Melatonin promotes cardiomyogenesis of embryonic stem cells via inhibition of HIF-1α stabilization.

Melatonin, a molecule involved in the regulation of circadian rhythms, has protective effects against myocardial injuries. However, its capability to regulate the maturation of cardiac progenitor cells is unclear. Recently, several studies have shown that melatonin inhibits the stabilization of hypoxia-inducible factors (HIFs), important signaling molecules with cardioprotective effects. In this study, by employing differentiating mouse embryonic stem cells, we report that melatonin significantly upregulated the expression of cardiac cell-specific markers (myosin heavy chains six and seven) as well as the percentage of myosin heavy chain-positive cells. Importantly, melatonin decreased HIF-1α stabilization and transcriptional activity and, in contrast, induced HIF-2α stabilization. Interestingly, the deletion of HIF-1α completely inhibited the pro-cardiomyogenic effect of melatonin as well as the melatonin-mediated HIF-2α stabilization. Moreover, melatonin increased Sirt-1 levels in a HIF-1α-dependent manner. Taken together, we provide new evidence of a time-specific inhibition of HIF-1α stabilization as an essential feature of melatonin-induced cardiomyogenesis and unexpected different roles of HIF-1α stabilization during various stages of cardiac development. These results uncover new mechanisms underlying the maturation of cardiac progenitor cells and can help in the development of novel strategies for using melatonin in cardiac regeneration therapy.

Molecular targets of the natural antioxidant pterostilbene: effect on protein kinase C, caspase-3 and apoptosis in human neutrophils in vitro.

OBJECTIVE: Pterostilbene, a naturally occurring phenolic derivative, exhibits various pharmacological effects, e.g. anti-cancerous, antioxidant, anti-inflammatory and anti-diabetic. Based on our previous study, we assessed the cellular and molecular effects of pterostilbene on human neutrophils and in cell free systems. Experimental and theoretical molecular descriptors of stilbene derivatives were also determined. METHODS: We assessed the antioxidant properties of pterostilbene using cell free system and computational methods. The effect of pterostilbene on protein kinase C activation/phosphorylation was detected by special anti-phospho protein kinase C antibodies. Membrane associated changes determining the life span of neutrophils and human recombinant caspase-3 assay were examined. RESULTS: Pterostilbene possessed comparable antioxidant properties as resveratrol in cell free system. Computational methods were used to establish the molecular characteristics of stilbene derivatives. The values of electronic parameters suggest a slight enhancement of electron donor properties of pterostilbene compared to resveratrol. Phosphorylation and thus activation of protein kinase C alpha/beta II in activated neutrophils was not decreased by pterostilbene. Pterostilbene in concentrations of 10-100 µM was found to inhibit the activity of human caspase-3 purified enzyme and did not influence cell viability significantly. CONCLUSION: Pterostilbene, an analog of resveratrol, was identified as a good natural antioxidant compound. However, reducing the oxidative burst of human neutrophils during their activation in vitro with pterostilbene does not include protein kinase C phosphorylation pathway. Pterostilbene showed dose dependent activation/inhibition of caspase-3 enzyme activity.

Formation of reactive oxygen and nitrogen species in the presence of pinosylvin - an analogue of resveratrol.

OBJECTIVE: Formation of reactive oxygen species in neutrophils of rats with adjuvant arthritis and generation of nitric oxide in RAW 264.7 macrophages were analysed in the presence of pinosylvin. METHODS AND RESULTS: The method of chemiluminescence was used for the detection of reactive oxygen species in blood of rats with adjuvant arthritis. Pinosylvin (50 mg/kg, daily, p.o.) and methotrexate (0.4 mg/kg, twice a week, p.o.) were applied separately or in a combination over a period of 28 days from the day of immunisation. Adjuvant arthritis was accompanied by a significantly increased number of neutrophils, by elevated concentration of oxidants in blood and by excessive responsiveness of neutrophils to stimulation with PMA. In rats treated with methotrexate, all these changes were significantly reduced and the inhibition became more pronounced when methotrexate was applied in the combination with pinosylvin; the monotherapy with pinosylvin did not induce any detectable changes in the parameters tested. Under in vitro conditions, pinosylvin inhibited formation of nitric oxide (NO) in macrophages, as demonstrated by the decreased concentration of nitrite - the end-product of NO metabolism (assessed by Griess' method), by the reduced expression of inducible NO synthase (detected by Western blot), and by the failure of pinosylvin to scavenge nitric oxide (measured amperometrically in cell-free system). CONCLUSION: The observed ability of pinosylvin to decrease concentration of reactive oxygen and nitrogen species, along with its capacity to enhance the efficacy of methotrexate in arthritis treatment may shed more light into the pharmacological potential of this prospective natural substance.

