Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst

Free Radical Biology & Medicine 45 (2008) 1084–1093

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst Madhumita Chatterjee a, Rohit Saluja a, Vipul Kumar b, Anupam Jyoti a, Girish Kumar Jain b, Manoj Kumar Barthwal a, Madhu Dikshit a,⁎ a b

Cardiovascular Pharmacology Unit, Division of Pharmacology, Central Drug Research Institute, Mahatma Gandhi Road, 226001 Lucknow, India Division of Pharmacokinetics and Metabolism, Central Drug Research Institute, Mahatma Gandhi Road, 226001 Lucknow, India

a r t i c l e

i n f o

Article history: Received 28 February 2008 Revised 11 June 2008 Accepted 25 June 2008 Available online 3 July 2008 Keywords: Scorbutic guinea pigs Neutrophils Nitric oxide Nitric oxide synthase Biopterin Phagocytosis Oxidative burst Free radicals

a b s t r a c t Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils. © 2008 Elsevier Inc. All rights reserved.

Neutrophils, as forerunners, provide a prompt immune response to pathogenic intrusion, inflicting the onset of inflammation and subsequently instigating a specific response from the adaptive immune brigade. These phagocytic granulocytes are one of the most vital depots for storing high concentrations of ascorbate [1–4], and activated neutrophils dynamically recycle it in the form of its oxidized derivative, dehydroascorbic acid (DHA) [1–5]. Ascorbate seemingly might be utilized to combat the oxidative load that these granulocytes are subjected to, or to quench the diffused extracellular oxidants from activated neutrophils into the extracellular milieu. Though a globally acclaimed antioxidant, ascorbate has been revealed to contribute to the generation of free radicals under both physiological and nonphysiological conditions [6–10], provoking quite a lot of controversy by posing the challenge of being a friend and at times a foe. Recently Chen et al. [10] have provided evidence that ascorbate is capable of generating ascorbate radical and hydrogen peroxide in the extracellular space in vivo and of its implication as a prodrug in cancer therapy. The beneficial role of ascorbate in supplementing the immune response has also long been suggested and variously demonstrated in

Abbreviations: NOS, nitric oxide synthase; BH4, tetrahydrobiopterin; DAF-2DA, diaminofluorescein diacetate; DCFDH2-DA, dichlorodihydrofluorescein diacetate; DHE, dihydroethidium; DPI, diphenyleneiodonium chloride; 7-NI, 7-nitroindazole. ⁎ Corresponding author. Fax: +91 5222623938. E-mail address: [email protected] (M. Dikshit). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.06.028

clinical studies as well as in in vitro laboratory explorations in animal models [11–13]. Overwhelming bacterial infection is a major cause of death in hospitalized patients, and ascorbate deficiency is common in these cases [14]. A new era in ascorbate biology has evolved, interpreting its contribution in influencing NOS activity [15–20] and also nonenzymatic generation of nitric oxide (NO) from its metabolites [7]. Ascorbate serves as one of the crucial plasma components causing release of NO from S-nitroso–albumin and S-nitroso-glutathione [8], the prominent NO reservoirs in plasma, to be implemented in antiplatelet and relaxant activities. Several in vitro studies have recorded that ascorbate augments endothelial (e) NOS activity in endothelial cells by ensuring the stabilization of tetrahydrobiopterin (BH4) [15–18] and boosts inducible (i) NOS activity in macrophages without altering the level of NOS expression [19,20]. It also exerts its overwhelming reduction potential to scavenge O2 −, and thereby prolong the availability of NO generated from the aortic endothelium under hypertensive conditions, and ameliorate NO-induced oxidative stress in endothelial cells [17,18]. A sustained level of NO generation in the presence of ascorbate also enables NO-mediated feedback regulation, decreasing NOS activity in endothelial cells [18]. NO as a multifaceted signal transducer modulates several functional aspects of neutrophils under diverse pathophysiological conditions [21]. Previous studies from this lab have documented an enhanced NO generation and respiratory burst potential in rat, monkey, and human neutrophils after in vitro treatment with ascorbate [22,23].

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Furthermore we also reported that ascorbate enhances NO-mediated inhibition of platelet aggregation in the presence of neutrophils [24]. These outcomes led us to explore the scenario in an in vivo situation during ascorbate deficiency in scorbutic guinea pigs. This study explores the means of NOS and oxidative burst regulation as offered by ascorbate in neutrophils and thereby defines a purpose for its enormous deposits in these granulocytes. Materials and methods

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reagent. For ascorbate uptake studies neutrophils (2 × 106) were treated with 1 mM ascorbate for 30–40 min after which the neutrophils were washed and lysed by sonication. Neutrophil lysate and plasma were treated with 10% trichloroacetic acid to precipitate the proteins and centrifuged at 13,000 rpm for 20 min. The supernatant was treated with DTC and incubated at 37°C for 3 h with constant shaking; 65% chilled sulfuric acid was added to the reaction system and it was further incubated at room temperature for 30 min [28]. Optical density readings were recorded for each sample at 520 nm on a spectrophotometer (UV 1201; Shimadzu, Japan).

