INF-γ Enhances Nox2 Activity by Upregulating phox Proteins When Applied to Differentiating PLB-985 Cells but Does Not Induce Nox2 Activity by Itself.

Background: The cytokine and drug interferon-γ enhances superoxide anion production by the antimicrobicidal Nox2 enzyme of neutrophils. Because mature neutrophils have a short lifespan, we hypothesized that the effects of interferon-γ on these cells might be mediated by its prolonged exposure to differentiating neutrophil precursors in the bone marrow rather than its brief exposure to mature circulating neutrophils. Effects of INF-Γ on NOX2 activity: To address this possibility we exposed the myeloid PLB-985 cell line to interferon-γ for 3 days in the presence of dimethyl sulfoxide which induces terminal differentiation of these cells.

Phox activity of differentiated PLB-985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6-PLA2.

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged.

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity.

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system.

Generation of neutrophil priming activity by cell-containing blood components treated with pathogen reduction technology and stored in platelet additive solutions.

Background: Storage of cell-containing blood components such as platelet concentrates (PCs) and red blood cells (RBCs) results in generation of biologically active compounds, many of which may be associated with adverse transfusion events. Priming of the neutrophil oxidase activity is a common characteristic of many of the biologically active compounds found in stored blood. We evaluated the priming activity of pathogen reduction technology (PRT)-treated PCs stored in plasma or platelet additive solution (PAS) and PRT-treated RBCs.

Long term G-CSF-induced remission of ulcerative colitis-like inflammatory bowel disease in a patient with glycogen storage disease Ib and evaluation of associated neutrophil function.

We present a 23-year-old female with Glycogen storage disease Ib (GSD Ib) who was diagnosed with ulcerative colitis-like inflammatory bowel disease (IBD) at 7 years of age. G-CSF therapy reversed the IBD, was required to maintain IBD remission and was well tolerated. Neutrophil functions at time of diagnosis showed impaired chemotaxis but normal superoxide anion production and bactericidal activity.

Lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet light-treated platelet concentrates.

Background: Pathogen inactivation technologies provide a potential solution to donor screening and blood testing strategies reducing the risk of transfusion-transmitted infectious diseases. The Mirasol pathogen reduction technology (PRT) system (CaridianBCT) uses riboflavin and UV light to introduce modifications in nucleic acids, reducing the infectious pathogen load in blood components. This study evaluated serum of patients who received PRT-treated platelet (PLT) concentrates over a time period of 28 days for the appearance of antibodies to neoantigens on PLTs.

Oxidase activity in cord blood neutrophils: a balance between increased membrane associated cytochrome b558 and deficient cytosolic components.

Introduction: Newborn infants are prone to develop life-threatening pyogenic infections. Alterations in the function of neonatal phagocytes, including the activity of the neutrophil NADPH oxidase, have been suggested as one cause of increased susceptibility to such infections.

Methods: In the present study, comprehensive analysis of NADPH oxidase enzyme system was performed in cord blood neutrophils from vaginally and cesarean section (CS) delivered, healthy, full-term infants.

CP-64131, an aminobenzazepine with cytokine-like properties, stimulates human neutrophil functions through the p38-MAPK pathway.

CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood.

NADPH oxidase activity of neutrophil specific granules: requirements for cytosolic components and evidence of assembly during cell activation.

Neutrophils and other phagocytic cells support host defense by ingesting microbes and destroying them with reactive oxygen species or oxygen independent mechanisms. Production of ROS is initiated by the phagocyte NADPH oxidase (phox), an enzyme system composed of several constituents. During activation of the cell cytosolic phox proteins (p47phox, p67phox, p40phox, and Rac2) translocate to the plasma membrane and specific granules fuse with the plasma membrane increasing the amount of flavocytochrome b(558).

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils.

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca2+ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105kDa protein (Rel-1) requires Ca2+-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10(-9) to 10(-6)M), becoming maximal after 30s.

A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity.

Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity.

In vivo treatment with granulocyte colony-stimulating factor does not delay apoptosis in human neutrophils by increasing the expression of the vacuolar proton ATPase.

Background: Neutrophils die by apoptosis, and in vivo administration of granulocyte colony-stimulating factor (G-CSF) delays this apoptotic cell death. G-CSF administered in vitro correlates delayed apoptosis with upregulation of the vacuolar proton ATPase (v-ATPase). Because this enzyme requires assembly of membrane and cytosolic domains to function, we hypothesized that in vivo G-CSF would increase synthesis and assembly of v-ATPase components to delay apoptosis.