MODULATION of LEISHMANIA (L) AMAZONENSIS GROWTH in CULTURED MOUSE MACROPHAGES BY PROSTAGLANDINS and PLATELET-ACTIVATING-FACTOR

Research

Paper

Mediators of Inflammation 3, 137-141 (1994)

Ttm role of endogenously synthesized PAF and prostaglandins on theinfectionofmousemacrophages byLetsbmanta(L)amazonensiswasinvestigated,as well

asthe possible correlationbetween the effects of these inflammatorymediatorswithnitricoxide production. It wasfound thatpretreatmentofmacrophageswith10- M

ofthePAFantagonists,BN-52021orWEB-2086,increased macrophageinfectionby 17 and 59%,respectively.The

cyclooxygenaseinhibitor, indomethacin(10 g/ml),

in-duced a significant inhibition which was reversed by

addition of PGE (10-3_{M)tothe culture medium. These} results suggestedthat the infection ofmacrophages by leisbmanla is inhibited by PAF and enhanced by prostaglandinsandthat these mediators areproduced by macrophages duringthis infection.This was confirmed

byaddition ofthese mediators to the culture medium

before infection; PAF (10-6, 10-9 _{and 10-1Z M) reduced} significantly theinfectionwhereas

_PGEz

(10- M)induced

amarkedenhancement.Thiseffect ofexogenous PAFon

macrophageinfectionwas reversedbythe twoPAF an-tagonists usedin thisstudyaswellasbythe inhibitor of

nitricoxidesynthesis, L-argininemethylester(100mM).

Takentogetherthe data suggest thatendogenous produc-tion of PAF and

_PGE2

exert opposing effects on

Lesbmana-macrophageinteractionand thatnitricoxide

maybe involvedinthe augmented destruction of para-sitesinducedbyPAF.

Key words: Leishmaniasis, Leishmania (L.) amazonensis, Macrophage infection, PAF, Prostaglandins

Modulation

of

Leishmania

(L.)

amazonensis

growth

in cultured

mouse

macrophages by

prostaglandins

and

platelet

activating factor

M. V.

C.

Lonardoni,

C. L.

Barbieri,

M.

Russo

and

S. Jancar

,cA

Department

of Biochemistry, Universidade

Estadual de Maringd, Parand;

Department

of Microbiology, Immunology and

Parasitology, Escola Paulista de Medicina,

So

Paulo;

Department

of

Immunology,

Instituto de Cincias Biomdicas, Universidade

de

S.o

Paulo,

Av.

Prof. Lineu

Prestes

2415,

CEP

05508-900,

S&o

Paulo, Brazil

ca_{Corresponding Author}

Introduction

SpeciesfromtheLeishmaniagenus.are protozoans which present two forms during their life cycle,

promastigotes which liveinthe digestivetractof the

insect vector and amastigotes, obligate intracellular parasites which replicate in macrophages of their vertebrate hosts.

It has been shown that lymphokine activated mouse macrophages present an increased nitrite

production which correlates with increased leishmanicidal activity ofthese cells.1-3 _It

seerns

_that production of nitric oxide (NO) is more important than superoxide inthe intracellular parasite killing. Leishmania destruction wasshowntobecompletely

reversed bystructuralanalogues of t-arginine which inhibit NO production.>3_{Conversely, parasitekilling}

can proceed normally in a macrophage cell line

deficient in the respiratory burst and Kupffer cells whichdo not displayan oxidativeburstin response

toLeishmania. However,Andradeetal.showed that

intheearly phase of infection,averylow number of

T cells is present at the site of inoculation of Leishmania in contrast to a massive influx of

macrophages which rapidly take up the parasites.

