Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 μmol/l (9-fold difference), but not by calcium ionophore A23187 (15 μmol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.     

Effect of 4-hydroxynonenal on superoxide anion production from primed human neutrophils

HNE (4-hydroxy-2,3-trans-nonenal), an aldehydic product of lipid peroxidation, has been reported to modulate different functional parameters of human and rat neutrophils (PMNs), such as chemiluminescence, migration and some enzymatic activities, thus exerting effects that varied according to the concentration tested. Experiments were done to evaluate the effects of HNE on superoxide anion (O₂^?.) production from human PMNs, isolated from healthy volunteers. After having tested that HNE by itself was not able to activate the cells, comparisons were made between its effects on PMNs, stimulated by either a single stimulus, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or a combination of stimuli, such as FMLP and the neuropeptide substance P (SP; primed PMNs). In the concentration range tested (10^?12–10^?4 M ), HNE inhibited FMLP-evoked O₂^?. production with an IC₅₀ of 11·6 ± 1·5 × 10^?6 M ; at concentrations ≤10^?6 M , HNE enhanced O₂^?. production elicited by FMLP + SP, while higher concentrations were inhibitory. There was a bell-shaped dose–response curve to the enhancing effects of HNE, depending on the incubation time being recorded after only short periods (≤5 min) of the exposure of the cells to HNE; this was not shown by structurally-related aldehydes, such as 2-nonenal and nonanal. These results suggest that low concentrations of HNE may participate in the evolution of the inflammatory process, by contributing to the activation of PMNs. The effects of high concentrations of the aldehyde may represent a mechanism which contributes to the regulation of the extent of the inflammatory response.     

Influence of gluten-derived fractions on chemiluminescence production by human neutrophils

The effects of gliadin and glyc-gli on leukocyte chemiluminescence response were assessed in vitro. A dose-dependent increase in chemiluminescence response of neutrophils stimulated by zymosan was observed by using gliadin at concentrations ranging between 1 and 20 μg. By increasing glyc-gli concentration, a bimodal response was observed with an enhancement up to 50 μg/ml, followed by suppressive effects, which were again dose-dependent. The possible implications of these findings in human pathology are discussed.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.     

Measurement of chemiluminescence from neutrophils in a 96-well microplate using Lumi Box U-800 II

We have developed a microplate photon counting system based on a cooled charge-coupled device (Lumi Box U-800 II) jointly with Maikurotekku Nition Company (Chiba, Japan). The system makes it possible to quantify chemiluminescence (CL) in a 96-well microplate automatically and simultaneously in a single experiment. We studied the measurement conditions for a luminol-dependent CL assay from neutrophils stimulated with opsonized zymosan (OZ) using this system. Parameters examined included the effect of OZ dose per well, mixing speed, mixing time and detection time on CL responses. The results indicated that this system allows the measurement of CL from phagocytes on a large number of samples using small amounts of sample and regents. © 1997 John Wiley & Sons, Ltd.     

Ticlopidine augments luminol-dependent chemiluminescence in human neutrophils

Neutrophils contribute to the pathophysiology of various ischaemic states. Since many agents thought to be antiplatelet have also been shown to affect neutrophil function, it was of interest to examine the effect of ticlopidine (250 mg, p.o., b.i.d. for three doses), an antiplatelet agent, on fMLP (formyl-methionyl-leucyl-phenylalanine) stimulated neutrophil aggregation and luminol-dependent chemiluminescence in whole blood. Neutrophil aggregation did not significantly change from baseline values during ticlopidine administration. However, luminol-dependent chemiluminescence, an index of respiratory burst metabolism, was noted to be markedly increased during ticlopidine administration. Two hours following the final dose of ticlopidine, the chemiluminescent response (mean ± SEM, n = 5) was significantly increased from 6.27 ± 1.88 to 12.66 ± 2.19 units (p < 0.05). A return to baseline (6.68 ± 2.24 units) five days following the administration of ticlopidine was noted. It is concluded from this study that the acute oral administration of ticlopidine may affect neutrophil function as demonstrated by the significant increase in stimulated luminol-dependent chemiluminescence.     

