Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway

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Inhibition by Interferon-y of Human Mononuclear Cell-mediated Low Density Lipoprotein Oxidation

Participation ofTryptophan Metabolism along the Kynurenine Pathway

Stephan Christen, Shane R.Thomas, Brett Gamer,*andR.Stocker

Biochemistry and *Cell Biology Groups, The Heart Research Institute, Camperdown, New South Wales 2050, Australia

Abstract

Inthis study we examined the potential inhibition by inter-

feron-y

(IFN'y) of theearlystages of lowdensitylipoprotein (LDL)oxidation mediatedby humanperipheral blood mononu- clear cells (PBMC) and monocyte-derived macrophages (NMDMN) in Ham's F-10 medium supplementedwith physiologi- calamountsof L-tryptophan(Trp).We assessed LDLoxida- tion bymeasuringtheconsumption ofLDL's major antioxidant (i.e., a-tocopherol)and targetsfor oxidation (cholesteryllino- leate andcholesterylarachidonate), together with the accumu- lation of cholesterylester hydroperoxides and the increase in relativeelectrophoretic mobilityof the lipoprotein particle. Ex- posureof PBMCorMDMto

IFNy

induced thedegradation of extracellular Trp with concomitant accumulation of kynuren- ine,anthranilicand3-hydroxyanthranilic acid (3HAA)in the culturemedium. Formation of3HAA,butneitherTrp degrada- tionnorformation ofkynurenineandanthranilicacid,wasinhib- itedby low amountsofdiphenylene iodonium(DPI)ina con- centration-dependentmanner.IncontrasttooxidativeTrpme- tabolism,exposureofhumanPBMCorMDMtoIFN-yfailed toinduce degradationof arginine,and nitritewasnotdetected in the cell supernatant, indicatingthatnitric oxide synthasewas notinduced under theseconditions.IncubationofLDLin Trp- supplemented F-10 medium resulted inatime-dependentoxi- dationof thelipoproteinthatwasaccelerated in the presenceof PBMCor MDMbutinhibited strongly in the presenceofboth cells and IFNy, i.e., whenTrp degradation and formation of 3HAA were induced. In contrast, when IFNy was added to PBMCorMDMin F-10mediumthatwasvirtually devoid of Trp, inhibition ofcell-accelerated LDLoxidationwas notob- served.Exogenous 3HAA addedtoPBMCor

purified

mono- cytes in the absenceof IFNyalsostronglyand ina concentra- tion-dependentmannerinhibited LDLoxidation.Selective inhi- bition of IFNy-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this

cytokiine

on both PBMC- and MDM-mediated LDL oxidation. These results show that

IFN'y

^treatmentofhumanPBMCorMDMinvitro attenuates theextentofLDLoxidation causedbythesecells,

Dr.Christen'spresentaddressisDivision of Biochemistry& Molecular Biology,University of California, Berkeley, CA94720.

Addresscorrespondence to Dr. R. Stocker,BiochemistryGroup, The Heart Research Institute, 145 Missenden Road, Camperdown, NSW 2050, Sydney,Australia.

Receivedforpublication7May1993and in revisedform 19 Jan- uary1994.

andindicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity. (J. Clin.

Invest. 1994.93:2149-2158.) Key words: atherosclerosis*3- hydroxyanthranilic acid *indoleamine 2,3-dioxygenase*lipid hydroperoxide*macrophages

Introduction

Oxidative modification of LDL may be important in the in vivoformation of lipid-ladencells(i.e., foam cells) that signifi- cantly contribute to thedevelopment of atherosclerosis ( 1, 2).

Although the precise molecular mechanism(s) underlying oxi- dative LDLmodification in vivo remains unknown, in vitro studies oncell-mediated LDL oxidation are generally carried out in medium containing small amounts of the transition metals iron and/or copper. Under these conditions all cell types present in a lesion (i.e., endothelial cells [3], macro- phages [4], smooth muscle cells [5, 6] and lymphocytes

[7])

arecapableofoxidizingLDL.Whereinvestigated directly,in vitro cell-mediatedLDLoxidation has been shown to have an absoluterequirement for the presence ofiron and copper in the medium(3, 6);i.e., intheir absence,these cells do notoxidize LDLlipidssubstantially. Theresulting modified LDL can be takenupreadilybymacrophages, turningthemintofoamcells similartothoseobserved inatherosclerotic lesions in vivo (8).

Antioxidants such as probucol (9), ascorbate (10), or ubi- quinol-O0 ( 11), when addedtothese and other systems, sup- press orcompletely inhibit LDL oxidation, indicated by the decrease inthelipid hydroperoxidesformed orthe preserva- tion of LDL-associated a-tocopherol (a-TOH)

.'

Inhibition ofmurine macrophage-mediated LDL oxida- tionhas alsobeen observed by treatment ofthe cells with inter-

feron-y (IFN'y)

(12-14).

IFNy-induced

attenuation ofcell- mediated LDL oxidation correlatedwith induction of nitric oxide radical(NO) synthesis from arginine(12, 14). Inaddi- tion to NO formation, IFN-y can induce at least two other pathwaysinmononuclearphagocytesthat may attenuate LDL oxidation: oxidativedegradation ofheme and Trpinitiatedby heme oxygenase andindoleamine2,3-dioxygenase (IDO), re- spectively. Both pathways eliminate potential pro-oxidants while producingpowerful antioxidants ( 15-19) (SchemeI).

We areinterested in oxidative Trp degradation along the kynurenine(Kyn)pathway and theconcomitant formation of

1.Abbreviations usedinthis paper:CE-OOH,cholesterylesterhydro- peroxides;C18:2,cholesteryllinoleate; C20:4,

cholesterylarachidonate;

DPI,diphenyleneiodonium; 3HAA,3-hydroxyanthranilicacid; IDO, indoleamine2,3-dioxygenase;Kyn,kynurenine; NO,nitricoxide radi- cal; MDM, monocyte-derived macrophages; a-TOH, a-tocopherol;

Trp,L-tryptophan.

J.Clin.Invest.

© The AmericanSociety^forClinical

Investigation,

^Inc.

0021-9738/94/05/2149/10 $2.00 Volume93, May 1994,2149-2158

/s o~~CH2-jH-COOH

NH2

H

L-Tryptophan

02'@

Indoleamine 2,3-dioxygenase

(IDO)

CO-CH2-CH-COOH

|NH2

H2N tC ^OH

L-Kynurenine N

I ~~~~~~~NH2

H2N COOH

H2N NH2

L-Arginine

I NO

synthase

NG-Hydroxy-L-arginine

3-HydroxykueniA

rCOOH

NH2 OH

3-Hvdroxvanthranhlic

acid

I

N=O-

02, H20

L-Citrulline NO2 NO3 SchemeI. Interferon-y-mediatedinductioninmononuclearphagocytesofpathwaysthatmayprovideadditionalantioxidantprotection. Me- tabolites withreportedantioxidantactivityareunderlined.

the antioxidant 3-hydroxyanthranilic acid (3HAA) because thispathway isreadily induced byIFNy ^{in human} (20, 21) monocytes/macrophages but not murine monocytes/macro- phages (22) ^or human lymphocytes. In contrast, IFNy (to- gether with LPS) readily inducesNOformation in murinemac-

rophages whileatleast in vitro this pathwayisnotinducedin humanmonocytes/macrophages by this and othercytokines.