The Role of Dietary Phenolic Compounds in Epigenetic Modulation Involved in Inflammatory Processes.

A better understanding of the interactions between dietary phenolic compounds and the epigenetics of inflammation may impact pathological conditions and their treatment. Phenolic compounds are well-known for their antioxidant, anti-inflammatory, anti-angiogenic, and anti-cancer properties, with potential benefits in the treatment of various human diseases. Emerging studies bring evidence that nutrition may play an essential role in immune system modulation also by altering gene expression. This review discusses epigenetic mechanisms such as DNA methylation, post-translational histone modification, and non-coding microRNA activity that regulate the gene expression of molecules involved in inflammatory processes. Special attention is paid to the molecular basis of NF-κB modulation by dietary phenolic compounds. The regulation of histone acetyltransferase and histone deacetylase activity, which all influence NF-κB signaling, seems to be a crucial mechanism of the epigenetic control of inflammation by phenolic compounds. Moreover, chronic inflammatory processes are reported to be closely connected to the major stages of carcinogenesis and other non-communicable diseases. Therefore, dietary phenolic compounds-targeted epigenetics is becoming an attractive approach for disease prevention and intervention.

Serotonin and its metabolites reduce oxidative stress in murine RAW264.7 macrophages and prevent inflammation.

In this study, we focused on comparing the effects of serotonin and its metabolites on the functions of RAW264.7 cells (emphasis on oxidative burst and production of nitric oxide and cytokines), thereby expanding the scope of existing knowledge with advent of novel findings in this field. Changes in production of reactive oxygen species (ROS) by RAW264.7 cells after treatment with serotonin, N-acetylserotonin and melatonin were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all respective compounds were measured using TRAP and amperometrical method. Nitric oxide (NO) production was measured by Griess reagent and inducible NO synthase (iNOS) expression by Western blot. Cytokine production was assessed using the Mouse Cytokine Panel A Array kit and ELISA. We showed that all tested compounds were able to reduce oxidative stress, as well as inhibit production of inflammatory cytokines by macrophages. Of the tested compounds, serotonin and N-acetylserotonin were markedly better antioxidants than melatonin. In comparison, other effects of tested compounds were very similar. It can be concluded that antioxidant capacity of tested compounds is a major advantage in the early stages of inflammation. Since plasma concentrations of N-acetylserotonin and melatonin are lower than serotonin, it can be deduced that serotonin plays a key role in modulation of inflammation and the regulatory functions of immune cells, while also protecting cells against oxidative stress.

Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7.

In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.

Interactions of oxidatively modified calf skin collagen with platelets and phagocytes.

OBJECTIVES: The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation and the oxidative burst of phagocytes were examined in the framework of a general hypothesis that collagen, platelets and phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress. MATERIALS AND METHODS: Calf skin collagen type I was subjected to oxidative modification by hydrogen peroxide or hydroxyl radical. Thermal denaturation of collagen was performed in a spectrophotometer equipped with a temperature gradient device. The aggregation of isolated human platelets obtained after differential centrifugation was measured using a dual-channel aggregometer. The production of reactive oxygen species by human whole blood phagocytes was evaluated by luminol-enhanced chemiluminescence. RESULTS: Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature. Oxidatively modified samples showed a modified SDS-PAGE pattern, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity. CONCLUSION: The results suggest that reactive oxygen species were able to modify collagen. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes.

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.

Black chokeberry (Aronia melanocarpa) polyphenols reveal different antioxidant, antimicrobial and neutrophil-modulating activities.

The current study reports data on antioxidant, antimicrobial and neutrophil-modulating activities of different polyphenolic preparations from black chokeberry fruits: crude extract, purified extract standardized to 20% and 40% anthocyanins, and proanthocyanidins; as well as pure compounds (chlorogenic acid, cyanidin-3-O-galactoside, epicatechin, rutin and quercetin) present in black chokeberries. Minor phenolic components - quercetin and epicatechin showed the highest ORAC and TRAP antioxidant activity. Given the amount of individual phenolics in the fruits, proanthocyanidins are the major contributor to antioxidant activity of fresh black chokeberries. Studied polyphenols and preparations had no effect on the spontaneous chemiluminescence (CL) of human neutrophils and only mild effect on PMA-activated CL. Greater effects were observed on OZP-activated CL, being statistically significant (p < 0.05) for quercetin and rutin. The antimicrobial activity test against 10 pathogens showed that black chokeberry proanthocyanidins are the most potent antimicrobial agents in the fruit.

