Ascorbate in Aqueous Humor Augments Nitric Oxide Production by Macrophages

Kyle C. McKenna, Kelly M. Beatty, Rebecca C. Scherder, Fuwang Li, Huanbo Liu, Alex F. Chen, Arnab Ghosh and Dennis J. Stuehr
J Immunol January 15, 2013, 190 (2) 556-564; DOI: https://doi.org/10.4049/jimmunol.1201754

Abstract

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (Mϕs). Consistent with previous observations, we observed that although Mϕs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of l-arginine (l-Arg) into l-citrulline, was augmented in lysates of Mϕs stimulated in the presence of AqH. These data suggested that intracellular l-Arg may have been limited by AqH. However, we observed increased mRNA expression of the l-Arg transporter, cationic amino acid transporter 2B, and increased l-Arg uptake in Mϕs stimulated in the presence of AqH. Arginases were expressed by stimulated Mϕs, but competition for l-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H4B), an essential cofactor for NOS2 homodimerization, increased after Mϕ stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in Mϕs via ascorbate, which limited degradation of H4B. Therefore, AqH may augment NO production in macrophages by stabilizing H4B and increasing intracellular l-Arg.

Introduction

Ocular immune responses are required to preserve vision by eliminating pathogens that could damage delicate tissues of the visual axis. However, these immune responses can also pose a threat to vision if uncontrolled inflammation damages tissues that do not regenerate (1, 2). As an evolutionary adaptation, ocular immune responses are very tightly controlled to minimize immunopathology. This stringent immune regulation is exemplified in ocular immune privilege, which was first characterized by the persistence of foreign tissues placed in the anterior chamber (a.c.) of the eye (1, 2). Similarly, immunogenic tumors grow progressively when transplanted into the a.c., but are rejected by host immune responses when transplanted in the skin, a nonprivileged site (3, 4).

We have recently reported that rejection of E.G7-OVA tumors transplanted in the skin required the induction of tumoricidal NO production in intratumoral Mϕs by cytotoxic CD8+ T cells (CTL) (5). In contrast, progressive E.G7-OVA growth in the a.c. of the eye occurred despite tumor infiltration by CTL and was associated with impaired NO production by intratumoral macrophages (Mϕ) (5), a potential consequence of mechanisms that preserve ocular immune privilege. The a.c. is filled with aqueous humor (AqH) that contains multiple immunosuppressive factors that can inhibit NO production by Mϕ (reviewed in Ref. 6). For example, TGF-β2 and α-melanocyte–stimulating hormone (α-MSH) have been shown to decrease NO synthase (NOS)2 protein levels by interfering with NOS2 mRNA transcription/stability, limiting NOS2 translation, and/or accelerating NOS2 protein degradation (7, 8). However, we observed that Mϕs within ocular tumors expressed NOS2 protein in vivo, although they produced low nontumoricidal concentrations of NO (5). Similarly, Taylor et al. (9) showed that Mϕs stimulated in the presence of AqH expressed NOS2, but a corresponding increase in nitrite in culture supernatants, an indirect measure of NO production, was not observed. In combination, these data suggested that posttranslational regulation of NOS2 may primarily occur within the ocular microenvironment.

There are several posttranslational mechanisms that could inhibit NOS2 enzymatic activity. NOS2 generates NO and l-citrulline by metabolizing l-arginine (l-Arg). Therefore, limiting l-Arg availability within the cell by preventing entry via the cationic amino acid transporter 2B (CAT2B) (10) or by increasing arginase activity that has similar Vmax/Km values to NOS2 (11) would decrease NO production. NOS2 enzymatic activity is also dependent on the formation of NOS2 homodimers, which may fail to form if indispensable cofactors, including tetrahydrobiopterin (H4B) (12) and 1 iron protoporphyrin IX (heme) (13) are scarce. Alternatively, certain proteins, for example NAP110 (14) and Kalirin-7 (15), have been shown to bind to NOS2 monomers in a manner that prevents homodimerization. The cellular location of NOS2 is also important to its enzymatic activity. For example, NOS2 normally interacts with the cytoskeletal protein α-actinin 4 to localize NOS2 to the plasma membrane and/or submembranal zone (16). When this interaction is disrupted, NOS2 is dispersed throughout the cytoplasm and enzymatic activity is compromised (16).

To identify mechanisms of posttranslational regulation of NOS2 within the ocular microenvironment, we characterized NOS2 enzymatic activity in Mϕs stimulated in the presence or absence of AqH. Interestingly, we observed increased NOS2 enzymatic activity in cell lysates of Mϕs stimulated in the presence of AqH, although nitrite was profoundly reduced in culture supernatants. These incongruent observations were not explained by limited intracellular concentrations of l-Arg, abrogated expression of the enzyme GTP cyclohydrolase (GTP) that produces H4B, or inhibited formation of NOS2 homodimers. Rather, we demonstrate that high concentrations of ascorbate in AqH interfered with the Griess assay used to measure nitrite. After controlling for interference by ascorbate, we observed that AqH augmented NO production in Mϕs via ascorbate. AqH increased intracellular concentrations of L-Arg and limited degradation of H4B, suggesting two potential mechanisms for augmented NO production by Mϕs.

Materials and Methods

Reagents

Murine rIFN-γ (BD Pharmingen, San Diego, CA), ultrapure LPS from Escherichia coli (Invivogen, San Diego, CA), porcine TGF-β2 (R&D Systems, Minneapolis, MN), l-Arg (Sigma-Aldrich, St. Louis, MO), calcitonin gene-related peptide (CGRP; Sigma-Aldrich), α-MSH (Sigma-Aldrich), pigment epithelium-derived factor (PEDF; BioproductsMD, Middletown, MD), ascorbate (Sigma-Aldrich), ascorbate oxidase (Sigma-Aldrich), and neutralizing mAb against TGF-β2 (R&D Systems) were used in these studies.

Mice

Male and female C57BL/6 J (B6) or B6;129P2-Nos2tm1Lau/J (NOS2−/−) mice on a B6 background from The Jackson Laboratory (Bar Harbor, ME) were used. All experimental animals were bred and maintained in the animal facilities at the University of Pittsburgh under specific pathogen-free conditions. All experimental procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and were in adherence to the Association for Research in Vision and Ophthalmology guidelines for use of experimental animals in ophthalmology research.

