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*Compound via MeSH
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*2-AMINO-1,3,4-TRIAZOLE
*INDOMETHACIN
*NITRIC OXIDE
The Journal of Immunology, 2002, 169: 5889-5896.
Copyright © 2002 by The American Association of Immunologists

Characterization of Nitric Oxide Consumption Pathways by Normal, Chronic Granulomatous Disease and Myeloperoxidase-Deficient Human Neutrophils1

Stephen R. Clark*, Marcus J. Coffey*, Rhona M. Maclean{ddagger}, Peter W. Collins{ddagger}, Malcolm J. Lewis{dagger}, Andrew R. Cross§ and Valerie B. O’Donnell2,*

Departments of * Medical Biochemistry and Immunology, {dagger} Pharmacology, Therapeutics and Toxicology, {ddagger} Hematology, University of Wales College of Medicine, Cardiff, United Kingdom; and § Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H2O2 substrate since all superoxide (O2minusdu;) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O2minusdu; generation, significantly faster than expected from 1:1 termination of NO with O2minusdu; Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide regulates leukocyte recruitment and attachment through suppression of adhesion molecule activity, e.g., CD11/CD18 in neutrophils (1, 2, 3, 4). Therefore, strict control of NO is essential for regulating leukocyte trafficking in vivo. Neutrophils are the most abundant leukocyte and can consume NO in a partially superoxide dismutase (SOD)3-inhibitable manner (5). This indicates some involvement of NADPH oxidase, although the detailed mechanisms by which leukocytes control NO responses, particularly in response to physiological agonists, have not been characterized. NADPH oxidase-knockout mice show decreased parasite- or hypercholesterolemia-induced leukocyte adhesion and migration, suggesting that leukocyte superoxide (O2minusdu;) controls NO responses through causing its removal in vivo (6, 7). Finally, neutrophils contain several additional oxidases that can catalytically consume NO in vitro, including lipoxygenases (LOX), myeloperoxidase (MPO), and PGH synthase (PGHS), and the ability of these to modulate NO signaling within the cells has not been examined (8, 9, 10, 11, 12, 13, 14).

Herein, the mechanisms and consequences of NO consumption by isolated neutrophils from healthy, chronic granulomatous disease (CGD) or MPO-deficient humans activated with the bacterial peptide fMLP or the protein kinase C activator PMA were studied. The results reveal a critical role for NADPH oxidase with CGD neutrophils being unable to consume NO following stimulation. Rates of NO consumption by normal or MPO-deficient cells were severalfold faster than expected based on the 1:1 reaction between NO and O2minusdu;; however, there was no role for MPO due to insufficient formation of H2O2 substrate. Finally, NADPH oxidase-dependent NO removal effectively prevented activation of soluble guanylate cyclase (sGC). These data indicate that human neutrophil NADPH oxidase is a critical regulator of NO signaling in neutrophils, and reveal an important functional difference between CGD and either normal or MPO-deficient leukocytes that will alter their ability to overcome the inhibitory effects of NO on adhesion and migration in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Neutrophil isolation

Whole blood was obtained from healthy volunteers free from nonsteroidal anti-inflammatory drugs for over 14 days. MPO-deficient patients were identified by routine screening at University Hospital Wales (Cardiff, U.K.). Whole blood from CGD patient was a kind gift from L. Moreton (Great Ormond Street Hospital, London, U.K.). Ethical permission for all donations was obtained from the Bro Taf Local Research Ethics Committee. Human neutrophils were isolated as described previously, by dextran sedimentation and Ficoll centrifugation and resuspended in a small volume of PBS, counted, and kept on ice (15).

Neutrophil fractionation

For the preparation of subcellular fractions, neutrophils were obtained from normal subjects and CGD patients by leukapheresis (16) and purified as above with the omission of dextran sedimentation. Neutrophils were treated with 2.5 mM disopropyl fluorophosphate for 10 min at 4°C, disrupted in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1 mM ATP, 1.25 mM EGTA, 10 mM PIPES, pH 7.3) by N2 cavitation, and fractionated on discontinuous Percoll gradients (17, 18). This produced cytosol and plasma membrane fractions whose final concentrations were adjusted to 9 x 107 and 1.25 x 109 cell equivalents (cell eq) ml-1, respectively. Fractions were stored at -80°C for up to 1 year without loss of activity.

Purification, relipidation, and reflavination of flavocytochrome b558

Flavocytochrome b558 was purified from human neutrophils, using the methods described previously (19, 20). The final product contained 18–22 nmol heme mg-1 protein, based on a molar absorption coefficient of 21.6 mM-1 cm-1 (cytochrome heme) for the dithionite-reduced minus air-oxidized absorbance band at 559 nm (21). Typical activities are 120–150 mol O2- s-1 mol heme.