Luminol-enhanced chemiluminescence after reaction of hydroperoxides with opsonized zymosan

Luminol-enhanced chemiluminescence (LEC) is very sensitive in detecting free radicals but relatively insensitive for hydroperoxides (hydrogen peroxide, tert-butyl hydroperoxide). However, in the presence of opsonized zymosan (often used for stimulation of phagocytic cells) hydroperoxides also induce LEC, suggesting that free radicals are produced under these conditions. Therefore careful interpretation with respect to the nature of the reactive species is necessary when LEC is used for characterization of zymosan-induced phagocytosis. We studied the properties of zymosan-induced LEC under different test conditions and with various inhibitors. Typical radical scavengers, e.g. nordihydroguaiaretic acid and superoxide dismutase, are strong inhibitors, indicating the importance of the superoxide anion. This system is useful for drug testing with respect to antioxidative or radical scavenging activity.     

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O₂ ^–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca²⁺ was absent (0.56±0.13 nmol/min per 10⁶ cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca²⁺. Moreover, the O₂ ^– production by fMLP in the presence of extracellular Ca²⁺ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca²⁺ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O₂ ^– production of electropermeabilized neutrophils in which the intracellular Ca²⁺ concentration was fixed to about 100 nM. These observations suggest that not only Ca²⁺ influx but the inhibition of PKA is necessary for the maximum O₂ ^– production by fMLP and that the O₂ ^– production is partially suppressed by the activation of PKA induced by fMLP.     

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

A fast and sensitive chemiluminescence assay for the determination of H₂O₂ in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H₂O₂ concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H₂O₂ without influence of H₂O₂ decomposition by cellular enzymes or newly produced H₂O₂ due to dismutation of superoxide anion radicals. Concentrations of H₂O₂ were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H₂O₂ concentration upon stimulation could be observed at 3000 cell/mL.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

Pholasin chemiluminescence detects mostly superoxide anion released from activated human neutrophils

The bioluminescent oxygen metabolite indicator protein pholasin was characterized with respect to the type and location of reactive oxygen metabolites detected in suspensions of stimulated human neutrophils. Whereas pholasin detected reactive oxygen metabolites from neutrophil suspensions stimulated with soluble agents, particulate stimulants were apparently not effective triggering agents for pholasin-dependent neutrophil chemiluminescence. Neutrophils stimulated with fMet-Leu-Phe (1 to 100 nmol/l) showed maximum pholasin-dependent chemiluminescence 45 to 60s after stimulation. The time of maximum chemiluminescence was virtually independent of fMet-Leu-Phe concentration. In contrast, the time to reach maximum light emission increased from 60s with 100 nmol/l phorbol ester to 295s with 1 nmol/l phorbol ester. Significant inhibition of stimulated chemiluminescence was caused by both superoxide dismutase (20 μg/ml, 80% inhibition) and reduction of the oxygen concentration in the incubation medium to less than 0.5 μmol/l (95% inhibition). In contrast, the myeloperoxidase inhibitor sodium azide (0.1 nmol/l) afforded only 50% inhibition of the pholasin-dependent neutrophil chemiluminescence. Our results show that pholasin detects superoxide radicals released from cells stimulated by soluble stimulants but not intracellular oxidative activity elicited by particulate stimulants.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Covalent modifications of aminophospholipids by 4-hydroxynonenal

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH₂ groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.     

The effect of storage on whole blood chemiluminescence measurement of equine neutrophils

The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half‐bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra‐receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively. Whole blood CL was estimated at 2, 6, 24, 48, 72, 96 and 120 h after collection. The study demonstrated that temperature and duration of sample storage are factors that determine the quality of CL measurements of whole blood in horses. The study concluded that samples should be stored at 4 °C and the assay should be performed as early as possible. It was also shown that the viability period of horse blood for CL assays is relatively long. Material stored at room temperature for 24 h and even up to 48 h at 4 °C did not show any significant decrease in spontaneous or stimulated chemiluminescence. Copyright © 2012 John Wiley & Sons, Ltd.     