Becausealargeproportionoflymphocytes presentinathero- sclerotic lesionsappeartobeactivated, local releaseofIFNy could result(23-25). We therefore examined whether IFNy attenuated LDL oxidation mediated by human PBMC or

monocyte-derived macrophages (MDM) and, ifsotowhatex- tentoxidativedegradation of arginine and/or Trp contributed tothisactivity. Oxidative modification of LDL^wasassessed by measuring the consumption of both a-TOH and polyunsatu- ratedesterified lipids, aswell asformation of cholesterylester hydroperoxides (CE-OOH) and changes innetsurface charge of the lipoprotein. Induction of IDO and NO synthase (Scheme I)wasdetermined by measuring Trp metabolites and nitrite, respectively, in the cell culturesupernatants.The results obtainedindicatethatinduction of IDO butnotNOsynthase participates in the observed inhibition by IFNy of human PBMC-orMDM-mediated LDL oxidation.

Methods

Materials. 3HAA,anthranilic acid, Kyn, EDTA, PBS, HBSS, (both Ca2+-andMg2+-free) and RPMI 1640 (powder)wereobtained from SigmaChemicalCo. (St. Louis,MO). L-Trp,chloroaceticandtrichlo- roacetic acid (TCA)^werefromMerck (Darmstadt, FRG),Ham's F-l0 medium(controlNo. 19K6422) fromGibco (Grand Island,NY), hu-

man recombinant IFN-y and chloramphenicol from Boehringer- MannheimGmbH (Mannheim,FRG), and potassium bromide from British DrugHouse(BDH, Poole, England). Diphenylene iodonium (DPI)^wasgenerously provided by Prof. O.T.G.Jones(Universityof Bristol, UK) and giventous as agiftby Dr. W. Jessup (Heart Research Institute); itwasstoredat-20°Cas a 1 mMstocksolution in50%

ethanol(vol/vol). Tert-butyl alcohol and ethanolwerefrom Rh6ne- Poulenc(Paris, France) andBDH,respectively(bothanalyticalgrade), and all otherorganicsolvents (HPLC quality) from Mallinckrodt,Inc.

(Chesterfield, MO). LiquiPure water (MODULAB) ^was used for buffersandaqueoussolutions, whichweresubsequentlytreated with Chelex-100(Bio-Rad Laboratories, Richmond, CA)toremove con-

taminatingtransition metals.Buffers andmediausedfor cell isolation, elutriation andculture (except for F-10 media)were sterile-filtered through Zetapor membranes (CUNO, Meriden, CT) and stored in heat-treated (250°C for3 h) glassware to minimize contamination withendotoxin(LPS), testedforregularly usingachromogenicLimu- lus amebocyte lysate test(26) (Associates ofCape Cod/American Diagnostica,Greenwich,CT) andfoundtobe^<50 pg/ml.

2150 Christenetal.

Heme

*-I Hemeoxygenase Co

41 Anthranilic

acid

N'-Oxo-L-arginine

iNH2

I

PreparationofLDL. Human LDL(d 1.06 g/ml)^wasisolated fromanticoagulated (EDTA-K3 vacutainers, Becton Dickinson & Co., Mountain View, CA) plasma obtained from nonfasted, healthy, and normolipidemic donors byrapid ultracentrifugation (2 h, 150C) (TL- 100.4 rotor inaTL-l00benchtop centrifuge, Beckman Instruments, Inc., Fullerton,CA)asdescribed (27). The isolated LDL wasthen dialyzedfor 18hagainstfourchangesof 100 vol of deoxygenated 10 mM PBS, pH7.0, containing0.1 mg/i chloramphenicol and 1 g/l Chelex-100. Chelex wasomitted in the lastdialysis step. For some experiments, the isolatedLDL wasgel-filtered(PD-bI column, Phar- macia) rather thandialyzed.Inall cases, the LDLwasthenfilter steril- ized (0.45

Am)

^andusedimmediately in experiments. LDL prepared in this wayconsistently contained only low levels of cholesterylester hy- droperoxides(CE-OOH),i.e.,45±38pmolCE-OOH/mgLDLprotein (mean±SD, ^{n =} 10), correspondingto 1 CE-OOH molecule for every 43^LDLparticles. LDLproteinconcentrationwasdetermined with BSA as the standard and using either the bicinchoninic acid method(Sigma Chemical Co.)orthemodified procedure of Peterson (28)(Sigma KitP5656).

Isolationofcells. Where feasible, PBMC were isolated from blood obtainedfrom thesamedonor usedfor the LDL preparation. Blood wasobtained under the guidelines and approval of the local human ethicscommittee. For isolation of large numbers ofPBMC, monocytes orMDM, white blood cell concentrates ("buffy coats," kindly pro- vided by the N.S.W. Red Cross Blood Transfusion Service, Sydney) were used. PBMC wereisolated from diluted plasma or buffy coats by centrifugationonLymphoprep (NYCOMED, Oslo, Norway) and cul- tured under nonadherent conditions (29) at 5X106 cells/ml in serum- free Ham's F-10 medium containing either10% (vol/vol) heat-inacti- vatedFCS (MultiSer, Cytosystems, Castle Hill, Australia) or 15% (vol/

vol) human AB serum (Red Cross).

Human monocytes(usedat 1 ^X 106cells/ml) andlymphocytes werepurified from PBMC (prepared from buffy coat) using counter- flowcentrifugalelutriation (30) carriedout at21'C. Theelutriation system consisted ofaBeckman JE-6rotorinaBeckman J2-21M/E centrifuge equipped with strobe assembly. Flow through the system wascontrolledwith a model 7545 Masterflex pump (Cole-Parmer In- strumentCo., Chicago, IL)equipped witha7021-20 head. PBMC, washed oncewith elutriation medium, were loaded into the elutriation chamberinafinal volumeof 50ml( ^1X109cells)^atarotorspeedof 2,020 rpm andan initial flowrate of 9.0 ml/min that was then in- creasedby 0.5mlevery 10 min. Platelets elutedimmediately,followed by lymphocytes (ataflowrateof 11.0to12.5ml/min)andamixed lymphocyte/monocytefraction(upto 15ml/min),and

finally

puri- fied monocytes(at 15.0ml/min). Aftercompleteelution of this mixed cellpopulation, the flow rate wasincreased to40 ml/min to elute the remaining purified monocyte fraction. Thisprocedure re- sulted in lymphocyte (>99% purity) and monocyte preparations (> 95%purity,asjudgedbynonspecific esterasestaining, SigmaKit 91-A) of>99%viability (trypan blueexclusion).

