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Expression of lipoprotein lipase mRNA and

secretion in macrophages isolated from human

atherosclerotic aorta.

L Mattsson, … , G Bondjers, O Wiklund

J Clin Invest.

1993;

92(4)

:1759-1765.

https://doi.org/10.1172/JCI116764

.

The expression of lipoprotein lipase (LPL) mRNA and the LPL activity were studied in

macrophages (CD14 positive) from human atherosclerotic tissue. Macrophages were

isolated after collagenase digestion by immunomagnetic isolation. About 90% of the cells

were foam cells with oil red O positive lipid droplets. To analyze the mRNA expression,

PCR with specific primers for LPL was used. Arterial macrophages were analyzed directly

after isolation and the data showed low expression of LPL mRNA when compared with

monocyte-derived macrophages. To induce the expression of LPL mRNA in macrophages,

PMA was used. When incubating arterial macrophages with PMA for 24 h we could not

detect any increase in LPL mRNA levels. Similarly, the cells secreted very small amounts of

LPL even after PMA stimulation. In conclusion, these studies show a very low expression of

LPL mRNA in the CD14-positive macrophage-derived foam cells isolated from human

atherosclerotic tissue. These data suggest that the CD14-positive cells are a subpopulation

of foam cells that express low levels of lipoprotein lipase, and the lipid content could be a

major factor for downregulation of LPL. However, the cells were isolated from advanced

atherosclerotic lesions, and these findings may not reflect the situation in early fatty streaks.

Research Article

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(2)

Expression of Lipoprotein Lipase

mRNA and

Secretion

in

Macrophages

Isolated from Human

Atherosclerotic Aorta

LillemorMattsson, Helena Johansson, Malin Ottosson, G6ran Bondjers, andOlov Wiklund

The WallenbergLaboratory for Cardiovascular Research,DepartmentofMedicineI, Universityof Gothenburg, Gothenburg, Sweden

Abstract

The expression oflipoprotein lipase (LPL) mRNA and the LPL activity were studied in macrophages (CD14 positive)

fromhumanatherosclerotic tissue. Macrophages were isolated

after collagenase digestion by immunomagnetic isolation. About90% ofthecells were foam cells with oil red 0 positive

lipid droplets. To analyze the mRNA expression, PCR with

specific primers forLPL was used.Arterial macrophages were analyzed directlyafterisolation and the data showed low ex-pression of LPL mRNA when compared with monocyte-der-ived macrophages. Toinducethe expressionof LPL mRNA in macrophages, PMAwas used. When incubating arterial macro-phageswith PMAfor24h we could notdetect any increase in LPL mRNA levels. Similarly, the cells secreted very small amountsofLPL evenafter PMA stimulation. In conclusion, thesestudiesshow a very lowexpression ofLPL mRNA in the

CD14-positive macrophage-derived foam cells isolated from human atherosclerotic tissue. These data suggest that the

CD14-positive cells are asubpopulation of foamcells that ex-press low levels of lipoprotein lipase, and the lipid content could be amajorfactor for downregulation ofLPL.However,

the cells wereisolated from advanced atherosclerotic lesions, and these findingsmay not reflect the situationinearlyfatty

streaks. (J. Clin. Invest. 1993.

92:1759-1765.) Key

words:

polymerase chain reaction*

cytokines

* foam cells * cell

isola-tion*atherosclerosis

Introduction

Lipid laden cells, foam

cells,

are ahallmarkofthe

early

athero-sclerotic lesions. Most ofthese cellsseem tobederivedfrom

macrophages (1).Macrophagesarebest known fortheir

pha-gocytotic

activity

butarealso

secreting

cells that

produce

sev-eral

biologically

active substances suchas

growth

factors,

cyto-kines, and reactive oxygen intermediates. The exact

mecha-nism for foam cell formation has not been settled.

Macrophagesdonotbecome foam cellswhenincubatedwith

nativeLDLbutincubation with

hypertriglyceridemic

VLDL or modified

(oxidized, acetylated)

LDL has been shown to result in foamcell formation in vitro(2-4).Lipoprotein lipase

Addresscorrespondenceto Dr.0.Wiklund, Wallenberg Laboratory,

Sahlgren'sHospital,S-413 45Gothenburg,Sweden.

Receivedfor publication17November1992andinrevisedform6

May 1993.

(LPL)'

is responsible for the catabolism of triglyceride-rich

lipoproteins,i.e.,chylomicronsand VLDLs. LPLenzyme activ-ity has been found in the intima oflarger arteries in experimen-tal atherosclerosis (5, 6). LPL could create remnants locally in the tissue and increase the cholesterol deposition in the arterial wall. In agreement, studies by Lindquist et al. (7) suggest that triglyceride-rich lipoproteins may contribute to foam cell

for-mationthrough the localdegradation of lipoproteins by

lipo-protein lipase,and to theuptake ofremnant particles into mac-rophages. Aviramet al. (8) demonstrated that LPL also can

modify LDL so that it is taken up more avidly by cultured

macrophages.Anothermechanismis proposed by Beisiegel et al.(9). Theyshow thatlipoprotein lipaseenhancesthe binding ofchylomicronsand,B-VLDL to cells via the LDL receptor-re-lated protein, which could be a mechanism for foam cell

for-mation. In vitrostudies show secretion of LPL in

monocyte-derivedmacrophages(10),mouseperitonealmacrophages, or

monocyte-derivedcell lines(1 1-14). In immunohistochemi-cal studies on human plaquesJonasson et al. (15) found no LPLassociated with CD 14-positivecells. On the other hand,

protein was observedin relation to smooth muscle cells.

Re-cently Yla-Herttualaetal. (16)found, by in situ hybridization andimmunocytochemistry,that asubfraction of lesion

macro-phages detected with a mAb against human macrophages

(HAM-56)and smoothmuscle cellsinhuman andrabbit

ath-erosclerotic aortaexpressedLPL mRNA. O'Brienetal. (17) demonstratedthatmacrophage-derived foam cells(HAM-56 positive)rather than smoothmusclecells are theprimary cellu-lar sourceofLPL, detectedbyinsituhybridization,in human coronary plaques. Thus, data are to some extent conflicting

andtherole of LPL inatherosclerotic tissue isnotclarified. Inthepresentstudyweanalyze the expression ofmRNA

for

lipoprotein lipase

andsecretion of theenzyme in

macro-phage (CD14-positive) -derived foamcellsfromhuman

athero-sclerotic tissueatbasal

levels,

aswellasthe

possibility

toinduce

synthesis

andsecretion.

Methods

Isolationofcellsfromtissue.Atherosclerotic aortic tissuewasobtained fromabdominalaorta(n =9)andfemoral arteries(n=2)from pa-tients (age 69-78 yr)undergoingsurgery, duetoaortic aneurysmor

intermittent claudication. Thespecimensof human atherosclerotic

tis-suedisplayed largevariation inmorphology,withsevere atherosclero-sisincludingcalcification and thrombosis. Afterexcision,thebiopsies

wereimmediatelytransferredtoa50-mltesttubecontainingHBSS. Small segments fromthetissuespecimenswerefixed in4% paraformal-dehydeandparaffin-embedded forhistologicalcharacterization. The adventitiawasremovedand intima mediapreparationswereused for cellisolation. Intimamedia,0.5-4. l-gtissuewetwt,wasminced and

1.Abbreviationsused in thispaper:FID, flame ionizationdetection; HMDM, humanmonocyte-derived macrophages; IM, immunomag-netic; LPL, lipoprotein lipase.

J.Clin.Invest.

©TheAmericanSocietyfor Clinical

Investigation,

Inc. 0021-9738/93/10/1759/07 $2.00

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digested withcollagenase solutionaspreviouslydescribed( 18). Iso-lated cellsweresuspendedin medium RPMI1640, supplementedwith 1%BSA, and the cellsuspensionwaskepton aslurryof ice andwater

after isolation.

This studywasapproved by the Ethics Committee of Research of theUniversity of Gothenburg, Sweden.

Immunomagnetic (IM) fractionation of macrophages. Macro-phageswere isolatedby using monoclonal antibodies andmagnetic

microspheresasdescribed(19). Briefly,theheterogeneouscell suspen-sion waspreincubated with uncoated dynabeads (Dynal AS, Oslo,

Norway) for 10 mintoget rid ofcollagen and elastin fibers. The cell

suspension wasthenincubated with amAb, anti-Leu-M3

(Becton-Dickinson andCo.,Sunnyvale, CA)thatrecognizestheCD 14antigen

presentonhumanmacrophages.Anti-HLA-DR andanti-Leukocyte

CD45 antibodies were also from (Becton-Dickinson andCo.). The antibodiesweredialyzed extensivelybeforeuse.Therosetting proce-durewascarriedoutbymixing pretreatedcellswithmagneticbeads coated withsheepanti-mouseIgG(Dynal AS).The target cellswere

thenisolatedbyapplyingamagnet. Rosetted cellswerewashed several times withPBS,andinspectedfor the presence of nonrosetted cellsby lightmicroscopy.The rosetted isolated cellswerequantitatedby

count-ing ina hemocytometer and the cell viability wasjudged byadye

exclusiontestwithTrypan-blue.Atotalof 0.4X 105-1.2 x 106 CD

14-positivecellswererecovered per gramtissue,withacellviabilityof 74±15%(mean±SD).

