Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2469-2477 ◽  
Author(s):  
Elahe Mollapour ◽  
David C. Linch ◽  
Pamela J. Roberts
Keyword(s):  
Nadph Oxidase ◽  
Phospholipase A2 ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Superoxide Production ◽  
Dual Inhibitor ◽  
Nadph Oxidase Activity ◽  
Gm Csf ◽  
Tnf Α ◽  
Arachidonate Release

Abstract Arachidonic acid (AA) generated by phospholipase A2(PLA2) is thought to be an essential cofactor for phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Both enzymes are simultaneously primed by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor–α (TNF-α). The possibility that either unprimed or cytokine-primed responses of PLA2 or NADPH oxidase to the chemotactic agents formyl-methionyl-leucyl-phenylalanine (FMLP) and complement factor 5a (C5a) could be differentially inhibited by inhibitors of the mitogen-activated protein (MAP) kinase family members p42ERK2 (PD98059) and p38SAPK(SB203580) was investigated. PD98059 inhibited the activation of p42ERK2 by GM-CSF, TNF-α, and FMLP, but it did not inhibit FMLP-stimulated superoxide production in either unprimed or primed neutrophils. There was no significant arachidonate release from unprimed neutrophils stimulated by FMLP, and arachidonate release stimulated by calcium ionophore A23187 was not inhibited by PD98059. In contrast, PD98059 inhibited both TNF-α– and GM-CSF–primed PLA2 responses stimulated by FMLP. On the other hand, SB203580 inhibited FMLP-superoxide responses in unprimed as well as TNF-α– and GM-CSF–primed neutrophils, but failed to inhibit TNF-α– and GM-CSF–primed PLA2 responses stimulated by FMLP, and additionally enhanced A23187-stimulated arachidonate release, showing that priming and activation of PLA2 and NADPH oxidase are differentially dependent on both the p38SAPK and p42ERK2 pathways. Studies using C5a as an agonist gave similar results and confirmed the findings with FMLP. In addition, methyl arachidonyl fluorophosphonate (MAFP), the dual inhibitor of c and iPLA2 enzymes, failed to inhibit superoxide production in primed cells at concentrations that inhibited arachidonate release. These data demonstrate that NADPH oxidase activity can be dissociated from AA generation and indicate a more complex role for arachidonate in neutrophil superoxide production.

scholarly journals Raised Neutrophil Phospholipase A2 Activity and Defective Priming of NADPH Oxidase and Phospholipase A2 in Sickle Cell Disease

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3423-3429 ◽  
Author(s):  
Elahe Mollapour ◽  
John B. Porter ◽  
Richard Kaczmarski ◽  
David C. Linch ◽  
Pamela J. Roberts
Keyword(s):  
Steady State ◽  
Nadph Oxidase ◽  
Phospholipase A2 ◽  
Oxidase Activity ◽  
Neutrophil Function ◽  
Cell Disease ◽  
Nadph Oxidase Activity ◽  
Gm Csf ◽  
Tnf Α ◽  
Phospholipase A2 Activity

Abstract Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% ± 0.5% of total cell radioactivity) were raised relative to control values (2.0% ± 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% ± 17% of control values (n = 10,P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-α was, respectively, 65% ± 11% (n = 7, P = .03) and 57% ± 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% ± 9% and 25% ± 3% of control for GM-CSF and TNF-α, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.

scholarly journals Raised Neutrophil Phospholipase A2 Activity and Defective Priming of NADPH Oxidase and Phospholipase A2 in Sickle Cell Disease

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3423-3429
Author(s):  
Elahe Mollapour ◽  
John B. Porter ◽  
Richard Kaczmarski ◽  
David C. Linch ◽  
Pamela J. Roberts
Keyword(s):  
Steady State ◽  
Nadph Oxidase ◽  
Phospholipase A2 ◽  
Oxidase Activity ◽  
Neutrophil Function ◽  
Cell Disease ◽  
Nadph Oxidase Activity ◽  
Gm Csf ◽  
Tnf Α ◽  
Phospholipase A2 Activity

Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% ± 0.5% of total cell radioactivity) were raised relative to control values (2.0% ± 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% ± 17% of control values (n = 10,P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-α was, respectively, 65% ± 11% (n = 7, P = .03) and 57% ± 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% ± 9% and 25% ± 3% of control for GM-CSF and TNF-α, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.

scholarly journals Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation

2021 ◽  
Vol 12 ◽  
Author(s):  
Ning Xia ◽  
Stefan Tenzer ◽  
Oleg Lunov ◽  
Martin Karl ◽  
Thomas Simmet ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Trichostatin A ◽  
Oral Treatment ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Superoxide Production ◽  
Sirtuin 1 ◽  
Binding Potential ◽  
Free System ◽  
Nadph Oxidase Activity

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2653-2661 ◽  
Author(s):  
Marianne O. Price ◽  
Linda C. McPhail ◽  
J. David Lambeth ◽  
Chang-Hoon Han ◽  
Ulla G. Knaus ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Enzyme Activation ◽  
Superoxide Production ◽  
Reduced Form ◽  
Human Neutrophils ◽  
Nadph Oxidase Activity ◽  
High Level

Abstract The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91phox, p22phox, p47phox, and p67phox. COS-7 cells express the essential small guanosine 5′-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid–induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)–induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47phox, p67phox, and Rac1. Expression of p47phox Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47phox or the p47phox Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47phoxphosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67phox, previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67phox(Val204Ala) mutant.

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.

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