Chemiluminescence Of Human Neutrophils Research Articles

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H 2O 2-dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H 2O 2 and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst.

Antioxidant activities of natural 9- cis and synthetic all- trans β-carotene assessed by human neutrophil chemiluminescence

Much attention has been focused on the biological activity of geometrical isomers of β-carotene. In the present study, the in vitro antioxidant activities of a natural 9- cis β-carotene purified from Dunaliella bardawil and synthetic all- trans β-carotene were studied using enhanced human neutrophil chemiluminescence upon stimulation with opsonized zymosan. Lucigenin-dependent chemiluminescence was used for largely measuring superoxide anion (O 2) production, luminol-dependent chemiluminescence with sodium azide (NaN 3) and horseradish peroxidase (HRP) was used for measuring hydrogen peroxide (H 2O 2) production, and luminol-dependent chemiluminescence without oxidant metabolism modulator was used for determination of myeloperoxidase (MPO)-mediated production. With β -carotene concentration of 1.4 μ M, neutrophils showed a decrease in lucigenin-dependent chemiluminescence to 75% and 78% of controls (p<0.05) by natural 9- cis β -carotene and synthetic all- trans β -carotene, respectively. With β -carotene concentration of 1.86 μ M, neutrophils also showed a decrease in luminol-dependent chemiluminescence in the presence of NaN 3 and HRP to 86% and 81% of controls (p<0.05) by natural 9- cis β -carotene and synthetic all- trans β -carotene, respectively. On the other hand, neutrophils did not show a statistically significant difference in any chemiluminescence assay by the same concentration of natural 9- cis β -carotene and synthetic all- trans β -carotene. These results suggest that, up to the concentration of 1.86 μ M, natural 9- cis β -carotene and synthetic all- trans β -carotene exert equal antioxidant activities assessed by enhanced human neutrophil chemiluminescence.

Effects of Helicobacter pylori in the stomach on neutrophil chemiluminescence in patients with gastric cancer.

Hypochlorous acid, which is one of the reactive oxgen species (ROS) produced from human neutrophils, is converted to cytotoxic NH(2)Cl after reaction with ammonia produced by urease in Helicobacter pylori (HP), increasing gastric mucosal injury and with potential development to gastric cancer. We compared the effects of HP on the production of ROS by human neutrophils between two groups-22 patients with gastric cancer and 16 patients without gastric cancer (control group), in whom HP was isolated from stomach biopsy tissues-using a luminol- and lucigenin-dependent chemiluminescence (LmCL and LgCL, respectively). It was very similar in the mean value or variance of mean maximal chemiluminescence intensities (MCI) and peak time in LmCL and LgCL between the two groups. MCI of LmCL was highly correlated with that of LgCL in both groups. These results indicate that there were no differences in the behaviour of HP on human neutrophil chemiluminescence between two groups. The progression from non-malignant mucosa to cancer may be associated with the time-dependent effects of HP via ROS produced by neutrophils on the gastric mucosa.

Effect of decaglycerol mono-oleate on chemiluminescence of human neutrophils.

Decaglycerol mono-oleate (DGMO), a type of food additive (a polyglycerol ester of fatty acids), was evaluated for its in vitro effect on reactive oxygen species (ROS) generation by human neutrophils using a luminol-dependent chemiluminescence assay. Neutrophils were isolated by the Histopaque-1077/1119 double density gradient technique. Opsonized zymosan (OZ) and phorbol 12-myristate 13-acetate (PMA) were employed as artificial stimuli. DGMO inhibited the chemiluminescence response of OZ-stimulated neutrophils by 58% (p < 0.01) at a concentration of 0.5 mg/mL. DGMO inhibited the PMA-induced chemiluminescence by 40% (p < 0.01) at a concentration of 0.01 mg/mL. The inhibition phenomena could not be attributed to light absorption by DGMO alone, because the percentage chemiluminescence in the presence of DGMO is lower than the percentage transmittance of DGMO at 425 nm. These results suggest that DGMO downregulates ROS generation by human neutrophils.

