Luminol-dependent Chemiluminescence Activity Research Articles

Хемилюминесцентная и энзиматическая активность нейтрофильных гранулоцитов у больных распространенным гнойным перитонитом в зависимости от исхода заболевания

Aim was to study the chemiluminescent activity and the activity of NAD- and NADP-dependent dehydrogenases of neutrophils depending on the outcome of the widespread purulent peritonitis (WPP). 51 patients with a mean age of RSE 54.2 ± 19.2 years were observed. As a control 75 healthy people of similar age range were examined. Lucigenin- and luminol-dependent chemiluminescence activity and activity of NAD(P)-dependent dehydrogenases peripheral blood neutrophils were studied. In patients with a favorable outcome of WPP the maximum intensity increased and the magnitude of the activation index of lucigenine-dependent spontaneous chemiluminescence reduced. Regardless to the outcome of WPP in patients with increased activation index and maxima of the luminol-dependent spontaneous and zymosan-induced chemiluminescence. At the unfavorable outcome of the disease in neutrophils NAD- dependent isositrate dehydrogenase and anaerobic lactate dehydrogenase reaction activity increased. Regardless to the outcome WPP in patients neutrophils aerobic reaction of lactate dehydrogenase, glucose-6-phosphate dehydrogenase and NADP-dependent glutamate dehydrogenase activity reduced but levels of NADH-dependent reaction of malate dehydrogenase, NADH-dependent glutamate dehydrogenase and NADP-dependent isocitrate dehydrogenase activity increased. With the reduction of the intensity ofplastic processes and imbalances enzymatic activity in nitrogen metabolism in patients with WPP at unfavorable outcome of the disease increases the activity of enzymes that characterize the level of anaerobic and aerobic respiration. In the absence of marked changes in the activity of enzymes that characterize the level of energy processes in cells of patients with favorable outcome of the WPP, increases the intensity of spontaneous lucigenin-dependent chemiluminescence and reduced neutrophil activation index.

Reduced ability of neutrophils to produce active oxygen species in streptozotocin-induced diabetic rats.

We investigated a possible alteration in the ability of neutrophils to produce active oxygen species in streptozotocin (STZ)-induced diabetic rats. The production of superoxide of the neutrophils was assessed by luminol-dependent chemiluminescence (LDCL) after the stimulation by opsonized zymosan. Four days after STZ (60 mg/kg, ip) injection, blood glucose level increased by 399 +/- 9 mg/dl and the LDCL activity was significantly reduced in diabetic rats (control group: 5.12 +/- 1.53 KC/min2/10(6) cells, STZ group: 1.10 +/- 0.07 KC/min2/10(6) cells, p less than 0.01). At Day 17, blood glucose level was maintained high (598 +/- 8 mg/dl) and the LDCL activity (1.01 +/- 0.39 KC/min2/10(6) cells) was almost at the same level as that of diabetic rats at Day 4. Subcutaneous injection of insulin for 10 consecutive days increased the LDCL activity of diabetic rats in a dose-dependent manner (vehicle: 0.82 +/- 0.27 KC/min2/10(6) cells, 0.04 U/day: 1.61 +/- 0.09 KC/min2/10(6) cells, 0.40 U/day: 1.99 +/- 0.47 KC/min2/10(6) cells, 4.00 U/day: 3.33 +/- 0.43 KC/min2/10(6) cells). The data obtained herein indicate that an increased susceptibility to bacterial infection in diabetic rats results from impaired neutrophil function to produce active oxygen species.

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

A protease inhibitor attenuates gastric erosions and microcirculatory disturbance in the early period after thermal injury in rats.

The effects of camostat mesilate, a synthetic serine protease inhibitor on gastric microcirculation and active oxygen species generated by leucocytes from the gastric and jugular veins in the early period after thermal injury were assessed. Male Wistar rats were anaesthetized and a 30% full skin-thickness dorsal burn was inflicted. Camostat mesilate (100 mg/kg) was dissolved in distilled water and administered orally to rats 40 min before thermal injury (the camostat group). The control animals (the vehicle group) were administered distilled water orally. Rolling leucocytes as well as Monastral blue B deposits in venules were observed using in vivo microscopy. Active oxygen species were measured by chemiluminescence. Camostat mesilate decreased the total length of gastric erosion, venular deposits of Monastral blue B, and rolling of leucocytes in venules, and relatively increased luminol-dependent chemiluminescence activity generated by zymosan-stimulated leucocytes 15 min after thermal injury. These results suggest that serine proteases are involved in the formation of gastric erosions and gastric microcirculatory disturbance in the early period after thermal injury.

Effects of aspirin and garlic on cyclooxygenase-induced chemiluminescence in human term placenta.

The spontaneous emission of chemiluminescence appears to reflect changes in the intracellular steady-state concentration of oxygen radicals. Stimulation of arachidonic acid metabolism in human platelets also induces chemiluminescence, which indicates the formation of highly reactive species by cyclooxygenase andlor lipooxygenase [1,2]. It was observed that arachidonic acid, linoleic acid and linolenic acid can induce luminol-induced chemiluminescence in human term placental villi. The luminol-dependent chemiluminescence activity after addition of these fatty acids was investigated after incubation of villi with aspirin and garlic extract.

Possible role of uric acid as an antioxidant in premature infants.

Uric acid (UA) is a water-soluble antioxidant in human body fluids. In umbilical cord blood of 172 neonates with gestational ages between 22 and 42 weeks, UA concentrations were between 50 and 990 mumol/l. There was no correlation with gestational age. Infants with an umbilical cord pH < 7.0 had markedly lower UA concentrations in cord blood than neonates without severe acidosis. UA levels increased during the first 24 h of life and subsequently declined within 2 weeks to lower levels than in older children. In tracheal aspirates (TA), UA at concentrations of about 10(-4) mol/l was found capable of effectively scavenging free oxygen radicals. Luminol-dependent chemiluminescence activity of isolated TA phagocytes or alveolar macrophages was suppressed by UA dose dependently, with a 100% reduction at UA concentrations of 10(-3) mol/l. On the assumption that oxidative stress contributes to the development of chronic lung disease (CLD), in premature infants the ratio of the antioxidant UA and malondialdehyde (MDA) as a marker of oxidative injury was measured in serially obtained TA of 102 ventilated premature infants with birth weights < 1,500 g. At the age of 3-14 days, infants who later developed severe CLD had significantly lower TA UA/MDA ratios than infants without CLD development. In infants with moderate CLD, UA/MDA ratios were comparable to those in the non-CLD group until day 5. During the next 2 weeks, decreasing ratios of UA/MDA paralleled lung injury with increased oxygen requirement and microvascular permeability. Serum UA concentrations did not differ between groups. It is speculated that UA may be physiologically important as an antioxidant in the epithelial lining fluid of the respiratory tract in neonates during the 1st week of life.

挫滅去勢あるいは副腎皮質刺激ホルモン投与ウシの血清がウシリンパ球および好中球機能に及ぼす作用

The effects of sera from calves receiving Burdizzo castration or intravenous ACTH injection (100 IU a head) and then developing an acute plasma cortisol rise on the bovine lymphocyte and neutrophil function were examined with a view to elucidating whether these treatments can induce the animals to develop a serum immunomodulatory effect on those cells. When incubated with bovine lymphocytes, the sera from ACTH-treated calves induced a significant suppression (p<0.05) of concanavalin A-induced blastogenesis. The sera from castrated animals showed similar suppression but not significantly. It can be supposed that the serum lymphocyte suppression might have been related, at least in part, to elevated cortisol concentrations due to the treatments. Those sera, however, did not affect the luminol-dependent chemiluminescence activity, a bactericidal parameter, of bovine neutrophils.

Oxidative stress in gastric mucosal injury: role of platelet-activating factor-activated granulocytes.

Temporal and spatial changes due to oxidative stress in the rat gastric mucosa were visualized and quantified during the process of mucosal hemorrhagic change. The fluorescence associated with dichlorofluorescein (DCF), a hydroperoxide-sensitive fluorochrome, increased 30 min after repeated electrical stimuli to the gastric artery. The increase in the fluorescence was enhanced in the area between two adjacent collecting venules. The content of platelet-activating factor (PAF), the activity of myeloperoxidase (MPO) in the gastric mucosa, the area of mucosal lesions, and the luminol-dependent chemiluminescence activity in zymosan-treated blood samples, obtained from the gastric vein, were measured and found to increase significantly 30 min after the stimuli. The intravenous injection of CV-6209, a PAF antagonist, 5 min prior to the stimuli significantly inhibited the DCF activation, the increases in PAF level and MPO activity, the mucosal hemorrhagic change, and the elevation in chemiluminescence activity. In addition, continuous infusion of superoxide dismutase also inhibited all these changes, except for chemiluminescence activity. These results suggest that oxygen radicals derived from PAF-activated granulocytes induce oxidative stress, and that oxidative changes are actually implicated in the pathogenesis of gastric mucosal injury.

