Pharmacological intervention with oxidative burst in human neutrophils.

In this study we investigated the effect of five therapeutically used drugs and four natural polyphenolic compounds on the mechanism of oxidative burst of human neutrophils concerning their participation in the generation of reactive oxygen species (ROS). The compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin > quercetin > curcumin > arbutin > dithiaden > carvedilol. The generation of intracellular reactive oxygen species in isolated neutrophils decreased in the same rank order, while carvedilol was ineffective. Scavenging of extracellular oxygen radicals followed the rank order of potency: N-feruloylserotonin > curcumin > quercetin > dithiaden. Arbutin and carvedilol had no effect. All compounds tested increased the activity of caspase-3 in cell-free system indicating a positive effect on apoptosis of neutrophils. Activation of protein kinase C was significantly decreased by dithiaden, curcumin, quercetin and N-feruloylserotonin. Carvedilol, dithiaden, quercetin and arbutin reduced activated neutrophil myeloperoxidase release more significantly compared with their less pronounced effect on superoxide generation The presented results are indicative of pharmacological intervention with neutrophils in pathological processes. Of particular interest was the effect of natural compounds. Intracellular inhibition of oxidative burst in isolated neutrophils by the drugs tested and natural antioxidants has to be further analysed since ROS play an important role in immunological responses of neutrophils.

Arbutin and decrease of potentially toxic substances generated in human blood neutrophils.

Neutrophils, highly motile phagocytic cells, constitute the first line of host defense and simultaneously they are considered to be central cells of chronic inflammation. In combination with standard therapeutic procedures, natural substances are gaining interest as an option for enhancing the effectiveness of treatment of inflammatory diseases. We investigated the effect of arbutin and carvedilol and of their combination on 4ß-phorbol-12ß-myristate-13α-acetate- stimulated functions of human isolated neutrophils. Cells were preincubated with the drugs tested and subsequently stimulated. Superoxide (with or without blood platelets, in the rate close to physiological conditions [1:50]) and HOCl generation, elastase and myeloperoxidase release were determined spectrophotometrically and phospholipase D activation spectrofluorometrically. The combined effect of arbutin and carvedilol was found to be more effective than the effect of each compound alone. Our study provided evidence supporting the potential beneficial effect of arbutin alone or in combination with carvedilol in diminishing tissue damage by decreasing phospholipase D, myeloperoxidase and elastase activity and by attenuating the generation of superoxide and the subsequently derived reactive oxygen species. The presented data indicate the ability of arbutin to suppress the onset and progression of inflammation.

Effect of stilbene derivative on superoxide generation and enzyme release from human neutrophils in vitro.

Neutrophils represent the body's primary line of defense against invading pathogens. They most rapidly reach the site of injury or infection, liberate antimicrobial proteins, proteases and produce reactive oxygen species. Prolonged or excessive liberation of these very effective and toxic substances could intensify the inflammatory process and enhance tissue damage in many diseases, such as allergies, infections and rheumatoid arthritis. Pterostilbene belongs to stilbenoids, structural analogues of resveratrol, which act as natural protective agents in defending the plant against viral and microbial attack. It possesses anticancerous, antidiabetic and anti-inflammatory properties.The study provides new information on the effect of pterostilbene [0.01-100 µmol/l] on superoxide generation in and myeloperoxidase (MPO) release from azurophil granules of isolated human neutrophils. PMA [1µmol/l], which activates NADPH-oxidase via protein kinase C, was used for stimulation of neutrophils Unstimulated cells showed neither superoxide generation nor myelopereoxidase release after preincubation with the drug studied. Pterostilbene dose dependently decreased superoxide generation in and MPO release from stimulated human neutrophils, however a significant decrease was recorded only in the concentration 100 µmol/l. The effect of pterostilbene was more pronounced on superoxide generation in comparison to MPO release. Our results suggest that the effect of pterostilbene may prove beneficial in controlling inflammation.

Quercetin inhibits degranulation and superoxide generation in PMA stimulated neutrophils.

Activated neutrophils represent the main source of myeloperoxidase (MPO), superoxide (SO) and subsequently derived oxygen metabolites. They have important microbicidal activities, however in inflammatory conditions they may secondarily attack surrounding tissues. Overproduction of reactive oxygen species, prolonged or excessive liberation of MPO and other effective yet also toxic substances from neutrophils may participate in disturbed apoptosis, intensify the inflammatory processes and result in serious human diseases. The inhibitory effect of quercetin on PMA stimulated SO generation in isolated human neutrophils was found to be dose-dependent, without affecting the activity of intact isolated neutrophils. At comparable conditions, quercetin was more potent in inhibiting MPO release than SO generation. Our results indicate that quercetin could support resolution of inflammation through decreased activity of neutrophils, i.e. respiratory burst and degranulation.

