Superoxide production by normal and chronic granulomatous disease (CGD) patient-derived EBV-transformed B cell lines measured by chemiluminescence-based assays

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

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X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽

10.1182/blood.v82.7.2196.bloodjournal8272196 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2196-2202 ◽

Cited By ~ 1

Author(s):

CD Porter ◽

MH Parkar ◽

RJ Levinsky ◽

MK Collins ◽

C Kinnon

Keyword(s):

Gene Therapy ◽

Nadph Oxidase ◽

B Cell ◽

Chronic Granulomatous Disease ◽

Cell Lines ◽

Large Subunit ◽

Granulomatous Disease ◽

Barr Virus ◽

Inherited Immunodeficiency ◽

Epstein Barr

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

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X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽

10.1182/blood.v82.7.2196.2196 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2196-2202 ◽

Cited By ~ 29

Author(s):

CD Porter ◽

MH Parkar ◽

RJ Levinsky ◽

MK Collins ◽

C Kinnon

Keyword(s):

Gene Therapy ◽

Nadph Oxidase ◽

B Cell ◽

Chronic Granulomatous Disease ◽

Cell Lines ◽

Large Subunit ◽

Granulomatous Disease ◽

Barr Virus ◽

Inherited Immunodeficiency ◽

Epstein Barr

Abstract Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

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Correlation between membrane potential changes and superoxide production in human granulocytes stimulated by phorbol myristate acetate. Evidence for defective activation in chronic granulomatous disease.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)85963-x ◽

1980 ◽

Vol 255 (5) ◽

pp. 1874-1878

Author(s):

J.C. Whitin ◽

C.E. Chapman ◽

E.R. Simons ◽

M.E. Chovaniec ◽

H.J. Cohen

Keyword(s):

Membrane Potential ◽

Chronic Granulomatous Disease ◽

Phorbol Myristate Acetate ◽

Superoxide Production ◽

Granulomatous Disease ◽

Myristate Acetate ◽

Human Granulocytes

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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

Blood ◽

10.1182/blood.v95.11.3548 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3548-3554 ◽

Cited By ~ 51

Author(s):

Antonio Condino-Neto ◽

Peter E. Newburger

Keyword(s):

B Cell ◽

Chronic Granulomatous Disease ◽

Cell Line ◽

Interferon Gamma ◽

Granulomatous Disease ◽

First Intron ◽

Cybb Gene ◽

Nuclear Rna ◽

Ifn Γ

Abstract 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-γ) 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-γ produced a 3-fold increase in CYBBtotal 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-γ 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-γ, showed a similar improvement in splicing. We conclude that IFN-γ partially corrects a nuclear processing defect due to the intronic mutation in theCYBB 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.

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Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Blood ◽

10.1182/blood.v89.1.41 ◽

1997 ◽

Vol 89 (1) ◽

pp. 41-48 ◽

Cited By ~ 96

Author(s):

Helga Björgvinsdóttir ◽

Chunjin Ding ◽

Nancy Pech ◽

Mary A. Gifford ◽

Ling Lin Li ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Aspergillus Fumigatus ◽

Chronic Granulomatous Disease ◽

Host Defense ◽

Bone Marrow Cells ◽

Superoxide Production ◽

Granulomatous Disease ◽

Wild Type ◽

Marrow Cells

Abstract The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

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A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽

10.1182/blood-2009-07-231498 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3309-3315 ◽

Cited By ~ 287

Author(s):

Juan D. Matute ◽

Andres A. Arias ◽

Nicola A. M. Wright ◽

Iwona Wrobel ◽

Christopher C. M. Waterhouse ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Autosomal Recessive ◽

Premature Stop Codon ◽

Superoxide Production ◽

Human Neutrophils ◽

Granulomatous Disease ◽

Recessive Mutations ◽

Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

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Improvement of superoxide production in monocytes from patients with chronic granulomatous disease by recombinant cytokines

Blood ◽

10.1182/blood.v81.8.2131.bloodjournal8182131 ◽

1993 ◽

Vol 81 (8) ◽

pp. 2131-2136

Author(s):

V Jendrossek ◽

AM Peters ◽

S Buth ◽

J Liese ◽

U Wintergerst ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Respiratory Burst ◽

Interleukin 1 ◽

Superoxide Production ◽

Granulomatous Disease ◽

Necrosis Factor Alpha ◽

Ifn Gamma ◽

Phorbolmyristate Acetate

Cytokines have been shown to modulate the respiratory burst of polymorphonuclear leukocytes and monocytes from normal controls. We have examined whether monocytes from children with chronic granulomatous disease (CGD) can be primed by cytokines other than interferon-gamma (IFN gamma), which has been demonstrated to improve the production of reactive oxygen species in vivo and in vitro. Monocytes isolated from peripheral blood were cultured without and with IFN gamma (500 U/mL), tumor necrosis factor-alpha (500 U/mL), interleukin-1 beta (IL-1 beta) (100 U/mL), and IL-3 (100 U/mL). After 3 days of culture, the phorbolmyristate acetate (2 ng/mL) and the formyl- methionyl-leucyl-phenylalanine (0.1 mumol/L)-stimulated superoxide- production was determined in a microtiter system. In nearly all of the 14 patients examined (5 autosomal, 5 X-chromosomal, and 4 of unknown inheritance), an improvement of superoxide production could be demonstrated. The most impressive effect with the cytokines newly tested was seen with monocytes from autosomal CGD patients treated with IL-3 and stimulated by phorbolmyristate acetate. In single patients cultivation of monocytes with IL-6 and granulocyte-macrophage colony- stimulating factor resulted in only slight improvement of superoxide production. Our findings indicate that cytokines other than IFN gamma can positively modulate the defective respiratory burst in CGD and that each patient reacts with an individual pattern to different cytokines.

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