Superoxide In Response Research Articles

Long-Term Cyclic Strain Downregulates Endothelial Nox4

Endothelial cells in vivo are constantly exposed to mechanical forces such as cyclic strain. In endothelial cells, Nox4-containing NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analyzed the effect of cyclic strain on endothelial ROS formation by electron paramagnetic resonance spectroscopy, cytochrome c assay, and dihydroethidium fluorescence, on NO formation by Griess reaction and on gene expression by RT-PCR and Western blot. Primary cultures of human umbilical vein endothelial cells were exposed to 2-18% cyclic strain for up to 24 h using the Flexercell system. Long-term application of 5-12% cyclic strain downregulated Nox4 expression and ROS formation in a time-dependent manner. Downregulation of Nox4 was further confirmed by promoter analysis using dual-luciferase assay. Cu/Zn SOD, MnSOD, and catalase expression was decreased after application of chronic 12% cyclic strain. In contrast, endothelial NO formation and eNOS were increased by cyclic strain. Strain-dependent Nox4 downregulation was abolished by eNOS inhibition with L-NAME. In conclusion, physiological levels of cyclic strain downregulate Nox4 expression and superoxide anion formation. This novel mechanism might contribute to a vasoprotective balance between NO and superoxide anions in response to physiological mechanical stimulation of endothelial cells.

What happens with ageing: decline or remodeling of opsonin‐independent phagocyte oxidative activity?

Changes that occur with age in the opsonin-independent oxidative activity of peripheral phagocytes in whole blood were examined by means of luminol chemiluminescence (LCL). The chemiluminescence was registered simultaneously by non-stimulated and stimulated cells and the age-related alterations of total and extracellular generation of reactive oxygen species (ROS) were studied using model systems. It was found that the rate of phagocyte activation by the glass surface of the measuring chambers, assessed by the time of the peak appearance after the start of LCL response, increased. However, the maximum oxidative activity and the integral oxidative capacity of the cells during adhesion, evaluated by the maximum LCL intensity and the area under the LCL curve, respectively, declined. No age-dependence of formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated oxidative cellular activity for total ROS generation was detected. The maximum oxidative activity and the integral oxidative capacity of peripheral phagocytes to generate extracellular superoxide in response to fMLP was decreased. The likely causes for the observed alterations in phagocyte function are discussed and an analysis of the obtained results is given on the background of the contradictory data published on phagocyte oxidative activity age-related changes.

Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1-4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.

Colchicine suppresses neutrophil superoxide production in a murine model of gouty arthritis: a rationale for use of low‐dose colchicine

When used to treat gouty arthritis, colchicine is believed to work by inhibiting microtubule-dependent cell infiltration. However, in vitro, colchicine also reduces monosodium urate (MSU)-induced superoxide production by neutrophils. Our study aimed to compare the effects of colchicine on neutrophil superoxide production and infiltration in an in vivo model of acute gouty inflammation. In vitro: Human and murine peritoneal neutrophils were incubated with MSU with and without colchicine, and superoxide production was measured. In vivo: Mice were treated with colchicine followed by an intraperitoneal injection of MSU to induce acute inflammation. After 4h, the peritoneal cells were recovered to measure superoxide production and neutrophil infiltration. Sera were tested for liver and renal toxicity. Colchicine dose-dependently inhibited MSU-induced superoxide production by both human and murine neutrophils in vitro. Oral colchicine inhibited MSU-induced superoxide production by neutrophils in vivo at doses 100 times lower than those required to inhibit neutrophil infiltration and without acute liver or renal toxicity. Neutrophils treated with colchicine in vivo still produced superoxide in response to another stimulus, 4-beta-phorbol-12-myristate-13-acetate. These results show a beneficial effect of colchicine for the treatment of MSU-induced superoxide production in vivo at sub-toxic doses without compromising superoxide production by other physiological processes. This is the first in vivo data to provide a biological rationale that supports the implementation of low dose, non-toxic, colchicine therapy for the treatment of gouty arthritis.

Staphylococcus and the Healing Power of Pus

Staphylococcus aureus infections induce formation of neutrophil-rich abscesses filled with debris from dead phagocytes. Corbin and colleagues report that this pus has antimicrobial powers through the activity of calprotectin. Calprotectin, a member of the S100 family of proinflammatory proteins, acts through chelation of manganese. As manganese is an essential cofactor for several enzymes in S. aureus, this impacts bacterial growth and the bacterium's ability to withstand oxidative stress.

