Nitrolinoleate Inhibits Superoxide Generation, Degranulation, and Integrin Expression by Human Neutrophils
Novel Antiinflammatory Properties of Nitric Oxide–Derived Reactive Species in Vascular Cells
Nitration of unsaturated fatty acids such as linoleate by NO-derived reactive species forms novel derivatives (including nitrolinoleate [LNO2]) that can stimulate smooth muscle relaxation and block platelet activation by either NO/cGMP or cAMP-dependent mechanisms. Here, LNO2 was observed to inhibit human neutrophil function. LNO2, but not linoleic acid or the nitrated amino acid 3-nitrotyrosine, dose-dependently (0.2 to 1 μmol/L) inhibited superoxide (O2·−) generation, Ca2+ influx, elastase release, and CD11b expression in response to either phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. LNO2 did not elevate cGMP, and inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one did not restore neutrophil responses, ruling out a role for NO. In contrast, LNO2 caused elevations in intracellular cAMP in the presence and absence of phosphodiesterase inhibition, suggesting activation of adenylate cyclase. Compared with phorbol 12-myristate 13-acetate–activated neutrophils, N-formyl-Met-Leu-Phe–activated neutrophils were more susceptible to the inhibitory effects of LNO2, indicating that LNO2 may inhibit signaling both upstream and downstream of protein kinase C. These data suggest novel signaling actions for LNO2 in mediating its potent inhibitory actions. Thus, nitration of lipids by NO-derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease.
Nitric oxide is a free radical signaling mediator generated by the healthy endothelium to maintain vascular homeostasis through the regulation of blood pressure and leukocyte-platelet activation. When generated at elevated levels during inflammation by inducible NO synthase in a variety of cell types, NO is readily transformed into potent nitrating and nitrosating species, including peroxynitrite (ONOO−), nitrogen dioxide (NO2), and nitrous acid (HONO). These species can react with unsaturated lipids, forming both oxidized and nitrated products, including nitro, nitrito, and nitroepoxy derivatives.1–4⇓⇓⇓ Several recent studies have demonstrated in vivo generation of nitrated lipids. For example, bovine cardiac muscle and human plasma contain species with chromatographic and mass spectral properties identical to those of nitrated arachidonate and linoleate, whereas plasma from rats exposed to liver ischemia/reperfusion contains nitrolinoleate5,6⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). Treatment of LDL with nitrating/nitrosating and oxidizing species that are generated during myeloperoxidase oxidation of nitrite (NO2−) causes its modification to a form recognized by the macrophage scavenger receptor CD36.7,8⇓ Also, macrophage uptake of LDL treated with nitrating/nitrosating species stimulates cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation.9,10⇓ Finally, synthetic nitrated lipids derived from either arachidonate or linoleate inhibit multiple indices of platelet activation and cause smooth muscle relaxation5,11⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). These observations raise the possibility that nitrated lipids could modulate atherogenesis and/or inflammation in vivo.
Activation of phagocytic leukocytes, including neutrophils, is a central feature of inflammatory disease.12–16⇓⇓⇓⇓ For example, neutrophil rolling and adhesion to the endothelium are enhanced in apoE and double apoE/LDL receptor–knockout mice, whereas neutrophils from septic patients show alterations in integrin expression and O2·− generation.13–16⇓⇓⇓ Also, mice deficient in adhesion molecules or chemokines and their receptors show impaired immune responses and diminished atherosclerosis.12,17–19⇓⇓⇓ Neutrophil function in vivo can be regulated through NO- and eicosanoid-dependent mechanisms.20–22⇓⇓ In this regard, nitrated unsaturated lipids formed by reaction with NO-derived reactive nitrogen species are of potential interest, inasmuch as they could influence neutrophil activation through either reactivity and may lead to the development of novel therapeutic strategies for inflammation.
