Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity.

When phagocytic leukocytes, e.g. neutrophils, monocytes and macrophages, interact with soluble or particulate stimuli, the cells respond with an increased production of reactive oxygen metabolites. This production can be measured with the luminol-amplified chemiluminescence (CL) technique. In the present study, the CL reaction induced in monocyte-derived macrophages was investigated and compared to the responses of neutrophils and monocytes. In systems without additives the CL response of macrophages to soluble stimuli (FMLP, PMA and ionomycin) was very low. Addition of a peroxidase (HRP) to the reaction mixtures resulted in a pronounced increase in CL activity. The cellular CL response in macrophages is thus limited by the amount of peroxidase available. The macrophage response differs qualitatively from the responses of neutrophils and monocytes, in that the intracellular phase of the response is missing.     

Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

The influence of age and sex on phagocyte chemiluminescence

The process of ageing is associated with increased susceptibility to infection. Phagocytes form the primary defence mechanism against infecting microorganisms, but the influence of ageing on phagocyte function remains controversial. In this study we have applied a microtitre plate phagocyte chemiluminescence (CL) assay suitable for clinical use to compare phagocyte oxidative metabolism in younger healthy subjects (age 20–60 years) and healthy older (60–70 years) subjects. Polymorphonuclear leukocytes (PMNL) and monocytes were stimulated using phorbol myristate acetate (PMA), serum opsonized zymosan (SOZ), and non-opsonized zymosan (ZYM) in the presence of both lucigenin and luminol. Monocytes showed a higher luminolenhanced CL response to PMA in males compared with females in the younger age group. No PMNL differences were observed between the sexes. Although no difference were found in relation to age when cells were stimulated with PMA and SOZ, significantly lower background (unstimulated) CL was obtained from PMNL with luminol. PMNL luminol-enhanced CL responses were also lower in response to ZYM. The findings suggest a reduced response of PMNL from older subjects to minimal stimulation. This could be related to abnormalities in the triggering of the respiratory burst or myeloperoxidase release due to ageing. The influence of age and sex should be taken into account in clinical studies of phagocyte CL.     

Luminol‐, isoluminol‐ and lucigenin‐enhanced chemiluminescence of rat blood phagocytes stimulated with different activators

Luminol‐, isoluminol‐ or lucigenin‐enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol‐12‐myristate‐13‐acetate (PMA), calcium ionophore A23187 (Ca‐I) or N‐formyl‐Met‐Leu‐Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic pro?les of luminol‐ and isoluminol‐enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA‐ and OZ‐stimulated CL were comparable. The presence of plasma increased OZ‐activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the ?uorescent OZ using a ?ow cytometer. In contrast, the presence of plasma decreased PMA‐activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol‐enhanced CL was reliable. Blood volumes over 5 µL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 µL whole blood is suf?cient for routine luminol‐enhanced CL analysis of whole blood oxidative burst in rats. Copyright © 2004 John Wiley & Sons, Ltd.     

The activation of the early chemiluminescent response of human neutrophils by joint treatment with tumor necrosis factor (TNF-alpha) and the calcium ionophore A-23187]

The influence of recombinant human tumor necrosis factor-alpha (TNF-alpha) and calcium ionophore A23187 on luminol- and lucigenin-dependent chemiluminescence capacity (CL) of human polymorphonuclear leukocytes (PMN) has been studied. The CL response of TNF-alpha treated PMN is amplified by lucigenin, but not luminol. TNF-alpha and A23287 synergistically induced both the luminol- and lucigenin-dependent early CL response. The combination of A23187 and activator of protein kinase C--phorbol (myristoyl-13-acetyl)--also provoked early CL response. While the combination of TNF-alpha and A23187 decreased late CL response compared to A23187 alone. The obtained results suggests that synergistic CL response of PMN induced by TNF-alpha and A23187 is connected with activation of protein kinase by TNF-alpha.     