Purification and assay of MPO

MPO (specific activity = 0.49 ± 0.03 mol guaiacol min-1 mol) was purified from neutrophil azurophil granule preparations made as a byproduct during neutrophil fractionation described above, as described (22). MPO activity was assayed by the H2O2-stimulated rate of guaiacol oxidation at 37°C using {epsilon}470 = 26.6 mM cm-1 (23, 24).

NO consumption

Anaerobic solutions of NO were prepared as described previously and measured electrochemically using an NO sensor (Harvard AmiNO700 or World Precision Instruments 2-mm probe with inNO meter; Sarasota, FL) (9). Where NO loss was not linear, rates are given as first order rate constants (Kobs). For all Kobs, the square of the Pearson product moment correlation coefficient (r) of the slope of the replotted data was >0.9, confirming that the reaction was first order. Where NO consumption was linear, NO disappearance was determined and the background rate of NO loss subtracted. For measurement of neutrophil NO consumption, NO (1.9–3.8 µM) was added to 0.5 ml PBS, 2.5 mM CaCl2, and 1.2 mM MgCl2 with neutrophils at 37°C with stirring. Once the electrode response had stabilized, 1 µg ml-1 PMA or 1 µM fMLP was added. NO consumption by isolated neutrophil membranes was measured following addition of 160 µM NADPH to 0.5 ml relaxation buffer containing 5.4 x 106 cell eq of membrane extract ml-1, 1.5 x 107 cell eq of cytosol ml-1, 10 µM GTP-{gamma}-S, and 100 µM SDS, in the chamber of the NO electrode at 37°C with stirring. To allow assembly of NADPH oxidase components, all constituents (except NADPH) were preincubated at 25°C for 2 min, followed by 37°C for 3 min before addition of 3.8 µM NO and/or NADPH.

RIA of cGMP

Neutrophils (4 x 106 cells ml-1) in 500 µl PBS, 2.5 mM CaCl2, and 1.2 mM MgCl2 were placed in the chamber of the NO electrode with stirring at 37°C. NO (1.9 µM) was added, then 1 µM fMLP with/without 3 µM oxyHb. In other experiments, 1 µg ml-1 PMA was added, with the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX; 1 mM). Samples were incubated for 5 min then aliquots removed for cGMP analysis using a commercial RIA (Amersham Pharmacia Biotech, Bucks, U.K.).

O2- generation assay

Neutrophil O2- generation was assayed by the SOD-inhibitable reduction of cytochrome c (cyt. c) measured at 37°C with stirring using {epsilon}550 = 21.1 mM cm-1 (25). fMLP (1 µM) was added to 0.25 x 106 ml-1 neutrophils (106 ml-1 for CGD neutrophils) in 2 ml PBS with 2.5 mM CaCl2, 1.2 mM MgCl2, and 50 µM cyt. c with/without SOD (300 U ml-1; Oxis, Portland, OR). O2- generation by isolated membranes was measured on addition of 160 µM NADPH to 0.75 ml relaxation buffer containing 5.4 x 106 cell eq of membrane extract ml-1, 1.5 x 107 cell eq of cytosol ml-1, 10 µM GTP-{gamma}-S, 100 µM SDS, 50 µM cyt. c. To allow assembly of NADPH oxidase components, all constituents (except NADPH) were preincubated at 25°C for 2 min, followed by 37°C for 3 min before addition of NO and/or NADPH.

O2 consumption assay

Human neutrophil O2 consumption was measured electrochemically using a Clark-type O2 electrode (Rank Brothers, Cambridge, U.K.). Calibrations were performed by addition of H2O2 to PBS with 34 U ml-1 catalase. A total of 1 µM fMLP was added to 0.5 ml PBS with neutrophils (2 x 106 cells ml-1), 2.5 mM CaCl2, 1.2 mM MgCl2, 300 U ml-1 CuZn-SOD, and 34 U ml-1 catalase at 37°C with stirring. Excess SOD and catalase were routinely included during neutrophil O2 uptake measurements to ensure full reduction of O2- to H2O, according to the following equation:

Measured rates of O2 consumption were multiplied by four to give true O2- generation rates.