Modulation of metabolic activity of phagocytes by antihistamines.

The purpose of the study was to investigate the effects of H(1)-antihistamines of the 1(st) generation (antazoline, bromadryl, brompheniramine, dithiaden, cyclizine, chlorcyclizine, chlorpheniramine, clemastine) and the 2(nd) generation (acrivastine, ketotifen, and loratadine) on the respiratory burst of phagocytes. Reactive oxygen species generation in neutrophils isolated from rat blood was measured using luminol-enhanced chemiluminescence. Changes in nitrite formation and iNOS protein expression by RAW 264.7 macrophages were analysed using Griess reaction and Western blotting. The antioxidative properties of drugs in cell-free systems were detected spectrophotometrically, luminometrically, fluorimetrically, and amperometrically. The majority of the H(1)-antihistamines tested (bromadryl, brompheniramine, chlorcyclizine, chlorpheniramine, clemastine, dithiaden, and ketotifen) exhibited a significant inhibitory effect on the chemiluminescence activity of phagocytes. H(1)-antihistamines did not show significant scavenging properties against superoxide anion and hydroxyl radical, thus this could not contribute to the inhibition of chemiluminescence. H(1)-antihistamines had a different ability to modulate nitric oxide production by LPS-stimulated macrophages. Bromadryl, clemastine, and dithiaden were the most effective since they inhibited iNOS expression, which was followed by a significant reduction in nitrite levels. H(1)-antihistamines had no scavenging activity against nitric oxide. It can be concluded that the effects observed in the H(1)-antihistamines tested are not mediated exclusively via H(1)-receptor pathway or by direct antioxidative properties. Based on our results, antihistamines not interfering with the microbicidal mechanisms of leukocytes (antazoline, acrivastine and cyclizine) could be used preferentially in infections. Other antihistamines should be used, under pathological conditions accompanied by the overproduction of reactive oxygen species.

In vivo effect of pinosylvin and pterostilbene in the animal model of adjuvant arthritis.

OBJECTIVE: The aim of this study was to evaluate the effects of pinosylvin (PIN) and pterostilbene (PTE), natural substances from the stilbenoid group, on the development of adjuvant arthritis in rats. METHODS: Adjuvant arthritis (AA) was induced by a single intradermal injection of Mycobacterium butyricum in incomplete Freund's adjuvant in male Lewis rats. Our experiments included healthy intact animals as reference controls, arthritic animals without any drug administration, and arthritic animals with administration of PIN and PTE in the oral daily dose of 30 mg/kg b.w. The treatment involved administration of the substances tested from day 0, i.e. the day of immunization, to the experimental day 28. The following parameters were monitored: change of the hind paw volume (HPV) on day 14, 21 and 28, luminol-enhanced chemiluminescence (CL) of the joint and myeloperoxidase (MPO) activity in hind paw joint homogenates (day 28). RESULTS: Arthritic animals treated with PIN showed a decrease in HPV, significantly on days 14 and 28. PIN decreased CL of the joint as well as MPO activity of the joint homogenate, in comparison with untreated animals. PTE had no effect on HPV and MPO activity in hind paw joint homogenates and exerted only a partial effect on luminol-enhanced CL. CONCLUSIONS: On the basis of our results we conclude that the effect of PTE on CL was only partial. PIN, on the other hand, had a beneficial anti-inflammatory and antioxidant effect on oxidative stress induced biochemical changes occurring in AA, as determined by all three functional parameters.

H1-antihistamines and oxidative burst of professional phagocytes.

OBJECTIVES: We analysed and compared the effect of five H1-antihistamines on stimulated oxidative burst at extra- and intracellular level of isolated and stimulated human polymorphonuclear leukocytes. DESIGN: Oxidative burst of isolated human neutrophils was studied by means of luminol and isoluminol enhanced chemiluminescence. RESULTS: The following rank order of potency for H1-antihistamines to decrease chemiluminescence was evaluated extracellularly: dithiaden> loratadine> chlorpheniramine> brompheniramine> pheniramine and at intracellular site: loratadine> dithiaden. CONCLUSION: H1-antihistamines differ substantially according to their chemical structure in suppressing oxidative burst both at extra- and intracellular site of isolated stimulated human neutrophils.

Inhibition of superoxide generation and myeloperoxidase release by carvedilol after receptor and nonreceptor stimulation of human neutrophils.