Obesity-induced nucleosome release predicts poor cardio-metabolic health.

OBJECTIVE: While circulating nucleosome levels are high in obese mouse models, it is unknown where these nucleosomes originate from and whether they are a marker of cardio-metabolic health in humans. Here, we aimed to determine whether an association exists between circulating nucleosomes and the risk of developing obesity, metabolic syndrome (MetS) and/or a dysfunctional cardiovascular performance. METHODS: We randomly selected 120 participants of the Kardiovize Brno 2030 study across three BMI strata: BMI 18-25, 25-30, and > 30. We assessed the association between circulating nucleosome levels and the risk of obesity, MetS, and poor cardiovascular health. We then cultured human neutrophils, adipocytes, and hepatoma cells to study nucleosome origins in a fat-rich environment. RESULTS: Circulating nucleosome levels positively correlated with BMI (R = 0.602, p < 0.05), fatty liver index (R = 0.622, p < 0.05), left ventricular mass (R = 0.457, p < 0.05), and associated with MetS (p < 0.001) and poor cardiovascular health (p < 0.001). Incubating neutrophils with 1-10 µM free fatty acids triggered nucleosome production without concomitant cell death. Nucleosomes were not produced during pre-adipocyte differentiation or upon incubation of hepatic cells with palmitic acid. CONCLUSIONS: Neutrophils are a bona fide source of circulating nucleosomes in an obesogenic environment and in overweight/obese patients. High nucleosome levels are associated with MetS and cardiovascular performance, and might represent novel candidate biomarkers for cardio-metabolic health.

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.

Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure.

Electropolymerization regime of meso-tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin is optimized to yield films possessing both electrocatalytical and permselective properties towards nitric oxide oxidation. The sensor composed of electrochemically oxidized carbon fiber, covered solely with nickel porphyrin derivative layer electropolymerized using our method, is characterized by high selectivity towards nitrite (1:600), ascorbate (1:8000) and dopamine (>1:80), determined by constant potential amperometry at 830 mV (vs. Ag/AgCl). Selectivity for ascorbate and dopamine as well as detection limit for NO (1.5 nM at S/N=3) is 5-10 times better than parameters usually reported for Nafion coated porphyrinic sensors. Nafion coating can further enhance selectivity properties as well as aids to the stability of the sensors' responses.

The effects of berberine on reactive oxygen species production in human neutrophils and in cell-free assays.

The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity - ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 µM and 197 µM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.

The common lavender (Lavandula angustifolia Mill.) pectic polysaccharides modulate phagocytic leukocytes and intestinal Peyer's patch cells.

Two pectic (chPS-L1, chPS-L2) and one polyphenolic (chPP-L) fractions were obtained from lavender flowers after boiling water extraction, exhaustive removing of alcohol-soluble molecules and SEC. chPS-L1 (52.4kDa) contains mainly low-acetylated and high-methoxylated homogalacturonans (HG), and smaller rhamnogalacturonan (RG) I backbone fragments rich in 1,3,5-branched arabinan and arabinogalactan (AG) II side chains. chPS-L2 (21.8kDa) contains predominantly similarly esterified HG, followed by RGI with AGII structures and RGII. The prevalence of catechin and epicatechin in chPP-L indicates that they form weak interactions with pectins. chPS-L1 and chPS-L2 enhanced ß2-integrin expression on neutrophils, inducing ROS generation and macrophage NO production. Both the effects on ß2-integrin and high complement fixation activity of chPS-L1 were proposed for its inhibitory action against PMA- and OZP-activated ROS formation. This, together with suppression of NO generation after co-stimulation with chPS-L1 and LPS, suggested anti-inflammatory activity of studied pectins. Lavender polysaccharides expressed intestinal Peyer's patch immunomodulating activity.

Acidic polysaccharide complexes from purslane, silver linden and lavender stimulate Peyer's patch immune cells through innate and adaptive mechanisms.

