Role of ascorbate in the regulation of nitric oxide generation by polymorphonuclear leukocytes

We have recently demonstrated that NO-mediated polymorphonuclear (PMN)-dependent inhibition of rat platelet aggregation is significantly enhanced in the presence of ascorbate. Consequently, the present study was undertaken to elucidate the underlying mechanisms involved in ascorbate-mediated potentiation of NO synthesis in PMNs. We observed that ascorbate or its oxidized product, dehydroascorbate (DHA), enhanced NOS activity, as measured by nitrite content, diaminofluorescein fluorescence or conversion of L-[3H]arginine to L-[3H]citrulline in rat, monkey, and human PMNs. The increase in NO generation following ascorbate treatment was due to the intracellular ascorbate as iodoacetamide-mediated inhibition of DHA to ascorbate conversion attenuated the DHA-mediated increase in NO synthesis. The augmentation of NOS activity in the PMN homogenate by tetrahydrobiopterin was significantly enhanced by ascorbate, while ascorbate alone did not influence the NOS activity. Ascorbate-mediated enhancement of NOS activity in the cultured PMNs was significantly reduced in the presence of biopterin synthesis inhibitors. Ascorbate, thus, seems to regulate the NOS activity in the PMNs through tetrahydrobiopterin.     

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.     

Augmented nitric oxide generation in neutrophils: Oxidative and pro-inflammatory implications in hypertension

Abstract

The present study explores expression of NOS and pro-inflammatory cytokines, NOS catalysis and NO mediated modulation of oxidative response and apoptosis in neutrophils from spontaneously hypertensive rats (SHR). Neutrophils from SHR showed ～3-fold increments in iNOS expression, 1.5-fold increments in nOS expression and calcium independent NOS catalysis, whereas GTPCH expression was unaltered. Although phagocytic potential was comparable, neutrophils from SHR demonstrated augmented oxidative burst, which was reduced by NOS inhibition or in the presence of NO scavenger. SHR neutrophils also exhibited enhanced MPO catalysis and [Ca²⁺]_i levels. Levels of TNF-α and IFN-γ were comparable, but IL-1β and CRP levels in SHR plasma were (p<0.05) elevated. This study evidenced significantly enhanced expression of IL-1β in SHR neutrophils whereas those of TNF-α and IFN-γ were unaltered. Moreover neutrophils from SHR exhibited (p<0.01) delayed apoptotic response and sustained NO generation, as evident from elevated nitrite levels in neutrophil culture supernatant above the control levels. Results obtained indicate an augmented NO generation from neutrophils during hypertension which might fortify their attribute to the oxidative and inflammatory stress in SHR, emphasizing the importance of neutrophils in hypertension.     

Phagocytosis, Oxidative Burst, and Produced Reactive Species are Affected by Iron Deficiency Anemia and Anemia of Chronic Diseases in Elderly

Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide (^?NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p?<?0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p?<?0.05). There was an overproduction of ^?NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and ^?NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.     

L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.     

Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain

The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 m M of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate production. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells – presumably complex II – plays important role in the regeneration of ascorbate from its oxidized form, DHA.     

Rooting hastened in onions by ascorbate and ascorbate free radical

Treatment of onion bulbs with ascorbate or its free radical hastened root emergence on the basal plate in relation to treatments with water or dehydroascorbate. This stimulation was accompanied by a significant increase of DNA synthesis per primordium. After a 24-h imbibition, ascorbate and ascorbate free radical also increased cell length. Ascorbate and ascorbate free radical apparently activated the onset of cell proliferation in root primordia, resulting in a shortening in G₁-S transition. The possible action of the ascorbate system at the plasma membrane level is discussed.Abbreviations ASC ascorbic acid - AFR ascorbate free radical - DHA dehydroascorbate     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation

Previous studies have shown that a Ca(2+)-dependent nitric-oxide synthase (NOS) is activated as part of a cellular response to low doses of ionizing radiation. Genetic and pharmacological inhibitor studies linked this NO signaling to the radiation-induced activation of ERK1/2. Herein, a mechanism for the radiation-induced activation of Tyr phosphorylation-dependent pathways (e.g. ERK1/2) involving the inhibition of protein-Tyr phosphatases (PTPs) by S-nitrosylation is tested. The basis for this mechanism resides in the redox-sensitive active site Cys in PTPs. These studies also examined oxidative stress induced by low concentrations of H(2)O(2). S-Nitrosylation of total cellular PTP and immunopurified SHP-1 and SHP-2 was detected as protection of PTP enzymatic activity from alkylation by N-ethylmaleimide and reversal by ascorbate. Both radiation and H(2)O(2) protected PTP activity from alkylation by a mechanism reversible by ascorbate and inhibited by NOS inhibitors or expression of a dominant negative mutant of NOS-1. Radiation and H(2)O(2) stimulated a transient increase in cytoplasmic free [Ca(2+)]. Radiation, H(2)O(2), and the Ca(2+) ionophore, ionomycin, also stimulated NOS activity, and this was associated with an enhanced S-nitrosylation of the active site Cys(453) determined by isolation of S-nitrosylated wild type but not active site Cys(453) --> Ser SHP-1 mutant by the "biotin-switch" method. Thus, one consequence of oxidative stimulation of NO generation is S-nitrosylation and inhibition of PTPs critical in cellular signal transduction pathways. These results support the conclusion that a mild oxidative signal is converted to a nitrosative one due to the better redox signaling properties of NO.     