For MDM preparations, elutriated monocyteswere washed and resuspended inRPMI-1640, and adhered for 90 minat37°C in 22-mm diam tissue culture wells(Costar,Cambridge, MA)at aconcentration of 1-3 X 106 cells/well. Afteradherence,themediumwasreplaced with 1.5 ml RPMI-1640with 10% (vol/vol) heat-inactivated human serum, 20mMglutamine,100IU/mlpenicillinand 100I g/mlstrepto- mycin,andthecells lefttodifferentiate for 6 dat37°C in 5%CO2in air with media changesondays 3 and 5. On day6, MDM were>99%

esterasepositiveandroutinelyyielded 200 sgprotein/106 cells.

Oxidativemodfi^cationofLDL. Ham'sF-10mediumwasusedfor allexperiments;the medium contains 3,uM Trpand unless stated otherwise,wassupplemented with 75-100AML-Trp,^aconcentration similartothat in humanplasma,i.e.,60±15

AM

(31).ForPBMC and purifiedmonocytesexperiment,LDL( 2.5_mgprotein/ml)(1 vol) wasincubated inHam'sF-10 medium(10vol) for upto24h at37°C inahumidified atmosphere (5%CO2inair)in the presenceorabsence of cells.Aliquots (300

AI)

^{ofcell-freemediumorcellsuspensionwere}

withdrawnatvarious timesand, where needed,cells removed by cen- trifugation (400 g for 10 minat4VC). For MDM experiments, 0.6 ml of LDL(100Mg/ml F-10 medium)wasadded per well and incubated for 24 h before the supernatant (600Ml)wasremoved forlipid and antioxidantanalyses. Where indicated, human IFNY was added at 1,000 U/ml. In thecaseofMDM, cellswerepreincubated for 20 h with 100or250U/ml ofIFNybefore addition of LDL. The relative electro- phoreticmobilityof oxidized LDL ( 1-2Mg)^wasdeterminedusing precast agarosegels(Ciba-Coming,PaloAlto, CA)subjectedtoelectro- phoresisinbarbitone buffer (pH8.6)at90Vfor 45 min and stained with Fat Red 7B stain (32). Before electrophoresis, the LDL contained in theculture mediumwasconcentrated bycentrifuge-assisted ultrafil- tration (Lida, Kenosha, WI).

Determinationofa-TOH,lipids, and CE-OOH by HPLCanalysis.

Lipid-solubleantioxidants (including a-TOH), neutral lipids (mainly free cholesterol and cholesterylesters) and CE-OOH were extracted quantitatively from the LDL-containingcell culture supernatantas described(10,27, 33, 34), and theextracts werestoredat-70'C for up to 24 h before analysis. Where indicated, cholesterylbenzoate was addedas aninternal standardtotheorganic solvent before extraction (27). Inallother cases,lipid-solublecomponentsofLDLwere stan- dardizedinternally againstcholesterol. The hexaneextracts weredried undervacuum and redissolved in ethanol (180 ul) for analyses by various HPLC methods. a-TOH and other lipid-soluble antioxidants, including ubiquinol-1O, weredetermined by reversed-phase HPLC with electrochemical detection (10). In some experiments, a-TOH was determined in thelipidHPLC assay byfluorescence detection (292/

330nm) (Spectra Physics,San Jose, CA; FL2000). Unoxidized neutral lipidsandCE-OOH were separated on an LC- 18 column (Supelco Inc., Bellefonte, PA; 25X0.46 cm with 5-cm guard column,

5-,um

^particle size),elutedwith tert-butyl alcohol/methanol (1: 1, vol/vol) at 1 ml/

min anddetected byultraviolet absorbance at 210 nm and post-co- lumn chemiluminescence respectively, as described originally (33), with the modification described (27).

AssessmentofTrp and arginine metabolism. For Trpmetabolism, theculturemedium(z ¹ ml)^wasdeproteinizedwith TCA(4%final concentration,wt/vol) after cells had been removed by centrifugation.

Trp, Kyn, andanthranilic acid in the resulting supernatant were sepa- ratedon aVeloSepRP-18column(AppliedBiosystems, Inc., Foster City, CA; 10 x0.32cmwithi-cm guard column, 3-,um particle size) with 100mMcholoroacetic acid/acetonitrile (pH 2.2) (8:2, vol/vol) and detectedphotometrically(Kyn, 365 nm; Trp, 280 nm) or fluori- metrically (anthranilic acid, 328/422 nm) (35). 3HAA was deter- minedbyelectrochemical detection (+0.5 V), eitheron-~linewith the above HPLC system(using^anLKB/Pharmacia model 2143 detector with glassy carbon electrode) or after extraction into ethyl acetate and separation on an LC-18-DB column (Supelco, 25 X 0.46 cmwith guardcolumn, 5-Mm particle size) (29). Degradationofargininewas assessed by measuring the nitrite present in the supernatant of the LDL-modifyingcell culturesusingtheGriess reagent (36); and/or by HPLCamino acidanalysis usingthe o-phthalaldehyde method(37).

Statistical dataanalysis.ThepairedStudentttest(one-tailed)^was usedtoevaluatedifferences in the absolute values of the various groups oftreatments.Significancewasaccepted^attheP<0.05 level,unless statedotherwise.Owingtotherelativelylarge variations in antioxidant andlipid contentofLDLpreparations from differentdonors(38), a-TOH,andcholesterylesterscontainingpolyunsaturatedfatty acids, i.e., cholesteryllinoleate (C 18:2)andcholesterylarachidonate (C20:4), wereexpressedaspercent values relativetotheinitialconcentration andpresentedasmean±SDorSE,andrange. CE-OOHvalueswere

givenin absolute concentrations.

Results

Ashas beenreported by^others(20, 39),incubation of PBMC orMDM in RPMI 1640 + 10% FCS and in thepresenceof

IFNy

resulted in induction of IDO as assessed by the time-de- pendent decrease of Trp and concomitant accumulation of Kyn,anthranilic acid,and 3HAA in the culture medium (not shown). Inthe absence of

IFNy,

degradationofTrpand for- mation of Kynpathway metaboliteswere muchsmaller, and exposure ofpurified lymphocytes to

IFNy

in the absenceof monocytes did not result in significant Trpdegradation (not shown).These resultsdemonstrate that

IFNy

induces Trpdeg- radation inhuman monocytes/macrophages but not lympho- cytes. Furthermore, the presenceof lymphocytes doesnot af- fect the extent of

IFNy-induced Trp

degradationbymonocytes (not shown, 39).

Studies on "cell-mediated" oxidative LDL modification aremostly carriedout in serum-free Ham's F-l0(e.g., 3, 6),a medium thatcontains added transition metals. Where investi- gateddirectly, the transitionmetalshavebeendemonstratedto berequiredfor the oxidation process toproceed(3, 6).In stud- iesusing transition metal-poorRPMI 1640

medium,

Cathcart and co-workershave shownthat activated butnotunactivated monocytesoxidizeLDL asassessedbythe

comparatively

less specific (tothe methods usedhere)thiobarbituric acid reactive substances assay(e.g., 4,40).Tostudyapotentialeffect of

Trp

metabolism along the Kyn pathway on monocyte/macro- phage-mediated LDL oxidation, we therefore

initially

exam- inedtheefficacy with which IFNy induces oxidative

Trp

^metab- olisminPBMC culturedin Ham's F-10,amediumpermissive for cell-mediatedLDLoxidation. TableIshows thatIFNy^also induced

Trp

degradation and metabolite formation in F-10 medium, with 3HAA andKynplus anthranilic acidaccount-

ing

^{for 2} and 63% of the

Trp ^lost, respectively. Although

detected, 3-hydroxykynurenine,^aprecursor of3HAA,didnot accumulateto concentrations >0.2

,tM

(not shown). Ithas beenreportedrecently (41 )that

quinolinic

acid

(which

wedid not measureandwhichdoes notcontainantioxidant moieties) accountsfora

significant proportion

oftheadditional

Trp

^de- graded byhumanMDM;(see,however, reference

20).

Addi- tion ofLDL toPBMC culturedin F-10didnotaffect theextent of

IFNy-induced Trp degradation

andmetabolite formation (TableI).Cellviability (measured bytrypanblue

exclusion)

in the

(serum-free)

F-10 mediumwas2 95%after24 hofincuba- tionandtherefore didnotaffectour

experiments

onLDLoxi- dation (see below) whichwerecarriedoutwith cells cultured forup to 24 honly.

IncubationofPBMC or MDMinF- 10medium inthe ab- sence orpresenceof IFNy for24 hdidnotresult in detectable

formation (2 0.5 nmol) of extracellular nitrite (not shown).

Treatment of these cells with IFN-y also did not result in a decrease in extracellular arginine, as assessed by amino acid analysis ofthecellsupernatant(not shown). These resultsindi- catethat the nitric oxide pathway was not induced under our conditions,consistent with data reported by others for human MDM(42), butin contrast to the situation observed with mu- rine macrophages (see reference 12 and references cited therein).

Table IIsummarizes the resultsobtained for LDL oxida- tion in F- 10 medium in the absence or presence of human PBMC orpurified monocytes, and theeffects ofadded

IFNy

(z I03 U/ml) or3HAA (10 ,M) on it. LDL oxidation was assessedby threedifferent methods: the loss of a-TOH, quanti- tatively the major lipid-soluble antioxidant associated with LDL(38); the accumulation of CE-OOH, the major initial lipidoxidationproductformedinoxidizingLDL(10,34, 43);

and the increase in LDL's relative electrophoretic mobility, an index of the surface charge of the LDL particle. As can be seen, incubation of LDL in Ham's F-10 medium for 24 h in the absence of cells resulted in oxidation ofthe lipoprotein, as indi- catedbyalteration of all three oxidation parameters. Signifi- cantLDLlipid peroxidation was observed in the presence of a-TOH; in fact, on average some 36 molecules ofCE-OOH were formed foreach moleculeofa-TOH consumed (Table II).Sincethe ratesofa-TOHconsumptionand CE-OOH for- mationareindicative forthe ratesofinitiationand propagation oflipid peroxidationin LDLrespectively,these results demon- stratethat cell-free LDL peroxidation in F-10 medium pro- ceeded via achain reaction, despitethepresence ofa-TOH.

Thisis consistent with recentfindings(44, 45) that indicated that underthese conditions LDLlipidperoxidation isactually mediated by a-TOH.Cell-freeLDLperoxidationwasinhibited almostcompletely bytheaddition of10MM3HAA.

IncubationofLDLinthe presenceofeitherPBMC orpuri- fiedmonocytesmarkedlyacceleratedlipoprotein oxidation,as indicatedby agreater lossofa-TOH and extent ofCE-OOH accumulationaswellasincrease in relative electrophoreticmo- bility (Table II). As wasthe case in thecell-freesystem, LDL lipid peroxidation occurred despite the presence of significant levels of a-TOH. Onamolarbasis, relativelymoreCE-OOH were detected than a-TOH lost. The simplest explanation for this is that cell-mediated LDL lipid peroxidation also pro- ceeded via a chain reaction. Co-incubation of PBMC with

IFNy

significantly decreasedthe extentofLDL oxidation (Ta-

TableLEffects^ofIFNy on Extracellular Levels of Trp and Kyn Pathway Metabolites Produced by Human PBMC Incubated in

F-O0

Mediuminthe Absence (-)orPresence(+)ofLDLfor24hat370C

Metabolite* Trp Kyn AA 3HAA

LDLV - + ^- + - + ^- +

Cell-free

(,gM)

^{95.8±5.7 102.5±7.3 0.28±0.4 0.40±0.3} ND ND <0.01 0.02±0.04

PBMC

(nmo1

105.9±13.2 104.1±10.2 0.69±0.5 0.56±0.3 ND ND 0.07±0.1 0.03±0.04

PBMC/rIFN-y(_103U/ml) 56.4±12.9 56.3±7.0 30.7±3.8 29.7±8.0 1.44±0.5 1.52±0.8 0.96±0.4 0.75±0.4

*Values represent concentrationinmicromolar (cell-free) or nanomoles per 5^X 106cellspresented as

mean±SD

of threeto seven independent experimentsperformed induplicatesorsingle determinations. $ Dialyzed.

ND,notdetectable.

2152 Christenetal.

TableII.a-TOHand CE-OOH Levels, and Relative Electrophoretic Mobility ofLDLafter Incubationin Ham'sF-JOfor24h intheAbsenceorPresenceofPBMCorPurified Monocytes*

LDL only LDL+PBMC LDL + monocytes

Control Noaddition +3HAAt Noaddition rIFN-y +3HAA* Noaddition +3HAAt

F-10 (6) (6) (4) (5) (4) (3) (2) (2)

a-TOH (%)§ 100a 82.1±17.7b 87.7±17.88.c 16.6±23.8d 46.2+34.6bc 87.6±8.7ac 41.7 88.4

CE-OOH (gM)11 0.12±0.21a 12.7±10.7b 0.05±0.04ab 28.0±10.3c 14.6+15.4 ab o.oo+o.oab 41.6 0.09 Relative electrophoretic

mobility 1.008 1.24+0.18 1.08±0.17a- (5) 1.47±0.18c 1.24±0.20b 1.14±0.13a 1.44 (1) 1.11 (1) Resultsare expressed as means±SD (numbers in parentheses represent number of independent experiments). Values within rows sharing the sameletter(s) are notsignificantly different from each other.

*Cells(5^{X 106}PBMC/ml^or106monocytes/ml) preincubatedin RPMI 1640+ 15%humanserumfor 2-3 hat

370C

before 24-h exposureto LDLwith and without additives. ^*Final concentration was 10MM(includesdata from experiments in F-10 medium without added Trp).

§ Initial concentration (= 100%) was 1.95±0.84MM(mean±SD) ranging from 0.9 to 2.91 ,uM. 1' CE-OOH of control LDL was determined after sterile-filtrationpriortotheincubation. The level of CE-OOH of the freshly prepared LDL before gel-filtration and any further treatment was 0.007±0.01 MM(mean±SD, n=6),consistent with the very low concentrations found in rapidly isolated LDL (10, 34).

bleII). An even greater effect was observed, when the potent antioxidant3HAA(17)was added to the cells at the beginning of the incubation and in the absence of

IFNy.

In this case, concentrations of CE-OOHanda-TOH as well as LDL's rela- tiveelectrophoretic mobilitywereindistinguishable from the corresponding control values. Monocyte-mediated oxidative modification ofLDL was prevented similarlyby theaddition of 3HAA (Table II). These results demonstrate that LDL (per)oxidation in F-10 mediumproceedsviaachain reaction that is accelerated by human mononuclear cells, but canbe inhibited stronglybyeither addition of micromolaramountsof 3HAA or treatmentofthe cellswith

IFNy.

Since the concentrations of 3HAA used in Table II ex- ceeded thoseformed by PBMC activated by IFNy (cf.TableI) weexaminedtheconcentration-dependent inhibition ofLDL oxidationby3HAA. As can be seen inFig. 1,3HAAinhibited LDLoxidationatverylowconcentrations, in thepresence or absenceof cells.Inbothcases,accumulation of

CE-OGH

was stronglyinhibitedby3HAAconcentrationsaslow as 0.5

,M,

where

only

10-20% of the extracellular a-TOH was lost.

Addition ofKyn (50 MM) together withAA (2 MM) to the cell-free orPBMC-containingsystem atconcentrationssimilar tothoseproduced

by

IFNy-treated cells after24 h

(cf.

Table

I)

didnotreduceLDLoxidation

significantly (data

^not

shown).

Similarly,

addedascorbic acid (0.5

MM)

didnot

significantly

inhibit PBMC acceleratedLDLoxidation (not shown).These results showthat 3HAAat aconcentration similartothatpro- ducedby

IFNy-treated

PBMC

strongly

inhibitsLDL

lipid

per- oxidation.

One approachto assesstheextentof

participation

ofoxida- tive Trp

degradation

in theobserved

IFNy-induced

inhibition of PBMC-mediated LDL oxidation in general and that of 3HAAinparticular,would be to

selectively

inhibit 3HAA for- mationinstimulated mononuclear cells. Thelatteris depen- dent onthe

monooxygenase-catalyzed hydroxylation

of

Kyn

(20) (SchemeI). Since monooxygenases areoften flavin-de- pendentenzymes, wetestedwhether theflavin analogue^DPI couldinhibit 3HAAformation. Indeed,DPIinhibited IFNy- induced formation of 3HAA by PBMC very

effectively

but showedno

significant

effecton

degradation

of

Trp

andforma-

100 80

E ₆₀

x

40

0

0%

0i>

Jc

I-O x 00

9i

100 80

60 40 20 0

0 0.1 1 10

3HAA

(jlUM)

Figure 1. Inhibition of LDLoxidation by 3HAA.LDL(dialyzed)was incubatedfor 24 hat37°CinF-10medium only (o ),orin the pres- enceof PBMC(5X 106cells/ml,*).Variousamountsof 3HAAwere addedtotheLDL-containingculturesatthebeginningof the incuba- tion.LDLoxidationwasmeasuredbydetermination of extracellular a-TOH andCE-OOHasdescribed in Methods. Each value represents themean±SEoftriplicateincubationsofatypical(ofthree cell-free andtwocell-containing) independentexperiment. (A)^a-TOHcon- centrationremainingafter 24 h of incubation relativetothat present priortoincubation(i.e.,0.8MM⁼ 100%). (B)CE-OOHconcentra-

tion, expressedrelativetothat detected after 24hin the absence of 3HAA(i.e.,3.5and10.7MMin the absence and presence of cellsre-

spectively).

tion of Kyn and anthranilic acid by these cells at concentra-

tionsup to sM(Fig. 2). Comparableresultswereobtained

with MDM and whetherLDLwaspresentornot(not shown).

Wenexttested whether the inclusion ofDPIataconcentra-

tion that "selectively" inhibited 3HAA formation could re- verseIFN-y-mediated inhibition of PBMC-facilitatedLDLoxi- dation. The time-dependent analysis of such examination is shown in Fig. 3. Incubation of LDL in thepresenceof PBMC resulted in rapid consumption of extracellulara-TOHafteran initiallag phaseof 6 h(Fig. 3A). ^Thiswasparalleledby^a

gradual loss from the medium ofC18:2, the major polyunsatu- ratedcorelipid and therefore substrate for oxidation inLDL (Fig. 3 B). Concomitant with the decrease in a-TOH and C18:2, extracellular CE-OOH increased (Fig. 3^C),accounting for82.7±43.6%(mean±SD,n=4)ofthe C 18:2 lost. CE-OOH representthoselipid hydroperoxides thatarederived from poly- unsaturated fatty acids in LDL esterifiedtocholesterol. How-

ever, since the cholesteryllinoleate hydroperoxide detected during oxidation of human LDL usually accounts for more

than 85%of the total CEOOH formed (33) the above calcula- tion(i.e., CE-OGH formedperC1 8:2 lost) givesareasonably accurateestimate ofthe degree of conversion of nonoxidized

core lipids into the corresponding hydroperoxides. Impor- tantly,inthe absence ofIFNy, addition of DPIdidnotalter cell-mediated LDL oxidation, as seen from the rates ofa-

TOH/C18:2 consumption and CE-OOH formation (Fig. 3).