Humanmonocyte-derivedmacrophages(HMDM)wereobtained byculturingmononuclearcells isolated frombuffycoatsbythe Ficoll-Hypaqueprocedure(20).Mononuclear cellswereplatedat adensityof

107cells/dish.After removalof nonadherent cells the adhered

mono-cyteswerecultured in RPMI1640supplementedwith 100U/ml peni-cillin, 100Ag/ml streptomycin, 10% FCS, and 10% human serum.

After 6 days the cellswerecultured in RPMI 1640 medium with 10% FCS. TheexperimentswereperformedwithHMDM 7dafterplating.

The cellswerescraped from thepetridish,and the cell number and cell viabilitywerequantitated. When indicated anti-Leu-M3 (CD 14)

-posi-tive cells,CD45-positivecells, andHLA-DRpositivecellswereisolated from HMDM by thesameprocedure thatwasusedfordigestedcells from tissue. Endotoxin levelswereanalyzed in the reagents and cell culturemediausingCoatestEndotoxin(Coatech Lab.,Ljungby,

Swe-den)andwerefoundtobe<0.08ng/ml.

Preparation andanalysis ofRNA.Total cellularRNA wasisolated from the cells byamicromodification ofguanidinium-hydrochloride/ isothiocyanateextraction and CsCl gradientultracentrifugation(21 ).

Briefly,cellswerelysed with 1 ml of 6Mguanidinium-hydrochloride/ isothiocyanatesolution. The cell homogenatewaslayeredontop of 1.2

ml5.7MCsCl beforecentrifugationfor 21 hat35,000 rpm. The

follow-ing oligonucleotideprimers forLPL weresynthesizedon aDNA synthe-sizer(Applied Biosystems, Inc., FosterCity,CA) 5'-GAGATTTCT-CTGTATGGCACC-3' and 5'-CTGCAAATGAGACACTTTCTC-3'. The 5'-primer spanned thejunction of the first two exons and the 3'-primerspanned thejunction ofthenext twoexonsfor LPL (22). For

comparison wealso amplified mRNA for

IL-1j3,

using the primers describedbyWangetal.(23). The5'-primerwaslabeled in the 5'-end with32P-gamma-ATPusing polynucleotide kinase. RNA was reversed, transcribed together withaninternal standard (AW 108) into cDNA, and the cDNA wasthen amplified by PCR. AW108 is a synthetic

cRNA thatcontains5'primers of12target mRNAs followed by the

complementarysequences ofthe3'primers. Quantitative analysis was

performed usingthe PCR asdescribed by Wang et al. (23) using a Gene Amp RNA PCR Kit (Perkin-Elmer Cetus Corp. Norwalk, CT).

Briefly,the reverse transcription mixture containing 1 ng total cellular

RNAfrom HMDM up to 100ng total RNA from tissue macrophages

correspondingto5X 102-5X I04cells, 1X 104-1 X I05molecules of AW108 cRNA, and random hexamer primer were incubated at room temperature for 10min,42°C for 15min, heated to 99°C for 5

min,

andthen in5°Cfor5min.After reverse transcription, serial 1:2 or 1:3 dilutions of thecDNAmixture were amplified by using the 3'-primer with the

32P-labeled

5'-primer.

The PCR

amplification protocol

in-volved denaturationat950C for 2min in cycle 1, followed by 950C for 1min,annealing at 60C for 1min and extension at 60C for 7minin 35 cycles for theanalysis of mRNA for LPL. All incubationsweredone in a Perkin-Elmer Cetus DNA ThermalCycler. The PCR products were thenseparatedon a4% Nusieve GTG agarose gel (FMC

Bioprod-uctsCorp., Rockland, ME). 10 Ml of each PCR reaction mixturewas

electrophoresed with 10 Ml from aPCR reaction mixture without a radioactive primer. Theelectrophoresiswas runformorethan5 h and visualized with ethidium bromide (E. Merck AG Darmstadt, Ger-many). Appropriate bandswere cutout from thegel and melted, and the radioactivity was determined by liquid scintillation counting. The

amountsofradioactivity recovered from the excised gel bands were plottedagainst the template concentrations.

Induction of macrophagedifferentiation with PMA. The IM iso-lated cells were resuspended in medium RPMI 1640 containing 10% FCS and 10nM PMA(Sigma Chemical Company, St.Louis, MO)and seeded at adensity of 105 cells/dish. The control incubations were

performed inmedium containing0.1% ethanol, the solvent usedto

dissolve PMA. The HMDM werewashed 3 times with PBS before adding the RPMI mediumcontaining PMAtothe cells. Heparin 10 U/ml

(LMwens,

Malmo,Sweden)wasaddedtothe cells 30 min before

collectingthemedium. To studyLPLactivity,themediumwas imme-diately frozen and storedat-700C until the day of assay. No sample wasstored longer than 14dbefore assay.

Determination ofLPL activity. Heparin-releasable LPL activity wasdetermined accordingtoNilsson-Ehleetal.(24).Inbrief, 100 Ml medium was incubated with 100Mlfatty acid-labeled triacylglycerol

emulsion,as asubstrate,at370C. The substrate with[3H]triolein (De-partment of Medical andPhysiological Chemistry, Universityof Lund, Sweden) and unlabeled trioleinwasemulsified with lecithin and

con-tained humanfastingserum as a sourceof apo CII and albuminas a fattyacid acceptor, pH 8.0. After 30min,released [3H]oleic acidwas

isolated from the acylglycerols byaliquid-liquid partition procedure. Liberated [3H ] oleic acid in the upper phase was quantified with liquid scintillation.Allsampleswereassayed induplicate(coefficient ofvaria-tion 2.2%).Lipolyticactivitywasexpressed as milliunits (mU) per 106 cells. One mUof enzymeactivity representsthe releaseof 1 nmol of fatty acid/min.

Lipid analyses. Lipids were extracted from the arterial macro-phageswith hexane/isopropanol (3:2, vol/vol). The sample was ap-pliedon aquartz rod coatedwith silica gel, ChromarodS111 (Newman-Howells,Winchester, Hampshire, UK). Separation of lipids was per-formed with the use oftwo sequential solvents: hexane/ethyl ether (9:1 vol/vol), hexane/ethylether/acetic acid (72:18:1). After each sol-vent,FIDwas used toseparate and quantify the lipids. The measure-ments wereperformed on latroscan TH- 10 MK2 (Newman-Howells),

andsignals were integrated on a Hewlett-Packard computer. Quantifi-cation of cholesterol ester, triglycerides, and free cholesterol was made by standard comparison.

Results

Characterization oftissue macrophages.Aphotomicrograph of

ahematoxylin-eosin stained section from one ofthe

atheroscle-rotic lesions includedin the study is shown in Fig. 1. The aortic tissue showintimal thickening with numerous cells and a cen-trallipidcoreregion. 5to34% of all cells digested from tissue

withcollagenase were CD 14-positive. In two experiments an

estimationof 40%oflesion mononuclear cells were recovered

asCD14-positivecells. Mostofthe CD14-positive cells isolated from atherosclerotic lesion were foam cells, as characterized by their appearance as big round cells with foamy cytoplasm,

whichstained positively with oil red0 ( 19). The frequency of oil red 0-stained CD 14-positive cells was 88±-11%

(mean+SD,

n = 8). In separate experiments CD 14-positive cellswere isolatedfrom tissuessimilar to those used for LPL measurements, andwere

analyzed

forthe

lipid

contentwitha

(4)

Figure 1. Intima media from one of the analyzed lesions. Tissues were fixed in formaldehyde solution and paraffin-embedded. Hematoxylin/

eosin staining. x50.

combination ofTLC and FID (25). In Table I the

concentra-tion of lipids isshownforCD

14-positive

cellsisolated from 11

specimens of human atherosclerotic aorta. The results show

accumulation ofcholesterol ester,freecholesterol, and

triglyc-erides in these cells.

Preparationandanalysis ofRNA. To show the expression

ofmRNAinhuman cells, RNA wasisolatedfrom

monocyte-derived macrophages and from macrophage-monocyte-derived foamcells

(CD14-positive) fromhumanatherosclerotic tissue. After re-verse transcription, cDNA wasamplified by PCR. We have

used theAW108 internal standardtodeterminetheamountof

LPLmRNAinthese cells. Forquantification ofmRNAit is of

importance that theamplification curves are parallel forthe

standardand the target mRNA. Theefficiency of the

amplifi-TableI.Lipid DistributioninIsolated Cells fromHumanAtheroscleroticIntimaMedia

Lipid class CD14-positivecells

pg/mgcell protein

Cholesterolester 408±349 Triglycerides 133±103 Freecholesterol 333±200

Values aregivenasmean±SD (n= 1 1). Lipidswereextracted from

CD14-positivecellsisolated from atherosclerotic tissue andanalyzed

byacombination of TLC and FID.

cationwasfoundto be the sameforLPL mRNAandAWl08

cRNA. Whenaliquotes ofthe cDNA mixture were amplified with increasing number of cycles, the exponential phase of

am-plificationwasfound tobebetween 24 and 40 cycles. In the present experimentsall samples were amplified for 35 cycles.

An example ofPCR products of LPL mRNA from

macro-phagesandtheinternal standardinserial 1:3 dilutions of the cDNAmixtureisshowninFig. 2. Reactionproducts were

re-solvedbygel electrophoresisandvisualizedbyethidium

bro-mide staining. Thesize ofthe LPL PCR productfrom AW108

cRNAis 300 bp and can beeasilyseparated from thetarget mRNAPCRproduct,whichhas asize of277 bp,forLPL.The

amountsofradioactivity recovered from the excised gel bands

were plottedagainst the template concentrations. As shown in

Fig.3, RNAfrom 2,000 arterialmacrophages and 1,000

mole-culesof AWl08 cRNA gave the sameamountsofLPLPCR

product, which gives 0.5 molecules ofLPL mRNA/cell. The

cell numberisbasedon viablecellsasjudged bytrypan blue

staining. To study the reproducibility ofthe

quantitation

of

LPL mRNA, two RNApreparations wereanalyzed in

tripli-cate, and thecoefficient of variationwas7.0%.Separatecontrol

experiment inwhichreversetranscriptasewasexcludedorin which mRNA was excluded showed no detectable signal.

These results show that quantification ofLPL mRNA with PCRin isolatedcellsisspecificand

reproducible

(Fig. 3).

Expression

of

mRNA

for

LPL inisolated cells. We have

analyzed arterial CDl4

positive

macrophages

directly

afterIM

(5)

1

2

3

4

5

6

7

Figureanalvsis2.of LPL mRNAQuantitative

inmacrophages.

Ethi-dium bromide staining of PCR products

sepa-rated in4% agarosegel. Lanes 1-5indicate LPL mRNAPCR products from 1:3 dilutions ofa

sample containing I ng total RNA from

macro-300 phages plus I X IO'

277 molecules of AW108

cRNAafter amplifica-tionfor 35cycles. Lane 6shows thecontrol re-action withouttemplate amplified for35 cycles. Lane 7 is the molecular

weightmarker.

II). When blood monocytesattach topetridishes and

differen-tiate intomacrophages, theyshow atranscriptional activation of mRNA for LPL. Theexpressionof LPL mRNA in the total

HMDMpopulation culturedfor7d was - 1,600molecules/

cell. To study the CD14-positive subpopulation of HMDM,

RNA from Leu-M3-positive cellswasisolated fromHMDM culturedfor7d. Lowlevels ofLPL mRNA weredetected in the Leu-M3-positive macrophages (Table II). It is unlikelythat

thedifference is duetoRNAdegradation, since the cellswere treated in paralleland storedonice forthesametime. When totalRNAwas runonagarosegel,thepreparationsshowed the

samepatterns. Adifference in viabilitybetween total HMDM and CD14-positive cells may exaggeratethedifference, since only 70% of totalHMDM were viable while - 90%ofCD14

positive cells wereviable. In order to exclude the possibility

thatcollagenase treatment ofthe cells decreases the levels of LPL mRNA, control experiments wereperformed in which

20o00

Iractges

* 10000 AW108rIce

0

E

co 5000

2000-E

z 1000

500

500 1000 2000 3000 5000

CPM

Figure 3. Quantitative analysis of LPL mRNA levels in macrophages isolated from human atherosclerotic aorta. After separation of the PCR products with agarose gel electrophoresis the bands were cut out

andradioactivity was determined by liquid scintillation counting.

Thevariable template concentrations of the internal standard AW 108

cRNA(A) and the number of macrophages (o) were plotted against theradioactivity of their PCR products.

TableII. LPL mRNALevels in ArterialMacrophages

and Human Monocyte-derived Macrophages

Cells LPL mRNA

molecules/cell

ArterialM.CD 14 3.4±2.6

HMDM 1,578+695

HMDMCD14 33±44

Total RNAwasisolated frommacrophage-derived foam cells isolated

fromatherosclerotictissue(arterialM.CD14), HMDM cultured for

7d, and Leu-M3positive cells isolated from HMDM (HMDM

CD14). LPL mRNAmolecules/cell is basedonviable cellsasjudged

by trypan bluestaining.Data representmeanvalue±SDof four experiments.

HMDMweretreated withcollagenasesolution. After

incuba-tion with collagenase, the

expression

ofLPL mRNAwas554 LPL mRNAmolecules/cell, comparedto496 molecules/cell inthe nontreatedcells. Anotherexperimentwasperformedto

determineifbinding of anti-Leu-M3 antibodiestothe CD14

antigen decreasesthe LPL mRNAexpressionin HMDM. No

differencewasfound in LPL mRNA levels between cells

incu-bated with anti-Leu-M3 and the control cells. Thus,the cell

isolation procedure didnotseem todecrease the

expression

of LPLmRNA.

Induction ofLPL mRNA andLPL activitybyPMA. To

examineifthedownregulated expressionof LPL mRNA in the

arterialmacrophageswasreversible,the induction of LPL

pro-ductionwith PMAwasstudied. PMA have been shownto mod-ulatedifferentiation ofmacrophagesandincrease LPL

secre-tion ( 14). LPL mRNAlevelswerequantitated after culturing ofthe IMisolated cells withPMA. Inoneexperiment, arterial macrophageswereincubatedfor 6hwithPMA. No change was observedinmRNAlevels, 0.4LPL mRNAmolecules/cell vs.

0.2LPL mRNAmolecules/cellin thecontrols, withoutPMA.

After 12 hofincubation withPMA,themRNAlevelswere0.3 LPL mRNAmolecules/cell, compared to 0.1 molecules/cell

withoutPMA(n=2). Whenincubatingarterial macrophages

withPMAfor24 h, wecouldnotdetect anyincrease in LPL mRNA levelsascomparedtocontrol cultures, forwhich 1.0 and 1.6 LPL mRNA molecules/cell were obtained,

respec-tively

(Table III).To evaluatetheeffect ofPMA incultured

monocyte-derived macrophages, RNA was isolated after

in-ductionof cells withPMA.InductionofLPL mRNA

expres-sion appeared after6 hincubationofHMDMwith PMA. After 12 h ofincubation with PMAthe LPL expression reached a

maximum, 10,937 LPL mRNA molecules/cell as compared to 675 molecules/cellwithoutPMA(mean value, n=2). Infive

experiments a 24-htreatment ofthe HMDM with PMA in-creased thelevelsofLPL mRNAalmosttwofold(Table III). In

oneexperiment,weisolatedCD 14, CD45 and HLA-DR

posi-tivecellsfromthesameHMDM. LPL mRNA levels were nine

timeshigher in CD45-positive cells and 6.5 times higher in HLA-DR positive cells compared with CD 14-positive cells (Table IV). Thissuggeststhat macrophages that express the CD 14antigenare asubpopulation of cells that express lower levels ofLPL mRNA. To evaluate if culturing of cells with beadschanged LPLexpression,the CD 14, CD45, and HLA-DRpositive cellswereculturedwithorwithout PMA for 24h,

after whichLPL mRNAexpressionwas measured. The beads

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TableIII. LPL mRNA Levels (Molecules/Cell) in PMA-induced

and Noninduced Arterial Macrophages andinMonocyte-derived

Macrophages

Cells -PMA +PMA

Arterial M.CD14 1.6 1

HMDM 1,093±623 1,940±671

HMDMCD14 18 192

Thearterial macrophages (Arterial M), HMDM, and leu-M3 positive cellsisolated from HMDM (HMDM CD 14) were grown in either the presence or absence of 10 nM PMA for 24hbefore RNA extraction andquantification with PCR. LPL mRNA molecules/cell is based on viable cells as judged by trypan blue staining. Data represent mean value of two experiments from arterial macrophages, and of five ex-periments from HMDM (mean±SD), and mean value of two experi-mentsfrom HMDM CD 14.

were stillpresent and attached to the cells during the culture

experiments. Culturing withbeadsincreasedthe LPL mRNA.

TheLPLmRNAexpression in CD14-positive cells increased from 31 to 328 LPL mRNA moleculespercellafter incubation

withPMA (Table IV). These results strongly suggest that the mRNAforLPLinmacrophagesisolated fromhuman athero-sclerotic aorta could not be induced with PMA, while a10-fold increase inmRNAwas seen in CD14-positiveHMDM. Torule outthatthefailureof tissue macrophagestorespond to PMA was due to collagenase treatment, control experimentswere

performedinwhichHMDM weretreatedwithcollagenase

be-foreimmunomagneticisolation and induction with PMA. A

24-h treatment with PMAshowed aninefold increase of the LPL mRNAexpression in the CD 14-positive cells, compared with collagenase-treated cellsnotexposedtoPMA. Thus, the abilityof CD14-positivecellsto respondto PMA was not

af-fectedby collagenasetreatment.

Wealso exploredthesecretion ofLPLfrom CD 14-positive foam cellsand HMDM. For that purpose, cellswerecultured6, 12,and 24 hwithorwithout PMA,

heparin

wasadded,and the

medium

wascollected and assayed forthe

heparin-releasable

LPLactivity.Asshownin Table V,nodetectable induction of

LPLactivity was seen in arterial macrophages. Low

activity

wasseen inarterial macrophages after 6-handafter12-h

stimu-TableIV. LPL mRNALevels

(Molecules/Cell)

inPMA-induced

andNoninduced

Subpopulations of Monocyte-derived

Macrophages

Oh 24h 24h

Cells -PMA +PMA

CD14 13 31 328

CD45 107 197 236

HLA-DR 88 151 244

TotalRNA wasanalyzedinCD14,CD45,andHLA-DRpositivecells after isolation from HMDM cultured for7d.TheCD14, CD45,and

HLA-DRpositivecells isolated from HMDMweregrown in either presenceorabsence of 10nMPMA for 24 h beforeRNAextraction

and quantification with PCR. LPL mRNA molecules/cell is based on viable cellsasjudged by trypan bluestaining.

TableV LPL Activity inPMA-induced and Noninduced Macrophages

Arterial CD14 HMDM Incubation

time -PMA +PMA -PMA +PMA

6 h 0.05 0.02 0.86 0.66

12 h 0.02 0.04 0.72 2.68

24 h 0.06±0.06 0.05±0.03 1.14±0.23 2.94±0.13

The effect of PMA on the LPL activity in the culture media of

mac-rophage-derivedfoam cells isolated from atherosclerotic tissue (arte-rial CD14) and 7-d-old HMDM. Cells were grown for 6, 12, or 24 h in the presence or absence of 10 nM PMA. Heparin 10 U/ml was added30 minbefore collecting the media. LPL activity was measured in the medium and is expressed as mU/106 cells. The data from 24-h incubation represent the mean value of sixexperimentsfrom tissue macrophages and of three experiments from HMDM. The other timepointsaremeanvalues of twoexperiments.

lation with 10nMofPMA. Wefoundthat the LPL activity was

0.05

mU/

106 cells for foam cells after24 hof incubation inthe presenceofPMA, and the controlswithoutPMA showed 0.06

mU/

106.

Forcomparison, in HMDM no difference in LPL activitywasseenafter 6-h incubation withPMA; however, the LPLactivitywas40-fold higherthan in arterial macrophages. A 3.5-fold increasewasdetectedafter12 hof incubation with

PMA. InHMDM, the LPLactivity increased from1.14 to2.94 mU/

106

cellsaftera24-htreatmentwith 10 nM PMA.

Inconclusion, thedata areconsistent withthe observed low

levels

of

LPL mRNA in CD14-positive arterialmacrophages. LPLactivitywas lowinnoninducedas well asininduced cells.

InHMDM,treatment withPMAfor 24h resulted in -

2.5-fold increase inLPLactivity.

Discussion

Inthisstudyweshow that CD

14-positive

macrophage-derived

foam cells isolated from human atheroscleroticaortaexpress very lowlevelsofmRNAfor LPL, and the

lipolytic activity

of

secretedLPLis

extremely

low. Theobservationson

tissue-de-rived cellswerecompared with studieson HMDM. These data suggest thatwithin the bloodmonocyte

population

CD

14-posi-tive cellsmay representa

subpopulation

with lower

expression

ofLPL. However, even inthese cells the

expression

of LPL mRNA wasabout tenfold higherthan that

for

tissue-derived cells.

Furthermore,

theCD

14-positive

HMDM could be in-ducedto about a

10-fold

increase inLPL mRNAafter incuba-tion withPMA.Theseobservationssuggestthat thelow levelof

LPL mRNA and the lack ofresponseto PMAisnot dueto

downregulation

or

degradation

ofmRNA

during

theisolation

procedure orduringco-culture withmagneticbeads(butdoes not ruleit out). Strayetal.

(26)

have shown that treatment

with

actinomycin

Dfor2 hdidnot

change

LPL

synthesis,

and

after 24 h

only

a

partial

inhibition was

observed,

suggesting

that the mRNAforLPL hadarather

long

half-life. This sug-gests that the low levelofmRNAand the low

synthesis

found

inmacrophages isolated from atherosclerotic

tissue,

isin agree-mentwithotherstudies in whichLPLcouldnotbe

detected,

or

wasdetectedin lowamounts, inCD14-positive

macrophages

(7)

is basedonthe expression of CD 14. The expression of CD14 antigen is restricted to cellscapableofphagocytosis,whereas

CD14-negativecells have the ability to mediate extracellular cytolysis withoutexhibiting anyphagocytosing activity (27).

Thisis a subpopulation of themacrophagespresent in athero-sclerotic tissue and it is possible that othersubgroupsexpress LPL athigher levels,asshown for

CD45-positive

orHLA-DR positive cells. The data from in situ hybridizations done by

Yla-Herttuala ( 16), showing that 10-20%of

macrophages

in

humanlesionsexpressLPL, and datafromO'Brienetal. ( 17), whofound thatLPL mRNAisassociatedwiththemost

lipid-laden cells of human coronary plaques, suggestthat LPL is expressed by a subpopulation of cells. The discrepancy be-tween ourresults and those ofYla-Herttuala et al.

