Stimulation Of Oxidative Burst Research Articles

INVOLVEMENT OF MITOGEN-ACTIVATED PROTEIN KINASES P38 AND ERK1/2 AS WELL AS PROTEIN KINASE B AKT1/2 IN THE FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS

Neutrophils release decondensed nuclear chromatin or Neutrophil Extracellular Traps (NETs) in response to a great number of physiological stimuli to protect the host from pathogens. However, NETs have recently been shown to play an important role in the pathogenesis of autoimmune, infl ammatory, and malignant diseases. Therefore, understanding the molecular mechanisms underlying NETs formation, usually leading to the neutrophil death (NETosis), is extremely important to control the aberrant release of chromatin. Mitogen-activated protein kinases (MAP-kinases) are involved in various cellular functions such as oxidative burst, chemotaxis, degranulation, adhesion, and apoptosis, but their role in NETosis is not well understood. Three families of MAP-kinases, p38, ERK1/2, and JNK, have been described in human neutrophils, and we investigated the contribution of p38, ERK1/2, and protein kinase B Akt1/2 in oxidative burst and NETosis using inhibitory analysis. We have shown that MAPkinase p38 as well as protein kinase B Akt1/2 are activated upon stimulation of oxidative burst and NETosis with calcium ionophore ionomycin. However, these kinases are not involved in the oxidative burst induced by diacylglycerol mimetic phorbol 12-myristate 13-acetate (PMA) but are involved in PMA-induced NETosis.

High-Density Lipoprotein from Chronic Kidney Disease Patients Modulates Polymorphonuclear Leukocytes.

The anti-inflammatory properties of high-density lipoproteins (HDL) are lost in uremia. These HDL may show pro-inflammatory features partially as a result of changed protein composition. Alterations of polymorphonuclear leukocytes (PMNLs) in chronic kidney disease (CKD) may contribute to chronic inflammation and high vascular risk. We investigated if HDL from uremic patients is related to systemic inflammation by interfering with PMNL function. PMNL apoptosis was investigated by assessing morphological features and DNA content. CD11b surface expression was quantified by flow cytometry. Oxidative burst was measured via cytochrome c reduction assay. Chemotaxis was assessed by using an under-agarose migration assay. We found that HDL from CKD and hemodialysis (HD) patients significantly attenuated PMNL apoptosis, whereas HDL isolated from healthy subjects had no effect on PMNL apoptosis. The use of signal transduction inhibitors indicated that uremic HDL exerts anti-apoptotic effects by activating pathways involving phosphoinositide 3-kinase and extracellular-signal regulated kinase. Healthy HDL attenuated the surface expression of CD11b, whereas HDL from CKD and HD patients had no effect. All tested isolates increased the stimulation of oxidative burst, but did not affect PMNL chemotactic movement. In conclusion, HDL may contribute to the systemic inflammation in uremic patients by modulating PMNL functions.

Effects of energy supply and nicotinic acid supplementation on phagocytosis and ROS production of blood immune cells of periparturient primi- and pluriparous dairy cows

Effects of energy supply and nicotinic acid (NA) supplementation on the phagocytic activity of polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC), and on ROS production in PMN of periparturient cows differing in parity were examined. 29 pluriparous and 18 primiparous cows were allocated to four different feeding groups from 42days prepartum until 100days postpartum. They were fed either a ration with a low concentrate proportion of 30% (LC) or a high concentrate proportion of 60% (HC). After parturition all animals received 30% concentrate which was increased to 50% either within 16 (LC) or within 24days (HC). The different concentrate feeding strategies aimed at triggering differences in postpartum lipolysis. Half of the animals per group were supplemented with 24g per day of NA from 42days prepartum until 24days postpartum. All investigated parameters varied significantly over time compared to parturition (p<0.05). Numbers of phagocytosing PMN and PBMC increased in the course of the experiment, whereas the amount of engulfed bacteria per cell decreased between 42 and 11days prepartum. Percentage of basal ROS producing PMN decreased strongly before parturition and reached initial values only at 28days in milk again. Mean fluorescence intensity (MFI) in these ROS producing cells, however, increased before parturition. Oxidative burst stimulation in PMN was reduced around parturition but the amount of ROS produced in the stimulated cells was increased. Pluriparous cows exhibited higher numbers of basal ROS producing PMN and phagocytic PBMC. NA supplementation influenced phagocytosis in blood leukocytes.

Imatinib Does Not Inhibit Neutrophil Oxidative Burst Function in Patients with Chronic Myelogenous Leukemia (CML).