Chemiluminescence Response of Human B-Cell Lines

Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cells share certain properties with monocytes: they are capable of presenting protein antigens to antigen-specific T-lymphocytes and of releasing an Interleukin 1-like factor. It was our interest to study whether transformed B-cells resemble monocytes by generating toxic oxygen radicals. Human B-cell lines were developed from human peripheral blood lymphocytes by EBV-transformation. The induction of the respiratory burst in the B-cells was assessed by chemiluminescence (CL) in the presence of lucigenin. B-cells were stimulated with phorbol-myristate-acetate (PMA), zymosan particles, the chemotactic peptide f-met-phe, the complement split product C5a and with a recently described granulocyte activating cytokine (GRAM). Stimulation with PMA elicited a distinct CL-response in the tested B-cell lines. The CL-signal was significantly reduced by superoxide dismutase, but not by D-mannitol and catalase. No significant response to any of the other stimuli was detected. Furthermore, none of the stimuli induced a luminol-enhanced CL signal, which, in contrast to lucigenin, is dependent on the presence of peroxidase.

Our results indicate that EBV infected B-cells were able to generate significant amounts of reactive oxygen species, particularly superoxide. It appears that virus transformation uncovers genetic information which is usually not expressed in non-transformed B-cells.     

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.     

Anti-My-26: a monoclonal antibody inhibiting human phagocyte chemiluminescence

Anti-My-26, a mouse monoclonal IgG1 antibody, was raised against human granulocytes and has been shown to inhibit luminol-enhanced, glucose-independent chemiluminescence (CL) of human granulocytes (or monocytes) responding to the soluble secretagogues A23187 or ionomycin (calcium ionophores) and phorbol myristate acetate (PMA). Anti-My-26 inhibition of CL was reversible and was dependent on both secretatogue and monoclonal antibody concentration. This inhibition appeared to be directed at the component of granulocyte CL that is independent of NAD(P)H-oxidase-catalyzed formation of superoxide anion, because neither opsonized zymosan-stimulated CL nor the PMA-induced decrease in NAD (P)H-associated autofluorescence was affected by anti-My-26. In addition, ionomycin, over a wide concentration range, failed to generate any decrease in granulocyte autofluorescence. The A23187-induced CL inhibited by anti-My-26 was correlated with its depression of oxygen consumption. Furthermore, anti-My-26 was not cytotoxic and did not itself induce oxidative metabolism when used as a stimulant. Binding of anti-My-26 to phagocytic cells was not decreased by pre-exposure of cells to either A23187 or PMA. Evidence is presented to suggest that the binding of anti-My-26 to the granulocyte surface inhibits the oxidative response to calcium ionophore and PMA by blocking a common pathway(s) stimulated by these different secretagogues.     

Chemiluminescence Response of Human B-Cell Lines

Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cells share certain properties with monocytes: they are capable of presenting protein antigens to antigen-specific T-lymphocytes and of releasing an Interleukin 1-like factor. It was our interest to study whether transformed B-cells resemble monocytes by generating toxic oxygen radicals. Human B-cell lines were developed from human peripheral blood lymphocytes by EBV-transformation. The induction of the respiratory burst in the B-cells was assessed by chemiluminescence (CL) in the presence of lucigenin. B-cells were stimulated with phorbol-myristate-acetate (PMA), zymosan particles, the chemotactic peptide f-met-phe, the complement split product C5a and with a recently described granulocyte activating cytokine (GRAM). Stimulation with PMA elicited a distinct CL-response in the tested B-cell lines. The CL-signal was significantly reduced by superoxide dismutase, but not by D-mannitol and catalase. No significant response to any of the other stimuli was detected. Furthermore, none of the stimuli induced a luminol-enhanced CL signal, which, in contrast to lucigenin, is dependent on the presence of peroxidase.

Our results indicate that EBV infected B-cells were able to generate significant amounts of reactive oxygen species, particularly superoxide. It appears that virus transformation uncovers genetic information which is usually not expressed in non-transformed B-cells.     

Is lysosomal fusion required for the granulocyte chemiluminescence reaction?

