Helen Lundqvist

ISOLUMINOL-ENHANCED CHEMILUMINESCENCE : A SENSITIVE METHOD TO STUDY THE RELEASE OF SUPEROXIDE ANION FROM HUMAN NEUTROPHILS

A very sensitive chemiluminescence (CL) method was developed for the determination of respiratory burst products generated by the NADPH-oxidase in human neutrophils. Despite the fact that the CL reaction is peroxidase dependent, hydrogen peroxide was found not to participate in the light generating reaction. Phagocytic cells were mixed with isoluminol, a chemiluminescence substrate that detects extracellularly released oxygen species only. Owing to the fact that the availability of released cellular myeloperoxidase is a limiting factor in the reaction, extra peroxidase (horseradish peroxidase; HRP) has to be added to the measuring system. From the fact that the response to phorbol myristate acetate (PMA), as well as to formyl-methionyl-leucyl-phenylalanine (fMLP) was almost totally inhibited by superoxide dismutase (SOD), we conclude that O2.- is the reacting oxygen species measured. The assay system described is well suited for real-time studies of superoxide anion release from activated neutrophils. With the technique, the release of O2.- can be detected from as few as 250 neutrophils.

Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told.

The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles in the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA‐induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule‐deficient HL‐60 cells and cord blood neutrophils, we suggest that the scavenger‐insensitive part of the NADPH oxidase activity in normal neutrophils re‐sides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.

Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium.

The mobilization of intracellular calcium plays an important role in regulating neutrophil activation. With this in mind we investigated the effect of intra‐ and extracellular calcium on the ability of human neutrophils to kill complement‐opsonized Staphylococcus aureus. We found that a rise in intracellular calcium is necessary for efficient killing of phagocytosed S. aureus. In the presence of extracellular calcium, killing of ingested bacteria in calcium‐buffered neutrophils compared with normal cells was slightly reduced. Calcium buffering had no effect on phagocytic uptake by the neutrophils, but did decrease the generation of toxic oxygen metabolites, measured as chemiluminescence (CL). In non‐depleted and calcium‐depleted cells, removal of extracellular calcium did not affect ingestion but did cause a marked decrease in the ability to kill the bacteria. In parallel, the CL response was substantially reduced or completely blocked. These data show that calcium is not a prerequisite for phagocytosis of S. aureus by human neutrophils, but does play a vital role in the post‐ingestion killing of the bacteria by regulating the generation of toxic oxygen metabolites.

Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis

Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the beta(2)-integrins, i.e. anti-CD11b (the alpha subunit of CR3) and anti-CD18 (the beta subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the alpha subunit of LFA-1) and to a lesser extent anti-CD11c- (the alpha subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of beta(2)-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.

Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: Effect of an association of the ligand-receptor complex to the cytoskeleton

The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15 degrees C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.

A simple chemiluminescence assay for the determination of reactive oxygen species produced by human neutrophils

We show that phagocyte production of reactive oxygen species can be measured using a microtitre plate based chemiluminescence blotting technique. The production of reactive oxygen species is determined by their ability to catalyze the oxidation of luminol or isoluminol, resulting in light emission which is recorded on a photographic film. The method permits the determination of NADPH oxidase activity from as few as 9000 cells. It could be used to detect NADPH oxidase defects in neutrophils (e.g. from patients suffering from chronic granulomatous disease), and to screen pharmaceuticals with scavenging activity for reactive oxygen species.

Phagocytosis following Translocation of the Neutrophil b‐Cytochrome from the Specific Granule to the Plasma Membrane is Associated with an Increased Leakage of Reactive Oxygen Species

The effect of neutrophil b‐cytochrome translocation on the respiratory burst activation generated during phagocytosis of yeast particles was investigated. Secretion of neutrophil specific granules was induced by the calcium ionophore ionomycin prior to phagocytosis. The secretory process is associated with a translocation from the specific granules to the plasma membrane of the respiratory burst b‐cytochrome. Respiratory burst activity was measured as release of hydrogen peroxide in the absence of azide (extracellular leakage) and in the presence of azide (total produclion). The subcellular localization of the b‐cytochrome was found to affect the extracellular release of hydrogen peroxide in that a plasma membrane localization was associated with a significantly increased release during phagocytosis. It should be pointed out, however, that most of the hydrogen peroxide, both in control and in ionomycin‐treated cells, is produced intracellularly, probably in the phagosomes.

Translocation of annexin XI to neutrophil subcellular organelles

In an earlier study, annexin XI was found to be present in the cytosol of neutrophil granulocytes (Blood (1996) 87, 4817). The protein was isolated by calcium-dependent translocation to specific granules and was found to be a 42-kDa truncated form of annexin XI. Using human autoantibodies directed against annexin XI we have now reinvestigated the ability of full size annexin XI to translocate to different neutrophil organelles isolated by subcellular fractionation. The autoantisera used recognised a protein of 55-kDa in neutrophil cytosol and comparison with a whole cell lysate indicated that the larger portion of the cellular content of this protein is localised to the cytosol. Azurophil granules, specific granules and secretory vesicles/plasma membrane were isolated by subcellular fractionation on Percoll gradients, mixed respectively with neutrophil cytosol and the calcium concentration was raised. Immunoblotting showed that annexin XI translocated to specific granules and secretory vesicles/plasma membrane at 100 micromol/l calcium. When raising the concentration of calcium to 1 mmol/l, annexin XI translocated to the azurophil granules as well. Periphagosomal translocation of annexin XI occurred during phagocytosis of yeast particles, implying that this protein plays a role in the events associated with the phagocytic process.

Okadaic acid inhibits the signal responsible for activation of the NADPH-oxidase in neutrophils stimulated with serum-opsonized yeast.

The effects of the phosphatase inhibitor okadaic acid (OA) on human neutrophil phagocytic activity were investigated. The chemiluminescence response was found to be greatly reduced in OA‐treated neutrophils during phagocytosis of serum opsonized yeast particles in comparison to control cells. However, the OA‐treated neutrophils phagocytosed yeast particles to the same extent as control cells and the engulfment of the prey was accompanied by phagolysosomal formation in both OA‐ treated and nontreated cells. We thus found that the OA effect was selective in the sense that it inhibits the NADPH‐oxidase activity in neutrophils phagocytosing yeast particles but not uptake of the prey or phagolysosomal formation. Based on the fact that the NADPH‐oxidase activity induced by the chemoattractant formyl‐methionyl‐leucyl‐phenylalanine was not reduced but elevated in OA‐treated neutrophils, we conclude that the state of phosphorylation has no direct effect on the oxidase, but is of importance for the NADPH‐oxidase activating signal(s) generated during phagocytosis of the yeast particles.