Thus, it isunlikelythatmacrophageactivation

medi-atedby Tcells wouldoccuratthis stageof infection. It is more plausible to think that macrophage re-sponsesinducedbytheparasitewould influence the course of the disease. Indeed, Barcinski etal. pre-sented evidence that granulocyte-macrophage

colony stimulating factor, a macrophage product,

increasestheinfectivityof Leishmaniaamazonensis

by protecting promastigotes from heat-induced death. A possiblerole forprostaglandinsinthe exac-erbation of the disease inL. major-infectedBALB/c

mice was suggested by Farrell and Kirkipatrick. It

has been shown that an increased production of PGE2, PGF,.=, LTC4,

TXB.

and

PGD2

occursduring the course of murine infection withL. donovani.9,1

The lipid inflammatory mediators derived from

arachidonic acid metabolism (eicosanoids) and PAF

(platelet activating factor) may exert important

autocrineeffectsonmacrophages. Prostaglandins of theEseries increasecANIP11q3_and

bythismechanism

theymodulate several macrophage functions.14 _PAF is an important amplifier of biological processes in

several cell types. Inmacrophages, PAF inhibits ex-pression of class II molecules,5 _{modulates IL-1}

pro-ductioninLPS-stimulatedmonocytes16_{andstimulates}

TNF production.7 _{To date, PAF involvement in}

Leishmania infection has not been reported.

The purpose of the present study was to

investi-gate the role of endogenously produced

prostaglandins and PAFin in vitroinfectionofmouse

macrophageswithL.(L.)amazonensis.Macrophages

of BALB/c mice were infected with L. (L.)

amazonensisamastigotes and the phagocytic index

was determined in presence and absence of PAF antagonists,WEB-2086orBN-52021, andaninhibitor

ofprostaglandin synthesis, indomethacin.The effect

ofPAF and PGE addedtothe culture medium was also investigated. The possible involvement of NO was studied by using t-arginine methyl ester (t-NAME) and an inhibitor ofNO synthesis.

Materials

and Methods

Animals:BALB/cmice(20-30g)and outbred Golden hamsters2-3months old fromourownanimal

facil-ities were used.

Isolation

_of

L. (L.)amazonensisamastigotes

_from

ham-ster

_foot

pads: L. (L.) amazonensis amastigotes (107

parasites/ml)weretransferred every4 to6weeksto

hamsters by inoculation into foot pads. Amastigote suspensions were prepared by homogenization of excised lesions in RPMI 1640 medium containing 10% foetal calf serum (FCS) with a Poter glass

homogenizer and theparasitesseparated by centrifu-gation at 1 400x g for 10 min.

Macrophagecultures and L. (L.)amazonensis

_infection:

Macrophages were collected in phosphate buffered

saline(PBS) fromperitonealcavitiesofBALB/cmice.

About 4 x105cells were allowed toattach to round

13mmglasscoverslips. The non-adherent cellswere

removed by rinsing the coverslips with PBS. Coverslips were placed in 16mm diameter wells of Costar plates containing 0.5 ml of RPMI 1640 plus

10% FCS, 100U of penicillin and 100/g of strepto-mycin per ml and kept in a 5% CO,. humid

atmos-phere at 37C. After 24 h, L. (L.) amazonensis

amastigotes were added to the macrophage

monolayersat a cellratioof 3 parasites/macrophage

at

37C.

At different times after infection, the

coverslips were washedwith PBS, the cellsfixed in

absolute methanol for 10min and stained with Giemsa, dried, mounted on glass slides and

exam-ined microscopically.

Resultswereexpressed byphagocyticindex which

is the product of the percentage of infected

macrophages times the average number of

amastigotes per macrophage.

Evaluation

_of

nitric oxide production: Nitrite is an oxidationproductformedbynitricoxideinan aque-oussolution.Thenitrite concentrationwasmeasured

in the culture supernatant media by the method previously described by Green etal. Briefly,

50/1

aliquots ofsamples intriplicatewere addedto

_50/1

ofGriessreagentin

96-well

fiat-bottomedplates.The absorbancewas readat 550nm(DynatechMR5000) after 10min ofreaction and NOconcentration was deduced fromastandardcurve usingconcentrations

from 1 to

_5/M

ofsodium nitrite in culture media. Drugtreatments: Indomethacin (10/g/ml) prepared

in Tris-HCl (1M, pH8.0) was added to the

macrophage culture medium 24h before infection.

The PAF antagonists,

WEB-2086

and BN-52021, wereaddedtothe culture medium 1 h before

infec-tionand every12h thereafteratconcentrations rang-ing from 10-5 _{to 10-TM. The diluent for BN-52021}

was provided by the drug company and

WEB-2086

was dissolved in PBS. The agonists PAF (10

-6,

10-9 and 10-12M) and

_PGE2

(10-5M) were added to the culturemediumatthemomentof infection. PAF and PGE,. were supplied in ethanol and were further diluted in saline containing 0.25% BSA and PBS,

respectively. The concentration of ethanol in the

culture medium neverexceeded0.1%. The effectof alldiluentsused wasassayed. At the concentrations

used, the drugs did not significantly affect

macrophageviabilityasassessedbythe

Trypan

blue

exclusion test.

