Microbiol.

Immunol.,

39(10),

809-815,

1995

Differential Prostaglandin Murine

Hiroshi Department

Effects

of

Prostaglandin and Cell

and Kikuo Sciences,

ESH1SS and Differentiation Line,

Onozaki* Nagoya City University, Nagoya, Aichi 467, Japan

ESH2SS on Growth Myeloid Leukemic

Hayashi, Faculty

of

M1

Sawamura, of Hygienic

Hidetoshi Chemistry,

of Pharmaceutical

Received

March

10,

1995;

in revised

form,

May

26,

1995.

Accepted

June

28,

1995

NH

AbstractNS:Effects leukemic inhibited cells into cell nor line

of prostaglandins M1 were the cells studied.

(PGs)

of

the

E series

on

growth

and the

differentiation growth of M1 the not

of cells.

murine

myeloid

PGESH1SS, but not effect by interleukin

PGESH2SS, inhibited

PGESH2SS neither of M1 effect on the

augmented

antiproliferative induced

of PGESH1SS. augmented PGE, 6. PGESH2SS, however, cAMP only level did

differentiation any whereas

macrophage-like

exhibit cells,

differentiation. no effect. These

PGESH1SS a marked caused results however, numbers or of indicate revealed receptors or also a role that M1 that

increase cells there low are

in intracellular able no to respond

in M1

PGESH2SS had PGESH1SS bind that the cells

to PGESH1SS. Radiolabeled in M1 for cells, suggesting

ingexperiments, express iloprost, low

was affinity

specific receptors the

binding specific growth of

very

PGESH1SS. Stable cells. These

agonists

of PGISH2SS, suggest that

cicaprost as

carbacyclin,

potently in the

inhibited differentiation

of M1

findings lineage

PGESH1SS as well

PGISH2SS may play

monocyte-macrophage

cells.

NH

Key

wordsNS: PGESH1SS, Myeloid PGISH2SS,

leukemic

cell,

Cell

growth

Prostaglandins active tuallyall PGs, shown in Refs. compounds tissues especially

(PGs) produced from all the

are

a family by many

of cell fatty

biologically types acids have in vir (30). been

polyunsaturated E the For series immune example,

(PGE1/E2), response PGE2 (IFN-),

to down-regulate 9 and 27).

(reviewed inhibits the

interleukin factor liferationof and suppresses

2 (IL-2), (TNF-) murine natural inhibits and

interferon- lymphotoxin and killer human (NK)

tumor

necrosis and 6, 8, (10). of as well pro 12), In IL-1 as can

production T cells cell (1, activity

addition, and tumor

PGE2

macrophage factor of Ia of IgG2a PGE2 isotype (TNF) (31). IFN- Fc

production (20, 21),

necrosis expression the

macrophage also CD8+ cells IL-4 increase

However, receptors on the of of factor

PGE2 on

expression and

human

lymphocytes (5, on 41). In

receptors

WEHI-3 effect of and

addition,

potentiates switching

receptors on the cell surface which similarly recognize PGE1and PGE2(2, 30). PGE2 (PGE1) receptors are pharmacologicallysubclassedinto three types, EP1 EP2 and EP1 3), and these subtypesare suggestedto be dif (2, ferentin their signal transduction; i.e., they are pre sumedto couple to stimulationof phospholipaseC, and stimulation or inhibition of adenylate cyclase, respec tively(2, 11,32, 40). Recently,cDNAs for each type of receptors have been cloned (13, 34, 37). We have observed that murine myeloid cell lines (M1) were inducedinto macrophage-likecells by a vari etyof cytokines, includingIL-1, IL-6,TNF and IFNs(2325). During previous studies we investigatedthe possi bleinvolvement of PGE in cytokine action (26). In this study, we report the differential actions of PGE1 and PGE2on growth and differentiationof M1 cells. Materials and Methods

immunoglobulin IL-2-induced colony effects suppressive involved of these and effects of

