina O-Acetiltransferasa/metabolismo , Diencéfalo/enzimología , Diencéfalo/inmunología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Mesencéfalo/enzimología , Mesencéfalo/inmunología , NADPH Deshidrogenasa/inmunología , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa , Ratas , Ratas Wistar , Telencéfalo/enzimología , Telencéfalo/inmunologíaRESUMEN
The neuronal distribution of argininosuccinate synthetase (ASS) was mapped in the rat brain. Argininosuccinate synthetase is one of the enzymes of the arginine metabolic pathway and catabolizes the synthesis of argininosuccinate from aspartate and citrulline. Since arginine is the precursor of nitric oxide, argininosuccinate synthetase may act as part of the nitric oxide producing pathway. Argininosuccinate is also suggested to have a messenger function in the nervous system. Therefore, the localization of ASS is of great interest. Polyclonal antisera against purified rat liver argininosuccinate synthetase revealed a characteristic distribution pattern of argininosuccinate synthetase-like immunoreactivity: (1) many neurons with strong argininosuccinate synthetase-like immunoreactivity were observed in the septal area, basal forebrain, anterior medial and premammillary nuclei of the hypothalamus, anterior and midline thalamic nuclei, dorsal endopiriform nucleus of the amygdala, basal nucleus of Meynert, subthalamic nucleus, laterodorsal tegmental nucleus, raphe nuclei, nucleus ambiguus, and the area postrema, (2) neuropile staining was dense in the septal areas, hypothalamus, area postrema, nucleus of the solitary tract, and the laminae I and II of the caudal subnucleus of the spinal trigeminal nucleus and the spinal dorsal horn, (3) relay nuclei of the specific sensory systems such as the dorsal lateral geniculate nucleus and the ventral nuclei of the thalamus were devoid of argininosuccinate synthetase-like immunoreactivity, (4) no staining was seen in the large white matter structures such as the internal capsule, corpus callosum, and the anterior commissure, and (5) most of the neurons stained were small or medium in size and appeared to be interneurons. The results suggest that argininosuccinate synthetase affects the widely distributed, neuromodulatory system in the brain.
Asunto(s)
Argininosuccinato Sintasa/análisis , Encéfalo/enzimología , Animales , Arginina/metabolismo , Argininosuccinato Sintasa/química , Argininosuccinato Sintasa/inmunología , Encéfalo/anatomía & histología , Química Encefálica/fisiología , Mapeo Encefálico , Cerebelo/anatomía & histología , Cerebelo/enzimología , Diencéfalo/anatomía & histología , Diencéfalo/enzimología , Inmunohistoquímica , Masculino , Bulbo Raquídeo/anatomía & histología , Bulbo Raquídeo/enzimología , Mesencéfalo/anatomía & histología , Mesencéfalo/enzimología , Terminaciones Nerviosas/enzimología , Terminaciones Nerviosas/inmunología , Neuronas/enzimología , Óxido Nítrico/metabolismo , Puente/anatomía & histología , Puente/enzimología , Ratas , Ratas Endogámicas , Médula Espinal/anatomía & histología , Médula Espinal/enzimología , Telencéfalo/anatomía & histología , Telencéfalo/enzimologíaAsunto(s)
Argininosuccinato Sintasa/aislamiento & purificación , Ligasas/aislamiento & purificación , Hígado/enzimología , Animales , Argininosuccinato Sintasa/inmunología , Argininosuccinato Sintasa/metabolismo , Humanos , Punto Isoeléctrico , Cinética , Sustancias Macromoleculares , Peso Molecular , Fragmentos de Péptidos/análisis , RatasRESUMEN
Properties of argininosuccinate synthetases (ASS), including immunological properties and heat stability, from young and old rats were quite similar. However the degradation rates of ASS from young and old rats were found to be different as shown in the experiment using the double labeling technique. Three forms of this enzyme from young and old rat livers were separated by DEAE-Sephadex A-50 column chromatography, and they were called ASS 1, 2 and 3 in order of elution. The degradation rate of ASS 1 from young and old rat livers was very similar, but the rate of degradation of ASS 2 form old rat livers was greater than that from young rat livers.
Asunto(s)
Envejecimiento , Argininosuccinato Sintasa/inmunología , Isoenzimas/inmunología , Ligasas/inmunología , Hígado/enzimología , Animales , Cromatografía por Intercambio Iónico , Calor , Inmunodifusión , Ratas , Ratas EndogámicasRESUMEN
Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.