• No results found

Monocyte chemoattractant protein 1 in human atheromatous plaques

N/A
N/A
Protected

Academic year: 2020

Share "Monocyte chemoattractant protein 1 in human atheromatous plaques"

Copied!
8
0
0

Loading.... (view fulltext now)

Full text

(1)

Monocyte chemoattractant protein-1 in human

atheromatous plaques.

N A Nelken, … , D Gordon, J N Wilcox

J Clin Invest.

1991;

88(4)

:1121-1127.

https://doi.org/10.1172/JCI115411

.

Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and

as a potential source of growth factors mediating intimal hyperplasia, but the chemical

messages which stimulate the influx of monocytes into human atheroma remain unknown.

Monocyte chemoattractant protein-1 (MCP-1) is a recently described molecule with powerful

monocyte chemotactic activity expressed by monocytes, vascular endothelial cells, and

smooth muscle cells in culture. To begin to address the role of MCP-1 in vivo, we examined

10 normal arteries and 14 diseased human arteries for MCP-1 expression by in situ

hybridization. MCP-1 mRNA was detected in 16% of 10,768 cells counted in human carotid

endarterectomy specimens with highest expression seen in organizing thrombi (33%) and in

macrophage rich areas bordering the necrotic lipid core (24%) as compared to the fibrous

cap (8%) and the necrotic lipid core itself (5%). Based on immunohistochemical staining of

serial sections and on cell morphology, MCP-1 mRNA appeared to be expressed by

vascular smooth muscle cells (VSMC), mesenchymal appearing intimal cells (MICs), and

macrophages. By contrast, few cells expressing MCP-1 mRNA were found in normal

arteries (less than 0.1%). These data suggest a potential role for MCP-1 in mediating

monocytic infiltration of the artery wall.

Research Article

(2)

Monocyte

Chemoattractant Protein-1

in

Human Atheromatous Plaques

Nicolas A. Nelken,*Shaun R.Coughlin,*DavidGordon,9andJosiah N.Wilcoxil

Cardiovascular Research Institute and *Division of Vascular Surgery and

*Department of

Medicine, University

of

California, San Francisco,SanFrancisco, California 94143; University of Washington, Department

of

Pathology, Seattle,

Washington 98195;andI1GenentechCorporation, South San Francisco, California 94080

Abstract

Monocytes appear tobe central toatherogenesis bothasthe

progenitors of foam cells and as a potential source of growth factors mediating intimalhyperplasia, but thechemical mes-sages whichstimulatetheinflux ofmonocytesintohuman ath-eromaremainunknown. Monocyte chemoattractantprotein-i

(MCP-1)is arecently described moleculewithpowerful

mono-cytechemotactic activity expressed bymonocytes,vascular

en-dothelialcells,and smoothmuscle cells inculture.To

begin

to

address the role of MCP-1 invivo, we examined 10 normal

arteriesand 14diseased human arteriesfor MCP-1 expression byinsituhybridization.MCP-1 mRNA wasdetectedin16%of

10,768cells counted inhumancarotidendarterectomy

speci-menswithhighest expressionseeninorganizing thrombi (33%)

and inmacrophage richareasborderingthenecroticlipidcore

(24%)ascompared to thefibrouscap(8%) andthenecroticlipid

coreitself (5%).Based onimmunohistochemical staining of

se-rialsectionsand oncell morphology, MCP-1mRNAappeared

tobeexpressedbyvascular smoothmuscle cells

(VSMC),

mes-enchymal appearing intimalcells (MICs),andmacrophages.

Bycontrast,fewcellsexpressing MCP-1mRNA werefoundin normalarteries(< 0.1%). Thesedata suggest apotential role forMCP-1 in mediating monocytic infiltration of the artery wall.(J. Clin. Invest. 1991.

88:1121-1127.)

Keywords: Insitu hybridization-vascularsmoothmuscle cells *vascular

disease-macrophages*cytokines

Introduction

Monocyte-derived

foam cellsare a

major

component of

ather-oma,comprisingasmuch as 60% of cellsfoundinthenecrotic

lipidcore,and 10-20%ofcells in thefibrouscap(1). Thesecells havebeendemonstratednotonlyin matureplaques but also in early

fatty

streaks in animal models of

atherogenesis

and in human

disease

(2). Ithas been suggested that growth factors produced by plaque macrophages may be

important

interms ofplaque progression (3).Thesignalswhichattractmonocytes

Reprint requests should be addressedtoJosiah N.Wilcox,Division of Hematology/Oncology, Emory UniversitySchool ofMedicine, 308 Woodruff MemorialBuilding,DrawerAR, Atlanta, GA30322.

Receivedforpublication8November1990and in revisedform16

April1991.

1.Abbreviations usedinthispaper:MCP-1, monocytechemoattractant

protein-i;

MIC, mesenchymal intimalcells;PCR, polymerasechain

reaction; VSMC,vascularsmooth muscle cell.

into these lesionsremain unknown. Monocyte

chemoattrac-tantprotein-l

(MCP-1)I

isasecreted molecule withpotent che-motactic activity for monocytes(4). MCP-l is expressed by

culturedgliomacells(4),fibroblasts(5), monocytes(6),

lym-phocytes (7), endothelial cells (8), and smooth muscle cells (9). TNFa (5, 8, 10), IL-1j (5, 8, 10),

yIFN

(8), and endotoxin (5,

10) have all beenshowntoincrease MCP-l mRNAlevels in culturedendothelial cells and fibroblasts. In addition, increases in MCP-l-like activity and MCP-l mRNA have been docu-mented in supernatants of human umbilical vein endothelial cells (HUVE's) stimulated with serum enriched with mini-mallyoxidized LDL and

ft

VLDL (1 1, 12). These in vitro stud-ies suggest a potential role for MCP-l in therecruitment of

monocytesinto the vessel wall in the setting ofinflammationor

hyperlipidemia.

Toaddress whether MCP-l may play a role in recruiting

monocytes tothe vessel wall in vivo, we set out toidentify

MCP-l mRNA production in human atheroma usingin situ

hybridization. Atherosclerotic plaques exhibited marked hy-bridization, especially in macrophage-rich inflammatory

re-gions. Bycomparison, minimal expressionwas noted in con-trol arteries without atherosclerotic disease.

To identify possible regulators ofMCP-1 production in cellsderived from human atheroma, vascular smooth muscle cells (VSMCs) grown from human carotid endarterectomy

specimenswerestimulated with variouscytokinesand MCP- 1 mRNA levels were determinedbyNorthern blot. Tumor

ne-crosis factor (TNFa),gammainterferon

(yIFN),

and superna-tants from mixedlymphocyteculture were shown to stimulate

productionofMCP-l mRNA, whereas 10%FCSdidnot. These data suggest a potential in vivo role for MCP-l in mediating monocytic infiltration of the artery wall, and a role forinduction of MCP-lexpression byinflammatorymediators in modulating this process.

Methods

MCP-1probe isolation.The cDNA for MCP-Iwas clonedfrom stimu-lated humanlung fibroblastsusingpolymerase chain reaction(PCR) amplificationof cDNA made fromcytoplasmicRNA. Human lung

fibroblastswereobtained fromexplantsobtainedatautopsy. Cultures weremaintained in DMEM supplemented with 10%FCS, 1% penicil-lin/streptomycin,and25 mM Hepes, buffer pH 7.4. To increase MCP-1 mRNA levels, cellswerestimulated withphorbolmyristicacetate

(100ng/ml for 48h),thenwith E. coliendotoxin(10pg/ml,Sigma Corp.,St.Louis, MO)in the presence of 1 zg/mlcycloheximide (Sigma

ChemicalCorp.)for 6 h(13).TotalRNA wasisolatedusing guanidium thiocyanatelysisandCsCldensity gradientcentrifugation (14).cDNA wasthen producedusing12-18meroligodTprimers (PharmaciaFine

Chemicals, Piscataway, NJ) and reverse transcription followed by

RNAseAdigestion.30cyclesofPCRwereperformed(15)using33mer

oligonucleotideprimerswhichprimedthe 5' and 3' ends of thecoding regionfor MCP-1 and encodedprotrudingXhoI(5'primer)and Eco RI(3'primer)restrictionsites.The PCRproductwassubcloned into

Monocyte ChemoattractantProtein-i in Human Atheroma 1121 J.Clin. Invest.

© The American Society for ClinicalInvestigation,Inc.

0021-9738/91/10/1121/07 $2.00

(3)

.

*::.: " \ s.:<

ws )

OX SC}*: :....

P ..

to .*.:.

..E :.

i ..

A

./p

d

id__

4.5

-'Na

,

e:+

.,:* > . .,j3

I

-4-A..

.1

.W.'. :., I ... 4.,A: .-%%

:v

.:W:l

-40.

%

i

-J%

IL'.

..

X

... :..

fr.

'It.

(4)

the Xho I and EcoRIsites of Bluescript SK-plasmid and its sequence determined by dideoxy nucleotide sequencing (16). Primer sequences were 5' CCC CTC GAG TCC AGC ATG AAA GTCTCT GCC CTT for the 5' sequence, and 5' CCC GAA TTC TGT TCA AGT CTTCGG

AGT TTGGGT for the 3' complementary sequence.

In situhybridization. Human carotid endarterectomy specimens wereobtained directly from the operating room, fixed for 2 h at4VCin

4% paraformaldehyde immediately upon surgical excision, cryopre-served in 15% sucrosefor3 h andfrozen in OCT at -70'C. To con-struct riboprobes, Bluescript KS+ plasmid containing a full-length MCP-Iinsert was made linear by restriction digestion with Eco RI or Xho I(forsenseandantisense probes, respectively), then transcribed using T3 or T7 RNA polymerase (Stratagene Corp., La Jolla, CA) in the presenceof"S-UTP.Insituhybridizationwasperformed on 10Itm

frozen histologic sections as previouslydescribed (17, 18). After coating withphotographic emulsion, sections were exposed for 4-12 wk. Ex-posedsections were counterstained with hematoxylin and eosin.

The proportion of cells expressing MCP-1 was determined by counting 10,768 individual cells identified by nuclear staining at 400x in the14atherosclerotic specimens hybridized to theMCP-lriboprobe (Fig. 1, eandf).Acell was considered positive if the number of silver grains associated with stained nuclei exceeded five times background

overequivalentcell-sizedareaswithoutnuclei.Formostsectionswe

screened, cells withseven or moregrains were considered positive.

Immunohistochemistry.Serial microscopic frozen sections immedi-atelyadjacent to sections hybridized with theMCP-l probe were used forimmunohistochemical identification ofcells in three atheroscle-rotic specimens. Primary monoclonal antibodies included HAM56 (Dako Corp.,Carpinteria, CA) which reacts with - 95%ofthe human monocyte/macrophage cellpopulation ( 19). Cross-reactivity ofthis an-tibody has been shown to capillary endothelium, hence, all slides stained with HAM56 were compared with sequential slides stained with Ulex europaeus lectin (Vector Laboratories, Inc., Burlingame, CA), which selectively stainsendothelium (20). Vascular smooth

mus-cle cellswere identified with HHF35 (Enzo Pharmaceuticals, New York, NY)tomuscle actin whichspecificallyreactswithcardiac, skele-tal, and smooth muscle cells, but not fibroblasts, endothelium, neural

tissue,orepithelium (21).Tlymphocyteswereidentifiedwith UCHL1 (Dako Corp., Santa Barbara, CA) which reacts with CD45RO

ex-pressedbyprestimulated andasubset of restingTcells (22). This

anti-bodydoesnotreactwithBcellsorNKcells,althoughthereissome

cross-reactivitywith cellsofmyeloidand monocytic lineage(PMNs wererarein thesespecimens). Appropriate biotin-conjugated second-ary antibodieswereused anddetected with avidin-alkaline

phospha-tase(ABC-AP kit;VectorLaboratories,Inc.)accordingtothe

manufac-turer's instructionsas reportedpreviously (17, 18). Counterstaining was donewith 0.4% methyl green (Sigma Corp.).

Cell culture. VSMCs were culturedin collagen matrix (Vitrogen; Collagen Corp., Palo Alto, CA) from human atheroma obtained at carotid endarterectomy using published techniques (23). Medium was changed twice weekly, and cells passed before reaching confluence. All cellswereusedwithin the first six passages.

Smooth muscle cell phenotype wasconfirmed by immunohisto-chemical staining using HHF35 andfluoresceinsecondary antibody. Cultured cells stained positivefor actin in a spiculated pattern typical for smooth muscle cells in 83±5% (95% confidence interval) of 195 cellscounted (not shown).

Northern blotting. VSMCs grown to 90% confluence in 150 cm2 flasksweremadequiescent by incubatingfor 48 h with serum-free mediumafter washing 3x with PBS. Thereafter, theywereeither stimu-latedfor4or 24 h with the cytokines or growthfactors used in each

specific experiment.Total cellular RNAwasisolatedusingthe guanid-iumisothiocyanatemethod (14). 10 ugof glyoxal-treated RNA was thenrunina1%agarosegel and transferedtonylon reinforced nitro-cellulose (Schleicher & Schuell Corp., Keene, NH)asdescribed (14).

32P-labeled MCP-l probewasproducedusing the random primer tech-nique andT7DNA polymerase (StratageneRandom Primer kit; Strat-ageneCorp., La Jolla, CA). Nitrocellulose membraneswere prehybri-dized for4h andhybridizedat420overnightat aconcentrationof 106 cpm/ml in buffercontaining50%formamide. Two washesusing2X SSC with 0.1% SDSat550 andtwohigh-stringencywashesusing0.1 x SSC with 0.1% SDSat550wereperformedfor 15 min each. Nitrocellu-lose was then exposed to Kodak X-OMAT film with anintensifying

screen at -70° for variableperiodsof timedependingonestimated

radioactivity of the nitrocellulose membranes.

Results

Insitu hybridization. 14 carotid endarterectomy specimens, eightnormal coronaryarteries, and two normal internal mam-maryarterieswereexamined byinsitu hybridization.Positive hybridization for MCP-l was clearly present in all atheroma examined(Fig. 1, a, c,e).No cellularhybridizationwasseen in control slides probed with MCP-l sense riboprobe (data not

shown).

Expression of MCP-I in histologically

defined

regions of human atheroma. Positive hybridization was detected in 16% of10,768 cells counted. Random high-power fieldsamong

rep-resentativehistologic regionswerescoredusingacounting grid

Figure 1. (a)Aportion of atherosclerotic plaqueshowingextensivehybridizationof MCP-I antisense probeat8 wk exposure.Areaof inflam-matory cellinfiltrate inorganizingthrombushybridizesmoststrongly in this section. Darkfieldpolarized lightepiluminescenceviewenhances silvergrains correspondingto areasofhybridization (8-wk exposure;magnification, 100). (b)Samefieldas ainbrightfieldstained with

hema-toxylinand eosinshowing fibrouscaparea(right) adjacenttoorganizingthrombus inintraplaque hemorrhage(left).Infiltration ofinflammatory

cellscanbeseenbetween thesetwo areas(magnification, 100). (c) Darkfield polarized-light epiluminescenceviewof carotid atherosclerotic plaqueshowinghybridizationfor MCP-Iin theinflammatorycell-richareaunderlyingthenecroticlipidcoreabuttingthearterial media(above) (8-wkexposure; magnification, 100).(d) Adjacent frozen sectionto cstainedwithantimacrophagemonoclonalantibody (HAM56) showing

correlation ofimmunohistochemicalstaining for macrophageswithareasof intensehybridizationseeninc. Positivestainingappears red

com-paredwithmethylgreencounterstain(magnification,100). (e)High-power darkfieldpolarized light epiluminescenceview ofanatherosclerotic plaqueshowing MCP-1 hybridizationinaVSMC in thefibrous capregion.Discrete vascular smooth muscle cellswereshowntohybridize

strongly andwereoften surroundedby morphologicallysimilar cells which didnothybridize (8-wkexposure,magnification, 500).

(f)

Same field

as einlightfieldview forcomparison, showinganareawithahighpercentage of smooth muscle cells(positiveHHF35stainingonserialsection notshown). Note typical elongated nucleus and smooth musclemorphologyinhybridizedcellseenine.(g)Section of normal internalmammary

artery obtained from coronarybypasssurgical specimens hybridizedwith MCP-Iantisenseprobe. Verylowlevels ofhybridizationwere seen

in normal vessels.Examinationof this section under high power revealed only four cellshybridizingto

MCP-l

probe (arrows). The otherbright

areasin theadventitiarepresentrefractile fibrous tissue,anormal feature of the adventitia when viewedbyepiluminescence(8-wkexposure;

magnification, 100). (h)Section of control coronary arteryobtainedfromcardiomyopathy patient duringhearttransplantation(native heart)

hybridizedwith

MCP-l

antisense probe. Hybridizationwas rareinthesespecimens.One smoothmuscle-appearingcellcanbeseenin themedia withhigherthanbackgroundhybridization(arrow).Concentric fibrous intimalthickeningwas auniversalfindingin adult coronaryspecimens,

however, itwasnotassociated withhybridizationof probe (8-wk exposure;magnification, 125).

(5)

14

vW

'p

.%... A

i

4'-rS

/W

-*~~~~~~~~~~~~

.:I A/

r

JOY

t: ~ ~ ~ ~ ~F:.~

Ak'

-1

i

4ko /

4.B;

.0

4s

*:0F : l;.

,wt

A.

:%

044

:

*Si; !. .:t

*t ?a@

.:.,

41,

*:<:

.. i::

*. iris

S

?

4w

-_

b.

:t

A..

:

`.

, .1

W. :,.:

..:. "

i.:.: Ak.

.41 z,

\w

..I

A .,*.

'IN:.

"

.,"

--:

(6)

TableL ComparisonofPercentage Nuclei Associated wtInS

Hybridizationto MCP-I mRNA

Positivecells/totalcells

Morphologicsubdivision

Fibrous cap (uniform portion) 76/1471 (5%)

Fibrous cap(pleomorphic portion) 288/2871 (10%) Inflammatory area underlying necrotic core 482/1999 (24%)

Necrotic core 28/475 (6%)

Organizingthrombus 342/1035 (33%) Immunohistochemical subdivisic

HAM56positive cells 439/1163 (38%)

HHF35positivecells 82/ 1754 (5%)

Total 1737/10768 (16%)

Acell wasconsidered positive if the number of silver grains associated with stained nuclei exceeded five times background over equivalent cell-sized areas. For mostsections we screened, cells with seven or moregrains were considered positive. Both morphologic and immu-nohistochemical criteria were used for subdivision of tissues. For the morphologic subdivisions, uniform and pleomorphic portions of the fibrous cap were seen to have different morphologic features and to hybridize to different degrees (see text for details), and were separated onthosegrounds. For immunohistochemical subdivisions, HAM56 identified cells of monocyte lineage (primarily macrophages), and HHF35identified VSMCs. Results show a preponderance of hybrid-ization to macrophages, inflammatory areas underlying the necrotic

lipidcore, andorganizingthrombus.Vascular smooth muscle, how-ever,wasalso seentohybridizebuttoalesserdegree.

and thepercentageof hybridizedcellstallied (TableI).For the purposesof this analysiswecategorizedareasofthe 14

athero-sclerotic plaques into five histologically identifiable regions:(a)

fibrous cap containing uniform elongated "pancake" nuclei arranged inaprimarily unidirectional orientation, (b) fibrous

cap containing pleomorphic nuclei without ordered

orienta-tion,

(c)

inflammatory

areasborderingthenecroticlipidcore,

(d)

necrotic lipid core, characterized by cellular debris, and cholesterol

clefts,

and (e)

organizing

intraplaque thrombus characterized by the presenceofmorphologically identifiable fibrin

deposits

or

hemosiderin containing

macrophages.

Fibrouscap. Inthefibrouscap,8%of4,342 cells hybridized

tothe MCP-l riboprobe (Fig. 1, eandf). Significantly more

MCP-

1-positive

cells were found in areas of the fibrous cap

regions predominantly containing cells with pleomorphic

nu-clei arranged inarandom orientation(10% of 2,871 cells)

com-pared with fibrouscapregions

containing predominantly

uni-form

spindle

shaped cellstypical of VSMCs (5% of1,471cells,

P<0.001, chi square). The in situ signal intensitywas generally

lowerovercellsinthefibrouscapcomparedwith the intense

signals

seenin thesamesectionsmostnotably in inflammatory

areasbordering the necrotic

lipid

core(seebelow).

Regions of the fibrous cap with uniform spindle-shaped

cellsgenerally showed uniform staining with the HHF35 anti-body. Fibrous cap regions with pleomorphic nuclei often exhib-ited fewer VSMCs staining with HHF35 and frequently

con-tained HAM56and afewUCHLl-positive cellsaswell. MCP-1-positive cells in the fibrous cap included a population of

mesenchymalappearing intimal cells (MIC) often in pleomor-phic zones which did not co-stain with any of the antibodies used in thisstudy (Fig. 2). These cells had large pale nuclei

distinct from the typical spindle-shaped smooth muscle cell nucleus, a stellate cytoplasm, and were morphologically similar to theplatelet-derived growth factor (17) ortissue factor(18)

mRNA-containing MICs described previously in

atheroscle-rotic plaques. Manyareas within the atherosclerotic plaques

examined containednumerouscells meeting thesecriteria espe-cially in the pleomorphic regions ofthe fibrous cap and areas of

organizing thrombi.Itisdifficulttoestimate theproportion of positive MICsbecausetheir detection is bya processof

exclu-sion rather thandirect identification.

Organizing thrombus. Many cells

hybridized

strongly to theMCP-l probe inareasoffocal organizing thrombi

(33%

of

1,035 cells; Fig. 1, a andb). These regions often contained

many HAM56-positive cells with some UCHLl-positive T

cells. Spatial correlationof HAM56-positive cells and cells

hy-bridizing for MCP-l strongly suggeststhat macrophages ex-press MCP-1. We cannot exclude thepossibilitythat some

ex-pression in theseareasisby MICs becausethese cellsareoften found inareasof focalorganizing thrombus (24, 25).

Necrotic

lipid

core. Discrete cells in the necroticlipidcore were markedlypositive forMCP-1 mRNA (6% of475cells) andwereoftenadjacenttononhybridizingcells. The necrotic coresexhibited diminished cellular

density

andcontained

pre-dominantly necrotic debris, foam cell deposits, cholesterol

clefts,

and calcification. In agreement with previous studies macrophageswerethe predominantcell typefound in the

ne-croticcorealthough UCHLl-positive cellswerealsoidentified in theseregions (1).

Inflammatory

regions

bordering the

necrotic

lipid

core. Of all the

regions

studied the most

intensely hybridizing

MCP-l-positive

cellswerefound in

inflammatory regions

bor-dering

the necrotic core. Positive cellswere

relatively

abun-dant,

representing

24%

of 1,999

cellscounted. These

inflamma-tory

regions

containedmany

HAM56-positive

cellswithafew

scattered

UCHLl-positive

cells.

Histologically

identified foam cells wereoften observedto

hybridize

tothe

MCP-l

probe in theseareas.

Expression

of

MCP-J

by

cell type.Toidentify specificcell typesassociated with MCP-l

production,

serial

microscopic

sections

immediately adjacent

tosections

hybridized

withthe

MCP-1 probe were used for

immunohistochemical

staining

with HAM56, UCHL1, HHF35, and Ulexlectin inthree

repre-sentative

specimens. Carefully

chosen

regions

which stained

exclusively

with HAM56werefoundtobe

strongly

positive

for

Figure 2.(a) Brightfield epiluminescentviewof cellular area withahighproportionofimmunohistochemicallyunstainedmesenchymal

ap-pearingintimal cells(MICs)incarotidatheroscleroticplaque (arrows). Nucleiarevisibleasthedarkhematoxylin-stainedbodies surroundedby

brightsilvergrains. Hematoxylinand eosinsection;magnification,200.(b)Adjacentsectionto astainedwithHAM56,showinglackof tissue

macrophagesin this section(typical cross-reactivitytoendotheliumby thisantibodyis alsoillustrated). (c)Serial section stained with HHF35 foractin-containingVSMCs. Somepositivecellswerenoted, but donotappeartoberelatedtothosehybridizingforMCP-1inthissection.

(d)

Serial section stained withUCHL1 showingabsenceof T cells in this section. Positivestainingfor each monoclonalantibodyappearsred,

(7)

MCP- 1 (38%of1,754 cells). Cells meeting histologic criteria for macrophages, including large pale nucei with foamy

cyto-plasm, clearly hybridizedwithMCP-1 riboprobe, whereas

he-mosiderin containingmacrophages did not. Fewer MCP-l

posi-tive cells were found in regions staining exclusively with HHF35 (5% of1,163 cells). These areas showed no HAM56

staining. Despite

the low percentage

of

cells hybridizing in theseareas, individual cells clearly meeting histologiccriteria for VSMCs,includingspindle-shapednuclei and elongated

cy-toplasm, hybridized for MCP-l (Fig. 1, eandf).Itisnot possi-bleto comment on the colocalization ofTcell staining with MCP-l hybridization because no region was found to stain

exclusively with UCHL1 in the absence ofHAM56 staining.