NADPH oxidation assay

NADPH oxidation was determined at 340 nm, following addition of 160 µM NADPH to 0.75 ml relaxation buffer with 1.5 x 107 cell eq of cytosol ml-1, 10 µM GTP-{gamma}-S, 100 µM SDS, and 2.7 x 106 cell eq ml-1 neutrophil membranes from a normal subject, a patient with partially functional flavocytochrome b558 designated X91+ (26), or purified flavocytochrome b558, at 25°C. To allow assembly of NADPH oxidase components, all constituents (except NADPH) were preincubated at 25°C for 5 min, before NADPH addition. Absorbance was measured before and after addition of 345 µM 2-(N,N-diethylamino)-diazenolate-2-oxide (DeaNONOate) which releases 30 µM NO min-1 (27).

Assays of neutrophil MPO

Western blotting was performed as described (28, 29, 30). Briefly, samples (105 cell eq) were probed with rabbit polyclonal anti-human MPO Ab (Calbiochem, San Diego, CA) (1:1000) and visualized using ECL (Amersham Pharmacia Biotech). MPO activity was assayed as described for purified MPO in the absence or presence of 2 mM aminotriazole (ATZ) or 1 mM azide to enable MPO vs eosinophil peroxidase to be determined (24).

Kinetic simulations

To understand the chemical behavior of reactive intermediates generated by activated neutrophils, simulations were performed using the Euler method, with software written by F. Neese (Unversität Konstanz, Germany). This algorithm uses numerical methods to solve the simultaneous differential equations generated from the reactions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of NO loss in the electrode system

NO (2–4 µM) decay in aerobic buffer at 37°C followed first order kinetics, with Kobs depending on probe (Kobs = 2.8 ± 0.3 x 10-3 sec-1 r = 0.99, or 7.9 ± 0.9 x 10-3 sec-1 r = 0.99, for World Precision Instruments 2 mm or Harvard AmiNO700 probes, respectively). This indicates that NO oxidation by the electrode and diffusion into the gas phase cause NO decay in these experiments, rather than autoxidation to form NO2- which is second order. Using these Kobs, rates of background NO loss were subtracted from all experiments. For accurate determination of NO consumption rates by cells, bolus additions of NO were used instead of donor compounds.

Activated human neutrophils consume NO

In the presence of resting neutrophils, NO disappearance from aerobic buffer did not increase over background and was still first order (Kobs = 2.8 ± 0.1 or 2.4 ± 0.5 x 10-3 sec-1 for buffer alone, or with cells, respectively). Neutrophil activation with 1 µM fMLP caused an immediate increase in NO loss (8.7 ± 0.8 nmol min-1 106 cells; Fig. 1GoA). Similar results were obtained using PMA (1 µg ml-1) as stimulus (data not shown).



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  		FIGURE 1. fMLP-activated neutrophils consume NO through NADPH oxidase turnover. A, i, NO (1.9 µM) was added to 0.5 ml PBS, with neutrophils (0.25 x 106 cells ml-1), 2.5 mM Ca2+, and 1.2 mM Mg2+ and its removal measured as described in Materials and Methods. ii, fMLP (1 µM) was added after NO as shown by arrow, and a second bolus of NO once concentrations had reached zero. iii, Cells with 3,000 U ml-1 SOD and 20 µM DPI, activated with fMLP as shown by arrow. B, NO consumption determined as shown in A. Samples contained 20 µM DPI, 3,000 U ml-1 SOD or 20 µM MnTE-2-PyP5+ (MnP) as shown (n >= 3, mean ± SD). **, p < 0.01 vs control, ***, p < 0.001 vs control using one-way ANOVA and Tukey post hoc test. All results are shown of a representative experiment, repeated with at least three different donors. C, NO was added to 0.5 ml PBS containing neutrophils from a CGD patient (1 x 106 cells ml-1), 2.5 mM Ca2+, and 1.2 mM Mg2, as described in Materials and Methods. fMLP (1 µM) was added, as indicated by arrow. Inset, fMLP (1 µM) was added to 2 ml PBS containing CGD neutrophils (1 x 106 cells ml-1), 50 µM cyt. c, 2.5 mM Ca2+, and 1.2 mM Mg2 and O2- generation determined as described in Materials and Methods. A control trace from a healthy volunteer is included for reference.

 
NO removal is dependent on O2- generation by NADPH oxidase

To determine the involvement of O2- in NO consumption, 3000 U ml-1 SOD or 20 µM diphenyleneiodonium (DPI) were added. Either agent alone significantly inhibited neutrophil NO consumption, however in combination, NO removal was totally blocked (Fig. 1GoB). Incubation with the SOD-mimetic manganese porphyrin MnTE-2-PyP5+ (MnP, 20 µM) also attenuated NO consumption (Fig. 1GoB). In further support of the central role of O2- in mediating NO consumption, neutrophils from a CGD patient did not consume NO (Kobs for NO decay = 7.0 ± 1.3 x 10-3 sec-1, vs 7.5 ± 0.4 x 10-3 sec-1 before or after fMLP addition, respectively; Fig. 1GoC).