Aqueous humor

Rabbit AqH, obtained by paracentesis of the a.c., was generously provided by the Charles T. Campbell Ophthalmic Microbiology Laboratory (University of Pittsburgh), or was purchased from Pel-Freeze Biologicals (Rogers, AR). Rabbit AqH was centrifuged, and the supernatant was filtered through a 0.2-μm filter and then used fresh or frozen at −85°C until use.

Mϕ culture

Mϕs were obtained from B6 or NOS2−/− mice by peritoneal lavage 3–4 d after i.p. injection of 3 ml thioglycolate broth (BD Biosciences, San Diego, CA). Peritoneal exudate cells were plated overnight in standard growth medium (SGM; RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 50 μM 2-ME, gentamicin, penicillin, and streptomycin) or in serum-free X-Vivo 10 medium (Lonza), and then washed extensively to remove nonadherent cells. The remaining cells were predominantly mature F4/80+ Mϕs (Supplemental Fig. 1). RAW 264.7 Mϕs were obtained from American Type Culture Collection (Manassas, VA). Mϕs maintained in SGM or X-Vivo 10 medium were stimulated with a combination of IFN-γ (100 U/ml) and LPS (100 ng/ml) for indicated periods of time, at 37°C and 5% CO2 atmosphere in a humidified incubator. To stimulate Mϕs in the presence of AqH, the media was diluted with AqH at a 1:1 ratio. In some experiments, AqH and SGM were pretreated with neutralizing anti–TGF-β2 Abs, or AqH was treated with ascorbate oxidase (10 U/ml) for 1 h at room temperature.

RNA extraction and RT-PCR

Mϕs activated in the presence or absence of AqH were harvested at indicated time points utilizing a cell scraper in the presence of RLT buffer (Qiagen, Germantown, MD). Total RNA was then extracted using the RNeasy minikit from Qiagen following the manufacturer’s protocol. cDNA was prepared using a high-capacity cDNA reverse-transcription kit (Applied Biosystems, Carlsbad, CA), and expression of arginase 1 (ARG1; primer Mm00475989_m1), arginase 2 (ARG2; primer Mm00477592_m1), CAT2B (primer Mm00432032_m1), GTP cyclohydrolase 1 (GTPCH1; primer Mm00514993_m1), NOS2 (primerMm00440485_m1), and pyruvate carboxylase (primer Mm00500992_m1) was determined by quantitative real-time PCR using TaqMan PCR universal mix (Applied Biosystems) and an ABI StepOne Plus thermocycler (Applied Biosystems). All primers were obtained from Applied Biosystems. Relative expression was determined using the 2ΔΔCT method. First, cycle threshold (CT) values for experimental gene expression were normalized to the CT values of the housekeeping gene pyruvate carboxylase within the same sample (ΔCT). The ΔCT from Mϕ-stimulated cultures was then subtracted from the ΔCT of nonstimulated Mϕ cultures (ΔΔCT) to determine fold differences from controls = 2ΔΔCT.

l-Arg uptake assay

Triplicate primary Mϕ cultures (5.0 × 106 cells in 2 ml SGM contained within a 60-mm petri dish) were untreated or stimulated with IFN-γ and LPS in the presence or absence of AqH. Eighteen hours later, plates were washed with warm (37°C) PBS and then incubated with 0.1 μM [3H] l-Arg (Perkin Elmer, Boston, MA) in warm PBS for 2 min. Plates were then washed twice with warm PBS, and cells were scraped and homogenized in lysis buffer and transferred to a tube containing scintillation fluid in which radioactive cpm were measured.

NOS2 protein expression

Stimulated and nonstimulated Mϕ cultures (5.0 × 106 cells in 2 ml SGM contained within a 60-mm tissue culture-treated petri dish) were washed with PBS, and then cells were scraped from plates in Complete Lysis M buffer (Roche, Manheim, Germany) to prepare cell lysates. Cell lysates were homogenized by pipetting and sonication. Protein concentration was determined by a Bradford assay (Bio-Rad, Hercules, CA), and 10–30 μg total protein was loaded onto a 6% polyacrylamide gel. Following electrophoresis, separated proteins were transferred to a polyvinylidene difluoride membrane that was blocked with Odyssey Blocking Buffer (LI-COR, Lincoln, NB), and then primary Abs were added at the following dilutions: anti-NOS2, 1:1000; anti-ARG1, 1:1000; and anti–β-tubulin 1:20,000 in 3% nonfat dried milk in PBS-Tween-20. Secondary Abs were then used at the following dilutions: goat anti-rabbit IRDye680 1:20,000 and goat anti-Mouse IRdye 800CW 1:20,000. Protein expression was resolved by imaging on the Odyssey Infrared Imager (LI-COR).

NOS2 activity measurements

NO production was measured indirectly by measuring nitrite levels in supernatants from triplicate cultures (5.0 × 105 cells in 0.2 ml medium contained within an individual well of a 96-well plate, or 2.5–5.0 × 106 cells in 2.0 ml medium contained with a 60-mm petri dish) by the Griess assay (Promega, Madison, WI), according to the manufacturer’s instructions. In some experiments, culture supernatants were incubated with 10 U/ml ascorbate oxidase for 1 h at room temperature prior to use in the Griess assay. To measure NOS2 enzymatic activity, the conversion of supplemented l-Arg into l-citrulline was measured using a NOS activity assay kit, according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).

Determination of the presence of NOS2 monomers and dimers

Size exclusion chromatography of Mϕ lysates was performed, as described previously (17), at 4°C. A column prepacked with Superdex 200 (S200; Amersham, Pharmacia Biotech) was equilibrated at 0.5 ml/min with 40 mM EPPS buffer (3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid) (pKa of 8, pH 7.6–8.6), containing 3 mM DTT, 10% glycerol, and 150 mM NaCl. Equal amounts of lysates were diluted to 200 μl prior to injections. The proteins in the column effluent were detected at 280 nm using a flow-through detector. Aliquots of each column fraction were subjected to SDS-PAGE and Western blotted to detect NOS2. The m.w. of the protein fractions were estimated relative to gel filtration m.w. standards. Band intensities on NOS2 Western blots from column fractions were measured using Image J quantification software. Values from fractions containing monomers or dimers were summed, and then the monomer:dimer ratio was determined by simple division.