Drugs and reagents: Cell culture medium (RPMI

1640),

indomethacin, t-glutamine, penicillin, strepto-mycinandN-omega-nitro-t-argininemethylester

hy-drochloride(t-NAME)wereallpurchasedfromSigma Chem. Co.(USA); PGE fromUpJohnCo. (USA) and PAF from Bachem (Switzerland). BN-52021 and

WEB-2086 were kindly supplied by Institut Henri

Beaufour (France) and Boehringer-Ingelheim

(Ger-many), respectively.

Statisticalanalysis:Student’s t-testforpaired samples

and analysis ofvariance were used to evaluate the significance of the data (p<0.01).

Results

Effect of

endogenous PAFandprostaglandinsonL. (L.)

amazonensis

_infected

macrophages: In orderto

inves-tigate the role ofendogenously generated PAF and

prostaglandins

in L. (L.)amazonensis macrophage

infection, themacrophages were treatedwith either thePAFantagonists(BN-52021 orWEB-2086) orthe

cyclooxygenase inhibitor (indomethacin) before

in-fection, and the phagocytic index was determined

48 h later. The two antagonists were added to the culture medium 1 h beforeinfection and every 12 h thereafter while indomethacin was added 24h before infection. The resultsobtained (Fig. 1) show that pre-treatment ofmacrophageswithBN-52021 or

WEB-2086

significantly increased the macrophage

infection (17.3 and

59.4%

increase, respectively).

Indomethacin,however,induced asignificant

inhibi-tion. Addition of

_PGE2

(10-5M) to indomethacin-treated macrophages reversed this inhibition.

modulated by lipid

800

700

600- 500-X

o

400-o

o

300-200

100-0

Treatment BN WEB Indo Indo+PGE

FIG. 1. Effect of endogenous PAF and prostaglandins on L. (L.)

amazonensis infected macrophages. Mouse peritoneal macrophages

treated with indomethacin(10#g/ml), BN-52021 (10-SM), WEB-2086

(10-M)and indomethacin +PGE (10-M) beforeinfection with L. (L.)

amazonensisamastigotes.Indomethacinand thePAFantagonistswere

addedtothe culture medium24and hbefore infection,respectively. Thephagocytic indexwasdetermined 48hlater.Thecontrolsrepresent the values obtained in non-treated macrophages. The results were

obtainedbycountingatleast 200macrophages perduplicatecoverslip.

Errorbarsshow standarddeviations of three experiments(*p<0.01

comparedwiththenon-treatedgroup).

Table 1 shows that

WEB-2086

increasedthe

infec-tiondose dependently, amaximumpotentiation

be-ing achieved with

10-SM,

although at 10-6 _and

10-7Mthe drugwas also significantly effective. The controlgroup treatedwiththe diluentsofthedrugs

did not differ from the non-treated group.

Effect of

exogenous PAF and

_PGE2

on L. (L.)

amazonensis

_infected

macrophages:The results

ob-tained with the PAF antagonists and indomethacin suggest that PAF and prostaglandins endogenously

generatedmodulate the in vitroinfection. In orderto

evaluate whether addition of PAF or PGE,. to the culturemediuminfluencethe infection, these

media-tors were added to the culture medium at the

mo-ment of infection and the phagocytic index deter-mined48 h later. The resultsobtainedshow thatPAF (10

-6,

10-9 _{and 10-12M) reduced the infection dose}

dependently whereas PGE,. (10-5M) induced a

marked enhancement (Fig. 2). In some experiments

PAFwasaddedtoculturemedium24h before

infec-tion. In this caseitcaused

26.5

and

74.6%

of

inhibi-Table 1.Effect ofWEB-2086 on theinfectionof macrophagesby L.(L)amazonensis

Concentration of WEB-2086

Timeafterinfection(h)

(M) 24 48 72

Control 249.31 +_9.03 408.91 +/-5.34 658.08+/-9.83

10-7 _{388.83+/-7.27* 505.06+/-21.80" 563.71 +/-11.85"}

(55.96) (23.51 (14.34)

10-6 _{412.97+/-5.80* 577.30+/-7.18" 722.26+/-8.80*}

(57.95) (36.08) (18.62)

10-s 492.43+ 14.71" 696.72+/-16.51" 850.87+/-17.90"

(88.34) (64.23) (39.76)

Phagocyticindex obtained in macrophages infected with L. (L)

amazonensis inthe absence(Control)orpresenceof WEB-2086, added to the culturemedium hbefore infection.Data represent

the mean +/-S.E.M. of three experiments in duplicate. *p<0.01 comparedwiththegrouptreatedwiththe diluent.Datainbrackets

representthepercentageofenhancement.

O 1 4OO Q.

0

Treatment 12

PAF (-log M) PGE

FIG. 2. Effect of exogenous PAFand _PGE2on L. (L.) amazonensis infectedmacrophages.Mouseperitoneal macrophagestreated withPAF (10-1,10-9and10-8M)and_PGE2(10 M)at the momentof infection withL. (L.)amazonensis amastigotesandphagocytic index determined

48hlater. Non-treatedmacrophages were usedascontrol.The results

were obtained by counting at least 200 macrophages per duplicate coverslip. Error bars show standard deviations of six experiments

(*p<0.01 comparedwiththenon-treatedgroup).

Table 2. Effect ofPAFantagonistsonthePAFinduced inhibition of

macrophagesinfectionbyL. (L)amazonensisamastigotes

Group Treatment AgonisP Phagocytic

(10-6_{M) index}

425.57+/-10.26

PAF 125.17+/-2.36*

III BN-52021 (10-SM) PAF 223.88+/-7.76**

IV WEB-2086(10-s M) PAF 181.95+/-4.44**

aThe antagonistsweregiven hbefore infection.

bPAF wasaddedatthemoment of infection.

CThe phagocytic index was determined 48h after infection of

macrophageswithL.(L.)amazonensis.

Data representthemean+S.E.M.of three experimentsinduplicate

*p<0.01 comparedwithgroupI. **p<0.01 comparedwithgroupI1.

tion at10-9_and

_10-6M,

_{respectively. At 10-12Mitwas}

nolongereffective. The controlgroups treatedwith

diluentsofPAF and PGE,.didnotdiffer from the non-treated group.

Effect of

PAFantagonists onPAFinduced inhibition

_of

L. (L.)amazonensis macrophage

_infection.

The ability ofPAF antagoniststoinhibitthe PAFinduced effect

250

200

X (1)

1

50-0

Treatment PAF PAF+L-NAMEL-NAME

FIG.3.EffectofL-NAMEonPAFinduced inhibition ofL.(L.)amazonensis

macrophageinfection.Mouseperitonealmacrophages pretreatedfor h withL-NAME (100pM) followedby administration ofPAF (10 M)at

the moment of infection with L. (L.) amazonensis amastigotes. Phagocytic indexwasdetermined48 h later. The resultswereobtained

bycountingatleast200macrophages pertriplicate coverslip.Errorbars

show standarddeviations of three experiments(*p< 0.01 compared with the non-treatedgroup).

was investigated. Macrophages were treated with 10-5_{MoftheantagonistsBN-52021or}

_WEB-2086,

_{1 h} beforeadditionofPAFat10-6_{Mconcentration tothe} culturemediumandthe phagocyticindexwas deter-mined 48 h later. Results presented in Table 2 con-firmed thatPAF induced amarked inhibition of the

phagocyticindexandshowed that both PAF

antago-nists were able to significantly reverse this effect.

Effect of

z-NAMEonPAFinduced inhibition

_of

L. (L.)

amazonensis macrophage

_infection:

The effect of i:

NAME was studied in combination with PAF. The

macrophages weretreated with

t-NAME,

1 h before

additionofPAF (10-6_{M) and}

parasitestothe culture medium and the phagocytic index was determined 48 h later. Controls were carried out bytreating the

macrophages with eithert-NAME or PAF alone. Fig.