The rate of NO removal is greater than O2- production

NO removal by fMLP-activated healthy neutrophils was consistently faster than O2- generation, measured as SOD-sensitive cyt. c reduction in the absence of NO (e.g., for one representative donor, NO consumption and O2- generation were 8.7 ± 0.8 and 3.4 ± 0.8 nmoles min-1 106cells, respectively; Fig. 2GoA). Similar results were obtained using PMA (data not shown). All experiments were conducted in HEPES- and glucose-free PBS (supplemented with CaCl2 and MgCl2) to ensure that artifactual radical reactions did not cause NO consumption, although comparisons in Kreb’s Ringer buffer yielded identical data (data not shown). To confirm that cyt. c reduction was an accurate measure of O2- generation, three additional approaches were taken.



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  		FIGURE 2. NO consumption by fMLP-stimulated human neutrophils is faster than O2- generation. A, left bar, O2- production measured by the CuZn-SOD-inhibitable reduction of cyt. c at 550 nm. fMLP (1 µM) was added to 2 ml PBS containing human neutrophils (1 x 106 cells ml-1), 50 µM cyt. c, 2.5 mM Ca2+, and 1.2 mM Mg2+ as described in Materials and Methods. Right bar, NO consumption was measured following fMLP (1 µM) addition to 0.5 ml PBS containing NO (3.8 µM) neutrophils (1 x 106 cells ml-1), 2.5 mM Ca2+, and 1.2 mM Mg2+ as described in Materials and Methods. Results shown are of a representative experiment from a singe donor repeated with at least three donors (n >= 3, mean ± SD). ***, p < 0.001 vs NO uptake using an unpaired Student t test. B, right bar, NO consumption was measured following addition of NADPH (160 µM) to 0.5 ml relaxation buffer containing NO (3.8 µM), 5.4 x 106 cell eq of membrane extract ml-1, 1.5 x 107 cell eq of cytosol ml-1, GTP-{gamma}-S (10 µM), and SDS (100 µM) as described in Materials and Methods. Left bar, O2- production was measured by CuZn-SOD-inhibitable reduction of cyt. c at 550 nm. NADPH (160 µM) was added to 0.75 ml relaxation buffer as above but with the inclusion of 50 µM cyt. c. (n >= 3, mean ± SD). ***, p < 0.001 vs NO uptake using an unpaired Student t test. C, NADPH oxidation by purified flavocytochrome was monitored at 340 nm following addition of NADPH (160 µM) to 0.75 ml relaxation buffer with 2.7 x 106 cell eq of membrane extract ml-1, 1.5 x 107 cell eq of cytosol ml-1, 10 µM GTP-{gamma}-S, and 100 µM SDS as described in Materials and Methods. Rates of NADPH oxidation were measured before and after addition of 345 µM DeaNONOate which gives an NO release rate of 30 µM NO min-1 (t1/2 = 16 min, 22–25°C; Ref. 27 ). D, NADPH oxidation by isolated membranes from a CGD patient with functional NADPH oxidase flavin (X91+) was measured following addition of NADPH (160 µM) to 0.75 ml relaxation buffer with 2.7 x 106 cell eq of membrane ml-1, 1.5 x 107 cell eq of cytosol ml-1, 10 µM GTP-{gamma}-S, and 100 µM SDS as described in Materials and Methods. NADPH oxidation was measured before and after addition of 345 µM DeaNONOate. NADPH oxidation by healthy neutrophil membranes is included for reference (n >= 3, mean ± SD).

 
Cell-free reconstitution system. Intact cells contain intracellular phagolysosomal compartments that might cause underestimation of O2- generation. Therefore, premixing isolated cytosol and membrane with SDS and GTP-{gamma}-S allows NADPH oxidase to be assayed in a detergent-containing cell-free system, where all O2- is accessible (22). Using membranes prepared from healthy neutrophils, rates of NO consumption were significantly faster than O2- generation (1.97 ± 0.08 vs 0.95 ± 0.14 nmol min-1 106 cell eq, respectively; Fig. 2GoB), similar to intact cell experiments (Fig. 2GoA).

O2 consumption. During O2- generation, stoichiometric amounts of O2 are consumed through reduction by NADPH oxidase. Rates of fMLP-stimulated O2 consumption were not significantly different to those measured using cyt. c reduction (13.8 ± 1.8 vs 15.4 ± 0.2 µM min-1 for cyt. c reduction and O2 consumption, respectively).

