Burst In Human Neutrophils Research Articles

Glutathione Reductase Facilitates Host Defense by Sustaining Phagocytic Oxidative Burst and Promoting the Development of Neutrophil Extracellular Traps

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.

5‐hydroxy‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐3,7‐dimethoxy‐4H‐chromen‐4‐one (MSF‐2) suppresses fMLP‐mediated respiratory burst in human neutrophils by inhibiting phosphatidylinositol 3‐kinase activity

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2), a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP-induced respiratory burst in human neutrophils and suggests a possible therapeutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.

The effect of olive extract on human neutrophils

The neutrophil respiratory burst, which is essential for the host's ability to kill microorganisms, can be stimulated by some natural agents. Olea europaea (Oleaceae) is used as anti hypertensive, hyperglycemia, and hypercholestremia in traditional medicine. Here in this study, we evaluate the stimulatory effect of olive oil extract on oxidative burst in human neutrophils in vitro. Buffy coat was separated from heparinized blood from normal volunteers by sedimentation in 1% W/V dextran then overlaid on Ficoll-Hipaque and centrifuged for 30 min. After washing, the neutrophils were cultured in 96 well plates containing 0.2 ml RPMI1640 as medium. After treatment with different doses of olive oil extract, the wells were washed with HBSS then incubated with 0.1 ml NBT (1 mg/ml) and 0.1 ml RPMI1640. The reduced NBT was solubilised by adding DMSO then the OD of 630 nm was read using micro ELISA reader. Statistical analysis by ANOVA, tukey test showed that there is dose response effect in the range of doses that examined (p

Inhibition of fMLP‐stimulated respiratory burst in human neutrophils by 5‐hydroxy‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐3,7‐dimethoxy‐4H‐chromen‐4‐one (MSF‐2), a natural lignan extract from Melicope Semecarpifolia: specific inhibition of phosphatidylinositol 3‐kinase activity

We examined the underlying inhibitory mechanisms of a lignan, MSF‐2, extracted from the fruit of Melicope. Semecarpifolia in human neutrophils.To clarify the effect of MSF‐2 on fMLP‐induced neutrophil activation, intracellular signals induced by fMLP were evaluated. MSF‐2 inhibited the fMLP‐induced superoxide anion production and cathepsin G release in a concentration‐dependent manner with respective IC50 values of 22.8±4.8 and 14.2±1.7 μM. MSF‐2 was found to suppress fMLP‐induced phosphorylation of on Akt and intracellular calcium mobilization, however, it was not observed to inhibit the phosphorylation of ERK and p38 induced by fMLP. Moreover, MSF‐2 inhibited the PDK1, which subsequently phosphorylated on Akt. This could be its underlying inhibitory mechanism of phosphorylation because. In addition, MSF‐2 demonstrated inhibitory effects on PI3K activity and it was found to inhibit phosphorylation of PLCγ2 which was activated by PI3K. MSF‐2 inhibited fMLP‐induced superoxide anion production by inhibiting fMLP‐induced Akt activation.

Ketamine reduces inducible superoxide generation in human neutrophilsin vitroby modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47(phox), one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl-methionyl-leucyl-phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 microM) reduced fMLP-induced superoxide anion generation and p47(phox) phosphorylation in neutrophils in a concentration-dependent manner (y = -0.093x + 93.35 for S(+)-ketamine and y = -0.0982x + 95.603 for R(-)-ketamine, respectively). While treatment with 50 microM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 microM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60.5 +/- 8.3% and 60.0 +/- 8.5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47(phox) and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47(phox) phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47(phox) phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.

Roles of phospholipase D in phorbol myristate acetate-stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10-fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA-stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA-stimulated respiratory burst. Using 1-butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA-stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA-stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA-stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA-stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2-dioctanoyl-sn-glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA-stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA-stimulated respiratory burst.

Effects of polychlorinated biphenyls on the neutrophil NADPH oxidase system

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.