Nutrient Uptake Changes in Ascorbate Free Radical-Stimulated Onion Roots

Long-term treatments with ascorbate free radical-stimulated glucose, fucose, sucrose, and nitrate uptake in Allium cepa roots. Glucose and fucose showed saturation kinetics in untreated roots, but after treatment with the ascorbate free radical, uptake was linear with time. Although the rates of nitrate and sucrose uptake increased after treatment with ascorbate free radical, the kinetics were similar to those observed in the controls. Ascorbate and dehydroascorbate inhibited nutrient uptake. The uptake rates for all nutrients increased throughout the 48-h period of pretreatment with ascorbate free radical. During the treatment an increase in the vacuole volume and tonoplast surface area also occurred. These results show the relationship between an increase in vacuolar volume and stimulated nutrient uptake from ascorbate-free radical, resulting in enhanced root elongation. These results suggest that activation of a transplasma membrane redox system by ascorbate-free radical is involved in these responses.     

Tetrahydropteridines suppress gene expression and induce apoptosis of activated RAW264.7 cells via formation of hydrogen peroxide

Tetrahydrobiopterin, a redox-active cofactor, is essential for nitric oxide (NO) biosynthesis. Previous work showed that intracellular tetrahydrobiopterin levels modulate activity of nitric oxide synthases (NOSs). The 4-amino analog of tetrahydrobiopterin is an effective inhibitor of all three purified NOS isoforms that, in intact cells, preferentially targets the inducible isoenzyme. In vivo, 4-amino-tetrahydrobiopterin prolonged allograft survival and rescued rats from septic shock. Here we investigated the effects of tetrahydrobiopterin and its 4-amino analog on RAW264.7 murine macrophages activated with lipopolysaccharide. Surprisingly, both tetrahydropteridines inhibited NO formation. This was caused by downregulation of inducible NOS expression rather than by affecting enzyme activity. In addition, expression of tumor necrosis factor-alpha was impaired, and apoptosis, as characterized by quantifying DNA content and caspase-3 activation and being associated with the formation of a 33 kDa fragment of nuclear factor-kappaB p65, was induced. The effects of tetrahydropteridines were scavenged by catalase or glutathione but not by superoxide dismutase. Like tetrahydropteridines, hydrogen peroxide at concentrations comparable to those found in tetrahydropteridine-treated cultures affected gene expression and cell survival, whereas increasing intracellular tetrahydrobiopterin levels by sepiapterin did not. Thus, extracellular tetrahydropteridines suppress gene expression and induce apoptosis in RAW264.7 cells via hydrogen peroxide formed in the culture medium during autoxidation.     

Nitric oxide- and oxygen-derived free radical generation from control and lipopolysaccharide-treated rat polymorphonuclear leukocyte.

Previous studies from this lab have shown NO-mediated modulation of free radical generation from polymorphonuclear leukocytes (PMNs), following hypoxic-reoxygenation as well as in the normoxic cells. The present study is an attempt to investigate further the regulation of NO and free radical generation in the lipopolysaccharide (LPS)-treated PMNs. PMNs were isolated from the rat blood and peritoneal cavity, 4 h after LPS (1 mg/kg, i.p.) treatment. Nitric oxide synthase (NOS) activity and nitrite content were increased in the peripheral and peritoneal PMNs following LPS treatment. An increase in the apparent V(max) for l-arginine uptake was also observed in the LPS-treated peripheral PMNs, while peritoneal PMNs exhibited increase in both apparent V(max) and affinity for l-arginine. Synthesis of nitrite did not augment after increasing the availability of substrate to control PMNs, however, peripheral and peritoneal PMNs from LPS-treated rats utilized l-arginine more efficiently for nitrite synthesis. NOS activity, l-arginine uptake, and its utilization were maximal in the peritoneal PMNs. Arachidonic acid (AA, 1 x 10(-6) M)-induced free radical generation from PMNs was also enhanced significantly after LPS treatment. Preincubation of PMNs with nitrite elevated the free radical generation and myeloperoxidase (MPO) release. MPO and antioxidant enzyme activity in the PMNs was significantly augmented after LPS treatment. NOS inhibitors, aminoguanidine and 7-nitroindazole, inhibited arachidonic acid-induced free radical generation from LPS treated PMNs. The results obtained thus indicate that augmentation of free radical generation from rat PMNs following LPS treatment appears to be regulated by NO and MPO.     

