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(Circulation Research. 2002;91:375.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Nitrolinoleate Inhibits Superoxide Generation, Degranulation, and Integrin Expression by Human Neutrophils

Novel Antiinflammatory Properties of Nitric Oxide–Derived Reactive Species in Vascular Cells

Barbara Coles, Allison Bloodsworth, Stephen R. Clark, Malcolm J. Lewis, Andrew R. Cross, Bruce A. Freeman, Valerie B. O’Donnell

From the Department of Medical Biochemistry (B.C., S.R.C., V.B.O.) and the Department of Pharmacology, Therapeutics, and Toxicology (M.J.L.), University of Wales College of Medicine, Cardiff, UK; the Department of Molecular and Experimental Medicine (A.R.C.), The Scripps Research Institute, La Jolla, Calif; and the Departments of Anesthesiology, Biochemistry, and Molecular Genetics and the Center for Free Radical Biology (A.B., B.A.F.), University of Alabama at Birmingham.

Correspondence to Valerie O’Donnell, PhD, Wellcome Trust RCD Lecturer, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. E-mail o-donnellvb{at}cf.ac.uk

	Abstract

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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.

Key Words: nitric oxide • nitrolinoleate • neutrophils • atherosclerosis • peroxynitrite

	Introduction

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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 nitrolinoleate^5,6 (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 relaxation^5,11 (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

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Materials
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 4.6x250-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%.

Neutrophil Isolation
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.

Neutrophil Fractionation
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 9x10⁷ and 1.25x10⁹ cell equivalents per milliliter, respectively. Fractions were stored at -80°C for up to 1 year without loss of activity.

Superoxide Generation
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 ( 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 5.4x10⁶ cell equivalents of membrane extract per milliliter, 1.5x10⁷ cell equivalents of cytosol per milliliter, 10 µmol/L GTP--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 [R_max] and minimum fluorescence [S_b]), followed by 5 mmol/L MnCl₂ (to give minimal fluorescence ratio [R_min] and maximum fluorescence [S_f]) to fura 2-AM–loaded neutrophils. [Ca²⁺_ic] was calculated using the following equation: [Ca²⁺_ic]=K_d · S_f · (R-R_min)/S_b · (R_max-R), where K_d is the dissociation constant of fura 2 under intracellular conditions (224 nmol/L); S_f and S_b are the maximum and minimum values of fluorescence, respectively, at 380 nm; and R_max and R_min 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 (M integrin, MAC-1) was determined by flow cytometry.²⁹ Briefly, isolated neutrophils (2x10⁶/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 2x10⁶/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 (2x10⁶/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 (4x10⁶/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).

	Results

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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.

View larger version (28K): [in this window] [in a new window]   Figure 1. LNO2 inhibits neutrophil O2·- generation in response to either PMA or fMLP. O2·- generation was measured using cytochrome c reduction as described in Materials and Methods. Neutrophils (106) in 2 mL Krebs buffer containing 50 µmol/L cytochrome c, with stirring at 37°C, were activated by 1 µmol/L fMLP or 1 µg/mL PMA, and absorbance (550 nm) was monitored. LNO2, LA, or 3-NT was added 2 minutes before activation. A, Time course of O2·- generation by fMLP-activated neutrophils with/without LNO2 is shown. B, Dose-dependent inhibition of fMLP-stimulated neutrophil O2·- generation by LNO2 is shown. C, LA and 3-NT do not inhibit fMLP-stimulated neutrophil O2·- generation. D, Dose-dependent inhibition of PMA-stimulated neutrophil O2·- generation by LNO2 is shown. For all data, values are mean±SD (n=3). *P<0.05 vs no LNO2 after ANOVA and Bonferroni post hoc test.

View larger version (22K): [in this window] [in a new window]   Figure 2. LNO2 does not block O2·- generation in isolated neutrophil membranes, and inhibition of neutrophil O2·- generation does not involve sGC activation. A, O2 generation was determined as described in Materials and Methods after the addition of 160 µmol/L NADPH to 0.75 mL relaxation buffer containing 5.4x106 cell equivalents (cell eq) of membrane extract per milliliter, 1.5x107 cell eq of cytosol per milliliter, 10 µmol/L GTP--S, 100 µmol/L SDS, and 50 µmol/L cytochrome c. To allow assembly of NADPH oxidase components, all constituents (excluding NADPH) were preincubated at 25°C 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 (middle bar) or immediately before NADPH (right bar). B, O2·- generation of neutrophils in response to 1 µmol/L fMLP was determined as in Figure 1 after preincubation with/without 3 µmol/L LNO2. Where used, 4 µmol/L ODQ was preincubated with neutrophils for 20 minutes before assay. C, cGMP synthesis by neutrophils was determined as described in Materials and Methods after 5 minutes of incubation with 10 µmol/L LNO2 and 10 µmol/L DEANONOate (DEANO) with/without 1 mmol/L IBMX. For all data, values are mean±SD (n=3), and all experiments were repeated at least 3 times with neutrophils obtained from different donors (B). *P<0.05 vs control after ANOVA and Bonferroni post hoc test (C). Bars marked with asterisks are not significantly different from each other.

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.

View larger version (23K): [in this window] [in a new window]   Figure 3. LNO2 attenuates fMLP-stimulated neutrophil CD11b expression. Cells (2x106/mL) were incubated with/without 3.6 µmol/L LNO2 for 2 minutes and then activated by 1 µmol/L fMLP for 5 minutes at 37°C. Anti-human CD11b-FITC or isotype control was then added, and cells were incubated for 30 minutes at 4°C. Cells were washed once with PBS and then fixed with 0.1% paraformaldehyde/PBS for FACScan analysis, as described in Materials and Methods. A, Fluorescence shift induced by fMLP in control neutrophils is shown. B, LNO2 inhibits fMLP-induced CD11b expression. C, Percentages of cells in population designated M1 in panels A and B are shown. For all data, values are mean±SD (n=3), and all experiments were repeated at least 3 times on different donors. *P<0.05 vs fMLP after ANOVA and Bonferroni post hoc test.

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.

View larger version (20K): [in this window] [in a new window]   Figure 4. LNO2 inhibits fMLP-stimulated neutrophil degranulation. Elastase release was measured by hydrolysis of MeO-Suc-Ala-Ala-Pro-Val-MCA as described in Materials and Methods. A, Cells (2x106) were incubated with 1 µg/mL cytochalasin B and 40 µmol/L MeO-Suc-Ala-Ala-Pro-Val-MCA at 37°C, with stirring, for 5 minutes before activation using 1 µmol/L fMLP, as indicated by arrow. In some experiments, 3 µmol/L LNO2 was added 2 minutes before fMLP. B, Rates of fluorescence change after fMLP activation of neutrophils with/without 3 µmol/L LNO2 were determined. For all data, values are mean±SD (n=3), and all experiments were repeated at least 3 times with neutrophils obtained from different donors. *P<0.05 vs fMLP after ANOVA and Bonferroni post hoc test.

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.

View larger version (19K): [in this window] [in a new window]   Figure 5. LNO2 attenuates fMLP-stimulated Ca2+ mobilization in neutrophils in the presence of extracellular Ca2+. Neutrophils labeled with fura 2-AM as described in Materials and Methods were stimulated with 1 µmol/L fMLP, as indicated by arrows, in the presence of 1 mmol/L Ca2+, and fluorescence was monitored at 37°C with stirring. A, Ca2+ mobilization of neutrophils with/without 2-minute preincubation with 3 µmol/L LNO2. B, Control experiments showing no effect of 3 µmol/L LNO2 on control neutrophils. C, Maximum Ca2+ elevations recorded on addition of 1 µmol/L fMLP to control or 3 µmol/L LNO2–treated neutrophils. [Ca2+ic] indicates intracellular Ca2+ concentration. For all data, values are mean±SD (n=3), and all experiments were repeated at least 3 times with neutrophils obtained from different donors. *P<0.01 vs fMLP alone after independent t test.

View larger version (28K): [in this window] [in a new window]   Figure 6. LNO2 does not alter fMLP-stimulated Ca2+ mobilization in neutrophils in the absence of extracellular Ca2+. Neutrophils labeled with fura 2-AM as described in Materials and Methods were stimulated with 1 µmol/L fMLP, as indicated by arrows, in the presence of 100 µmol/L EGTA, and fluorescence was monitored at 37°C with stirring. A, Ca2+ mobilization of neutrophils with/without 2-minute preincubation with 3 µmol/L LNO2. B, Control experiments showing no effect of 3 µmol/L LNO2 on control 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.

View larger version (24K): [in this window] [in a new window]   Figure 7. LNO2 elevates cAMP in neutrophils. Neutrophils (2x106/mL) in Krebs buffer were prewarmed to 37°C for 3 minutes. LNO2 (3.6 µmol/L) with/without 1 mmol/L IBMX was then added, and samples were incubated for 1 minute at 37°C. cAMP was determined as described in Materials and Methods using a commercial radioimmunoassay kit. For all data, values are mean±SD (n=3), and all experiments were repeated at least 3 times on different donors. *P<0.005 vs control or IBMX after independent t test.

	Discussion

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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 atheroma^5,6,31 (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 relaxation^5,11 (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 (M 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 NO^5,11 (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 dependent^5,11 (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 effects^5,11 (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.

	Acknowledgments

 
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.

Received April 19, 2002; revision received July 15, 2002; accepted July 22, 2002.

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