B cells

enhances macrophage 29). limited cell types Many *Address Hygienic City Co., Thus, to

production stimulating PGE2 activities, interactions are to Dr. mediated Kikuo on

granulocyte (28, are not

(GM-CSF) function according other through

immune but vary with

to the

cytokines. specific of

correspondence Chemistry, 3-1 Faculty Tanabe, Tsuruga 914,

Onozaki,

Department Sciences, Aichi 467, Nagoya

of Pharmaceutical Mizuho, Institute Nagoya,

Materials. PGE1, PGE2, RPMI 1640medium,polymyx inB sulfate and bovineserumalbumin(BSA, fractionV) were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). PGI2 and carbacyclin were from Cay manChemical Co. (Ann Arbor, Mich., U.S.A.). Ilo - prost, cicaprost and sulprostone were generous gifts
Abbreviations: buffered saline; BSA, bovine serum albumin; FBS, fetal bovine PBS, phosphate serum; IL, interleukin; LPS, lipopolysaccharide;

University, Present Ltd.,

Japan. Toyobo

address: Tsuruga

of Biotechnology,

Japan.

PG, prostaglandin.

809

810

H. SAWAMURA

ET AL

from Dr. F.M. McDonald of Schering (Berlin, Germany). Fetal bovine serum (FBS) was obtained from Bocknek (Toronto, Canada), latex particles (diameter, 0.81 E.tM) from Difco Laboratories (Detroit, Mich., U.S.A.) and 5,6-3H(N)-prostaglandin (specificactivE, ity; 2,220.0 GBq/mmol) from Dupont (Boston, Mass., U.S.A.). Purifiedhuman recombinantIL-6, expressedin Escherichia coli, was kindly provided by Dr. Y.Akiyama (Ajinomoto Co., Ltd., Yokohama,Japan). PGE,, PGE2,PGI2and carbacyclinwere dissolved in ethanol at 10-3M. Cell cultures. Ml cells were provided by Dr. K.S. Akagawa of the National Institute of Health (Tokyo). The M1 cell clone, M1/436-7 differentiates into macrophages but not neutrophils (16). A subclone of M1/436-7, Ml-3b-a, which differentiates in response to IL-6, was cloned by limiting dilution and used in this study (25). Ml cells were maintained in RPMI 1640 medium containing 10 mm HEPES (pH 7.4), 100 units/ml of penicillinG and 100 tg/liter of streptomycin and 5% heat-inactivatedFBS. Proliferationof Ml cells was determined by counting the viable cell number using a hemocytometeror by MTT methods (22). TIG1, a human lung fibroblastcell line, and P815, a murine mastocytomawere obtained from the Japanese Cancer Research Resources Bank-Cell (Tokyo). These cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivatedFBS, HEPES and antibiotics. Assayfor differentiationof M1 cells. The differentiation of M1 cells was determined by measurement of phagocyticactivity.asdescribedpreviously(23). Briefly, 100 i of Ml cell suspension (2.5 X 105 cells/ml) in RPMI 1640 supplementedwith HEPES, antibioticsand 5% heat-inactivatedFBS were cultured in wells of flatbottomedmicrotiter plates(Falcon,N.J.,U.S.A.)at 37 C in CO, in air. Although the medium did not contain endotoxin by the Limulus amoebocyteassay (sensitivitylimit of 0.1 ng/ml), we usually added polymyxin B (5 tg/ml) to culturesto inhibitthe effect of <0.1 ng/ml endotoxin that is able to induce cell differentiation.After 3 days of culture, cells were incubated with 0.5% polystyrene latex particles for another 15 hr. Then, the percentageof cells ingestingmore than 10 particleswas determinedby counting more than 200 cells in a hemocytometer. CyclicAMP assay. Four hundred tl of Ml cells (4 X 106 cells/ml) were incubated in RPMI 1640 supplemented with 5% FBS with or without PGEs at 37 C for 15 min. Cells were collected by centrifugation( 200 X g, 5 min), and incubated with 400 tl of ice-cold 5% (w/v) trichloroaceticacid at 4 C for 30 min. After centrifugation,the supernatantswere extracted 3 times with 4 volumes of water-saturated ether. Levels of cAMP were then measuredby radioimmunoassay usinga CAMP

assay kit from Yamasa Shoyu Co., Ltd. (Chiba, Japan). cAMP contents of TIG-1 cells were measured as described previously(35). Briefly,TIG-1 cells in a subconfluent state were cultured in 6-well culture plates (Falcon) for 30 min at 37 C. After aspiration of media, cAMPwas extracted with 400 [l of 0.1 N HC1for 30 min and levels of cAMP measured as described above. j'H]PGE, binding assay. M1 or P815 cells were washed twice with binding buffer (Ca", Mg'-free Hanks' solution, pH 7.2, containing 0.25% BSA) and then resuspended with binding buffer. Cells (4 X 106) were incubatedwith [3H]PGE, the absence (total bindin ing) and presence of the unlabeled PGE, (nonspecific binding)for 20 min at 37 C. After incubation,150 .tlof aliquot was collected and filtered through glass fiber filters (GC 50, Advantec, Tokyo), which were washed twice with ice-cold washing buffer (phosphate-buffered saline [PBS], pH 7.2, containing 0.25% BSA), dried and taken up in 5 ml of aqueous counting scintillant, ACS II (Amersham Co., Ltd., Buckinghamshire,U.K.). Radioactivity countedin a liquidscintillation was counter (LSC1000, Aloka Co., Ltd., Tokyo). Specific binding was determinedas the differencebetween total and nonspecificbinding (determinedin the presence of a 1,000fold excessof unlabeledPGEs). In competitionexperiment,cellswere incubatedfor 20 min at 37 C with 10-9M [3H]PGE, the presence or absence of increasing conin centrationsof unlabeledPGE, or PGE2. Statistics. Each experiment was performed 3 times. Data are expressed as the mean standard deviation of triplicate experiments. As shown in Table 1, the twotailed Student'st-testfor comparisonof unpairedsamples was performed. Results Effects of PGEs on the Growth of MI Cells We determined the effects of PGE, and PGE2on the growth of murine myeloid leukemic Ml cells. Cells were cultured for 3 days in the presence of PGEI or PGEI at varying concentrations. The results in Fig. 1 demonstratethat PGE, inhibitsthe growth of Ml cells in a dose-dependent manner. PGE2,however, exhibited inhibition only at the concentration of 10-5 M. This growth inhibitionmay be caused by a nonspecificeffect. Vehicle (ethanol) alone did not exhibit any effect on cellproliferation.In order to determinewhether there are synergisticor inhibitoryeffects between PGEI and PGE2, cells were culturedwith varying concentrationsof PGEI or PGE2 (10-9-10-5 M) in the presence or absence of PGE2 PGE, (10-6M),respectively.There were no synor ergisticeffectsbetweenPGE, and PGE2 (data not shown).

SELECTIVE

EFFECTS

OF PGE1 ON M1 CELLS

811

Fig. were PGE2

1.

Effects

of PGE1 for 3 days

and

PGE2

on growth in the

of M1

cells. of PGE1

M1 cells () or cell is the

Fig. tionin 30min 2. Effects M1 at cells. 37C in of PGE1 M1 the and cells presence After as the described mean PGE2 (1.6~106 of PGE1 on intracellular cells) () the were or PGE2 cAMP incubated ( ) of forma for at var cAMP Each

cultured ()

at 37C

presence After

at various was determined of triplicate

concentrations. by MTT

culture, Each

viable value

number mean}S.D.

method.

determinations.

iousconcentrations. were determined represents

incubation, in with "Materials range

contents and Methods."

Table cells

1.

Effects

of

PGE1

and

PGE2

on

differentiation

of

M1

value tions.

of

triplicate

determina

induced

by IL-6

exhibited induced pmol/105 cells. PGE2 intracellular PGE1 M1 absence phagocytic Methods." a) b) Percent Significant of phagocytic to medium; cells}S.D. *P<0.05, (n=4). **P<0.01. cells were cultured at 37C or with PGE2. IL-6 After (50ng/ml) 3 days in the of culture, and cAMP of 3B). the PGE1 Only or presence activity of PGE1 was at

quite only cells

a weak a two-fold versus

augmenting increase; PGE2 (10-5M):

effect; medium:

at

10-5M

it

0.21}0.02

0.42}0pmol/105

(10-6M) cAMP 10-7-10-5M accumulation was not a slight

neither

augmented of In M1

nor cells

inhibited induced intracellular by to 5~10-7M 10-6M observed to determine

the by

accumulation (Fig. in affected M1 by 3A). cells PGE2

addition, induced at up

determined

as described

in "Materials

(Fig. at

augmentative of 10-5M. were PGEs

effect In due on fibroblast in Fig. accumulation manner. 4, order to

was

concentration these the effects cell

whether PGE2,

differences of type, As these human shown

inactivation accumulation cell PGE1 line and TIG-1,

of

cAMP

Effects of PGEs on Differentiationof M1 Cells It is known that differentiationof M1 cells accompa niesthe inhibition of cell growth (17). Thus, we inves tigatedwhether PGEs induced differentiation of M1 cells into macrophagesas assessed by phagocyticactiv ity.PGE1 or PGE2alone at 10-6M did not induce dif ferentiation(Table 1). We have shown that IL-6 is a potent inducer of differentiationof M1 cells (25). PGE1 augmentedthe differentiationactivity of IL-6 (Table1). PGE2,however, only slightly augmented this differenti ation.Adhesion of M1 cells to the plate induced by IL-6 was also augmented by PGE1 but not by PGE2 (data not shown). Effects of PGEs on Intracellular Levels of cAMP The effects of these PGEs on the intracellularcAMP level were studied. As shown in Fig. 2, PGE1at more than 10-7M caused a marked and dose-dependent increase in cAMP levels in M1 cells. In contrast, PGE2

of were

another

investigated. intracellular

PGE2 cells

caused in

cAMP

in TIG-1

a similar

dose-dependent

Binding The tionsites ascertain Unexpectedly, was between presence of

of

[3H]PGE1 suggest PGE1

to M1 that but not

Cells there for are PGE2 assay specific in M1 recogni cells. performed. binding difference in the site To

results for this,

[3H]PGE1 no specific 5),

binding saturable i.e., no

was

[3H]PGE1 significant

observed total of

(Fig. and 1,000-fold to M1

nonspecific

binding of found. was of shown). to identify

(measured unlabeled In addition,

concentration cells of was [3H]PGE1

PGE1) no upon PGE1

[3H]PGE1

significant addition or PGE2 In binding of up

inhibition various to 10-5M we on were murine

observed unlabeled

concentrations (data able not

contrast, sites

specific cell line

PGE1 P815

mastocytoma

812

H . SAWAMURA

ET AL

Fig.

4.

Effects

of PGE1 lung 30min

and

PGE2

on cell

intracellular line TIG-1.

cAMP TIG-1 of PGE1 and

accu cells () the

mulationin were and incubated PGE2 of

human for After

fibroblast at 37C

in the cAMP as

presence was

( ). cAMP

incubation, determined represents

extracted

amount and

was value

described with

in "Materials range of trip

Methods."

Each

the mean

licatedeterminations.

Fig. cells with

3.

Effects

of PGE2 (A) M1

on intracellular cells were

cAMP for

formation 30min ( ) incubated ( )

in M1 at 37C or pres with Fig. were 5. Binding of [3H]PGE1 for 20min in the to M1 at 37C absence cells. with (total M1 cells (4~106 cells)

by PGE1. varying

incubated

concentrations of PGE2 (10-6M).

of PGE1 (B) M1

in the absence cells were

ence() various () of triplicate

concentrations PGE1 (5~10-7M). determinations.

of PGE2 Each

in the value

absence is the

or presence incubated increasing binding; ) excess as described concentrations or a pres of unlabeled in "Mate of of labeled [3H]PGE1

mean}S.D.

ence(nonspecific PGE1. [3H]PGE1 Methods."

binding; ) binding was

of a 1,000-fold determined

(data not shown). From the Scatchard analysis, 2 class esof PGE1binding sites were demonstrated:high and low affinitysites,Kd=2.93nM and 8.34nM with number 4,527/cell and 8,627/cell, respectively, are almost the same as those previously described by Yatsunami et al (39). In addition,unlabeledPGE1and PGE2 caused sig nificantinhibitionof [3H]PGE1 binding to P815 cells in a dose-dependentmanner. Effects of PGI2 and Its Agonists on the Growth of M1 Cells PGE1is known to bind to the PGI2receptor (4). To ascertain that the PGE1 response is mediated through the PGI2 receptor,growth inhibitoryactivitiesof PGI2and

rialsand

its agonistswere studied. PGI2 not inhibitthe growth did of M1 cells even at 10-5M(Fig. 6). However,stable ago nistsof PGI2,carbacyclin,iloprost and cicaprost, potent lyinhibited the growth of M1 cells (Fig. 6). In contrast, a stable agonistof PGE, sulprostone(EP1,EP3type ago nist)had no effect against M1 cells. The stable agonists of PGI2 also augmented differentiation the inducedby IL6 (data not shown). Discussion The present study shows that PGE1and PGE2exhibited

SELECTIVE

EFFECTS

OF PGE1 ON M1 CELLS

813

out cAMP

because levels

both in

PGE1 the

and

PGE2 fibroblast

increased cell A second because we have line

intracellular TIG-1 in a is con

human

similar that M1

dose-dependent cells are refractory PGE2. that (26). but PGE2 PGE2

manner. to

possibility they previous

PGE2

stitutivelyproduce lyobserved M1 M1 the cells cells,

However, not produced was which failed effects to

was

in unstimulated induced by IL-6 in

production

indomethacin, of PGE2,

completely inhibit of IL-6 the

inhibited antiprolif These did not

production

erativeand results contribute effects sitizationto Fig. cells 6. Growth inhibitory for activities 3 days at 37C of PGI2 in the or its agonists. of M1 PGE1 or Therefore, actions activities Honma of glandinsin ferentiationof thacin-mediated on of of suggest

differentiative that to IL-6 the and PGE2 the M1 PGE1 et M1 al

(26). PGE2

endogenously antiproliferative that by there constitutive between intrinsically is no

induced and

differentiative of of and to desen PGE2. PGE2

possibility production PGE1 due

differences cells and reported cells the cells are

were

cultured

presence

different

(, PGI2 sulprostone by the MTT

(), iloprost (). After method.

( ), cicaprost (), carbacyclin () culture, cell proliferation was determined Each value represents the mean}S.D.

PGE2. that production associated of with (15). by PGE2 is prosta the Indome PGE1 predomi cells in IL-6and dif

triplicate

determinations.

is closely by

dexamethasone was counteracted and

inhibition not by PGF1 in also observed the or

markedly different effects on the growth of and cAMPaccumulation M1 cells. There are many reports in showing that PGE1 and PGE2exhibit similar effects. The structureof PGE1and PGE2are quite similar. Com paredto PGE1, PGE2 possesses only one additional unsaturatedbond. Therefore, it is likely that the recep torsfor PGEs are able to bind both PGE1and PGE2. Currently receptors for PGEs are classified into three types:EP1,EP2and EP3(2, 3). All of these receptorsbind equally both PGE1 and PGE2. EP1 is thought to be responsiblefor the generation of inositol 1,4,5-triphos phate(IP3)and diacylglyceride (40). In contrast,EP2and EP3are thought to mediate the function of PGE1 and PGE2to increase or decrease intracellularcAMP levels throughregulation of adenylatecyclase activity,respec tively(11, 32). As shown in Fig. 1, PGE1at more than 10-8Minhibited the growth of M1 cells. The apparent increase in cAMP level was also observedwith PGE1at more than 10-7M. This difference may be due to dif ferentculture conditions. cAMP was determined after 30-min stimulation, while cell proliferation was deter minedafter 3 days of culture. As reported previously, M1 cell proliferationwas inhibitedby cAMP-generating agents, cholera toxin, dibutyryl cAMP, 8-bromo CAMP and forskolin(26). Therefore,the antiproliferative effect of PGE1seemsto be mediated by cAMP. In considering function, the effects of PGE1in M1 cells are consid eredto be mediated by EP2. However, as described above, EP2binds both PGE1 PGE2 and (37) thus negating this possibility. The possibility that the refractoriness of M1 cells to PGE2 was due to the inactivation of PGE2 was ruled

PGE2

but

PGF2,

nantlyproduced (14). induced previously, tributeto due cells to do the not the We

terminally the production (26). PGE2

differentiated of PGE2 as appear

differentiated endogenous

cells

However, did not

described to may since K. con be our et al,

differentiation. clones of

This M1

discrepancy cells used, (Onozaki,

different respond data). possibility growth PGE1 (EP1, exhibits (4). cicaprost, did

to dexamethasone

unpublished A and cells. PGE while to PGI2 iloprost M1 cells. third induces Recently, receptors PGE2

is

that

PGE1 and been

binds

PGI2

receptors of not M1 only

inhibition has EP2 weak Stable or

differentiation to also bind PGI2 binding of PGI2, the

shown but

EP3) or

receptor, activity

negligible

receptor and

agonists

carbacyclin, growth presumably quite unstable to (4). or most of

potently not exhibit and agonists, activity

inhibited such activity,

PGI2

because (2). possess Another negligible specific a stable nisticactivity lysuggest are One

it is chemically of potent agonist, activity PGI2 agonist for that agonist for M1 selective the PGI2 agonist

metabolically iloprost, for however, the

was EP1 exhibits and

reported receptor weak is the

cicaprost, for yet EP1 cell PGE

receptor (33), (2),

described and EP3

and exhibited

sulprostone, no ago

growth. effects of

These PGE1

results on M1

strong cells

mediated Recently,

through it has

PGI2 been of

receptors. reported that PGE1 caused production peptide a

marked and in edT

stimulation secretion of T-cell (18).

intracellular

cAMP

parathyroid leukemia In murine virus

hormone-related type I (HTLV-I)-infect

human cells

neuroblastoma

N1E-115

814

H. SAWAMURA

ET AL

cells,PGE, increasedintracellularlevelsof cAMPand IP3 formation(19). Although that studyexaminedthe effect of PGEs at limited doses of 10-6 Mfor the T cells and 10-5and 10-'m for ME-115 cells, PGE, exhibitedlittle effect. Furthermore, a stable analogue of PGIZ,carbacyclin, enhanced IL-1(3-induced iNOS mRNA levels in rat mesangial cells. In contrast, PGE2inhibited IL-1 action, and forskolin, an activator of adenyl cyclase, mimickedthe effectof prostacyclin not PGE2.Therebut fore, in mesangial cells prostacyclinand possibly PGE,, but not PGE2, were suggestedto enhancethe effectof IL1 throughgenerationof intracellularcAMP(36). Therefore, the differential effects between PGE, and PGE, are commonin various cell types,and presumablydue to the response through the PGIZ receptor. To ascertain the presence of specific receptors for PGEI on Ml cells, we examined ['H]PGE, binding. However,saturatingspecificbindingof ['H]PGE,was not observed in Ml cells. Under the same conditions, we were able to detect 2 classes of PGE, binding sites on murinemastocytoma P815 cells. The numbersand affinities of these specificbinding sites were almost the same as those previously described by Yatsunamiet al (39). Therefore, the inability to detect specific PGE, binding was not due to technicalproblems. There are possibilities of very rapid breakdownor internalizationof PGE, in Ml cells; however,we failed to observespecificbinding even at low temperature(4 C) or using a membrane fraction of Ml cells (data not shown). Presumably,Ml cells express extremelylow numbers of saturablePGE, receptors,or specificreceptors with extremelylow affinity for PGE,, that could not be detected by conventional binding assay. The affinityof PGE, to PGIZ receptors in various cell types was reported to be very low (below 1/10) compared with that of PGIZ. Ml cells are a good model of myeloidcell differentiation into macrophages. Murine bone marrow progenitor cells that lead to macrophagecolonies in semisolid agar were sensitive to PGEz, and this sensitivity was dependent on the maturation stage (38). PGEz preferentially inhibits the more immature cells of monocytemacrophagelineage(7). PGIZ alreadyknownto play was an importantrole in vascularsystems. Therefore,PGI2 as well as PGE, may also play an important role in the regulation of proliferation and differentiationof monocyte-macrophagelineage cells. Theauthors wishto thankKoichi Katoof Nagoya UniCity versity hisexcellent for technical assistance Dr.DeYang and for helpin preparing manuscript. this

References 1) Betz, M., and Fox, B.S. 1991. Prostaglandin E2 inhibits production of Thl lymphokines but not of Th2 lymphokines. J. Immunol. 146: 108-113. 2) Coleman, R.A., Kennedy, I., Humphrey, P.P.A., Bunce, K., and Lumley, P. 1990. Prostanoids and their receptors, p. 643-714. In Hansch, C., Sammes, P.G., Taylor, J.B., and Emmett, J.C. (eds), Comprehensive medical chemistry, Vol. 3, Pergamon Press, Oxford. 3) Coleman, R.A., Kennedy, I., and Levy, G.P. 1987. Evidence for the existence of three subtypes of PGE2 sensitive (EP) receptors in smooth muscle. Br. J. Pharmacol. 91: 323P. 4) Dong, Y.J., Jones, R.L., and Wilson, N.H. 1986. Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues. Br. J. Pharmacol. 87: 97-107. 5) ElMasry, M.N., and Rich, R.R. 1989. Prostaglandin E2selectively increases interferon gamma receptor expression on human CD8+ lymphocytes. J. Clin. Invest. 83: 1436-1440. 6) Ferreri, N.R., Sarr, T., Askenase, P.W., and Ruddle, N.H. 1992. Molecular regulation of tumor necrosis factor-a and lymphotoxin production in T cells: inhibition by prostaglandin E2.J. Biol. Chem. 267: 9443-9449. 7) Gentile, P., Byer, D., and Pelus, L.M. 1983. In vivo modulation of murine myelopoiesis following intravenous administration of prostaglandin E2. Blood 62: 1100-1107. 8) Goodwin, J.S., Bankhurst, A.D., and Messner, R.P. 1977. Suppression of human T-cell mitogenesis by prostaglandin: existence of a prostaglandin-producing suppressor cell. J. Exp. Med. 146:1719-1734. 9) Goodwin,J.S., and Ceuppens,J. 1983. Regulation of immune response by prostaglandins. J. Clin. Immunol. 3: 295-315. 10) Goto, T., Herberman, R.B., Maluish, A., and Strong, D.M. 1983. Cyclic AMP as a mediator of prostaglandin E-induced suppression of human natural killer cell activity. J. Immunol. 130:1350-1355, 11) Halushka, P.V.,Mais, D.E., Mayeux, P.R.,and Morinelli, T.A. 1989. Thromboxane,prostaglandin and leukotriene receptors. Annu. Rev. Pharmacol. Toxicol. 29: 213-239. 12) Hasler, F., Bluestein, H.G., Zvaifler, N.J., and Epstein, L.B. 1983. Analysis of defects responsible for the impaired regulation of EBV-induced B cell proliferation by rheumatoid arthritis lymphocytes: II. Role of monocytes and the increased sensitivity of rheumatoid arthritis lymphocytes to prostaglandin E. J. Immunol. 131: 768-772. 13) Honda, A., Sugimoto, Y., Namba, T., Watanabe, A., Irie, A., Negishi, M., Narumiya, S., and Ichikawa, A. 1993. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2subtype. J. Biol. Chem. 268: 7759-7762. 14) Honma, Y., Kasukabe, T., and Hozumi, M. 1979. Inhibition of differentiation of cultured mouse myeloid leukemia cells by nonsteroidal antiinflammatory agents and counteraction of the inhibition by prostaglandin E. Cancer Res. 39: 21902194. 15) Honma, Y., Kasukabe, T., Hozumi, M., and Koshihara, Y. 1980. Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells. J. Cell. Physiol. 104: 349-357.

SELECTIVE

EFFECTS

OF PGE, ON Ml CELLS

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