Ascorbate measurement

Ascorbate was measured in AqH using the Ascorbate Assay Kit (Cayman Chemical), according to the manufacturer’s instructions.

H4B measurement

H4B (20 μM) was spiked into 2.0 ml solutions of SGM, SGM diluted 1:1 with AqH, or SGM diluted 1:1 with AqH that was pretreated with ascorbate oxidase (10 U/ml for 1 h). Solutions were then incubated at 37°C at 5% CO2 atmosphere in a humidified incubator. Aliquots (50 μl), removed at multiple time points, were added to pH 3 HPLC grade water (450 μl; Fisher Scientific) containing 100 μM dithioerythritol (Sigma-Aldrich) and 100 μM diethylenetriaminepentaacetic acid (Sigma-Aldrich). Samples were stored at −85°C, before analysis. For analysis, the thawed samples were measured by HPLC with fluorescence detection, as described previously (18). Briefly, protein was removed by adding 10 μl of a 1:1 mixture of 1.5 mol/l HClO4 and 2 mol/l H3PO4 to 90 μl samples, followed by centrifugation at 13,000 × g for 5 min at 4°C. To determine total biopterin (H4B, dihydropterin [H2B], and oxidized biopterin) by acid oxidation, 10 μl 1% iodine in 2% potassium iodine solution was added to the protein-free supernatant (90 μl). To determine H2B and oxidized biopterin by alkali oxidation, 10 μl 1 M NaOH was added to 80 μl sample, and then 10 μl 1% iodine in 2% potassium iodine solution was added. Samples were incubated at room temperature for 1 h in the dark. Alkaline-oxidation samples were then acidified with 20 μl 1 M H3PO4. Iodine was reduced by adding 5 μl fresh ascorbic acid (20 mg/ml). Samples of 90 μl were injected into a 250-mm–long, 4.6-mm inner-diameter Spherisorb ODS-1 column (5 μm particle size; Alltech Associates, Deerfield, IL) isocratically eluted with a methanol-water (5:95, v/v) mobile phase running at a flow rate of 1.0 ml/min. Fluorescence detection (350 nm excitation, 450 nm emission) was performed using a fluorescence detector (RF10AXL; Shimadzu). H4B concentrations were calculated by subtracting H2B plus oxidized biopterin from total biopterins.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA). ANOVA with Tukey’s posttest comparison of selected groups was used. The p values <0.05 were considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.

Results

Influence of aqueous humor on NOS2 enzymatic activity in Mϕs

To identify mechanisms that inhibit NOS2 enzymatic activity within the ocular microenvironment, we used a relevant in vitro system in which Mϕs were stimulated with LPS and IFN-γ in the presence of AqH (9). NO production was monitored indirectly by measurement of nitrite in Mϕ culture supernatants (Griess assay) as nitrite is a stable breakdown product of NO. Nitrite increased when primary peritoneal Mϕs (Fig. 1A) or the RAW 264.7 Mϕ cell line (Fig. 1B) was stimulated. In contrast, nitrite did not accumulate in culture supernatants of stimulated Mϕs from NOS2-deficient mice (Supplemental Fig. 2), which indicated that NO was produced by NOS2. The addition of AqH profoundly suppressed nitrite accumulation in supernatants of stimulated Mϕs (Fig. 1A, 1B), despite NOS2 protein expression (Fig. 1C) that reproduced previous observations (9).

FIGURE 1.
FIGURE 1.

Influence of aqueous humor on NOS2 activity. Nitrite concentrations in culture supernatants of primary peritoneal Mϕs (A) or RAW 264.7 Mϕ cells (B) that were untreated or stimulated with LPS and IFN-γ in the absence or presence of AqH. Nitrite was measured 18 h after treatment. Data shown are representative experiments of 2–11 that were performed with similar results. (C) NOS2 and α-tubulin (loading control) protein expression in cell lysates of primary Mϕ cultured as indicated. Data are representative of two experiments performed. (D) NOS2 enzymatic activity in lysates of Mϕs treated as indicated. Each line represents measurements from an independent experiment.

To directly evaluate NOS2 enzymatic activity, we monitored the conversion of l-Arg into l-citrulline in cell lysates of Mϕs stimulated in the presence or absence of AqH. As shown in Fig. 1D, NOS2 enzymatic activity in Mϕs increased upon stimulation, which was consistent with increased nitrite in culture supernatants. However, NOS2 enzymatic activity was even greater in lysates of Mϕs stimulated in the presence of AqH (Fig. 1D), although nitrite levels in these culture supernatants were reduced (Fig. 1A).

TGF-β2, α-MSH, CGRP, or PEDF does not inhibit NO production by Mϕs

Certain molecules present in normal AqH, including TGF-β2, α-MSH, and CGRP, have been shown to inhibit NO production in activated Mϕs (7–9). Therefore, we tested whether these molecules at concentrations reported to be present in normal AqH (9, 19, 20) were able to significantly decrease NO production by primary Mϕs activated with IFN-γ and LPS. TGF-β2, CGRP, or α-MSH alone or in combination was not suppressive (Fig. 2A). Superphysiological concentrations of these molecules (10 ng/ml TGF-β2, 20 ng/ml CGRP, or 300 ng/ml α-MSH) were also incapable of suppressing nitrite accumulation by stimulated Mϕ (percent inhibition: <20%) (data not shown). Neutralization of TGF-β2 in culture medium or in AqH increased NOS2 protein levels slightly; however, a corresponding increase in NO production was not observed (data not shown). Another factor present in the AqH that has been shown to inhibit NO production by Mϕs is PEDF (21, 22). However, the addition of PEDF to stimulated Mϕ cultures also did not inhibit NO production (Fig. 2B). Taken together, these data indicated that TGF-β2, CGRP, α-MSH, and PEDF within AqH did not mediate suppression of NO production in Mϕs under our experimental conditions.

FIGURE 2.
FIGURE 2.

Influence of TGF-β2, α-MSH, CGRP, or PEDF on NOS2 activity. Nitrite concentration in culture supernatants of primary peritoneal Mϕs stimulated in the presence of AqH, TGF-β2, α-MSH, and/or CGRP (A) or PEDF (B) for 18 h. Plots are individual experiments representative of two to three experiments performed.

AqH increases l-Arg transport via CAT2B

The measurement of NOS2 enzymatic activity in cell lysates involved supplementation with l-Arg and H4B. Therefore, our incongruent measurements of NO production made by Griess assays and NOS2 enzymatic activity assays could have been due to AqH limiting the concentration of intracellular l-Arg in intact Mϕs. To test this possibility, we first evaluated the mRNA expression of the l-Arg transporter, CAT2B, in Mϕs stimulated in the presence or absence of AqH. In comparison with control unstimulated Mϕs, CAT2B expression increased after Mϕ stimulation in two independent experiments (Fig. 3A). The addition of AqH further increased CAT2B expression at 18 h (experiment 1, 1.7-fold greater; experiment 2, 1.3-fold greater in comparison with Mϕs stimulated without AqH), suggesting that l-Arg uptake via CAT2B was not impaired in AqH-treated Mϕs. Nevertheless, to evaluate the functional activity of CAT2B, we directly measured l-Arg uptake by Mϕs 18 h after stimulation and 2 min after addition of [3H]l-Arg (Fig. 3B) when l-Arg transport was linear (23). l-Arg uptake was significantly greater in stimulated Mϕs than in unstimulated Mϕs, and greatest in Mϕs stimulated in the presence of AqH, which was consistent with their higher CAT2B mRNA expression (Fig. 3A). These data indicated that the concentration of intracellular l-Arg was increased rather than inhibited by AqH treatment.

FIGURE 3.
FIGURE 3.

AqH does not inhibit l-Arg transport. (A) mRNA expression of the CAT2B at 6 and 18 h after LPS and IFN-γ stimulation of primary Mϕ in the presence or absence of AqH, under TGF-β2 neutralizing conditions. Data are presented as fold change in comparison with unstimulated Mϕs. Data from two independent experiments are shown. (B) [3H]l-Arg uptake by primary Mϕs cultured as indicated. One experiment, which is representative of two experiments performed, is shown.

Arginase activity does not contribute to AqH-mediated inhibition of NO production by Mϕ

Intracellular l-Arg can be depleted by competition with arginases (ARG1 and ARG2) that have similar Vmax/Km values to NOS2 (11). Therefore, NOS2, ARG1, and ARG2 mRNA expression were measured by quantitative RT-PCR (Fig. 4A–C). Upon Mϕ stimulation in the presence or absence of AqH, NOS2 mRNA expression increased dramatically relative to unstimulated control Mϕs in two independent experiments (Fig. 4A). ARG1 and ARG2 mRNA expression also increased after stimulation, albeit to a much lesser extent than NOS2, and were further increased by AqH at 18 h (Fig. 4B, 4C) (Arg1: experiment 1, 5.7-fold greater; experiment 2, 2.4-fold greater; Arg2: experiment 1, 2.0-fold greater; experiment 2, 2.5-fold greater, in comparison with Mϕs stimulated without AqH). However, this analysis did not compare copy number of NOS2, ARG1, and ARG2 mRNAs. In nonstimulated Mϕ cultures, the cycle titration number for NOS2 was higher than ARG1 and ARG2 (data not shown). Assuming equal efficiency of PCRs, these data suggested that the total mRNA copy number for ARG1 and ARG2 exceeded NOS2 copies. Therefore, we evaluated ARG1 and NOS2 protein expression to better evaluate the absolute concentration of these enzymes in intact Mϕ. As shown in Fig. 4D, resting primary Mϕs expressed ARG1, but not NOS2 protein, which was consistent with previous reports (24). Upon stimulation, ARG1 expression was maintained and NOS2 expression increased equivalently in Mϕ cultures stimulated with or without AqH. As Mϕ cultures stimulated in the absence of AqH produced NO despite ARG1 expression, which was equivalent to Mϕ cultures stimulated in the presence of AqH, these data suggested that arginase activity did not contribute to AqH-mediated suppression of NO production. In addition, Mϕs were cultured in RPMI 1640 medium that contains l-Arg at a concentration (1.15 mM) that was too high to promote competition between arginases and NOS2 for l-Arg (25).

FIGURE 4.
FIGURE 4.

Influence of AqH on arginase and NOS2 expression. mRNA expression of NOS2 (A), arginase (ARG1) (B), and ARG2 (C) at 6 and 18 h after LPS and IFN-γ stimulation of primary Mϕs in the presence or absence of AqH, under TGF-β2 neutralizing conditions. Data are presented as fold change in comparison with unstimulated Mϕs. The dashed line indicates no change over control. Data from two independent experiments are shown. (D) NOS2, ARG1, and α-tubulin (loading control) protein expression in cell lysates from Mϕ stimulated in the presence or absence of AqH. One representative experiment of two experiments performed is shown.

Effect of AqH on GTPCH1 expression

NOS2 enzymatic activity requires the formation of homodimers that are stabilized by H4B and l-Arg (13, 26). H4B is produced by the coenzyme GTPCH1. Therefore, we measured GTPCH1 mRNA expression by quantitative RT-PCR to determine whether differences in expression of GTPCH1 might explain inhibited NO production in Mϕs activated in the presence of AqH. In two independent experiments, GTPCH1 mRNA increased in Mϕ stimulated in the presence or absence of AqH (Fig. 5), suggesting that inhibited coenzyme activity did not explain AqH-induced suppression of NO production in Mϕ.

FIGURE 5.
FIGURE 5.

GTPCH1 expression in stimulated Mϕs. mRNA expression of GTPCH1 was evaluated at 6 and 18 h after stimulation of primary Mϕs in the presence or absence of AqH, under TGF-β2 neutralizing conditions, by quantitative RT-PCR relative to unstimulated Mϕ. The dashed line indicates no change over control. Data from two independent experiments are shown.

AqH does not interfere with NOS2 homodimerization

As NOS2 enzymatic activity requires the formation of NOS2 homodimers (17), we examined whether AqH interfered with NOS2 homodimerization within cell lysates from RAW 264.7 cells stimulated in the presence of AqH. Cell lysates were fractionated by size exclusion chromatography and obtained fractions electrophoresed on a denaturing gel and immunoblotted with anti-NOS2 Abs (Fig. 6A, 6B). Consistent with previous observations (17), Mϕs stimulated in the absence of AqH showed NOS2 protein molecules comprised of homodimers (eluting in fractions 7–13 at ∼250 kDa) and monomers (eluting in fractions 14–20 at ∼140 kDa) (Fig. 6A) at approximately a 1:1 ratio (Fig. 6C). Mϕs activated in the presence of AqH presented a different pattern, showing high m.w. NOS2 aggregates (eluting in fractions 1–6 at ∼700 kDa [Fig. 6B]). However, these aggregates did not interrupt the formation of NOS2 dimers (Fig. 6C). A decreased concentration of monomers was also observed.

FIGURE 6.
FIGURE 6.

Influence of AqH on NOS2 homodimerization. Measurement of NOS2 monomers and dimers in cell lysates from RAW 264.7 Mϕs stimulated in the absence (A) or presence (B) of AqH under TGF-β2 neutralizing conditions. Approximate m.w. of individual fractions shown based on elution of known standards. (C) Band intensities for individual fractions. Data presented are from an individual experiment of three performed. High m.w. NOS2 aggregates were observed in two of three experiments.

Ascorbate in AqH interferes with the Griess assay

As our accumulated data provided no evidence of inhibited NOS2 activity by AqH, we tested whether a factor within AqH interfered with the Griess assay used to measure nitrite in culture supernatants. Titrated nitrite standards were prepared in media or media supplemented with AqH at the same concentration as was used in Mϕ cultures. As shown in Fig. 7A, AqH completely inhibited measurement of nitrite by the Griess assay, confirming our hypothesis.

FIGURE 7.
FIGURE 7.

Ascorbate within AqH interferes with the Griess assay. (A) Griess assay on titrated nitrite standards in SGM supplemented with AqH treated with vehicle, or SGM supplemented with AqH that was treated with ascorbate oxidase (AO). (B) Griess assay with titrated nitrite standards in SGM, 1 mM ascorbate in SGM treated with vehicle, or 1 mM ascorbate in SGM that was treated with AO. AO was prepared in 4 mM sodium phosphate buffer (pH 5.6) with 0.05% BSA (vehicle). (C) Griess assay on serial dilutions of AqH in SGM spiked with 50 μM nitrite. (D) Griess assay on serial dilutions of ascorbate in SGM spiked with 50 μM nitrite. Each panel represents an independent experiment that was performed twice with equivalent results.

The Griess assay is very susceptible to reducing agents as it involves an oxidation reaction. Therefore, we tested whether the antioxidant ascorbate, which is present in very high concentrations within AqH (27), was responsible for interference with the Griess assay. Ascorbate was measured in four lots of rabbit AqH, and the average concentration (1.0 ± 0.6 mM) was comparable to concentrations observed in a previous report (28). For comparison, the concentration of ascorbate in rabbit serum was 30 μM (28). As shown in Fig. 7B, a 1 mM ascorbate solution also completely inhibited measurement of nitrite standards by the Griess assay. To determine the threshold concentration of ascorbate that would cause interference, titrated ascorbate solutions (1 mM–488 nM) were added to a 50 μM nitrite standard solution and the Griess assay was performed. Ascorbate concentrations as low as 0.1 mM completely inhibited nitrite measurements (Fig. 7D). We also tested the threshold of interference by AqH. Complete interference was observed with AqH diluted 1:16 in medium (Fig. 7C). The ascorbate concentration at this dilution would be ∼0.1 mM based on a 1.0 mM concentration in AqH. To confirm that interference was due to ascorbate, ascorbate was oxidized by incubation with ascorbate oxidase. As shown in Fig. 7A and 7B, ascorbate oxidation restored measurements of nitrite standards. These data clearly indicated that ascorbate in AqH interfered with the Griess assay, resulting in artifactual low nitrite measurements.

Ascorbate in aqueous humor augments NO production in Mϕs

To determine the true concentration of nitrite in Mϕ cultures stimulated in the presence of AqH, Mϕ culture supernatants were treated with ascorbate oxidase prior to performing the Griess assay. As shown in Fig. 8, nitrite concentrations in supernatants from Mϕs stimulated in the presence of AqH were greater in comparison with Mϕ cultures stimulated without AqH. Importantly, this difference was only observed when ascorbate was oxidized in culture supernatants. These data indicated that AqH augmented NO production in Mϕs, which was consistent with our observations made with NOS2 enzymatic activity assays (Fig. 1D).

FIGURE 8.
FIGURE 8.

AqH augments NO production in stimulated Mϕs. (A) Nitrite concentrations in culture supernatants treated with ascorbate oxidase (AO) or vehicle from primary peritoneal Mϕs cultured as indicated. Data shown are a representative experiment of three independent experiments performed with similar results. (B) Nitrite concentrations in culture supernatants treated with AO from primary peritoneal Mϕs stimulated as indicated in the presence of ascorbate (1 mM), AqH, or AqH that was pretreated with AO before addition to Mϕ cultures. Data shown are a representative experiment of two experiments performed.

Nakai et al. (29) previously demonstrated that ascorbate increases NO production in Mϕs, and we reproduced this finding with macrophages stimulated with LPS and IFN-γ in the presence of 1 mM ascorbate (Fig. 8B). Hence, we reasoned that ascorbate in AqH was responsible for augmented NO production in Mϕs. To test the influence of ascorbate in AqH on NO production, ascorbate was oxidized in AqH by incubation with ascorbate oxidase prior to addition to Mϕ cultures. As shown in Fig. 8B, oxidation of ascorbate in AqH abrogated the enhancement of NO production by AqH, which confirmed that ascorbate in AqH was responsible for increasing NO production. Similar results were observed when ascorbate was removed from AqH by dialysis (data not shown).

