RESUMEN

Interferon-γ (IFN-γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN-γ/interleukin (IL)-12 axis. Using transcriptome profiling (RNA-seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein-Barr virus-transformed B lymphocytes (B-EBV) cells from CGD patients, IFN-γ receptor deficiency patients, and normal controls, treated in vitro with IFN-γ for 48 hours. Our results show that IFN-γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN-γ-induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome-mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN-γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre-mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl-transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN-γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN-γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN-γ treatment. These data support the concept that IFN-γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.

Asunto(s)

Linfocitos B/metabolismo , Expresión Génica/efectos de los fármacos , Enfermedad Granulomatosa Crónica/sangre , Enfermedad Granulomatosa Crónica/genética , Interferón gamma/farmacología , Receptores de Interferón/deficiencia , Linfocitos B/virología , Línea Celular , Exones/genética , Enfermedad Granulomatosa Crónica/patología , Herpesvirus Humano 4 , Humanos , Empalme del ARN/genética , ARN Mensajero/genética , RNA-Seq , Transducción de Señal/efectos de los fármacos , Triptófano-ARNt Ligasa/genética , Receptor de Interferón gamma

RESUMEN

The human CYBB gene encodes the gp91-phox component of the phagocyte oxidase enzyme complex, which is responsible for generating superoxide and other downstream reactive oxygen species essential to microbial killing. In the present study, we have identified by sequence analysis a putative NF-κB binding site in a DNase I hypersensitive site, termed HS-II, located in the distant 5' flanking region of the CYBB gene. Electrophoretic mobility assays showed binding of the sequence element by recombinant NF-κB protein p50 and by proteins in nuclear extract from the HL-60 myeloid leukemia cell line corresponding to p50 and to p50/p65 heterodimers. Chromatin immunoprecipitation demonstrated NF-κB binding to the site in intact HL-60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF-κB binding site and the CYBB promoter region. Inhibition of NF-κB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of κB "super-repressor" showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF-κB site linked to the CYBB 5' flanking promoter region revealed enhanced expression, augmented by treatment with interferon-γ. These studies indicate a role for this distant, 15 kb upstream, binding site in NF-κB regulation of the CYBB gene, an essential component of phagocyte-mediated host defense.

Asunto(s)

Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , NADPH Oxidasas/química , NADPH Oxidasas/genética , FN-kappa B/metabolismo , Fagocitos/metabolismo , Análisis de Secuencia de ADN/métodos , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica , Células HL-60 , Humanos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , FN-kappa B/antagonistas & inhibidores , Regiones Promotoras Genéticas , Unión Proteica , Salicilatos/farmacología

RESUMEN

This work investigated the functional role of nuclear factor-kappaB (NF-kappaB) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-kappaB (IkappaBalpha-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorder of NF-kappaB function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91(0) CGD). NCF1 gene expression in EDA-ID S32I cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A47(0)) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-kappaB site 5' to the CYBB gene in U937 cells treated with NF-kappaB inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-kappaB repressor. These studies show that NF-kappaB is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system.

Asunto(s)

Displasia Ectodérmica/inmunología , Leucocitos/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidasas/metabolismo , FN-kappa B/fisiología , Línea Celular Transformada , Células Cultivadas , Expresión Génica , Enfermedad Granulomatosa Crónica/patología , Humanos , Leucocitos/patología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Fagocitos/metabolismo , Fagocitosis

RESUMEN

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P=0.036). Monocytes from SCD patients showed higher levels of p47(phox) phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P=0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P>0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Asunto(s)

Anemia de Células Falciformes/metabolismo , Interferón gamma/biosíntesis , Leucocitos/metabolismo , NADPH Oxidasas/metabolismo , Regulación hacia Arriba , Adolescente , Adulto , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , NADPH Oxidasas/genética , Fosforilación , ARN Mensajero/genética , Transcripción Genética/genética , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by early onset of recurrent and severe infections. The molecular defects causing CGD are heterogeneous and lead to absence, low expression, or malfunctioning of one of the phagocyte NADPH oxidase components. The aim of this study was to analyze the clinical features and to investigate the molecular genetic defects of Latin American patients with CGD. PROCEDURES: The study included 14 patients. The diagnosis was based on a history of recurrent severe infections, impaired respiratory burst, and the demonstration of an underlying mutation by single strand conformation polymorphism (SSCP) or RT-PCR analysis, followed by genomic DNA or cDNA sequencing. RESULTS: Seven unrelated patients were found to have the X-linked form of CGD (X-CGD). Heterogeneous mutations affected the CYBB gene: two insertions, one substitution, and four splice site defects; two of them are novel. Seven patients presented with one of the autosomal recessive forms of CGD (A47-CGD); all had the most common mutation, a DeltaGT deletion in exon 2 of the NCF1 gene. Pneumonia was the most frequent clinical feature, followed by pyoderma, sinusitis, otitis, and liver abscess. Patients with X-CGD were more likely to have initial infections before age 2 years and to have inflammatory obstructive granulomas later. None of the patients had severe adverse reactions to BCG immunization. CONCLUSIONS: X-CGD patients from Latin America showed a high degree of molecular heterogeneity, including two novel mutations. Their clinical characteristics included early onset of infections and eventual obstructive granulomas. A47-CGD represented 50% of the reported cases, a higher prevalence than reported in other series.

