Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

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Control

of

Interferon

Synthesis:

Effect of

Diethyl-aminoethyl-Dextran

on

Induction

by

Polyinosinic-Polycytidylic Acid

JAN VILCEK, SANDRA L. BARMAK, AND EDWARD A. HAVELL

Departmentof Microbiology, New York UniversitySchool of Medicine, New York, New York 10016

Received for publication24July1972

Interferonproductionincultures ofrabbit kidney cells (RKC) stimulated with 10

to250,igofpolyinosinic-polycytidylic acid (polyI polyC) permlpeakedat3to4

hraftertheexposureofcellstoinducerand rapidly declined thereafter. On the other hand, RKC stimulatedwithpoly I poly C (10or2 jig/ml) inthepresenceof

diethyl-aminoethyl (DEAE)-dextran (100or20 jig/ml,respectively) producedaprotracted

interferonresponse, with the release of interferon continuing for over24 hr. The

kinetics of interferonproduction inRKC stimulated with lower concentrations of

the mixture ofpolyI polyCandDEAE-dextranwere similartotheresponse

pro-ducedbypolyI-polyCalone(10to250,ug/ml). Only theresponsesthatterminated earlywereparadoxically enhanced bytreatmentwithlowdoses ofactinomycinD or

withcycloheximide.Cellsstimulated with 50 Mgof poly I polyC/mni showed

hypo-responsiveness to a secondinterferoninduction with poly I-poly Cwhen restimu-lated 7 hrafter primaryinduction. Thishyporesponsiveness could beovercome by

restimulatingwithhigherconcentrations ofthe polyI polyC-DEAE-dextran

com-plex. Theresults are compatible with thehypothesis that the early termination of

interferonproductionandhyporesponsivenesstorepeated inductionwith polyI* poly

C areduetoa cellularrepressor exerting negative controlon interferonsynthesis,

and that the increased cellular uptakeofpoly I -poly C in thepresence of

DEAE-dextranmay effectively neutralizethe repressor. These results also suggested that the often observed different kineticsandthevaried effects of inhibitors ofribonucleic

acidorprotein synthesisoninterferonresponsesin variouscellsand in cells

stimu-lated with different inducers (suchaswith virusesascompared withpolynucleotides)

need notimply the existenceoffundamentally different mechanisms ofinterferon production.

Polyinosinic-polycytidylic acid (polyI-poly C)-induced interferon

production

in rabbit kidney

cells(RKC)hasbeenwidely

employed

asamodel

system for the study of cellular control

mecha-nismsoperating in interferon

synthesis.

The

sys-tem ischaracterized byarapid synthesis and

re-lease ofinterferon, whichpeaksin3 to4hrafter

the exposure of cells to the inducer. Interferon

production declines rapidly thereafter; concur-rently with the cessation of the interferon

re-sponse, cells develop a state of hyporesponsive-ness to

repeated

induction (3, 14,

15).

Judicioustreatmentof induced cells withsome

inhibitors of ribonucleic acid (RNA) or protein synthesishas beenfoundtointerferewith the

nor-mal shut-off process of interferon synthesis and

to result in a

paradoxical

enhancement of inter-feronproduction.These resultssuggestedthatthe

earlytermination ofinterferon

production

is due

to the synthesis of a regulatory protein (repres-sor). Theparadoxical enhancement ofinterferon production by some metabolic inhibitors is

thoughtto bethe result ofa

preferential

inhibi-tion of repressorsynthesisoverinterferon

synthe-sis

(12,

14).

Similar experiments with some other types of

cell culture, such as with diploid human cell strains (8, 10; E. A. Havell and J. Vilcek, Bac-teriol. Proc., p. 195, 1971), largely upheldthe

re-sults and the conclusions of the experiments in RKC. However,poly

I.

polyC-induced interferon

production in cultures ofmouse Lcells (11,

14)

and chickembryo cells(9) couldnotbe enhanced

bytreatmentwith metabolicinhibitors, seemingly

suggesting thatthecontrolof interferon

synthesis

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CONTROL OF INTERFERON SYNTHESIS

in those lattercells is somehow different from the controlexertedin RKC.

The present study wasinspired by the fortuitous observation that interferon production in RKC stimulated by the complex of poly I- poly C and

diethylaminoethyl (DEAE)-dextran neither dis-played the early shut-offphenomenonnor could it be paradoxically enhanced by treatment with metabolicinhibitors. Further experiments offered anexplanation of theseobservations that is fully compatible with the hypotheses advanced earlier onthemechanism ofinterferon induction by

poly-nucleotides in RKC(lOa, 14, 16). Moreover, this study suggested that the differences in the re-sponse of poly I-poly C-induced interferon

pro-ductiontometabolicinhibitors, such as observed between RKC and some other types of cells, may not necessarily be due to fundamental

dis-similarities in the underlyingmechanisms of the control of interferon synthesis, and could be causedby less essential differences in the mode of

deliveryanduptake ofthe inducer.

MATERIALS AND METHODS

Cell cultures. Cultures of RKC were prepared by trypsinization of kidneys from 2- to 4-week-old rabbits.Allexperiments were done in secondary RKC cultures grown to confluency in 60-mm petri dishes (Falcon Plastics, Los Angeles, Calif.) at 36 C in a

humidified incubator provided with 5% CO2. Eagle

minimal essential medium (MEM) with 10% heated fetalcalfserum or2% gammaglobulin-freefetalcalf

serum was employed as growth medium or

main-tenancemedium, respectively.Alltissueculture media andserawerepurchasedfromGrandIslandBiological

Co., GrandIsland, N.Y.

Interferon induction. RKC cultures were washed

oncewithEarlebalanced salt solution(EBSS)and in-cubatedat36Cwith theappropriateconcentration of

poly

I.-poly

C (suppliedby theAntiviral Substances Program, National Institutes of Health, Bethesda,

Md.) orof the mixture ofpolyI-poly C and DEAE-dextran (molecular weight, 2 X 106, Pharmacia, Uppsala,Sweden),diluted inwarm(37C)

phosphate-bufferedsaline (PBS; 0.13 MNaCl, 7 mmphosphate, 0.9mimCaC12,and 0.5 mMMgCl2-6H20; pH 7.4). The mixture of poly I-poly C (10 sg/ml) and DEAE-dextran(100,g/ml) in PBSwasfirstincubated for 30 minat37Cinawaterbath; furtherdilutions of this mixture were prepared in warm (37 C) PBS as

re-quired. RKC were incubated with 1 ml of the ap-propriate inducer for 30 or 60 min, as indicated, andwerethoroughlywashedwith EBSS thereafter.

Interferon titrations. A modification of the

semi-micromethod described by Armstrong (1) was

em-ployed.IndividualwellsofMicro Test II tissueculture plates (FalconPlastics,LosAngeles,Calif.)werefirst

filledwith50Aliters of serum-free MEM, bufferedwith

0.155%sodiumbicarbonate,6.6mm N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid, and 3.3 mm

tricine to pH 7.6, and containing 100 units ofpenicillin, 100jugofstreptomycin, 1001Agofgentamicin, and 2.5 ,ug ofFungizone(amphotericin B) per ml. Portions of 25 or 50jliters of the interferon samples were then added to the first well. Series of two- or threefold dilu-tions were then prepareddirectly in the microplates by serially transferring 25 or 50;&literswith anEppendorf automaticmicropipette (Brinkmann Instruments Inc., Westbury, N.Y.). Each well was then seeded with 40,000 RKC(obtainedbytrypsinizationofprimary or secondarycultures) in100uliters of MEM containing buffers and antibiotics as indicated above, plus7.5% heated gammaglobulin-free fetal calf serum. After 18 hr ofincubation at 36 C in an atmosphere of 5% C02, each well was inoculated with 1,000plaque-forming

units (PFU) of the Indiana-type vesicular stomatitis virus in 50 ,Aliters of serum-free MEM with additions asabove. Severalwells on eachplate served as virus controls and cell controls.