Chemicals Estimation of tetrahydrobiopterin in neutrophils Dextran, Histopaque 1083 and 1119, L-ascorbic acid (Asc), DHA, 2,4, dinitrophenylhydrazine, diaminofluorescein diacetate (DAF-2DA), dichlorodihydrofluorescein diacetate (DCFDH2-DA), dihydroethidium (DHE), nitroblue tetrazolium (NBT), diphenyleneiodonium chloride (DPI), 7-nitroindazole (7-NI), D-glucose, iodoacetamide, ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N-tetraacetic acid (EGTA), BAPTA-AM, fMLP, flavin adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), Dowex 50WX (T-400), N-naphthyl ethylenediamine, sulfanilamide, orthophosphoric acid, cadmium pellets, and Trizol reagent were purchased from Sigma–Aldrich (USA), calmodulin was from Calbiochem (USA), and L-[3H]arginine was procured from Amersham Biosciences (UK). BH4, nicotinamide adenine dinucleotide phosphate (NADPH), and dithiothreitol (DTT) were from RBI (USA), iodine was from Qualigens (India), potassium iodide was from Merck (Germany), and RevertAid H− First-Strand cDNA Synthesis Kit was from Fermentas Life Sciences (Canada). Animals Male English albino guinea pigs were procured from the national animal house facility of the Central Drug Research Institute and scorbutic models were prepared by providing an ascorbic-aciddeficient diet for a period of 2 weeks, whereas the control set was maintained on a normal guinea pig diet consisting of grass and green vegetables supplemented with ascorbate at a recommended dose of 30 mg/animal/day [25]. Symptoms of weight loss and difficulties in movement were observed among the scorbutic models within the treatment period. All animal experimentations were approved and performed as per the ethical guidelines of the institute. Preparing stock solutions of ascorbate and dehydroascorbate

BH4 stock solution was prepared fresh in 0.1 N HCl supplemented with 1 mM DTT. BH4 content in neutrophils was estimated by subjecting neutrophil lysates (2 × 107 cells) to acidic and alkaline oxidation [29–32]. The fluorescent derivatives were analyzed by fluorescence detection in RP-HPLC using a Lichrosphere RP-18 column (Merck), with 50 mM ammonium acetate (pH 3.5) as mobile phase at the rate of 0.8 ml/min and run for 30 min. Concentrations in the biological samples were depicted from the standard BH4 subjected to acidic or alkaline oxidation. NO generation by flow cytometry NO generation in neutrophils (2 × 106cells/ml) was measured by using the cell-permeable dye DAF-2DA (5 μM). The cells were incubated for 5 min with the dye at 37°C and further incubated in the presence of ascorbate (1 mM) and DHA for 30 min. Samples were acquired on a flow cytometer (Becton–Dickinson, FACSCaliber with 488 nm argon laser) and mean fluorescence intensity was recorded for at least 10,000 neutrophils [17,18]. Data were analyzed using CellQuest Pro software. To ensure that the ascorbate-mediated effect on NO generation potential was due to NOS activity and not solely due to the interaction of DHA and ascorbate with DAF as reported earlier, we verified ascorbate-mediated effects in the presence of the NOS inhibitor 7-NI (1 mM). In these sets neutrophils were preincubated with 7-NI before the incubation with the dye and subsequently with ascorbic acid [22,23]. NOS activity in neutrophil lysates using L-[H3]arginine NOS activity was assessed by the formation of L-[3H]citrulline from 7 L-[ H]arginine as reported earlier [17]. Neutrophils (2 × 10 cells) were sonicated in incubation buffer (Hepes 25 mM, NaCl 140 mM, KCl 5.4 mM, MgCl2 1 mM, pH 7.4) and incubated in the presence of cofactors BH4 (10 μM/100 μM), NADPH (1 mM), FAD (5 μM), FMN (25 μM), and calmodulin (10 μg/ml). Neutrophil lysates were also treated with ascorbate (300 μM/1 mM) and calcium chloride (2 mM) and/or EGTA (5 mM) in the specified reaction systems. Reaction was initiated by the addition of L-[3H]arginine, continued for 30 min at 37°C, and stopped by the addition of ice-cold stop buffer (NaCl 118 mM, KCl 4.7 mM, KH2PO4 1.18 mM, NaHCO3 1 mM, EDTA 4 mM, Nω-nitro-L-arginine methyl ester 2 mM, pH 5.5). The mixture was passed through Dowex 50WX (T-400) columns. Radioactivity in the eluent was measured using a β-scintillation counter (LKB Wallace; liquid scintillation counter). NO synthesis in the neutrophils is reported as pmol L-[3H]citrulline/30 min/107 cells [27]. 3