Ascorbate in AqH stabilizes H4B

Ascorbate has been shown to augment NO production in Mϕs by limiting H4B oxidation, which extends the t1/2 of this molecule (29). Therefore, we evaluated the degradation of H4B added to medium, medium diluted in AqH, and medium diluted in AqH that was pretreated with ascorbate oxidase. As shown in Fig. 9, degradation of H4B in medium was rapid and complete after 4 h at 37°C in a 5% CO2 atmosphere. In contrast, AqH limited the degradation of H4B. The stabilization of H4B was due to ascorbate in AqH as pretreatment of AqH with ascorbate oxidase abrogated the stabilizing effect.

FIGURE 9.
FIGURE 9.

Ascorbate in AqH stabilizes H4B. Percentage of H4B spiked in SGM medium, SGM supplemented 1:1 with AqH that was treated with vehicle, or SGM supplemented 1:1 with AqH that was treated with ascorbate oxidase (AO) after incubation at 37°C at 5% CO2 atmosphere for indicated periods of time. This experiment was performed twice with equivalent results.

Discussion

In this study, we have demonstrated that ascorbate in AqH augments NOS2 enzymatic activity and NO production in stimulated Mϕs. These data contradict the interpretations of a previous study by Taylor et al. (9), which indicated that AqH inhibited NO production by Mϕs based on their observations that nitrite did not accumulate in culture supernatants of Mϕs stimulated in the presence of AqH. We reproduced those observations, but show that the low nitrite levels in these cultures were erroneous. High concentrations of ascorbate in AqH interfered with nitrite measurements made by Griess assays. When interference by ascorbate was controlled for, we observed increased nitrite in culture supernatants of Mϕs stimulated in the presence of AqH. The augmentation of NO production by Mϕs was mediated by ascorbate, as treatment of AqH with ascorbate oxidase abrogated this augmentation. Our interpretation that AqH augments NO production in Mϕs was further supported by direct measurement of NOS2 enzymatic activity, which showed greater conversion of l-Arg into l-citrulline in lysates of Mϕs stimulated in the presence of AqH.

It is important to note that the previous study (9) relied entirely on nitrite measurements in culture supernatants and did not indicate that interference by ascorbate in AqH was controlled for. However, what remains puzzling is that this study also showed that nitrite levels were restored in AqH-treated Mϕ culture supernatants if AqH was incubated with neutralizing Abs to CGRP (9). Ascorbate would still remain in this treated AqH sample, which should have interfered with the Griess assay and resulted in artifactual low nitrite readings. One potential explanation is that the addition of Abs produced false-positive readings. In support of that interpretation, certain molecules, including proteins, have been shown to cause both false-positive and false-negative readings in the Griess assay (30). False-negative readings could also explain other observations showing that CGRP or PEDF alone inhibited nitrite levels in stimulated Mϕ culture supernatants (9, 22). However, we were unable to reproduce inhibitory effects with these molecules. Regardless, the sensitivity of the Griess assay to interference by many different molecules requires careful controls in which nitrite standards are measured in the presence of tested aqueous solutions.

There were some methodological differences between our study and the previous study. For example, in our study, AqH was stored frozen before use and Mϕs were cultured in serum-containing medium. In contrast, Taylor et al. (9) used fresh AqH and cultured Mϕs in serum-free medium. It has been suggested that the suppressive activity of AqH toward T cells is destroyed by freezing AqH, and that proteases in serum degrade immunosuppressive peptides (19, 31). However, we also observed augmented NO production by Mϕs that were stimulated in serum-free medium in the presence of fresh AqH (Supplemental Fig. 3). These data argue against the loss of immunosuppressive activity in AqH due to freezing of AqH or our culture conditions. In fact, the augmentation of NO production by Mϕs stimulated in the presence of AqH was greater when fresh AqH and serum-free medium were used. Therefore, we find no evidence of an immunosuppressive factor within AqH that inhibits NO production in Mϕs.

Why Mϕs stimulated with fresh AqH in serum-free conditions produced even more NO is not completely understood. One simple explanation is that freezing AqH decreased the concentration of ascorbate, thereby minimizing its enhancing effect on NO production. However, we directly compared fresh and frozen AqH, and, although the concentration of ascorbate was slightly lower in frozen AqH, mM concentrations were still observed (data not shown). Another potential explanation is that the serum-free media we used, which did not contain ascorbate, reduced the basal ascorbate concentration in Mϕs. Hence, the effect of ascorbate supplementation was magnified. In support of that explanation, May et al. (32) showed that freshly prepared thioglycolate-elicited Mϕs contained ∼3 mM ascorbate. After overnight culture in ascorbate-free medium, this concentration was reduced to 1–2 mM. Our peritoneal Mϕ cultures were allowed to adhere overnight in serum-free medium. Therefore, the intracellular ascorbate concentration would have been reduced. The nitrite concentrations in supernatants of control Mϕs stimulated without AqH were noticeably lower than in similar cultures stimulated in serum-containing, and thus ascorbate-containing, RPMI 1640 (compare Fig. 1A with Supplemental Fig. 3). These data could suggest that ascorbate is a critical cofactor for optimal NOS2 enzymatic activity under normal physiological conditions.

It is important to note that a previous study also demonstrated that ascorbate increases NO production in RAW 264.7 Mϕs (29), which supports our observations. Interestingly, their automated method (33) for measuring nitrite in culture supernatants did not require the addition of ascorbate oxidase to eliminate interference by ascorbate in the Griess assay. This is most likely due to differences in sample preparation. In their procedure, culture supernatants were first deproteinized by precipitation with sulfosalicyclic acid and centrifuged, and then an aliquot of the supernatant was treated with NH4Cl and NaOH. These steps would dilute the original ascorbate concentration and potentially oxidize ascorbate. In our study, and in the study by Taylor et al. (9), which we refute, undiluted Mϕ culture supernatants with high concentrations of ascorbate were analyzed. Therefore, treatment with ascorbate oxidase was necessary to prevent interference with the Griess assay, which requires an oxidation reaction.

