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.     

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.     

Mild hypothermia protects against postischemic hepatic endothelial injury and decreases the formation of reactive oxygen species

The ability of mild hypothermia (MH; 34 degrees C) to protect against postischemic endothelial injury and decrease reactive oxygen species' (ROS) formation was studied using lucigenin and luminol enhanced chemiluminescence (CL). Lucigenin CL is largely specific for superoxide, while luminol reacts with many ROS. Isolated rat livers perfused under constant flow in a non-recirculating system were exposed to 2.5 h of ischemia after 0.5 h perfusion with Krebs-Henseleit buffer at either normothermia (38 degrees C) or mild hypothermia (34 degrees C) (n = 5, all groups). CL (cps), vascular resistance (Woods units), O2 consumption, and potassium efflux were measured at the end of perfusion, and at 0 min reperfusion, and every 30 min during reperfusion. For both the lucigenin and luminol groups, CL and vascular resistance increased significantly (repeat measures ANOVA, P <0.05) for normothermia (NT, 38 degrees C) but not mild hypothermia. Potassium efflux did not change significantly for the mild hypothermia groups. In the luminol enhanced group, oxygen consumption was greater in the mildly hypothermic group at 1 h and 1.5 h of reperfusion. Mild hypothermia decreased postischemic ROS production. Increased vascular resistance in the normothermia group may indicate an endothelial injury. Mild hypothermia appears to protect against this injury.     

Influence of neopterin on the generation of reactive oxygen species in human neutrophils

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.     

Mild hypothermia protects against postischemic hepatic endothelial injury and decreases the formation of reactive oxygen species

Abstract

The ability of mild hypothermia (MH; 34°C) to protect against postischemic endothelial injury and decrease reactive oxygen species' (ROS) formation was studied using lucigenin and luminol enhanced chemiluminescence (CL). Lucigenin CL is largely specific for superoxide, while luminol reacts with many ROS.

Isolated rat livers perfused under constant flow in a non-recirculating system were exposed to 2.5 h of ischemia after 0.5 h perfusion with Krebs-Henseleit buffer at either normothermia (38°C) or mild hypothermia (34°C) (n = 5, all groups). CL (cps), vascular resistance (Woods units), O₂ consumption, and potassium efflux were measured at the end of perfusion, and at 0 min reperfusion, and every 30 min during reperfusion.

For both the lucigenin and luminol groups, CL and vascular resistance increased significantly (repeat measures ANOVA, P <0.05) for normothermia (NT, 38°C) but not mild hypothermia. Potassium efflux did not change significantly for the mild hypothermia groups. In the luminol enhanced group, oxygen consumption was greater in the mildly hypothermic group at 1 h and 1.5 h of reperfusion.

Mild hypothermia decreased postischemic ROS production. Increased vascular resistance in the normothermia group may indicate an endothelial injury. Mild hypothermia appears to protect against this injury.     

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.     

Lucigenin luminescence elicited by microsomes and its modulation by nitroazole compounds

The lucigenin luminescence elicited by rat liver microsomes and its modulation by the nitroazole compounds metronidazole and sanazole (drug AK-2123), as well as the rates of lucigenin-dependent NADPH consumption and cytochrome c reduction, were studied. The obtained data suggest that the luminescence can be the result of univalent lucigenin reduction by microsomal NAD(P)H-reductases, generation of superoxide anion radical in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of cytochrome P450 heme on dioxetane decomposition, followed by light emission.     

Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa

Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.     

