Ticlopidine augments luminol-dependent chemiluminescence in human neutrophils

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

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.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.     

Pholasin chemiluminescence detects mostly superoxide anion released from activated human neutrophils

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

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

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

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

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

Synergy of human neutrophils with fluconazole in killing Candida species

The killing of Candida species by human neutrophils in a long-term 24-h assay and possible synergy with fluconazole (FCZ) for killing was investigated. The test medium (TM) consisted of RPMI-1640, penicillin and streptomycin (P/S), and 10% fresh autologous serum. TM alone was highly fungistatic for Candida species compared to TM without serum. When neutrophils were cocultured in TM with Candida species for 24 h the inoculum colony-forming units (CFU) were always significantly reduced (killing) by 58 to 99%. FCZ was tested over a range of 1–500 g/ml, and though almost always fungistatic itself, it synergized with neutrophils for significantly increased killing of C. albicans (isolate Sh27) (P<0.01) and C. albicans (isolate 94-20) (P<0.05). Killing of non-albicans Candida species was so efficient in the absence of FCZ that demonstration of synergy with FCZ was difficult.     

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

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

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

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

Potassium permanganate–glutaraldehyde chemiluminescence system catalyzed by gold nanoprisms toward selective determination of fluoride

Gold and silver nanoparticles (NPs) are shown to exert a positive effect on the chemiluminescence (CL) reaction of permanganate aldehydes. Interestingly, between various shapes examined, Au nanoprisms have the highest beneficial effect. This effect is even more notable in the presence of sodium dodecyl sulfate (SDS) surfactant. UV‐vis spectra and transmission electron microscopy were used to characterize the NP shapes and sizes. Furthermore, it was observed that iron(III) ions can slightly increase CL emission of this system. This intensification is very effective in the presence of fluoride ions (F^–). These observations form the basis of the method for the high sensitive determination of F^– in the 6–1200 nmol L^–1 concentration range, with a detection limit of 2.1 nmol L^–1. The proposed method has good precision and was satisfactorily used in the selective determination of low concentrations of fluoride ion in real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Reactions of copper complexes with oxygen radicals generated by human neutrophils

The intensity of the chemiluminescence of unstimulated human neutrophils in the presence of luminol was used to investigate the effects of low-molecular-weight copper complexes at the cellular level. In different models (superoxide dismutase mimetic activity, inhibition of haematoporphyrin derivative/light-induced lysis of cells), the biological activity of the complexes exceeded the activity of the ligands alone.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Microparticles released by human neutrophils adhere to erythrocytes in the presence of complement

The release of cell surface-derived microparticles, or ectosomes, has now been described for many different cell types. In various diseases characterized by systemic inflammation, the numbers of ectosomes released from specific cell-types are found increased manifold in the circulation. Their pro-inflammatory and pro-coagulant functions make them potentially important actors in disease establishment and/or progression. Until now, ectosomes have been believed to be free in the circulation. Herein, we provide evidence for sequestration of ectosomes derived from human polymorphonuclear neutrophils to erythrocytes, similarly to immune complexes. We show that ectosomes activate and bind complement in vitro. In whole blood, opsonization of ectosomes by complement mediated their immune adherence to erythrocytes through complement receptor 1. Taken together, our data suggest an important role for complement and erythrocytes in the sequestration, and possibly clearance, of blood-borne ectosomes stemming from neutrophils. The immune adherence described here may modify the biological activity and function of ectosomes.     