Thetitrationswerescoredmicroscopically24to 48 hr after virus inoculation, when the virus controls showed

completedestruction by the virus. Thehighestdilution of thetitratedsamplecausingan atleast 50% protec-tion ofcells was considered the end point. In most

experiments,eachsample was titrated in duplicate. An internal laboratory rabbit interferon standard was

included with each titration. This internal standard had been calibrated against areference rabbit inter-feron standard prepared by thelaboratoryof Monto Ho, University of Pittsburgh, Pittsburgh, Pa., and received from the Reference Reagents Branch, Na-tional Institutes of Health, Bethesda, Md. The calibra-tion of the internal standard against the reference standard was done by the plaque assay (16) with the

useof2ml of each interferon dilutionperplate. All

interferontiters were corrected tothis standard. Thus,

although the actual volume of diluted interferon samples employed in the microplate assay was 100

,Iiters, all interferon yields are expressed per 2 ml.

Disregarding the volume used in the assay, the

sen-sitivityof themicroplateassay wasabouthalf that of the plaque assay. The internal laboratory standard calibratedtocontain 1,000referenceunits/2ml in the

plaque assayaveraged about 500 units in the micro-plate assay.

Chemicals. Cycloheximide (Acti-dione) was ob-tained from TheUpjohnCo.,Kalamazoo, Mich.,and actinomycin DwaspurchasedfromCalbiochem, Los

Angeles,Calif.

RESULTS

Kinetics of interferon production in cultures stimulated with various concentrations of poly

I-poly C and of the poly I -poly C-DEAE-dextran mixture. Interferonproduction in RKC cultures stimulated with various concentrations of poly

I-polyC,rangingfrom 10 to 250 ,ug/ml, peaked

at 3 to 4hr after theexposure of cells to the in-ducer and declined rapidly thereafter. Although

RKCstimulated with higher doses ofpolyI-poly Cproducedmoreinterferon,thekinetics of

inter-feron release werenot strikingly different.

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616 VILCEK, BARMA

ever,cultures of thesamebatchof RKCexposed

toamixture of poly I * poly C and DEAE-dextran (10 and 100

Ag/ml,

respectively) produced a

markedly different type ofresponse: the rateof interferonproduction continuedto riseuntil5 hr after induction andremained high throughout the duration of the experiment (Fig. 1).

Todeterminewhether theprotracted interferon

response wascharacteristic of thepolyI-poly

C-DEAE-dextranmixture, or whether it was

essen-tiallyafunction of the dose ofinducertaken up by cells, RKC werestimulated withvarious dilu-tionsof themixtureofpolyI*polyCand

DEAE-dextran. A mixture of 10 and 100 jug of poly

I*poly C andDEAE-dextran perml,respectively, producedaresponsesimilartotheoneobserved with thesamedoseofinducer inthepreceding

ex-periment.Afivetimeslower concentration of this

mixture also produced a protracted response, but

the rate ofdecline of interferon

production

was faster than with the

higher

dose.Cellsstimulated witha25-fold dilution of the

original poly

I*

poly

C-DEAE-dextran

mixture

produced

a response

whichwasquite similartothat

produced by poly

I-poly C uncomplexed with DEAE-dextran, in

that itreached a peak between 3 and4hr after induction andrapidly

declined

thereafter

(Fig.

2).

That theprotracted interferonresponse was not

theresult ofadirect action of DEAE-dextranper se on RKC cultures was shown by the results summarized in Fig. 3. RKC cultures were first treatedwithpolyI *polyCfor1hr,and thenwere

washed and incubated for 30 min with various concentrations of DEAE-dextran alone. Such

separate treatment with DEAE-dextranfailed to

alter the kinetics of interferon

production

charac-teristicof polyI*polyCalone.