Imatinib mesylate is a competitive Bcr-Abl tyrosine kinase inhibitor which has been used in treatment of chronic myelogenous leukemia (CML). In addition to the inhibition of the Bcr-Abl protein tyrosine kinases, it also shows activity against other protein kinases such as platelet-derived growth factor receptor (PDGF-R), c-Kit and protein kinase C (PKC) in higher doses (Manley et al., 2002). Myelosuppression has been reported up to 60% of patients with CML under imatinib therapy (Deininger et al., 2003). Microbicidial activity of the neutrophils is mainly generated by the enzyme NADPH oxidase which produces superoxide anion. Several protein kinases including protein kinase C (PKC) that phosphorylate components of the NADPH oxidase and regulate oxidase activity have been identified. Little has been known about the effect of imatinib on neutrophil function, so far. Therefore, we aimed to evaluate the neutrophil respiratory burst activity using Phorbol Myristate acetate (PMA), a PKC activator, in vitro, in patients with CML achieving imatinib. Heparinized peripheral blood samples were collected from 14 patients with CML under imatinib therapy (400 mg daily) for 9.4 ± 4 months and 13 voluntary healthy controls. Oxidative burst was evaluated using a standard method defined previously by flow cytometry (Robinson et al., 1993) Oxidative burst stimulation index was calculated as the ratio of mean value of fluorescence intensity of DCFH-DA labelled neutrophils before and following PMA stimulation. It has been found that the stimulation index of neutrophils in patient achieving imatinib was 14.5±5 which was comparable to healthy control group 14±6.6. Furthermore, no serious infection disease was clinically detected during 8 month follow up period. So this study suggest Imatinib which has ability to inhibit bcr-abl fusion protein selectively and also can inhibit some others kinases, however has no inhibitory effect on the neutrophil function in patients with CML.

New cytokine dressings. II. Stimulation of oxidative burst in leucocytes in vitro and reduction of viable bacteria within an infected wound

Recently, we have developed the new dressings containing rhG-CSF or rhGM-CSF. In the present study we investigated either in vitro or in vivo biological activity of the dressings. Human whole blood samples were incubated with extracts from the collagen- or polyurethane-based dressings containing rhG-CSF or/and rhGM-CSF and phagocytic and oxidative metabolic activities were quantitated using Phagotest or Bursttest kits. The results indicate that both the number of phagocyting cells and the intensity of phagocytosis per cell, as well as the level of the oxidative burst in particular, were stimulated by one or both of the cytokines extracted. Next, the experimental skin wounds in mice were infected with 10 7 CFU of Pseudomonas aeruginosa strain ATCC 27853 and treated locally with the rhG-CSF-containing dressing. The analysis of the biopsies taken from the wounds indicated that in mice treated with the cytokine-containing dressing on the third day the log of CFU per biopsy was 5.0 vs. 6.2 in the control ( P<0.001), and on the 8th day was lower than 4 vs. 5.4 in control ( P<0.0001). Our findings clearly suggest that the newly designed dressings containing the incorporated CSFs can be used as effective topical cytokine-delivery system in the treatment of bacterial infections in wounds.

Highly significant role of Helicobacter pylori urease in phagocytosis and production of oxygen metabolites by human granulocytes.

The contribution of Helicobacter pylori urease, the vacuolating cytotoxin VacA, and the 128-kDa protein CagA to the stimulation of human granulocytes in terms of phagocytosis and oxidative burst was evaluated. Blood was incubated with H. pylori strains and corresponding isogenic mutants lacking either the large urease subunit (UreB) or an accessory urease protein (UreG) or VacA or CagA. Phagocytosis and oxidative burst were monitored by flow cytometry. The UreB-lacking mutant was phagocytosed more efficiently (P < .001) and induced significantly less oxidative burst (P < .001) than its parental strain or the UreG-lacking mutant, which produces an enzymatically inactive urease. Values of the other mutants did not differ greatly from those of their parental strain. These data indicate inflammatory effects of H. pylori urease causing inhibition of phagocytosis and stimulation of oxidative burst by a pathway being largely independent of ammonia production.

Immunotoxicological effects on piglets of feeding sows diets containing aflatoxins

Three groups of four Large White sows were fed diets containing either 800 ppb purified aflatoxin Bi (group 1), 800 ppb purified aflatoxin Gl (group 2) or 400 ppb Bi...

Deactivation of macrophage oxidative burst in vitro by different strains of Histoplasma capsulatum.

It was previously reported that Histoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi, Candida albicans and Cryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.