Likewise, LDL oxidation in F-l0 medium alonewasnot al- teredby the addition of DPI (not shown). Incontrast,incuba- tion of LDLwithPBMC in thepresenceof

IFNy

preventedor

almostcompletely inhibited the consumption of C1 8:2 and accumulation ofCE-OOH, and reduced the loss of a-TOH by

50% (Fig. 3). Insome casestheamountsof CE-OOH de-

tectedinthepresenceof

IFNy

^{weresimilartoor evenless than}

~0

Is

¹⁰⁰

0

0 - ^_ 80

C

L. <e _

4mX ⁶⁰

00 40

q ²⁰

Yd ⁰

0 0.001 0.01 0.1 ¹ 10

DPI (pM)

Figure2.DPI-mediated "selective"inhibition ofIFNy-induced for- mationof 3HAA by PBMC. Cells (5^x 106/ml)^wereincubated in thepresenceofIFN-'y (I03 U/ml)for48 hat37°Cinthe absenceor presenceofvarious concentrations ofDPIbeforesupernatantswere

analyzedforTrp (.), Kyn (A),AA(*)and 3HAA(v)^asdescribed inMethods. Ethanolatthehighest concentration usedassolventfor DPI(0.5%) hadnosignificanteffectonIFNy-induced Trpmetabo- lism. Valuesrepresentmeans±SD of threetosevenindependent^ex- perimentsandareexpressedaspercentage of the controlincubation, i.e.,intheabsence ofDPI. The absolute control values(i.e.,100%

values)forTrp and its metabolitesareindicatedinTable^I.

_- lUV 0

60

20

100

N

CO

,5

80

60~

24

0

9 LU

16

8

0

0 6 12 18 24 ⁰ 6 12 18 24

Time(h)

Figure3.Time-dependent analysisof theinhibitionofcell-mediated LDLoxidationbyIFNyand itspartialreversalbyDPI. LDL(dia- lyzed)wasincubatedin the absence(o),^orpresence(solidsymbols) of untreated(A-C)^orIFNy-treated⁽103U/ml, D-F)PBMC(5

X 106 cells/ml, 2 ml final volume)^at37°Cinthe absence(o)or presence(A&)of 20 nM DPI added^atthebeginning^{of the}incubation.

Aliquotsof the medium^wereanalyzedfor a-TOH(AandD),C18:2 (BandE),andCE-OOH(CandF) using cholesterylbenzoate^asthe internalstandardasdescribed in Methods. Initial concentrations (means±SE)fora-TOH, C18:2,and CE-OOHwere1.25±0.3(rang- ing from0.55 to 1.83),95.6±19.0(from54.9to 140.8),and 0.008±0.005ALM,respectively. Each value represents themean±SE of 4independent experiments performedinduplicates. (Insets)CE-

OOHaccumulationduringcell-free(C)andIFNy-inhibitedcell-me- diated(F)LDLoxidationpresented usinganenlargedscale.*Signifi- cantlydifferenttocorrespondingvalue of untreated cells(minus IFN-y).**SignificantlydifferenttoIFNy-treatedcellsinthe absence of DPI.

those observedintheappropriate cell-free incubation (Fig. 3, E andFinsets). Addition of 20 nM DPI significantly reversed part of theinhibitory effectofIFNy^onconsumption of C 18:2 andaccumulationof CE-OOH (Fig. 3, E and F). Since only partial reversalwasobservedwith thisverylowconcentration of DPI, ^we tested whether higher inhibitor concentrations caused^{a more}pronounced effects. Indeed, addition of 100nM DPI, aconcentration thattotally inhibited 3HAA formation (Fig. 2), reversed the ability of IFNy to inhibit PBMC-me- diatedLDLoxidation between 51% and 100%, dependingon

the parameter of LDL oxidationmeasured(TableIII).

As ^asecondapproachto assesswhether Trp metabolism along theKyn pathway contributedtothe observedinhibitory action of IFNy on cell-mediated LDL oxidation, ^we used

2154 Christenetal.

A D

I~~

E2

F

0 12 24

U..j I

--

B _T

6

MDM inHam's F- l0 medium that was not supplemented with

Trp.

Commercial F- 10 medium contains only low micromolar concentrations of

Trp,

so that significant amounts of 3HAA can notbe formed by cells even when IDO is induced by treat- mentwith

IFNy.

Indeed,addition of

IFNy

to MDM cultured in nonsupplemented medium did not result in detectable Kyn or 3HAA (data not shown) and had no effect on MDM-me- diated LDL oxidation (Fig. 4). In contrast,

IFNy

treatment of MDMin Tr-supplemented F-IO medium resulted in forma- tion of micromolar amounts of 3HAA (ranging from 1 to 30

AM; depending

^{on the}cell numbers and

IFNy dosage used)

and almost complete inhibition of LDL oxidation (Fig. 4).

Similar results were obtained with PBMC(not shown). Addi- tionof

Trp

⁽⁷⁵

,M)

^{alone to}either the MDM or cell-free incu- bationsonly marginally affected LDL oxidation, whereas in- clusion of DPI in the

MDM/Trp/IFNy

system caused sub- stantial inhibition of

Trp

metabolism and reversion of the cytokine's inhibitory effect on LDL oxidation (not shown).

Together, these results strongly support an important role of 3HAAformation in

IFNy-mediated

inhibition of LDL oxida- tion by human monocytes/macrophages.

Discussion

We have examined the potential inhibition by IFNy of the early stages of human monocyte and macrophage-mediated LDLoxidationin Ham's F- I0mediumcontainingphysiologi- calamountsof added

TmP,

measuringtheconsumption ofthe lipoprotein's major antioxidant (i.e.,a-TOH) andtargets for oxidation (C 18:2andC20:4)togetherwiththeaccumulation ofCE-OOH, the primary and major oxidation product. Our resultsshow that as with murine macrophages,

IFNy

strongly attenuates cell-mediated LDL oxidation by human mono- cytes/macrophages. Unlike the murinesystemhowever,

IFNy-

mediated inhibition ofLDL oxidation by human

monocytes/

macrophages does not appear to belinkedto NObiosynthesis, asthispathwayisnotinduced,at least under theexperimental conditions used in thisstudy. In contrast, our resultsindicatea

TableIII. ReversaloftheInhibitory

Effect of

IFN-y onPBMC-mediatedOxidation

of

LDL

by

^DPI

a-TOH* CE-OOHP CEPUFAS

%remaining AM ^%degraded

PBMC"1

0 29.5±2.8 79.5±2.8

PBMC+IFNy 20.7±4.4 5.7±2.5 10.8±10.0

PBMC+IFNy+DPI 0 18.0±2.5 67.0±5.0

F-l0alone 5.0±5.1 13.5±5.8 39.8±3.5

DPIat 100nMinhibited 3HAA formationby 100%consistentwith results shown inFig. 2. 3HAA level in PBMC+IFN'ywas0.95 MM±0.2.Resultsarepresentedasmeans±SD ofanindividualexperi- ment carriedoutintriplicateandrepresentativeof 2indpendent experiments.