A synthetic nitrated lipid, nitrolinoleate (LNO2), which is structurally similar to that generated by ONOO−- or NO2-induced linoleate nitration, is reported in the present study to inhibit N-formyl-Met-Leu-Phe (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-mediated activation of human neutrophils.1 Superoxide generation, degranulation, and CD11b expression were inhibited at LNO2 concentrations <1 μmol/L and occurred in concert with attenuated elevations in intracellular Ca2+ and increased intracellular cAMP. Compared with PMA-activated neutrophils, fMLP-activated neutrophils were more susceptible to LNO2 inhibition, indicating that LNO2 may inhibit signaling upstream and downstream of protein kinase C. In aggregate, these data reveal that nitrated lipids potently inhibit leukocyte activation and demonstrate novel mechanisms by which NO-derived nitrated biomolecules may attenuate tissue inflammatory responses.
Materials and Methods
Lymphoprep was obtained from Nycomed Pharma. Unless otherwise stated, all other reagents were obtained from Sigma-Aldrich Ltd.
Synthesis and Purification of LNO2
The synthesis and purification of LNO2 have recently been described elsewhere and have been adapted from previous strategies for the synthesis of conjugated nitroalkenes via nitroselenylation of alkenes.11,23,24⇓⇓ Briefly, linoleic acid (LA), HgCl2, PheSeBr, and AgNO2 (1.0:1.3:1.0:1.0 [mol/mol]) were combined in THF-acetonitrile (1:1 [vol/vol]), with a final concentration of 0.15 mol/L LA. The mixture was degassed and stirred at 25°C in the dark for 1.5 hours, the supernatant was recovered, and solvent was removed in vacuo. Lipid was solvated again in THF, and a 10-fold molar excess of H2O2 was added to the lipid mixture after cooling to 0°C. After 20 minutes, the solution was warmed to ≈25°C until gas evolution was noted. The solution was cooled to 10°C, followed by stirring at ≈25°C for 30 minutes. Lipids were extracted and then chromatographed on a silica gel column (250 to 400 mesh) using a hexane-CHCl3 step gradient (5% increments from 0% to 30% CHCl3), with LNO2 predominantly eluting in the 20% to 30% CHCl3 fraction, as determined by negative-ion-mode mass spectroscopic monitoring of fractions. Further purification was accomplished by high pressure liquid chromatography using 0.1% acetic acid in 50% to 90% CH3OH gradient on a 4.6×250-μm reverse-phase C18 column. LNO2 yields were quantified by elemental analysis of nitrogen content after pyrolysis by chemiluminescent nitrogen detection (Antek Instruments), with caffeine used as a standard. After purification and quantification, LNO2 was stored in CH3OH under inert gas at −80°C. For all cell experiments, appropriate controls using methanol were carried out to exclude solvent effects, and methanol concentrations were always <0.1%.
Human neutrophils were isolated from 20 mL citrate anticoagulated whole blood as described.25 Approval for blood donations from healthy volunteers was given by the Bro Taf Local Research Ethics Committee, and all donors gave written consent. Briefly, blood was mixed 1:1 with 0.8% trisodium citrate (wt/vol) and 2% dextran (wt/vol) in PBS (containing 137 mmol/L NaCl, 2.68 mmol/L KCl, 8.1 mmol/L Na2HPO4, and 1.47 mmol/L KH2PO4) and allowed to sediment for 45 minutes at 20°C. After this procedure, the upper plasma layer was underlaid with ice-cold Lymphoprep (2:1 for plasma/Lymphoprep) and centrifuged (800g, 20 minutes, 4°C). The pellet was resuspended in ice-cold PBS and 0.4% trisodium citrate (wt/vol) and centrifuged (400g, 5 minutes). Contaminating erythrocytes were removed using 3 cycles of hypotonic lysis. Finally, cells were resuspended in a small volume of Krebs buffer (100 mmol/L NaCl, 50 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L NaH2PO4, 1 mmol/L CaCl2, 2 mmol/L d-glucose, and 5 U/mL heparin, pH 7.4), counted, and kept on ice.