Activation of Phospholipase D by Interleukin-8 in Human Neutrophils

AbstractInterleukin 8 (IL-8), a member of the C-X-C branch of the chemokine superfamily, stimulated the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine (E3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) in human polymorphonuclear leukocytes (PMN) in the presence of cytochalasin B. In addition, the mass of diradyl-PA was increased with similar kinetics. In the presence of ethanol, 1-O-[3H]alkyl-2-acyl-phosphatidylethanol ([3H]EAPEt) was formed at the expense of [3H]EAPA formation, indicating the activation of phospholipase D by the cytokine. The effect was time- and concentration-dependent, reaching a plateau at 30 seconds with the maximally activating concentration of 120 nmol/L IL-8. Preincubation of cells with 1 µg/mL Bordetella pertussis toxin inhibited the breakdown of [3H]EAPC and [3H]EAPA formation, indicating a role for a pertussis toxin-sensitive guanosine triphosphate-binding protein. Formation of phosphatidic acid (PA) correlated with activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the oxidative burst enzyme, with both events occurring in the same concentration range. Inhibition of PA formation, by the presence of ethanol, also inhibited the oxidative burst stimulation by IL-8. Pretreatment of PMN with 10 nmol/L platelet-activating factor potentiated both [3H]EAPA accumulation and activation of NADPH oxidase by IL-8. Collectively, these data show that IL-8 stimulates the metabolism of choline-containing phosphoglycerides in human PMN and support a role for PA in the signaling mechanisms used by IL-8 to stimulate PMN function.

Phagocytosis of Salmonella typhimurium by differentiated U937 cells and oxidative burst stimulation: A flow cytometric and luminometric study

Phagocytosis of Salmonella typhimurium by differentiated U937 cells and oxidative burst stimulation: A flow cytometric and luminometric study

Stimulation of oxidative burst in human monocytes by lipoteichoic acids

Lipoteichoic acid isolated from Streptococcus faecalis or Streptococcus pyogenes caused direct activation of the respiratory burst in human peripheral blood monocytes. This activity appears to be related to the ability of lipoteichoic acid to bind to the monocyte membrane and trigger the polarization of receptors (capping).

Nitroblue tetrazolium reduction in monocytes and monocyte‐derived macrophages

The ability to mount an oxidative burst (OB) in response to medium, zymosan and phorbol myristate acetate (PMA) was assessed in human blood monocytes cultured for 1 day (MO) and monocyte-derived macrophages cultured for 10 days (MDM). Further, the effect of recombinant interferons (IFNs) on OB generation was examined. The OB was measured as a reduction of nitroblue tetrazolium (NBT). Unstimulated and stimulated NBT reduction per cell nucleus and the ratio of stimulated/unstimulated NBT reduction was not significantly different in cells cultured for 1 and 10 days. In MO, IFN-gamma stimulated the OB when co-stimulated with zymosan or PMA. IFN-alpha reduced MO adherence. When the lower adherence was corrected for, IFN-alpha enhanced NBT reduction. In MDM, a high concentration of IFN-gamma stimulated the OB without co-stimulation, in lower concentrations the presence of a co-stimulant was necessary for OB stimulation. IFN-alpha/beta enhanced the OB in response to PMA, suggesting that IFN-alpha/beta has a role in macrophage activation.

The Oxidative Burst and Related Phenomena in Mouse Macrophages Elicited by Different Sterile Inflammatory Stimuli

Mouse peritoneal macrophages (MPM) elicited by paraffin oil (PO) or thioglycollate (TG) were compared in their capacity to generate oxidative burst (OB) products such as superoxide ion (O 2 −) and hydrogen peroxide (H 2O 2) and lyse target erythrocytes. Strong OB stimulants such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), opsonized zymosan, and wheat-germ agglutinin (WGA) were found to stimulate O 2 − and H 2O 2 production by both PO- and TG-elicited MPM. PO-elicited MPM exhibited a vigorous OB response compared to TG-elicited MPM and showed a higher degree of self iodination in the presence of 125I. Macrophage-mediated cytolysis of 51Cr-labeled red blood cells, following OB stimulation, was revealed by PO-elicited MPM triggered by TPA, opsonized zymosan, WGA, and Concanavalin A. TG-elicited MPM failed to respond to TPA and opsonized zymosan. In the presence of horseradish peroxidase, both PO- and TG-elicited MPM exhibited augmented cytocidal activities upon stimulation with TPA, whereas catalase abrogated the capacity of TPA-stimulated MPM to lyse red blood cells, which may suggest the involvement of H 2O 2 in the lytic process.