(16)

and

O'Brienetal.(17)could beexplained bydifferences involved

innotusing thesamemacrophage

antibody

fordetection. Several factorsmayberesponsiblefora

downregulation

of LPL expression in

macrophages,

for

example

bacterial LPS and cytokines such asTNF,

IFN-,y,

and IL- 1

(28-30).

The

regulationof LPLbycytokinesis

complex, depending

on the cell system that is used. Treatment with LPS lowered both LPL

activity and LPL mRNA in

HMDM,

but TNF hadnoeffect

(28). However,mouseperitoneal macrophageswereshown to

respondto TNFwithareductionin LPL

activity

(29).In heart cell culturesthesuppressionof LPL

activity by

LPSwas me-diatedbyTNF(30). Querfeldetal.(31)have shown that

IFN-,y and IL-l inhibitproduction of LPLbyHMDM. IL-l

sup-pressesLPLactivity, butnotexpression ofLPL mRNAinrat

heart cells(32).Wehavenotfoundanystudies inthe literature

about the effect of IL-l on the

expression

of LPL mRNA in

macrophages. Activated

macrophages

produceTNFandIL-1,

but only IL-l has been shown to reduce LPL

activity

in HMDM. Wehave detected mRNA forIL-I in macrophages isolated from human lesions (not shown).IFNisanactivator of macrophages.Jonassonetal.

(33)

havedemonstratedthat

IFN-y suppressed synthesisand secretion ofLPLby

macro-phagesat IFNdoses that upregulated expression of the activa-tion marker HLA-DR.Wehave inearlier studies showedthat most of the isolated macrophage-derived foam cells also ex-pressed HLA-DR(19).ActivatedTlymphocytesare presentin theatherosclerotic plaque, and

activated

Tcells produce

IFN-y

(for

reviewseereference

34).

Inhuman macrophages, treat-mentwithIFNreducedLPL mRNAalongwith mediumLPL

activity, suggesting

that the

inhibitory effects

occur atthelevel of

transcription

or mRNA stabilization (30). Therefore, the

downregulation

ofLPL in arterial macrophages maybe

ex-plained byalocal production ofIFN and IL- 1 inthe lesion.

Addition ofPMA to cellshadanaugmenting effecton LPL

secretion by macrophages in vitro ( 14, 34). This is in

agree-ment with ourresults. Whenmonocyte-derived macrophages

aswell as CD14-positiveHMDM were incubatedwith PMA, the LPL mRNAexpressionwasincreased. In contrast, PMA had noeffectonCD14-positive macrophages isolatedfrom

ath-eroscleroticaorta; nochangewasobservedinLPLmRNA lev-els or in LPLactivity.It has been observed that the LPL

expres-sion

increased

when monocytes differentiate into

macro-phages,

and when promonocyticcelllines, such as THP- 1 cells, arestimulatedby phorbol esters (35),suggestingthat LPL

ex-pression is linkedtothedifferentiation, rather than to

activa-tion ofthecells. Because differentiatedcells express high LPL

activity

in vitro, and mature and quiescent cells show

sup-pressed

activity

(13, 14),

it

might

be

speculated

that

newly

recruited monocytes ingrowing plaquesexpresshighlevels of

LPL,

and that the

activity

islater

suppressed

asthe cells

be-comefoam cells. It hasrecentlybeen shownbySofer et al.

(36)

that accumulation of

triglycerides

decreasedthe LPL secretion ina

dose-dependent

mannerin

macrophages.

It is

possible

that the

suppressed

LPLexpression found in thisstudy isdue to

triglyceride accumulation, since CD14-positivecells from

hu-manaortacontainedhighlevels oftriglyceridesaswellasfree

cholesterolandcholesterolester.Further investigationwillbe

requiredto assessthe role of LPLexpression inlipid loaded

macrophages,

andin

macrophages

indifferentstatesof activa-tionin humanatherosclerotictissue.

In

conclusion,

these studies showavery lowexpressionof LPL mRNA in the CD14-positive macrophage-derivedfoam cellsisolatedfromhuman atherosclerotictissue. The low LPL

activity

ortheexpressionofLPL mRNA could not beinduced

with PMAtothe levels found in HMDM. These datasuggest

that the CD 1

4-positive

cellsare asubpopulationoffoam cells that express low levels oflipoprotein lipase, andthe cell

lipid

contentcouldbeamajorfactor fordownregulationofLPL.

Acknowledgments

Wethankthe Drs.Kent Lundholmand Folke Nilssonatthe

Depart-mentsofSurgeryandThoracic Surgery and Dr. Bo Risberg andPeter Falk atOstraHospitalforprovidinguswithhuman arterialtissue.

Thisstudywassupported bygrantsfrom theSwedish Heart-Lung Foundation,theMedical Research Council, and the Medical Facultyof

Gothenburg.

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