Simulations. To examine the fate of NO and oxidant species generated in our experiments, reactions were simulated using rate constants in Table IGo. This showed that under our conditions all O2- directly reacted with cyt. c, without dismutation to H2O2. Although rate constants for spontaneous O2- dismutation and the reaction of O2- with cyt. c are similar, dismutation of O2- is second order. Therefore, in the presence of 50 µM cyt. c, continuous removal of O2- and stiochiometric reduction of cyt. c occurs.


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  		Table I. Rate constants used for kinetic simulation of cyt. c assay (see main text for results)

 
Collectively, these data indicate that O2- generation rates are accurately determined in our experiments and confirm that NADPH oxidase-dependent NO consumption is considerably faster than the simple 1:1 reaction expected from termination of NO and O2-.

NO does not stimulate NADPH oxidase

To determine whether the fast rates of NO consumption were due to direct stimulation of electron flux through NADPH oxidase by NO (31), NADPH oxidation was determined using the cell-free reconstitution system as above where the enzyme is already active before NO addition. In this study, NADPH oxidation by highly purified flavocytochrome b558 was not stimulated by NO (Fig. 2GoC). Also, NADPH oxidation by membranes from a CGD patient with partial NADPH oxidase activity, designated X91+, (26) was not stimulated by NO (Fig. 2GoD). This mutant enzyme cannot directly reduce O2 but can transfer electrons from NADPH to the flavin center and thence to artificial electron acceptors, and along with the purified flavocytochrome b558 was used to reveal whether NADPH oxidase could directly reduce NO via the flavin or heme cofactors. For comparison, NADPH oxidation by normal membranes is shown (Fig. 2GoD).

Effect of inhibitors and scavengers on NO consumption

Addition of 100 µM diethylenetriaminopentaacetic acid, 20 µM indomethacin, 2 mM ATZ, 1 mM azide, or 2 mM urate were without effect on NO consumption, ruling out a role for Fenton chemistry, PGHS, or MPO (Fig. 3Go). Although urate is an effective scavenger of peroxynitrite (ONOO-), its oxidation to radicals that can potentiate secondary oxidation processes do not rule out a role for ONOO- in mediating NO consumption (32, 33).



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  		FIGURE 3. Effect of enzyme inhibitors and scavengers on NO consumption. NO consumption was measured following addition of fMLP (1 µM) to 0.5 ml PBS containing 3.8 µM NO, 106 cells ml-1 neutrophils, 2.5 mM Ca2+, and 1.2 mM Mg2+ as described in Materials and Methods. 2 mM urate, 100 µM diethylenetriaminopentaacetic acid, 2 mM ATZ, 1 mM azide, or 5 min preincubation with 20 µM indomethacin were added as shown (for all experiments, n >= 3, mean ± SD).

 
The rate of NO consumption by MPO-deficient neutrophils is greater than the rate of O2- production

Neutrophils were isolated from a patient with MPO activity that was 9% of healthy controls (Fig. 4GoA). Western blotting of these cells showed virtually undetectable MPO at 60 kDa, and the heme spectrum at 472 nm was absent (Fig. 4GoA, inset and data not shown). NO consumption by MPO-deficient neutrophils was considerably faster than O2- generation (14.59 ± 2.26 vs 4.45 ± 0.50 nmol min-1 106 cells, respectively; Fig. 4GoB). These differences are even greater than for healthy neutrophils (Fig. 2GoA). Addition of purified MPO to MPO-deficient cells at concentrations found in healthy subjects (11.25 pmols/106 cells, calculated from guaiacol oxidation rates and heme spectra) did not further stimulate NO consumption (14.90 ± 3.67 nmol min-1 106 cells; Fig. 4GoC). However, with 100 µM H2O2 substrate, this concentration of MPO consumed NO at easily detectable rates (4.2 µM min-1, which would be equivalent to 5.26 ± 0.70 nmol min-1 106 cells, data not shown). This indicates that unlike exogenous H2O2, fMLP-activated neutrophils cannot support MPO-dependent NO consumption.



