effect of prostaglandins and cyclic adenosine 3',5'- monophosphate

Vol. 27, No. 1INFECTION AND IMMUNITY, Jan. 1980, p. 158-1670019-9567/80/01-0158/10$02.00/0

Effect of Prostaglandins and Cyclic Adenosine 3',5'-Monophosphate Modulators on Herpes Simplex Virus Growth

and Interferon Response in Human CellsKENNETH F. TROFATTER, JR., AND CHARLES A. DANIELS*

Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

Mechanisms whereby prostaglandins and other cyclic adenosine 3',5'-mono-phosphate (cAMP) modulators might enhance the growth of herpes simplex virus(HSV) in human skin fibroblasts were explored. Prostaglandins Al, B1, El, E2,and F2a, as well as isoproterenol, imidazole, carbamylcholine, and dibutyrylcAMP had no effect on HSV growth. On the other hand, the phosphodiesteraseinhibitors 1-methyl-3-isobutylxanthine and theophylline delayed the growth,suppressed the cell-to-cell spread, but inhibited neither the adsorption nor thepenetration of the virus. Although none of the cAMP-elevating reagents directlyenhanced HSV growth, they were found to inhibit dose dependently the antiviralaction of both type I and HSV antigen-induced human interferon preparations.Furthermore, these reagents suppressed the production of HSV antigen-inducedinterferon by immune human mononuclear leukocytes. These data support thehypothesis that prostaglandin elaboration in vivo could contribute to exacerba-tions of HSV infections by compromising the host's interferon defense system.

Primary infections with herpes simplex virus(HSV) occur in 30 to 100% of the population,depending on the socioeconomic group surveyed(34). In the course of a primary infection, HSVcommonly invades the regional sensory ganglia(3, 13). Evidence indicates that HSV, latent inthe ganglionic neurons, is the endogenous sourceof virus for periodic recrudescent herpetic dis-ease (44).The events leading to reactivation of the la-

tent virus and the establishment of a currentinfection are poorly understood; however, recentstudies suggest that prostaglandins (PG) mayplay an important role (4, 21, 46). Most, if notall, of the factors that precipitate recrudescentherpes in patients, exposure to ultraviolet light(50), trauma (6, 24), and fever (26, 49), are as-sociated with a local or systemic increase in PG(1, 7, 11, 12, 15, 29, 33, 51). Recognition of thisrelationship led Hill and Blyth to propose thatPG enhance the local growth of HSV (22). Insupport of their hypothesis, they later showedthat PGE2, injected into mice at previous inoc-ulation sites, induced exacerbations of the latentinfection as frequently as ultraviolet light (4) ormild trauma (23). Recently they have shownthat PGE2 and PGF2a increase HSV growth inVero cells (21), and others have demonstratedthat PG inhibitors suppress the growth of thevirus in mouse Ly cells (35).The mechanisms by which PG might enhance

the local replication of HSV in human cells havenot been adequately studied. PGE2 is known to

increase intracellular levels of cyclic adenosine3',5'-monophosphate (cAMP) (34), and reagentsthat elevate levels of this cyclic nucleotide incells have been shown to stimulate the replica-tion of human cytomegalovirus (49), ribonucleicacid tumor viruses (42, 50), and adenovirus (26)under certain conditions. Conceivably, HSVgrowth could be enhanced by PG via elevationof cAMP in infected human cells.

Alternatively, PG could indirectly cause anincrease in local HSV growth by suppressingdefense mechanisms that ordinarily curtail virusreplication. Interferon (IF) is one such defensemechanism that has been shown to limit HSVinfections both in vitro (28) and in vivo (16, 17).At least two types of IF are produced in responseto HSV or the virus antigen. Type I IF is syn-thesized by a variety of human cells (14, 18, 47);type II IF is produced by specifically immune Tlymphocytes in the presence of autologous mac-rophages (18). In humans, these cell populationshave been shown to be susceptible to the cAMP-elevating effects of PG (41), but little is knownconcerning the effect PG have on the ability ofthe cells to synthesize or respond to IF. If PGeither suppressed the production or inhibitedthe action of IF, HSV growth could be enhancedlocally and an exacerbation might result.

In this study, we determined the effects PGand other cAMP-modulating reagents had on:(i) the growth ofHSV in a human skin fibroblastcell line, (ii) the ability of IF to protect humancells from an HSV infection, and (iii) the syn-

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PROSTAGLANDINS AND GROWTH OF HSV 159

thesis of IF in immune human mononuclearleukocytes (MNL). We found that PG did notstimulate HSV replication, but did have a det-rimental effect on the action and production ofIF in human cells.