Age-Related Changes in Eosinophil Function in Human Subjects

Aging results in changes in immune cell function that have been described for T-cells, macrophage, neutrophils, and dendritic cells but not for eosinophils. We sought to define age-related changes in eosinophil function and their potential implications for asthma. We recruited human subjects with asthma in two age groups: a younger group (20 to 40 years), and an older group (55 to 80 years). Lung function, induced sputum, and peripheral blood were obtained from each subject. Eosinophils isolated from the peripheral blood were examined for in vitro functional activities including degranulation, superoxide anion production, adhesion, and chemotaxis. Eosinophil degranulation in response to interleukin-5 stimulation was significantly decreased in the older group (p = 0.025). Eosinophil production of superoxide anions in response to phorbol myristate acetate was lower in the older group but did not achieve statistical significance (p = 0.097). Eosinophil adhesion, eosinophil chemotaxis, lung function, and the percentage of sputum eosinophils were similar in the two groups. Airway eosinophilia is comparable in younger and older asthma subjects. However, there are age-related changes in peripheral blood eosinophil "effector" functions. Diseases such as asthma, in which eosinophils are thought to play a pathophysiologic role, may exhibit important clinical differences in the elderly due to age-related changes in inflammatory cell function that affect the manifestations of the disease and/or responsiveness to specific classes of medications.

20-Hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.

Implication of cytosolic phospholipase A 2 (cPLA 2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A 2 (cPLA 2) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA 2 inhibitors prior to IL-1β or TNF-α treatment. Total cellular AA concentrations and PLA 2 activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA 2 inhibitors inhibited both IL-1β and TNF-α-induced superoxide production in RA and OA cells. Basal PLA 2 activity was significantly more important in RA cells than in OA cells; PLA 2 activity was increased in IL-1β and TNF-α pre-treated RA cells, and cPLA 2 inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1β and TNF-α, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47- phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H + channel.

Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers

During 30-months of storage at 4 degrees C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18-24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca(+2) declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25-30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.

NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca 2+ elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca 2+ ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower (∼70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca 2+ activation.

Estrogen-TNF interactions and vascular inflammation

cardiovascular disease (CVD), especially coronary heart disease (CHD), is by far the leading cause of death in women ([29][1], [40][2]). As the prevalence of these diseases increases after menopause, much effort has been devoted to determining whether endogenous estrogen confers a protective effect

NADPH oxidase but not myeloperoxidase protects lymphopenic mice from spontaneous infections

The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91 phox-deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other’s deficiency in providing resistance to spontaneous bacterial infections.

Nitric Oxide Generation by NO Donors Is Enhanced Following Balloon Injury in the Porcine Coronary Artery

Vasospasm is a complication of cardiological procedures such as balloon angioplasty and may be related to vascular oxidant stress. Although nitric oxide donor drugs are often administered to prevent vasospasm, the response to these drugs in balloon-injured arteries has not been studied. Pig coronary arteries were balloon-injured in vitro and relaxations to nitric oxide (NO)-donating and NO-independent vasodilators studied. Generation of superoxide in response to injury was assayed using dihydroethidium. NO formation on addition of the NO donor drugs was studied using an amperometric sensor. Expression of nitrotyrosine, a peroxynitrite marker, was probed using immunocytochemistry. In vitro injury enhanced sensitivity to the NO donors SNAP and SpermineNONOate but blunted the response to isoprenaline or chromakalim. With both donors, NO formation was significantly enhanced in the presence of an injured vessel. Vascular superoxide generation was also increased throughout the vessel wall and a small increase in nitrotyrosine was detected in the injured vessel media following addition of SNAP. In conclusion, injured vessels were more sensitive to NO donors and this appears to be due to enhanced NO generation by the donor molecule. Increased formation of peroxynitrite within the injured vessel may contribute to the enhanced relaxation in injured vessels.

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1β and tumour necrosis factor-α

to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), and to study the NADPH oxidase involvement in this production. Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1beta and TNF-alpha, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4).

Simultaneous measurement of superoxide generation and intracellular Ca 2+ concentration reveals the effect of extracellular Ca 2+ on rapid and transient contents of superoxide generation in differentiated THP-1 cells

We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca 2+ concentration ([Ca 2+]i) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca 2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated superoxide response occurred without change in [Ca 2+]i. The cells treated with TMB-8, an intracellular Ca 2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca 2+]i elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca 2+]i elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca 2+ influx from the extracellular domain. Verapamil, voltage-dependent Ca 2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca 2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca 2+ release or extracellular Ca 2+ influx, respectively. Taking these results together, the characteristic extracellular Ca 2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca 2+]i.

Cutting Edge: MyD88 Controls Phagocyte NADPH Oxidase Function and Killing of Gram-Negative Bacteria

MyD88 is an adaptor protein for the TLR family of proteins that has been implicated as a critical mediator of innate immune responses to pathogen detection. In this study, we report that MyD88 plays a crucial role in killing Gram-negative bacteria by primary macrophages via influencing NADPH oxidase function. Peritoneal macrophages from MyD88-/- mice exhibited a marked inability to kill Escherichia coli (F18) or an attenuated strain of Salmonella typhimurium (sseB) in vitro. This defect in killing was due to diminished NADPH oxidase-mediated production of superoxide anion in response to bacteria by MyD88-/- phagocytes as a consequence of defective NADPH oxidase assembly. Defective oxidase assembly in MyD88-deficient macrophages resulted from impaired p38 MAPK activation and subsequent phosphorylation of p47phox. Together these data demonstrate a pivotal role for MyD88 in killing Gram-negative bacteria via modulation of NADPH oxidase activity in phagocytic cells.

Effects of uraemia and dialysis modality on polymorphonuclear cell apoptosis and function

Previous studies have reported that incubation of polymorphonuclear cells (PMN) in uraemic plasma or with different haemodialysis membranes and peritoneal dialysis solutions increases apoptosis in this cell type. In addition, PMN harvested from uraemic patients show a reduced ability to generate superoxide in response to stimuli as well as impaired phagocytosis, chemotaxis and degranulation. The aim of the current study was to investigate the effect of uraemia and dialysis modality on apoptosis and function in freshly harvested non-incubated PMN. Polymorphonuclear cells were harvested from 14 chronic haemodialysis (HD) patients, from 14 continuous peritoneal dialysis patients (CAPD), 28 chronic kidney disease (CKD), pre-dialysis patients and from 14 healthy subjects (Controls). In these in vivo experiments, PMN apoptosis was studied by means of flow cytometric analysis of annexin V binding to freshly isolated cells. Polymorphonuclear cell phagocytosis and production of reactive oxygen species by unstimulated or stimulated (S.aureus, fMLP, PMA) cells were also studied by flow cytometry using whole blood. We observed increased PMN apoptosis in CKD patients. CAPD and HD patients displayed PMN apoptosis rates similar to controls. In the HD group, PMN exhibited decreased phagocytosis rates. In contrast, phagocytosis rates in PMN from CAPD were not significantly different from controls. In the CKD and HD groups, apoptosis was inversely correlated with respiratory burst activity and phagocytosis. Our results suggest that both uraemia and treatment modality may interfere with PMN apoptosis and function. Dialysis appears to normalize the increased PMN apoptosis rates observed in CKD patients.

Microglial NADPH Oxidase Mediates Leucine Enkephalin Dopaminergic Neuroprotection

Abstract: Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti‐inflammatory properties. Mesencephalic neuron‐glia cultures pretreated with femtomolar concentrations of LE (10−15‐10−13 M) protected DA neurons from lipopolysaccharide (LPS)‐induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des‐tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10−15‐10−13 M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10−15‐10−13 M) reduced LPS‐induced superoxide production from microglia‐enriched cultures. Further, both LE and DTLE (10−14, 10−13 M) reduced the LPS‐induced tumor necrosis factor‐alpha (TNFα) mRNA and TNFα protein from PHOX+/+ microglia, as determined by quantitative real‐time RT‐PCR and ELISA analysis in mesencephalic neuron‐glia cultures, respectively. However, both peptides failed to inhibit TNFα expression in PHOX−/− cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10−14, 10−13 M) failed to show any neuroprotection against LPS in PHOX−/− cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS‐mediated amplification of TNFα gene expression in microglia.

Microglial NADPH Oxidase Mediates Leucine Enkephalin Dopaminergic Neuroprotection

Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti-inflammatory properties. Mesencephalic neuron-glia cultures pretreated with femtomolar concentrations of LE (10(-15)-10(-13) M) protected DA neurons from lipopolysaccharide (LPS)-induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des-tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10(-15)-10(-13) M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10(-15)-10(-13) M) reduced LPS-induced superoxide production from microglia-enriched cultures. Further, both LE and DTLE (10(-14), 10(-13) M) reduced the LPS-induced tumor necrosis factor-alpha (TNFalpha) mRNA and TNFalpha protein from PHOX+/+ microglia, as determined by quantitative real-time RT-PCR and ELISA analysis in mesencephalic neuron-glia cultures, respectively. However, both peptides failed to inhibit TNFalpha expression in PHOX-/- cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10(-14), 10(-13) M) failed to show any neuroprotection against LPS in PHOX-/- cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS-mediated amplification of TNFalpha gene expression in microglia.

Effects of p47phox C Terminus Phosphorylations on Binding Interactions with p40phox and p67phox: STRUCTURAL AND FUNCTIONAL COMPARISON OF p40phox and p67phox SH3 DOMAINS

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.