When phagocytic leukocytes interact with soluble or particulate stimuli, the cells increase their production of oxidative metabolites. This increased production can be measured as luminol amplified light emission or chemiluminescence. From the literature it can be concluded that the chemiluminescence reaction is dependent on oxygen radicals produced by the cells and on the enzyme myeloperoxidase. Since the radical producing system and the peroxidase are localized to different subcellular compartments, it is proposed that a lysosomal fusion, bringing the two reactants together into the same subcellular compartment, is a prerequisite for the chemiluminescence reaction.     

抗体包被瘤细胞膜引发人血多形核白细胞化学发光

用抗体包被K562细胞膜碎片(Ab-M)刺激人血多形核白细胞(PMN)产生化学发光,对其发光动力学及活性氧代谢特征进行了比较,结果表明Ab-M引发PMN发光动力学与酵母多糖不同.用秋水仙碱干扰PMN膜结构完整性可抑制其发光产额,Ca²⁺可促增PMN发光,提示PMN氧代谢的调控与Fc受体和Ca²⁺动员有关;活性氧系PMN实施细胞毒效应的重要物质.     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Influence of selenium on innate immune response in kids

The effect is described of selenium supplemented in an inorganic and organic form on the innate immune response of goats. Though the phagocytic activity (as a marker of the immune function) was found to be lower in organic-Se-treated group than in control (54.5 ± 4.32 vs. 60.2 ± 9.15 %), it did not generally exhibit any significant differences; similarly, no differences were found in the phagocytic index. The production of reactive oxygen species (ROS) was determined using the luminol-enhanced chemiluminescence (CL) (estimated as peak CL, integral CL and a peak time after addition of calcium ionophore A23187, opsonised zymosan (OZP) and phorbol-12-myristate-13-acetate as effectors. A significant ROS increase reflected in integral CL and a peak time was found in the inorganic-Se-treated group when OZP was used as activator; other parameters did not exhibit significant changes. The supplementation of Se in inorganic form can thus be seen to influence positively the innate immune system of kids.     

Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.     

Effects of Ph CL 28A on eicosanoid synthesis in rat isolated hearts.

The effects of Ph CL 28A, a derivative of sulphasalazine, on cardiac function and eicosanoid synthesis were examined in rat isolated hearts perfused via the coronary circulation. Two stimuli for eicosanoid synthesis were used: exogenous arachidonic acid (AA) or the calcium ionophore, A23187. Following exogenous AA, coronary perfusion pressure (CPP) and cardiac developed tension (CDT) increased transiently; only the CPP response was diminished by Ph CL 28A. The output of TxB2 but not that of 6-oxo-PGF1 alpha from the heart after exogenous AA was inhibited by Ph CL 28A. The ionophore, A23187, increased CPP with minor changes in CDT; Ph CL 28A did not affect either response. The ionophore released 6-oxo-PGF1 alpha, TxB2 and LTC4 from the heart but only LTC4 output was decreased by Ph CL 28A. We conclude that, although Ph CL 28A did not increase output of 6-oxo-PGF1 alpha from rat heart with either of the stimuli used, its inhibition of the output of vasoconstrictor eicosanoids could be of benefit to the coronary circulation.     

Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Different effects of exogenous horseradish peroxidase on luminol-amplified chemiluminescence induced by soluble and particulate stimuli in human neutrophils

The chemiluminescence (CL) technique with scavengers for superoxide anion (superoxide dismutase) and hydrogen peroxide (catalase) was used to characterize the generation of reactive oxygen species (ROS) inside and outside the human neutrophil after stimulation with both soluble (formyl-methionyl-leucyl-phenylalanine, FMLP) and particulate (urate crystals, zymosan, oxidized LDL) stimuli. Depending on the stimulus used, ROS generation differed in composition and absolute amounts. The ratio between extracellularly and intracellularly produced ROS ranged from 0.3 (zymosan) to 4.2 (FMLP). While enhancing substantially FMLP-stimulated CL, horseradish peroxidase inhibited CL induced by particulate stimuli by 40–80%. Furthermore, an azide-insensitive and therefore peroxidase-independent part of CL was found in FMLP-, LDL- and zymosan-stimulated cells. The results indicate that different agonists may lead through distinct chemical pathways to neutrophil luminol-amplified light generation. © 1998 John Wiley & Sons, Ltd.