Luminol chemiluminescence and active oxygen generation by activated neutrophils.

Upon stimulation by various ligands and membrane perturbers, neutrophils produce various active oxygen species. Since luminol chemiluminescence (LCL) in neutrophils can be blocked by azide, an inhibitor of myeloperoxidase, LCL has been believed to reflect mainly the myeloperoxidase-catalyzed reaction. When cells were stimulated by formyl-methionyl-leucyl-phenylalanine, LCL was strongly inhibited by superoxide dismutase (SOD) and uric acid, a scavenger for hydroxy radical (.OH) and singlet oxygen, whereas it was stimulated by azide. LCL was also inhibited by .OH scavengers, such as mannitol, ethanol, and dimethylsulfoxide. However, when stimulated by phorbol myristate acetate or opsonized zymosan, LCL was strongly inhibited by azide but not by uric acid, and the inhibitory action of SOD was low. Thus, the qualitative and quantitative aspects of reactive oxygen generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the metabolic fate of active oxygens in neutrophils and, hence, their effect on microorganisms and the surrounding tissues might differ depending on the stimulus.     

What happens with ageing: decline or remodeling of opsonin‐independent phagocyte oxidative activity?

Changes that occur with age in the opsonin‐independent oxidative activity of peripheral phagocytes in whole blood were examined by means of luminol chemiluminescence (LCL). The chemiluminescence was registered simultaneously by non‐stimulated and stimulated cells and the age‐related alterations of total and extracellular generation of reactive oxygen species (ROS) were studied using model systems. It was found that the rate of phagocyte activation by the glass surface of the measuring chambers, assessed by the time of the peak appearance after the start of LCL response, increased. However, the maximum oxidative activity and the integral oxidative capacity of the cells during adhesion, evaluated by the maximum LCL intensity and the area under the LCL curve, respectively, declined. No age‐dependence of formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐stimulated oxidative cellular activity for total ROS generation was detected. The maximum oxidative activity and the integral oxidative capacity of peripheral phagocytes to generate extracellular superoxide in response to fMLP was decreased. The likely causes for the observed alterations in phagocyte function are discussed and an analysis of the obtained results is given on the background of the contradictory data published on phagocyte oxidative activity age‐related changes. Copyright © 2009 John Wiley & Sons, Ltd.     

Superoxide Production by Stimulated Neutrophils: Temperature Effect

Activation of neutrophils results in a one-electron reduction of oxygen to produce the superoxide anion and other oxygen-derived, microbicidal species. Evidence from many kinetic studies of oxygen-derived radicals generated by stimulated neutrophils in vitro shows that radical production is optimal at 37°C but only lasts several minutes and then rapidly subsides. These findings support the widely held perception that the neutrophil's “oxidative burst” is a transitory event that peaks within minutes of stimulation and ends shortly thereafter. However, while some studies have shown that under controlled conditions stimulated neutrophils can generate superoxide continuously for several hours, others have observed that the superoxide formation by neutrophils stimulated in buffer at 37°C does not persist. To reconcile the conflicting findings and to better understand neutrophil function, we have reinvestigated the effect of temperature on the kinetics of radical generation by PMA-stimulated cells. Electron paramagnetic resonance spectroscopy coupled with spin-trapping and SOD-inhibitable ferricytochrome c reduction were used to monitor superoxide production by neutrophils stimulated at either 25°C or 37°C in RPMI 1640 medium or in Hank's balanced salt solution. When oxygen was supplied continuously, neutrophils stimulated at 25°Cin buffer or in medium generated superoxide for several hours but at 37°C. particularly in HBSS, O₂-formation strikingly and rapidly decreased. This cessation of superoxide generation was reversible by lowering the temperature back to 25°C. These data imply that in vivo neutrophils may be capable of generating oxy-radicals for prolonged periods. In part, our results may also explain the often observed termination of neutrophil-derived radical formation in vitro and help to dispel the perception that neutrophil-derived oxy-radical production is an ephemeral phenomenon.     

Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^-) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^?) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Effect of n-3 and n-6 polyunsaturated fatty acids and their ethylesters on stimuli-dependent superoxide generation in neutrophils

Polymorphonuclear leukocytes from healthy volunteers (HPMN) generated superoxide (O2*-) following treatment with various stimuli, such as phorbol myristate acetate (PMA), opsonized zymozan (OZ) and arachidonic acid (AA). Other types of n-3 polyunsaturated fatty acids (PUFAS), such as docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), also stimulated O2*- generation. The free form of DHA enhanced the generation of O2*- induced by PMA but inhibited that induced by OZ. In contrast, the ethylester of DHA (DHA-E) inhibited O2*- generation induced by PMA but stimulated that induced by OZ. Similar effects were also observed with ethylesters of EPA (EPA-E), DPA (DPA-E) and AA (AA-E). High concentrations of DHA-E reduced the PMA-induced formation of superoxide without affecting the cellular activity of protein kinase C (PKC). Similar phenomena were also observed with oral neutrophils from healthy volunteers (OPMN). These results indicate that PUFAS and their esters affect 02*- generation in human PMN via different pathways, thereby modulating inflammatory reactions.     

Platelet activating factor is a potent stimulant of the production of active oxygen species by human monocyte-derived macrophages

Platelet activating factor (PAF; C16), 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) stimulated the production of active oxygen species by human monocyte-derived macrophages in culture. An optimal response was observed at a concentration of 13 microM PAF with half-maximal stimulation at 5 microM. The generation of superoxide ion (O2-) and hydrogen peroxide (H2O2) in response to PAF was inhibited specifically by a PAF-antagonist (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N,-trimethyl) hexanolamine; such generation varied with the degree of maturation of cultured monocytes into macrophages. Production of active oxygen species increased progressively to reach a maximal level between days 4 to 6 of culture and remained maximal to day 12, after which it decreased progressively. Phorbol 12-myristate-13-acetate (PMA) and opsonized zymosan also stimulated generation of O2- and H2O2. PAF was however distinguished by its potent capacity to stimulate O2- and H2O2 production even at late stages of macrophage maturation (18 days), at which time both PMA and zymosan lacked significant effect. These findings suggest that PAF is a factor of potential relevance to the inflammatory role of the macrophage in atherogenesis.     

Isolation of human salivary polymorphonuclear leukocytes and their stimulation-coupled responses.

A simple method was developed to isolate viable human salivary polymorphonuclear leukocytes (SPMN) from the oral cavity, and stimulation-coupled responses of these cells were examined. From morphological characteristics and the presence of neutrophil-specific annexin protein (39-kDa protein), we found that these cells seemed to be very similar to human peripheral polymorphonuclear leukocytes (PPMN), although they were in rather young stages. Stimulation-coupled responses of these cells were observed in terms of superoxide (O2.-) genration, luminol chemiluminescence response (LCL), membrane depolarization, and changes in intracellular calcium ion concentration ([Ca2+]i). The rates of superoxide generation by various stimuli, such as formylmethionylleucylphenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) were different. Superoxide generation and strong chemiluminescence response were observed without addition of any stimuli. This endogenous LCL was inhibited by azide and superoxide dismutase (SOD), but not by uric acid (UA). The intensity of the endogenous LCL decreased with time after isolation from the oral cavity. This decrease was accompanied by the appearance of a FMLP-coupled response. Furthermore, the endogenous activity which produced active oxygen species was maintained in the medium at 4 degrees C for a long period after isolation. From these results, it is suggested that SPMN have the ability to show characteristic responses to various stimuli, and that SPMN play important roles in the defense mechanisms in the oral cavity.     

Inhibitory and enhancing effects of piroxicam on whole blood chemiluminescence.

The effects of piroxicam on the production of reactive oxygen species by stimulated phagocytes was studied in whole blood by a chemiluminescence (CL) technique in relation to maximum activity, localization and kinetics of radical generation. We found that piroxicam dose-dependently inhibited total (intra- and extracellular) zymosan-stimulated luminol CL (LCL) at a high stimulant concentration (p = 0.0001). Piroxicam additionally decreased cytochalasin B-reduced LCL, which shows that the effect of the drug should be sought in the extracellular component of the response. Piroxicam inhibited the first phase of extracellular LCL in a dose-dependent manner (p = 0.0001) and revealed itself as an enhancing agent of CL in later time intervals after the start of respiratory burst, in a model system containing horseradish peroxidase (HRP) and sodium azide. It enhanced LCL of a cell-free system, i.e. influenced the CL due to HRP-catalysed decomposition of hydrogen peroxide. It also dose-dependently inhibited the early extracellular superoxide production, evaluated by lucigenin CL (p = 0.022). Piroxicam inhibited the total fMLP-stimulated LCL by 70% approximately and, only by about 30%, the first phase of fMLP-stimulated extracellular LCL, which presupposes an effect on myeloperoxidase-catalysed formation of hypochloric acid. Piroxicam slightly increased the intracellular LCL by phagocytes (p = 0.02), an effect that is probably connected with its ability to induce the release of secondary messengers in signal transduction. In conclusion, the anti-inflammatory effect of piroxicam is probably related to the inhibition of the extracellular generation of superoxide and hypochloric acid in the early stages of phagocyte activation.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Bimodal effect of exogenous hydrogen peroxide on human neutrophils: cytotoxic effect and modulation of respiratory burst in response to an agonist]

It has been found that high concentrations of exogenous hydrogen peroxide kill human neutrophils, the range of toxic concentrations being 100 times as high as that for human endothelial cells. Whereas the H2O2 doses of 30-100 mM induce a fast massive death of neutrophils, 10 mM hydrogen peroxide induces appreciable death only within several hours after treatment. H2O2 used at 30 mM decreases superoxide anion generation by neutrophils stimulated with PMA or FMLP. This decrease is commensurate in value with cell death, thus indicating a high functional resistance of survived cells. In the dose of 10 mM hydrogen peroxide potentiates FMLP (but not PMA-)-induced generation of superoxide anions. Augmentation of superoxide anion generation by H2O2-primed neutrophils in response to FMLP amounts to 200% of the control value. Hydrogen peroxide alone is incapable of inducing superoxide anion generation. It is concluded that exogenous oxidants can alter the functional activity of leukocytes freshly recruited in inflammatory and ischemic tissues.     

Oxygen metabolism and the microbicidal activity of macrophages.

Like neutrophils, phagocytizing macrophages undergo a "respiratory burst" in which significant quantities of oxygen are drawn into the cell. The consumed oxygen is not used in oxidative phosphorylation but, rather, in the formation of superoxide anion (O2) and H2O2. These oxygen metabolites and the products of their interaction, in particular hydroxyl radical (OH), have been implicated in the killing of ingested bacteria by neutrophils. Their role in macrophage microbicidal activity has not been fully defined. However, activated macrophages, which mediate increased resistance to infection in vivo, have a markedly increased capacity to generate O2 and H2O2 in vitro when stimulated by phagocytosis or surface perturbation. The enhanced capacity of activated macrophages to generate highly reactive oxygen metabolites during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of these cells.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes.

Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.     

On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

Antioxidative properties and degradation of serum proteins by active oxygen forms (O2-., OCl-) generated by stimulated neutrophils

The ability of major serum proteins (albumin, immunoglobulin G) and free radical scavenger proteins (ceruloplasmin, superoxide dismutase, transferrin) to interact with O2-. and OCl- was studied. The interaction between serum proteins and OCl- was shown to be nonspecific and cause protein degradation. During SDS polyacrylamide gel electrophoresis ceruloplasmin and transferrin were degraded in the highest degree. Protein damage was also recorded by fluorescence changes. It is suggested that the damaging influence of active oxygen species secreted by stimulated neutrophils into the extracellular space can be abolished only by ceruloplasmin.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.