*Initial a-TOH concentration(100%)was1.7MM±0.3. ^*CE-OOH level before additiontoculture mediumwas0.01 MM. ^f

CEpuFu

refertoC 18:2plusC20:4. 11 Cells(5^X 106PBMC/ml)wereincubated in Ham'sF-10,supplemented^{with 75}MML-Trp,for 24 hr in the presence of LDL and the presenceorabsenceofIFNy(1000 U/ml).

Figure4. Inhibition of

100 A MDM-mediated LDL oxi-

dationbyIFNy^isdepen-

a 80 dent onthe presence of

Trp

E2 60 in the culture medium.

RS T _ LDL (100 Mg protein/ml)

cNj 40 wasincubated for 24 h in

u) 20 ~ _ _ Ham's

FlO

medium sup-

plemented (75MM)ornot

0 supplemented with

Trp

^and

B inthe absenceand_presence

1 2 of MDM(3^{X 106}cells/

well)andIFNy(1,000 U!

9 ml)before theconcentra-

tionsofcholesteryllinoleate

o (A),CE-OOH(B),^anda-

TOH(C)^{in themedium}

weredetermined (see

0

Methods).

^Beforeexposure

toLDL,^MDMwerepre- cultured for 20 h in RPMI

- 80 1640 mediumcontaining

_ 10%heat-inactivated hu-

60 man serum in the presence

orabsenceofIFNy (250

0 . U/ml). The results show

Zs020

the means±SD oftriplicate determinations from one

0 outof three

independent

MDM MDM MDM MDM

experiments.

Inall three Trp IFNy ^IFNy experiments

IFNy-induced

Trp inhibition of MDM-me- diated LDLoxidation was dependentonTrpsupplementation ofthemedium. In the absence ofIFNy additionofTrptoMDMhad small butvariableeffects on cholesteryllinoleate consumptionandCE-OOHformation,butdid notaffect a-TOH consumption. Initial concentrations(100%values) of C18:2 and a-TOHareindicated in thelegendtoFig.3.

protectiverolefor oxidative

Trp

^metabolism and 3HAA for- mation in thisprocess.

Theevidence for theparticipation ofoxidative

Trp

^metabo- lism and 3HAA formation in

IFN'y-mediated

inhibition of hu- manmonocyte/macrophage-mediatedLDLoxidation is based on the

following: Firstly,

exposure

of

these cells ^to IFNy- induced oxidative

Trp

metabolism along the Kyn pathway that resulted inthe releaseof low micromolaramountsof 3HAA.

Secondly, this

aminophenolic

metabolite isa

powerful

antioxi- dant( 17,46): it strongly inhibitedLDLoxidationwhen added toboth

cell-free

and

cell-containing

systemsin the absence of

IFN'y

and atconcentrations similartothoseformed by

IFN-y-

treated mononuclear cells invitro.

Thirdly,

incubation ofLDL andIFNy with PBMCor MDMunderconditions that resulted in oxidative

Trp

degradation with concomitant formation of 3HAA, inhibited LDLoxidation to an extentsimilartothat observed withexogenously added 3HAA. Fourthly, "selective"

inhibition of IFNy-induced formation of 3HAA by inclusion of the

flavoprotein

inhibitorDPI(47,48) reversedtheinhibi- tory action of this

cytokine

on mononuclear cell-facilitated LDL oxidation. Finally, the inhibitory activity of IFNy on MDM-mediatedLDL oxidation wasdependent onthepres- enceof

physiological

amountsof

Trp

^{in the}medium that did

allowdetectablelevelsof 3HAAtobeformed. Togetherthese results show that 3HAA is amajor contributing factorinthe

IFNy-mediated

inhibition of monocyte/macrophage-me- diated LDL oxidation in transition metalcontainingmedium.

Previousworkbyothers hasinvestigatedamodulatoryrole of

IFNy

oncell-mediated LDL oxidation. Cathcart et al.(49) treatedhumanmonocyte-derived macrophageswith this cyto- kineto examineapossible contributory role of NADPH-oxi- dase on LDLoxidation, but failed to detect an oxidation-en- hancing effect. Hurt-Camejo and colleagues (50) showed (though didnotdiscuss)results consistent with aninhibitory action of

IFNy

on LDLlipid oxidation mediatedbyhuman MDM in RPMI 1640 medium supplemented with 5

1AM CuS04 (their

^Table

IV).

This mediumcontains 25

,gM Trp,

most ofwhich would beexpectedtobedegradedunder their conditionswith formation of 3HAA(cf.TableI). This raises thepossibility that IDO-initiated Trp degradation ^toomight havecontributedtotheinhibition ofLDLoxidation observed by these authors.

IFN'y

isanimportant modulator of theimmune response, affectingmany cellular functions ofmonocytes/macrophages (51).Amongthese effectsare anincreased

capacity

of

phago-

cytes toproducereactive oxygenspeciesuponappropriatestim- ulation and, conversely, the induction ofSeveral metabolic pathwaysthat can beregarded^asprovidingadditional antioxi- dantprotection within and

surrounding

the cells

(Scheme I).

Here weinvestigated induction ofIDO, the initial and

02--

requiringenzyme(52) oftheKyn

pathway

thatcan

give

riseto hydroxylated, antioxidant active metabolites

( 17) (Scheme I).

Withone

exception (i.e.,

A498

kidney

carcinoma

cells),

hu- man

monocytes/macrophages

arethe

only

cells knowntopro- duceand release 3HAAupon

IFNy

stimulation

(20, 21).

^As mentioned above, evidence forthe

participation

ofTrp metabo- lismand3HAAformation in

particular,

^{in the}

IFNy-mediated

inhibition ofLDL

(per)oxidation

is based in partonthe fact that inclusion of submicromolaramountsof DPI reversed the inhibition in

consumption

of a-TOH,C20:4 andC

18:2,

and formation of CE-OOH. The extent of thisreversal increased withincreasing concentrations ofDPIused

(cf. Fig.

^{3 and Ta-} ble4).

WehaveusedDPIat lowconcentrationsin an attempt to inhibit formation of 3HAA without affectingotherenzymatic reactions thatareknowntobeinhibitedbyhigherconcentra- tions ofDPI andmay affect LDLoxidation. Thus, thefinal concentration ofDPIused(20-100nM)issometwo ordersof magnitude lowerthan thatrequired for inhibition ofNADPH oxidase (53), the superoxide anion radical-producingenzyme suggestedtobeinvolvedinhumanPBMC-mediatedLDL oxi- dation (54). Also, inhibition ofNOsynthase inmurine macro- phages requires somewhat higher DPI concentrations than used in ourexperiments( 12, 14, 48). In any case,an involve- mentof thisenzyme in the downregulation of LDL oxidation by IFNy asreported for mouse peritonealmacrophages ( 12, 13), isunlikelyto be important in the humansystem, as nei- theraccumulation ofnitrite nor degradation ofarginine from the culture medium were observed. In contrast, our studies

using

Trppoormediumclearly demonstrate arequirement for Trp metabolism inthe inhibitory activity seen byIFNythat is

affected

by DPI.