For the preparation of subcellular fractions, neutrophils were purified as described above with the omission of the dextran sedimentation step. Neutrophils were treated with 2.5 mmol/L diisopropyl fluorophosphate for 10 minutes at 4°C, disrupted in relaxation buffer (100 mmol/L KCl, 3 mmol/L NaCl, 3.5 mmol/L MgCl2, 1 mmol/L ATP, 1.25 mmol/L EGTA, and 10 mmol/L PIPES, pH 7.3) by N2 cavitation, and fractionated on a discontinuous Percoll gradient.26,27⇓ This method produces cytosolic and plasma membrane fractions whose final concentrations were adjusted to 9×107 and 1.25×109 cell equivalents per milliliter, respectively. Fractions were stored at −80°C for up to 1 year without loss of activity.
Neutrophil O2·− generation was determined using superoxide dismutase–sensitive cytochrome c reduction.28 Cells (106) were added to 2 mL Krebs buffer containing 50 μmol/L ferricytochrome c, with stirring, at 37°C. Cells were activated using either 1 μmol/L fMLP or 1 μg/mL PMA, and absorbance was monitored at 550 nm (ε 21.1 cm−1 · mmol/L for ferrocytochrome c). To confirm O2·− generation, superoxide dismutase (300 U/mL) was added in control experiments. Where used, the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 4 μmol/L) was preincubated with neutrophils 20 minutes before the assay. Superoxide generation by isolated neutrophil membranes was measured after the addition of 160 μmol/L NADPH to 0.75 mL relaxation buffer containing 5.4×106 cell equivalents of membrane extract per milliliter, 1.5×107 cell equivalents of cytosol per milliliter, 10 μmol/L GTP-γ-S, 100 μmol/L SDS, and 50 μmol/L cytochrome c. To facilitate the assembly of NADPH oxidase components, all constituents (excluding NADPH) were preincubated at 25° for 5 minutes before the addition of NADPH. LNO2 (10 μmol/L) was added in ethanol (final concentration 0.1%) either during NADPH oxidase assembly or immediately before NADPH addition.
Measurement of Neutrophil [Ca2+ic]
Neutrophils prepared as described were incubated at 107/mL with 5 μmol/L fura 2-AM (Molecular Probes) in Ca2+-free Krebs buffer at 37°C for 20 minutes. Then, cells were centrifuged at 400g for 10 minutes, resuspended in Ca2+-free Krebs buffer at 107/mL, kept on ice, and studied within 90 minutes. Fluorescence of neutrophils (106/mL) was measured using a Perkin-Elmer LS 50B fluorescence spectrophotometer at 37°C with stirring. Excitation wavelengths were 340 and 380 nm, with an emission wavelength of 509 nm. Corrections were applied for autofluorescence (unloaded neutrophils), and calibrations were performed by adding 1 μmol/L ionomycin, 1 mmol/L CaCl2 (to give maximal fluorescence ratio [Rmax] and minimum fluorescence [Sb]), followed by 5 mmol/L MnCl2 (to give minimal fluorescence ratio [Rmin] and maximum fluorescence [Sf]) to fura 2-AM–loaded neutrophils. [Ca2+ic] was calculated using the following equation: [Ca2+ic]=Kd · Sf · (R−Rmin)/Sb · (Rmax−R), where Kd is the dissociation constant of fura 2 under intracellular conditions (224 nmol/L); Sf and Sb are the maximum and minimum values of fluorescence, respectively, at 380 nm; and Rmax and Rmin are the maximum and minimum values of the ratio (340 nm/380 nm) under Ca2+-saturating and Ca2+-free conditions, respectively. Fluorescence of neutrophils was monitored on the addition of 1 μmol/L fMLP alone, 5 μmol/L LNO2, or fMLP after a 2-minute preincubation with LNO2 in the presence of either 1 mmol/L CaCl2 or 100 μmol/L EGTA (using Ca2+-free Krebs solution).
Determination of CD11b Expression
Expression of CD11b (αM integrin, MAC-1) was determined by flow cytometry.29 Briefly, isolated neutrophils (2×106/mL) were incubated with and without LNO2 (3.6 μmol/L) for 2 minutes at 37°C. After this procedure, fMLP (1 μmol/L) was added, and samples were incubated for a further 5 minutes at 37°C. Anti-human CD11b-FITC (10 μL, Serotec) or isotype control was then added, and samples were incubated for 30 minutes at 4°C. Finally, cells were washed once with ice-cold PBS and then resuspended in 0.1% paraformaldehyde-PBS until flow cytometry analysis. Neutrophils were analyzed on a FACScan flow cytometer (Becton Dickinson) and identified by forward and side scatter and FITC.