These data suggest that both VSMCs and macrophages can express MCP-l in vivo. In addition, cells not staining with HAM56orHHF35, meetingthehistological criteria forMICs were also seen to hybridize strongly for MCP-1.

We failed to detect

MCP-l

hybridization to endothelial cellslining thevasavasorum or onthe lumenal surface ofthe

plaques. Given the factthatthetotalamountofendothelium seenin thesespecimenswassmall duetotransversesectioning, lack

of

endothelium in

portions

ofthe plaque and insome cases loss of endothelium in preparation, conclusions regarding

MCP-l

expressed by the endothelium in vivocannotbe drawn fromthisstudy.

Normal, nondiseased internalmammary arteryspecimens obtained fromcoronarybypass

operations

exhibited little

pro-duction

ofMCP-l mRNA asdeterminedby in situ

hybridiza-tion.

Hybridizing

cells, ifpresent, tendedto be foundin the adventitia(Fig. 1g).

Occasional

macrophage stainingwas seen

in theadventitia suggestingthathybridizingcells may be

scat-tered tissue macrophages. Like the internalmammaryarteries, normalcoronary vessels

obtained

fromexplanted hearts also showedonly minimal MCP-I

expression (Fig.

1

h).

These

speci-mens wereobtained from

nonatherosclerotic

coronaryarteries in

patients

with

idiopathic cardiomyopathies undergoing

heart

transplantation.

Asmallamountof concentric intimal thick-ening whichwas

invariably

presentin these

specimens

butno

advanced atherosclerotic lesionswereseen.

Hybridization

was

found

in <0.1% of cells countedinnormal vessels.

Cytokine stimulation of

VSMCsin

culture.

The

finding

that VSMCs expressed

MCP-l

invivosuggesteda

possible

rolefor smooth muscle-produced MCP-l in

initiating

or

enhancing

monocytic

infiltration

of

thevessel wall. To

begin

toexamine factors

potentially responsible for regulation

of

MCP-1

expres-sion by VSMCs,

Northern blot

analysis

was

performed

on

cyto-kine-treated human VSMCs in culture. Northern blot

analysis

of10

gg

of total cellularRNA per

sample

revealedaverylow levelof detectable

MCP-l

RNAin

resting

cellsaswellasthose stimulated with10%FCS.

Supernatants

fromamixed

lympho-cyteculture(donated byBruce

Hall, Stanford University,

Palo Alto, CA) however, produced a marked increase in MCP-1 mRNAdetectedat 4 and 24 h

(data

not

shown). Inflammatory

regulatorsTNFa(200 U/ml) and yIFN (50 U/ml)werethen

investigated

alone and in thepresence of5%FCS. Both

cyto-kines stimulated marked increases inthelevelof MCP-1 RNA

(Fig.

3). TNFa

elicited

greaterincreasesat24 hthan at 4

h,

whereasyIFNgenerated maximalresponse at 4 hwith diminu-tionovertime. The

differing

temposof MCP-1induction after

TNFa and yIFN

stimulation

presumably

reflect different mechanisms

of

action. Therewasno

appreciable difference

in

OC)-\~~~~~~~~~~~~~~~.

-\I

288-188

Figure 3. Northern blot of whole cell

lysate

RNA (10

ytg

per lane) hybridized with 32p labeled

MCP-l1

probe. Lanes include duplicate sample control cells incubated in serum-free medium for 48 h;

-yIFN,

50 U/ml for 4 and 24 h;

yIFN,

50 U/ml with 10% FCS for 4 and 24 h;TNFa, 200 U/ml for 4 and 24 h; TNFa, 200 U/ml with 10% FCS for 4 and 24 h. Very low level baseline production is seen in control lanescompared with peaks at 4 h for

eyIFN

and 24 h for TNFa. FCS had no effect on production of MCP-1 mRNA either in cytokine

treated

cells (above) or otherwise unstimulated cells (not shown).

theresponses to cytokines seen in the presence or absence of fetal calf serum.

Discussion

Human monocyte chemoattractant

protein-l1

(MCP- 1) is a se-creted peptide which exhibits powerful monocyte chemoat-tractant properties at nanomolar concentration. It has been detected in smooth muscle cells, endothelium, stimulated monocytes, lymphocytes, fibroblasts, and various tumor cell lines (6). Because the signals which attract monocytes to the atherosclerotic plaque remain unknown, we examined arteries for

MCP-l

production in vivo. We have shown that there is easily detectable

MCP-l

expression by multiple cell types in various regions of the atherosclerotic plaque, but little

expres-sion in normal vessels.

The majority of MCP- 1 production was seen in macro-phages. MCP- 1 was noted in all regions ofthe plaque with high concentrations of macrophages, especially underlying necrotic lipid cores and in organizing thrombi. The production of MCP-1 by macrophages may represent an amplification mechanism forthe recruitment ofadditional macrophages to the atheroscle-rotic plaque.

(8)

bothwith andwithout HAM56 staining.Becausesignalswhich

stimulate MCP-l production in VSMCs are not known and because MCP-l production by VSMCs may beinvolvedin the

initial recruitment ofmacrophages to the vessel wall, we

ex-posed cultured atheroma-derived VSMCs TNFa and

yIFN,

thendetermined MCP- I mRNAlevelsby Northern blot. The increase inMCP-1 expressioninduced by

yIFN

and superna-tantsof mixed lymphocyte culturesuggest apossible rolefor activated T lymphocytes in stimulation ofvascularsmooth

muscle MCP-l production.

Theproduction ofMCP- 1 by MICs adds to the increasing evidencesuggestingthat thesecellssecrete anumberof bioac-tive factors (17,18). Theorigin ofMICsand their rolein

ather-ogenesis

remain unknown.

Theidentification ofVSMCs and MICs as sourcesof MCP-1in atherosclerotic plaquesuggests thatthismoleculemay po-tentiallyplay a roleinmonocyteinfiltration inthe early devel-opment of the plaque. Mediators stimulatingmonocyte infil-tration into early fatty streaksareunknown, but theselesions have beenidentified aspossibleprecursors to mature

athero-sclerotic plaques (3). It istemptingto speculate thatMCP-l production by VSMCsorMICsmaybeelicitedbylipoproteins (11)orotherstimuli in early atherosclerotic lesions. With infil-tration of monocytes, a positive feedback mechanism might exist whereby

infiltrating

monocyte/macrophages secrete MCP- 1 in largeamounts, thereby attractingmoreperipheral bloodmonocytes tothedeveloping atherosclerotic plaque.

An-tagonists

of MCP-1action will be neededtoevaluateany causal

role forMCP- I in vivo.

In summary, we have shown that

MCP-l

mRNA is ex-pressedin atherosclerotic plaques in vivo.To ourknowledge,

this is thefirst studytodemonstrate theproduction ofaspecific

monocytechemotacticagentin human atheroma.

Acknowledgments

We thank R.Stoney, W.Ehrenfeld,J.Goldstone, and J. Rappof the Divisionof Vascular Surgery,UniversityofCalifornia,SanFrancisco,

D. E. Strandnessof the Division of VascularSurgery, Universityof

Washington, forprovidinguswith thetissues used in thisstudy,and Dr. LoisEpsteinof theUniversityofCalifornia, SanFrancisco,fora

generousgiftof gamma interferon.

N. A.Nelkenwassupportedbyagrant from the Pacific Vascular

Foundation and National Institutes of Health National Research Ser-vice Award grant F32HL-08241.D. Gordonwassupportedby

ROl

HL-42119.This workwassupportedinpart by

ROl HL-43322.

References

1.Jonasson,L., J. Holm,0.Skalli,G.Bondjers,andG. K. Hannson. 1986.

Regional accumulations ofTcells,macrophages,and smoothmuscle cells in the

humanatherosclerotic plaque. Arteriosclerosis.6:131-138.

2.Gerrity,R.1981. The roleofthemonocyte inatherogenesis.I.Transitionof

blood-borne monocytes into foam cells infattylesions.Am.J. Pathol. 103:181-190.

3. Ross, R. 1986. Thepathogenesis ofatherosclerosis. N. Engl.J. Med. 314:488-500.

4.Robinson,E. A.,T.Yoshimura,E. J.Leonard,S.Tanaka,P. R.Griffin,J.

Shabanowitz,D.F.Hunt,and E.Appella.1989.Completeaminoacid sequence

of a human monocyte chemoattractant, a putative mediator of cellular immune reactions. Proc.Natl. Acad. Sci. USA. 86:1850-1854.

5.Strieter, R. M., R. Wiggins, S. H. Phan, B. L. Wharram, H. J. Showell, D. G.

Remick,S. W.Chensue,and S. L. Kunkel. 1989.Monocyte chemotacticprotein

geneexpressionby cytokine-treated human fibroblasts and endothelial cells. Bio-chem. Biophys. Res. Commun. 162:694-700.

6.Yoshimura, T., N. Yuhki, S. K. Moore, E. Appella, M. I. Lerman, and E. J. Leonard. 1989. Human monocyte chemoattractant protein-l (MCP-1). Full-length cDNAcloning, expression in mitogen-stimulated blood mononuclear leu-kocytes, and sequencesimilarity to mouse competence gene JE. FEBS(Fed. Eur. Biochem. Soc.) Lett. 244:487-493.

7. Kaczmarek,B., B. Calabretta, and R. Baserga. 1985. Expression of cell-cy-cle-dependent genes in phytohemagglytinin-stimulated human lymphocytes. Proc. Nail. Acad. Sci. USA. 82:5375-5379.

8.Rollins, B. J., T. Yoshimura, E. J. Leonard, and J. S. Pober. 1990. Cyto-kine-activated human endothelial cells synthesize and secrete a monocyte che-moattractant,MCP-1/JE. Am. J. Pathol. 136:1229-1233.

9. Valente, A. J., D. T. Graves, C. E. Vialle-Valentin, R. Delgado, and C. J. Schwartz. 1988.Purification of a monocyte chemotactic factor secreted by non-human primate vascular cells in culture. Biochemistry. 27:4162-4168.

10.Sica, A., J. M. Wang, F. Colotta, E. DeJana, A. Mantovani, J. J. Oppen-heim, C. G. Larsen, C. 0. C. Zachariae, and K. Matsushima. 1990. Monocyte chemotactic and activating factor gene expression induced in endothelial cells by IL-I and tumor necrosis factor. J. Immunol. 144:3034-3038.

11.Berliner, J. A., M. C. Territo, A. Sevanian, S. Ramin, J. A. Kim, B. Bamshad, M. Esterson, and A. M. Fogelman. 1990.Minimally modified low density lipoprotein stimulates monocyte endothelialinteractions.J.Clin.Invest. 85:1260-1266.

12.Cushing, S. D., J. A. Berliner, A. J. Valente, M. C. Territo, M. Navab, F. Parhami, R.Gerrity, C. J. Schwartz, and A. M. Fogelman. 1990. Minimally modified low density lipoprotein induces monocytechemotactic protein1 in humanendothelial cells and smooth muscle cells. Proc.Nail. Acad.Sci. USA. 87:5134-5138.

13. Furutani,Y., H. Nomura, M. Notake, Y. Oyamada, T. Fukui, M. Ya-mada, C. G. Larsen, J. J. Oppenheim, and K. Matsushima. 1989. Cloning and sequencingof the cDNA for human monocyte chemotactic and activating factor (MCAF). Biochem.Biophys.Res.Commun. 159:249-255.

14.Sambrook,J., E. F. Fritsch, and T. Maniatis. 1981. MolecularCloning.A

Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY. 7.19-7.22.

15.Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B.Mullis, and H. A.Erlich. 1988. Primer-directed enzymatic amplification of DNAwith a thermostable DNA polymerase. Science (Wash. DC).

233:1076-1078.

16.Sanger, F., S.Nicklen, andA.R.Coulson. 1977. DNAsequencingwith

chain-terminatinginhibitors. Proc.Natl. Acad.Sci. USA. 74:5463-5469.

17.Wilcox,J. N., K. M.Smith, L. T. Williams,S. M. Schwartz,andD.

Gordon. 1988. Platelet-derived growth factor mRNA detection in human athero-scleroticplaques byinsituhybridization.J.Clin.Invest.82:1134-1143.

18.Wilcox, J. N.,K.M.Smith, S. M. Schwartz, andD.Gordon. 1989. Local-ization of tissue factor in the normal vessel wall and in theatherosclerotic plaque.

Proc.Natl. Acad.Sci. USA.86:2839-2843.

19. Gown,A.W.,T.Tsukada, and R. Ross. 1986. Humanatherosclerosis.II. Immunocytochemicalanalysis of the cellularcomposition ofhuman atheroscle-roticlesions. Am. J. Pathol. 125:191-207.

20.Holthofer, H., 1.Virtanen,A.L.Karimiemi,M.Hormia,E.Linder,and A.Miettinen. 1982.UlexeuropaeusIlectinas amarkerfor vascular endothelium in humantissues.Lab.Invest. 47:60-66.

21. Tsukada, T., D.Tippens,D. Gordon,and R. Ross. 1986.HHF35,a

muscle-actin-specificmonoclonalantibody.Am.J.Pathol. 126:51-60.

22. Terry, L. A., M. H. Brown, and P. C. L.Beverley. 1988. The monoclonal

antibodyUCHLIrecognizesa180 kD component ofthe humanleucocyte

com-mon antigen, CD45.Immunology.64:331-339.

23.Libby, P., S. J. Warner, R. N.Salomon,and L.K.Birinyi.1988. Produc-tion ofplatelet-derived growthfactor-likemitogen bysmooth-muscle cells from human atheroma. N.Engl.J. Med.318:493-498.

24.Wilcox, J.N.1991.Analysisoflocal geneexpressionin human atheroscle-rotic plaques by in situhybridization. Trends Cardiovasc. Med.1:17-24.

25.Wilcox, J. N., S. M.Schwartz,and D. Gordon.1990. Localproductionof platelet-derived growth factor in the human atheroscleroticplaque.InGrowth Factors, DifferentiationFactors, andCytokines.A.Habenicht,editor.

Springer-Verlag, Berlin.305-323.

References

Related documents

Summary: Isolated human peripheral lymphocytes were treated in vitro with styrene-7, 8–oxide (SO) and the kinetics of the repair of induced DNA damage was assessed by comet

Conflicts are universal part of ones life and they take place everywhere within every kind of setting, but the individuals are required to possess certain skills and attributes in

externalizing issues. 24 In sum, ratios of students to school counselors are correlated with a number of measurable student outcome variables. 19,21,22 A higher ratio of

In order to achieve maximum conversion rate (98.35%) of Ti, modeling suggested optimum values of four independent process variables, namely, temperature, KOH

Blade in c.s.: about 230 µm thick, epidermis uniseriate, amphistomatic, pubescent upper and lower epidermis, bifacial with palisade parenchyma in 2-3 rows, vascular

PMR agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated

These results further support our hypothesis that patients do differentiate be- tween the level of perceived autonomy support from each provider type, as the single second-order

Objective: To evaluate the effect of varying concentrations of brilliant blue green (BBG) and their different biochemical characteristics on retinal pigment epithelial (RPE)