OBJECTIVES: To compare three stimuli which activate human neutrophils with different signal transduction mechanisms, in order to better localize the effect of the beta-adrenoceptor antagonist carvedilol (CARV) on superoxide generation (O2*-) and myeloperoxidase release (MPO). The effect of CARV [0.1-100 micromol/l] on O2*- generation and MPO release from isolated human neutrophils was studied after specific receptor activator N-formyl-methionyl-leucyl-phenylalanine (fMLP) and nonreceptor phorbol-12-myristate-13-acetate (PMA) and calcium ionophor (A23187) stimuli. METHODS: O2*- generation was measured as superoxide dismutase inhibitable reduction of cytochrome c and MPO release as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A at respective 550 and 463 nm. RESULTS: CARV had no effect on O2*- generation and MPO release in nonstimulated cells. In the concentration 10 and 100 micromol/l, it significantly decreased fMLP and PMA stimulated O2*- generation and MPO release. Incubation of neutrophils with CARV [100 micromol/l] caused significant inhibition of O2*- generation and MPO release induced by A23187. Wortmannin, a specific inhibitor of 1-phosphatidylinositol-3-kinase, inhibited significantly only fMLP stimulated O2*- generation. CARV [100 micromol/l] with wortmannin [50 nmol/l] further decreased O2*- generation after the same stimulus. CONCLUSION: CARV decreased O2*- generation and MPO release from isolated human neutrophils both by membrane-operating stimulus - fMLP and membrane bypassing activators - PMA and A 23187. This fact, together with effect the of wortmannin, indicates that the inhibition may be attributed to the non-specific action of CARV and its interference with phospholipase D signaling pathway, which plays only a minor role in proteinkinase C stimulated O2*- generation.

Effect of the extract from salivary glands of Lucilia sericata on human neutrophils.

OBJECTIVES: There is incomplete information about host immune response to maggot therapy, nowadays increasingly used to clean chronic wounds from necrotic debris and infection. Maggots are applied to the wound during the inflammatory phase. At the same time neutrophils infiltrate the inflammatory site as the first defense line of the organism. Myeloperoxidase (MPO) and reactive oxygen species, generated during the respiratory burst by neutrophils, are the key players participating in microbial killing as well as in signalling pathways. AIM: We studied the effect of an extract from salivary glands (SGE) of Lucilia sericata (L. sericata) on opsonized zymosan stimulated whole blood chemiluminescence (CL), superoxide generation and MPO release from human neutrophils. METHODS: Formation of reactive oxygen species in whole blood was determined by luminol-enhanced CL. superoxide generation was measured as superoxide dismutase inhibitable reduction of cytochrome c, MPO activity as the oxidation of o-dianisidine in the presence of hydrogen peroxide. RESULTS: Crude SG extract of L. sericata had no significant effect either on superoxide generation and MPO release from isolated unstimulated human neutrophils or on activity of isolated enzymes. Crude SG extract of L. sericata in the highest concentration used significantly decreased opsonized zymosan (0.5 mg/ml) stimulated blood CL, superoxide generation and MPO release. CONCLUSION: On the basis of our results as well as from the literature we suggest that the beneficial effects of maggot therapy might involve the decrease of generation and release of proinflammatory factors, while neither phagocytosis nor subsequent apoptosis is disturbed.

Effect of carvedilol on the production of reactive oxygen species by HL-60 cells.

OBJECTIVES: The generation of reactive oxygen species (ROS) by phagocytes is one of the irreplaceable microbicidal tools of innate immunity. It has been reported in our previous studies that short-term treatment by carvedilol ex vivo inhibits ROS generation. The purpose of this study was to investigate the long-term effect of carvedilol on phagocytes. METHODS: Human leukemia HL-60 cells differentiated into granulocyte-like cells were used as the model. Final concentrations of carvedilol were 0.1-100 micromol/l. The production of ROS by HL-60 cells was measured using luminol-enhanced chemiluminescence (CL). RESULTS: Carvedilol in concentrations 0.1-10 micromol/l did not exhibit any toxic effect on cells (measured using bioluminescent bacteria Photorhabdus luminescens subsp. thracensis). One hour's treatment with 10 micromol/l carvedilol significantly decreased both spontaneous and activated CL of cells. Conversely, no inhibitory effects on CL were observed in 10 micromol/l carvedilol after 48 h incubation; lower concentrations of carvedilol even slightly increased the CL activity of HL-60 cells. A significant increase in spontaneous CL activity was detected in cells incubated with 10 micromol/l carvedilol in comparison with the control. Powerful antioxidative properties of carvedilol against peroxyl radical (ORAC assay) were proved. No scavenging of nitric oxide (electrochemical method) was observed. CONCLUSIONS: Long-term influence of carvedilol can induce an increase in the generation of phagocyte-derived ROS and potentially also other inflammatory mediators. The increased ROS production is compensated for by antioxidative properties of carvedilol although the increased production of inflammatory mediators could affect the proper function of immune system.

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.

In vitro effect of carvedilol on professional phagocytes.