Inhibitions of platelet-activating factor production and granulocyte oxidative activation by Rikkunshi-To in the process of gastric mucosal injury

Abstract The effects of Rikkunshi-To on gastric mucosal injury were investigated with attention to the mechanisms of oxidative stress. Repeated electrical stimulation (RES) on the gastric artery of male Wistar rats decreased mucosal blood flow, which was measured by laser Doppler flowmetry, and subsequently produced hemorrhagic lesions in the gastric mucosa. At 30 min after the completion of RES, increased platelet-activating factor (PAF) content and myeloperoxidase (MPO) activity were observed in the gastric mucosa. Luminol-dependent chemiluminescence activity in blood samples taken from a gastric vein was also elevated after the stimulation. Oral administration of Rikkunshi-To (50 mg/kg) attenuated the increases in luminol-dependent chemiluminescence activity, mucosal PAF content and MPO activity, and the decrease in mucosal blood flow, and prevented mucosal injury induced by the electrical stimulation. These results support the concept that PAF is involved in granulocyte-mediated oxidative stress and gastric mucosal injury, and suggest that Rikkunshi-To prevents mucosal microvascular injury and formation of hemorrhagic lesion by modulating PAF production and oxidative granulocyte activation.

Phagocytosis associated chemiluminescence of hemocytes in Mytilus edulis (Bivalvia)

The respiratory burst associated with phagocytosis by Mytilus edulis hemocytes was investigated by measurement of luminol-dependent chemiluminescence (LDCL). After experimental parameters (number of cells, quantity of stimulus) were determined, the biochemical mechanisms involved in the chemiluminescent process were investigated using inhibitors of oxygen radicals and enzymes. In particular, catechol-like phenols suggested the involvement of NADPH-oxidase and peroxidase in oxidative metabolism of mussel hemocytes. The variability of LDCL response observed among individuals and separated hemocyte subpopulations strongly suggests a variable immunocapacity depending on hemogram composition. Using a specific monoclonal antibody to discriminate different hemocyte types, the eosinophilic granulocytes appeared to exhibit the highest LDCL activity.

Visualization of oxidative processes at the cellular level during neutrophil-mediated cytotoxicity against a human hepatoma cell line, HCC-M.

Human neutrophil-mediated oxidative processes against a human hepatoma cell line, HCC-M, was visualized at the cellular level by using a silicon-intensified target camera and subsequently processing with a computer-assisted digital-imaging processor. Neutrophils were activated by a streptococcal preparation, OK-432. A hydroperoxide-sensitive tracer, dichlorofluorescein diacetate, was loaded in HCC-M and temporal and spatial changes of lipid peroxides in this cell after addition of stimulated neutrophils were analyzed. The luminol-dependent chemiluminescence activity of neutrophils was significantly enhanced and continued for at least 2 hr by stimulation with OK-432, and its activity was shown to be accumulated at the site where a neutrophil attached with HCC-M. The intensity of dichlorofluorescein fluorescence in HCC-M rapidly increased after adding stimulated neutrophils, and their reaction was significantly attenuated by superoxide dismutase. The number of non-viable cells was increased as the dichlorofluorescein fluorescence increase. It is suggested that oxidative stress may play an important role in neutrophil-mediated tumor-cell damage.

Mechanism of inhibitory action of ketone bodies on the production of reactIve oxygen intermediates (ROIS) by polymorphonuclear leukocytes

We determined an effect of acetoacetic acid (AcAc) and 3-hydroxybutyrate (3-OHB) on the production of reactive oxygen intermediates (ROIs) in polymorphonuclear leukocytes from healthy volunteers. Both AcAc and 3-OHB inhibited the luminol-dependent chemiluminescence (LDCL) activities assessed with initial slope and the inhibition rates were about 42%, 44% respectively by AcAc and 3-OHB when the leukocytes were preincubated with 10 mM AcAc or 3-OHB for 60 minutes. The LDCL activity was reduced by 16% and 42% following the addition of 1mM and 10mM AcAc. The similar reduction of the LDCL activity was observed in the addition of 3-OHB. Either 3-OHB or AcAc failed to show a significant reduction of myeloperoxidase (MPO) activity. However, both 3-OHB and AcAc dose-dependently inhibited superoxide anion (O 2 −) production, measured by using cytochrome c. These data provided evidence that both 3-OHB and AcAc suppress neutrophil oxidative metabolism with respect with O 2 − production.