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  		FIGURE 4. MPO-deficient neutrophils reveal no role for MPO in mediating neutrophil NO consumption. A, Neutrophils identified as peroxidase negative on routine hematology screening were examined for MPO activity as determined as described in Materials and Methods (n >= 3, mean ± SD). ***, p < 0.001 vs healthy neutrophils (control) using unpaired Student t test. Inset, Western blot of control and MPO-deficient neutrophils, showing 60-kDa MPO subunit. B, left bar, O2- generation measured by SOD-inhibitable reduction of cyt. c at 550 nm. fMLP (1 µM) was added to 2 ml PBS containing human neutrophils (0.4 x 106 cells ml-1), 50 µM cyt. c, 2.5 mM Ca2+, and 1.2 mM Mg2+ at 37°C with stirring as described in Materials and Methods. Right bar, NO consumption was measured following addition of fMLP (1 µM) to 0.5 ml PBS containing NO (3.8 µM), neutrophils (0.4 x 106 cells ml-1), 2.5 mM Ca2+, and 1.2 mM Mg2+ as described in Materials and Methods (n >= 3, mean ± SD). ***, p < 0.001 vs NO uptake using an unpaired Student t test. C, NO consumption was measured as above, but with/without addition of purified MPO at a concentration equivalent to the enzyme activity of normal cells, determined by heme spectroscopy and guaiacol oxidation rates (11.25 pmol/106 cells) (n >= 3, mean ± SD).

 
Kinetic simulations of NO consumption by O2- and MPO

Reactions for NO included its diffusion-limited reaction with O2- forming ONOO-, the first-order background rates of NO decay calculated herein, and the slow rate of NO reaction with ONOO- (Table IIGo, equations 2, 4, and 6), but the second-order autoxidation of NO was omitted since this did not appreciably contribute to NO decay in our system. Although neutrophils contain intracellular SOD, O2- is generated extracellularly in these experiments, so dismutation that occurs will be spontaneous (Table IIGo, equation 3). Some simulations also included MPO, with NO consumption occurring via reduction of either compound I or II and using either H2O2 or ONOO- as oxidants (equations 10–13). MPO oxidation by ONOO- directly forms compound II with no detectable compound I (34). The rate constant for NO oxidation by compound I has not been determined; however, this reaction is considerably faster than compound II reduction by NO (equation 13; Ref. 12). Initial modeling experiments used several values from that equal to equation 13, up to diffusion limited (109 M-1 s-1) for equation 12, and found that the rate of H2O2-dependent NO consumption by MPO was independent of the value of this rate constant. Therefore, our final model uses the value of equation 13 to model the complete set of NO consumption reactions by MPO.


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  		Table II. Kinetic simulation of NO consumption

 
Initial simulations using equations 1–4 (Table IIGo) at O2- generation rates of 1.7 µM min-1 (i.e., equivalent to 3.4 nmol min-1 106 cells), where NO only reacted with O2-, revealed that NO consumption rates were identical with O2- generation, with all O2- forming ONOO-, and virtually no dismutation to H2O2 (Fig. 5GoA). Including reactions of MPO caused no change in the rate of NO decay (Fig. 5GoA, Table IIGo, equations 10–13). The lack of MPO-catalyzed NO consumption results from insufficient generation of H2O2 substrate (<1 pM). Although ONOO- could potentially stimulate MPO-dependent NO consumption, its concentration remained <100 nM (34). The complete reaction sequence incorporating equations 1–9 shows the formation and decay of NO, O2-, H2O2, and ONOO- during NADPH oxidase-dependent NO consumption and clearly shows that at these O2- generation rates, H2O2 does not form until all NO has been consumed (Fig. 5GoB).



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  		FIGURE 5. Kinetic simulations of NO consumption by activated human neutrophils. Kinetic simulations of the reactions occurring during the activation of neutrophils in the presence of NO, using the Euler method, were performed as described in Materials and Methods using reactions and rate constants as in Table IIGo. A, Progress curves for NO consumption curves were generated using: i, initial reactions only, equations 1–4; ii, initial and MPO-dependent reactions, equations 1–4 and 10–13. B, Simulations using equations 1–9 showing progress curves for H2O2, O2-, NO, and ONOO- generation and decay. The concentrations of H2O2 and ONOO- do not exceed 1 fM and 80 nM, respectively, during rates of O2- generation occurring in our neutrophil experiments in the presence of NO. Once NO is consumed, H2O2 and O2- levels increase to micromolar levels, and ONOO- disappears.

 
NO consumption prevents neutrophil sGC activation

cGMP generation was determined following a 5-min incubation of neutrophils with 1.9 µM NO, with or without fMLP, or oxyHb as NO scavenger (Fig. 6GoA). Experiments were repeated with the phosphodiesterase inhibitor IBMX which inhibits cGMP hydrolysis. IBMX blocks agonist-induced neutrophil activation (via cAMP elevation); therefore, in this experiment, cells were stimulated with PMA (Fig. 6GoB). Following incubation with 1.9 µM NO, elevations in neutrophil cGMP were found; however, this was effectively inhibited by simultaneous generation of O2- (Fig. 6Go). Addition of 3 µM oxyHb to scavenge NO also fully blocked NO activation of sGC (Fig. 6Go).