Our data suggest two possible mechanisms by which AqH augments NO production in Mϕs. First, we demonstrate that AqH increases l-Arg uptake in Mϕs, providing more available substrate for NOS2 to generate NO and l-citrulline. Second, ascorbate in AqH stabilized H4B. Therefore, increased intracellular concentrations of l-Arg and H4B could increase the formation of functional NOS2 dimers that would thereby increase total NOS2 enzymatic activity. In support of that interpretation, we did observe fewer NOS2 monomers and more NOS2 dimers in lysates of Mϕs stimulated in the presence of AqH (Fig. 6). However, it is important to note that increasing intracellular H4B does not directly correlate with increased NOS2 enzymatic activity. For example, Nakai et al. (29) artificially elevated intracellular H4B in Mϕs by either ascorbate or sepiapterin supplementation. Sepiaterin generates H4B via a salvage pathway, whereas ascorbate is thought to reduce H4B oxidation and thereby limit its degradation. Although H4B was concentrated 20-fold greater in sepiaterin-treated Mϕs in comparison with ascorbate-treated Mϕs, ascorbate-treated Mϕs produced more NO. Supplementation of ascorbate and sepiaterin had a synergistic effect, producing even more NO. Hence, the redox state of H4B may be more critical than the total H4B concentration in terms of influencing NOS2 enzymatic activity.

The formation of high m.w. NOS2 aggregates in Mϕs stimulated in the presence of AqH suggests unique interactions between NOS2 and other proteins that may inhibit or activate NOS2 enzymatic activity in these holoenzyme complexes. As our data show that AqH increased NOS2 enzymatic activity, this suggests that negative regulatory proteins, including NO-associated protein of 110 kDa (NAP-110) (14) and Kalirin-7 (15), which bind to the N terminus of NOS2 and prevent homodimerization, are not involved. However, we cannot exclude the possibility that AqH promoted association of NOS2 with proteins that enhanced its activity. Along those lines, Daniliuc et al. (16) showed that NOS2 association with α-actinin 4 was critical for enzymatic activity. In addition to changing the cellular location of NOS2, α-actinin 4 appeared to be a critical cofactor for NOS2 function, as NOS2 dimers without α-actinin 4 were not enzymatically active. Future experimentation involving immunoprecipitation of proteins bound to NOS2 will be necessary to evaluate whether AqH influences NOS2 enzymatic activity by changing the proteins associated with NOS2.

At first glance, it is hard to understand why an immune-privileged site would contain a molecule that augments proinflammatory NO production in Mϕs. However, ascorbate also minimizes inflammation by neutralizing reactive oxygen species. For example, Rosenbaum et al. (27) showed that ascorbate in AqH inhibited myeloperoxidase activity in neutrophils. NO combines with reactive oxygen species to form peroxynitrite, which induces cell death (34). Therefore, ascorbate may limit the toxic effects of NO by limiting peroxynitrite formation.

In summary, our data overturn a long-standing paradigm of AqH-mediated immunosuppression of Mϕ NO production by demonstrating that AqH augments proinflammatory NO production in Mϕs via ascorbate. High concentrations of ascorbate in AqH interfere with Griess assays, highlighting the need to carefully control for interference when evaluating the influence of aqueous solutions on NO production in cultured cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Stephen A.K. Harvey for excellent technical assistance; Eric Romanowski and Kathy Yates from the Charles T. Campbell laboratory (University of Pittsburgh) for providing rabbit aqueous humor; Dr. Sidney Morris, Jr. (University of Pittsburgh) for providing anti-ARG1 Ab and helpful discussions; and Dr. Robert Hendricks (University of Pittsburgh) for review of the manuscript.

Footnotes

  • This work was supported by National Institutes of Health Grants EY018355 (to K.C.M.), HL76491 (to D.J.S.), and EY08098 (to K.C.M.); the Eye and Ear Foundation of Pittsburgh; and an unrestricted grant from the Research to Prevent Blindness.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    a.c.
    anterior chamber
    AqH
    aqueous humor
    CAT2B
    cationic amino acid transporter 2B
    CGRP
    calcitonin gene-related peptide
    CT
    cycle threshold
    GTPCH1
    GTP cyclohydrolase 1
    H2B
    dihydropterin
    H4B
    tetrahydrobiopterin
    l-Arg
    l-arginine
    Mϕ
    macrophage
    α-MSH
    α-melanocyte–stimulating hormone
    NOS
    NO synthase
    PEDF
    pigment epithelium-derived factor
    SGM
    standard growth medium.

  • Received June 22, 2012.
  • Accepted November 13, 2012.

