Kinetics of the reaction of nitroblue tetrazolium reduction by human blood neutrophils

Kinetics of Nitro blue tetrazolium (NBT) reduction to diformasan by neutrophils was investigated using 27 samples of human blood. Analysis of alteration in the share of activated neutrophils (ANP) and activated neutrophil index (ANI) was done in relation to the reaction time. The former reaction is an irreversible reaction of zero (pseudozero) order, while the latter is an irreversible reaction of the first (pseudofirst) order. It has been found out that an induced NBT reduction occurs in parallel with a spontaneous reaction, and that neutrophils have essentially different oxidizing power. The kinetic approach enabled us to discover some indices (NBT quantity involved in the reaction, and reaction speed constant of the first order) which in different samples varied within broader limits than ANP or ANI (within the limits of an order), i.e. provided a possibility to make a more delicate analysis of processes in neutrophils.     

Co-activation of protein kinase C and NADPH oxidase in the plasma membrane of neutrophil cytoplasts

Enucleated, granule-free neutrophil cytoplasts, which in hypotonic media fully release cytosolic components and generate ghosts, have been used to study the cell localization of protein kinase C (PK-C). Treatment of cytoplasts with phorbol myristate acetate, a potent activator of neutrophil functions, triggers translocation of PK-C from the cytosol to the plasma membrane, with an activity recovery of 83 ± 16%. In the ghost fraction, PK-C catalyzes the phosphorylation of polypeptides with an apparent mol. wt. of 115K, 89K, 79K, 62K, 47K and 19K. From the plasma membrane PK-C can be extracted in an active form by Triton X-100 but not by EGTA. Translocation of PK-C is already evident at 5 sec and plateaus at about 50 sec. Activation of plasmalemmal, O₂⁻ generating NADPH oxidase by the phorbol ester is delayed by about 20 sec with respect to the activation of PK-C. Dose/response experiments show that the pattern of activation of O₂⁻ generation by cytoplasts strictly superimposes with the pattern of PK-C translocation.     

Increased nitroblue tetrazolium dye reduction by human neutrophils stimulated by interferon in vitro

Human leukocyte interferon enhanced nitroblue tetrazolium dye (NBT) reduction by human neutrophils (PMNs). Increase in NBT reduction paralleled increase in interferon dose. When human leukocyte interferon was heated to 60 C or 80 C for 30 min, both the antiviral activity and the effect on NBT reduction decreased. Human leukocyte interferon neutralized with anti-human leukocyte interferon serum showed no effect on NBT reduction. A human fibroblast interferon preparation also enhanced NBT reduction. The species dependency of interferon was shown in NBT reduction as well as in antiviral activity.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins: A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Separation of human neutrophil plasma membrane from intracellular vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis.

A putative reservoir of functional plasma membrane proteins, the secretory vesicle identified by latent alkaline phosphatase and tetranectin, has previously been demonstrated based on indirect evidence (Borregaard, N., Miller, L. J., and Springer, T. A. (1987) Science 237, 1204-1206; Borregaard, N., Christensen, L., Bjerrum, O. W., Birgens, H. S., and Clemmesen, I. (1990) J. Clin. Invest. 85, 408-416). Difficulties in separating plasma membranes from this entity by density gradient centrifugation has prohibited discriminative dynamic and quantitative studies of secretory vesicles and plasma membranes. By combining density centrifugation with free flow electrophoresis we overcame this obstacle. Freshly prepared unperturbed human neutrophils were subjected to nitrogen cavitation followed by density centrifugation on Percoll gradients. Light membrane fractions containing plasma membranes and secretory vesicles were applied to high voltage free flow electrophoresis on an Elphor VaP 22. Plasma membrane vesicles, identified by HLA class I antigen mixed enzyme-linked immunosorbent assay (Bjerrum, O. W., and Borregaard, N. (1990) Scand. J. Immunol. 31, 305-313) and 125I applied to cells before cavitation, were clearly separated from secretory vesicles. Electron microscopy revealed a morphology typical of plasma membranes in the former fraction and a population of vesicles with markedly different appearance in the latter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles demonstrated distinct differences in protein patterns between the two fractions. Superoxide generating capacity induced by sodium dodecyl sulfate and cytosol, an entity traditionally ascribed to the plasma membrane, was largely confined to fractions containing secretory vesicles. Thus, the majority of membrane-bound NADPH oxidase components of light membranes of human neutrophils colocalize with secretory vesicles.     

Inhibition of aromatase and NADPH cytochrome c reductase activities in human endometrium by the human placental NADPH cytochrome c reductase antiserum

The cross-reactivity of human placental microsomal NADPH-cytochrome c reductase antiserum, REDFBIV, against the endometrial reductase alone and as a component of the endometrial aromatase was investigated. Human endometrial particulate fractions were incubated with various amounts of REDFBIV for 1 h at 4 degrees C and both enzyme activities were measured at the end of incubation. The extent of inhibition of these endometrial enzymes was compared with the ability of this antiserum to inhibit the placental microsomal reductase and aromatase activities. The antiserum effectively inhibited the activities of both enzymes in both tissues in a dose dependent manner with aromatase activity inhibited to a greater extent than reductase activity. These results indicate the antiserum to the placental microsomal NADPH-cytochrome c reductase component of aromatase recognizes the reductase component of the aromatase enzyme system in endometrium.     

Characteristics of the functional activity of human blood neutrophils using the nitroblue tetrazolium reduction test]

A study was made of the test of restoration of the nitroblue tetrazolium (NBT) by the intact blood neutrophils and the neutrophils stimulated with various endotoxin doses of Gram-negative bacteria in 70 healthy persons. A high sensitivity of the test with the NBT for the quantitative assessment of the neutrophil response under conditions stimulating the phagocytic activity was demonstrated. A functional nonhomogeneity of the neutrophil population, the most distinct with the minimal stimulating action, was noted.     

Ability of granulocytes to reduce nitroblue tetrazolium in uremic patients treated conservatively

NTB reduction test, both spontaneous and stimulated with E. coli endotoxin, was performed in peripheral blood granulocytes of 40 individuals of both sexes aged between 18 and 64 years treated conservatively at the nephrologic outpatient clinic. Serum creatinine, urea and uric acid were assayed at the same time. A control group included 40 healthy individuals of both sexes aged between 20 and 56 years. Statistically significant increase in spontaneous reduction of NTB was achieved in the group of the uremic patients in comparison with the control group. Moderately positive correlation between creatinine level and percentage of NTB-positive cells in the spontaneous test was shown. Possibility of granulocyte stimulation by uremic toxins is being considered.     

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins. A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.     

Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.     

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. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.     

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.