The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis.
AuthorsSuh CI, Stull ND, Li XJ, Tian W, Price MO, Grinstein S, Yaffe MB, Atkinson S, Dinauer MC
JournalJ Exp Med
PubMed ID16880255
'Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed ... More
Severe impairment in early host defense against Listeria monocytogenes in mice deficient in acid sphingomyelinase.
AuthorsUtermöhlen O, Karow U, Löhler J, Krönke M
JournalJ Immunol
PubMed ID12594290
'The phagolysosomal compartment is crucial for the defense against infection with intracellular pathogens. Within this compartment, the TNF- and IFN-gamma-responsive acid sphingomyelinase (ASMase) generates the signaling molecule ceramide, resulting in the activation of proteases like cathepsin D. To investigate the possible role of ASMase as a mediator of the antibacterial ... More
Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123).
AuthorsTrayner ID, Rayner AP, Freeman GE, Farzaneh F
JournalJ Immunol Methods
PubMed ID7594627
'It is well established that the fluorescent probes dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydrorhodamine 123 (H2R123) can be used to detect the respiratory burst response of mature myeloid cells. We describe a simple, fast and quantitative assay for myeloid differentiation based on the oxidation of these probes, which can be performed ... More
A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex-stimulated neutrophils.
AuthorsSeetoo KF, Schonhorn JE, Gewirtz AT, Zhou MJ, McMenamin ME, Delva L, Simons ER
JournalJ Leukoc Biol
PubMed ID9307071
'Receptor-mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N-formyl-methionyl-leucyl-phenylalanine (fMLP)l with or without ... More
Fluorescent probes for living cells.
AuthorsJohnson I
JournalHistochem J
PubMed ID10188922
'The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellular ion ... More
Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis.
AuthorsRoepstorff K, Rasmussen I, Sawada M, Cudre-Maroux C, Salmon P, Bokoch G, van Deurs B, Vilhardt F,
JournalJ Biol Chem
PubMed ID18160398
The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1 dominant-positive mutants enhanced, whereas dominant-negative mutants ... More
Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations.
AuthorsElbim C, Lizard G,
JournalCytometry A
PubMed ID19358285
Flow cytometric analysis provides a rapid screen for abnormalities of polymorphonuclear neutrophils (PMN) function and reflect their behavior in vivo more accurately. This review summarizes the major fluorescent probes used to study PMN oxidative burst and apoptosis using flow cytometry (FCM). We also provide examples of FCM studies in physiological ... More
Simultaneous detection of free radical release and membrane current during phagocytosis.
AuthorsHolevinsky KO, Nelson DJ
JournalJ Biol Chem
PubMed ID7713941
Stimulation of macrophages induces the "respiratory burst" response which is associated with the generation of superoxide (O2-), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O2- was temporally related to changes in ... More
Changes in neutrophil functions in astronauts.
AuthorsKaur I, Simons ER, Castro VA, Mark Ott C, Pierson DL
JournalBrain Behav Immun
PubMed ID15265537
Exploration class human spaceflight missions will require astronauts with robust immune systems. Innate immunity will be an essential element for the healthcare maintenance of astronauts during these lengthy expeditions. This study investigated neutrophil phagocytosis, oxidative burst, and degranulation of 25 astronauts after four space shuttle missions and in nine healthy ... More
Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro.
AuthorsHardy MM, Flickinger AG, Riley DP, Weiss RH, Ryan US
JournalJ Biol Chem
PubMed ID8034601
In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr ... More
Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked.
AuthorsGewirtz AT, Seetoo KF, Simons ER
JournalJ Leukoc Biol
PubMed ID9665282
Neutrophils phagocytize high-valency immune complexes (HIC) by an Fc receptor-mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as ... More
Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils.
AuthorsRyan TC, Weil GJ, Newburger PE, Haugland R, Simons ER
JournalJ Immunol Methods
PubMed ID2165099
Immune complex stimulation of human neutrophils elicits, among other events, the formation of phagocytic vacuoles into which the products of the stimulus activated oxidative burst and degranulation are released. In order to monitor burst activity in the phagocytic vacuole, we have developed a fluorochrome-coupled derivative of this neutrophil agonist. The ... More
Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways.
AuthorsKim C, Dinauer MC
JournalJ Immunol
PubMed ID11145705
Rac2 is a hematopoietic-specific Rho family GTPase implicated as an important constituent of the NADPH oxidase complex and shares 92% amino acid identity with the ubiquitously expressed Rac1. In bone marrow (BM) neutrophils isolated from rac2(-/-) mice generated by gene targeting, we previously reported that PMA-induced superoxide production was reduced ... More
Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages.
AuthorsHolevinsky KO, Nelson DJ
JournalBiophys J
PubMed ID9788954
We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average ... More