Determination of Neutrophil Degranulation by Elastase Release
Degranulation of azurophilic granules was determined by elastase release.30 Briefly, isolated neutrophils were resuspended at 2×106/mL Krebs buffer containing 1 μg/mL cytochalasin B and 40 μmol/L MeO-Suc-Ala-Ala-Pro-Val-MCA at 37°C with stirring. After 5 minutes, 1 μmol/L fMLP was added, and fluorescence was monitored (excitation wavelength 380 nm, emission wavelength 460 nm). In some experiments, 3 μmol/L LNO2 was added 2 minutes before fMLP.
Determination of cAMP and cGMP
For cAMP, neutrophils (2×106/mL) in Krebs buffer were prewarmed to 37°C for 3 minutes. Then, 3.6 μmol/L LNO2 with or without 1 mmol/L 3-isobutyl-1-methylxanthine (IBMX) was added, and samples were incubated for 1 minute at 37°C. Then, proteins were precipitated using ice-cold 6% trichloroacetic acid, which was subsequently removed by washing samples several times with water-saturated diethyl ether. For cGMP, neutrophils (4×106/mL) in Krebs buffer were incubated for 5 minutes at 37°C with or without 1 mmol/L IBMX, 10 μmol/L LNO2, or 10 μmol/L 2(N,N-diethylamino)-diazenolate-2-oxide (DEANONOate). Then, proteins were precipitated using ice-cold 66% ethanol. Cyclic nucleotides were determined by radioimmunoassay (Biotrak, Amersham).
LNO2 Inhibits fMLP- or PMA-Induced O2·− Generation by Intact Neutrophils but Not by Reconstituted NADPH Oxidase
Addition of LNO2 (0.5 to 5 μmol/L) to stirred neutrophils in suspension did not stimulate O2·− generation (not shown). However, LNO2 added to neutrophils 2 minutes before agonist activation using either 1 μmol/L fMLP or 1 μg/mL PMA led to concentration-dependent inhibition of O2·− generation (Figures 1A, 1B, and 1D). In contrast, similar concentrations of the parent fatty acid, LA, or the nitrated amino acid, 3-nitrotyrosine (3-NT), were without effect (Figure 1C). This indicates that inhibition of neutrophil activation was specific to LNO2 and was not a feature of all unsaturated lipids or nitro-containing compounds. Neutrophil membranes were isolated and assayed for O2·− generation in a reconstituted system after the addition of NADPH. Preincubation of neutrophil membranes during assembly with 10 μmol/L LNO2 slightly suppressed O2·− generation rates, although this was not significant (Figure 2A). Also, the addition of LNO2 to the enzyme that had been allowed to preassemble in vitro had no effect on O2·− generation. These data indicate that LNO2 does not inhibit O2·− generation through a direct effect on NADPH oxidase assembly or turnover.
cGMP Synthesis Is Not Involved in Inhibition of Neutrophil O2·− Generation by LNO2
To examine whether NO was involved in the inhibitory effects of LNO2, the sGC inhibitor ODQ was added. This did not restore neutrophil O2·− generation in response to fMLP (Figure 2B). Separately, cellular cGMP levels were found to be unchanged after a 5-minute incubation with LNO2 in contrast to DEANONOate, which was used as a positive control (Figure 2C). These data rule out a role for NO activation of sGC in the inhibitory actions of LNO2.
Effect of LNO2 on CD11b Exposure in Response to fMLP
Basal neutrophil expression of CD11b, as evidenced by the difference in fluorescence between cells stained with either anti-CD11b antibody or isotype control, was not affected by LNO2 (Figure 3). fMLP activation of neutrophils induced a further significant increase in CD11b expression (Figures 3A and 3C), which was fully inhibited by preincubation of cells with 3 μmol/L LNO2 (Figures 3B and 3C). This indicates that LNO2 can inhibit neutrophil functions associated with adhesion.