Pentoxifylline attenuates fMLP‐induced oxidative burst in human neutrophils through PKA dependent and independent mechanisms: Effect on Ras, Raf, and MAPK activation

Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP‐induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the respiratory burst by both PKA dependent and independent mechanisms.Human PMNs (1 x 106 cells/sample) were isolated and stimulated with fMLP (1μmol/L) and PTX. PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580 10 mM), ERK 1/2 (PD98059 50 mM), and PKA inhibitors (H89 10 mM). Western blot analysis of Ras, c‐Raf, p38 MAPK, and ERK 1/2 was performed in PMNs exposed to fMLP and PTX.PTX produced a decrease in the reduction of cytochrome C that was diminished but not abrogated by H89 exposure. The reduction in Ras activation seen with PTX was not effected by the presence of H89. The attenuation in phosphorylation of c‐Raf p38 MAPK, and ERK 1/2 observed with PTX was only partially reversed with H89.PTX attenuates fMLP induction of oxidative burst through effects on Ras, c‐Raf, p38 MAPK, and ERK 1/2. The attenuation of early events by PTX in the cascade leading to production of the oxidative burst is PKA independent, while the effects of PTX on signaling distal to c‐Raf phosphorylation rely on both PKA‐dependent and independent mechanisms.Source of Support: Department of Surgery, University of California San Diego

Fungal Transformation of Dydrogesterone and Inhibitory Effect of Its Metabolites on the Respiratory Burst in Human Neutrophils

The biotransformation of the synthetic hormone dydrogesterone (1) by a number of fungal strains - including Cephalosporium aphidicola, Rhizopus stolonifer, Cunninghamella elegans, and Fusarium lini - afforded ten different metabolites, compounds 2-11. From a structural point of view, these transformations involved a combination of hydroxylation, oxidation, reduction, and/or epoxidation. The pregnane-based metabolites 10 and 11 are new compounds. All the known compounds were obtained for the first time from 1 by fungal transformation. The metabolites 3, 5, and 8 showed more-potent respiratory-burst inhibitory activity than the substrate 1 in a neutrophil-based cellular assay (Table 3).

Inhibition of respiratory burst in human neutrophils and lipoxygenase enzyme by compounds fromHaloxylon griffithii

Secondary metabolites, ferulic acid (1), 2,6-dimethoxy-4-hydroxy acetophenone (2), herniarin (3), p-hydroxy acetophenone (4), methyl 3,4-dihydroxycinnamate (5), and methyl 4-hydroxy-3-methoxycinnamate (6) were isolated from Haloxylon griffithii, a member of the family Chenopodiaceae. The structures of compounds 1-6 were identified with the help of spectroscopic techniques. These compounds were isolated for the first time from this plant. The lipoxygenase and respiratory burst inhibitory activities were determined. Compound 5 was found to be the most potent inhibitory activity against respiratory burst in human neutrophils among all the compounds as well as exhibited moderate lipoxygenase inhibitory activity from this plant.

Pityriarubins, Novel Highly Selective Inhibitors of Respiratory Burst from Cultures of the Yeast Malassezia furfur: Comparison with the Bisindolylmaleimide Arcyriarubin A

Pityriasis versicolor is the most common skin mycosis in humans worldwide. Yeasts of the genus Malassezia, particularly M. furfur, a saprophyte occurring widely on human skin, are generally regarded as the causative agents. M. furfur is able to convert tryptophan into a variety of indole alkaloids, some of them showing biological properties that correlate well with certain clinical features of pityriasis versicolor. This suggests a possible role for these compounds in the pathophysiology of the disease. We here report that the novel pityriarubins A, B and C, isolated from cultures of the yeast, inhibit respiratory burst in human neutrophils, activated by various agents, in a highly selective, unexpected manner. The release of 5-lipoxygenase products after challenge of neutrophils with the calcium ionophore A23187 is also inhibited in a dose-dependent manner. These activities reflect the close structural relationship of pityriarubins to bisindolylmaleimides, which have recently gained great interest as protein kinase inhibitors.

Non-specific effects of 4-chloro- m-cresol may cause calcium flux and respiratory burst in human neutrophils

We examined the effects of 4-chloro- m-cresol (4-CmC, a potent and specific activator of ryanodine receptors) on Ca 2+-release/influx and respiratory burst in freshly isolated human PMN as well as HL60 cells. 4-CmC induces Ca 2+ store-depletion in a dose-dependent manner at concentrations between 400 μM and 3 mM, however no dose-dependent effect on Ca 2+-influx was found. 4-CmC depleted Ca 2+ stores that were shared with the GPC agonists such as fMLP and PAF, and therefore 4-CmC presumably depletes Ca 2+ from ER. Since the authentic ligand for RyR is cyclic ADP-ribose (cADPR), we assessed the functional relevance of RyR in PMN by studying the presence and function of membrane-bound ADP-ribosyl cyclase (CD38) in PMN. First, expression of CD38 was confirmed by RT-PCR using cDNA from HL60 cells. Second, PMN from trauma patients showed significantly enhanced CD38 expression than those from healthy volunteers. In addition, although no chemotaxis effect was detected by 4-CmC, it stimulated respiratory burst in PMN in a dose-dependent manner. Our findings suggest that RyRs exist in human PMN and that RyR pathway may play an active role in inflammatory PMN calcium signaling. 8-Br-cADPR and cyclic 3-deaza-ADP did not have inhibitory effects either on 4-CmC-induced Ca 2+ store-depletion or on respiratory burst, on the other hand, PLC inhibitor, U73122, completely attenuated both 4-CmC-induced Ca 2+ store-depletion and respiratory burst. Although it has been used as a specific activator of RyR, 4-CmC has non-specific effects which cause Ca 2+ store-depletion and respiratory burst at least in human PMN.