Ten millimolar stock solutions of L-ascorbic acid and DHA were prepared fresh in Milli-Q water just before the experiment as described earlier [23]. Isolation of peripheral blood neutrophils Peripheral blood neutrophils were isolated as reported earlier [26,27]. Briefly, blood was collected by cardiac puncture and subjected to dextran sedimentation to remove the majority of erythrocytes, followed by differential centrifugation through Histopaque gradients 1119 and 1083. Neutrophils were collected from the interface of the two gradients, washed twice, and finally resuspended in Hanks' balanced salt solution (HBSS) (NaCl, 138 mM; KCl, 2.7 mM; Na2HPO4, 8.1 mM; KH2PO4, 1.5 mM; and diethylene triaminepentaacetic acid 0.1 mM; pH 7.4). Cell viability was estimated by Trypan blue (2 mg/ml) exclusion assay and always found to be N98%. Estimation of ascorbic acid content in plasma and neutrophils Ascorbic acid content in neutrophils (2 × 106) and plasma (500 μl) was estimated using DTC (3.0 g of 2,4-dinitrophenylhydrazine +0.4 g of thiourea +0.05 g copper sulfate to 100 ml of 9 N sulfuric acid)

iNOS and nNOS expression in neutrophils assayed by RT-PCR RNA was isolated from neutrophils (1 × 107) by Tri reagent (Sigma) as per the manufacturer's instructions. Total RNA (5 μg) was reverse transcribed with MMLV reverse transcriptase using random hexamer primers as per the manufacturer's instructions. The cDNA was amplified in two separate PCRs for the iNOS gene using the primers

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F, 5′–CAGCGCTACAACATCCTGGA-3′, and R, 5′-GAGGGTACATGCTGGAGCC-3′, as mentioned previously [33], and for nNOS, F, 5′-CCGGCCGACTGGGTGTGGAT-3′, and R, 5′-CGTTGCCGAAGGTGCTGGTCA-3′. The expression of the β-actin gene was checked using the genespecific primers F, 5′-GACGAAGCCCAGAGCAAAAGAGGT-3′, and R, 5′-TCGGCCGTGGTGGGAAACTGTAG-3′. Cycling conditions were 94°C for 30 s for denaturation, 65°C for 1 min for annealing in the case of iNOS and 52°C for 1 min for nNOS and β actin, and 72°C for 1 min for extension through 35 cycles for iNOS and 30 cycles for nNOS and β actin. The PCR-amplified product was 739, 418, and 449 bp for iNOS, nNOS, and β-actin, respectively. Assay for phagocytic activity in neutrophils Phagocytosis was assayed in neutrophils using flow cytometry as previously described [34]. Escherichia coli were heat inactivated at 60°C for 30 min and labeled with FITC (50 μg/ml) in the dark at room temperature for 1 h with continuous shaking. Labeled bacteria were washed twice in HBSS, declumped with repeated pipetting, and resuspended in HBSS. Neutrophils were incubated in the presence of bacteria at ratios of 1:50 and 1:30 for 30 min and analyzed immediately by flow cytometry. To differentiate between phagocytosed and adherent bacteria trypan blue (20 μl of a stock solution of 2 mg/ml for 5 min) was added to the suspension to quench the fluorescence of the adherent bacterial population and the samples were reanalyzed on flow cytometer (FACSCalibur; Becton-Dickinson). Free radical generation assessed by flow cytometry The generation of ROS and reactive nitrogen species (RNS) and superoxide radical in neutrophils (2 × 106) was measured by using cellpermeable dyes such as DCFDH2-DA (for ROS/RNS, 10 μM) and DHE (for O2 −, 10 μM). The cells were incubated for 5 min with the dye at 37°C and further incubated in presence of ascorbate (1 mM) for 15 min and subsequently with neutrophil stimulants such as bacterial peptide fMLP (3 μM) and E. coli bacteria (in a 1:50 ratio with neutrophils) for 30 min. Interventions such as iodoacetamide (1 mM), 7-NI (1 mM), or DPI (1 mM) were used as a pretreatment before subjecting the cells to stimulation or treatment with ascorbate or DHA. Glucose (1 mM) was added just before the addition of DHA. Samples were acquired on a flow cytometer (FACSCalibur; Becton-Dickinson) and mean fluorescence intensity was recorded for at least 10,000 neutrophils [26,27]. Data were analyzed using CellQuest Pro software.