Contribution of nitric oxide synthase to human neutrophil chemiluminescence

A chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. Primed neutrophils produce ROS and nitric oxide (NO) upon induction of nitric oxide synthase (NOS) activity. NO and superoxide (O(2)(-)) form peroxynitrite (ONOO(-)), and emit CL. We examined the involvement of NOS in the CL response of neutrophils using a method based on the modulation of enzyme activity of NOS by the substrate L-arginine and an inhibitor; L-NAME. We used lipopolysaccharide (LPS) as the neutrophil-priming agent. Addition of sodium azide (NaN(3)) with horseradish peroxidase (HRP) to luminol-dependent CL, gave a CL response that was significantly enhanced when 10 mmol/L L-arginine was present (p <0.05), suggesting that NOS activity contributed to the CL response of human neutrophils. LPS-primed luminol-dependent CL was significantly inhibited by L-NAME compared with D-NAME. The proportion of the difference between the two inhibitors in luminol-dependent CL was 12.3 +/- 15.0%. Therefore, approximately 12% of the LPS-primed luminol-dependent CL decrease induced by L-NAME indicated the contribution of NOS activity to the CL response.

Contribution of nitric oxide synthase to human neutrophil chemiluminescence

Contribution of nitric oxide synthase to human neutrophil chemiluminescence

Contribution of nitric oxide synthase to human neutrophil chemiluminescence.

A chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. Primed neutrophils produce ROS and nitric oxide (NO) upon induction of nitric oxide synthase (NOS) activity. NO and superoxide (O(2)(-)) form peroxynitrite (ONOO(-)), and emit CL. We examined the involvement of NOS in the CL response of neutrophils using a method based on the modulation of enzyme activity of NOS by the substrate L-arginine and an inhibitor; L-NAME. We used lipopolysaccharide (LPS) as the neutrophil-priming agent. Addition of sodium azide (NaN(3)) with horseradish peroxidase (HRP) to luminol-dependent CL, gave a CL response that was significantly enhanced when 10 mmol/L L-arginine was present (p <0.05), suggesting that NOS activity contributed to the CL response of human neutrophils. LPS-primed luminol-dependent CL was significantly inhibited by L-NAME compared with D-NAME. The proportion of the difference between the two inhibitors in luminol-dependent CL was 12.3 +/- 15.0%. Therefore, approximately 12% of the LPS-primed luminol-dependent CL decrease induced by L-NAME indicated the contribution of NOS activity to the CL response.

Effect of linear polarized near-infrared ray irradiation on the chemiluminescence of human neutrophils and serum opsonic activity.

The purpose of this study was to investigate the in vitro effects of linear polarized near-infrared ray irradiation on neutrophil chemiluminescence (CL) and serum opsonic activity. We used luminol- and lucigenin-dependent CL to detect the affected reactive oxygen species production process of human neutrophils and measured serum opsonic activity based on luminol-dependent CL. The linear polarized near-infrared ray irradiation suppressed a maximum light emission (peak height) of luminol- and lucigenin-dependent CL in a dose-dependent manner. The findings suggested that the linear polarized near-infrared ray irradiation suppressed the superoxide anion and hypochlorite production of human neutrophils. The serum opsonic activity was decreased by linear polarized near-infrared ray irradiation, and this suppressive effect might be caused by inhibiting the activation of the classical and alternative complement pathway. Therefore, it is suggested that near-infrared ray irradiation may have an inhibitory effect against chronic pain via reduction of reactive oxygen species production and opsonic activity.

PADMA-28, a traditional tibetan herbal preparation inhibits the respiratory burst in human neutrophils, the killing of epithelial cells by mixtures of oxidants and pro-inflammatory agonists and peroxidation of lipids

Both aqueous and methanolic fractions derived from the Tibetan preparation PADMA-28 (a mixture of 22 plants) used as an anti-atherosclerotic agent, and which is non-cytolytic to a variety of mammalian cells, were found to strongly inhibit (1) the killing of epithelial cells in culture induced by 'cocktails' comprising oxidants, membrane perforating agents and proteinases; (2) the generation of luminol-dependent chemiluminescence in human neutrophils stimulated by opsonized bacteria; (3) the peroxidation of intralipid (a preparation rich in phopholipids) induced in the presence of copper; and (4) the activity of neutrophil elastase. It is proposed that PADMA-28 might prove beneficial for the prevention of cell damage induced by synergism among pro-inflammatory agonists which is central in the initiation of tissue destruction in inflammatory and infectious conditions.

2,4-(Hydroxyphenyl)-ethanol, an antioxidative agent produced by Candida spp., impairs neutrophilic yeast killing in vitro

Culture supernatants of Candida albicans were examined for factors with inhibitory activity against the chemiluminescence of human neutrophils. By high resolution gel chromatography, a low-molecular-mass chemiluminescence inhibitor was isolated. The compound was identified as 2,4-(hydroxyphenyl)-ethanol. Half-maximum inhibition (IC 50) of the chemiluminescence response of neutrophils phagocytizing opsonized zymosan or C. albicans occurred at 38.1±2.3 μM and 19.9±8.3 μM, respectively. As shown by flow cytometry, the compound protected C. albicans against phagocytic killing (IC 50=73.8±16.9 μM). Substantially higher concentrations of the inhibitor were produced by C. albicans and C. tropicalis than by C. parapsilosis and C. glabrata, suggesting a potential role in pathogenicity ranking.

Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils

A panel of non-steroidal anti-inflammatory drugs commonly used for therapeutic purposes was assessed for their effects on the respiratory burst of isolated human polymorphonuclear neutrophils. Cells were stimulated with opsonised yeast and the production of reactive oxygen species was measured by amplified chemiluminescence with luminol and lucigenin which are two luminogenic agents measuring different cellular events. A special attention was devoted to the establishment of dose–effect curves and calculation of ED 50. Some of the drugs tested (acemetacine, diclofenac, flufenamic acid and niflumic acid) were able to decrease both luminol and lucigenin chemiluminescence in a dose-dependent manner reflecting an inhibitory effect on the respiratory burst. The most potent derivative was flufenamic acid (ED 50 8 and 78 μM, respectively, with luminol and lucigenin), followed by diclofenac (21 and 98 μM), niflumic acid (97 and 227 μM) and acemetacine (585 and 427 μM). In contrast, several other drugs (flurbiprofen, ibuprofen, ketoprofen, piroxicam) stimulated both luminol and lucigenin chemiluminescence, suggesting a pro-oxidant activity. Acetylsalicylic acid (up to 1250 μM) was a modest inhibitor (maximum 25% inhibition) showing no dose-dependant effect and tolmetin (up to 125 μM) had no significant effect in both systems. The results were in agreement using both luminogenic agents, except for indomethacin, naproxen and tenoxicam which showed different kinds of effects. The unspecific and complex nature of the measurement systems used did not allow to give a complete mechanistic interpretation of the results, but the comparison with literature data gave some pertinent explanations for both anti- and pro-oxidant effects.

Phagocyte activation with anti-ICAM-3 monoclonal antibodies

Monoclonal antibodies ICO-60 clustered as CDw50 (anti-ICAM-3) drastically boosted the chemiluminescence of human neutrophils, depending on the dose of antibodies, initial chemiluminescence, and individual human reactivity. The contact-adhesion interactions of neutrophils treated with ICO-60 and of intact neutrophils were associated with intensification of chemiluminescence. ICO-60 sharply increased the adhesion and aggregation of human neuroblasts and suppressed these phenomena induced by phorbol ester. The expression of ICAM-3 on neuroblasts is proposed.

Luminol-enhanced, whole blood chemiluminescence of human neutrophils evaluated by means of an automated, computer-assisted, and high-sensitivity luminescence analyzer.

Luminol-enhanced whole blood chemiluminescence of human neutrophils was studied using opsonized zymosan as a stimulus. Heparinized blood (0.5 microliters) was used, and the chemiluminescence signals were recorded by a very sensitive, automated, and computer-assisted luminometer (LB 950, Berthold, Wildbad, Germany). The following parameters were provided: integral values over the total measuring time, peak values, the time to reach maximum value, and the time to reach half maximum value. Normal subjects, neutropenic patients, subjects with total or partial myeloperoxidase deficiency, patients with recurrent infections, phagocytic defects, thrombocythemic patients and those with non-Hodgkin's lymphomas undergoing therapy with recombinant human granulocyte colony-stimulating factor were studied. The integral response of chemiluminescence and the time of reach half maximal value were useful indicators of chemiluminescence defects; the assay, was able to detect chemiluminescence responses in neutropenic subjects with neutrophil levels as low as 0.6 x 10(9)/l; differences between cellular and plasma defects could be identified; the quenching effect exerted by erythrocytes was negligible.