AIM: Superfluous reactive nitrogen and oxygen species generation is implicated in the damage of tissues at sites of inflammation where activated neutrophils and macrophages are involved. This study was conducted to investigate whether the beneficial effects of carvedilol involve modulation of respiratory burst, degranulation-myeloperoxidase release and inducible nitric oxide synthase (iNOS) expression. METHODS: Spectrophotometry and chemiluminescence were used to evaluate the effect of carvedilol on opsonized zymosan (0.5 mg/ml)- or N-formyl-methionyl-leucyl-phenyl-alanine (fMLP, 0.1 micromol/l)-stimulated superoxide generation and myeloperoxidase release in human neutrophils. Western blot analysis was used for iNOS expression and Griess reagent for nitric oxide production in RAW 264.7 macrophages (lipopolysaccharide (0.1 microg/ml) stimulated). RESULTS: Carvedilol (10 and 100 micromol/l) significantly decreased opsonized zymosan- and fMPL-stimulated superoxide generation and myeloperoxidase release. Carvedilol (100 micromol/l) enhanced the effect of wortmannin (50 nmol/l), a specific inhibitor of 1-phosphatidylinositol 3-kinase and decreased iNOS expression and nitric oxide production. CONCLUSION: Carvedilol appears to have a non-specific effect on membranes and to interfere with the phospholipase D signaling pathway, with subsequent inhibition of reactive oxygen species generation and myeloperoxidase release, without affecting iNOS expression.

On the pharmacology and toxicology of neutrophils.

OBJECTIVES: To study the effect of H1-antihistamines dithiaden (Dit) and loratadine (Lor) and compare it with that of histamine (His) on phorbolmyristate acetate (PMA) stimulated chemiluminescence (CL) of whole blood, isolated neutrophils, release of myeloperoxidase (MPO), and on superoxide (SO) generation. METHODS: Luminol- and isoluminol-enhanced CL was applied for measuring the oxidative burst, spectrophotometry was used for determination of MPO (o-dianisidine) and SO generation (superoxide dismutase inhibition of cytochrome c). RESULTS: Dit and Lor dose-dependently inhibited CL of whole blood and significantly decreased oxidative burst both at the extra- and intracellular sites of neutrophils. Release of MPO was decreased with both drugs tested in 10-times lower concentrations than was SO inhibition. Histamine (His) was much less effective in the inhibition of the parameters investigated. CONCLUSIONS: Histaminergic drugs of the 1st generation inhibited oxidative burst of human phagocytes in whole blood and in isolated neutrophils. The rank order of potency to inhibit CL, MPO release and SO generation in PMA stimulated phagocytes was: Dit>Lor>His.

Effect of carvedilol on reactive oxygen species and enzymes linking innate and adaptive immunity.

OBJECTIVES: Neutrophils and macrophages are critical components of our innate host defenses. They are a potential source of nitric oxide (NO) and superoxide, known to play an important role in many physiological processes including defense, immune and inflammatory responses. Myeloperoxidase (MPO), a major NO scavenger and a marker of oxidative stress, as well as increased inducible nitric oxide synthase (iNOS) expression, may affect the NO-superoxide balance, critical for cellular redox balance in the cardiovascular system at physiologic conditions. The effect of carvedilol was studied on stimulated superoxide generation, MPO release and iNOS expression in phagocytes by using receptor operating stimuli [N-formyl-Met-Leu-Phe (FMLP), lipopolysaccharide (LPS) and specific inhibitors (wortmannin, propranolol). METHODS: Superoxide generation was measured as superoxide dismutase inhibitable reduction of cytochrome c (550 nm), MPO activity as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A (463 nm). Expression of iNOS (Western-blot analysis) in RAW 264.7 cell line (murine macrophages) was stimulated by lipopolysaccharide (LPS). RESULTS: Carvedilol dose-dependently decreased superoxide generation and MPO release from intact neutrophils stimulated by FMLP. In the highest concentration tested, carvedilol pronounced the effect of wortmannin [inhibitor of phospholipase D (PLD) regulatory pathway] and significantly decreased LPS stimulated iNOS expression in RAW 264.7 cell line. CONCLUSION: The conceivable cumulative non-specific membrane effect of carvedilol and its effect on PLD signalling pathway contribute to the decrease of both superoxide generation and MPO release, thus supporting the restoration of NO-superoxide balance.

Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils.

Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage. We studied the effect of carvedilol (CARV) [0.1-100 micromol/l], an antihypertensive and cardiovascular drug with antioxidative properties, on superoxide generation (SO) and myeloperoxidase (MPO) release from isolated human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus. Unstimulated cells showed neither SO formation nor MPO release after preincubation with drug. CARV decreased fMLP and PMA stimulated MPO release and SO generation dose dependently. The inhibitory effect of CARV may attributed to non-specific action since its effect was not influenced by the type of stimulation. It might inhibit SO generation as well as MPO release either by membrane-operating stimulus (fMLP) or membrane bypassing activator (PMA).