Incubation ofLDL in the presence of PBMC resulted in detectable extracellularCE-OOHafteran initiallag period of

6

h,

^when

significant

levels of a-TOHwerestillpresent

(Fig.

3). The observation that a-TOHwasconsumed

during

^rather than before theonset oflipid peroxidation is consistent with ourpreviousobservationson

cell-free,

radical-mediated LDL oxidation( 10, 12,44)and also transition metal-induced oxida- tion

(45).

It is alsoconsistentwitharole for a-TOH-mediated

peroxidation (45) during

PBMC-mediatedLDLoxidation

(in

theabsence of suitable reductants for

a-tocopheroxyl radical)

and LDLlipid peroxidationproceedinginachain reaction. In

fact, preliminary

studiesshowed that enrichment of LDL with a-TOH enhanced MDM-mediated

lipid hydroperoxide

forma- tion

during

^the

early

stages of

lipoprotein

oxidation inF-10 medium (Dr.

Thomas,

Dr. Jiri

Neuzil,

^{and Dr.}

Stocker,

^un-

published observations).

A

striking finding

ofour

study

^wasthe

efficacy

^{with which}

very low concentrations of 3HAA inhibited LDL oxidation.

Although it is difficultto

directly

compare our results with those from othergroups, 3HAA is

clearly

oneof themosteffi- cientLDL antioxidants known atpresent.

Hence,

^anunder-

standing

of the

mechanism(s)

involved in this inhibition will be

important.

^3HAA

likely

inhibited LDL oxidationatan

early

stage ofradical-induced chain oxidation of the

lipoprotein,

whether cellswerepresentor not.The

possibility

^that3HAA chelatesand makes the transition metals in Ham's F- 10 me-

dium unavailableto

participate

^{in LDL}

oxidation,

^{canbe ruled}

out for severalreasons:

Firstly,

separatein vitro studies

(Dr.

Christenand Dr.

Stocker, unpublished observations)

^showed that3HAA

(5 ,M)

^{is unabletoinhibit F-}10

medium-catalyzed (aut)oxidation

of ascorbate.Even whenpresent^atuptoa50- fold molarexcess overthe

metal,

^{3HAA doesnotinhibit}

Cu2+

orFe3

-catalyzed

^oxidationof ascorbate in

phosphate buffer,

pH 7.0.

Secondly,

neither the

absorption

^nor fluorescence spectra of 3HAA in solution are altered

by

the addition of eitherCu2+^or

Fe3", suggesting

^{that the}

aminophenol

^doesnot

complex

^{withthesemetalsunder}these conditions. This iscon-

sistent with

previous

studies

demonstrating

that stable com-

plexes

of 3HAA with

Cu2+

or

Fe3"

areformedatalkaline

pH only, i.e.,

^{whenboth theamino and}

hydroxyl

groups of 3HAA are

deprotonated (the pK.

^values for amino and

hydroxyl

groupsare5.2 and10.

1, respectively) (

⁵⁵

). Thirdly,

the results in

Fig.

^{1 showthatLDL}oxidation isinhibited

by

^concentra- tions of 3HAA that are

clearly

below

(i.e., submicromolar)

thoseof the transition metalspresentin Ham'sF-10.

(i.e.,

³

,uM

iron

plus

0.01 tM

copper).

Alternatively,

3HAAmay protect LDLfrom oxidation

by reducing

(or

eliminating)

the tocopheroxyl radical produced from

a-TOH, and/or maintaining (LDL-associated)

ubi-

quinol-10

in the

reduced,

antioxidant active form. Eitheror bothof these reactionswould

provide powerful

antioxidation

(10,

11, 44, 45). ^A reaction of tocopheroxyl radical with aqueousreductants

(antioxidants)

has beenreported forascor- bate andbilirubin

(56, 57).

Whilewearecurrently

investigat- ing

these

possibilities,

^itis clear fromourresultsthat if 3HAA operates in one or both ofthe lattermodes, components of F-10 mediumwouldhavetomaintain 3HAA in thereduced statebecauseitisnotconsumed

substantially

while

providing

antioxidation. Thisopensthe

intriguing possibility

that IFNy modulatesthe

production

and/or releaseby monocytes/mac-

rophages

ofa

"catalyst"

forLDLantioxidation. Lowconcen- trations ofsucha

catalyst

would besufficientto

provide

sub- stantial additional

protection by maintaining

^LDL'santioxi-

2156 Christenetal.

dant(s) in the reduced state through common and readily available reductants which themselvesare not efficientantioxi- dants,andcan notinteractwithLDL's a-TOH.

Inaddition to the protective antioxidant action of

IFNy,

this cytokine is likelytohave other effects relevanttoatheroscle- rosis. Thus, treatment of human MDMwith IFNycauses inhi- bition of synthesis and secretion oflipoprotein lipase (58)as wellasexpression of the scavenger receptor and thedevelop- mentof foam cells in vitro (59). Thesuppressiveeffect of

IFNy

on production ofplatelet-derived growth factorbymononu- clearphagocytes(60) may also be importantinthe control of smooth muscle cellproliferation,which isamajor^eventin the developmentof the atherosclerotic plaque. It has been demon- strated that

IFNy

administration regulates vascular smooth muscleproliferation andinhibitslipase activityinvivo(61 ).

Lymphocytes and monocytes/macrophages are major cell typespresent in thehealthy intimaatabout thesameratioas in circulation (25). As a fatty streak forms and progressesintoa lesion, therelative contributionofmonocytes/macrophagesin- creases as does the absolute number ofcells(25).IFNyislikely to be present in atherosclerotic lesions, as evidenced by the presenceof MHC classIIantigensandotherspecific activation markerson cells foundinthoseareas (23, 25). Inductionof IDO with release of 3HAA thus appearsfeasibleand may either alone,ortogetherwith theaforementioned

activities,

influence the development of atherosclerosisaswell asotherinflamma- tory processes.

Acknowledgments

We thank Dr. P. Bannon for his help with the elutriation of monocytes andperformingLPS tests,Dr. W.Jessup for thegiftof DPI, and Dr. W.

Sattlerfor the synthesis and purification of cholesterylbenzoate. We also thank the many donors for blooddonations;AleksJovanovich and Drs.P.Bannon,L.Kritharides,and W. Laufor numerous and painless bloodtaking; andDr. W.Jessup forhelpfulandstimulatingdiscus- sions.

This workwassupported in part byanOverseas Postgraduate Re- search Scholarship and Sydney University Postgraduate Research AwardtoDr.Christen,anAustralian Postgraduate Research Awardto Dr. Garner, and the National Health & Medical Research Council Grant No. 920371 toDr.Stocker.

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