Modulation Of Oxidative Burst Research Articles

Modulation of oxidative burst with exposure to cytokines in neutrophil cell activation

Abstract Neutrophils are essential for killing microorganisms and have a significant role in regulation of inflammatory response. Generation of reactive oxygen species (ROS) is one of the critical events that modulate the immune response in phagocytic cells. Stimulated neutrophils activate membrane-associated NADPH oxidase (NOX2) resulting in a powerful oxidative burst during which a large amount of oxygen is consumed generating ROS. The Agilent Seahorse XF analyzer is used to quantify oxygen consumption rate (OCR) as a direct non-invasive measure of neutrophil activation. In this study, neutrophil activation in the presence of cytokines known to be expressed within microenvironments in normal and disease states is evaluated. Treatment of neutrophils with the inflammatory cytokine TNFα induces oxidative burst within an hour of treatment, as observed in real time using the XF analyzer. Interestingly, pretreatment with TNFα reduces the oxidative burst observed when neutrophils were activated with phorbol myristate acetate (PMA). In contrast, treatment with GM-CSF and IL1 β had no acute effect in inducing oxidative burst. Rather, pretreatment of neutrophils with GM-CSF and IL1 β primed them for an enhanced response to PMA and a reduced elapsed time to reach maximal activation. Of note, the oxidative burst observed is associated with simultaneous increased of proton efflux rate (PER), indicative of the dependence on glycolysis and pentose phosphate pathway for production of NADPH during oxidative burst. These results, show the utility of the XF neutrophil activation assay to study the effect of modulators such drug treatments, microenvironment and disease progression during innate immune response.

Stem bark and flower extracts of Vismia cauliflora are highly effective antioxidants to human blood cells by preventing oxidative burst in neutrophils and oxidative damage in erythrocytes

Context: Vismia cauliflora A.C.Sm. [Hypericaceae (Clusiaceae)] is an Amazonian plant traditionally used by indigenous population to treat dermatosis and inflammatory processes of the skin. Previous research on V. cauliflora extracts suggests its potential to neutralize cellular oxidative damages related to the production of reactive oxygen and nitrogen species.Objective: To determine the activity of stem bark and flower extracts of V. cauliflora on the modulation of oxidative burst in human neutrophils, as well as its potential to inhibit oxidative damage in human erythrocytes.Materials and methods: The modulation of neutrophil’s oxidative burst by the ethanolic extracts (0.3–1000 µg/mL) was determined by the oxidation of specific probes by reactive species. Additionally, the potential of these extracts to inhibit oxidative damage in human erythrocytes was evaluated by monitoring its biomarkers of oxidative stress.Results: Vismia cauliflora extracts presented remarkable capacity to prevent the oxidative burst in activated human neutrophils (IC50 < 15 µg/mL). However, the maximum percentage of inhibition achieved against hydrogen peroxide was 45%. Concerning the oxidative damage in human erythrocytes, the extracts were able to minimize the tert-butyl hydroperoxide-induced hemoglobin oxidation and lipid peroxidation in a very low concentration range (2.7–18 μg/mL). Furthermore, only stem bark extract (100 µg/mL) was able to inhibit the depletion of glutathione (13%).Discussion and conclusion: These results reinforce the therapeutic potential of stem bark and flower extracts of V. cauliflora to heal topical skin disease, namely in the treatment of neutrophil-related dermatosis and skin conditions related to oxidative stress, including skin aging.

P73 - Stem bark and flower extracts of Vismia cauliflora: modulation of oxidative burst in human neutrophils’ and inhibition of oxidative damage in human erythrocytes

