VCAM-1 is a receptor for encephalomyocarditis virus on murine vascular endothelial cells.

0022-538X/94/$04.00+0

CopyrightC) 1994, AmericanSocietyforMicrobiology

VCAM-1 Is a Receptor for Encephalomyocarditis Virus

on

Murine Vascular

Endothelial Cells

SALLY A. HUBER*

Departmentof Pathology, Universityof Vermont, Burlington, Vermont 05405 Received 13 September 1993/Accepted 16 November 1993

MurineVCAM-1 hasbeenidentified as a receptor for theDvariant of encephalomyocarditis (EMC-D) virus onvascular endothelial cells from the heart. Monoclonal antibodies to VCAM-1 inhibited infection and

lysis

of endothelial cells with EMC-D virus. CHO cells transfected with the VCAM-1 gene were susceptible to EMC-D virus lysis, while control CHO cells transfected with the ELAM-1 gene were resistant. Similarly,

35S-labeledEMC-Dvirusbound toCHO-VCAM cells, and binding was inhibited with anti-VCAM-1 antibodies. Little or no radiolabeled virus bound to CHO-ELAM-1 cells.

Vascular endothelial cells (VEC) provide an important interface between the vascular circulation and the underlying parenchymal tissue. Growing evidence indicates that the VEC of different organs may have distinct characteristics which influence localization of nutrients, hormones, and cells to

particularorgans(3, 12, 16, 17, 21, 30). While the differences in VEC may play an important role in normal physiological processes, these same characteristics might also function in

pathogenicstates.Forexample, Auerbach et al. demonstrated that invitro tumor cell adhesion to VEC derived from different tissues resembles the propensity for the tumor cells to metas-tasize to particular organs in vivo (1). Additionally, various investigators have shown that the VEC may play an important role in virus tropism (9).

Picornaviruses use cell surface molecules as receptors for virus infection(24).These receptors contribute to the tropism ofthe viruses forparticularcells ortissues(7, 19,26).Todate,

only a few picornavirus receptors have been identified.

ICAM-1 acts as a receptor for a number ofrhinovirus sero-types (11, 29).Anotherimmunoglobulin superfamilymember acts as the receptor for poliovirus (24), and VLA-2 is the receptorfor echovirus (4).This report presents evidence that another immunoglobulin superfamily molecule, vascular cell adhesion molecule 1 (VCAM-1), acts asareceptor for the D variant ofencephalomyocarditis (EMC-D) virus on VEC de-rived from the heart.

MATERIALSAND METHODS

Mice. InbredBALB/cCUM micewere originally purchased

from CumberlandFarms,Clinton, Tenn. Breeding colonies of these animals are maintained attheUniversityof Vermont.

Virus.EMC-D viruswas kindly supplied byGlenn Wilson,

Department of Anatomy, University of South Alabama, Mo-bile. The virus titer is determinedbytheplaqueformingassay on L929 cells (15). Coxsackievirus B3 (CVB3) was originally

obtained from J. F.Woodruff. This virus is grown and its titer is determined in HeLa cells(10).

Radiolabeling of EMC-D virus. Radiolabeled virus was prepared by infecting a confluent monolayer ofL cells in a

225-cm2

tissue culture flask with approximately 10 PFU of

EMC-D virus per cell. After incubation for 1 h at 37°C, the monolayerwaswashed and freshmediumcontaining 6.5mCi

*Mailing address: Department of Pathology, University of

Ver-mont, Burlington,VT05405. Phone:(802)656-8940.

ofTrans-35S-label (ICN Laboratories)wasaddedtothe flask. The flask was cultured overnight at 37°C until greater than

90%cytopathiceffectwasobserved. The cells and supernatant were alternately frozen and thawed three times to release maximum virus and centrifugedat 300 xgto removecellular

debris.Cold methanol (-20°C)wasaddedtothe supernatant to a final volume of33%, and the mixture was incubated at -20°C for 20 h. The precipitatewasretrievedby centrifugation at8,000 xgfor 30 min,resuspendedin 0.2MP04 buffer(pH