LNO2 Attenuates fMLP-Induced Degranulation
Pretreatment of neutrophils with 3 μmol/L LNO2 blocked fMLP-induced azurophilic degranulation, as measured by elastase release, whereas the addition of LNO2 to unstimulated cells had no effect (Figures 4A and 4B). This indicates that neutrophil functions associated with tissue damage and bacteriolysis can be attenuated by LNO2.
LNO2 Attenuates Ca2+ Mobilization in Response to fMLP
To examine the mechanisms of LNO2 inhibition of neutrophil activation, Ca2+ mobilization in response to fMLP was monitored by fura 2-AM fluorescence. On the addition of 1 μmol/L fMLP to Ca2+-containing samples, a characteristic increase in [Ca2+ic] was observed (Figure 5A). Preincubation of neutrophils with 3 μmol/L LNO2 significantly attenuated fMLP-induced Ca2+ mobilization and accelerated the subsequent rate of Ca2+ removal, whereas the addition of LNO2 to unstimulated cells had no effect (Figure 5). In the absence of external Ca2+, LNO2 did not alter the kinetics of Ca2+ mobilization (Figure 6), indicating that LNO2 primarily exerted an effect on external Ca2+ fluxes in neutrophils.
LNO2 Elevates cAMP in Neutrophils
LNO2 inhibits platelet activation via a cAMP-dependent pathway.11 To determine whether similar mechanisms are operative in neutrophils, cAMP was determined after incubation with LNO2. After a 1-minute incubation with 3.6 μmol/L LNO2, a 40% increase in cAMP was observed (Figure 7). However, in the presence of the phosphodiesterase inhibitor IBMX, LNO2 caused a 3-fold elevation in neutrophil cAMP levels. This indicates that LNO2 elevates cAMP synthesis through the activation of adenylate cyclase in neutrophils.
Nitrated lipid has recently been found in human plasma, bovine cardiac tissue, and plasma from a rat model of liver ischemia/reperfusion, with extensive nitration and oxidation of biomolecules also occurring in human atheroma5,6,31⇓⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). Nitrated lipids, including LNO2, possess vascular protective properties through the inhibition of platelet activation and promotion of smooth muscle relaxation5,11⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). In the present study, the influence of LNO2 on human neutrophil functional responses was examined to characterize their bioactivity toward proinflammatory leukocytes, which are central to the pathogenesis of atherosclerosis. LNO2 (0.1 to 1 μmol/L) inhibited neutrophil activation in response to PMA or fMLP, as indicated by the inhibition of O2·− generation, Ca2+ mobilization, azurophilic degranulation, and CD11b expression. This reveals that lipid nitration results in the formation of an antiinflammatory and vascular-protective product that potently attenuates multiple leukocyte functions.
Neutrophil cytotoxicity requires efficient degranulation to release proteases and other degradative enzymes. In the present study, LNO2 effectively blocked fMLP-induced azurophilic degranulation, indicating that events associated with bacterial killing and cell damage are attenuated by nitrated lipids (Figure 4). The cell surface glycoprotein, CD11b (αM integrin, MAC-1), is expressed as a heterodimer with CD18 (C receptor type 3) on the surface of leukocytes in response to activating agonists such as fMLP and is involved in adhesion to endothelial cells and in transendothelial migration to inflammatory sites.32,33⇓ Recruitment of leukocytes to the arterial wall is an important event in atherogenesis and plaque rupture.12,17–19,32,33⇓⇓⇓⇓⇓ This is mediated via leukocyte integrin receptors, including CD11b, and is modulated by a variety of both endothelium- and leukocyte-derived lipid oxidation products and free radicals, including O2·−, NO, prostaglandins, and isoprostanes.34–42⇓⇓⇓⇓⇓⇓⇓⇓ Inhibition of fMLP-induced CD11b expression by LNO2 indicates that nitrated lipids also modulate leukocyte responses associated with the development of vascular disease (Figure 3).