Hypothalamic Proline-Rich Polypeptide Is a Regulator of Oxidative Burst in Human Neutrophils and Monocytes

Objective: The effects of proline-rich polypeptide (PRP) isolated from neurosecretory granules of bovine neurohypophysis produced by nuclei supraopticus and paraventricularis on phagocytosis, bacterial intracellular killing and oxidative burst induction in normal human cells and inflammatory cells from patients with Behçet’s disease (BD), i.e. peripheral blood neutrophils and monocytes, were investigated. Methods: Intracellular killing of Staphylococcus aureus by neutrophils and monocytes of normal controls and BD patients, phagocytic activity as well as spontaneous and N-formyl-Met-Leu-Phe (fMLP)- or phorbol 12-myristate 13-acetate (PMA)-induced activation of their respiratory burst were determined by quantitative flow cytometry using highly specific fluorescence probes. Results: PRP does not affect human peripheral blood neutrophil and monocyte phagocytosis but dramatically enhances spontaneous or fMLP- and PMA-induced oxidative burst as well as the intracellular killing of S. aureus. PRP induced the upregulation of the spontaneous or fMLP- and PMA-induced oxidative burst in normal PMNs and monocytes; the number of inflammatory BD cells did neither increase further nor undergo spontaneous or PMA-stimulated oxidative burst. In BD patients, increased spontaneous production of reactive oxygen intermediates (ROIs) by neutrophils and monocytes is characterized by impaired intracellular protein-kinase-C (PKC)-dependent oxidative burst regulation as well as overregulation of chemotaxis/inflammation-mediated respiratory burst induction. PRP restores rather the impaired intracellular PKC-dependent regulation of ROI production in inflammatory diseased cells than the chemotaxis/induction of the inflammation-mediated respiratory burst. Conclusion: We demonstrated the regulatory role for PRP on oxidative burst in neutrophils and monocytes from normal controls and BD patients. Our results suggest that PRP differentially affects both chemotaxis- and PKC-dependent oxidative burst in normal and inflammatory cells from patients.

Staphylococcus aureus strains that express serotype 5 or serotype 8 capsular polysaccharides differ in virulence.

Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5(+) strain showed a significantly higher bacteremia level than mice challenged with the CP8(+) strain. Similarly, the CP5(+) strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5(+) strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5(+) and CP8(+) strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.

Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes.

We have shown previously that in listeric encephalitis of cattle and rats, nitrotyrosine was produced in microabscesses, implying that both superoxide anion (O(2) (-)) and nitric oxide (NO) are present and react with each other. Evidence of local synthesis of NO by macrophages was provided, but the source of O(2) (-) remained unknown. Here we have examined whether phagocytes exposed to viable and heat-killed Listeria monocytogenes (LMDelta) produce O(2) (-) and, if so, whether this results from direct interaction of phagocytes with the bacterial surface of L. monocytogenes or whether prior opsonization is required. Using lucigenin-enhanced chemiluminescence (LCL) for the measurement of O(2) (-), we show that LMDelta induces an oxidative burst in human neutrophils, monocytes and monocyte-derived macrophages (Mphi). Viability is not required, and opsonization by antibodies and/or complement does not enhance the LCL signal. As Toll-like receptors (TLR) were shown recently to mediate an oxidative burst, TLR agonists representative for pathogen-associated molecular patterns (PAMPs) were tested for their ability to elicit an oxidative burst. These included lipoteichoic acid (LTA), bacterial peptidoglycan (PGN), recombinant flagellin, CpG-containing DNA and double-stranded RNA. Only PGN and flagellin consistently elicited an LCL signal resembling that induced by LMDelta with regard to the kinetics and cell spectrum stimulated. However, flagellin was unlikely to be responsible for the LMDelta-mediated burst, as a flagellin-deficient mutant showed no decrease in LCL. We therefore assume that in LMDelta, core PGN acts as a PAMP and directly induces an oxidative burst in all phagocyte populations. We conclude that in cerebral lesions superoxide anion is generated locally by phagocytes recognizing bacterial PGN.

Suppression of respiratory burst in human neutrophils by new synthetic pyrrolo-benzylisoquinolines

Reactive oxygen species produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, the inhibition of superoxide anion (O 2 −) generation in human neutrophils by new synthetic pyrrolo-benzylisoquinoline derivatives was determined. We found that KW-2, KW-5, and KW-7 (8,9-dimethoxyl-1-( R-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione; where R is 3-chloro, 3-bromo, and 4-methoxy, respectively) were the most effective inhibitors of formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP)-induced O 2 − release in human neutrophils. KW-2, KW-5, and KW-7 displayed no antioxidant or O 2 −-scavenging ability. The inhibition of O 2 − generation was reversed by the protein kinase (PK)A inhibitor, N-(2-(( p-bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide (H89), but not by the PKG inhibitor (8 R,9 S,11 S)-(−)-2-methyl-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta(cde)trinen-1-one (KT5823), or the soluble guanylate cyclase (sGC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). KW derivatives increased cellular cyclic AMP concentrations through the inhibition of phosphodiesterase (PDE) activity but not the elevation of adenylate cyclase (AC) activity. These results indicate that inhibition of FMLP-induced respiratory burst in human neutrophils by KW derivatives are cyclic AMP/PKA-dependent and are due to inhibition of PDE. The new chemical skeleton of PDE inhibitors may protect against the progression of inflammation.

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5 ′-AMP-activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5 ′-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or with 5 ′-AMP significantly attenuated both phorbol 12-myristate 13-acetate (PMA) and formyl methionyl leucyl phenylalanine-stimulated superoxide anion (O 2 − ) release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47 phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA-dependent H 2O 2 release, and induced the phosphorylation of c-jun N-terminal kinase 1 (p46), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5 ′-AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.

Attenuation of Inflammatory-induced Oxidative Burst in Human Neutrophils by Albuterol Is Enantiomer-specific

Attenuation of Inflammatory-induced Oxidative Burst in Human Neutrophils by Albuterol Is Enantiomer-specific

C242T CYBA Polymorphism of the NADPH Oxidase Is Associated With Reduced Respiratory Burst in Human Neutrophils

Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox-based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22phox is identical to neutrophil p22phox, we studied the association between the C242T, A640G, and -930A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7+/-0.7, 7.9+/-0.6, and 5.9+/-1.2 micromol/L per minute per 10(6) neutrophils for the C242T CC, CT, and TT genotypes, respectively (P<0.05). In contrast, the A640G and the -930A/G polymorphisms did not alter the neutrophil respiratory burst. Phagocytic respiratory burst activity in homozygous individuals with the T allele of the C242T CYBA polymorphism is significantly lower than of wild-type carriers and heterozygous individuals. Because p22phox exists in both the neutrophil and vessel wall, vascular oxidative stress is likely diminished in individuals with this polymorphism.

Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst in human neutrophils by adrenaline: inhibition of Phospholipase A 2 activity but not p47phox phosphorylation and translocation

The polymorphonuclear neutrophil (PMN)-respiratory burst plays a key role in host defense and inflammatory reactions. Modulation of this key neutrophil function by endogenous agents and the mechanisms involved are poorly understood. This study was designed to analyze the mechanisms involved in the effect of adrenaline on neutrophil superoxide anions production. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, we report here that the β-adrenergic agonist, adrenaline at physiologic concentrations (5–100 nM) inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated but not phorbol-myristate-acetate (PMA)-stimulated PMN superoxide anion production. The inhibitory effect of adrenaline runs in parallel with an increase in intracellular levels of cAMP which was reversed by the protein kinase A (PKA) inhibitor H-89, suggesting a role for PKA in mediating the inhibitory effect of adrenaline on fMLP-induced superoxide production. Adrenaline at physiological concentrations did not inhibit the fMLP-stimulated membrane translocation of the NADPH oxidase components p47phox and p67phox, nor the fMLP-stimulated phosphorylation of p47phox. However, adrenaline strongly depressed the activity of the cytosolic isoform of Phospholipase A 2 (cPLA 2). We suggest that adrenaline inhibits fMLP induced superoxide production upstream of the NADPH oxidase via a mechanism involving PKA and cPLA 2.