7.4), incubated at37°C for 30 min with 0.5% ox-chymotrypsin (Sigma), andfinally subjected to sequential centrifugation of the supernatantat29,750 xgand144,743 xg.Thepelletwas retrieved after the secondcentrifugation, resuspended in 0.1M P04buffer(pH 7.4),andcentrifugedon a0/15/30% discontin-uous sucrose gradientfor 20h at72,128 xg.Thefinalpellet

containing the viruswasretrieved, resuspended in phosphate-buffered saline (PBS), titered for PFU, and quantitated for countsperminute.

VEC. Hearts were removed, minced, anddigested in 0.2%

collagenase (GIBCO, Grand Island, N.Y.)and0.01% DNase

(Sigma Chemical Co., St. Louis, Mo.) for 1 h at 37°C. After

digestion, the tissue was vigorously pipetted to obtain a single-cell suspension and then placed on a discontinuous Percoll gradient (Pharmacia Co., Piscataway, N.J.) as de-scribedpreviously(14).Theendothelial cell-enriched fraction was removed, washed, and cultured in Dulbecco modified Eagle medium (DMEM)-F12 medium (GIBCO) containing

5% fetal bovineserum(FBS; Sigma),20 Uofheparinperml,

10 ,ug ofendothelial cellgrowthfactor(Sigma)perml, 5

jig

of insulin(Sigma)perml, 5

jig

oftransferrin(Sigma)perml, 5 ng of selenium (Sigma) per ml, and 200 ,ug of Endo-Gro (VEC

TEC Inc., Schenectady, N.Y.) per ml. The endothelial cells weresubculturedby exposingthem for2minto0.01% trypsin (GIBCO) at37°C,whichpreferentiallydetaches the endothe-lial cells butnotfibroblasts. Subculturesaregreaterthan90%

endothelial cells

(16).

Transfected CHO cells. CHO (Chinese hamster ovarian)

cellswhichweretransfected with either the murine VCAM-7D or human ELAM-1 gene were most graciously provided by

RoyLobb, Biogen, Inc., Cambridge, Mass.The techniquefor theproduction of the transfected cells has been described in detailpreviously(5).The animal cell

expression

vectorusedin the production of these cells was a derivative of

pJOD-S,

pMD901. These cellsare maintained inalphaminimal essen-tial medium containing 10%

dialyzed

FBS

(GIBCO),

4 mM glutamine, and 200nM methotrexate (Sigma).

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MAbs. Hybridoma M/K-2 making a monoclonal antibody

(MAb)

to VCAM-1 was most

graciously supplied

byCharles G. Orosz,

Therapeutic

Immunology Laboratories, Ohio State

University College

of

Medicine,

Columbus.

Hybridomas

YN1/

1.7.4

(anti-ICAM-1),

KM 201 (anti-PgP-1), 2E6 (anti-CD18),

R1-2

(anti-LPAM-1),

and MEL-14

(anti-lymphocyte

surface receptor for

endothelium)

wereobtained from the American TypeCulture

Collection, Rockville,

Md. The

hybridomas

were grown in DMEM-10% FBS

containing glutamine, penicillin,

and

streptomycin.

The supernatants were

retrieved, dialyzed

against PBS, lyophilized,

and reconstituted at

1/10

their

origi-nalvolume

prior

to use.

Sepharose

S-200

purification

of anti-VCAM-1 antibody.