Stimulation of neutrophils by fMLP or PMA causes translocation of at least 3 different cytosolic proteins (p67-phox, p47-phox, and p21-rac1) to the membrane, where they interact with 2 membrane-bound proteins (gp91-phox and p22-phox) to form the active O2·−–generating NADPH oxidase complex. To determine whether LNO2 exerted its inhibitory actions directly on NADPH oxidase or prevented complex assembly, neutrophil membranes were isolated and assayed for O2·− generation in a reconstitution system after the addition of NADPH. The lack of inhibition by LNO2 in this assay indicates that LNO2 exerts its inhibitory effects upstream of NADPH oxidase (Figure 2A).
There are at least 2 potential mechanisms by which nitrated lipids may exert cell signaling. The inhibition of platelet activation by LNO2 requires cAMP synthesis, whereas stimulation of smooth muscle relaxation by LNO2 or nitrated arachidonate involves cellular metabolism of the nitro functional group to NO5,11⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). An elevation in either cGMP or cAMP can inhibit neutrophil function and is consistent with the observed effects of LNO2 on agonist-induced Ca2+ elevations.34–39⇓⇓⇓⇓⇓ However, cGMP was ruled out because (1) elevations in response to LNO2 did not occur and (2) inhibition of sGC did not restore neutrophil activation (Figure 2). In contrast, LNO2 increased intracellular cAMP, in the presence and absence of IBMX, suggesting an involvement of this metabolite (Figure 7). Previous studies have shown that cAMP inhibits fMLP- but not PMA-stimulated neutrophil O2·− generation. This indicates that cAMP acts upstream of protein kinase C.40 Therefore, inhibition of PMA-stimulated O2·− generation by LNO2 suggests an additional cAMP-independent mechanism of action. In support, fMLP-activated neutrophils were considerably more susceptible to the inhibitory effects of LNO2 than when they were stimulated with PMA (Figures 1B and 1D).
This preparation of LNO2 is a mixture of 4 positional isomers identified using nuclear magnetic resonance and IR spectroscopy (D.G. Lim, B.A. Freeman, unpublished data, 2002), and this preparation was used because it can be synthesized with a relatively high yield and purity. Levels of nitrated lipid in vivo are too low to allow purification of amounts sufficient for biological studies. Similarly, nitration of linoleate through oxidant pathways (eg, ONOO− and NO2) in vivo will also form a mixture of LNO2 isomers. Metabolism to release NO by smooth muscle cells is unlikely to be isomer specific; however, activation of adenylate cyclase through receptor-dependent pathways may be more effective with ≥1 specific isomer.
Data from the present study and others indicate that lipid nitration results in the formation of compounds that can signal through either NO- or cAMP-dependent pathways, with the biological mechanism of action being highly cell type dependent5,11⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). For example, LNO2 mediates smooth muscle relaxation through exclusively cGMP-dependent mechanisms, whereas in platelets and neutrophils, there is no role for NO, with its biological effects being at least partly cAMP dependent11 (D.G. Lim, B.A. Freeman, unpublished data, 2002, and data in the present study). The reasons for this may involve (1) different cellular rates of metabolism of LNO2 to NO and/or (2) differences in the expression of eicosanoid receptors that activate adenylate cyclase.
In summary, the present study shows that LNO2 potently blocks multiple neutrophil proinflammatory responses, including degranulation, O2·− generation, and integrin expression. This indicates that nitrated lipids will attenuate leukocyte migration and subsequent activation in atherosclerotic lesions, demonstrating a further vascular protective property of these compounds in addition to antiplatelet and vasorelaxing effects5,11⇓ (D.G. Lim, B.A. Freeman, unpublished data, 2002). These data reveal novel mechanisms by which nitrated biomolecules modulate cell activation and show how the presence of the nitro functional group can alter the bioactivity of lipids through the formation of vascular protective and antiinflammatory products.
This study was supported by the British Heart Foundation (Drs Lewis, O’Donnell, and Clark) and NIH grants RO1-HL-64937, RO1-HL-58115, P6-HL-58418 (Dr Freeman), and RO1-A124838 (Dr Cross). Dr O’Donnell is a Wellcome Trust RCD Fellow.
Original received April 19, 2002; revision received July 15, 2002; accepted July 22, 2002.
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