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Fig. 1. Bar diagram representing ascorbate content in the plasma (500 µl) and neutrophils (2×106 cells) from control and scorbutic guinea pigs at the end of the 2-week treatment period. Values are presented as means ±SEM of 10 independent experiments. ⁎⁎⁎p b 0.008 in comparison to the respective controls.

and in plasma to 80% compared to the control was observed (Fig. 1). The ascorbate uptake potential of scorbutic neutrophils, however, remained unaffected. Scorbutic neutrophils accumulated intracellular ascorbate when exposed to ascorbate (1 mM) in vitro significantly (p b 0.05) above the basal levels. The basal levels of ascorbate in untreated neutrophils from control and scorbutics were 9.89 ± 0.38 and 2.5 ± 0.26 μM/2 × 106 cells, respectively. Within 30–40 min of incubation the control and scorbutic neutrophils showed 2 ± 0.06-and 3 ± 0.35-fold increase above the basal levels, respectively. Availability of BH4 during ascorbate deficiency A significant difference in the amount of tetrahydrobiopterin was observed in scorbutics compared to the controls (Fig. 2A). Tetrahydrobiopterin content in the neutrophils isolated from control and scorbutic guinea pigs were 5.7 ± 0. 2 and 2.3 ± 0.1 ng/1× 107 cells, respectively. But the absolute levels of tetrahydrobiopterin in scorbutic neutrophils could not be ascertained in 6 out of 12 of the samples as they went below the detection level in the scorbutic group. These data reflect the limiting levels of BH4 for NOS catalysis among the scorbutics. NOS activity in neutrophils

Superoxide generation by nitroblue tetrazolium assay Neutrophils (1 × 107) were incubated with 10 μM NBT in the presence or absence of ascorbate (1 mM) for 30 min; the blue formazan thus formed was subsequently dissolved in 2 M KOH and DMSO (100 μl), and the absorbance was read at 620 nm [35]. Statistical analysis Results are expressed as means ± SEM of at least five independent experiments in each group. Comparison between two groups was performed using an unpaired Student t test and multiple comparisons were made by one-way ANOVA followed by Newman–Keuls postanalysis test. Results were considered significant at p b 0.05. Results Ascorbate levels in neutrophils and plasma The ascorbate-deficient diet provided to guinea pigs for 2 weeks led to a significant (p b 0.008) ascorbate deficiency in the scorbutic guinea pigs. A decline in ascorbate content in neutrophils to 78%

NO generation potential was deciphered by flow cytometry using DAF-2DA. The scorbutic animals were compromised in terms of NO generation potential as evident from the significant difference (p b 0.005) in mean fluorescence for DAF-2T in resting neutrophils (Fig. 2B). Ascorbate (1 mM) in vitro significantly enhanced NO generation (p b 0.05) in neutrophils from both groups in a timedependent manner but the level in scorbutic animals never matched the control level. DHA, the oxidized derivative of ascorbate and preferably accumulated by neutrophils to recycle ascorbate, had similar effects on NO generation potential. Ascorbate-mediated enhancement in NO generation was counteracted by the NOS inhibitor 7-NI (p b 0.05 for ascorbate-treated (cells + Asc) vs 7-NI-pretreated group (cells + Asc + 7-NI) among both control and scorbutic neutrophils) as presented in Fig. 2C, suggesting the involvement of NOS in the ascorbate-mediated enhancing effect. NOS catalysis was evaluated in neutrophil lysate supplemented with the essential cofactors FAD, FMN, NADPH, calmodulin, and BH4. As reported earlier [33], that guinea pig iNOS is calcium dependent, we also observed most of the NOS activity in guinea pig neutrophils to be calcium dependent, as chelation of calcium in the presence of EGTA (5 mM) completely abrogated catalysis in scorbutics, whereas there

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was only marginal activity in controls (Fig. 3A). Increasing the concentration of ascorbate (300 μM and 1 mM) significantly (p b 0.05) enhanced NOS catalysis in vitro (Figs. 3B and 3C). Addition of

Fig. 2. (A) Bar diagram representing tetrahydrobiopterin content as evaluated in neutrophils (1 × 107 cells) from control and scorbutic guinea pigs. Values are presented as means ± SEM of 15 samples in controls and 6 of 12 samples in scorbutics. ⁎⁎p b 0.01 in comparison to the control. (B) Bar diagram representing NO generation in terms of DAF fluorescence in neutrophils after treatment with ascorbate (1 mM) and DHA (1 mM) for 30 min at 37 °C. Values represent mean fluorescence for DAF ± SEM, ⁎p b 0.05 in comparison to the basal level of DAF fluorescence and #p b 0.005 for scorbutics in comparison to control guinea pigs. (C) Bar diagram representing NO generation as DAF mean fluorescence in neutrophils from control and scorbutic guinea pigs in presence of ascorbate (1 mM) and NOS inhibitor 7-NI (1 mM). Values shown represent DAF mean fluorescence ± SEM, ⁎p b 0.05 for sets having 7-NI treatment vs vehicle, $p b 0.05 for ascorbate-treated cells in comparison to basal level, and #p b 0.05 for scorbutic vs control guinea pig neutrophils.