References

  1. ↵
    1. Streilein J. W.
    2003. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nat. Rev. Immunol. 3: 879–889.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Niederkorn J. Y.
    2006. See no evil, hear no evil, do no evil: the lessons of immune privilege. Nat. Immunol. 7: 354–359.
    OpenUrlCrossRefPubMed
  3. ↵
    1. McKenna K. C.,
    2. P. W. Chen
    . 2010. Influence of immune privilege on ocular tumor development. Ocul. Immunol. Inflamm. 18: 80–90.
    OpenUrlCrossRefPubMed
  4. ↵
    1. McKenna K. C.,
    2. J. A. Kapp
    . 2004. Ocular immune privilege and CTL tolerance. Immunol. Res. 29: 103–112.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Vicetti Miguel R. D.,
    2. T. L. Cherpes,
    3. L. J. Watson,
    4. K. C. McKenna
    . 2010. CTL induction of tumoricidal nitric oxide production by intratumoral macrophages is critical for tumor elimination. J. Immunol. 185: 6706–6718.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Taylor, A. W. 2007. Ocular immunosuppressive microenvironment. Chem. Immunol. Allergy 92: 71–85.
  7. ↵
    1. Vodovotz Y.,
    2. C. Bogdan,
    3. J. Paik,
    4. Q. W. Xie,
    5. C. Nathan
    . 1993. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta. J. Exp. Med. 178: 605–613.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Star R. A.,
    2. N. Rajora,
    3. J. Huang,
    4. R. C. Stock,
    5. A. Catania,
    6. J. M. Lipton
    . 1995. Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone. Proc. Natl. Acad. Sci. USA 92: 8016–8020.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Taylor A. W.,
    2. D. G. Yee,
    3. J. W. Streilein
    . 1998. Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor. Invest. Ophthalmol. Vis. Sci. 39: 1372–1378.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Nicholson B.,
    2. C. K. Manner,
    3. J. Kleeman,
    4. C. L. MacLeod
    . 2001. Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J. Biol. Chem. 276: 15881–15885.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Mori M.
    2007. Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling. J. Nutr. 137(Suppl. 2): 1616S–1620S.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Bertholet S.,
    2. E. Tzeng,
    3. E. Felley-Bosco,
    4. J. Mauël
    . 1999. Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production. J. Leukoc. Biol. 65: 50–58.
    OpenUrlAbstract
  13. ↵
    1. Baek K. J.,
    2. B. A. Thiel,
    3. S. Lucas,
    4. D. J. Stuehr
    . 1993. Macrophage nitric oxide synthase subunits: purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J. Biol. Chem. 268: 21120–21129.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Ratovitski E. A.,
    2. C. Bao,
    3. R. A. Quick,
    4. A. McMillan,
    5. C. Kozlovsky,
    6. C. J. Lowenstein
    . 1999. An inducible nitric-oxide synthase (NOS)-associated protein inhibits NOS dimerization and activity. J. Biol. Chem. 274: 30250–30257.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Ratovitski E. A.,
    2. M. R. Alam,
    3. R. A. Quick,
    4. A. McMillan,
    5. C. Bao,
    6. C. Kozlovsky,
    7. T. A. Hand,
    8. R. C. Johnson,
    9. R. E. Mains,
    10. B. A. Eipper,
    11. C. J. Lowenstein
    . 1999. Kalirin inhibition of inducible nitric-oxide synthase. J. Biol. Chem. 274: 993–999.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Daniliuc S.,
    2. H. Bitterman,
    3. M. A. Rahat,
    4. A. Kinarty,
    5. D. Rosenzweig,
    6. L. Nitza
    . 2003. Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4. J. Immunol. 171: 3225–3232.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Albakri Q. A.,
    2. D. J. Stuehr
    . 1996. Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability. J. Biol. Chem. 271: 5414–5421.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Du Y. H.,
    2. Y. Y. Guan,
    3. N. J. Alp,
    4. K. M. Channon,
    5. A. F. Chen
    . 2008. Endothelium-specific GTP cyclohydrolase I overexpression attenuates blood pressure progression in salt-sensitive low-renin hypertension. Circulation 117: 1045–1054.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Cousins S. W.,
    2. M. M. McCabe,
    3. D. Danielpour,
    4. J. W. Streilein
    . 1991. Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest. Ophthalmol. Vis. Sci. 32: 2201–2211.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Taylor, A. W. 2003. Modulation of regulatory T cell immunity by the neuropeptide alpha-melanocyte stimulating hormone. Cell Mol. Biol. 49: 143–149.
  21. ↵
    1. Yoshida Y.,
    2. S. Yamagishi,
    3. S. Ueda,
    4. K. Yoshimura,
    5. S. Okuda,
    6. R. Yamakawa
    . 2007. Aqueous humour levels of asymmetric dimethylarginine (ADMA) are correlated with pigment epithelium-derived factor (PEDF) in patients with uveitis. J. Int. Med. Res. 35: 892–895.
    OpenUrlPubMed
  22. ↵
    1. Zamiri P.,
    2. S. Masli,
    3. J. W. Streilein,
    4. A. W. Taylor
    . 2006. Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation. Invest. Ophthalmol. Vis. Sci. 47: 3912–3918.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Racké K.,
    2. C. Hey,
    3. J. Mössner,
    4. R. Hammermann,
    5. C. Stichnote,
    6. I. Wessler
    . 1998. Activation of L-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages. J. Physiol. 511: 813–825.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Salimuddin A.,
    2. A. Nagasaki,
    3. T. Gotoh,
    4. H. Isobe,
    5. M. Mori
    . 1999. Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide. Am. J. Physiol. 277: E110–E117.
    OpenUrlPubMed
  25. ↵
    1. Stickings P.,
    2. S. K. Mistry,
    3. J. L. Boucher,
    4. S. M. Morris,
    5. J. M. Cunningham
    . 2002. Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans. Nitric Oxide 7: 289–296.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Tayeh M. A.,
    2. M. A. Marletta
    . 1989. Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate: tetrahydrobiopterin is required as a cofactor. J. Biol. Chem. 264: 19654–19658.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Rosenbaum J. T.,
    2. E. L. Howes Jr..,
    3. D. English
    . 1985. Ascorbate in aqueous humor protects against myeloperoxidase-induced oxidation. Am. J. Pathol. 120: 244–247.
    OpenUrlPubMed
  28. ↵
    1. Lam K. W.,
    2. P. Lee,
    3. R. Fox
    . 1976. Aqueous ascorbate concentration in hereditary buphthalmic rabbits. Arch. Ophthalmol. 94: 1565–1567.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Nakai K.,
    2. M. Urushihara,
    3. Y. Kubota,
    4. H. Kosaka
    . 2003. Ascorbate enhances iNOS activity by increasing tetrahydrobiopterin in RAW 264.7 cells. Free Radic. Biol. Med. 35: 929–937.
    OpenUrlCrossRefPubMed
  30. ↵
    Kleinbongard, P., T. Rassaf, A. Dejam, S. Kerber, and M. Kelm. 2002. Griess method for nitrite measurement of aqueous and protein-containing samples. Methods Enzymol. 359: 158–168.
  31. ↵
    1. Taylor A. W.,
    2. J. W. Streilein,
    3. S. W. Cousins
    . 1994. Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor. J. Immunol. 153: 1080–1086.
    OpenUrlAbstract
  32. ↵
    1. May J. M.,
    2. L. Li,
    3. Z. C. Qu,
    4. J. Huang
    . 2005. Ascorbate uptake and antioxidant function in peritoneal macrophages. Arch. Biochem. Biophys. 440: 165–172.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Green L. C.,
    2. D. A. Wagner,
    3. J. Glogowski,
    4. P. L. Skipper,
    5. J. S. Wishnok,
    6. S. R. Tannenbaum
    . 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126: 131–138.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Szabó C.,
    2. H. Ischiropoulos,
    3. R. Radi
    . 2007. Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat. Rev. Drug Discov. 6: 662–680.
    OpenUrlCrossRefPubMed
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