Metabolic Transformation of Neuropeptide Carnosine Modifies Its Biological Activity

1. The ability of carnosine and carnosine-related compounds (CRCs) to interact with several free oxygen radicals is analyzed.2. Carnosine, the CRCs (imidazole, histidine, anserine), and ergothioneine were found to be equally efficient in singlet oxygen quenching. During generation of hydroxyl radicals from hydrogen peroxide in the Fenton reaction, carnosine was found to be more effective than the CRCs tested.3. By measuring the chemiluminescence produced by carnosine and CRCs in rabbit leukocytes in the presence of luminol or lucigenin, we conclude that carnosine and other CRCs play a stimulating role in superoxide oxygen production while suppressing the myeloperoxidase system.4. ADP-induced aggregation of human platelets is slightly stimulated by carnosine but is inhibited by acetylanserine.5. The following rank order of efficiency of CRCs was demonstrated while measuring the oxidation of human serum lipoproteins: acetylcarnosine < acetylanserine < homocarnosine = ophidine < carnosine < anserine.6. The results obtained demonstrate that metabolic transformation of carnosine into CRCs in tissues may play an important role in regulating the native antioxidant status of the organism.     

What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H₂O₂ is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe²⁺ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co²⁺ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co²⁺. Cu²⁺ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration–reaction curve is not as steep. NaOCl enhances H₂O₂/Fe²⁺-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10⁴ (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.     

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO₂ and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO₂ and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H₂O₂ did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H₂O₂, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H₂O₂ also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H₂O₂ had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H₂O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used. © 1997 John Wiley & Sons, Ltd.     

Influence of bioactive glasses on the respiratory burst metabolism of polymorphonuclear neutrophils]

BACKGROUND: Bio-glasses are bioactive materials used to coat implants. The immunological reaction to wear particles from such coatings has hardly been investigated. The generation of reactive oxygen species by polymorphonuclear neutrophils (PMN) on phagocytosis of particles might elicit further immune reactions. METHODS: The production of reactive oxygen species was investigated in whole blood in the form of chemiluminescence using the probes luminol and lucigenin. RESULTS: Bioglass particles stimulated PMN to generate free radicals as a function of the bioactivity of the composition. This activation was significantly reduced by prior soaking of the particles. Extracts of the bioactive glasses also inhibited the liberation of free radicals upon the stimulus opsonized zymosan. CONCLUSION: Our results show that despite the proven good biocompatibility of bioactive glasses, further in vivo checks in the early stages of the reaction are needed.     

Detection of Superoxide and Peroxynitrite in Model Systems and Mitochondria by the Luminol Analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10–100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.     

Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.     

Evaluation of the total antioxidant capacity by using a multipumping flow system with chemiluminescent detection

An automated flow-based procedure for assessment of total antioxidant capacity was developed. It involved a multipumping flow system, a recent approach to flow analysis, and exploited the ability of selected compounds to inhibit the chemiluminescence reactions of luminol or lucigenin with hydrogen peroxide. The system included several discretely actuated solenoid micropumps as the only active components of the flow manifold. This enabled the reproducible insertion and efficient mixing of very low volumes of sample and reagents as well as the transportation of the sample zone toward a flow-through luminometer, where the chemiluminometric response was monitored. With luminol as the chemiluminogenic reagent, linearity of the analytical curves was noted up to 3.2x10(-4), 1.1x10(-3), and 8.8x10(-8) molL-1 for Trolox, ascorbic acid, and resveratrol, respectively. With lucigenin, linear calibration plots up to 2x10(-5) molL-1 of Trolox and 5.7x10(-5)molL-1 of ascorbic acid were obtained. As favorable analytical figures of merit, the measurement precision (RSD typically between 0.2 and 2.0%, n=10), low operational costs, low reagent consumption, sampling rate (160 and 70 h-1), and versatility should be highlighted. The proposed system can be used in distinct analytical circumstances without requiring physical reconfiguration.     

Reactive oxygen forms and luminescence of intact microspore cells

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.     

Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Effect of High-Pressure Helium on Latex-Induced Activated Chemiluminescence of Human Blood Leucocytes

High-pressure helium reduces the latex-induced activated chemiluminescence of diluted human blood. This effect is more noticeable, when lucigenin rather than luminol is used as the activator of chemiluminescence. The effect lessens in the presence of Mg²⁺ but not Ca²⁺. The data suggest the association of this effect with actin polymerization in leucocytes phagocytosing the latex particles.     

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.     

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.