Fig. 3. (A) Histograms representing NOS activity in neutrophils from control and scorbutic guinea pigs without external calcium supplementation or chelation (cells), in the presence of calcium (2 mM) and EGTA (5 mM). NOS activity in the presence of EGTA could not be detected in the case of scorbutics. Augmentation in NOS activity as ascertained by increased generation of L-[3H]citrulline from L-[3H]arginine after treatment with (B) 10 μM BH4 and 300 μM ascorbate or (C) BH4 (10 or 100 μM) in combination with 1 mM ascorbate. NOS activity is reported as pmol L-[3H]citrulline synthesis/30 min/107 neutrophils. Values represent the means ± SEM for at least three independent experiments. ⁎⁎p b 0.01 for sets treated with increasing concentrations of BH4 and/or ascorbate with respect to the basal level in the presence of 10 μM BH4 for both control and scorbutic groups, and #p b 0.05 and ##p b 0.01 for scorbutic vs control.

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increasing concentrations of BH4 (10 or 100 μM) also produced the same augmenting effect on NOS catalysis (p b 0.05) as shown in Fig. 3C. But neither ascorbate nor BH4 supplementation could restore the NOS activity in scorbutics to match the control level. Despite ascorbate and BH4 supplementation there was a respective 64% (1 mM ascorbate + 100 μM BH4) and 60% (100 μM BH4 alone) difference in NOS activity in scorbutic neutrophils in comparison to the controls. This could presumably be attributed to an alteration in the expression of NOS. Expression of iNOS and nNOS in neutrophils RT-PCR analysis revealed a significant (p b 0.01) decline in iNOS expression by 2.7-fold, or ~70% in terms of iNOS:β-actin ratio, in neutrophils (Figs. 4A and 4C). The expression of nNOS also showed a significant (p b 0.05) decline among scorbutics (Figs. 4B and C). Ascorbate deficiency thus possibly accounted for the lack of a restoring effect of ascorbate or BH4 on NOS catalysis as demonstrated in Figs. 3B and 3C. Phagocytic potential of neutrophils during ascorbate deficiency Because neutrophils are the professional phagocytes of the innate immune brigade their phagocytic response during ascorbate deficiency was evaluated. No significant difference in the phagocytic activity between neutrophils from the two groups, with either a higher (1:50) or a lower (1:30) ratio of neutrophil:bacteria, was observed as presented in Fig. 5.

Fig. 5. Histogram representing phagocytic activity in terms of phagocytosis of FITClabeled bacteria by neutrophils from control and scorbutic guinea pigs. Values represent FITC mean fluorescence ± SEM for six independent experiments.

Oxidative burst potential of neutrophils in scorbutics Oxidative burst potential in terms of superoxide generation was assessed by using DHE dye in fMLP (3 μM)-activated neutrophils from both groups and also in the presence and absence of ascorbate (1 mM) (Fig. 6A). Ascorbate-mediated increment in superoxide generation

Fig. 4. Expression of (A) iNOS and β-actin (lanes 1, molecular weight markers; 2 and 3, RT-PCR products of β-actin; 4 and 5, RT-PCR products of iNOS from control and scorbutic neutrophils, respectively) and (B) nNOS and β-actin (lanes 1, molecular weight markers; 2 and 3, RT-PCR products of nNOS; 4 and 5, RT-PCR products of β-actin from control and scorbutic neutrophils, respectively) in neutrophils from control and scorbutic guinea pigs. The data are representative of 10 independent experiments showing similar results. (C) Graphical representation of densitometric analysis of iNOS/nNOS:β-actin ratio from control and scorbutics. Data represent the means± SEM, ⁎p b 0.05 and ⁎⁎p b 0.01 for scorbutic vs control.

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Fig. 6. (A) Histogram representing superoxide generation as assessed by DHE fluorescence in neutrophils of control and scorbutic guinea pigs. (B) Bar diagram representing superoxide generation in nitroblue tetrazolium assay showing absorbance at 620 nm. (C) Bar diagram showing ROS/RNS generation in terms of DCF response from resting and activated neutrophils of control and scorbutic guinea pigs and enhancement of oxidative burst after treatment with ascorbate (1 mM). Ascorbate-mediated enhancement of free radical generation was counteracted by pretreatment of cells with (D) DPI (1 mM), a dual inhibitor of NOS and NADPH oxidase, and (E) 7-NI (1 mM), a NOS inhibitor. (F) DHA-mediated enhancement of free radical generation in neutrophils and its inhibition in the presence of iodoacetamide (1 mM) or D-glucose (1 mM). Values represent mean fluorescence ± SEM, ⁎p b 0.05 for quiescent neutrophils vs fMLP-(3 μM) and/or ascorbate-(1 mM) treated neutrophils, #p b 0.05 control vs scorbutic, $p b 0.05 for vehicle-treated neutrophils vs treated with DPI or 7-NI, ⁎⁎p b 0.01 for neutrophils treated with DHA vs D-glucose-or iodoacetamide-treated neutrophils.