Activation energy determinations suggest that thiols reverse autooxidation of tetrahydrobiopterin by a different mechanism than ascorbate

In neutral aqueous solutions tetrahydrobiopterin is oxidized by dioxygen in a reaction that is succinctly described as autooxidation. Ascorbate and thiols moderate this reaction by reversing the oxidative process. In the present study the effect of various thiols on the apparent Arrhenius activation energy of tetrahydrobiopterin autooxidation was characterized and compared to that of ascorbate determined previously. We observed that - in sharp contrast to ascorbate - the efficiency of thiols to protect tetrahydrobiopterin decreased with the elevation of temperature from 22 to 37 degrees C. Accordingly, the apparent Arrhenius activation energies (in kJ/mol) measured in the presence of thiols were consistently greater than the value determined with tetrahydrobiopterin alone (59.6 +/- 1.4) or in the presence of ascorbate (59.9 +/- 2.8). Thus, the energy values were 88.8+/-1.1 with glutathione, 87.6 +/- 2.1 with N-acetylcysteine, 79.2 +/- 1.6 with cysteine, 75.1 +/- 2.4 with dithiotreitol and 70.3 +/- 0.9 with homocysteine. Since thiols are as potent reducing agents as ascorbate, these findings suggest that thiols and ascorbate protect tetrahydrobiopterin from oxidation acting at different steps of the oxidation process. It is likely that thiols reduce quinoidal dihydrobiopterin, whereas ascorbate scavenges the trihydrobiopterin radical to tetrahydrobiopterin. Furthermore, the results indicate that thiols are excellent tools to protect tetrahydrobiopterin from autooxidative decomposition in laboratory experiments conducted at relatively low temperatures, whereas the protective effect diminishes at 37 degrees C, i.e. under physiological conditions.     

Radical reactions of nitric oxide synthases

NOSs (nitric oxide synthases) are flavohaem enzymes that function broadly in human health and disease. We are combining mutagenesis, crystallographic and rapid kinetic methods to understand their mechanism and regulation. The NOSs create a transient tetrahydrobiopterin radical within the enzyme to generate their free radical product (NO). Recent work is revealing how critically important this process is at all levels of catalysis. This article will synthesize four seemingly disparate but related aspects of NOS tetrahydrobiopterin radical formation: (i) how it enables productive O2 activation by providing an electron to the enzyme haem, (ii) what structural features help to regulate this electron transfer, (iii) how it enables NOS to synthesize NO from its diamagnetic substrate and (iv) how it allows NOS to release NO after each catalytic cycle instead of other nitorgen oxide-containing products.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

The phosphatidylinositol 3-kinase signaling pathway exerts protective effects during sepsis by controlling C5a-mediated activation of innate immune functions

The PI3K/Akt signaling pathway has been recently suggested to have controversial functions in models of acute and chronic inflammation. Our group and others have reported previously that the complement split product C5a alters neutrophil innate immunity and cell signaling during the onset of sepsis and is involved in PI3K activation. We report in this study that in vivo inhibition of the PI3K pathway resulted in increased mortality in septic mice accompanied by strongly elevated serum levels of TNF-alpha, IL-6, MCP-1, and IL-10 during sepsis as well as decreased oxidative burst activity in blood phagocytes. PI3K inhibition in vitro resulted in significant increases in TLR-4-mediated generation of various proinflammatory cytokines in neutrophils, whereas the opposite effect was observed in PBMC. Oxidative burst and phagocytosis activity was significantly attenuated in both neutrophils and monocytes when PI3K activation was blocked. In addition, PI3K inhibition resulted in strongly elevated TLR-4-mediated generation of IL-1beta and IL-8 in neutrophils when these cells were co-stimulated with C5a. C5a-induced priming effects on neutrophil and monocyte oxidative burst activity as well as C5a-induced phagocytosis in neutrophils were strongly reduced when PI3K activation was blocked. Our data suggest that the PI3K/Akt signaling pathway controls various C5a-mediated effects on neutrophil and monocyte innate immunity and exerts an overall protective effect during experimental sepsis.     

Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis.

There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, NG-nitro-L-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L-arginine, suggesting an involvement of the L-arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS(-/-)) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS(-/-) mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS(-/-) mice, in which these mechanisms were potentiated and reverted by nitro and L-arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils.