Activation of mouse lymphocytes by vesicular stomatitis virus.

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0022-538X/80/09-0757/09$02.00/0

Activation of

Mouse Lymphocytes by Vesicular Stomatitis

Virus

GAIL W. GOODMAN-SNITKOFF ANDJAMES J. McSHARRY*

Departmentof Microbiology and Immunology, Albany Medical College of Union University, Albany, NewYork 12208

Vesicular stomatitis virus (VSV) is a mitogen for mouse spleen cells, and

infectious virusis not required for mitogenesis. At concentrations between 10 and

100

,tg

per culture, VSV stimulated DNA synthesis and blast transformation.

Maximal activation by VSV occurred48 hafter culture initiation. Spleen cells

depleted ofT-lymphocytes bytreatmentwith anti-Thy 1.2 and complement and

those obtainedfromcongenitallyathymic BALB/c nu/nu mice were activated by

VSV, suggesting that VSV is a B-cell mitogen. Activation of spleen cells was

independentof the host in which the viruswas grown, since VSV grown in

BHK-21,HKCC,orMDBKcellswasmitogenic. The mitogenesis was specific forVSV,

since MDBKcell-grown WSN influenza viruswas not amitogen in this in vitro

activation system, VSV-specific antibodyprevented VSV mitogenesis, and VSV

was mitogenic for spleen cells from C3H/HeJ mice which were resistant to

mitogenesis by endotoxin.

Vesicular stomatitis virus (VSV) isan

enve-loped RNA virus containingalipoprotein

enve-lope composed of repeating units of a single

internal membrane protein andasingle external

glycoprotein (8, 26, 41). The lipid envelope is

composed ofphospholipid, cholesterol, and

gly-colipids (22,28).Anexternallayerof

proteogly-can is associated with the virion (36). In vivo,

VSV is a potent activator of the immune

re-sponse,stimulatingtheproductionof both

neu-tralizing antibodies and cytotoxic lymphocytes

specificfor the viralglycoprotein (16, 18, 25, 27,

46,47).

Our interest in the activation of unprimed

lymphocytes by mitogens and the previous

dem-onstration that some viruses such as herpex

simplextypes 1and2(20, 29) and the Singapore

typeofinfluenza virusA(6)aremitogens ledus

to examine themitogenic potential ofVSV,its

isolated antigens, and itsassociated lipids,

gly-colipids, and proteoglycans. VSV is an ideal

modelsystem to studythe mitogenic potential

ofvirions, individual viral proteins, and the other

chemical constituents associated with the virion.

This viruscanbepropagatedinanumber ofcell

culture systems which affect thelipid and

car-bohydratecontentof the virions without

chang-ing the protein composition,allowingthe

mito-genic potential of the lipid and carbohydrate

constituents to be determined in the context of

theviralprotein (27). VSVcanbegrown in the

presence ofnoncytocidal viruses, such as the

paramyxovirus simianvirus 5, which alters the

protein composition of the hybrid viruses by

insertingadditionalglycoproteins into theviral

envelope andremoves allsialic acid from both

hybrids and nonhybrid bullet-shaped virions

without changing other lipid and carbohydrate

constituents (26). These bullet-shaped,

nonhy-brid virusparticleswhich lack sialic acidcanbe

used to study the role of sialic acid in viral

mitogenesis. Purified viral antigenscan be

ob-tained from the virus inlarge enough quantities

forchemical andbiologicalassays(25),allowing

themitogenicpotentialof individualproteinsto

bedetermined.Defectiveinterfering (DI)

parti-cles, suchasDI-011, which have all of the viral

proteins,lipids, andcarbohydratesinthe

appro-priate configurations, butlack all biosynthetic

activities, are available to determine whether

infectious virus isrequired for mitogenicity (23).

Finally, temperature-sensitivemutantswith

de-fects in various structuralproteins, particularly

G and M, are available to test the mitogenic

potential of various functional antigens. Thus

VSVrepresentsafullymanipulativesystemfor

studying the mitogenic potential of virions and

the various chemical constituents of the virus

particle.

In this initial report, we demonstrate that

VSV is a murine B-cellmitogenasmeasuredby

DNAsynthesisand blasttransformationin both

normal and B-cell-enriched lymphocyte

popu-lations.Infectious virusisnotrequiredfor

acti-vationof murinespleen cells.

MATERIALS AND METHODS

Mice.CBA/J andC3H/HeJfemale micewere ob-tained from Jackson Laboratories (Bar Harbor, Maine) and maintainedinourfacilityonPurinamouse 757

on November 10, 2019 by guest

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758 GOODMAN-SNITKOFF AND McSHARRY

chowand acidifiedwaterad lib. Female micebetween 8and 12weeks of agewereused for allexperiments. BALB/cnu/+and nu/nu female micewerepurchased fromGIBCO Animal Resources andweremaintained inasterilelaminar-flow hoodonsterilemousechow andwaterad lib. These micewereusedat6 to 7weeks of age.

Lymphocytecultures. Cell suspensionswere pre-pared from spleens which had been aseptically

re-movedandwere minced withforcepsin RPMI 1640

(GIBCO Laboratories); the cells were further dis-persed bygentle pipetting.Largepieceswereremoved bygravitysedimentation for10min, and the cellswere

washed twice in RPMI 1640 medium. The cellswere

counted forviability in ahemacytometer, using the techniqueof eosindyeexclusion(39),andresuspended

at 4x 106cells/mlinfresh RPMI1640medium sup-plemented with 100 U of penicillin and 100 ,ug of streptomycinperml withoutserumsupplementation. Thecellsuspension (0.1 ml/well)wasdispensedinto microtiterplate(FalconMicrotest II withlid),andan

equal volume ofmitogenwasadded(total volume,0.2

ml/well).Cellswerecultured inanatmosphereof 10% CO2inhumidified airat 37°Cfor various periodsof time. Twenty-fourhours before harvesting, 1 ,uCiof [3H]thymidine (42 Ci/mmol; New England Nuclear Corp.) wasaddedto each culture. At the end of the culture period,cellswereharvested onto glassfiber filters,usingaScatronmultiple harvestingapparatus (Flow Laboratories).

Fortubecultures,the cellswereresuspendedto a

concentration of2x106cells/ml,and1mlwasadded

toplastictubes(16 by75mm;Falcon);then0.1mlof mitogenwasadded,and the cellswereincubated and pulsed as described above. The cultures were then harvested by usingamultiple-samplemanifold (Mil-liporeCorp.)ontoglassfiberfilters(Whatman GF/C), andtrichloroaceticacid-insolublematerialwas precip-itated. Ineach case,[3H]thymidine incorporationwas

determined byliquidscintillationcounting.

Anti-Thy1.2treatment.Monoclonalanti-Thy1.2

wasobtained from New EnglandNuclearCorp. and used as previously described (12). All experiments

were done in triplicate and,unless otherwise stated, standard error of the mean was less than 10%. In addition,eachexperimentwasrepeatedaminimum of three times.

Blastogenesis. Cells were cultured in tubes as

described above; however, [3H]thymidine was not

added. Forty-eight hours after culture initiation, the

cellswerecentrifugedontoglass slideswith a cytocen-trifuge, stained with Wright-Giemsa stain,andscored forblastogenesis.

Mitogens. Salmonella typhosalipopolysaccharide endotoxin (LPS) 0901 waspreparedby the

phenol-watermethod ofWestphal etal. (43) and contained less than 1% protein. Concanavalin A (ConA) was

purchasedfromSigmaChemical Co.

Cells.Monolayersof the BHK-21-FlineofSyrian hamster kidney cells and the HKCC cell line were

growninFalconplastic tissue culture flasks (75cm2) or plastic roller bottles (490cm2) in Eagle medium

(GIBCOLaboratories) reinforcedwith109%ocalfserum

(GIBCO)and 10% tryptosephosphatebroth(17).The

MDBK line of bovinekidneycellswasgrowninplastic

flasks or roller bottles in reinforced Eagle medium with 10%fetal bovine serum (9).

Virus. Stocks of the Indiana serotype of VSV were initiated from single plaques in BHK-21-Fcells,grown in stationarycultures of MDBKcells, and assayed on BHK-21-F cell monolayers as described previously (10). TheWSN strain of influenza virus was obtained from L. A.Caliguiri, and stocks were grown in MDBK cells and plaqued on MDCK cells according to pub-lished procedures (7). Additionally, purified DI-Oll wasobtained from R.Lazzarini. DI-Oll is a defective interfering particle which contains RNA in a hairpin configuration and does not induce viral protein or RNAsynthesis (23).

Propagationandpurificationofvirus.Briefly,

confluent monolayers of BHK-21-F, HKCC, and MDBKcellsinrollerbottles were washed two times withwarm phosphate-buffered saline (PBS) and in-oculated withVSVat amultiplicity of infection of 0.05

to 0.1PFU/cell.Aftera1-hadsorption period at 37°C

on aroller apparatus, Eaglemedium reinforced with 1%calf serum(BHK-21-F and HKCC cells) or 1% fetal bovine serum (MDBK cells) was added. Incubation wascontinued for approximately 22 hon the roller apparatusat37°C. The WSN strain of influenza virus waspropagated in MDBK cells under similar condi-tions. In each case, released viruswasharvested and purified byclarificationat1,500 rpmfor10min, cen-trifugedonto a0.5-mi50% (wt/wt)potassium tartrate cushion (Beckman SW27, 23,000 rpm,60min, 4°C), diluted 1:10 with PBS,andisopycnically centrifuged in 15 to35% (wt/wt) potassiumtartrate gradients at 23,000 rpmfor2.5 h inaBeckman SW27 rotor. The virusbandwasharvested anddialyzedagainst PBS at 4°C overnight. Viruses were then concentrated by pelleting, resuspended in a small volume of sterile PBS, andfrozen at -70°Cuntil used. The purity of viruspreparationswasdeterminedbypolyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. Only preparations which showed no

contaminatingproteinswereusedfor mitogenassays. Antiserum. Antiserum topurified VSV was pre-pared in New Zealand white rabbits by immunizing withpurified VSV incomplete Freund adjuvant, with booster injections every 10 days. The rabbits were

bled from theearveinbeforereceiving their booster injection, andserum wasprepared.

Chemical analysis. Protein was determined by themethod of Lowryetal.(24).

PAGE. All virus preparations were examined by PAGE(2).

RESULTS

VSV-induced mitogenicity and

blasto-genesis.Theabilityof VSV to act as amitogen

forculturedmouse cellswasexaminedby both

DNA synthesis,asmeasured by[3H]thymidine

incorporation into insoluble material, andblast

transformation,asmeasuredbydirect counts of

Wright-Giemsa-stainedcells(Table 1).VSV

in-ducedsignificant DNAsynthesis (asmeasured

by astimulation index [SI] of3 or greater) at

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759

concentrations of 7, 35, and 70 ,Lg/culture.

Higher concentrations of virus (up to 150 ,ug/

culture) caused stimulations that were on the plateau or on the descending portion of the

dose-response curve (see Fig. 1). Thedose-response

curve of VSV-induced stimulation was broad

and wassimilar to those of B-cell mitogens such

asLPS orpurified protein derivative (PPD) of

tuberculin (13, 40) as opposed to the sharp dose-response curve characteristic of ConA, a T-cell

mitogen (42). Inconjunction with the

incorpo-rationof[3H]thymidine into insoluble material,

the actual number of cells undergoing blast

transformationincreasedatall concentrations of

VSV. At 35and 70

jig/culture,

23 and26% of the

cellswereblasts, and the number of blast cells

was 4.5and 5 timesbackground levels,

respec-TABLE 1. VSV-induced blastogenesisofmouse spleen cellsa

t3H]thymidine

Wright-Giemsa-stained

cells

Mito- uptake gen

and sa

amt cpm SI" lympho- cells

Totacellst

(ug/mi)

~~~cytes

ceces

None 1,260 2,150 118 2,268 5.2

LPS, 61,939 49.1 1,035 1,257 2,292 54.8 100

vsv

7 3,813 3.0 2,006 309 2,315 13.3 35 11,370 9.0 1,823 551 2,374 23.3

70 14,928 11.8 1,708 613 2,321 26.4

a

Cells

werecultured in tubes at a concentration of

2 x 106cells/ml for 48 h. For [3H]thymidine uptake, standard error was less than 10%.

bSI = experiment counts per minute divided by

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control counts perminute.

TABLE 2. Activationofmousesplenocytes by VSVa [3H]thymidineuptake" Amt Microtiter

Mitogen cultre plates Tubes

culture) pae

cpm SI cpm SI

None 1,149 5,065

LPS 10 43,439 37.8 149,847 29.6

50 19,400 16.9 154,285 30.5 100 20,263 17.6 172,945 34.1

BHK-21-F 6 3,124 2.7 7,746 1.5

VSV 12.5 5,997 5.2 8,553 1.7

25 7,939 6.9 12,323 2.4

50 15,535 13.5 17,624 3.5 75 19,729 17.2 22,947 4.5 100 19,866 17.3 27,869 5.5

aForty-eight-hour cultures, standard error equal to

orless than 10%.

bMicrotiterplateshad 0.2mlofmedium and 4 xlOs

cells. Tubes had1mlofmedium and 2 x106cells.

tively.LPS also stimulated bothmitogenesisand

blasttransformationinthesecultures.

Effectof culture conditionsonactivation

by VSV.To demonstrate that thestimulation

which wasobserved in mouse splenocytes

cul-tured with VSV was not due to the culture

conditions, the ability ofVSV to induce

mito-genesis in cells cultured in tubes or microtiter

wellswasexamined (Table 2). Under both

con-ditions,significant stimulation of the

incorpora-tionof

[3H]thymidine

intoprecipitable material

wasobtained. In the microtiter system, the

op-timal response was obtained at 75 ,tg/culture

and did not increasesignificantly at

100,ug/cul-ture,suggesting that the system was saturated.

In the test tube system, the optimal response

wasobtainedat 100,Lg/tube,thehighest

concen-trationused in thisexperiment.The lower SI for

mitogenesis in tubecultures reflects the higher

background due to an increased number ofcells

in the culture. The tube cultures did not have

anincreased number of counts as compared with

the microtiterwellsbecause thesameamountof

mitogen wasadded to each culture. Since there

were five times as many cells in the tube

cul-tures, there wasonly one-fifthasmuchmitogen

per cell. Thus,the tubecultureswerenot

satu-rated with mitogen as were the microtiter

cul-tures.Indeed,atconcentrations of10,ug/culture,

LPS gavemaximal activation in microtiterwells,

whereas 10 times that, or 100 ug of LPS per

culture, was needed togive asimilar SI in the

tube cultures. AlthoughtheSI valueswerenot

thesameduetodifferences in the ratio of virus

to cells between microtiter and tube

cultures,

VSVwasmitogenicineachculture system. Since

we were able to saturatethe microtitersystem

withsmallamountsofvirus,all of the

following

experimentsweredone inmicrotiter

plates.

Dose response of VSV-induced

activa-tion. To compare the

mitogenicity

of VSVwith

othermitogens,orwithitself in different

exper-iments,

it is

important

to be working at the

optimal concentration ofmitogen. Tothis

end,

we studied the effect of

varying

the

mitogenic

dose.Oneinitialobservation wasthat the

opti-mal concentration varied with each batch of

virus,and could vary from

approximately

10to

100

,ug/culture

(Fig.1and2).The

optimal

stim-ulation ofspleencellswasachieved with 25to

50

,tg

of HKCC VSVor 50,ugof BHK-21-F VSV

and thendecreasedslowly (Fig. 1).When

com-paredwithConA,which inourhandspeakedat

approximately 0.125 ,ug/culture and then

de-creasedtobackgroundlevelsby0.25 or0.5,tg/

culture,these curvesare very broad.The

dose-response curve for VSV

generally

peaks at

higher concentrations than thatfor ConA and

retains its response over a broader range of

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25-

20-2 15-E

10.

5-BHK21FVSV

*HKCC vSv

i 10 25 50 100 50O

VgVSV Protien per well

FIG. 1. Response ofmouselymphocytesto two dif-ferent VSVpreparations. Mouse lymphocytes were

cultured in microtiterwells ata concentration of4 x 105 cells/wellwith 0.1 mlofvirusatthe concentra-tions indicated. The cultureswereincubated at37°C

and 100%o humidity for 48 h. One microcurie of [3H]thymidinewasadded 24 hbefore harvestingthe cellsforscintillationcounting.

Ix0

E

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IJ Viral Protien per W*ll

FIG. 2. Response ofmouselymphocytestofour dif-ferent viralpreparations. Cellswereculturedas

de-scribedinFig.1.

doses. The dose-responsecurvefor other mito-gens,such as LPS or PPD, peaks at50 or 100

,ug/culture and then remains at high levels of

stimulation over a largerange ofmitogen

con-centrations (34, 40),sometimesnotapproaching

background [3H]thymidine incorporation until

concentrations of500 ug/culture are used (12).

Therefore, sinceVSV preparations stimulate a

mitogenicresponsewhichplateaus, rather than

peaking and returning rapidly to background

levels, the dose-response curves more closely

resemble thoseof LPSorPPD,B-cell mitogens,

than ConA,aT-cell mitogen (42).

Effect of host cell on VSV mitogenicity.

VSVgrown indifferent host cells has different

amounts of sialic acid and lipid (22, 28). For

thesereasons, we examined the ability of VSV

grown indifferent cell types to activatemouse

lymphocytes (Fig. 2). VSV preparations

propa-gated in MDBK, HKCC, or BHK-21-F cells

wereall stimulatory formousespleen cells. All

these virus preparations induced optimal

acti-vation between10and 25 ,ug/culture. The slight

butsignificantdifferences inthestimulation of

lymphocytesbyVSVgrownindifferent celllines

may be due to known differences in the sialic

acid content oftheviruses (27). However,

fur-ther studies will be required to determine

whether these differences are associated with

the observeddifferencesinmitogenicity. In

con-trast, WSN influenza virus grown in MDBK

cells andpurified inanidenticalmanner asVSV

was notstimulatory.Theselatter resultssuggest

that theobserved activation is specific for VSV

and isnotduetocontaminantsfrom the medium

or tocellular contaminants whichmaycopurify

with the virus, since the possibility of

contami-nation is equal for VSV and influenza virus

grownand purified under thesameconditions.

Kinetics of the mitogenic response. The

optimal time of a nonspecific lymphocyte

re-sponse can vary depending upon the mitogen.

Forexample,themaximalstimulation by ConA,

a T-cell mitogen,occursafter3 days of culture

(42), whereasB-cellmitogenssuch as LPS,PPD,

or endotoxin protein stimulate an optimal

re-sponsewithin2days of culture of mitogen

stim-ulation (12, 34, 39,40).To determine thetimeof

optimalresponse,themitogenic activityof VSV

was determined at various times after culture

initiation(Fig. 3).Themaximalresponse toVSV

grown inHKCCorBHK-21-Fcellswasobtained

in 2daysofculture;24hafter culture initiation,

[3H]thymidine uptake in VSV-stimulated

cul-tures wasequalto backgroundlevels (datanot

shown). After5days, stimulationwas nolonger

significant. Thisearlyresponse is typical of

B-cellactivators and suggests that the B-cell is the target ofVSV-directedactivation.

B-cell mitogenicity. To examine the

possi-bility that VSV is a B-cell mitogen, two

ap-proacheswere used. Thefirststudied the

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ion by VSV oflymphocytes which had been mitogen, ConA,decreased from 5.5 times

back-letedof T-cellsbytreatmentwithanti-Thy ground in the unseparated cells to 0.6 times

and complement. The data (Table 3) show background in theT-cell-depleted cultures, sug-LtVSVactivated both the unseparated spleen gesting that the T-cells were selectively

re-Is and the B-lymphocyte-enriched popula- moved. Furthermore, these data together with

n. Although the actual counts per minute thoseonthe kineticresponseareadditional

ev-relower in theanti-Thy 1.2-treated cells, this idence ofB-cell activationby VSV.

struefor both the control and theexperimen- A secondapproachtosupportthishypothesis cultures. The SI values for VSV and LPS, comparedtheresponseto VSV oflymphocytes B-cell mitogen control, were the same in from congenitally athymic BALB/c nu/nu

ated(8.8 and 10.7) and untreated (7.8 and 9.0) ("nude" orB-cell) mice with that of

phenotypi-tures,whereas the stimulation by theT-cell callynormalBALB/c nu/+mice (Table4). On the basis of eithercountsperminuteorSI, the

--.BH21F-VSV cellsfromnu/nu micewereasresponsive to VSV --HKCC-VSV as those fromnu/+mice. Thelymphocytesfrom

the nu/nu responded to both HKCCVSV and BHK VSV with optimal counts of 16,155 and 12,728,respectively, andtoLPS withanaverage cpmof68,760, butwerenotstimulatedby ConA.

This lack of stimulationby ConA demonstrates the absence of functional T-cells in these

ani-mals. The nu/+ mice responded to both VSV preparations, LPS and ConA. These data with B-cellsare consistent with ourhypothesis that VSV isactingasaB-cellmitogen.

Specificity.ThatVSVand notacontaminant

isthemitogenic moiety issuggested bythe fact

thatthe WSN strain of influenzawasnotactive.

DAYS Further evidence insupportof this conclusionis

JIG. 3. Kineticsofthe response ofmouse spleen shown in Tables 5 and 6.Lymphocyte activation 's to two VSVpreparations. Mouse lymphocytes byVSVwascompletelyinhibitedbyrabbit

anti-*ecultured in microtiterplatesataconcentration VSVantiserum,but notbynormalrabbitserum

!x105cells/well with 0.1ml of10pgof BHK-21-F (Table 5). These sera did not significantly

en-V or 25 pg ofHKCC VSV. The cultures were hancebackgroundproliferationasmeasured by

ibatedat37°Cand100%humidityforthe number [3H]thymidine incorporation, nordid they

sup-laysindicated. One microcurieof[3H]thymidine pressactivationbytwounrelatedmitogens,LPS

addedforthefinal24hofculture. and ConA (datanot shown). This antiserumat

ABLE 3. Activation a dilution of

1:1,000

was able to reduce virus

VSVa infectivity5.5logsfrom 1010to 5x 104PFU/ml.

vat

dep

1.2 tha

cell

tior

wei

wai

tal the trei

culi

5-

4-

3-F

celb

wer

of4

VSi

inch

ofd

was

[t'H]thymidineuptake

Amt Unseparated Anti-Thy1.2

Mitogen (ug/ cells +complement-culture) treatedcells

cpm SI cpm SI

None 1,961 1,007

ConA 0.25 2,919 1.5 920 0.9

0.5 6,326 3.2 433 0.4

1.0 10,874 5.5 632 0.6

2.0 9,807 5.0 531 0.5

LPS 1.0 11,832 6.0 7,056 7.0

10 17,659 9.0 10,715 10.7 50 12,109 6.2 7,466 7.5

BHK-21-F 20 4,214 2.1 2,277 2.3

VSV 50 7,631 3.9 5,296 5.3

100 15,312 7.8 8,853 8.8

aMicrotiter plates, 48-h cultures, standard error

equalto orlessthan 10%.

TABLE 4. Activation oflymphocytesfromBALBIc nu/nu and nu/+ mice by VSVa

['H]thyrnidineuptake

Amt

Mitogen (Rg/ nu/nu nu/+

culture)

cpm SI cpm SI

None 2,034 2,666

LPS 50 68,760 22.8 81,878 30.7

ConA 0.12 1,583 0.8 95,058 35.7 BHK-21-F 0.1 7,231 3.5 9,848 3.6

VSV 1 12,728 6.3 13,965 5.2

5 8,819 4.3 6,889 2.5

HKCC VSV 1 4,588 2.3 5,242 2.0

10 16,155 7.9 17,360 6.5 25 13,055 6.4 16,611 6.2

aMicrotiterplates,48-hcultures, standard error less than orequalto10%v.

VSV MITOGENESIS 761

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Thus,theability of this antiserumtoneutralize

virus infectivity and mitogenicity is additional evidence that itwasthe VSV whichwasacting as alymphocyte mitogen.

Furthermore, thatthis activationwascaused by LPSis ruled out in theexperimentspresented in Table 6. Lymphocytes from C3H/HeJ mice aregenetically unresponsivetopurifiedLPSand respondtotheLPS-protein complexless vigor-ously than do CBA/Jmice(39,40).Whenspleen cellsfrom thesemicewerechallengedinparallel

with VSV, the stimulation was essentially the same. Since the C3H/HeJ mice were able to respondtothemitogenic signalof VSV butwere

notresponsive to LPS, we mayconclude that VSVwasnotcontaminated with LPS andthat VSVwasthe activemoiety.

Infectivity. The role of VSV infectivity in

lymphocyte activation was examined. The

ne-cessity of infectious virus for mitogenicity ap-pears to be virus specific, since HSV-induced lymphocyte activation requires infectious virus (20,30), but mitogenicity duetoinfluenza virus does not (6). The role of VSV infectivity in mitogenicity was examined in two ways. The

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firstwas by using UV-inactivated virus (Table

TABLE 5. Inhibitionof VSV-induced activation by rabbit anti-VS Vserum0

Mito- [IH]thymidineuptake±standarderror(cpm) %

In-

hibi-gen No NRS a-VSV tion

None 2,849±66

VSV 29,102±2,512 32,871±2,843 2,460±63 100I

None 4,329±308

VSV 34,525±3,043 32,690±174 2,227±163 100c

aCellswereculturedaspreviouslydescribed.VSVwas

in-cubatedwitha1:10 dilution ofheat-inactivatedserumat37°C

for 30 min and then diluted and added at 25 iLg/culture. Neitherserumpreparationinhibited[ 'H]thymidine incorpo-rationbyLPSorConA,nordidtheycausesignificant changes

inbackground. NRS, Nornalrabbitserum.

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a-VSV reducesincorporationtobackground levels.

TABLE 6. Activation of C3H/HeJspleen cellsby VSVa

['H]thymidineuptake+standarderror

Mito- Amt C3H/HeJ CBA/J

gen (ug/cul- lymphocytes lymphocytes

cpm SI cpm SI

None 1,991 113 0 1,835 128 0

ConA 0.06 58,093±5,608 29.2 60,498 3,617 33.0

LPS 50.0 2,517+244 1.3 40,215 1,771 21.9

VSV 1 2,405±248 1.2 2,949 271 1.6

5 7,093+285 3.6 7,241 688 3.9 10 10,178±227 5.1 9,690±439 5.2

25 9,383±647 4.7 11,230±365 6.1

50 7,213+697 3.6 6,360±112 3.5

aCells were cultured in microtiter wells for 48 h. This experimentisrepresentativeof fourseparateexperiments.

7). UV inactivation reduced the infectivity of

VSVgreaterthan9logstoless than1PFU/ml. However, when compared with untreated virus

from the same lot, the lymphocyte activation

elicitedwasonly reduced byapproximately 30%

atoptimal concentrations of VSV and essentially

unchanged at suboptimal concentrations,

sug-gestingthatinfectivity of VSVwas notrequired

forlymphocyte activation.

A second approach to this problem was

through theuseof DIparticles. DI-Oll contains

all the viralproteins andsomeviral nucleicacid,

butcannot direct virusbiosynthesis (23).

How-ever,because DIparticles do containallthe viral

proteins, theirpresentationtothecell is

essen-tially the same as an infectious virus particle.

When these particlesweretested for their ability

toactivatelymphocytes,itwasfound thatthey

werehighly stimulatoryforCBA/J spleen cells

(Table 8). Similar results were also seen with

C3H/HeJ lymphocytes (data not shown). In

fact,onthe basis ofmicrogramsperculture,the

TABLE 7. Activationofmousespleen cells by UV-inactivatedVSV0

Amt

[:3H]thymidine

uptake ±

stand-Amt,

arderror

Mitogen

(Ag/cul-ture) cpm SI

None 1,775+122 0

VSV 1 1,738± 132 1.0

10 4,993±461 2.8 25 9,859±684 5.6

50 13,480±728 7.6

UVVSVb 1 1,378±160 0.7

10 4,709 +205 2.7

25 6,447±315 3.6

50 9,968±285 5.6

aCBA/Jspleen

cells,

48-h microtiter

wells.

Asingle representativeexperiment is presented.

bVSVwasUV inactivatedwithagermicidal lamp (GeneralElectric)at5 cmfor15min.Infectivitywas

decreasedtoless than1PFU/ml.

TABLE 8. ActivationofCBA/Jspleencellsby

DI-olIa

[I3H]thymidineuptake ± standard Mitogen Amt_culture)

(urgl

error

cpm SI

None 2,307±376 0

VSV 1 2,834+67 1.2

5 7,052+530 3.1

10 9,377±811 4.0

25 7,632±627 3.3

DI-Oll 0.15 13,385±427 4.8

0.75 29,732+1,762 11.9

1.5 11,246± 1,142 4.8

3.6 15,734±1,378 6.8 Cultureconditionswere aspreviouslydescribed.

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lymphocyte stimulation by DI-Oll was greater

than that of the infectious VSV preparation to

which itwas compared. Thesetwo approaches

demonstratethatinfectivity isnot arequirement

for VSV-induced mitogenicity.

DISCUSSION

The data presented here demonstrate that

VSV ismitogenic formouseB-lymphocytes.The

stimulationof bothcellular DNAsynthesisand

blasttransformationaregreatest 48h after

cul-ture with the virus. Infectious virus is not

re-quiredforactivationof murinespleencells.

The VSV preparationsused in these

experi-ments contained no detectable contaminating

protein as determined by PAGE. Coomassie

blue-stainedgelscontainedonlythe fourmajor

proteins(L,G, N,andM)knowntoexist in VSV

(41). The NS protein does not resolve into a

separate bandonourgelsystem. Thepresence

of smallamountsofcontaminating material such

as serum proteins, cellular material, or lipids

which could bemitogenic but arenotdetected

byourstained gelsystem canbe ruled outby

ourexperimentswith influenzavirus,whichwas

propagated in tissue culture cells(MDBK) and

purified in exactly the same manner as VSV.

Since thesevirusesareenvelopedandwere

pre-pared similarly, any contaminating mitogen

wouldbeexpectedtobepresentin all

prepara-tions of virus. However, influenza virus is not

mitogenicin ourassay system.This result

sug-gests that the

mitogenic

activityobserved with

purified VSV is specific to that virus and not

duetosomecontaminatingmaterial. Inaddition,

VSV-induced lymphocyte activation was

in-hibited byincubating the virus with anti-VSV

serum,againindicatingspecificity.AlsoanLPS

contaminantwasruledoutbytheabilityof VSV

to activate LPS-unresponsive C3H/HeJ

lym-phocytes.Thesedata,inadditiontothose which

showednocellularproteincontamination of the

VSV preparations used in this study, support

ourhypothesisthat it is the VSV which is

stim-ulatoryformousespleencells.

The kinetics oflymphocyte activationbyVSV

aswellasthe shape ofthedose-response curve

suggestedto usthat VSVwasactivatingB-cells.

The maximalstimulationat 48 h was similar to

that seen with LPS and otherB-cell mitogens

(12, 13, 34, 39, 40), butdifferentfrom thatseen

withConA,aT-cellmitogen,where the optimal

response is seen after 72 h ofculture with

mito-gen (42). Also, the shape of the dose-response

curvegenerated by stimulation with VSV was oneinwhich there was a broad range of concen-trations which were stimulatory and there

ap-peared to bea plateauaround the optimal

re-sponse.Again,this is similartoB-cell mitogens (12, 13, 34, 39, 40), butcontrastswith the very

sharpplateaulesscurve seenincellsstimulated

by ConA. In both experimental approaches used

here, T-cell-depletedlymphocytes and lympho-cytes from nude mice, VSV was stimulatory.

ThelackofT-cells in both of thesesystems was

demonstratedbyalack of responsivenesstothe

T-cell mitogen, ConA, whereas theunseparated

or nu/+ lymphocytes were responsive to this

mitogen. In all the experiments, the

B-cell-en-richedpopulationwasasresponsiveasthe

nor-mal cells when SI valueswere compared, thus

confirming our hypothesis that VSV was a

B-cell activator.

The data presented here show that virus

in-fectivity isnotrequired for lymphocyte

activa-tionby VSV. Both UV-inactivated VSV and DI

particleswere able tostimulate mitogenesis in

murinespleencells. This result is similartothat

of Butchko et al. (6), who showed that

UV-inactivated influenza virions could induce

lym-phocytemitogenesis, andcontrastswith results

reported by Kirchneretal. (20) and Mochizuki

and co-workers(30), who demonstrated that

in-fectivity wasrequired for herpessimplex

virus-induced mitogenesis. That noninfectious VSV

can act as a mitogen for mouse lymphocytes

suggests that it may be a component of the

virion, such as the glycoprotein or some other

viral protein,which is the active moiety.

There-fore, we plan to examine the mitogenicity of

virions from which the glycoprotein has been

enzymatically removed, as well as the purified

glycoprotein,in ordertobettercharacterize the

activemoiety.

As a mitogen, VSV has many of the same

characteristics as the other viral mitogens.

Herpes simplex virus has been shown to be

maximally

stimulatoryafter48 (30)or 72 (20)h

ofculture with the cells. Influenza viruses of the

H2N2subtype induceanoptimal mitogenic

re-sponse after being in culture with the cells for

48h(6). Furthermore,thelevels of stimulation

obtained are similar when SI values are

com-pared(6, 20,30).Inallcases,stimulation is lower

than that for ConAorLPS. Mochizukietal.(30)

and Kirchneretal. (20)havedemonstrated that

herpessimplexvirus isaB-lymphocytemitogen,

whereas influenza virus stimulates mitogenesis

inboth B- andT-cells(6). Therefore itappears

thatVSV, like other viral mitogens,stimulates

early

[3H]thymidine

incorporationatlevels

gen-erally lower than those ofLPS or ConA.

Fur-thermore, viruses canactivate eitherB-cellsin

theabsenceof T-cells(20, 30)orboth B- and

T-cell-enriched lymphocyte subpopulations (6).

The nonspecific effect of VSV on T-cells and

macrophages requires further study. Since

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cells may be infected by VSV whichproliferates

and lyses the cells when theyare exogenously

activated byamitogensuchasConA(4),itmay

be that infectious VSV willnotbestimulatory

for T-cells but noninfectious VSV or apurified

componentwill bestimulatory. Additionally,our

results with WSN influenza virus (HON1)arein

agreementwith the data of Butchko etal. (6),

who showed that onlyinfluenza viruses of the

H2N2 subtype could act as lymphocyte

mito-gens.

For many years it has been recognized that

certain substances,such asConA,

phytohemag-glutinin, and bacterialLPS, cannonspecifically

activatelymphocytestoblastogenesis and

mit-ogenesis (33, 41). Recently, the list of

lympho-cyte mitogens hasexpandedtoinclude not only

plantlectins andLPS,butalsooutermembrane

components of both gram-positive and

gram-negative bacteria, mycobacteria, mycoplasma,

andyeast (1, 5, 11-13, 18, 32, 36, 38, 40).

Fur-thermore,low-molecular-weight chemicals such

as2-mercaptoethanol orperiodate orpolymers

such as polyinosinic-polycytidylic acid have

been shown to be stimulatory to lymphocytes

(14, 19, 37). We have now shown that VSV is

mitogenic for mouse lymphocytes. Preliminary

studies indicate that theparamyxovirus,simian

virus 5, and the togavirus,Sindbisvirus, are also

mitogenic for murine spleen cells. Therefore,

viruses as well as bacteria and their products

and plant lectins can nonspecifically activate

cells.

The biologicalimportance of thistype of

ac-tivation can be speculated on; perhaps it is a

mechanism for expanding the

immunocompe-tency of the organism, since the immune

re-sponse ofgermfree animals is qualitatively

dif-ferent from that of conventional animals.

An-otherpossibility is that nonspecific mitogenesis

isimportantinthepathogenic mechanism of the

virus as suggested by Butchko et al. (6). This is suggested because a large number of viruses can

grow inmitogen-activated lymphocytesbut not

in resting cells (3, 4, 21, 44, 45). Furthermore,

activatedlymphocytes can express endogenous

type C RNAviruses (15, 32, 35) which are latent

innormalcells.In short,although the repertoire

oflymphocyte mitogens has grown, the question

of thein vivobiologicalrelevance of this activity

hasyetto beexamined.

ACKNOWLEDGMENTS

We thankL.A. Caliguiri, D. A. Lawrence, M.Schron, and L. S.Sturmanforhelpful discussions, Marti B. McSharry for technicalassistance, and KathyCavanagh for help in prepar-ingthismanuscript.

This workwassupported in part by the New York State HealthResearchCouncil grant 1572. G. G.-S. is a National

Needspostdoctoralfellow and issupported byNational Sci-enceFoundationgrant SP179-14819.

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