Sepharose

S-200 beads

(Pharmacia)

were

equilibrated

inPBS

(pH 7.4) and pouredinto acolumn (1.5 by76 cm). The void volume as determined with blue dextran (molecular weight

[MW],

2 x

106; Pharmacia)

was approximately40 ml. Other standards used to calibrate the column and their respective

elution volumes were mouse

immunoglobulin

G (MW,

150,000;

58

ml; Cappel Laboratories, Downington, Pa.)

and ovalbumin

(MW, 45,000;

102

ml;

Sigma). Approximately

5ml of the anti-VCAM-1 supernatant was added to the

column,

and 2-mlfractionswerecollected. Protein concentrationswere determined at 280 nm

by using

a Perkin-Elmer spectropho-tometer

(Perkin-Elmer Corp.,

Newton

Center,

Mass.)

and a standard

protein

concentration curve

using

bovine serum albumin

(Sigma).

Virus cytotoxicity assay. Endothelial cells or transfected CHO cellswere

plated

at

104

cells per well in 96-well tissue culture

plates

(Falcon,

Lincoln

Park, N.J.).

After incubation

overnight

in a

37°C,

5%

C02-95% air,

humidified

incubator,

the cell

monolayers

were washed with medium and overlaid with 100

RI

ofDMEM-10% FBS

(endothelial cells)

or

alpha

minimal essential medium-10%

dialyzed

FBS

(CHO cells)

aloneor medium

containing

either 104 PFU of EMC-D virus or107 PFU of CVB3 per ml. After incubation for 1hat37°C, the

monolayers

werewashed andreculturedin their

respective

media for 48 h. At this

time,

100

[lI

of10%formalinwasadded toeach

well,

and the

plates

wereincubatedat room tempera-turefor 15 min. The formalinwas

removed,

and 100

RI

ofa5%

crystal

violet solution in 20% ethanolwas added. The

plates

were incubated for 5 min and then washed

thoroughly

with water. The

plates

were

dried,

and 100 ,ul of a 50% ethanol solution was added to each well to redissolve the

dye.

The

plates

wereread in aBio-Tek

enzyme-linked

immunosorbent

assay

plate

reader

(Bio-Tek, Winooski, Vt.)

at599nm.Wells without cellsweretreatedwith

crystal

violetasdescribedabove to provide

background

absorbance.

Lysis

of the

monolayer

results in decreased absorbance.

Binding

of radiolabeled virus.

Approximately

104 CHO-VCAM-1 or CHO-ELAM-1 cells were cultured in wells of 96-well tissue culture

plates.

The supernatant was removed,

andthe

plates

wereincubatedonicefor 5 min.Fiftymicroliters of medium

(4°C) containing

104 PFU of labeled virus was addedto each

well,

andtheplateswere incubatedon ice for thetimes indicated. The mediumwas removed,and the cells

were washed with 50 ,ul of cold PBS. The wash and medium were combinedandcounted asunboundvirus. The cellswere

subsequently

lysedwith 50

RI

of0.5 N NaOH, and the wells werewashedwith 50

RI

of1% Triton X-100. This wash and the cellular

lysate

were combined and counted as virus bound. Percent virus bound was

determined

by the formula [cpm

bound/(cpm

unbound + cpmbound)] x 100.

Statistical

analysis.

The Studentt testwasusedtoevaluate

statisticaldifferences between groups.

Medium

EMC-D+anti-LPAM-1

EMC-D+anti-MEL-14

EMC-D+anti-CD18

EMC-D+anti-PgP-1

EMC-D +anti-ICAM-1

EMC-D+anti-VCAM-1

EMC-Donly

0.0

-1

-I

I1

I1

-I

0.1 0.2 0.3

ADSORBANCE 599 nm

FIG. 1. Lysis of cardiac VEC with EMC-D virus. VEC cultures were isolated from hearts of BALB/cmice; 104cellswere dispensed into wellsandexposedtomedium aloneormedium containingoneof severaldifferentMAbs(100

jig/ml)

for30 minpriortotheadditionof 0.1PFUof EMC-Dvirus per cell.After2days ofincubation, thecells werestainedwithcrystal violet and washed.Theamountofdyeuptake

as an indication of remaining cell monolayer was determined by

spectralanalysis.Results representmeanabsorbance+standarderror of themeanoftriplicate culturesin oneoftwoexperiments.

RESULTS

Inhibition of EMC-D virus-induced VEC lysis with anti-VCAM-1 antibody. VEC were isolated from the hearts of BALB/c mice and infected with 0.1 PFU of EMC-D virus per cell. Insomecultures, 50

[lI

of medium containing100 ,ugof MAbtooneof several cellular adhesion molecules per ml was added 30 minpriorto addition of the virus. After incubation for 1 h, thecellswerewashed and recultured in fresh medium

for48 h. Lysiswas determinedby crystalviolet uptakein the monolayers (Fig. 1). The results show that EMC-D virus is highly effective in lysing endothelial cells in vitro. Of the various MAbs, only the one to VCAM-1 was capable of

partially inhibitingcelllysis(absorbanceinthe

virus-plus-anti-VCAM-1 groupwasgreaterthan in thevirus-onlygroupatP < 0.05). Next, endothelial cellswereincubated for 30 minat room temperature with serial twofold dilutions of the

Sephacryl S-200-purified anti-VCAM-1 antibody and then

exposedtothe EMC-D virusasdescribedabove (Fig. 2).The results indicate that the anti-VCAM-1 antibody was effectiveat concentrations aslow as25

RI/ml.

No substantial differences wereobserved inblocking activity between103and105PFUof virus per well(0.1 and 10 PFU percell,respectively).

UseoftransfectedcellstoconfirmVCAM-1 astheEMC-D virus receptor. In the previous experiment, anti-VCAM-1

might inhibit virus interaction with theendothelialcell either

by directly binding to the virus receptoror perhapsby steric inhibition. To confirm that VCAM-1 is a relevant receptor, CHO cells transfected with either the murine VCAM-1orthe human ELAM-1 genewere exposedto 100PFUof CVB3 or 0.1 PFUof EMC-Dvirus per cell(Fig. 3). Control wellswere the CHO cells cultured in medium alone. EMC-D viruswas highlylytictotheVCAM-1-transfected CHO cells (P<0.01)

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-0.05

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ANTIBODY TITER (ug/mi)

FIG. 2. Titration of anti-VCAM-1 blocking activity on EMC-D

virus-induced lysis ofendothelial cells. Endothelial cells (104) were

incubated for 30 min with various concentrations of anti-VCAM-1 antibody.The cellswerewashed and exposedfor1 hat37°Ctoeither

10 PFU (0.1 PFU percell; D)or 105 PFU (10PFUpercell; *)of

EMC-Dvirus. The wellswerewashed, and the cellswereculturedfor

2days in DMEM-10% FBS. The cellswerefixed with 10%formalin,

stained with0.5% crystal violet,and washed thoroughly,andA5.9was

read. Absorbance of wells cultured without virus was 0.2 + 0.02. Resultsrepresent meanabsorbance + standard errorofthe meanof

threereplicatesperpoint.

butwasunabletolyse the control transfected(CHO-ELAM-1) monolayers. CVB3was not lyticto eithercell population. To determine whether a higher concentration of EMC-D virus

might be lytictothecontrol cells,endothelial cells and thetwo

types oftransfected CHO cells were plated in 96-well tissue

cultureplates, and concentrations of virus ranging from 0.1 to

105 PFU perwell (approximately 10-4 to 10 PFU per cell) were added (Fig. 4). Higher concentrations of the EMC-D

viruspercellwerenotpossible because of the titer of the virus preparation used for these studies. The results indicate that both endothelial cell and VCAM-1-transfected CHO cell monolayerscan becompletely lysed by EMC-D virus (dotted

line indicates background absorbance to wells without cells). Generally, CHO cells transfected with the ELAM-1gene were

highly resistant tovirus-mediated lysis.Approximately 20% of the CHO-ELAM-1 cell monolayer was lysed at the highest

0.5-CHO-VCAM-1

~ ~ ~~~[ CHO-ELAM

-

U

1

a~~~~~~~~K7,1

UC0.3-/to

z

0.2

0

C,)

survvingcell

0.0

CV133

EMC-D

Control

FIG. 3. Viral infection of transfected CHO cells. CHO cells

trans-fected with the murine VCAMorhuman ELAMgenewere infected with either 0.1 PFU ofEMC-D virusper cellor100 PFU ofCVB3per

cell. Controlswere CHO cells cultured without virus. After 2 days,

survivingcellswere stainedwith

crystal

violet. Results representthe

meanabsorbance_standarderrorof themeanoftriplicateculturesin

oneoftworeplicate experiments.

VIRUS PFU/WELL

FIG. 4. Virustitrationonendothelialcells and CHOtransfectants. Cells (1(4) were incubated with various concentrations of EMC-D virus perwell of 96-well tissue culture plates. After 1 h of incubation at37°C,the cellswerewashed andreincubated in DMEM-l0% FBS for 2days. Thecultureswerefixed with10%formalin and stained with 0.5% crystal violet, and A599 was read. The dotted line indicates absorbanceinwellswithout cells. Results representmeanabsorbance + standarderrorof the meanoftriplicate samples. O, CHO-VCAM cells;E,endothelialcells;m,CHO-ELAM cells.

virus concentration in this experiment, but some replicate experiments showed nosignificant lysis.

Next, CHO-VCAM-1 and CHO-ELAM-1 cells were ex-posed to EMC-D virus (0.1 PFU percell)with or without 100 pLg of the various MAbs per ml(Fig.5).Aswith the endothelial cells,only the antibodytoVCAM-1 wascapable ofinhibiting CHO-VCAM-1 cell lysis by the EMC-D virus. To determine the sensitivity of viral lysis of the CHO-VCAM-1 cells to anti-VCAM-1 antibody-mediated blocking,various concentra-tions ofthe Sepharose S-200-purified antibodywere addedto the transfected cells. After incubation for 30 min at room temperature, the antibody was removed and the cells were exposed to either 0.1 or 10 PFU of virus per cell. Figure 6 demonstrates thatthe CHO-VCAM-1 cellswere morereadily blockedby the anti-VCAM-1 antibodythan endothelial cells were. However, again no significant differences in blocking wereobserved betweenculturesexposedto0.1and 10 PFU per cell.

Radiolabeled virus binding totransfected CHO cells. The transfected CHO cells were incubated with 50 ,ul ofmedium aloneor mediumcontaining 50 ,ug of anti-VCAM-1 antibody per ml for 30min. The cellswerewashed and incubated with 1 PFU ofsucrose-purified 35S-labeled EMC-D virus per cell. Figure 7 shows the amount ofradiolabel bound to the cells after times of incubation ranging from 0 to 60 min. In two replicatebindingexperiments,littleor noradioisotope binding toCHO-ELAM-1 cells above the values obtained at the zero time point was observed. In contrast, substantial binding to CHO-VCAM-1 cell monolayers wasobserved. In the experi-ment shown, anti-VCAM-1 antibody was highly effective in blocking radiolabeled virus binding to the CHO-VCAM-1 cells. These results strongly indicate that the VCAM-1 mole-cule isacting as areceptor for the EMC-D virus.

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Medium

EMC-D+anti-LPAM

EMC-D+anti-CD18

EMC-D+anti-MEL14

EMC-D+antiPgP-1 EMC-D+anti-ICAM-1 EMC-D+anti-VCAM-1

EMC-D only

0.0 0.1 0.2

0

;

10-o 0

0

0.

5-z

X0

[image:4.612.54.288.80.296.2]

ADSORBANCE 599 nm

FIG. 5. Inhibition of CHO cell lysis byMAbs. Transfected CHO cellswereexposed to100pgof the various MAbs per ml for 30 min prior to theadditionof 0.1 PFU of EMC-Dvirusper cell.After 2 days, thesurvivingcells were stained withcrystalviolet.Theresults repre-sentmean absorbance ± standard error of themean of one of two experiments.E1,CHO-VCAMcells;*, CHO-ELAMcells.

DISCUSSION

The surface of the picornavirus capsid is irregular and contains both raised regions and depressions or canyons. Evidenceindicates that thefloors of these canyons contain the

specific binding sites for attachment of the virus to the cell membrane (18, 24). The cellular molecule binding the virus and facilitating viral internalization into the cell is designated the virus receptor. Often, the virus receptorcaninfluence the

tropismof the virus forparticulartissues(7,19,26).Therefore, identification of cellular molecules which can be utilized by various virusesasreceptorscanbeimportant in understanding

E

r-z 0

0.2

-0.15

-0.1

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-ANTIBODY TITER (ug/ml)

FIG. 6. Titration ofanti-VCAM-1 blocking antibody on EMC-D virus-induced lysis of CHO-VCAM-1 cells. CHO-VCAM-1 cells (104) were treatedwith anti-VCAM-1 and EMC-D virus as described for Fig. 2. Absorbance of cells cultured without virus was 0.20 ± 0.02. Results represent mean absorbance ±standard error of the mean of threereplicatesperpoint. O,CHO-VCAM cellsat1,000 PFU per well; 0,CHO-VCAM cellsat100,000PFU per well.

.6

eq oT o4

CHO-VCAM

--0- CHO-VCAM+ANTI-VCAM

---{ CHO-ELAM

--O-- CHO-ELAM+ANTI-VCAM

INCUBATION TIME (MIN)

FIG. 7. 35S-EMC-D binding to transfected CHO cells. CHO-VCAM-1or CHO-ELAM-1 cells(104)wereincubated for 30 minwith anti-VCAM-1 antibody (10

jig/ml),

washed,andexposedat40Cto104 PFU of sucrose-purified labeled virus for the times indicated. The results represent the mean percent radioactivity bound ± standard errorof the mean offour replicate cultures per group. Significantly moreradioactivity bound to CHO-VCAM-1 in the absence ofantibody at all incubation times compared with time zero (P c 0.05). No significantbindingwasobserved in other groupscompared withtheir time zero values.

thepathogenesisof viral infections. Thepresentstudy provides evidence that a major receptor for EMC-D virus on cardiac VEC is VCAM-1, based ontheabilitytoinhibit virus-induced lysis ofendothelial cell cultures with theMAb tothismolecule andontheabilityofCHOcellstransfected with theVCAM-1 cDNA to be lysed by EMC-D virus. Thesame molecule does not actas the CVB3 receptor. Anti-VCAM-1 wasnot able to block CVB3 infection of cardiac endothelial cells (data not shown), and the transfected CHO cellswere notsusceptibleto this viruseventhough CHO cells cansupport viral replication and arelysed whenexposed to infectious CVB3 RNA (14a).

Although VCAM-1 is probably an important EMC-D virus receptor oncardiacVEC, it may not be the only receptor used by this virusinvivo.Picornaviruses canuse morethanonetype ofreceptor(7, 8, 16,31),andwhichreceptor isemployed may largely depend on the type of cell being infected (25). The present studies provide no evidence for other cell surface molecules on either CHO-VCAM-1 transfectants or cardiac VEC which act as classical virus receptors. Antibody to VCAM-1 completely blocks virus-mediated lysis of both cell types, sinceabsorbance incultures with100

[Lg

ofantibodyper ml and virus is identical to absorbance in cultures without virus. Furthermore, radiolabeled EMC-D virus shows no ap-parentbindingtoCHO-VCAM-1 cells in the presence ofthe antibody. One somewhat perplexing observation, however, is that at 10 PFU percell, a small reduction in absorbancewas observed with CHO-ELAM-1 cellmonolayers despite the fact that no radiolabeled virus bound tothese cells. Twopossible explanations may exist for these observations. First, some breakdown of virions might occur with release of infectious RNA. At high enough virus concentrations, sufficient infec-tious RNA might be available to infect some cells through receptor-independent mechanisms. Similarly, should the CHO cellspinocytize extracellular medium,somevirusmight addi-tionally be internalized through this mechanism without actu-allyrequiring interaction with areceptor.

Despite the failure to demonstrate alternative cell surface receptors on cardiac VEC or CHO-VCAM-1 cells, other receptors must, nonetheless, exist. EMC virus is a highly pathogenic agentwhichhasbeen associated with a number of experimental diseases, including insulin-dependent diabetes,

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paralysis indicative of central nervous systemdisease, myocar-ditis, and pancreatitis (6). In eachcase, virus can be demon-strated in the infectedtissues or virus variants associated with aparticulardisease can be shown to replicate in targeted cells of the affectedorgan(i.e., beta cells oftheisletsof Langerhans in diabetes). It is highly unlikelythat these parenchymal cells would be infected via the VCAM-1 molecule, since this particular protein is restrictedto endothelial cells (22). Thus, different cellular receptors should exist on parenchymal and endothelial cells for thesamevirus.

Useof the VCAM-1 moleculeas an EMC-D virusreceptor onVECmay, nonetheless,have important implications in the infection and injury of the underlying parenchymal tissues. VCAM-1 is poorly expressed on normal microvascular endo-thelia, but expression can be substantially augmented by exposure ofthe VECto avariety of cytokines associated with the inflammatory immune response, including

interleukin-l

(IL-1), tumor necrosis factor alpha, IL-4, and gamma inter-feron (20, 23, 27, 28). Limited infections of either the VECor underlyingcells maybeinadequateto cause substantial tissue injury. However, cytokines such as IL-1 released from cells associated with the focal infection could stimulate VCAM-1 expression on the adjacent endothelium. This could facilitate additional virus localization from the blood into the organ. Furthermore, since VCAM-1 is animportant adhesion mole-culepromoting lymphocyte migration, enhanced expression of the VCAM-1 molecule on VEC adjacent to infection sites should augment inflammatory cell migration into the affected tissue. The inflammatory response could both contribute to tissue injury through lysis of either infected or uninfected parenchymal cells and promote extended virus infection of morecellsby releasing cytokines which upregulate virus recep-torexpression. In both EMC virus-induced diabetes (13) and pancreatitis (2), pathogenicity is highly dependent on func-tional T-lymphocyte responses in vivo. In the experimental

model ofpancreatitis, the Tlymphocytes do not cause direct immuneinjurytothetissue but,rather,causeaugmented virus infection in the pancreas, leading to extensive virus-induced cell death. Elimination of theTcellsdramaticallyreduces virus infection, providing circumstantial evidence that in this dis-ease,cytokinesreleasedbytheactivatedTcells increase EMC virus infection, replication, andspread throughout the tissue. In contrast, in some murine models of EMC virus-induced diabetes, pathogenicity is associated with the presence ofT cells, butnocorrelation betweenseverityof diabetes and virus titers inthe pancreasis observed (15).In thiscase,

pathogen-esis may be related to the direct lytic effects of the immune lymphocytes on the beta cells.

ACKNOWLEDGMENTS

Ithank Charles G.Orosz, whowas most graciousinsupplyingthe M/k-2 hybridoma making anti-VCAM-1 MAb, and Roy Lobb and Laurelee OsbournofBiogen Inc., whomostgraciously supplied the CHO transfectant cells. I also thankLaurie Sabens andMarilynChates for expert secretarial work in the assembly and submission of the manuscript.

Thiswork wassupported by American Heart Association

grant-in-aid 90-0999.

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