PolyI *poly C is knowntoformacomplexwith

1 2 3 4 56 7

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FIG. 1. Kineticsof interferon production inrabbit kidneycells stimulated with various concentrations ofpoly

I-polyCorwithamixtureofpolyI-poly C and DEAE-dextran.Fourgroupsof cultureswere incubated for Ihr

withtheindicatedconcentrationsof inducer,washedfivetimesthereafter, and replenishedwith 2ml ofmaintenance

medium.Atthe indicated intervals,fluidswerecollected,andthe cultureswerewashedoncewith EBSS antd imme-diately replenishedwithwarm (37C) freshmaintenancemedium. Interferontitersweredetermined inthepooled

fluids from individualgroups.If the interval betweentwosamplingswas morethan Ihr, the determined interferon

yield (units/2 ml)wasdividedbythe number of hoursthat hadelapsedfrom the collection of the precedingsample.

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FIG. 2.Kineticsofinterferonproduction in rabbit kidney cellsstimulated withvarious concentrations ofthe

poly I-polyC-DEAE-dextran mixture. Fourgroupsofcultureswereincubatedfor30 min with the indicated

con-centrationsofthepolyI-poly C-DEAE-dextranmixtureorforI hrwithpoly I-poly C(250 jig/ml). Other

pro-cedureswere asintheexperimentshowni inFig.1.

DEAE-dextran (4). The formation of such a

complex isapparently responsible forthegreatly

enhanced cellularuptakeofpoly I-polyCinthe presenceof DEAE-dextran(2, 5). Theprotracted interferonproductioninRKCculturesstimulated

with high doses of the poly I-poly C-DEAE-dextran mixture is thus likely to be due to the moreefficientuptake ofthe inducerbycells.

Effects ofactinomycinD and cycloheximide on

interferonproduction stimulated by polyI-poly C

or poly I poly C-DEAE-dextran mixtures. RKC

cultureswerestimulatedeitherwithpolyI* polyC

(50

,g/ml)

or with the mixture ofpolyI polyC

and DEAE-dextran (10 and 100 ,ug/ml,

respec-tively). Immediately after a 30-min incubation with theinducer,groupsof cultures weretreated with various concentrations ofactinomycinDor

cycloheximide. Theyieldof interferonwas

deter-mined in culture fluids collected 24 hr after

induc-tion.Actinomycin D,atconcentrations of0.1and 0.3

Ag/ml,

increased theyieldof interferon

stimu-lated withpolyI polyCalone. No such increase

wasnotedin RKCstimulated with themixtureof

polyI-polyCandDEAE-dextran treated with the same doses of actinomycin D. An even more

significant difference was found in the effects of

cycloheximide on the two interferon responses:

the interferon yield in cultures stimulated with polyI.polyCalonewasincreased about fivefold,

but itwas decreased in cells exposedtothe poly

I-poly C-DEAE-dextranmixture(Table 1).

Cultures stimulated with decreasing

concentra-tions ofthe polyI-polyC-DEAE-dextran mixture

produced decreasing yields ofinterferon, but at

the sametime interferon production became

in-creasingly sensitive to paradoxical enhancement

byactinomycinDorcycloheximide (Table 2).

Hyporesponsivenesstorepeatedinterferon induc-tionin cultures restimulated with various dosesof

poly I poly C and of the poly I poly C-DEAE-dextran mixture. It was shown that RKC

stimu-lated with poly I poly C (50 jug/ml) produced

decreased interferon yields when restimulated with thesamedoseof inducer. Such hyporespon-160 *

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FIG. 3. Effect ofDEAE-dextran,addedtorabbitkidneycellsafterinduction withpolyI-poly C, onthe kinetics

of interferon production.Cultureswereincubatedfor Ihr withpoly I-polyC(250 pAg/ml)and washedfivetimes

thereafter. Threegroups werethen treatedfor 30min with the indicatedconcentrationsofDEAE-dextran while

onegroup receivednofurthertreatment.Otherprocedureswere asin theexperimentshown inFig.1.

TABLE 1. Effectsof actinomycinDandcycloheximideoninterferon productionstimulated with

poly I-poly Cor amixtureofpoly I-poly Cand DEAE-dextran Interferonyieldb

Treatmenta PolyI-poly Cc Poly I-poly C +DEAE-dextrand

Units/2ml Percent' Units/2ml Percent' None... 87 (100) 8,100 (100) Actinomycin D,0.1

,pg/ml

... 218 250 8,100 100

ActinomycinD,

0.3,pg/ml

... 150 172 4,050 50

Actinomycin D,

1.0,pg/ml

... 16 18 2,025 25

Cycloheximide, 1.0,pg/ml. ... 486 558 4,050 50

Cycloheximide, 10.0

pg/ml

... ... 486 558 1,620 20

aThe indicated concentrations ofinhibitors were added immediately after a 30-min exposure to

inducer. ActinomycinDwasleft on the cultures for 30 min; thereafter, thecultures were washed twice and replenished with drug-free maintenance medium. Cycloheximide was maintained in the cultures throughout the duration ofexperiment.

bInculture fluids collected 24 hr after exposure to inducer. Fluids from cycloheximide-treated

cul-tures weredialyzed priortointerferon assay.

cCultureswereincubated with 50 ug of polyI-polyC/ml for 30 min and washed five times thereafter.

dCultureswereincubated with a mixture of poly I- polyC(10,g/ml)andDEAE-dextran (100,g/ml)

for 30 min and washed five times thereafter. e Percentageof control.

jg

/ml

SYMBOL

DEAE-DEXTRAN

*

0

a 4

D

20

100 E

cm

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D

0

I

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TABLE 2. Effects of actinomycin D andcycloheximide onlinterferon production stimulated with various concentrations of poly I- poly C andDEAE-dextran

Interferonyieldb Inducer(.g/ml)a

ActinomycinDc

Cycloheximide,10Ag/mld

Polyl-polC dextran Control 0.3

ug/ml

0.1

jug/ml

PolyI-polyC DEAE- (units/2ml) 0. /m01pgm

dextran

Units/2ml Percent' Units/2 ml Percent Units/2 ml Percent'

10 100 8,747 2,916 33 4,374 50 1,458 16

2 20 1,458 972 66 972 66 1,458 100

0.4 4 162 972 600 2,187 1,345 2,916 1,800

0.08 0.8 18 162 900 1,458 8,100 2,916 16,200

a

Cultures

wereincubated for 30 min with a mixture of the indicated concentrations ofpoly I- poly C

and DEAE-dextran and washed four times thereafter. b Inculture fluids collected 24 hr after exposure to inducer.

cTreated with the indicated concentrations for 30 min immediately after the exposure to inducer.

Thereafter, thecultureswere washed twiceandreplenished with drug-free maintenance medium.

dCycloheximide was added immediately after the 30-min exposure of cultures to inducer and was

maintained in the culture fluids throughout the duration of the experiment. Prior to titrating the inter-feron content, the harvested fluids were dialyzed to remove the cycloheximide.

ePercentage of control.

siveness was shown todevelop concurrently with thecessation ofinterferon production and to last forabout48 hrafterthe first exposureofcellsto the inducer (3, 15; S. L. Barmak, Ph.D. thesis, NewYork University,1972).

Intheexperimentsummarized in Table3,RKC

werefirststimulated withpoly I polyC (50,ug/

ml)and wererestimulated7hrlaterwithvarious doses of poly I-polyC either complexed or un-complexed with DEAE-dextran. The interferon yields obtained on the second stimulation were

compared with yields from control, previously unstimulated culturesexposedtothe samedoses of inducer. The

degree

of hyporesponsiveness decreased with increasing doses of

poly

I-polyC

used for restimulation. Hyporesponsiveness was notdetectedincultures restimulated with2or 10

jig

of

poly

I

-poly

C per ml inthe presence ofa 10-foldexcessofDEAE-dextran.

DISCUSSION

The kinetics ofinterferonproductionin RKC

stimulated withthepolyI-poly C-DEAE-dextran complex weredifferent from those observed with poly I-

poly

C alone. Stimulation with various concentrations of the

polynucleotide

uncom-plexed with DEAE-dextran produced an inter-feron response thatpeakedat3to4hrafter

induc-tion and declined quiterapidlythereafter. Onthe other hand, cells stimulated with 2 or 10Ag of

polyI

poly

C per ml in the presenceofa 10-fold

excess of DEAE-dextran

produced

a much

pro-tracted response, with interferon

being

released

intothe culture fluid formorethan 24 hr.

Asnoted earlier (12,14),interferon

production

stimulated withpoly I poly C was paradoxically enhanced by treatment with certain concentra-tions of actinomycin D or by incubating stimu-lated cultures in the presence of cycloheximide.

Onthe other hand, the production ofinterferon stimulated with high concentrations of the poly

I-polyC-DEAE-dextran complex was suppressed by similar treatment withactinomycinDor cyclo-heximide.

These findings raised the question of whether interferon induction by the polyI *poly C-DEAE-dextrancomplexwasperhaps qualitatively

differ-entfromtheinterferon response elicitedby poly

I *polyCalone.Furtherexperimentsrevealedthat

interferon production in response to lower

con-centrations ofthe

poly

I-poly C-DEAE-dextran complex showed similar characteristics to inter-feronproduction after thestimulation with poly

I-polyCalone: it ceased early after inductionand it could be paradoxicallyenhancedbytreatment

withactinomycinD orcycloheximide.

The findings supportthe previously suggested notionthat arelationshipexists among theearly

shut-offof interferon

production,

theparadoxical

enhancement ofinterferonsynthesisbymetabolic inhibitors, and the hyporesponsiveness to re-peated interferon induction by

poly

I

poly

C

(15).Theearlycessation of interferonproduction

has been attributed to a labile repressorprotein

that exerts a negative controlon interferon

syn-thesis.Theparadoxicalenhancement of interferon

productionby metabolicinhibitors is the result of

protractedreleaseof interferonand is believedto

be duetopreferentialinhibition of repressor syn-thesis over interferon synthesis (12, 14).

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TABLE3. Effect of stimulation with various concen-trations of poly I-poly C and of the polyI-poly

C-DEAE-dextran mixture on hyporesponsive-ness to repeated interferon induction

Inducer(ug/ml)" Interferonyieldb Restimulated Control culturesd PolyI. polyC DEAE- culturesc_________

dextran (units/2ml)

Units/2 ml Percente

250 0 192 128 66

50 0 144 80 55

10 0 72 20 27

10 100 2,880 3,824 132 2 20 1,440 1,424 98

0.4 4 576 128 22

0.08 0.8 288 32 11

a

Cultures

were incubated for 1 hr witha

mix-ture of the indicated concentrations of poly

I.polyCand DEAE-dextran and washed fivetimes thereafter.

IInculture fluids collected 24 hr after exposure to inducer.

cAt 7 hr prior to stimulation, these cultures

were incubated with PBS for 1 hr, washed, and then incubated in maintenance medium until

stimulation.

dAt 7 hrpriorto restimulation, thesecultures

were incubated with poly I poly C (50 ,ug/ml)

for 1 hr, washed three times, and replenished withmaintenancemedium;6 hrlater,thecultures

werewashedtwice and restimulated by incubating with theappropriateinducerfor1hr. Thereafter, the cultures were washed four times and replen-ished with maintenance medium. Culture fluids collected7hrafterprimary stimulationcontained 192 units of interferon per 2 ml. The interferon

yields givenare aftersubtraction of the residual

amount of interferon (16units/2 ml)produced by

control unrestimulated cultures during the same experimentalperiod.

e Percentageof control.

responsiveness to repeated induction by poly I-poly C, which appears concurrently with the

cessation of interferon production (15), is also

likely to be attributable to the action of this repressor. The repressorapparently controls inter-feronproduction by suppressing itssynthesisat a

post-transcriptional level, mostlikely bydirectly

bindingtotheinterferon messenger RNA (16). A

similarmechanism was earlier implicated in the

control of steroidhormone-inducedenzyme syn-thesis in animalcells(13).

Ithasbeensuggested that the first step in

inter-feron induction by polynucleotides involves the

binding and neutralization ofthe repressor pro-tein by the inducer (lOa). According to thisas yetspeculative notion,a basallevel ofrepressor

is thought to be continuously synthesized in

uninduced cells; the rate of repressor synthesis would be stepped up after interferon induction

andtheincreasedrepressor concentration would inturn resultin the cessationof interferon syn-thesis and hyporesponsiveness to repeated in-duction.

The observations made in this study could be explained by the increased cellular uptake of inducer, resulting in a more efficient binding of

therepressor incells stimulated with higher con-centrations of the poly I-poly C-DEAE-dextran

complex. Efficient neutralization ofthe repressor

could account for the lack of theearly shut-off, as well as for the failure to increase interferon

production by treatment with metabolic inhibi-tors. As long as the cellular concentration of

inducer is sufficient to neutralize the repressor,

interferon synthesis could continue,and inhibition of repressor synthesis by metabolic inhibitors would not be expected to promote interferon

production. The overcoming of

hyporesponsive-ness to repeated induction by higher

concentra-tionsofthepoly I. poly C-DEAE-dextran complex

could be explained on the same basis: by the

uptake ofenough inducer to neutralize the in-creased concentrationofrepressorbelieved to be present inhyporesponsive cells.

Wehave alsoconsidereda somewhatdifferent explanation forthedissimilarresponsesproduced by poly I poly C or low concentrations ofthe

poly

I.poly C-DEAE-dextran mixtures as

com-paredtothe responseobtained with high doses of

thecomplexedinducer. It was shown earlier that

primarymousekidney cell culturesarecomposed ofheterogeneous cell populationswhichdifferin

theirresponse to stimulation with poly I polyC orNewcastledisease virus(11).It cannotbe ruled outthat RKC culturesmay also havecontained heterogeneous cellpopulations, with one type

of

cellsthat could be stimulatedtoproducetheearly

response, andanother celltype characteristically producing a later and protracted interferon re-sponse that could be stimulated only with high doses ofthepolyI-poly C-DEAE-dextran

com-plex. However, experiments similar to the ones

describedinthis paper,performed in culturesofa

serially passaged human diploid cell strainandof

acontinuous line of RKC (RK 13)which are not

composed of markedlyheterogeneous cell

popula-tions, yieldedresults quite comparable to those in RKC (E. A. Havell, L. Mozes, and J. Vildek, unpublisheddata). Thus, cell heterogeneity does not seem to be a likely basis for the observed

differencesin theinterferonresponses.

That the presence of DEAE-dextran greatly

enhances therateof cellular uptakeof poly I-poly Cwasdemonstratedby other investigators (2, 5).

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Whether the polycation actually increases the amountof inducer that penetrates across the cell membrane or only increases its rate of attachment to thecell surface has not been determined. It is also not clear what role, if any, the increased

resistance of the poly I-poly C-DEAE-dextran

complex to nuclease degradation (4, 6) plays in

promoting induceruptake and interferon

produc-tion.

Earlier studies failed to demonstrate paradoxi-calenhancement of polyI polyC-induced inter-feron production by inhibitors ofRNA orprotein synthesis in cultures ofL cells (11, 14) or chick embryo cells (9), and it was concluded that the control of interferon production in these cells is probably different from the control in RKC.

However, asthese studiesemployed high

concen-trations of the poly I-poly C-DEAE-dextran complex, the differences may, in fact, not have

been due to fundamental dissimilarities in the control of interferon synthesis but, rather, to

different conditions of induction. Ourresultsalso suggest thattheobserved differencesin the kinet-ics, andinthedegree of suppression or

enhance-ment by various metabolic inhibitors, of the interferon responses produced by viruses or by polyI polyC (7, 9, 15, 17) neednotimplythat

fundamentally different mechanisms operate in

interferon production stimulated by viruses or isolatedpolynucleotides.

ACKNOWLEDGMENTS

Wethank Fermina Varacalli and JuanCasanova for skilled technicalassistance.

This investigation was supported by Public Health Service grant AI-07057 and contract NIH-NIAID-70-2169 from the National Institute ofAllergyand InfectiousDiseases andby a

grant from the IrwinStrassburgerMemorialMedical Foundation. J. V.istherecipient ofPublic Health Service Career Develop-mentaward 5-K4-AI-38784. S. L. B. wassupported byPublic HealthServicetraininggrantGM-01290.

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