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  		FIGURE 6. NADPH oxidase-dependent NO removal prevents neutrophil sGC activation. NO (1.9 µM) was added to 0.5 ml PBS containing 4 x 106 cells ml-1 human neutrophils, 2.5 mM Ca2+, and 1.2 mM Mg2+ as described in Materials and Methods. Immediately following NO addition, 1 µM fMLP, 1 µg ml-1 PMA, or 3 µM oxyHb was added. Samples were incubated for 5 min at 37°C before analysis of cGMP by RIA. A, Neutrophils were activated using fMLP. B, Neutrophils were activated using PMA in the presence of 1 mM IBMX. Results are of a representative experiment from a single donor repeated with at least three donors (n = 3, mean ± SD). ***, p < 0.001 compared NO alone, using one-way ANOVA and Tukey post hoc test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, normal or MPO-deficient, but not CGD human neutrophils consumed NO through NADPH oxidase-dependent mechanisms at rates significantly faster than corresponding O2- generation (Figs. 1Go and 2Go). Also, NO consumption effectively blocked cGMP synthesis in the neutrophils (Figs. 1GoC and 5). This indicates that the ability of activated leukocytes to attenuate the bioactivity of NO is considerably greater than expected from the simple 1:1 termination between NO and O2-, and that NADPH oxidase deficiency in CGD will prevent neutrophils from attenuating the inhibitory effects of NO in vivo.

Herein, a combination of SOD and DPI fully inhibited NO consumption (Fig. 1GoB). The incomplete inhibition by SOD alone results from SOD-catalyzed O2- generation which occurs when H2O2 builds up in the presence of high concentrations of SOD (J. P. Crow and J. S. Beckman, unpublished observations). Similarly, DPI alone did not fully inhibit NO consumption (Fig. 1Go). DPI reacts with a catalytic intermediate formed during flavin turnover; therefore, some enzyme catalysis occurs before full inhibition (35). The combination of DPI and SOD was fully effective, as residual O2- produced during DPI inhibition was scavenged by SOD (Fig. 1GoB). Along with the lack of NO consumption by CGD cells, these data indicate that NADPH oxidase is absolutely required for fMLP or PMA-stimulated NO consumption by acutely activated human neutrophils (Fig. 1GoC).

Several additional oxidases could potentially consume NO in leukocytes, for example, PGHS-1, 15-LOX, and MPO (10, 11, 12, 14). However, these did not remove NO following acute activation of neutrophils. In particular, the lack of NO consumption by MPO was intriguing since this enzyme constitutes 5% of neutrophil protein, and was recently shown to catalyze H2O2-dependent NO consumption following either cellular overexpression or transcytosis of MPO into rat aortic endothelium (14). The inability of MPO to catalyze NO consumption following addition of exogenous purified enzyme to MPO-deficient cells shows that even when all MPO is extracellular, fMLP-stimulated neutrophils cannot support MPO-dependent NO consumption (Fig. 4GoC). Furthermore, azide and ATZ which effectively block consumption of NO by purified MPO and leukocyte-dependent nitration of tyrosine are without effect on neutrophil NO consumption (Fig. 3Go; Refs. 12 and 36). NO metabolism by purified or cellular MPO is critically dependent on exogenously added H2O2 (12, 14). In this study, kinetic simulations showed that all O2- generated by agonist-activated neutrophils in the presence of NO forms ONOO-, with no dismutation to H2O2 (Fig. 5GoB). In agreement, total inhibition of H2O2 generation by macrophages in the presence of NO was previously reported (37). These observations don’t exclude a role for MPO in catalyzing NO consumption when H2O2 is generated by NADPH oxidase-independent mechanisms. In this regard, LPS injection in vivo attenuates acetylcholine-dependent vasorelaxation in wild type, but not MPO-/- mouse aortic rings (14). In that system, H2O2 could form independent of O2- from diverse vascular and reticuloendothelial sources, including xanthine oxidase catalysis or mitochondrial leakage of electrons (38, 39).

Others have suggested that leukocyte-contained MPO may promote different reactions than MPO present in the extracellular milieu, following observations that neutrophil-associated MPO does not nitrate phagocytosed probes or bacterial proteins, in contrast to purified MPO (40, 41). In addition, critical differences are emerging regarding the relative rates of MPO/NO2-/H2O2-dependent reactions in different types of inflammation. For example, recent studies using MPO-/- mice showed that the contribution of MPO/NO2-/H2O2 to nitration reactions is highly disease model-dependent with no role found in leukocyte-rich acute inflammatory models (42).

In contrast to MPO, PGHS expression by freshly isolated neutrophils is low, suggesting that this pathway may not consume NO in resting neutrophils (43). Finally, while neutrophils constitutively express 5-LOX protein, its activation in response to fMLP is poor (44).