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neutrophils. The DHA-mediated effect was significantly (p b 0.01) counteracted in the presence of D-glucose (1 mM) by interfering with the intracellular uptake of DHA through GLUT-1/3 and iodoacetamide (1 mM), an inhibitor of glutaredoxin involved in the intracellular conversion of DHA back to ascorbate. The mean stimulation index [MSI= (mean fluorescence of DHA-stimulated cells)/(mean fluorescence of glucose-and iodoacetamide-treated cells further subjected to DHA)] after treatment with DHA was 4.83± 0.18 in case of control neutrophils, whereas it was found to be 0.95 ± 0.42 and 0.79 ± 0.21 (Fig. 6F) in presence of glucose-and iodoacetamide-pretreated neutrophils, respectively. In the scorbutic group the respective values were 3 ± 0.04, 0.92± 0.01, and 0.78± 0.01 (Fig. 6F). These MSI values corresponded to a 30 and 100% decrease in mean fluorescence among the controls and 100% decrease among scorbutic neutrophils in response to DHA in the presence of D-glucose and iodoacetamide, respectively. Discussion

Fig. 6 (continued).

was further confirmed by nitroblue tetrazolium assay, which showed a significant increment in both control and scorbutic cells but the level in scorbutics was markedly lower (p b 0.01) (Fig. 6B). Efficacy of control neutrophils in generating more ROS/RNS compared to their scorbutic counterparts was also evident from the DCF response in resting neutrophils (Fig. 6C) and after bacterial challenge triggering respiratory burst (p b 0.05). Ascorbate supplementation (1 mM) in vitro further enhanced the free radical generation status among the stimulated neutrophils (Fig. 6C). Ascorbate-mediated modulation of respiratory burst: contribution of NOS and NADPH oxidase Ascorbate-mediated enhancement of oxidative burst potential was dependent on NOS activity, as the phenomenon was counteracted in the presence of the NOS inhibitor 7-NI (1 mM) and the inhibitor of both NOS and NADPH oxidase, DPI (1 mM) (Figs. 6D and 6E), to a significant extent (p b 0.05 for NI and Asc + NI groups; p b 0.05 for DPI and Asc + DPI groups). DHA exerts effects on the respiratory burst that are similar to those of ascorbate Incubation of neutrophils with DHA (300 μM), the oxidized derivative of ascorbate, significantly boosted the generation of ROS/RNS in

Under physiological conditions neutrophils, one of the most vital and enormous storage for ascorbic acid [1–5], prefer to recycle ascorbate in its oxidized form, after bacterial phagocytosis or chemical activation, readily through the GLUT-1 transport systems [1–5]. Intracellular regeneration of ascorbate from DHA requires glutaredoxin catalysis [2–4] at the expense of GSH. Because ascorbate contributes to the enhancement of NOS activity in diverse cellular systems it is likely that ascorbate has more attributes to be explored than mere antioxidant defense in neutrophils. This study explored the neutrophil NO generation potential and oxidative burst activity during ascorbate deficiency in scorbutic guinea pigs. Because of the rapid turnover of the neutrophil repertoire in the peripheral circulation it was assumed that their ascorbate storage would be depleted within 2–4 weeks. Initially scorbutic models were maintained for 4 weeks, but we observed severe weight loss and mobility constraints within 3 weeks and occasional death after 4 weeks as also reported previously by Wells et al. [25]; the treatment schedule was therefore confined to 2 weeks, so as to assess the effects of ascorbate depletion on neutrophils within a short duration of ascorbate deficiency. There was a significant decline in neutrophil ascorbate content with a concurrent decrease in plasma, confirming ascorbate deficiency in the scorbutic models (Fig. 1). But despite deficiency, scorbutic neutrophils retained their ascorbate uptake potential when exposed to ascorbate. Ascorbate is documented to stabilize BH4 in endothelial cells and macrophages and thereby promote NOS activity [15–20,23]. Scorbutics in the present study showed a significant decline in BH4 (Fig. 2A). Earlier observations have documented that ascorbate has no influence on the biosynthesis of BH4 in endothelial cells and that the effects were solely mediated through stabilization of the cofactor in its reduced form [15,16]. The vital cofactor for NOS dimerization and electron transfer being limiting, this correlated with a decline in NOS activity from neutrophils of the scorbutic group as reflected in the NO generation potential ascertained by DAF (Fig. 2B). Oxidized derivatives of BH4 such as BH2 are not able to replace BH4 as cofactors; moreover, they interfere with NOS catalysis as reported earlier [36]. Furthermore, as demonstrated previously, ascorbate does not reduce 7,8-BH2 to BH4, nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH2 [37]. However, it can stabilize BH4 by preventing oxidation of BH4, which readily scavenges superoxide [37]. We have assessed the amounts of BH4 using BH4 standards [29–32,38] and by subjecting to differential oxidation simultaneous with biological samples; unlike others [37,39–40] who have used modified method of Fukushina and Nixon [41]. The main concern regarding the differential oxidation procedure is the stability of BH4, to compensate for which we prepared the BH4 stock in 0.1 N HCl and supplemented it with DTT (1 mM), as widely recommended. Ascorbate and DHA pretreatment in vitro for 30 min enhanced NO generation to a significant extent (Fig. 2B). Previous reports have evidenced interference of ascorbate and DHA in the DAF response [42].