Three polysaccharide complexes (PSCs) were isolated from the aerial parts of common purslane (Portulaca oleracea L.), and the flowers of common lavender (Lavandula angustifolia Mill.) and silver linden (Tilia tomentosa Moench) by boiling water extraction and ethanol precipitation. The chemical composition and immunomodulating effects of isolated PSCs were characterized. The chemical characterization revealed that the three samples contain mainly pectic polysaccharides. They exhibited ex vivo intestinal immunomodulating activity through the murine Peyer's patch-mediated bone marrow cell proliferation test at 100µg/ml concentration. At the same time, they stimulated ex vivo human blood T-cell populations (CD4+/CD25+ and CD8+/CD25+), phagocytic leukocytes (CD14+ and CD64+ cells) and induced IL-6 production from human white blood cells and Peyer's patch cells. The herbal PSCs stimulated ex vivo ROS production from whole blood phagocytes and showed unspecific in vitro anti-proliferative activity against normal and A549, HeLa and LS180 tumor cells. This is the first report on immunomodulating studies of linden flower pectins and chemical and biological activity characterization of lavender polysaccharides. Our study demonstrates that similarly to purslane, lavender and silver linden herbal materials contain immunomodulating polysaccharides that could be useful for support of compromised immune system.

Tilia tomentosa pectins exhibit dual mode of action on phagocytes as ß-glucuronic acid monomers are abundant in their rhamnogalacturonans I.

Silver linden flowers contain different pectins (PSI-PSIII) with immunomodulating properties. PSI is a low-esterified pectic polysaccharide with predominant homogalacturonan region, followed by rhamnogalacturonan I (RGI) with arabinogalactan II and RGII (traces) domains. PSII and PSIII are unusual glucuronidated RGI polymers. PSIII is a unique high molecular weight RGI, having almost completely O-3 glucuronidated GalA units with >30% O-3 acetylation at the Rha units. Linden pectins induced reactive oxygen species (ROS) and NO generation from non-stimulated whole blood phagocytes and macrophages, resp., but suppressed OZP-(opsonized zymosan particles)-activated ROS generation, LPS-induced iNOS expression and NO production. This dual mode of action suggests their anti-inflammatory activity, which is known for silver linden extracts. PSI expressed the highest complement fixation and macrophage-stimulating activities and was active on intestinal Peyer's patch cells. PSIII was active on non-stimulated neutrophils, as it induced ß2-integrin expression, revealing that acetylated and highly glucuronidated RGI exhibits immunomodulating properties via phagocytes.

Alterations in plasma antioxidants during reperfusion of the ischemic small intestine in rats.

The aim of this study was to evaluate the contribution of three plasma antioxidants (albumin, uric acid, SH groups) to the plasma total peroxyl radical-trapping antioxidant capacity (TRAP) in 2 and 4 h of intestinal reperfusion in rats. TRAP increased significantly both after 2 and 4 h of reperfusion. Neither albumin nor SH groups contributed significantly to this increase. TRAP was strongly influenced by the increase in uric acid concentration and also probably by the cell destruction caused by oxidative stress. Since the TRAP increase was accompanied by an increase in the level of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation), we can conclude that even such a large increase in TRAP is not sufficient to prevent the progression of lipid peroxidation and oxidative stress.

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.

Perioperative and postoperative course of cytokines and the metabolic activity of neutrophils in human cardiac operations and heart transplantation.

OBJECTIVES: The purpose of this study was to compare systemic inflammatory responses after heart transplantation and nontransplant cardiac operations, both involving cardiopulmonary bypass with a focus on the role of polymorphonuclear leukocytes. METHODS: Lipid peroxidation, blood phagocyte radical production, and interleukin 6, 8, and 10 plasma concentrations during surgical intervention and on the first and seventh postoperative days were evaluated in patients undergoing heart transplantation (n = 24) and in patients not undergoing transplantation (n = 30). RESULTS: Levels of interleukin 6, 8, and 10 increased in both groups of patients during early reperfusion. They normalized within the first postoperative day in the transplant group, whereas the nontransplant group's interleukin 6 and 8 levels remained increased on the seventh day after the operation. Interleukin 10 plasma levels were higher in the heart transplant group during reperfusion. Lipid peroxidation was increased after the operation in both groups of patients. Phagocyte activity was enhanced at reperfusion and at all other sampling times only in the nontransplant group. On the other hand, phagocyte activity oscillated around the preoperative level during heart transplantation, or it was even decreased. CONCLUSION: Both cardiac operations involving heart transplantation and those without transplantation are associated with increased oxidative stress and an enhanced production of proinflammatory and anti-inflammatory cytokines. Differences in interleukin 10 production and phagocyte activity could be caused mainly by the immunosuppressive therapy in heart transplant operations.

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes.

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 mumol/l) and by the H1-antagonist dithiaden (1-100 mumol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemiluminescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H1-antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A2) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.