The human NADPH oxidase: primary and secondary defects impairing the respiratory burst function and the microbicidal ability of phagocytes.

Phagocytes, such as granulocytes and monocytes/macrophages, contain a membrane-associated NADPH oxidase that produces superoxide leading to other reactive oxygen species with microbicidal, tumoricidal and inflammatory activities. Primary defects in oxidase activity in chronic granulomatous disease (CGD) lead to severe, life-threatening infections that demonstrate the importance of the oxygen-dependent microbicidal system in host defence. Other immunological disturbances may secondarily affect the NADPH oxidase system, impair the microbicidal activity of phagocytes and predispose the host to recurrent infections. This article reviews the primary defects of the human NADPH oxidase leading to classical CGD, and more recently discovered immunological defects secondarily affecting phagocyte respiratory burst function and resulting in primary immunodeficiencies with varied phenotypes, including susceptibilities to pyogenic or mycobacterial infections.

Local arterial nanoparticle delivery of siRNA for NOX2 knockdown to prevent restenosis in an atherosclerotic rat model.

Both atherosclerosis and arterial interventions induce oxidative stress mediated in part by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that have a pivotal role in the development of neointimal hyperplasia and restenosis. For small interfering RNA (siRNA) targeting of the NOX2 (Cybb) component of the NADPH oxidase to prevent restenosis, gene transfer with viral vectors is effective, but raises safety issues in humans. We developed a new approach using the amino-acid-based nanoparticle HB-OLD7 for local delivery of siRNA targeting NOX2 to the arterial wall. siRNA-nanoparticle complexes were transferred into the regional carotid artery walls after angioplasty in an atherosclerotic rat model. Compared with angioplasty controls, Cybb gene expression (measured by quantitative reverse transcriptase-PCR) in the experimental arterial wall 2 weeks after siRNA was reduced by >87%. The neointima-to-media-area ratio was decreased by >83%, and the lumen-to-whole-artery area ratio was increased by >89%. Vital organs showed no abnormalities and splenic Cybb gene expression showed no detectable change. Thus, local arterial wall gene transfer with HB-OLD7 nanoparticles provides an effective, nonviral system for efficient and safe local gene transfer in a clinically applicable approach to knock down an NADPH oxidase gene. Local arterial knockdown of the Cybb gene significantly inhibited neointimal hyperplasia and preserved the vessel lumen without systemic toxicity.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

An unusual intronic mutation in the CYBB gene giving rise to chronic granulomatous disease.

The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5' splice site and results in multiple abnormal mRNA products.

Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of 4 genes encoding phagocyte NADPH oxidase subunits. Unlike other CGD subtypes, in which there is great heterogeneity among mutations, 97% of affected alleles in patients previously reported with A47(0) CGD carry a single mutation, a GT deletion (DeltaGT) in exon 2 of the p47-phox gene, NCF-1. This unusually high incidence results from recombination events between NCF-1 and its highly homologous pseudogenes, in which DeltaGT originates. In 50 consecutive patients with A47(0) CGD, 4 were identified who were heterozygous for DeltaGT in NCF-1, and for the first time, 2 were identified whose DNA appeared normal at this position. To avoid co-amplification of pseudogene sequence and to enable the identification of mutations in these patients, allele-specific polymerase chain reaction was used to amplify alleles not containing DeltaGT. In each of the 4 patients who were heterozygous for DeltaGT, an additional novel mutation was identified. These were 2 missense mutations, G125 --> A in exon 2 (predicting Arg42 --> Gln) and G784 --> A in exon 8 (Gly262 --> Ser), and 2 splice junction mutations at the 5' end of intron 1, gt --> at and gtg --> gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125 --> A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574 --> A, which predicts Gly192 --> Ser but is likely to result in defective splicing because it represents the final nucleotide of exon 6.

Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation.

X-linked chronic granulomatous disease (CGD) derives from defects in the CYBB gene, which encodes the gp91-phox component of NADPH oxidase. We studied the molecular basis of the disease in a kindred with variant CGD, due to a single base substitution at the sixth position of CYBB first intron. The patients' phagocytes have been shown previously to greatly increase superoxide release in response to interferon-gamma (IFN-gamma) in vitro and in vivo. We examined CYBB gene expression in an Epstein-Barr virus (EBV)-transformed B-cell line from 1 patient in this kindred. These cells showed markedly decreased levels of CYBB transcripts in total RNA (5% of normal) and nuclear RNA (1.4% of normal), despite equal CYBB transcription rates in the CGD and control cells. Incubation with IFN-gamma produced a 3-fold increase in CYBB total messenger RNA (mRNA) levels in the patient's cells, and decreased nuclear transcripts to undetectable levels. Reverse transcriptase-polymerase chain reaction analysis of RNA splicing revealed a preponderance of unspliced CYBB transcripts in the patient's nuclear RNA. In vitro incubation with IFN-gamma increased by 40% the ratio of spliced relative to unspliced CYBB mRNA in nuclei from the CGD B-cell line. Total RNA harvested from the same patient's monocytes, on and off therapy with IFN-gamma, showed a similar improvement in splicing. We conclude that IFN-gamma partially corrects a nuclear processing defect due to the intronic mutation in the CYBB gene in this kindred, most likely by augmentation of nuclear export of normal transcripts, and improvement in the fidelity of splicing at the first intron.

NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system.

We investigated the NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in normal Epstein-Barr-virus (EBV)-transformed B lymphocytes, compared to EBV-transformed B lymphocytes from patients with X-linked chronic granulomatous disease (CGD), normal peripheral blood neutrophils or mononuclear cells, and the A301 or C8166 lymphoblastoid cell lines. CGD phenotypes included both "classic" disease with no detectable gp91-phox protein (termed X91(0)) and "variant" phenotype with reduced but detectable gp91-phox protein (X91(-)). Normal EBV-transformed B lymphocytes show a dose-dependent PMA-induced superoxide release. Culturing these cells with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days, caused a modest increase in their NADPH oxidase activity (P > 0.05 in all situations). Normal EBV-transformed B lymphocytes have lower NADPH oxidase activity and cytochrome b558 content than peripheral blood neutrophils or mononuclear cells (P < 0.05 in all situations). In contrast, they have higher NADPH oxidase activity and cytochrome b558 content than X91(-) CGD EBV-transformed B lymphocytes (P < 0.05 in all situations). A301 or C8166 lymphoblastoid cell lines and X91(0) CGD EBV-transformed B lymphocytes have barely detectable NADPH oxidase activity or cytochrome b558 content (P < 0.05 in all situations). Gene expression studies also show a modest increase in expression and transcription rates of gp91-phox and p47-phox genes in normal EBV-transformed B cells cultured with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days. We conclude that NADPH oxidase activity and cytochrome b558 content correlate with gp91-phox and p47-phox gene expression in EBV-transformed B lymphocytes.

Differential expression of Id genes in multipotent myeloid progenitor cells: Id-1 is induced by early-and late-acting cytokines while Id-2 is selectively induced by cytokines that drive terminal granulocytic differentiation.

Hematopoietic development is regulated by a complex mixture of cytokine growth factors that guide growth and differentiation of progenitor cell populations at different stages in their development. The genetic programs that drive this process are controlled at the molecular level by the type and number of transcriptional regulators coexpressed in the cell. Both positive- and negative-acting helix-loop-helix transcription factors are expressed during hematopoietic development, with the Id-type transdominant negative regulators controlling the net helix-loop-helix activation potential in the cell at any given time. It has been demonstrated that some of these Id factors are involved in the checkpoint at which undifferentiated progenitor cells make the commitment to terminal maturation. Therefore, we sought to determine whether these Id family factors are selectively induced or extinguished by cytokines that act at different points during hematopoiesis. NFS-60, a myeloid progenitor line that proliferates in response to multiple cytokines, was stimulated by treatment with SCF, IL-3, IL-6, G-CSF, and erythropoietin. Id-1 expression correlated tightly with cellular proliferation: it declined when growth factor stimulation was withdrawn and was quickly induced whenever the cell began to proliferate. The regulation of Id-2 was more complex: its expression was slightly upregulated in factor-deprived cells but only strongly reinduced after extended exposure to cytokines that drive granulocytic differentiation (IL-6, G-CSF, and TGFbeta). These data support a cell-cycle regulatory role for Id-1 in multipotent myeloid progenitor cells and a role for Id-2 during terminal granulocytic differentiation.

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase.

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.

Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid.

The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all-trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Selenium-regulated translation control of heterologous gene expression: normal function of selenocysteine-substituted gene products.

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a "selenium translation element" in the 3' untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3' untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor beta 1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution.

Expression of basic helix-loop-helix transcription factors in explant hematopoietic progenitors.

The basic helix-loop-helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl-1, E12/E47, and ld-1, in individual lineage-defined progenitors and hematopoietic growth factor-dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage-defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT-PCR. SCL and E12/E47 were expressed in the vast majority of tri-, bi-, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl-1 showed a more restricted pattern of expression, although expression was seen in some bi- and unilineage progenitors. No expression was detected in erythroid, erythroid-megakaryocyte-macrophage, macrophage-neutrophil, macrophage, or megakaryocytic progenitors. Id-1, an inhibitory bHLH transcription factor, was also widely expressed in all bi- and unilineage progenitors; only the trilineage erythroid-megakaryocyte-macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id-1, but not E12/E47, mRNA was increased in FDC-P1 and MO7E hematopoietic cell lines after exposure to IL-3 or GM-CSF. Id-1, E12, and lyl-1 showed marked variation at different points in cell cycle in isoleucine-synchronized FDC-P1 cells. These results suggest that SCL, lyl-1, E12/E47, and Id-1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle.

Partial reconstitution of the respiratory burst oxidase in lymphoblastoid B cell lines lacking p67-phox after transfection with an expression vector containing wild-type and mutant p67 -phox cDNAs: Deletions of the carboxy and amino terminal residues of p67-phox are not required for activity.

The respiratory burst oxidase of phagocytes and B lymphocytes in a multicomponent enzyme that catalyzes the reduction of oxygen by NADPH. It is responsible for O-(2) production in response to stimulation with phorbol 12-myristate 13-acetate (PMA). The study of patients with chronic granulomatous disease (CGD), an inherited disorder characterized by deficient of absent respiratory burst activity, has contributed greatly to our understanding of the NADPH-oxidase. The absence of any one of four components results in the clinical expression of CGD: the two membrane-bound components of the cytochrome b-558, gp91-phox and p22-phox, or the cytosolic factors, p47-phox and p67-phox. We used a system to investigate the activity of mutant p67-phox proteins expressed in a reconstitution assay. This system is characterized by the partial reconstitution of O-(2) production in an Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell line from a patient with p67-phox-deficient CGD by transfection with an expression plasmid containing the 67-phox cDNA in the sense orientation. No O-(2) production was detectable in p67-phox-deficient lymphoblastoid B cell lines transfected with an antisense plasmid or in untransfected p67-phox lymphoblastoid cells stimulated by PMA. We tested two mutants, pEBOp67delta1-22 and pEBOp67delta512-526, and found that both recombinant proteins are active in our system. Thus, we conclude that the first 22 amino acid residues and the last 14 amino acid residues are not critical for initiation of O-(2) production

Umbilical cord blood as a new and promising source of unrelated-donor hematopoietic stem cells for transplantation.

A rapidly accelerating number of transplantations of hematopoietic stem cells from human umbilical cord blood have been performed for malignancies and for congenital disorders. Umbilical cord blood presents multiple advantages over bone marrow as a source of stem cells. Harvesting presents no donor risk or discomfort, the product carries less likelihood of infectious disease transmission, and collection can be targeted to include minority groups underrepresented in bone marrow donor registries. Furthermore, the interval from initiation of a search to the transplantation procedure has been much shorter than for bone marrow, and the lack of mature T lymphocytes in cord blood reduces the incidence and severity of graft-versus-host disease in transplant recipients. Potential problems under current investigations include whether cord blood provides a sufficient quantity of stem cells for adult recipients or an effective level of "graft-versus-leukemia" effect.

RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA.

Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDS-polyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.