(This paper was presented in part at the 68thAnnual Meeting of the International Academyof Pathology, 5-9 March 1979, San Francisco,Calif.)

MATERIALS AND METHODSVirus, cells, and sera. Type 1 HSV, strain CHR-

HSV-3, was passaged in human embryonic skin fibro-blasts (HuF) (8) and was assayed as plaque-formingunits (PFU) on primary rabbit kidney (PRK) cells asdescribed previously (20). HuF were grown and allvirus dilutions were done in Eagle minimal essentialmedium (MEM) containing penicillin G (100 U/ml),streptomycin (50 jug/ml), and 10% (vol/vol) fetal bo-vine serum (EMFBS). Human HSV-immune serum(anti-HSV) was collected from a patient with recurrentherpes labialis, and after heating at 560C for 30 min,the serum had a neutralization titer of 1:256 (19). (Allsera were heat inactivated at 560C for 30 min beforeuse.) Human AB nonimmune serum was obtainedfrom a person with no history of HSV infection, anda 1:2 dilution of this serum was without neutralizingactivity. Preparation ofHSV antigen (HSV-Ag) for IFassays was based on the method of Rasmussen et al.(38). Briefly, EMFBS containing 1066 PFU of HSVper ml or cytoplasmic extracts from uninfected HuF(control) were heated at 560C for 2 h. After heatinactivation, no infectious virus was present in theundiluted materials as determined by plaque assay onPRK cells.

Reagents. The following compounds were obtainedfrom Sigma Chemical Co. (St. Louis, Mo.): (i) prosta-glandins A,, B., E1, E2, and F2a (all were synthetic);(ii) N6,02'-dibutyryl adenosine 3',5'-cyclic monophos-phoric acid (db-cAMP); (iii) 1-methyl-3-isobutylxan-thine (MIX), and 1,3-dimethylxanthine (theophyl-line); (iv) isoproterenol sulfate; (v) carbamylcholine;and (vi) imidazole. All reagents were dissolved inMEM, and dilutions were done in EMFBS.HSV growth assay. HuF were grown to conflu-

ence (5 X 104 cells) in 16-mm plastic wells (model 76-033-05, Linbro, Hamden, Conn.). After the monolayerswere washed with MEM, selected wells were incubatedfor 30 min at 370C with 0.2 ml of EMFBS containingthe various reagents or medium alone (control). (Un-less otherwise indicated, all incubations were done at37°C in a humidified atmosphere containing 5% CO2and 95% air.) Each well then received 0.2 ml ofEMFBS containing HSV diluted in the reagent ormedium alone as indicated. After a 2-h adsorptionperiod with HSV at the multiplicity of infection shown,the cells were washed (four times) with MEM, andthen 0.4 ml of EMFBS or medium containing theoriginal reagent was added to each well. At intervalsafter infection, monolayers were frozen at -70°C. Atthe end of the experiment, all samples were thawed,sonicated at 120 W for 10 s (Sonifier, model W185;Heat Systems Ultrasonics, Plainsview, N.Y.), centri-fuged for 15 min at 640 x g, and assayed for virus

infectivity. Experiments were done at least twice, anddata are expressed as the mean titer log1o PFU oftriplicate wells ± 1 standard deviation. The reagents,at the concentrations used in the experiments, neitherinactivated virus, affected HuF viability, nor influ-enced the HSV assay on PRK cells.HSV plaque size measurements. The effect of

the reagents on HSV plaque size was determined onconfluent monolayers of HuF in 35-mm plastic wells(model FC-6-TC; Linbro). HuF were inoculated with50 PFU of HSV that had been diluted in 1.0 ml ofeither EMFBS containing the reagent indicated ormedium alone (control). After a 2-h incubation, 0.05ml of anti-HSV was added to each well. The cultureswere incubated 48 h and then fixed and stained. Plaquesizes were determined by measuring the diameters ofthe magnified (x18), projected plaque images. Valuesare expressed as the mean diameter (in millimeters) of25 plaques ± 1 standard deviation.Adsorption and penetration studies. Confluent

monolayers of HuF in 35-mm dishes were placed onice and inoculated with 50 PFU of HSV diluted inEMFBS containing the reagents or medium alone.After a 2-h adsorption period at 4°C, the monolayerswere washed (four times) with cold (4°C) MEM toremove the unattached virus. The cells were overlaidwith 1.0 ml of EMFBS and incubated for 2 h at 37°Cto allow the adsorbed HSV to penetrate the HuF.After the incubation period, the cultures were overlaidwith 1.0 ml of EMFBS containing 5% (vol/vol) anti-HSV. After 48 h, the monolayers were fixed andstained and the plaques were counted. For the pene-tration assay, confluent HuF in 35-mm wells wereincubated for 2 h at 4°C with 250 PFU of HSV in 0.5ml of EMFBS. After washing (four times) with coldMEM to remove the unattached virus, the monolayerswere overlaid with 1.0 ml of the cold test reagent orEMFBS alone (control) and then warmed to 37°C. Atintervals after warming, overlays were replaced with2.0 ml of EMFBS containing 10% (vol/vol) anti-HSVto neutralize unpenetrated virus. Cultures then wereincubated for 48 h and the plaques were counted.cAMP assay. cAMP levels in HuF were deter-

mined by a radioimmunoassay (NEX-132; New Eng-land Nuclear Corp., Boston, Mass.) based on themethod of Steiner et al. (43). Confluent cultures ofHuF were trypsinized, washed twice with MEM, andresuspended in EMFBS to 4 x 105 cells/ml. Samples(0.5 ml) of this HuF suspension were distributed into80-mm2 flat-bottomed glass tubes and incubated for 3days. The medium overlays were removed, and thecells were inoculated with 0.5 ml ofEMFBS containing106" PFU of HSV or medium alone (control). Thecultures were incubated and at selected intervals wereremoved from the incubator and treated as follows:certain cultures were immediately heated for 2 to 3 sin a Bunsen burner flame, placed on ice, and thenfrozen at -70°C; other cultures were first incubatedfor 10 min with 0.5 ml of reagent in EMFBS or mediumalone and then harvested as described above. Imme-diately before assay, all samples were thawed, sonifiedat 120 W for 20 s, and centrifuged at 640 x g for 15min. Samples (0.1 ml) of the resulting supernatantswere used for cAMP determinations as described inthe test kit. Results are expressed as the mean cAMP

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160 TROFATTER AND DANIELS

concentration (picomoles/1060 cells) ± 1 standard de-viation of triplicate determinations.

IF preparations. Human type I IF was obtainedfrom the Medical Research Council, Mill Hill, London,England (Research standard B, 69/19). The materialhas an activity of 5,000 U/ml. One unit is defined asthat amount of material which reduces by 50% theplaque count of HSV on HuF (31). The procedure forpreparing IF in human MNL was based on the meth-ods of Haahr et al. (18). MNL from HSV-immuneindividuals were isolated on Ficoll-Hypaque gradients(5), and the washed cells were adjusted to a concen-tration of 8 X 106 MNL/ml in MEM containing 30%(vol/vol) nonimmune human AB serum (EMHS).Samples (0.2 ml) of the MNL suspension were distrib-uted into 16-mm wells, and to these were added 0.1 mlof: (i) EMHS containing the reagent or medium aloneand (ii) HSV-Ag or uninfected cell extract. After 3days, the cell suspensions were removed and centri-fuged (640 x g, 15 min) and the supernatants werecollected. These fluids were dialyzed twice for 24 hagainst 200-fold volumes of MEM and then werestored at -20oC. The amount of IF produced by theMNL was expressed as units per milliliter.

IF assays. Serial twofold dilutions of the IF prep-arations were done in EMHS alone or in the mediumcontaining various concentrations of the reagents.Samples (0.4 ml) of each IF dilution were incubatedfor 18 to 20 h on confluent HuF grown in 16-mm wells.HuF were then washed in MEM (three times), and toeach well was added 0.4 ml of EMFBS containing 50PFU of HSV. After a 2-h adsorption period, 0.05 ml ofanti-HSV was added. Cultures were incubated for 24to 48 h, fixed, and stained. The titer of IF was definedas the reciprocal of the greatest twofold dilution whichcontained at least 1 U of IF activity. Each assay wasdone in triplicate, and experiments were repeated atleast twice. Mean titers are shown. At the concentra-tions used in the experiments, the reagents did notinactivate the IF preparations. When added toEMFBS containing IF, the reagents could be removedby dialysis, thereby restoring the antiviral activity ofthe IF solution.

RESULTScAMP levels in HSV-infected cells. HuF

were incubated with HSV or medium alone (con-trol), and samples were assayed at intervals forcAMP (Fig. 1). Changing the medium overlayinvariably reduced the cAMP in the cell cul-tures. Consequently, after inoculating the mono-layers, the initial cAMP concentrations were low(0.07 pmol/106 HuF) in both the infected anduninfected cells, but the level of the cyclic nu-cleotide in the uninfected HuF rapidly increasedand by 12 h had reached a plateau (1.55 pmol/106 HuF). In contrast, the cAMP concentrationin the cells infected with HSV attained a maxi-mum (0.45 pmol/106 HuF) at only 4 h afterinfection and by the end of the experiment wasonly 25% of the control value. Experiments nextdetermined whether the cAMP level of HSV-

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I 120/0

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ECf 0.60.

Infected

0.40-

020

0 2 4 6 8 10 12 24

HOURS

FIG. 1. Effect ofHSV infection on the cAMP levelsin HuF. Cells were incubated with EMFBS contain-ing HSV (multiplicity of infection = 50:1) (0) ormedium alone (O), and cAMP levels were determinedat the times indicated. Values shown represent themean of triplicate determinations + 1 standard de-viation.

infected HuF could be increased by PGE2 orMIX.Effect of PGE2 and MIX on cAMP levels

in HSV-infected cells. HuF were incubatedwith HSV or medium (control), and at 2 or 20 hpostinoculation these cultures were exposed for10 min to PGE2, MIX, or medium. The cAMPresponse of the 2-h-infected cells to the reagentswas essentially identical to that ofthe uninfectedcontrol HuF (Table 1). When pulsed for 10 minwith fresh medium alone, both the uninfectedand infected cells had low levels of cAMP, 0.05and 0.07 pmol/106 HuF, respectively. Relativeto these values, MIX caused significant increasesof cAMP in both 2-h-uninfected (0.70 pmol/106HuF) and infected (0.90 pmol/106 HuF) cultures.The cAMP response to PGE2 of the 2-h-unin-fected (37.5 pmol/106 HuF) and infected (33.5pmol/106 HuF) cells was most dramatic, repre-senting 400- to 700-fold elevations in intracellu-lar cAMP concentrations above those achievedwith medium alone.

Uninfected cells that had been cultured for 20h showed similar increases in cAMP levels afteraddition of the MIX or PGE2. The 20-h-infectedHuF, however, were hyporesponsive to thesereagents. Relative to the cAMP level attained incultures incubated for 10 min with medium alone(0.07 pmol/106 HuF), the cAMP elevations in-duced in the 20-h-infected cells by MIX (0.24


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TABLE 1. Concentration ofcAMP in infected and uninfected HuF after incubation with PGE2 or MIX'

cAMP concn (pmol/10'HuF)bMaterial incubated with HuF 2-h-uninfected con- 2-h-infeted 20-h-uninfected con- 20-h-infected

trol 2--netdtrol20hifceMedium alone 0.05 i 0.03 0.07 ± 0.02 0.12 ± 0.01 0.07 ± 0.02MIX (10-3 M) 0.70 ± 0.10 0.90 ± 0.50 1.05 ± 0.06 0.24 ± 0.04PGE2 (3 x 10-5 M) 37.5 ± 15 33.5 ± 4.0 32.0 ± 9.7 7.7 ± 2.5

aHuF (2 x 105 cells) were inoculated with HSV (multiplicity of infection = 50:1) or medium alone (control).After 2 or 20 h of incubation, the inocula were replaced with PGE2, MIX, or EMFBS alone. The cultures wereincubated for 10 min at 370C and then were assayed for cAMP.

bValues shown represent the mean cAMP concentration ± 1 standard deviation of triplicate determinations.C Medium was EMFBS.

pmol/106 HuF) or PGE2 (7.7 pmol/106 HuF)were only 25% of those achieved by the unin-fected MIX-treated (1.05 6pmol/106 HuF) or

PGE2-treated (32.0 pmol/10 HuF) controls. Re-gardless, the infected cells did show a significant(P < 0.01) increase in cAMP after adding thereagents, and thus we proceeded to determinethe effect these and other cyclic nucleotide-mod-ulating reagents had on the growth of HSV inHuF.Growth ofHSV in the presence ofPG and

MIX. PGE2 had little effect on HSV synthesisin HuF compared with cells incubated with me-dium alone (Fig. 2). Infected cells incubatedcontinuously for 24 h with PGE2 (3 x 10-5 M)yielded the same amount of virus (107- PFU) as

did HuF incubated with medium. Identical re-

sults were obtained with 10-, 100-, and 1000-fold-lesser concentrations of PGE2. Furthermore, no

effect on HSV growth was noted with PGAl,-Bl, -El, or -F2a at concentrations of 3 x 10-5 to3 x 10-8 M. Other reagents that modulate cyclicnucleotide levels in human cells also were tested:isoproterenol (10-4 to 10-6 M), imidazole (10-5 to109 M), carbamylcholine (10-4 to 10-10 M), anddb-cAMP (10-3 to 10-5 M). These reagents nei-ther stimulated nor suppressed the growth ofHSV in the HuF (not shown).MIX (10-3 M), however, affected both the rate

of virus growth in HuF and the amount of HSVproduced (Fig. 2). As shown, the infectivity in-crease in the MIX-treated cultures lagged be-hind that in the medium control; after 24 h,significantly less (P< 0.01) virus (107.2 PFU) was

present. Significantly less virus (107-4 PFU) alsowas obtained in the presence of 10-4 M MIX;however, no difference was found between 10-5M MIX and the medium control. The inhibitoryeffect of this reagent was not related to a de-crease in virus adsorption. In a representativeadsorption study, no significant difference was

found between the quantity of HSV attached toHuF in cultures treated with MIX (45.3 6.7PFU) or medium alone (46.7 + 6.0 PFU). Simi-

8-or

7OFs

0

LU

I-

6 0-

* Medium alone

£PGE2 (3x10-5M)* MIX (10-3M)

501F

4 U 2 5 8 10 12 14 16 24HOURS AFTER INFECTION

FIG. 2. Growth of HSV in the presence of PGE2and MIX. HuF were infected with HSV (multiplicityof infection = 10:1) in the presence ofPGE2 (A), MIX(), or EMFBS alone (0). Cultures were harvestedand assayed for viral infectivity at the times indi-cated. Each point represents the mean titer of HSV(logo PFU) ± 1 standard deviation of triplicate de-terminations.

larly, compared with the medium control, MIXhad no effect on the rate at which the viruspenetrated the cells after adsorption (Fig. 3).Neither could a generalized toxic effect explainthe inhibition ofHSV growth, since the viabilityof the HuF in the presence of MIX at the con-centrations used was always >98%. The sup-pression appeared to be due to the reagent'smetabolic effect, for theophylline (5 x 10' M),another phosphodiesterase inhibitor, similarly

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2501

CL 200

D

0

150-

O0 MIX (Io -3 M)o-----o Medium alone

100

a_ 150 -

0 15 30 45 60 90 120 180

Time Incubated with Reagent (Min.)FIG. 3. Effect of MIX on the penetration of HSV

into HuF. Confluent HuF were incubated for 2 h at4°C with 250 PFU of HSV. After washing with cold(4°C) MEM, the monolayers were overlaid with coldMIX (10-3 M) (0) or medium alone (0) and thenwarmed to 37°C. At intervals thereafter, overlayswere replaced with EMFBS containing 10% (vol/vol)anti-HSV. Cultures then were incubated for 48 h, andthe plaques were counted. Values shown representthe mean number of plaques (PFU) ± 1 standarddeviation of triplicate determinations.

delayed virus growth and reduced HSV yield(data not shown).HSV plaque size in the presence of PG

and MIX. Another quantitative method for de-termining.virus growth is the measurement ofplaque size, which assesses the ability ofHSV tospread from cell to contiguous cell. Experimentswere done to determine whether PG or otheragents that elevate cAMP levels in cells wouldalter the rate at which infected HuF pass virusto neighboring cells (Table 2). HSV plaque sizein HuF in the presence of medium alone was1.80 mm; adding PGE2 (3 x 10-5 to 3 x 10-8 M)to the cultures did not significantly alter plaquesize. Furthermore, no effect on plaque size wasnoted when the other PG were tested at 3 x 10'to 3 x 10-8 M concentrations. Other reagentsthat elevate cAMP also were examined for theirinfluence on plaque size, and an effect was notedonly with the phosphodiesterase inhibitors.Both MIX (10-3 M) and theophylline (5 x 10-3M) caused a greater than 60% reduction inplaque size as compared with medium alone.Lesser concentrations of these reagents also in-hibited HSV plaque size in a dose-dependentfashion. Thus, by two independent methods, thephosphodiesterase inhibitors were shown to sup-press HSV growth, whereas PG had no effect onvirus replication. We next explored the possibil-

ity that these reagents could indirectly influenceHSV growth by inhibiting the action or induc-tion of IF in human cells.

Effect of cAMP-elevating reagents on theresponse of HuF to IF. Stock solutions ofhuman type I IF and HSV-Ag-induced IF weremade that contained 32 and 16 U of antiviralactivity per ml, respectively. The physical char-acteristics of these IF preparations were deter-mined. The titer of HSV-Ag-induced IF wasreduced from 1:16 to less than 1:2 by heat treat-ment (560C, 1 h) or by acidification (pH 2, 1 h),whereas the titer of the standard human type IIF remained constant (1:32) with similar treat-ments (Table 3). These IF preparations nextwere titered on HuF in the presence of cAMP-elevating reagents or medium alone (control).Figure 4 shows the effect of PGE2, MIX, and db-cAMP on the ability of HuF to respond to typeI IF. The titer of this IF preparation on cellstreated with medium was 32. Exposure of HuFto PGE2 (23 x 10-7 M), MIX (21 x i10' M), or

TABLE 2. Effect of PGE2, MIX, and theophylline onHSVplaque size on HuF monolayersa

Material incubated with Concn (mol/ Plaque diameterHuF liter) (mm)

Medium (control) 1.80 ± 0.22PGE2 3 x 10-5 1.84 ± 0.36

3 X 10-6 1.72 ± 0.253 x 10-7 1.78 ± 0.223 X 10-8 1.73 ± 0.31

MIX 1 X 10-3 0.73 ± 0.21c1 X 10-4 1.36 ± 0.17cX 10-5 1.61 ± 0.22

Theophylline 5 X 10-3 0.76 ± 0.14c1 X 10-3 1.26 ± 0.23c2 X 10-4 1.67 ± 0.29

' HuF were inoculated with HSV in EMFBS con-taining the reagents or EMFBS alone. After a 2-hadsorption period, 0.05 ml of anti-HSV was added toeach well. The cultures were incubated for 48 h, fixed,and stained.

' Values shown represent the mean diameter of 25plaques ± 1 standard deviation.

' Significantly (P < 0.05; Student's t-test) differentfrom the medium control.

TABLE 3. Physical characteristics of the IFpreparations

IF prepn Treatment Antiviral ti-

Type I None 1:32560C; 1 h 1:32pH 2; 1 h 1:32

HSV-Ag induced None 1:16560C; 1 h <1:2pH 2; 1 h <1:2

Dilution in EMFBS which reduced by 50% thenumber of HSV plaques on HuF.

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8

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TF +MEDIUM IF+ PGE2(m/L) IF + MIX (m/l) IF + db-cAMP (rn/L)

MATERIAL INCUBATED WITH HuFFIG. 4. Effect of PGE2, MIX, and db-cAMP on the response of HuF to type I IF. A standard type I IF

preparation (32 U/ml) was titered on HuF exposed to medium alone or various concentrations ofPGE2, MIX,or db-cAMP. Each assay was done in triplicate, and mean titers are shown. Asterisks represent values whichare significantly (P < 0.05; Student's t-test) different from the control (IF + medium).

db-cAMP (21 x 1O-4 M) significantly decreasedthe responsiveness of the cells to IF. AddingPGE2 (3 x 1O-5 M) 18 h after exposing HuF toIF had no inhibitory effect on the IF's antiviralactivity. Reagents that elevate cAMP also werefound to inhibit the action of HSV-Ag-inducedIF on HuF (Fig. 5). Whereas the titer of this IFpreparation on cells exposed to medium alonewas 16, titers on HuF incubated with PGE2 (23X 10-7 M) or db-cAMP (21 x 10-4 M) weresignificantly depressed. Thus, the experimentsestablished that reagents which elevate cAMPinhibit in a dose-dependent fashion the abilityof HuF to respond to both human type I andHSV-Ag-induced IF. The inhibition could notbe correlated directly, however, with the abso-lute levels of cAMP attained in the presence ofthe various reagents. PGE2 at concentrations of3 x 10-7 M was found to produce significantlygreater cAMP elevations (-7.3 pmol/106 HuF)than even the highest concentration of MIX(10-3 M; Table 1), yet was not as effective as thelatter in inhibiting the action of IF. Finally, weexamined the effect of these reagents on theability of immune human MNL to produce IF.Effect of cAMP elevating reagents on the

production of IF. MNL from a patient withrecurrent HSV were incubated with HSV-Ag inthe presence of cAMP-elevating reagents or me-dium alone (control). Figure 6 shows the effectsof PGE2, PGF2a, and MIX on the IF productionby human leukocytes. MNL incubated with

161

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4

IF + 3x10' 3xo-1 310-' 3a10-5 I0- 10'4 I0°'MEDIUM IF * PGE2(m/L) IF db-cAMP (m/l)

MATERIAL INCUBATED WITH HuFFIG. 5. Effect of PGE2 and db-cAMP on the re-

sponse ofHuF to HSV-Ag-induced IF. An IF (16 U/ml) preparation, produced by immune human MNLin the presence of HSV-Ag, was titered on HuF ex-posed to medium alone or to various concentrationsof PGE2 or db-cAMP. Each assay was done in trip-licate and mean titers are shown. Asterisks representvalues which are significantly (P < 0.05) differentfrom the control (IF + medium).

HSV-Ag in the presence of medium alone syn-thesized 8 U of IF. In a dose-response fashion,PGE2 (23 x 1O-7 M), PGF2a (23 x 10-6 M), and

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3x1O-03xO-r' 3x1o-63x1O-

HSV-Aq+ PGE2 (rm/L)

3xuK- 3x)0- 3x)Q4 l-lo- ,, 10-l

HSV-Ag + PGF2a (m/L) HSV-Ag+MIX (m/I)

MATERIAL INCUBATED WITH HUMAN MNL

FIG. 6. Effect of PGE2, PGF2a, and MIX on the production of IF by immune human MNL. MNL wereincubated for 3 days with HSV-Ag and with EMHS containing concentrations of PGE2, PGF2a, MIX, ormedium alone (control). The cell supernatants were dialyzed and assayed for IF. Values shown represent theaverage number of units produced by 1.6 x 106 MNL. Each assay was done in triplicate, and asterisksrepresent values which are significantly (P < 0.05) different from the control.

MIX (21 x 10-5 M) significantly inhibited theproduction of IF by the MNL.

DISCUSSION

Previous studies had demonstrated that re-

agents which increased cAMP could enhancethe in vitro growth of cytomegalovirus in humancells (52). We investigated whether cAMP ele-vation in HuF would directly stimulate HSVreplication. The virus infection itself was foundto interfere with the ability of HuF to producecAMP (Fig. 1). Although the response of HuFto the cAMP- elevating reagents PGE2 and MIXwas reduced by HSV infection, cells incubatedwith these substances still had significantlyhigher cAMP levels than cells incubated withmedium alone (Table 1). Despite this response,PGE2 neither enhanced HSV replication (Fig. 2)nor potentiated the cell-to-cell spread of thevirus in HuF (Table 2). In addition, PGAj, -B1,-El, and -F2a were tested at a wide range ofconcentrations; none augmented HSV growth,and neither did other substances known to in-crease cAMP (MIX, theophylline, isoprotere-nol), decrease cAMP (imidazole), or increasecyclic 3',5'-guanosine monophosphate (carba-mylcholine).The results we have obtained, using a human

cell line to study the effects of PG on the growthof HSV, differ from those previously reported,which used nonhuman cells. Harbour and col-leagues found that HSV yield at low multiplici-ties of infection was increased in Vero cells

incubated with PGE2 or PGF2a and attributedthese findings to enhanced cell-to-cell spread ofthe virus (21). Similarly, Newton has reportedincreased yields of HSV in mouse Ly cells incu-bated with PGE, (35). In support of their studyimplicating PG in enhanced HSV growth, Har-bour et al. also reported that inhibitors of PGsynthesis, mefenamic acid and indomethacin,decreased virus yield in Vero cells. This effectwas partly reversed by the addition of exogenousPGE2 (21). Newton, on the other hand, couldnot duplicate this result in mouse Ly cells (35).Thus, discrepancies exist between those whohave studied the effect of PG on HSV growthon nonhuman cell lines. This is not surprising,since the metabolic effects of PG have beenshown to differ depending on cell type (36).Differences may also exist in the effects PG haveon HSV growth in human cells with differenttissue origins; however, the results reported heredemonstrate no potentiating effect of PG on thegrowth of HSV in a human fibroblastoid cellline.On the contrary, significant suppression of

HSV growth (Fig. 2) and plaque formation (Ta-ble 2) was achieved by the methylated xanthinederivatives MIX and theophylline, known toelevate cAMP directly by inhibiting the cAMPphosphodiesterase. Judging from the pattern ofgrowth in the presence of MIX, HSV productionwas delayed by approximately 2 h, but once

infectious virus synthesis began, the slope of thegrowth curve paralleled that of the control (Fig.

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2). Since neither MIX nor theophylline inhibitedHSV adsorption or penetration (Fig. 3), the datasuggest that the reagents exerted a suppressiveeffect on a metabolic event(s) occurring duringthe latency period. The conclusion agrees withthe results of Stanwick et al., whose kineticstudy also indicated that phosphodiesterase in-hibitors affect an early event in HSV replication(42). Whatever the mechanism, inhibition ofHSV growth by MIX or theophylline was notattributable to cAMP elevation alone, sincePGE2 produced significantly highercAMP levelsand had no effect on virus replication (Table 1,Fig. 2).

Despite having no direct potentiating effecton HSV growth, the cAMP-elevating reagentswere found to interfere with the antiviral activ-ities of human IF preparations, thus indirectlyenhancing virus replication. PGE2, MIX, and db-cAMP dose dependently suppressed the viralinhibitory action of human type I (Fig. 4) andHSV-Ag-induced (Fig. 5) IF preparations. In arelated fashion, Degre (9) had reported earlierthat cholera toxin, a potent stimulator of adenylcyclase, also inhibited the action of a humanleukocyte-derived IF preparation.

In addition to suppressing the antiviral actionof IF, the cAMP-enhancing reagents inhibitedits production in response to a specific antigenicstimulus. Human MNL from HSV-immune in-dividuals previously had been shown to synthe-size IF in the presence of HSV-Ag (38). Wefound that when PGE2, PGF2a, or MIX wasadded with HSV-Ag to human MNL, the pro-duction of IF was suppressed (Fig. 6). Similarly,mitogen-induced synthesis of IF by mousespleen cultures has been found to be inhibitedby db-cAMP, MIX, and cholera toxin (25). Fur-thermore, Dianzani et al. (10) demonstrated thattreatment of murine cells with theophylline pluseither db-cAMP or adrenaline suppressed theproduction of type I IF induced by polyinosinic-polycytidylic acid or Newcastle disease virus.Others have demonstrated a similar effect ofthese same cAMP-elevating reagents on the in-fluenza virus-induced synthesis of IF by aviancells (40).The method of producing IF, using Ficoll-Hy-

paque-purified MNL from an immune individ-ual, resulted in an IF preparation with the phys-ical attributes of type II IF (Table 3). Othershave shown that IF, produced by Ficoll-Hy-paque purified leukocytes from immune or non-immune individuals, had the physical character-istics of type I IF, being heat and acid stable (18,39). Conceivably, subtle differences in method-ology could account for this discrepancy. Specif-ically, as relates to the prior published work ofHaahr et al. (18), we used Eagle rather than

McCoy medium, harvested our IF preparationat an earlier time, and did not subject the MNLto NH4Cl. Whether these or other unknownfactors could explain the differences in the phys-ical characteristics of IF obtained from immuneindividuals is at present unclear.The mechanism by which cAMP-elevating re-

agents inhibit IF production has not been eluci-dated; however, a hypothesis can be generatedfrom the scattered reports regarding IF andcAMP which have appeared in the literature. IFsynthesis has been proposed to be regulated bythe production of a repressor protein (48); db-cAMP has been shown to inhibit the productionof IF in chicken embryo fibroblasts; and thisinhibition can be reversed by inhibitors of pro-tein synthesis (10). Taken together, these threeobservations suggest that cAMP might regulateIF production by promoting the synthesis of therepressor protein. Since IF itself induces an in-crease in cAMP in mouse Ly cells before theestablishment of an antiviral state (30), we pro-pose that cAMP might play a negative-feedbackrole in both the production and action of IF.Such a hypothesis is supported by the recentfindings that the IF inducer polyinosinic-poly-cytidylic acid, as well as human IF, causes pro-duction of PGE in cultured human fibroblasts(54).

In summary, many of the factors known toprecipitate recurrent herpetic disease are corre-lated with elevated local or systemic PG levels.We have shown that PG and other cAMP-ele-vating reagents, although having no direct stim-ulatory effect on HSV replication, could indi-rectly enhance growth of the virus by inhibitingboth the action and the production of IF.IF is one of the few host defense mechanisms

which has been documented both in vitro (28)and in vivo (17, 32) to limit HSV infection. Howa breach in the IF defense system might lead toa HSV recurrence is worthy of brief speculation.Some patients who experience multiple recur-rent herpetic infections have lymphocytes thatare poor IF producers (38); one study has shownthat, as their leukocytes regain the ability tosynthesize IF, these subjects have less frequentattacks (37). IF could be responsible for main-tenance of the latent state by suppressing HSVsynthesis in ganglion cells (2). Alternatively, IFcould have little influence on the emergence ofHSV from its central dormant state, but mightdetermine whether this virus successfullyspreads in the periphery from cell to cell andproduces a recognizable clinical lesion.The experiments reported here show that PG

suppress the IF defense system. Prior studies inour laboratory have shown that PG also inhibithuman MNL from mediating antibody-depend-

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ent cell-mediated cytotoxicity against HSV-in-fected human cells (46). Together these obser-vations suggest that transient in vivo rises in PGmay shift the HSV-host balance in favor of thevirus and thereby result in exacerbation of her-petic disease.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants2TOlGM-00726 and DE04609 from the National Institutes ofHealth.We gratefully acknowledge the invaluable assistance of

Jessie Calder, Bill Boyarsky, Pat Burks, and Frances Slocumin the preparation of the manuscript.

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effect of prostaglandins and cyclic adenosine 3',5'- monophosphate

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