胞内Ca2+和小G-蛋白对人中性粒细胞胞吐的调控作用

中性粒细胞在机体抵抗细菌感染中起着非常重要的作用。本研究应用全细胞膜片钳微电极灌流和膜电容检测技术研究了细胞内钙及GTPγs对中性粒细胞胞吐的调控作用。结果表明,钙引起的细胞膜电容增加呈现两个不同的分泌相。第一相发生在钙浓度为0.2-14μmol/L区间,膜电容增加幅度为1.23 pF,钙的EC50值为1.1μmot/L。这部分膜电容增加可能对应于三级颗粒的释放。第二相发生在钙浓度为20-70μmol/L区间,膜电容增加幅度为6.36 pF,钙的EC50值为33μmol/L。这部分膜电容增加可能对应于一、二级颗粒的释放。细胞内Ca2+浓度可同时影响细胞分泌的平均速率和最终可达到的分泌程度。而用GTγs刺激细胞分泌时,只要作用时间足够长(>20 min),20-100μmot/L的GTPγs均可使中性粒细胞膜电容增加6 pF以上。GTPγs的浓度不影响细胞膜电容的最终可增加的幅度,但细胞分泌的平均速率随着GTPγS的浓度的增加而增加。     

4℃预处理对人中性粒细胞膜电流和极性的影响

对急性分离的人中性粒细胞采用4℃预处理是进行膜片钳实验前经常采取的步骤,但这一步骤对电生理记录结果有何影响尚无文献报道。本实验探讨这一步骤对电生理记录过程和实验结果的影响。结果显示,4℃预处理可以显著提高细胞的封接率,有利于对中性粒细胞进行电生理记录;封接率提高的原因与4℃预处理降低细胞的极性活动有关,但记录到的电压依赖性钾通道全细胞电流和大电导Ca^2＋依赖性K^＋单通道电流动力学没有显著的变化。这些结果表明,4℃预处理可能影响到细胞膜上与极性有关的脂膜变化,但对细胞膜上蛋白的功能影响较少。     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

Interactions of F1 fractions from different strains of shape Paracoccidioides brasiliensis with human complement and with human neutrophils

The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1 fractions from four different isolates of P. brasiliensis. Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage in guinea pigs. The four isolates of the fungus were processed for purification of F1 fractions and the activation of the human complement system was studied by a kinetic method of hemolytic activity measurement. The incubation of F1 fractions in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways. The F1 fraction from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18). Previous absorption of sera with F1 fractions completely abolished classical pathway activation. Using zymosan, instead of F1, in the absorption process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway activation. The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence. On the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared with the F1 fraction from the low virulence isolate. Whole P. brasiliensis yeast cells (WYC) from two distinct strains, 18 and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested. These differences in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory response generation and maintenance processes in paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

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

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

Intracellular production of reactive oxygen species in human neutrophils following activation by the soluble stimuli FMLP, dioctanoylglycerol and ionomycin.

The stimuli, sn-1, 2-dioctanoylglycerol; (DG8) the calcium specific ionophore, ionomycin, and the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) can interact with normal human neutrophils and activate their superoxide/hydrogen peroxide generating NADPH-oxidase. In response to the peptide as well as DG8, the neutrophils produced both superoxide (O2-) and hydrogen peroxide (H2O2). Since interaction between the cells and ionomycin was not associated with any notable superoxide production and hydrogen peroxide was induced only in the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, we conclude that this stimulus can generate oxygen metabolites intracellularly. Since the DG8-induced production of hydrogen peroxide was increased in the presence of azide, whereas the FMLP-induced response was largely unaffected, we concluded that the three stimuli differ in their capacity to generate oxygen metabolites intracellularly. The use of sn-1,2-didecanoylglycerol (DG10) as stimulating agent did not result in any detectable activation of the NADPH-oxidase. However, preincubation caused an increased (primed) response during stimulation with the chemotactic peptide FMLP. The response of primed neutrophils to FMLP proceeds with a time-course different from that seen in normal cells. From the results presented on FMLP-induced activity in the presence of azide, we conclude that FMLP causes normal cells to produce oxygen radicals which are released from the cells. However, the primed cells are also capable of generating oxygen metabolites that are retained inside the cells. In fact, measurement of the intracellularly generated metabolites discloses this to be the predominant part of the response.     

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H₂O₂) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H₂O₂ evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H₂O₂ resulted in a increase in [Ca²⁺] _i , indicating the release of calcium from intracellular stores. H₂O₂ abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H₂O₂-induced calcium release. We conclude that H₂O₂ induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.