Asunto(s)

Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , Mutagénesis Insercional , NADPH Oxidasas/genética , Fosfoproteínas/genética , Polimorfismo Conformacional Retorcido-Simple , Eliminación de Secuencia , Análisis Mutacional de ADN/métodos , Exones/genética , Femenino , Genes Recesivos/genética , Enfermedad Granulomatosa Crónica/complicaciones , Humanos , América Latina , Masculino , NADPH Oxidasa 2 , Sitios de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos

RESUMEN

The aim of this work was to analyze the effect of Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in human colostrum macrophages (CM), peripheral blood monocytes (PBM), and myelomonocytic THP-1 cells. We also investigated the effect of IFN-gamma on the release of TNF-alpha by these cells. Our results show that under basal culture conditions, CM release more superoxide than PBM and THP-1 cells (p < 0.05). The addition of IFN-gamma, alone or in combination with TNF-alpha, increased spontaneous superoxide release by PBM and THP-1 cells (p < 0.05) and increased phorbol myristate acetate (PMA)-stimulated superoxide release by CM, PBM, and THP-1 cells (p < 0.05). The NADPH oxidase activity of THP-1 cells consistently remained lower than that of CM or PBM, despite a dramatic response to IFN-gamma and TNF-alpha. Under basal conditions, gp91-phox gene expression was significantly higher in CM and PBM compared with THP-1 cells (p < 0.05). The addition of IFN-gamma alone or in combination with TNF-alpha caused a dramatic increase in gp91-phox gene expression in THP-1 cells (p < 0.05) but not in CM or PBM. Under basal conditions or in the presence of IFN-gamma, CM released more TNF-alpha than PBM or THP-1 cells (p < 0.05). In addition, PBM released more TNF-gamma than THP-1 cells (p < 0.05). IFN-gamma did not significantly augment the release of TNF-alpha by these cells (p > 0.05). Thus, IFN-gamma and TNF-alpha induced equivalent gp91-phox gene expression in THP-1 cells compared with CM or PBM but did not bring about equivalent NADPH oxidase activity. TNF-alpha release was higher in more mature cells. This partial divergence of gp91- phox gene expression, NADPH oxidase activity, and TNF-alpha release is probably a consequence of different events of myeloid cell biology and relates at least in part to cell differentiation state.

Asunto(s)

Calostro/inmunología , Interferón gamma/farmacología , Macrófagos/enzimología , Monocitos/enzimología , NADPH Oxidasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular , Calostro/citología , Femenino , Expresión Génica , Humanos , Macrófagos/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monocitos/inmunología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Embarazo , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease.

Asunto(s)

Anemia/complicaciones , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Enfermedad Granulomatosa Crónica/genética , Infecciones/complicaciones , Sustitución de Aminoácidos , Anemia/enzimología , Secuencia de Bases , Células Sanguíneas/enzimología , Niño , ADN/genética , ADN Complementario/genética , Expresión Génica , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/complicaciones , Enfermedad Granulomatosa Crónica/enzimología , Humanos , Infecciones/enzimología , Masculino , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Superóxidos/metabolismo

RESUMEN

The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Interferón gamma/farmacología , NADPH Oxidasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Biomarcadores/análisis , Ácido Butírico/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Linaje de la Célula , Células Clonales/efectos de los fármacos , Células Clonales/enzimología , Células Clonales/metabolismo , Activación Enzimática/efectos de los fármacos , Eosinófilos/citología , Eosinófilos/enzimología , Eosinófilos/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Interleucina-5/farmacología , Proteínas de Unión a Maltosa , Glicoproteínas de Membrana/genética , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Peroxidasa/genética , Peroxidasa/metabolismo