An unexpected finding was that NO consumption was considerably faster than O2- generation. To determine whether this resulted from either 1) enhanced NADPH oxidase assembly rates, or 2) stimulation of electron flux through NADPH oxidase by NO, a cell-free reconstitution system was used, where the complex preassembled in the absence of NO, and electron flux was directly measured by NADPH oxidation (31, 45, 46, 47). Using membranes from a normal subject, NO consumption was significantly faster than O2- generation, similar to agonist-activated cells, indicating that accelerated NO consumption does not require NO to be present during NADPH oxidase assembly (Fig. 2GoB). Next, NADPH oxidation was determined in the reconstitution system using either highly purified flavocytochrome b558 or membranes from a patient with partially functional oxidase (X91+; Ref. 26). In this study, NO did not enhance NADPH oxidation by either enzyme preparation, demonstrating that it does not stimulate electron flux through turning-over enzyme, nor accept electrons directly from reduced NADPH oxidase flavin (Fig. 2GoD). Collectively, these data indicate that the accelerated rates of NO consumption are not due to NADPH oxidase stimulation by NO.

ONOO- is a reactive species that crosses cell membranes, mediating oxidation of intracellular constituents including sugars, thiols, proteins, and lipid (48, 49, 50, 51, 52, 53). Its addition to macrophages forms electron paramagnetic resonance-detectable thiol and protein tyrosyl radicals that may directly consume NO (54). Inclusion of the ONOO- scavenger urate did not significantly attenuate neutrophil NO consumption. However, urate oxidation by ONOO- forms reactive radicals that can still consume NO (33). Therefore, although this could not be conclusively proven, oxidation of biomolecules by ONOO- in activated neutrophils may cause the accelerated NO consumption observed herein.

An important finding was the profound differences in NO-metabolizing capacity of CGD vs either MPO-deficient or healthy neutrophils. CGD is a rare and devastating condition, characterized by recurrent life-threatening infections, whereas MPO deficiency is relatively common (1:2000), with only a slight increased susceptibility to Candida infection (55, 56, 57). Reasons for this difference are unclear since both CGD and MPO-deficient phagocytes show similarly impaired bacterial killing in vitro (57). In the vasculature, phagocyte adhesion and migration is suppressed through the actions of endothelial-derived NO via cGMP-dependent inhibition of CD18/CD11 activity (1, 4, 58, 59). Therefore, the inability of CGD leukocytes to metabolize NO may render them more sensitive to its inhibitory effects. In vitro studies under NO-free conditions show that chemotaxis of neutrophils from CGD patients does not differ from healthy controls (60). However, decreased leukocyte adhesion and emigration in cholesterol-fed p47phox-/- mice and delayed monocyte or T cell migration into livers of Leishmania donovanii-infected gp91phox-/- mice indicate that lack of NADPH oxidase renders leukocytes less able to migrate during inflammation in vivo (6, 7). This is consistent with a critical role for NADPH oxidase in regulating leukocyte responses to NO, and would not be expected in MPO deficiency where NO consumption following agonist activation is preserved (Fig. 4Go).

In summary, human neutrophils consume NO at unexpectedly fast rates via NADPH oxidase turnover, effectively inhibiting NO signaling in the cells themselves (Fig. 6Go). These findings have implications for the role of NADPH oxidase in the development of inflammatory vascular disease, and for the pathophysiology of CGD where leukocyte NO consumption following agonist activation is absent.


   Acknowledgments
 
We thank Prof. B. A. Freeman for helpful suggestions and L. Moreton for gift of CGD neutrophils.


   Footnotes
 
1 This work was supported by grants from the Wellcome Trust (to V.B.O.), British Heart Foundation (to V.B.O., M.J.L., and S.R.C.), and National Institutes of Health (RO1 A124838; to A.R.C.). Back

2 Address correspondence and reprint requests to Dr. Valerie B. O’Donnell, Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K. E-mail address: o-donnellvb{at}cardiff.ac.uk Back

3 Abbreviations used in this paper: SOD, superoxide dismutase; O2·-, superoxide; PGHS, PGH synthase; MPO, myeloperoxidase; CGD, chronic granulomatous disease; ONOO-, peroxynitrite; cell eq, cell equivalent; DPI, diphenyleneiodonium; cyt. c, cytochrome c; LOX, lipoxygenase; IBMX, 3-isobutyl-1-methyl-xanthine; DeaNONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide; ATZ, aminotriazole; sGC, soluble guanylate cyclase. Back

Received for publication July 15, 2002. Accepted for publication September 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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