A comparative study of hemocytes from six different snails: Morphology and functional aspects

Hemocytes taken from six different gastropod snails, Achatina achatina, A. fulica, Biomphalaria glabrata, Bulinus natalensis, Helix aspersa, and Lymnaea stagnalis, were compared for morphology, peroxidase activity, and using methods developed for L. stagnalis, the ability to generate reactive oxygen intermediates upon phagocytic stimulation. Numbers of hemocytes per milliliter hemolymph and hemocytes' microscopical morphology showed some variation among the snail species. Peroxidase activity was demonstrated in all snail hemocytes except in those of B. glabrata and A. fulica. Hemocytes of all species generated superoxide upon phagocytic stimulation with zymosan (tested by superoxide dismutase-inhibitable reduction of nitroblue tetrazolium). When tested, hemocytes of A. achatina and of A. fulica displayed luminol-dependent chemiluminescence activity.

Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis

The kinetics of oxygen radical production by phagocytosing hemocytes of the pond snail Lymnaea stagnalis were investigated. After contact had been established between zymosan and hemocytes in monolayers at 0°C, phagocytosis was initiated by a shift to room temperature. Until the internalization phase of phagocytosis was completed, oxidative activity was detected mainly extracellularly (superoxide dismutase inhibitable cytochrome C reduction and peroxidase-catalyzed phenol red oxidation were used for the detection of superoxide and hydrogen peroxide, respectively). Thereafter, extracellular oxygen radical production by phagocytosing hemocytes decreased. Luminol-dependent chemiluminescence activity grew and, after the internalization phase of phagocytosis, remained at a high level, suggesting continued oxygen radical activity intracellularly. These results indicate that contact between zymosan and the hemocyte's plasma membrane stimulates a membrane-bound system to generate and release oxygen radicals. After internalization, this system appears to continue oxygen radical production inside the phagosome. So far, oxygen radical production in snail hemocytes shows many similarities to the mechanism in mammalian leucocytes.

急性胃粘膜病変における内皮 か粒球間相互反応とｐｌａｔｅｌｅｔ‐ａｃｔｉｖａｔｉｎｇ ｆａｃｔｏｒの関与

Gastric microcirculatory disturbances induced by intermittent electrical stimuli (irritation) on the gastric artery produced acute gastric mucosal lesions in the rat stomach. In this experimental model, participation of platelet-activating factor (PAF) in granulocyte-endothelium interaction were evaluated.Laser doppler flowmetry revealed that post-ischemic hyperemia was observed just after the irritation and the flow was significantly decreased after this hyperemic state. Mucosal tissue-type plasminogen activator (t-PA) activity was also elevated just after the irritation and still more increased thirty minutes after the stimuli. Moreover, both luminol-dependent chemiluminescence activity of regional blood sample and myeloperoxidase activity in gastric mucosa were remarkably increased thirty minutes after the irritation. A newly developed PAF-antagonist, CV-6209 attenuated the local fibrinolytic activity, granulocyte-mediated oxidative stress and the alteration of mucosal blood flow, and significantly diminished the gastric ulcer formation.The present study suggests that PAF may cause the excessive fibrinolytic activity and granulocyte-mediated oxidative stress on mucosal microvasculature during acute gastric ulcer formation.

934 INTERACTION OP POLYMORPHONUCLEAR LEUKOCYTES (PMN) AND RESPIRATORY SYNCYTIAL VIRUS (RSV): POSSIBLE ROLE IN PATHOGENESIS OF BRONCHIOLITIS

The effect of live RSV, RSV antigen-antibody complexes, and antibody to RSV on oxidative functions of human PMN was determined by employing luminol-dependent-chemiluminescence (LDCL). The presence of immune complexes was established by Raji cell radioimmune assay (RIA) and by the presence of immune aggregates on electron microscopy. The fresh PMN were obtained from healthy adult volunteers. RSV or antibody to RSV alone failed to induce significant degree of LDCL. However, RSV and RSV antibody mixtures generated significant degree of LDCL (P<0.001). The degree of LDCL activity induced by the virus-antibody mixtures was directly proportional to the presence and concentration of RSV-specific immune complexes as detected by RIA and EM. Removal of immune complexes by ultracentrifugation effectively eliminated the LDCL responses. LDCL activity was reduced four-fold after complement depletion. These data suggest a marked activation of oxidative metabolic pathway of PMN by RSV specific immune complexes. It is proposed that PMN mobilized to respiratory epithelium during the course of RSV infection, rather than the respiratory epithelium itself may serve as the targets for immune complex deposition. Such interaction may mediate the pathogenesis of RSV infection and bronchospasm via the possible release of metabolic products such as prostaglandins or thromboxanes from activated PMN.