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To overcome this drawback we preincubated neutrophils with the NOS inhibitor 7-NI (1 mM), known to inhibit both cNOS (constitutive NOS that is nNOS and eNOS taken together) and iNOS in neutrophils [21], before loading the cells with the dye, and it counteracted the subsequent ascorbate-mediated enhancement of NO generation (Fig. 2C). This clarifies that the ascorbate-mediated effect was not solely due to the resultant interaction with DAF but was being mediated through a boost in NOS activity. However, the 7-NI-pretreated cells, when further subjected to ascorbate treatment (cell + Asc + 7-NI), showed augmentation in DAF fluorescence to a significant extent compared to basal levels (cell) in the control (possibly due to an intracellular interaction of DHA with DAF), much unlike the scorbutic group, which showed complete inhibition of ascorbate-mediated augmentation. We further evaluated the ascorbate influence on NOS catalysis in neutrophil lysates, though we could not discriminate between calcium-dependent and-independent NOS catalysis because most of the NOS activity was found to be calcium dependent (Fig. 3A), supporting previous observations [33] that guinea pig iNOS is calcium dependent. Increasing concentrations of both ascorbate (300 μM and 1 mM) and BH4 (10 and 100 μM) enhanced NOS catalysis among both groups, but it was not sufficient to recover the NOS activity among scorbutics to match the control level (Figs. 3B and 3C). Therefore an alteration in NOS expression seemed likely during ascorbate deficiency, taking into consideration previous reports that ascorbate deficiency causes an increase in the levels of corticosterone [43] that is supposed to suppress iNOS expression [44]. RT-PCR analysis for iNOS as well as for nNOS expression in neutrophils revealed a significant decline in iNOS and nNOS expression in the scorbutic group (Figs. 4A, 4B, and 4C). This observation substantiates our hypothesis that ascorbate could exert a two-pronged effect modulating not only catalysis but also the expression levels of NOS. We report here for the first time this novel finding of an ascorbate-mediated modulation of NOS expression in vivo, which could bear profound implications under inflammatory situations. It is well documented that NO has a modulatory effect on diverse functional aspects of neutrophils [21], and ascorbate through its

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regulation of NOS activity might exert a NO-mediated modulatory effect on neutrophil immune response. So we explored the basic immune functions offered by these granulocytes, i.e., phagocytosis and respiratory burst activity. We did not encounter an alteration in phagocytic potential among the scorbutic neutrophils in comparison to control (Fig. 5), which clarified that neutrophils under ascorbate deficiency are capable of being subjected to equivalent bacterial load as their normal counterparts. Sequestration of bacteria by neutrophils either inside phagocytic vacuoles or in extracellular traps follows the microbicidal actions of granule proteases and a battery of free radicals delivered to the target site. Previous observations from this lab have reported NO-mediated augmentation of oxidative burst among neutrophils [26,27] and also in response to ascorbate in vitro [22,23]. Ascorbate also influences intracellular as well as extracellular H2O2 generation [9,10]. In the present study we have shown that a decline in NO generation potential furthermore substantiated to a compromised generation of ROS/RNS in neutrophils from the deficient group (Figs. 6A, 6B, and 6C). Most of the DCF fluorescence is reported to result from its interaction with peroxynitrite; therefore the augmented DCF response from the control group might have been owing to a dual boosting effect on NOS activity and respiratory burst offered by its ascorbate storage; the scorbutic group, being deficient and compromised in NOS activity, gave significantly less response. Moreover an up-regulation of free radical generation after pretreatment with ascorbate in vitro was dependent on NOS and NADPH oxidase activity and counteracted by the presence of 7-NI and DPI (Figs. 6D and 6E). Neutrophils generally prefer to recycle ascorbate in the form of its oxidized derivative DHA through GLUT over energy-driven ascorbate transport. Pretreatment of neutrophils with DHA had enhancing effects on free radical generation similar to those of ascorbate (Fig. 6F), counteracted by the presence of D-glucose in the cell suspension medium, preventing loading of neutrophils with DHA by blocking GLUT in a competitive manner, whereas iodoacetamide prevented glutaredoxin-catalyzed reduction of DHA back to ascorbate. The results of this study present the prospect of ascorbate storage in neutrophils not only as a defense against constitutive oxidative