Radical scavenger activity of different 3',4'-dihydroxyflavonols and 1,5-dicaffeoylquinic acid studied by inhibition of chemiluminescence.

To gain more insights into structure-activity relationships, four 3',4'-dihydroxyflavonols differing in the substitution of the A and C rings and 1,5-dicaffeolyquinic acid were evaluated for their ability to inhibit chemiluminescence of human neutrophils stimulated with opsonized zymosan or FMLP as well as in an enzymatic system with H2O2 and horseradish peroxidase. It could be shown that an additional o-dihydroxy structure in the A-ring, or a 6-methoxy group, respectively, has no significant influence, thus confirming the o-dihydroxy group of the B-ring as the most important structural feature for the radical scavenger activity. It can be supposed that the main effect of the tested flavonols is based on their inhibition of myeloperoxidase, besides inhibition of enzymes involved in activating the NADPH-oxidase, and a direct reaction with oxygen radicals. Inhibition of chemiluminescence by 1,5-dicaffeoylquinic acid was in the same order as those observed with the flavonols.

Thyroid hormones and regulation of cell reliability systems

Data and arguments are presented that provide evidence of a role played by thyroid hormones (TH) in cell reliability improvement. This role may be determined by synergistic TH action on the following key cell reliability systems: (1) reactive oxygen species (ROS) attack inhibition, and (2) genetic structure repair from injuries inflicted in the course of endogenous and induced mutagenesis. 1. (1)|New approaches to ROS oxidation defence were examined. It has been shown that Ca 2+-ATPase and, probably, regulatory proteins of cell membranes may be the main target for oxidative attack. Protein phosphorylation as well as use of dithiothreitol will lead to a protective action against Ca 2+ transport damaging in aorta smooth muscle sarcoplasmic reticulum under oxidation by HOCl, the most toxic ROS of activated neutrophils, whereas thyroxine (T 4) and 3,5,3′-triiodothyronine (T 3) validly inhibit chemiluminescence in human neutrophils activated by pyrogenal, a lipopolysaccharide from Salmonella typhi cell wall. As this takes place, TH most likely block neutrophil stimulation at the receptor-ligand interaction level. In this case L-T 4 and L-T 3 antioxidative effect is greater than that of dl-thyroxine and much greater than that produced by such a potent antioxidant as 4-methyl-2,6-diisobutyl phenol. 2. (2)|T 4 acts as reparogen in rat liver cells under X-ray irradiation when a dose measuring one-half of daily hormone production by the normally functioning thyroid gland is administered to animals. Ionizing radiation dose reduction factor reached 1.3–1.4 following T 4 administration. Reparogenic effect of T 4 persists for at least 2 months from the moment the hormone has been administered and can be reduced in the presence of dinitrophenol. It is important to note that antioxidant and reparogenic TH potential can manifest itself within the range of physiologic concentrations of these hormones. Therefore, stimulation of cell reliability systems with TH may prove to be important for correcting conditions caused by errors in energy- and Ca 2+-dependent DNA repair under extensive ROS attack. In particular, taking into account different responsiveness of normal and neoplastic tissues to TH, the use of TH reparogenic as well as antioxidant potential may contribute significantly to the improvement of antitumor radiotherapy efficacy.

Effect of the osmotic pressure of the medium on the chemiluminescence of human neutrophils

Effect of the osmotic pressure of the medium on the chemiluminescence of human neutrophils

The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations in vitro.

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.

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.

The effects of N‐ethylmaleimide on extracellularly and intracellularly generated chemiluminescence in neutrophils indicate that the rate of deactivation of NADPH‐oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane

Sustained generation of reactive oxygen metabolites following respiratory burst activation in neutrophils is a result of continued replenishment of a pool of active NADPH-oxidase. The sulphydryl-modifying reagent N-ethylmaleimide (NEM) has been shown to be without effect on the turnover of activated NADPH-oxidase but to inhibit the replenishment of active oxidase molecules (Akard et al., 1988). NEM was thus used to determine the rate of deactivation of extracellularly and intracellularly generated chemiluminescence in human neutrophils. We have shown that deactivation is more rapid when activation leads to a release of oxygen metabolites (extracellular chemiluminescence) than when the metabolites are generated intracellularly. The results indicate that the rate of deactivation of NADPH-oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane.