Prostaglandin E Modulation of the

Mitogenic Response of HumanT Cells

DIFFERENTIAL RESPONSEOF T-CELL SUBPOPULATIONS

JOHN D. STOBO, MICHAEL S. KENNEDY, and MARCE. GOLDYNE,Departm7Xenit ofNiedicine, Sectiotn of Rheumncatology atnd Clinical Imm7lutnology, Moffitt Hospital,

acnd Department of Dermtiatology, Sani Francisco General Hospital, Utniversity ofCalifornia, Sant Fratncisco, Sani Fratncisco, California 94143

A B S T RA C T Prostaglandins (PG) of the E series,PGE, and PGE2(PGEs), can induce elevations of intra-cellular cyclic AMP(cAMP) among thymus-derived (T)lymphocytes (T cells) and inhibit their reactivity. Forexample, 0.1 ,uM of PGEs induces a two- to threefoldincrease of intracellular cAMPamong human periph-eral blood T cells and a 20-30% suppression of theirblastogenic response to phytohemagglutinin. How-ever, this suppression actually represents the net reac-tivity of T-cell populations demonstrating quite differ-ent responses to PGEs. Fractionation of T-enrichedpopulations on a discontinuous density gradient yieldsa population of high density cells whose phytohemag-glutinin-induced blastogenic response is suppressed60%; a population of intermediate density cells whoseresponse is suppressed 20%; and a population of lowdensity T cells whose response is not suppressed, butis enhanced 20% by both of the PGEs. The diametri-cally opposite responses of low and high density T cellsto the PGEs is not related to any difference in theirintrinsic mitogen reactivity nor is it influenced by in-teractions with other T cells, bone marrow-derived (B)cells, or monocytes. Moreover, the distinct blastogenicresponse of low and high density T cells to PGEs doesnot simply correlate with PGE-mediated activationof adenylate cyclase. PGE2 induced comparable abso-lute and identical relative increases of intracellularcAMPamong the low and high density T cells. Choleratoxin, a potent activator of adenylate cyclase, and exog-enous 8-bromo cAMP mimicked the effects of thePGEson these two T-cell populations. These data dem-onstrate that T cells are heterogeneous with regard to

Dr. Stobo is an investigator of the Howard Hughes MedicalInstitute.

Received for publication 22 March 1979 and in revisedform 1 June 1979.

1188

their response to the PGEs. Thus, PGEs should beconsidered as potential regulators rather than as uni-versal suppressors for T-cell reactivity. Moreover, theeffect of PGEs on the blastogenic response of a givenT-cell population depends upon intracellular eventswhich occur subsequent to elevations of cAMP.

INTRODUCTION

Prostaglandins (PG)1 represent a family of 20 carbon,unsaturated fatty acids which may modulate functionin a variety of biologic systems (1, 2). Their potentialimportance as modulators of immune reactivity is sug-gested by two observations. First, cells of the mono-cyte/macrophage lineage are crucially involved inregulating immune function and also constitute one ofthe major PGE-secreting classes of immunocytes (3-5).Second, it has been directly demonstrated that PG ofthe E series (PGE, and PGE2 [PGEs]) can modulateimmune function, presumably through their ability toalter intracellular levels of cyclic nucleotides (6-8).Most of these studies suggest that the PGEs suppressimmune reactivity. For example, PGEs elevate intra-cellular cyclic AMP(cAMP) among thymus-derived (T)lymphocytes (T cells) and inhibit their mitogen-induced proliferation as well as their production ofcertain lymphokines (9-11). However, evidence alsoexists to suggest that PGEs may augment T-cell reac-tivity. Yoneda and Mundy (12) have recently demon-strated that PGE, enhances the mitogen-induced pro-duction of osteoclast-activation factor by T cells. It ispossible therefore, that PGEs may not be universally

IAbbreviations used in this paper: BSA, bovine serum al-bumin; cAMP, cyclic AMP; ETOH, ethanol; FCS, fetal calfserum; PBMC, peripheral blood mononuclear cells; PG,prostaglandins; PGEs, PGE, and PGE2; PHA, phytohemag-glutinin; 8-Br, 8-bromo.

J. Clin. Invest. ©3 The American Society for Clinical Investigation, Inc. 0021-9738/79/11/1188/08 $1.00Volume 64 November 1979 1188-1195

suppressive for T-cell reactivity. Instead, their im-munoregulatory effects may depend on the T-cell reac-tivity being assayed and thus reflect differential re-sponses of individual T-cell populations.

In this paper, we describe experiments which dem-onstrate that T cells are indeed heterogeneous withregard to their response to the immunomodulating ef-fects of PGEs. Although PGEs inhibit the phytohemag-glutinin (PHA) -induced blast transformation occurringamong whole peripheral blood T cells, this representsthe contribution by T-cell populations manifesting dis-tinctly different responses to PGEs. Fractionation ofT-enriched populations on a 5-step, discontinuous bo-vine serum albumin density gradient yields a popula-

%inhibition =

or as

%augmentation =

tion of high density T cells whose mitogen reactivityis suppressed by both PGEs. In contrast, both PGEsaugment the mitogen reactivity of low density T cells.PGE2 induces comparable increases of intracellularcAMPamong both low and high density T cells, andthe effect of PGE2 on their mitogen-induced blasto-genesis can be mimicked by other agents capable ofactivating adenylate cyclase as well as by exogenous8-bromo (8-Br) cAMP. These studies support the con-cept that PGEs function as modulators rather than asuniversal suppressors of T-cell reactivity and furthersuggest that this modulation results from intracellularevents occurring subsequent to activation of adenylatecyclase.

METHODSCell populationt and culture. Peripheral blood mononu-

clear cells (PBMC) (70±4% T, 4±2% immunoglobulin [Ig]bearing, and 20±8% esterase positive); populations enrichedfor T cells (93±5% T, 2±1% Ig bearing, and 3+2% esterasepositive); and populations enriched for bone marrow-derived(B) cells and monocytes (10+3% T, 40± 10% Ig bearing, and 40±8%esterase positive) were obtained as previously described(13). T-enriched populations were fractionated on a discon-tinuous bovine serum albumin (BSA) density gradient thatconsisted of layers of 10/23/26/29/33% BSA(Pathocyte 5, MilesLaboratories, Inc., Kankakee, Ill. [13]). The recovered gradi-ent-fractionated cells were divided into three populations bypooling cells sedimenting in the top two (10/23% and 23/26%BSA) interfaces; keeping cells of intermediate density sepa-rate (26/29%) interface; and by pooling cells sedimenting inthe bottom interface and pellet. This was done to obtain num-bers of cells sufficient for the experiments. 70% of the Tcells originally placed on the gradient were recovered with50±8.2 (mean±SE), 25±4.8, and 25±4.9% of the recoveredcells obtained in the three fractions, respectively.

To determine the effects of PGEs on T-cell proliferation,

PGEs (10 mglml in 100% ethanol [ETOHI) were added in afinal concentration of 0.1 ,uM and 10 niN to T cells suspendedin RPMI 1640 (Cell Culture Facility, University of California,San Francisco, San Francisco, Calif.) that contained 10% heat-inactivated (56°C for 30 min) fetal calf serum (FCS). Controlsconsisted of cell suspensions which contained an equivalentfinal concentration of ETOH(0.01 and 0.00 1%) without PGEs.50,000 cells of each mixture were cultured in triplicate, round-bottom microtiter wells at 37°C in a humidified atmosphereof 95% air, 5% CO2 for 4 d with medium only or with threeconcentrations of PHA(0.3, 1.0, and 3.0 ,ug/ml final concentra-tions; Burroughs Wellcome Co., Triangle Park, N. C.). Re-activity was determined by assaying the amount of tritiatedthymidine (2.0 Ci/mM, NewEngland Nuclear, Boston, Nlass.)incorporated into DNAduring the final 12 h of a 4-d culture(13). The mean change in counts per minute (counts per minutein stimulated cultures minus counts per minute in nonstim-ulated cultures) was calculated and results are expressed as:

[1 - (Acpm in cultures containing PGEs i 100

L /Acpmin cultures containing ETOH x1

L( Acpm in cultures containing PGEs 1-1 I x 100.

Acpm in cultures containing ETOH/ I

In initial experiments, it was aseertainied that the regulatoryeffects of PGEs as determined by incorporation of tritiatedthymidine correlated with those determined by mnorphologicevaluation of blast transformation.

Determiniatiotn of initracellular cAMP. To determiiine theability of PGEsto alter intracellular cANIP, cells to he assayedwere first incubated in 5% FCS in the presence 0.1 mnMof aphosphodiesterase inhibitor (RO-20-1724) for 45 min at 37°C.PGEs or appropriate concentrations of ETOH were thenadded, and the cells were incubatedl at 37°C in a humidifiedatmosphere of 95% air, 5%CO2. At various intervals, aliquotswere removed and centrifuged at 1,800 g for 30 s in a micro-fuge (Beckman Instruments, Inc., Spinco Div. Palo Alto,Calif.); and then the cell pellet was suspenided in 20 m11Msodiumacetate, 0.1 mMmethylisobutyl-xanthine, pH 4, and boiledfor 5 min. Cellular cAMPwas determined with the comiipet-itive protein binding assay of Gilman (14). Allowance wasmade for residual media in the pellet. Results are expressedas picomoles of cAMP/107 cells.

RESULTS

Inhibitiotn of proliferatiotn am11on0g wthole antd frac-tionated T cells by PGEs. In unpublished studies, wedemonstrated that adherent human peripheral bloodmonocytes, but not T cells, synthesize PGE.2 Quanti-tative gas-liquid chromatography/miiass spectrometryanalysis of culture fluids obtained from 1 x 106 ad-herent monocytes incubated for 48-72 h in 0.5 ml indi-cated the accumulation of 1.1 ng of PGE, and 16.9+2.1ng of PGE2. On the basis of these data, PGEs wereused in final concentrations of 0.1 ,uM and 10 nM to testtheir effects on the proliferative response of T-en-riched populations to three concentrations of PHA(these as well as all subsequent proliferative assays

2 Goldyne, NI., and J. Stobo. Mlanuscript submitted forpublication.

Prostaglandin E-induced Immunoregulationt 1189

were performed in the presence of 1 ,uM of indo-methacin to block endogenous PGE synthesis by thesmall number of monocytes present in the T-enrichedpopulation). The results (Fig. 1) are consistent withprevious reports demonstrating that PGEssuppress thePHA-induced proliferation among human peripheralblood T cells (11).

To investigate the possibility that distinct popula-tions of T cells respond differently to PGE, advantagewas taken of the fact that subpopulations of periph-eral blood T cells can be delineated by differencesin their density (13, 15). T-enriched populations werefractionated on a discontinuous BSA gradient, and therecovered three fractions were assayed for PHAreac-tivity in the presence of 0.1 A.M of PGE1 or PGE2 (Fig.2). Both PGEsmarkedly suppressed PHA-induced pro-liferation among high density cells. This suppression(-60%) was substantially greater than that noted whencomparable concentrations of PGEswere added to un-fractionated T cells (30-40%, Fig. 1). In contrast, nei-ther PGE i]low densitybut significzall three cosuppressiormediate delow and hil0.1 ,M an((Three repr

z0I-

z

w

0

z0

z

40 k

20 _-

to -2

4

FIGURE 1 Etionated T cEreactivity toin the preseitivity was mwith the resusuppressionthe presenccmean+SEot

8 SCz

21

0 4

Z 2

0~LOWDENYT

rtlil ra r

NIERMEDIATE DENSITY T HION DENSITY T

Jr GEoF2

0.3 1.0 3.0 0.3 1.0 3.0 0.3 1.0 3.0

PHACONC. (Ag/mI)

FIGURE 2 Effects of PGEs on the PHAreactivity of fraction-ated T cells. T-enriched populations were fractionated on adiscontinuous BSA density gradient, and the recovered cellswere pooled into three fractions. The reactivity of these cellsto three concentrations of PHAwas assayed in the presence of0.1 ,M PGE1 or PGE2. The results are presented as the meanpercent augmentation or percent suppression +SE noted forsix experiments.

nhibited the proliferative response among Several points concerning these studies should beZ T cells. Instead, PGEs effected a small, emphasized First, incubation of low density T cells inant (P < 0.01), augmentation of reactivity to concentrations of BSA comparable to those present in)ncentrations of PHA. PGEs caused -20% the high density fractions, and vice versa, did not alterl of the PHAreactivity among cells of inter- their subsequent response to PGEs (data not shown).nsity. The absolute counts per minute for Second, the differential effect of the PGEs on low andgh density cells tested in the presence of high density T cells was not dependent upon the time atd 10 nM PGE2 are presented in Table I. which DNA synthesis was determined. PGE2 alsoresentative experiments are shown.) augmented reactivity among low and suppressed

reactivity among high density cells when tritiatedthymidine incorporation was measured after 72 or after120 h of culture (data not shown). Third, PGE2-mediated modulation of low and high density T cells

A B was not substantially altered by the addition of B cells0.1 PLM lOnM and monocytes. PGE2 (0.1 ,uM) effected a 14% mean

augmentation of maximal PHA reactivity amongcultures containing 50,000 low density T cells and50,000 cells from autologous populations enriched for Bcells and monocytes. Similarly, PGE2 suppressed by47% the maximal PHAreactivity of cultures containingcomparable proportions of high density T cells and Bcells plus monocytes. (The maximal PHAreactivity of B

cells and monocytes tested alone in the presence orI4l= I oPGEI absence of 0.1 ,uM PGE2was meager: 8,752 and 9,934El PGE2 cpm, respectively.) Finally, PGE-imediated interac-

I I tions with high density T cells did not, in turn, result in0.3 1.0 3.0 0.3 1.0 3.0 influences capable of suppressing low density popula-

PHACONC. (Mg/ml) tions (Fig. 3). Addition of increasing proportions of high-ffects o density T cells to the low density population in theil fects of PGEs on the PHA reactivity of unfrac-prsneo G2(1 M)euldinaeatvyells. T-enriched populations were tested for their presence of PGE2 (0.1 ,uM) resulted in a relativelythe indicated three final concentrations of PHA linear decline in maximal PHA reactivity. This wasnce of 0.1 uM and 10 nM PGE, or PGE2. Reac- similar to the situation noted when the reactivity of theeasured by the incorporation of [3H]thymidine low density population was simply diluted by the

ilts presented as percent augmentation or percent intrinsically unresponsive B cells and monocytes. The(reactivity in the presence of PCE/reactivity in

e of ETOH). The data represent the arithmetic sum of these findings indicates the existence of T-cell)tained for 10 experiments. Conc., Concentration. populations manifesting distinct differences in their

1190 J. D. Stobo, M. S. Kennedy, and M. E. Goldyne

TABLE IEffect of PGE2 on the PHA Reactivity of Fractionated T Cells

Concentration of PHA

Source of T cells Media tgirnl ... 3 1 0.3

Low density cpmrl cpm+ ETOH 844 52,594 63,874 39,890

+258 ±+18,734 -+18,659 -+11,033+ PGE2, 0.1 ,m 760 62,321 79,112 52,762

+ 130 ±19,036 +21,994 ±14,308+ PGE2, 10 IIM 513 57,633 66,923 40,483

±227 ±+18,515 +19,546 ±19,513

High density+ ETOH 217 51,158 48,372 29,113

±91 ±7,840 ±13,040 ±8,378+ PGE2, 0.1 uMM 218 15,416 16,310 11,467

±91 ±3,760 +5,526 +6,999+ PGE2, 10 nMI 341 34,205 35,198 20,367

±124 ±6,673 ±10,728 ±6,164

T-enriched populations were fractionated on a discontinuous BSA densitygradient. Low density (fractions 1 and 2) as well as high density (fractions 4 and5) cells were tested for their reactivity to three concentrations of PHA in thepresence of ETOH (0.001%) or two concentrations of PGE2 (0.1 AM, 10 nM).The response of nonstimulated cells (media) is also shown. Reactivity wasmeasured by the incorporation of tritiated thymidine into DNAwith results pre-sented as mean counts per minute±SE for three experiments.

response to PGEs. Furthermnore, they suggest that thisdifferential response is not related to the BSA used inthe density gradient, occurs independently of the

1 00r

80

m0

x

E

60k

40k

20

I

LOWDENSITY T 1 3 1 1 0

HIGH DENSITY TOR

B+MONOCYTES 0 1 1 3 1

RATIO OF CELL POPULATIONSASSAYED

FIGURE 3 Effect of high density T cells or B cells and mono-cytes on the PGE2-modulated reactivity of low density T cells.Low density T cells were mixed in the indicated proportionswith either high density T cells (0) or with populations en-riched for autologous B cells and monocytes (0). 100,000 cellsfrom each mixture were then tested for their reactivity to threeconcenitrations of PHA in the presence of 0.1 iMM PGE2. Re-suilts are presented as maximal counts per minute noted inresponse to PHAand is representative of two experiments.

duration of culture, and is not altered by interactionswith other mononuclear cells.

PGE-induced modulation of intracellular levels ofcAMP among fractionated T cells. PG can increaseintracellular cAMP among intact lymphocytes, and itis presumbaly through this mechanism that they exerttheir inhibitory effects on lymphocyte proliferation(16, 17). One might postulate that the different PGE-initiated metabolic events occurring among low andhigh density T cells would be reflected by differencesin PGE-induced elevations of this cyclic nucleotide.To determine this, both cell populations were incu-bated with 0.1 AMPGE2, and intracellular cAMPlevelswere determined. The results of a typical experimentare graphically presented in Fig. 4. PGE2-induceddetectable increases of intracellular cANIP after 3 minof incubation (data niot shown) with maximal in-creases among both populations occurring after 6 min.PGE2 caused substantial absolute increases in cAMPamong both low and high density T cells (five experi-ments, 486-+140 and 238±t51 pmol cAMP/107 low andhigh density T cells, respectively). Moreover, the rela-tive (i.e., stimulated/nonstimulated) increase in cAMPassayed at 6 min was virtually identical among bothpopulations (2.6+0.48 vs. 2.5+0.34 [Fig. 4]). These datasuggest that the differential responses of low and highdensity T cells to the -PGEs reflect differences in intra-

Prostaglandin E-induced Immunoregulation 1191

400r K300 _-

(2.6* 0.48)

.1 ,5:034~

I

6

OLow Density T TABLE II* High Density T Effect of Cholera Toxin and 8-Br cAMPon the PHA-induced

Blastogenesis among Fresh or Cultured Low andHigh Density T Cells

60

TIME (min)

FIGURE 4 PGE2-induced elevations of intracellular cAMPamong low and high density T cells. 0.1 ,uM of PGE2 was

mixed with either low (0) or high density (0) T cells in thepresence of an inhibitor of phosphodiesterase. At the indicatedintervals, the cells were removed and assayed for intracellularcAMP. The results are presented as picomoles of cAMP/107cells and are representative of the actual values obtained forfive experiments (lines) or as the mean±SE relative increase(picomoles cAMP/107 cells at 6 min/picomoles cAMP/107 cellsat time 0) noted for five experiments. No significant change innonstimulated levels of intracellular cAMPamong low or highdensity cells occurred during 60 min of incubation.

cellular events occurring subsequent to increases ofintracellular cAMP. This is supported by the datapresented in Table II. Cholera toxin, another activatorof adenylate cyclase, and exogenous 8-Br cAMPaugmented PHA-induced proliferation among lowdensity and suppressed proliferation among highdensity T cells. Thus, both these agents mimicked theeffects of the PGEs on PHA-induced blastogenesisoccurring among the two T-cell populations.

Effect of PGE on cultured cells. It has been re-

ported that short-term (24-48 h) culture of PBMCyields a residual population which is resistant to thesuppressive effects of PGEs (18). This phenomenonis demonstrated by data presented in Fig. 5. The sub-sequent PHA response of T cells initially incubatedin 5% FCS for 4, 24, and 48 h was progressively more

resistant to PGE-mediated suppression; a process

which was abrogated if 24 ,ug/ml of cycloheximide, an

inhibitor of protein synthesis, was present during theinitial culture (Fig. 5, this concentration of cyclo-heximide inhibited protein synthesis of T cells by 90%,as determined by the incorporation of [3H]leucine intoTCA-precipitable protein; data not presented). Thedata presented in Fig. 6 indicate that this represents an

apparent, rather than an actual, loss of PGE-suppres-sible T cells. No significant difference was noted in theability of PGE2to suppress PHAreactivity among freshvs. cultured high density T cells. In contrast, whereasPGE2 induced a 5-8% increase in the reactivity offresh, low density cells, it caused a 30-40% augmenta-

Concentration of PHAPopulations

tested jug/ml... 0.3 1.0 3.0

Reactivity

Fresh T cellsLow density

Cholera toxin 19 t13 t108-Br cAMP T9 T10 116

High densityCholera toxin 153 147 1498-Br cAMP 127 124 122

Cultured T cellsLow density

Cholera toxin T38 T45 T338-Br cAMP 129 t48 T43

High densityCholera toxin 164 143 1498-Br cAMP 139 140 129

T-enriched populations were fractionated on a 5-step dis-continuous BSA density gradient. The reactivity of fresh lowdensity (fractions 1 and 2) and fresh high density (fractions 4and 5) T cells to three concentrations of PHAwas tested in thepresence of cholera toxin (5 ,g/ml, final concentration) and8-Br cAMP(1 ,uM, final concentration). The same low and highdensity T cells were then incubated for 24 h at 37°C in 5%FCS. The cells were then washed, resuspended in freshculture media, and tested for their PHA reactivity in thepresence of cholera toxin or 8-Br cAMP. Reactivity is pre-sented as percent augmentation (t) or as percent suppression(1) when compared with the reactivity of cells tested in theabsence of cholera toxin or 8-Br cAMP and represents themean of three paired experiments (i.e., the fresh and culturedpopulations were obtained from the same individual).

tion of reactivity among cultured low density T cells.These cultured low density cells were also more sensi-tive to the enhancing effects of cholera toxin and 8-BrcAMP, whereas the reactivity of cultured high densitycells was inhibited by both agents to an extent com-parable to that noted for fresh, high density populations(Table II). The culture-dependent change in the re-sponse of low density cells did not occur if cyclo-heximide was present during the initial incubation(Fig. 6).

DISCUSSION

Several investigators with a variety of systems haveindicated a dual role for PGEs in modulating biologicfunction. Bonta et al. (19) suggested that PGEs inhibitthe proliferative, but augment the exudative phase of

1192 J. D. Stobo, M. S. Kennedy, and M. E. Goldyne

-inav

0

0

E0-

200

100

0I

Q20

~ 0

60

0.3 1.0 3.0 0.3 1.0 3.0 0.3 1.0 3.0 0.3 1.0 3.0

PHACONC.(ug/ml)

FIGURE 5 PGE IodltliatioIn of the PHA reactivity of un-fractionated, culture(d T cells. T-eniriched populations wereincubated in either 5% FCS (bhars) or 5% FCS conitaining24 gg/ml of cyclohexamide (@). At the indicatedl intervals,the recovered cells were remiioved, wvashed, resuispenided infresh culture, mediated, anid tested for their reactivity to threeconcenitrationis of PHA in the presence of 0.1 ,M PGEI (0)or PGE2 (O). Results are presented as the mean±+SE. Percentaugmentation or percent suppression (reactivity in the pres-ence of PGE/reactivity in the presence of ETOH) notedfor twvo to six experimiients.

inflamnmatory reactionis. Whereas PGEs enhance pro-duction of collagenase by macrophages, they inhibittheir secretion of plasminogen activator (20).3 PGEsinhibit the liberation of some lymphokines (migrationinhibition factor, for example) but enhance the pro-duction of others (osteoclast-activating factor and afactor affecting vascular permeability [9, 10, 12, 21]).Our data suggest a similar dual role for PGEs inmodulating PHA-induced T-cell proliferation. PGEsaugment proliferation among a population of lowdensity T cells and suppress proliferation amonghigher density T cells. Novogrodsky et al. (22) alsosuggested the existence of populations of T cells differ-ing in their response to PGE modulation. Theseauthors demonstrated that PGE effected relativelyminor suppression among T cells responsive to somemitogens (PHA and concanavalin A) but marked sup-pression of T cells responsive to others (peanut ag-glutinin and soybean agglutinin).

At least three explanations might account for thenoted differences in the response of the T-cell popula-tions to PGE. First, it could simply be related tochanges induced by the fractionation procedure usedto obtain the T-cell populations. However, it was notpossible to alter the PGE-modulated response of T-cellpopulations by incubating them in different concentra-tions of BSA. Recombination of low or high density T

3 Werb, Z. Personal communication.

z0

I-

.iz

0

z

80O Low Density TO High DensityT

0.3 1.0 3.0 0.3 1.0 3.0

PHACONC.(.ug/mI)

FIGURE 6 PGE2 modulationi of the PHA reactivity amongculltured low., and high density cells. Low or high density Tcells were incuhated either in 5% FCS(hars) or in 5% FCScontaining 24 ,ug/ml of ecvlohexamide (a). At the indicatedintervals, the recovered cells were washed, resuspended infresh medlia, aind tested for their reactivity to three concentra-tions of PHA in the presence of 0.1 ,uM PGE2.Results are rep-resented as mean-+-SE. Percent augmentation or percentsuppression (reactivity in the presence of PGE/reactivity inthe presence of ETOH) noted for two to four experiments.

cells with B cells and monocytes did not substanitiallyalter their response to PGEs, although it did bluntPGE-meldiated suppression among the high densitypopulations (47% suppression among mixed popula-tions vs. 60% suppression among high density T cellstested alone). Additionally, high density T cells did notalter the PGE-mediated response of low density cells,at least when compared with the effects of B cells andmonocvtes. When considered together, these datasuggest that the distinct reactivity of fractionatedT cells to PCEs reflects intrinsic differences relevantto their response as they exist among whole PBMC.

The second explanation which might be pertinent tothe different responses to PGEs concerns the ability ofPGEs to modulate intracellular levels of cyclic nucleo-tides. Because agents which elevate intracellularcAMP inhibit T-cell proliferation, we hypothesizedthat PGE would increase intracellular cAMP amonghigh but not among low density T cells. Instead, PGE2initiated comparable absolute and identical relativeelevations of this cyclic nucleotide among both popula-tions. In data not presented, it could be demonstratedthat PGE2 also induced comparable relative increasesin cANIP among both populations when determinationswere performed in the absence of an iiihibitor of phos-phodiesterase. This result rules against possible grossdifferences in the catabolism of cAMPexisting amongthe two T-cell populations. Most importantly, themodulating effects of the PGEs on both T-cell popula-tions could be mimicked by another agent capable ofactivating adenylate cyclase (cholera toxini) as well as

Prostaglandin E-induced Immunoregulation 1193

by exogenous 8-Br cAMP. In preliminary experiments,there was no difference in PGE2-induced activation ofcAMP-dependent protein kinease among the T-cellpopulations.4 We did not assay for other metabolicevents which may be important in determining im-munocyte activation (intracellular cyclic guanosinemonophosphate, influx of divalent cations, and alter-ations among distinct pools of intracellular cAMP).Nonetheless, we feel that these data are consistent withthe thesis that the differential response of T-cell popu-lations to PGEis related to intracellular events occur-ring subsequent to elevations of intracellular cAMP.Novogrodsky et al. (22) also noted that T-cell popula-tions responded differently to agents which increasedintracellular cAMP.

The third explanation for the observed results couldbe that the differential response to PGEs is not a con-stant feature of distinct T-cell populations but rathera variable property related to different cell-cycle stagesexisting within a single T-cell line. Wecannot rule outthis possibility. Clearly, the responses of other homo-geneous cell lines-to PGEshave been observed to varywith cell-cycle stages (23). The experiments outlinedin Fig. 6 do not directly bear on this point becausenonstimulated cells may not move from one stage of thecell cycle to another. These experiments simply indi-cate that the differential response of low and high den-sity cells to PGEs is consistent even after they havebeen incubated for 24 (Fig. 6) or 72 (data not shown) h.Even if the differential PGE responsiveness reportedhere is related to a function of the cell cycle, it does notdetract from the notion that PGE is a modulator ratherthan a universal inhibitor of T-cell reactivity.

The experiments outlined in Fig. 5 emphasize theneed for considering this concept of modulation ininterpreting data concerning PGE-mediated regulationof heterogeneous cell populations. Goodwin et al. (18)recently reported that T-cell reactivity among culturedPBMCwas resistant to PGE-mediated suppression.The data presented in Fig. 5 confirm this. However,this only represents an apparent resistance arising froman increase in the enhancing effect of the PGEs on themitogenic response of low density T cells rather thanany real loss of PGE-suppressible high density cells(Fig. 6, Table II). In other words, it is crucial to con-sider the possibility that net PGE-mediated effects onwhole cell populations may represent the algebraicsum of different responses occurring among individualsubpopulations.

Recently, there has been much interest in the im-munosuppressive effects of PGEs. Likewise, recentdata suggest that excessive production of PGEs byadherent cells may be etiologically related to clinically

4Kennedy, M., and J. Stobo. Unpublished observations.

apparent T-cell dysfunction (24). Our data indicatesthat PGEs are not universally suppressive for T cellsand suggests that their immunoregulatory capabilitiesmay depend on the functional T-cell populations as-sayed. The pertinence of this in vitro modulation to invivo biologic consequences of PGE-mediated inter-actions between macrophages and regulatory or effec-tor T-cell populations remains to be determined.

ACKNOWLEDGMENTS

We thank Dr. John Pike for his generous gifts of PGE, andPGE2; Dr. Henry Bourne for the cholera toxin; and Dr. H.Sheppard of Hoffinann-LaRoche, Inc., for the inhibitor ofphosphodiesterase. The expert technical assistance of Ms.Marianne Newvton is gratefully acknowledged.

This work was supported by U. S. Public Health Servicegrants AI 14014 and Al 14752.

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4. Murota, S-I., M. Kawamura, and I. Morita. 1978. Trans-formation of arachidonic acid into thromboxane B2 by thehomogenates of activated macrophages. Biochim. Bio-phys. Acta. 528: 507-511.

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