Fig. 7. Diagrammatic presentation describing the recycling of ascorbate in the form of DHA after extracellular oxidation of ascorbate by reactive oxygen/nitrogen species or oxygen. DHA is taken up through the glucose transporter, competitively inhibited in the presence of D-glucose, and once taken up DHA is reduced back to ascorbate by glutaredoxin catalysis, inhibited by iodoacetamide. The role of ascorbate in modulating neutrophil NOS activity through BH4 stabilization and in the positive modulation of the respiratory burst (NADPH oxidase activity) is demonstrated. Ascorbate (Asc), dehydroascorbate (DHA), glucose transporter (GLUT), tetrahydrobiopterin (BH4), nitric oxide synthase (NOS), nitric oxide (NO), 7nitroindazole (7-NI), diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS), reactive nitrogen species (RNS).

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stress but also in boosting NOS catalysis and mounting the necessary immune functions to be performed by these granulocytes (Fig. 7). An oxidative influence of ascorbate in vivo that enhances the generation of NO is thus more beneficial in the long run than detrimental. Ascorbate-mediated modulation of neutrophil NOS and consequentially other functional aspects of neutrophils could have farfetched effects in both immunological [45,46] and cardiovascular pathologies [47]. Previously studies from this lab documented an ascorbate-mediated increment in the inhibition of platelet aggregation in the presence of neutrophils in a NO-mediated manner [24] with potential antithrombotic implications. Cardiovascular hemostasis might also be influenced by NO generated from neutrophils, which regulates their chemotactic migration and association with endothelium [48,49]. Ascorbate storage in neutrophils could also influence the instigation and resolution of the inflammatory response under physiological and pathological adversities in which neutrophils serve as an active accomplice. Furthermore, by fine-tuning the redox status of neutrophils ascorbate might regulate the oxidative stress attributed by these granulocytes in various ailments such as hypertension, arthritis, sepsis, endotoxemia. Because ascorbate stabilizes BH4, the ready availability of this cofactor would help to prevent uncoupling and the resultant production of superoxide from NOS [50,51]. On the other hand, among chronic granulomatous disease patients [52], ascorbate storage in neutrophils might serve as a boon in augmenting compensatory microbicidal NO generation under limiting and ineffective oxidative burst conditions. Ascorbate is already proposed in the treatment of patients with recurrent furunculosis and impaired neutrophil function [53]. Because NO has pro-as well as antiinflammatory effects, ascorbate needs to be evaluated in greater detail during such pathologies. Ascorbate supplementation of the immune response is documented vaguely [11–14], without clarification of the causative mechanism. A critical evaluation of the inherent mechanisms of ascorbate influence in modulating immune functions, as we show in the case of neutrophils, could have great clinical significance. This study for the first time also reports a decrease in NO and free radical generation due to ascorbate deficiency in guinea pig neutrophils. Acknowledgments The expert technical assistance of Mr. A.L. Vishwakarma and Mrs. M. Chaturvedi in the flow cytometry experiments is acknowledged. A fellowship grant to M.C. from the Indian Council of Medical Research and funding to M.D. from the Department of Biotechnology are also acknowledged. This is CDRI Communication No. 7392. References [1] Vera, J. C.; Rivas, C. I.; Fischbarg, J.; Golde, D. W. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature 364:79–82; 1993. [2] Welch, R. W.; Wang, Y.; Crossman Jr., A.; Park, J. B.; Kirk, K. L.; Levine, M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem. 270:12584–12592; 1995. [3] Rumsey, S. C.; Kwon, O.; Xu, G. W.; Burant, C. F.; Simpson, I.; Levine, M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J. Biol. Chem. 272:18982–18989; 1997. [4] Park, J. B.; Levine, M. Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin. Biochem. J. 315 (Pt 3):931–938; 1996. [5] Wang, Y.; Russo, T. A.; Kwon, O.; Chanock, S.; Rumsey, S. C.; Levine, M. Ascorbate recycling in human neutrophils: induction by bacteria. Proc. Natl. Acad. Sci. USA 94:13816–13819; 1997. [6] Podmore, I. D.; Griffiths, H. R.; Herbert, K. E.; Mistry, N.; Mistry, P.; Lunec, J. Vitamin C exhibits pro-oxidant properties. Nature 392:559; 1998. [7] Smith, J. N.; Dasgupta, T. P. Mechanisms of nitric oxide release from nitrovasodilators in aqueous solution: reaction of the nitroprusside ion ([Fe(CN)5NO]2−) with L-ascorbic acid. J. Inorg. Biochem. 87:165–173; 2001. [8] Scorza, G.; Pietraforte, D.; Minetti, M. Role of ascorbate and protein thiols in the release of nitric oxide from S-nitroso-albumin and S-nitroso-glutathione in human plasma. Free Radic. Biol. Med. 22:633–642; 1997.

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