Transient Association of the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunits p47phox and p67phox With Phagosomes in Neutrophils From Patients With X-Linked Chronic Granulomatous Disease

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3521-3530 ◽  
Author(s):  
Lee-Ann H. Allen ◽  
Frank R. DeLeo ◽  
Annabelle Gallois ◽  
Satoshi Toyoshima ◽  
Kensuke Suzuki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Nicotinamide Adenine Dinucleotide ◽  
Polymorphonuclear Leukocytes ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Actin Binding ◽  
Nicotinamide Adenine ◽  
Cell Periphery ◽  
Granulomatous Disease ◽  
Flavocytochrome B

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

Molecular Characterization of Autosomal Recessive Chronic Granulomatous Disease Caused by a Defect of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced Form) Oxidase Component p67-phox

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2505-2514 ◽  
Author(s):  
Pablo J. Patiño ◽  
Julie Rae ◽  
Deborah Noack ◽  
Rich Erickson ◽  
Jiabing Ding ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Molecular Characterization ◽  
Nicotinamide Adenine Dinucleotide ◽  
Autosomal Recessive ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Splice Mutation ◽  
Reduced Form ◽  
Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytes in which defective production of microbicidal oxidants leads to severe recurrent infections. CGD is caused by mutations in any of 4 genes encoding components of nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase, the multisubunit enzyme that produces the precursor of these oxidants, superoxide. Approximately 5% of CGD patients have an autosomal recessive form of disease caused by a severe deficiency of p67-phox, a 526-amino acid subunit of the oxidase that appears to regulate electron transport within the enzyme. Here we report the biochemical and molecular characterization of 6 unrelated kindreds with p67-phox deficiency. These studies show that, as in gp91-phox and p22-phox deficiencies, the p67-phox CGD patients show a high degree of heterogeneity in the genetic defects that underlie their disease. Five different mutant alleles were identified: (1) a nonsense mutation in exon 4 (C304 → T); (2) a 5-nucleotide (nt) deletion in exon 13 (nts 1169-1173); (3) a splice mutation in the first nucleotide of intron 4 (G → A); (4) a deletion of 1 nt in exon 9 (A728); and (5) a 9-nt in-frame deletion in exon 2 (nts 55-63). The splice mutation was seen in 3 unrelated kindreds, while the 5-nt deletion was seen in 2 apparently unrelated families (both of Palestinian origin). Homozygosity was present in 4 of the kindreds, 2 of which had consanguineous parentage. In the isolated neutrophils of each of the affected patients in the 6 kindreds, there was no measurable respiratory burst activity and no p67-phox protein detected by immunoblot analysis. The level of 67-phox mRNA was less than 10% of normal in the mononuclear leukocytes from 3 of the 4 patients analyzed by Northern blot studies. Thus, this heterogeneous group of mutations in p67-phox all lead to marked instability of mRNA or protein (or both) that results in the complete loss of NADPH oxidase activity.

NADPH oxidase promotes NF-κB activation and proliferation in human airway smooth muscle

2002 ◽  
Vol 282 (4) ◽  
pp. L782-L795 ◽  
Author(s):  
Sukhdev S. Brar ◽  
Thomas P. Kennedy ◽  
Anne B. Sturrock ◽  
Thomas P. Huecksteadt ◽  
Mark T. Quinn ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Nadph Oxidase ◽  
Airway Smooth Muscle ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nuclear Factor Κb ◽  
Nicotinamide Adenine ◽  
Phagocytic Cells ◽  
Diphenylene Iodonium ◽  
Reduced Nicotinamide Adenine Dinucleotide

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O[Formula: see text]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O[Formula: see text] as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22phox. Transfection with p22phoxantisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-κB (NF-κB), and overexpression of a superrepressor form of the NF-κB inhibitor IκBα significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22phoxregulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-κB.

scholarly journals NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease

2020 ◽  
Vol 4 (23) ◽  
pp. 5976-5987
Author(s):  
Suk See De Ravin ◽  
Julie Brault ◽  
Ronald J. Meis ◽  
Linhong Li ◽  
Narda Theobald ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Messenger Rna ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Functional Restoration ◽  
In Vivo Studies ◽  
Allogeneic Bone ◽  
Granulomatous Disease ◽  
Primate Model

Abstract Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

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