Vismia cauliflora is an Amazonian plant traditionally used to treat dermatosis and inflammatory processes of the skin by indigenous population. Our research group showed that stem bark and flower extracts of V. cauliflora are efficient in vitro scavengers of reactive oxygen and nitrogen species. In this study, we determined the activity of stem bark and flower extracts of V. cauliflora plant on the modulation of in vitro oxidative burst in human neutrophils and their potential to inhibit the oxidative damage in human erythrocytes. The oxidative burst in activated neutrophils were monitored by specific probes to detect the oxidizing effect of superoxide anion radical (MCLA), hydrogen peroxide (amplex red) and hypochlorous acid (APF), and both extracts were efficient to neutralize the oxidative burst (IC50 from 3 to 15µg/mL). These same extracts were also effective against oxidative damage in erythrocytes by inhibiting hemoglobin oxidation (IC50=18µg/mL) and lipid peroxidation (IC50=2.7 and 7.5µg/mL, flower and stem bark, respectively). In addition, stem bark extract (100µg/mL) inhibited the depletion of glutathione by 13%. These extracts have similar phenolic composition, but flower presents quercetin (14%) in its composition. Therefore, these results reinforce the potential therapeutic of stem bark and flower extracts of V. cauliflora to heal topical skin disease and requires further research targeted effectively to develop phytopharmaceutical drug based on this plant.

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

A Comparative Study on Invasion, Survival, Modulation of Oxidative Burst, and Nitric Oxide Responses of Macrophages (HD11), and Systemic Infection in Chickens by Prevalent Poultry Salmonella Serovars

Poultry is a major reservoir for foodborne Salmonella serovars. Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg are the most prevalent serovars in U.S. poultry. Information concerning the interactions between different Salmonella species and host cells in poultry is lacking. In the present study, the above mentioned Salmonella serovars were examined for invasion, intracellular survival, and their ability to modulate oxidative burst and nitric oxide (NO) responses in chicken macrophage HD11 cells. All Salmonella serovars demonstrated similar capacity to invade HD11 cells. At 24 h post-infection, a 36-43% reduction of intracellular bacteria, in log(10)(CFU), was observed for Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg, whereas a significantly lower reduction (16%) was observed for Salmonella Enteritidis, indicating its higher resistance to the killing by HD11 cells. Production of NO was completely diminished in HD11 cells infected with Salmonella Typhimurium and Salmonella Enteritidis, but remained intact when infected with Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg. Phorbol myristate acetate-stimulated oxidative burst in HD11 cells was greatly impaired after infection by each of the five serovars. When newly hatched chickens were challenged orally, a high rate (86-98%) of systemic infection (Salmonella positive in liver/spleen) was observed in birds challenged with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Kentucky, while only 14% of the birds were Salmonella Senftenberg positive. However, there was no direct correlation between systemic infection and in vitro differential intracellular survival and modulation of NO response among the tested serovars.

The effects of low-dose X-irradiation on the oxidative burst in stimulated macrophages

Purpose : Local irradiation with a dose of around 0.5 Gy is an effective treatment of acute necrotizing inflammations. The hypothesis that low doses of X-rays modulate the oxidative burst in activated macrophages, which plays a major role in the acute inflammatory process, was tested. Materials and methods : Murine RAW 264.7 macrophages were stimulated with LPS/ γIFN, PMA or zymosan and oxidative burst was measured using either DCFH-DA or by reduction of cytochrome-C. Radiation doses of 0.3-10 Gy were given shortly before or after stimulation. Results : Low X-ray doses of <1 Gy significantly reduced the oxidative burst in activated macrophages, whereas higher doses had little effect on oxidative burst. Conclusions : The modulation of oxidative burst by low radiation doses may contribute to the therapeutic effectiveness of low-dose radiotherapy of acute necrotizing inflammations.

Identification of a novel neutrophil membrane protein involved in modulation of oxidative burst

On the basis of selective recognition by antibodies directed against neutrophil membrane determinants, a new neutrophil protein (molecular mass 82 kDa) has been identified, and shown to be functionally correlated with the oxidative response evoked in these cells by agonist stimulation. The protein is present in neutrophil membrane fraction but only upon activation it becomes accessible to recognition by a specific monoclonal antibody. In these conditions a complete and selective inhibition of O 2 − production occurs. The presence of a new protein antigen in neutrophil membranes linked to the activation of the O 2 − producing multienzyme complex that becomes external to the cell surface in primed or activated cells, might be important for future approaches aiming at the control of neutrophil response and at the identification of the activated forms of these cells.