Lucigenin- and luminol-enhanced chemiluminescence in turkey monocytes

Monocytes from 10 week-old specific pathogen-free turkeys were isolated from peripheral blood by density centrifugation and assayed for their oxidative activity by means of a luminometer. Chemiluminescence (CL) properties after stimulation with different soluble and particulate stimuli were compared in lucigenin- and luminol-enhanced assays. A distinct response could be measured with 12-phorbol 13-myristate acetate (PMA) and Zymosan A, but only a weak signal was obtained with calcium ionophore A23187. No oxidative activity could be induced with N-formyl-methionyl-phenylalanine. Peak maxima for both lucigenin- and luminol-enhanced CL were ranked: PMA>Zymosan A>calcium ionophore. The velocity of the lucigenin- and luminol-enhanced responses induced by calcium ionophore were of similar magnitude, but the lucigenin-enhanced responses of Zymosan A and PMA-stimulated monocytes were respectively about 5 and 10 times higher than those obtained in luminol-enhanced assays. No peroxidase activity could be detected in the purified turkey monocytes. As luminol-enhanced CL primarily results from the peroxidase activity, this lack of myeloperoxidase may explain the observed lower responses to the different stimuli, in the presence of a luminol. In contrast, lucigenin-enhanced CL is not related to peroxidase activity, but is a selective probe of oxidase activity. Irrespective of the myeloperoxidase deficiency, different soluble and particulate stimuli induced a significant and reproducible CL response in turkey monocytes, in the presence of both chemiluminigenic probes, lucigenin and luminol. The possibility of measuring the phagocyte oxygenation activity of turkey monocytes represents a useful tool for the study of monocyte mediated host defence in the turkey. © 1997 John Wiley & Sons, Ltd.     

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.     

Temporal adaptation of human neutrophil metabolic responsiveness to the peptide formylmethionyl-leucyl phenylalanine: a comparison between human neutrophils and granule-depleted neutrophil cytoplasts

When polymorphonuclear leukocytes (neutrophils) and soluble or particulate matter interact, the cells produce superoxide anions (O2-) and hydrogen peroxide (H2O2). The chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) induced a very weak response in normal neutrophils. The cellular response was changed, however, as a result of in vitro aging of the cells, i.e. the magnitude of the response was increased following storage of the cells at 22 degrees C for up to 120 min, in the absence of any stimulus, and before the addition of the peptide. When phorbol myristate acetate was used as a stimulus, there was a pronounced production of O2- and H2O2, but no change in magnitude as a result of in vitro aging. When neutrophil cytoplasts (granule-free vesicles of cytoplasm enclosed by plasmalemma) were exposed to the peptide FMLP of PMA, the vesicles produced both O2- and H2O2. There was, however, no increase in oxidative metabolite production in cytoplasts as a result of in vitro aging when either FMLP or PMA was used as a stimulus. The results thus indicate that mere incubation at room temperature primed the cells to increase their production of oxidative metabolites as a result of spontaneous exposure of hidden receptors. The fact that no such effects were observed with cytoplasts indicates that spontaneous receptor recruitment is a granule-dependent process.     

Influence of different luminols on the characteristics of the chemiluminescence reaction in human neutrophils

In search for a luminol with very high output of light, 20 different luminol samples were tested for their ability to enhance the chemiluminescence reaction in phorbol myristate acetate activated human neutrophils. We found that the majority of luminols tested (17 samples) gave almost the same light output from neutrophils, and that the major part of the activity was from an intracellular origin. Owing to the fact that three isoluminol samples were unable to monitor respiratory burst activity taking place intracellularly, a very low level of chemiluminescence was obtained with these samples. Their light output was, however, greatly increased when horseradish peroxidase or myeloperoxidase was added, showing that the light-generating reaction with isoluminol as well as with luminol is peroxidase-dependent. The fact that isoluminol could also use myeloperoxidase as amplifying peroxidase, suggests that the lack of measurable intracellular activity in the presence of isoluminol is somehow related to a limited or restricted diffusion of the molecule to intracellular sites. The isoluminol system constitutes a sensitive system for measuring release of oxygen metabolites from phagocytic cells.     

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils

We have made simultaneous spectrophotometric and O₂ measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O₂ uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H₂O₂. Whereas a second addition of stimulus did not increase O₂ uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest thatin vivo myeloperoxidase reacts with H₂O₂ generatedvia the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.Abbreviations FMLP N-formylmethionylleucyl phenylalanine - MPO²⁺.H₂O₂ Myeloperoxidase compound II - MPO³⁺.H₂O₂ Myeloperoxidase compound I - {ei275-1} superoxide     

Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal

The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE.