Luminol-enhanced Chemiluminescence Response Research Articles

Evaluation of the intracellular radical generation by stimulated leukocytes by total luminol chemiluminescence

Luminol-enhanced chemiluminescence (LCL) is a widely used method for quantitative evaluation of reactive oxygen species (ROS) generated by stimulated leukocytes. The chemiluminescent kinetics registered (Total LCL) is an expression of simultaneously running processes of extra- and intracellular radical generation. In literature, model chemiluminescent systems, designed to separate extra- from the intracellular light, have been proposed. These systems, however, change cellular activity. The aim of the present study was to propose an approach for evaluating the intracellular ROS generation by stimulated leukocytes in whole blood. Heparin anti-coagulated blood from healthy volunteers was used. The chemiluminescent kinetics reflecting leukocyte activity to produce ROS were registered by a computerized chemiluminometer. To distinguish extra - from the intracellularly generated light by the cells, some model chemiluminescent systems were used. It was found that the intracellular light generated by stimulated leukocytes was a substantial part of Total LCL. The proportion between the extra- and intracellular light within the Total response depended on the concentration of the particulate stimulant. At a low zymosan concentration (0.25 mg/ml) Total LCL response was found to be intracellular. On the basis of the results obtained we conclude that Total LCL at a low stimulant concentration is of an intracellular origin, which could be used to study the effect of different compounds on the intracellular oxidative leukocyte activity. In the course of some pathological processes accompanied by suppurative foci formation, bacteriemia, ect., the proportion between the number of bacteria and leukocytes considerably changes. A change in the stimulant concentration allows to model these pathological states and to evaluate the extra- and intracellular radical generation by stimulated leukocytes. Scripta Scientifica Medica 2007;39(2):111-114

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 × 10 6 cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017–167 ng ml −1) for 1.25 × 10 6 neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.

Pro-Oxidative Interactions of Cobalt with Human Neutrophils

The primary objectives of this study were to investigate the effects of cobalt(II) chloride (Co, 1.5–25 μ M) on the reactivity of hydrogen peroxide (H2O2, 100 μ M) or oxidants generated by activated human neutrophils. The prooxidative interactions of Co with H2O2 or cells were measured by luminol-enhanced chemiluminescence (LECL), and according to the extent of oxidative inactivation of added alpha-1-proteinase inhibitor (API). Cobalt dramatically potentiated the oxidation of luminol and API by both H2O2 and neutrophils activated with phorbol 12-myristate 13-acetate (5 ng/ml), without affecting the assembly of NADPH oxidase or the magnitude of oxygen consumption by the cells. Using 5,5-dimethyl-pyrolline 1-oxide-based electron spin resonance spectroscopy we were unable to detect hydroxyl radical formation by Co in the presence of either H2O2 or activated neutrophils, while the corresponding LECL responses were unaffected by the hydroxyl radical scavengers benzoate and mannitol (50 mM). These observations indicate that Co potentiates the reactivity of neutrophil-derived oxidants, primarily H2O2, which if operative in vivo during exposure to the heavy metal may pose the risk of oxidant- and protease-mediated tissue injury.

Respiratory burst activity of rainbow trout ( Oncorhynchus mykiss) phagocytes is modulated by antimicrobial drugs

In vitro and in vivo effects of five different antimicrobial drugs on the chemiluminescence (CL) response of phagocytes isolated from the blood and from the head kidney of rainbow trout ( Oncorhynchus mykiss) were examined. Opsonized zymosan was used to stimulate the respiratory burst utilizing luminol and lucigenin as substrates. The drugs tested were: oxolinic acid (OA), oxytetracycline (OTC), florfenicol (FF) and sulphadiazine in combination with trimethoprim (SDZ/TMP) in ratio 5:1. In the in vitro test, the CL response was measured after a pre-incubation of head kidney and blood phagocytes for 2 and 24 h, with six different concentrations (0.1–100 μg ml −1) of each drug. The lucigenin-enhanced CL response of blood phagocytes was significantly suppressed by OA, OTC and SDZ/TMP at the highest concentration tested, but the luminol-enhanced response was unaffected. The lucigenin-enhanced CL response of head kidney phagocytes was significantly suppressed by SDZ/TMP at concentrations greater than 5 μg ml −1 and by OTC and FF at the highest concentration tested. For the in vivo tests, the fish were fed for 10 days with therapeutic doses of the drugs and the blood and head kidney phagocytes were isolated immediately after the drug exposure. SDZ/TMP stimulated the luminol-enhanced CL response of blood and head kidney phagocytes. FF had a suppressive effect on CL response with luminol and lucigenin on blood phagocytes. OA had a suppressive effect on lucigenin CL response in blood and head kidney phagocytes.

Inhibition of chemiluminescent response of olive flounder Paralichthys olivaceus phagocytes by the scuticociliate parasite Uronema marinum.

Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian parasite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (ROIs) generated by respiratory bursts of phagocytes using zymosan as a stimulant. Cytotoxic and antioxidative activities of excretory-secretory (ES) products of the parasite were evaluated as well. Live U. marinum and its ES products had a negative and dose-dependent effect on luminol-enhanced CL responses of zymosan-stimulated phagocytes of olive flounder. After CL assay, the number of phagocytes showing viability was significantly reduced in the cells incubated with live U. marinum at ratios of 2:1 and 1:1 phagocytes:ciliates or ES products with 0.3 mg protein ml(-1) compared to controls. Lysis of phagocytes by exposure to ES products was observed also. ES products from U. marinum showed considerably high activities of superoxide dismutase (SOD) and catalase. The results of this study suggest that U. marinum can protect itself against host's phagocytes mediated oxidative damage by destroying phagocytes and scavenging ROIs.

Antioxidant properties of alpha-crystallin.

alpha-Crystallin is a major chaperone lens protein to which has been ascribed antioxidant functions. In the present work we have evaluated the antioxidant and free radical scavenging properties of bovine alpha-crystallin in a series of in vitro models: zimosan-induced, luminol-enhanced chemiluminescence response of polymorphonuclear leukocytes, the autoxidation of brain homogenate, bleaching of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-derived radical cations, trapping of peroxyl radicals, and reactivity toward hypochloric acid. In all these systems, the reactivity of alpha-crystallin is higher than or similar to that of bovine serum albumin. It is concluded that, given the high concentrations of ol-crystallin in the lenses, its capacity to interact with free radicals and to remove hypochlorous acid could contribute to the maintenance of the lens functionality.

Ambient Air Particles: Effects on Cellular Oxidant Radical Generation in Relation to Particulate Elemental Chemistry

Epidemiologic studies have reported causal relationships between exposures to high concentrations of ambient air particles (AAP) and increased morbidity in individuals with underlying respiratory problems. Polymorphonuclear leukocytes (PMN) are frequently present in the airways of individuals exposed to particles. Upon particulate stimulation the PMN may release reactive oxygen species (ROS), which can result in tissue damage and injury. In this study a wide range of AAP samples from divergent sources (1, natural dust; 2, oil fly ash; 2, coal fly ash; 5, ambient air; and 1, carbon black) were analyzed for elemental content and solubility in relation to their ability to generate ROS. Elemental analyses were carried out in AAP and dH2O-washed AAP using energy dispersive x-ray fluorescence (XRF). Percent of sample mass accounted for by XRF-detectable elements was 1.2% (carbon black); 22–29% (natural dust and ambient air particles); 13–22% (oil fly ash particles); 28–49% (coal fly ash particles). The major proportion of elements in most of these particles were aluminosilicates and insoluble iron, except oil-derived fly ash particles in which soluble vanadium and nickel were in highest concentrations, consistent with particle acidity as measured in the supernatants. Human blood-derived monocytes and PMN were exposed to AAP and dH2O-washed particles, and generation of ROS was determined using luminol-enhanced chemiluminescence (LCL) assay. All the particles induced chemiluminescence response in the cells, except carbon black. The oxidant response of monocytes induced by AAP (with the exception of oil fly ash particles) was less than the response elicited by PMN. The LCL response of PMN in general increased with all washed particles, with oil fly ash (OFA) and one urban air particle showing statistically significant (p < 0.05) differences between dH2O-washed and unwashed particles. The LCL activity in PMN induced by both particles and dH2O-washed particles was significantly correlated with the insoluble Si, Fe, Mn, Ti, and Co content of particles (p < 0.05). No relationship between LCL activity in PMN and soluble transition metals such as V, Cr, Ni, and Cu was noted. Pretreatment of the particles with a metal ion-chelator, deferoxamine, did not affect LCL in PMN, suggesting that metal ions are not related to the induction of LCL in PMN. Particulate S content and acidity of the particles as measured in the supernatants did not relate to LCL activity in PMN. These results point to the possibility that the insoluble constituents of the particles are related to LCL in PMN. Since some of these dusts are capable of depositing in the lungs and can cause infiltration of PMN, the ability to activate those cells may contribute to particulate toxicity.

Oxidative activity of turkey monocytes, following the inoculation with Chlamydia psittaci

Chemiluminescence (CL) was used to investigate the competence of turkey monocytes to mount a respiratory burst response upon interaction with Chlamydia psittaci. The oxidative activity of purified turkey monocytes, following inoculation with the avian C. psittaci serovar D strain 92/1293, was studied using luminol- and lucigenin-enhanced CL. Purified turkey monocytes were inoculated with C. psittaci at multiplicity of infection (MOI) of approximately 100, 10 and 1. In the presence of luminol, no detectable CL or only a weak CL response was obtained, and if present it increased with increasing MOI. Either sham inoculated monocytes, or monocyte-free control assays supplemented with C. psittaci, gave no detectable luminol-enhanced CL responses. In the lucigenin-enhanced assays, monocytes inoculated with C. psittaci demonstrated an immediate CL peak, the height of which was proportional to the MOI used. Following inoculations at a MOI 1, a faint second peak was observed, when applying high concentrations of lucigenin. Sham inoculated monocytes gave no detectable lucigenin-enhanced CL responses. However, in the presence of lucigenin, the addition of C. psittaci to monocyte-free controls also resulted in an immediate CL peak, though no second peak was detected. This immediate lucigenin-dependent CL peak induced by C. psittaci was similar to the one observed in the presence of monocytes, and was not inhibited by superoxide dismutase. We demonstrated that this avian C. psittaci strain induces only a very weak respiratory burst response in turkey monocytes. In contrast, C. psittaci itself elicited an intense non-superoxide mediated lucigenin-dependent CL, indicating that in chlamydial research the detection of superoxide, using lucigenin, should be confirmed with a specific superoxide inhibitor.

Interaction between Salmonella typhimurium and phagocytic cells in pigs : Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes

Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related. Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC). When opsonized in normal serum the Salmonella bacteria, in the range of 2–5 × 10 7 cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD). Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs. Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer. Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response. In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.

Plasticizers and Inhibition of Leukocyte Function in Vitro

To evaluate the influence of the plasticizer metabolites of di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA) on various immune functions of polymorphonuclear blood leukocytes (PMNL) and monocytes (MN). MEHP, 2-EH, and PA are the main hydrolysis products of DEHP. Since DEHP is leached out of the plastic matrix, patients on hemodialysis and continuous ambulatory peritoneal dialysis are exposed to considerable amounts of DEHP and its metabolites. Teaching hospital, Department of Nephrology. Ten healthy volunteers. After incubation of leukocytes in solutions with different plasticizer concentrations, oxidative respiratory metabolism was determined by luminol-enhanced chemiluminescence (CL) after stimulation with phorbol myristate acetate (PMA). Furthermore, superoxide (O2-) generation was measured by cytochrome c reduction. At pH 5.4, a dose-dependent decrease of luminol-enhanced CL response was found in all assays. For MEHP and PA the level of significance was reached at 10 mg/L and 1 mg/L, respectively. Superoxide generation by PMNL and MN at pH 5.4 was also suppressed by MEHP and PA. At pH 7.4, only a slight suppression of oxidative metabolism at higher concentrations was observed. After incubation of the cells in a solution containing all DEHP metabolites (MEHP, PA, and 2-EH), a significant suppressive effect of CL at pH 5.4 could be observed at final plasticizer concentrations of 0.5 mg/L. A dose-dependent impairment of leukocyte oxidative metabolism at a low pH could be demonstrated. The suppressive effect was particularly marked after incubation of the cells in solutions containing a mixture of the main plasticizers. At pH 5.4, we observed a slight alteration even at concentrations very close to those that could be found in commercially available peritoneal dialysis fluids. These results might point toward a possible synergistic detrimental effect of the different DEHP metabolites on leukocyte function, with possible clinical relevance.

Plasma from patients exposed to ischemia reperfusion contains clastogenic factors and stimulates the chemiluminescence response of normal leukocytes

Clastogenic factors (CFs) are released by cells exposed to superoxide radicals and are found in various situations of oxidative stress. Certain of their components stimulate further superoxide production by competent cells, as shown with cytochrome c assay in previous work. In the present study, we report CF formation after ischemia reperfusion in patients undergoing coronary bypass surgery. Plasma ultrafiltrates, collected 20 min after reperfusion, had clastogenic properties in contrast to those collected before ischemia. We also show that the luminol-enhanced chemiluminescence response of neutrophils from healthy persons is increased when these cells are exposed to CF-containing postreperfusion samples from patients. Light emission was reduced to control values in the presence of superoxide dismutase. The burst of oxyradicals upon reperfusion is probably the initiating event of CF formation, which in turn leads to further oxyradical generation. This amplification process may explain why detectable levels of CF need a delay of at least 10 min. The activated state of neutrophils in ischemia reperfusion is at once a consequence and a source of CFs. Individual variation in the persistence of this clastogenic and leukocyte-activating material was observed. Therefore, antioxidants for prevention of ischemia reperfusion injury should be continued during the postoperative course.

Plasma levels of beta-carotene are inversely correlated with circulating neutrophil counts in young male cigarette smokers

Relationships between plasma levels of beta-carotene (BC) and the numbers and oxidant-generating activities of circulating neutrophils have been investigated in a group of asymptomatic young male cigarette smokers (N = 40) and in a group of nonsmoking control subjects. Plasma BC levels were measured using HPLC, while oxidant generation was measured using a phorbol myristate acetate (PMA) activated whole blood luminol-enhanced chemiluminescence (LECL) method. Relative to nonsmokers, the numbers of circulating neutrophils, as well as the LECL responses of these cells, were increased by 41% (p = 0.0001) and 47% (p = 0.004), respectively, while plasma BC levels were decreased by 24% (p = 0.01). In cigarette smokers, but not in nonsmokers, the numbers of circulating neutrophils, as well as the LECL responses of these cells, were significantly and inversely correlated with plasma BC levels (r = -0.36, p = 0.02; and r = -0.33, P = 0.04 respectively). Diminished plasma levels of BC in cigarette smokers probably reflect the increased numbers and prooxidative activities of circulating neutrophils. Intake of this antioxidant nutrient may be a determinant of susceptibility to smoking-related pulmonary dysfunction mediated by oxidants derived from smoke-activated phagocytes.

Effects of chronic copper exposure on the macrophage chemiluminescent response and gill histology in goldfish (Carassius auratusL.)

The effects of chronic copper exposure on the macrophage chemiluminescent response and gill histology in goldfish (Carassius auratusL.) were determined. Fish were exposed to sublethal concentrations (30, 65, 100, and 175ppb) of copper sulphate for 96h. Pronephric (head kidney) cells were collected and were comprised on lymphocytes, thrombocytes, monocytes/macrophages neutrophils and eosinophils/basophils (cell types grouped due to difficulty in identification). There were no significant differences in population profiles of cells used for chemiluminescence studies after exposure to 100ppb copper. Density gradient-separated cells were stimulated with zymosan, the luminol-enhanced chemiluminescence (CL) response was measured and the results were compared to non-copper exposed controls. There was a significant correlation between copper concentration and reduction in the time at which the peak CL response [time to peak (TTP) CL] occurred. In addition, there was a significant parabolic dose response curve for peak CL response, which suggests increased production of reactive oxygen intermediates by phagocytes from fish exposed to concentrations up to 100ppb, followed by inhibition at 175ppb. These results indicate that copper affects phagocyte function in a complex manner, stimulating or inhibiting production of reactive oxygen intermediates depending on copper concentration. Exposure to concentrations of copper also resulted in varying degrees of gill hyperplasia. The relative median severity score (a score assigned to each gill based upon the amount of hyperplasia seen in a histological section) correlated significantly with the copper concentration. In addition, there was a very high correlation between the degree of hyperplasia and the TTP CL response. Levels of copper in the head kidney of goldfish exposed to 100ppb were similar to controls as measured by atomic absorption spectroscopy. Thus, changes in the CL response of head kidney phagocytes of fish exposed to copper may be caused indirectly, via effects on other immunoregulatory cells or other physiological mechanisms. We conclude that addition of heavy metals such as copper to water can modify the CL responses of phagocytes and alter the histopathology of the gills inC. auratus.

Opsonic activity of sera and blister fluid from severely burned patients evaluated by a chemiluminescence method.

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol-enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin-index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.

Evidence of impaired polymorphonuclear leucocyte phagocytic functions and chemiluminescence response in rheumatoid arthritis

In this study, the phagocytic uptake of 3H-thymidine-labelled Staphylococcus aureus and bacterial killing (Bk) by polymorphonuclear leucocytes (PMN) from patients with rheumatoids arthritis (RA) were investigated and compared to the luminol-enhanced chemiluminescence response (LCL) to phorbolmyristic acetate (PMA), a receptor and second message-independent activator of respiratory burst activity in definite or classical rheumatoid arthritis (RA), with regard to the inflammatory activity of the disease process, and compared to patients with osteoarthritis (OA) and healthy controls. PMN of RA patients showed a significant reduction in uptake (57.8±4.4%) and less Bk capacity (27.3±4.2%) compared to OA patients (uptake 71.4±4.3%, Bk 20.6±2.9%; P<0.001) and controls (uptake 73.2±5.2%, Bk19.3±4.2%;P<0.001). In contrast, PMN LCL response was markedly enhanced in RA patients compared to OA patients (P<0.001) and controls (P<0.001). There was no significant influence of inflammatory activity on various PMN functions in RA and no difference was found between OA and control subjects. These data clearly demonstrated impaired PMN phagocytic functions (uptake, Bk) and enhanced LCL in RA, suggesting “priming” and/or activated peripheral blood PMN, which might be of clinical importance concerning altered host defence in these patients.

In vitro and in vivo intraleukocytic accumulation of azithromycin (CP-62, 993) and its influence on ex vivo leukocyte chemiluminescence.

The accumulation of azithromycin in phagocytic cells was studied both in vitro by using a radiolabelled drug and a bioassay and in vivo for 12 volunteers receiving 1.5 g (total dose) orally within 3 days. In vitro, neutrophils and unfractionated blood leukocytes accumulated azithromycin up to 160-fold the extracellular concentration within 1 h at 37 degrees C but less than 3-fold at 4 degrees C. Dead cells accumulated up to 30-fold azithromycin, whereas NaF-treated cells accumulated up to 60-fold arithromycin. The mean efflux from preloaded cells was at most 31.0% +/- 10.6% (standard error of the mean) of the cell-associated concentration within 4 h of incubation at 37 degrees C in drug-free buffer. In vivo, the azithromycin concentration was 45.2 +/- 6.1 mg/liter of intracellular fluid at 2 h after the third dose and 36.6 +/- 8.3 mg/liter at 1 week thereafter. The corresponding concentrations in serum were 0.2 +/- 0.1 (2 h) and less than 0.05 (1 week). The luminol-enhanced chemiluminescence response induced by phorbol myristate acetate, opsonized zymosan, and two opsonized strains of Haemophilus influenzae (a type b capsulated strain and a noncapsulated strain) was also studied ex vivo by using the blood leukocytes from the 12 test volunteers and 4 control volunteers at 2 and 6 h after the third oral dose of azithromycin and at 2, 4, and 7 days thereafter. Azithromycin did not influence this response despite high levels of cellular accumulation.

Hydroxyl radical generation by red tide algae

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) and N- t-butyl-α-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O 2 −) (DMPO-OOH) and that of hydroxyl radical (· OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me 2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me 2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me 2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.

Stimulation of human polymorphonuclear leukocytes by recombinant human interleukin-1 beta.

Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes. The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1 beta and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-1 beta elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8 x 10(-11)-8.8 x 10(-8) M. The effect could be blocked by prior treatment with the IL-1 receptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1 beta enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40%. The priming effect of rhIL-1 beta could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1 beta failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1 beta has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1 beta is a direct and specific, but probably weak stimulator of the PMN leukocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of eicosapentaenoic acid consumption on human neutrophil chemiluminescence

The effect of eicosapentaenoic acid (EPA) on the inflammatory potential of neutrophils was investigated by supplementing the diets of 12 subjects with 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a double blind crossover study. Neutrophil function as assessed by luminol enhanced chemiluminescence responses to platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) was significantly reduced after EPA but not after olive oil consumption in the subjects who consumed EPA first. In contrast, EPA had no significant effect on neutrophil chemiluminescence in the subjects who consumed olive oil first. Dietary supplementation with EPA inhibits neutrophil responses to inflammatory mediators such as PAF while other fatty acids appear to modify the effects of EPA.

Drugs effects on superoxide generation and chemiluminescence response of human leukocytes.

Luminol-enhanced chemiluminescence was used to determine the effects of diethyldithiocarbamate, dipyridamole, catechin and verapamil on the generation of reactive oxygen species in human leukocytes, and on superoxide generated by chemiluminescence of the hypoxanthine xanthine-oxidase reaction. These agents reduced the luminol enhanced chemiluminescence response of activated leukocytes, most probably by inhibiting the superoxide generation reaction. On the other hand, citrate and diethylcarbamazine, produced a slight increase of the luminol enhanced chemiluminescence of leukocytes.