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
Find the latest version:
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 * cellisola-tion*atherosclerosis
Introduction
Lipid laden cells, foam
cells,
are ahallmarkoftheearly
athero-sclerotic lesions. Most ofthese cellsseem tobederivedfrommacrophages (1).Macrophagesarebest known fortheir
pha-gocytotic
activity
butarealsosecreting
cells thatproduce
sev-eral
biologically
active substances suchasgrowth
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 lipaseAddresscorrespondenceto Dr.0.Wiklund, Wallenberg Laboratory,
Sahlgren'sHospital,S-413 45Gothenburg,Sweden.
Receivedfor publication17November1992andinrevisedform6
May 1993.
(LPL)'
is responsible for the catabolism of triglyceride-richlipoproteins,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 inmacro-phage (CD14-positive) -derived foamcellsfromhuman
athero-sclerotic tissueatbasal
levels,
aswellasthepossibility
toinducesynthesis
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 $2.00digested 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 syntheticcRNA 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 the32P-labeled
5'-primer.
The PCRamplification 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 beforecollectingthemedium. 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, andwereanalyzed
forthelipid
contentwithaFigure 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 11specimens 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
ofLPL 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
mRNAfor
LPL inisolated cells. We haveanalyzed arterial CDl4
positive
macrophagesdirectly
afterIM1
2
3
4
56
7
Figureanalvsis2.of LPL mRNAQuantitativeinmacrophages.
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. Anotherexperimentwasperformedtodetermineifbinding 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 inculturedmonocyte-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
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 themedium
wascollected and assayed fortheheparin-releasable
LPLactivity.Asshownin Table V,nodetectable induction ofLPLactivity was seen in arterial macrophages. Low
activity
wasseen inarterial macrophages after 6-handafter12-hstimu-TableIV. LPL mRNALevels
(Molecules/Cell)
inPMA-inducedandNoninduced
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 0.06mU/
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 withPMA. 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
ofsecretedLPLis
extremely
low. Theobservationsontissue-de-rived cellswerecompared with studieson HMDM. These data suggest thatwithin the bloodmonocyte
population
CD14-posi-tive cellsmay representa
subpopulation
with lowerexpression
ofLPL. However, even inthese cells the
expression
of LPL mRNA wasabout tenfold higherthan thatfor
tissue-derived cells.Furthermore,
theCD14-positive
HMDM could be in-ducedto about a10-fold
increase inLPL mRNAafter incuba-tion withPMA.Theseobservationssuggestthat thelow levelofLPL mRNA and the lack ofresponseto PMAisnot dueto
downregulation
ordegradation
ofmRNAduring
theisolationprocedure orduringco-culture withmagneticbeads(butdoes not ruleit out). Strayetal.
(26)
have shown that treatmentwith
actinomycin
Dfor2 hdidnotchange
LPLsynthesis,
andafter 24 h
only
apartial
inhibition wasobserved,
suggesting
that the mRNAforLPL hadarather
long
half-life. This sug-gests that the low levelofmRNAand the lowsynthesis
foundinmacrophages isolated from atherosclerotic
tissue,
isin agree-mentwithotherstudies in whichLPLcouldnotbedetected,
orwasdetectedin lowamounts, inCD14-positive
macrophages
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 byYla-Herttuala ( 16), showing that 10-20%of
macrophages
inhumanlesionsexpressLPL, 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)
andO'Brienetal.(17)could beexplained bydifferences involved
innotusing thesamemacrophage
antibody
fordetection. Several factorsmayberesponsibleforadownregulation
of LPL expression inmacrophages,
forexample
bacterial LPS and cytokines such asTNF,IFN-,y,
and IL- 1(28-30).
Theregulationof LPLbycytokinesis
complex, depending
on the cell system that is used. Treatment with LPS lowered both LPLactivity and LPL mRNA in
HMDM,
but TNF hadnoeffect(28). However,mouseperitoneal macrophageswereshown to
respondto TNFwithareductionin LPL
activity
(29).In heart cell culturesthesuppressionof LPLactivity by
LPSwas me-diatedbyTNF(30). Querfeldetal.(31)have shown thatIFN-,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 inmacrophages. 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)
havedemonstratedthatIFN-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 produceIFN-y
(for
reviewseereference34).
Inhuman macrophages, treat-mentwithIFNreducedLPL mRNAalongwith mediumLPLactivity, suggesting
that theinhibitory effects
occur atthelevel oftranscription
or mRNA stabilization (30). Therefore, thedownregulation
ofLPL in arterial macrophages maybeex-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 intomacro-phages,
and when promonocyticcelllines, such as THP- 1 cells, arestimulatedby phorbol esters (35),suggestingthat LPLex-pression is linkedtothedifferentiation, rather than to
activa-tion ofthecells. Because differentiatedcells express high LPL
activity
in vitro, and mature and quiescent cells showsup-pressed
activity
(13, 14),
itmight
bespeculated
thatnewly
recruited monocytes ingrowing plaquesexpresshighlevels of
LPL,
and that theactivity
islatersuppressed
asthe cellsbe-comefoam cells. It hasrecentlybeen shownbySofer et al.
(36)
that accumulation oftriglycerides
decreasedthe LPL secretion inadose-dependent
mannerinmacrophages.
It ispossible
that thesuppressed
LPLexpression found in thisstudy isdue totriglyceride accumulation, since CD14-positivecells from
hu-manaortacontainedhighlevels oftriglyceridesaswellasfree
cholesterolandcholesterolester.Further investigationwillbe
requiredto assessthe role of LPLexpression inlipid loaded
macrophages,
andinmacrophages
indifferentstatesof activa-tionin humanatherosclerotictissue.In
conclusion,
these studies showavery lowexpressionof LPL mRNA in the CD14-positive macrophage-derivedfoam cellsisolatedfromhuman atherosclerotictissue. The low LPLactivity
ortheexpressionofLPL mRNA could not beinducedwith PMAtothe levels found in HMDM. These datasuggest
that the CD 1
4-positive
cellsare asubpopulationoffoam cells that express low levels oflipoprotein lipase, andthe celllipid
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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