3 shows that theinhibitoryeffect ofPAFonthe L. (L.)

amazonensismacrophageinfection wasreversedby

100mMt-NAME.Thisfigurealsoshows that t-NAME

increasedthe phagocyticindexofmacrophages

treat-edwith or withoutPAF. In addition, weassayedthe NOreleaseinthe supernatants. Theconcentrationof nitrites/nitrates inthese supernatants wasbelow the detection limitallowedbythe assayemployed(data

not shown). The efficacy oft-NAME to inhibit NO production was tested on macrophages from BCG infected mice. It was found that BCG-activated

macrophages released0.929+

_0.054

nmol of NO/105

peritoneal cells after 48h in culture medium andthat pre-treatmentofmacrophageswith 100mM

of t-NAME significantly inhibited this production

(0.402+ 0.018nmol/105 peritoneal cells).

Discussion

In leishmaniasis, as in other intracellular parasite

infections, macrophages are both the host and the effector cell. Severalstudiesfocusedontheinfluence ofT cells and their products on macrophages acti-vationwhichwould leadeithertohealingor exacer-bation of the disease (for review see Locksley and Scott18). However, few studies have analysed the consequences of the initial interaction between

macrophageandparasites on parasitemultiplication.

In the present workwe showed that treatment of

macrophages with indomethacin, an inhibitor of

prostaglandins synthesis, before infectionwithL. (L.)

amazonensissignificantlyinhibited parasite growth.

This result indicatesthat prostaglandinsare released

by macrophages during the infection and that they

favour parasite survival possibly by inhibiting

acti-vation of the macrophage. Since the effect of indomethacin was reversedbythe addition of

_PGE2

to the culture medium, it is likely that this

cyclooxygenasemetaboliteistheoneresponsiblefor

the observed effect. Ourfindings are in agreement with those reported by Buchmtller-Rouiller et al.19

by lipid

showingthatPGE inhibitstheleishmanicidal activity

ofmacrophages activated with ionophore and LPS.

That macrophages are able to produce

prostaglandins when infected with Leishmaniahas been shown by studies in vitro and in vivo.9’1

Prostaglandin E isknowntoexertnegativefeedback

onmacrophageactivation,2_{aneffectprobably}

medi-atedbyanincreased intracellularlevelofcal[P.19_In

ourmodel, it is probablethat prostaglandins

gener-ated by the infected macrophages inhibited their capacitytocontrolparasitemultiplication.In support ofthis assumptionit has been shown thattreatment

ofsusceptible BALB/c mice infected with L. major

with indomethacinsignificantlyinhibitedthe number ofmetastatic lesions. However, thiseffectmightnot

rely entirely on inhibition of macrophage function

since prostaglandins also inhibit lymphocyte

prolif-eration induced by Con A and parasite antigens.

To study the role of PAF on this infection, two

specific PAF antagonist, BN-5202121 or

XVEB-208622

wereadded to the culture mediumbefore infection withL. (L.)amazonensis.Thesetreatmentsincreased

thephagocyticindexindicating that PAFisgenerated

endogenously byinfected macrophages andin turn

it exertsamarked inhibitionofparasitegrowth.This was confirmed by experiments where PAF was added exogenously to culture medium of infected

macrophages. In this case, a clear dose dependent

inhibition of parasite multiplication was observed. We also showed that the doses of the PAF

antag-onistsusedinthis studywereeffectivetoantagonize PAF receptors in murine macrophages since both antagonists wereabletoreversethe inhibitory effect of PAF.

It is known that macrophages can generate PAF

whenproperly stimulated;17 _{however, toour}

know-ledge thisis the firstreport to showthat PAF exerts

such a relevant effect onleishmanial infection. Another substance that is recognized as a potent leishmanicidal agent in vivo and in vitro is nitric

oxide (for review see Liew and C0x23). Thus, we reasoned whether the inhibitory effect of PAF on leishmanialgrowthcouldbemediatedbyNO.Tothis endweaddedaninhibitorofNOsynthesis(t-NAME)

to infectedmacrophages inthe presence orabsence ofPAF. In bothsituationst-NAME increased parasite

multiplication. These results, although indirect,

stronglysuggestthe participationof NOastheactive

molecule thatmediatesthePAFinducedmacrophage

leishmanicidal activity.

The present work showed that PGE has a stimulatory effect whereas PAF has an inhibitory effect on L. (L.) amazonensis infection in mouse

macrophages. It also showed that theleishmanicidal

effect ofPAFismediatedbyNO.Itwillbe ofinterest

to measure the levels of PGE, and PAF released by

infected macrophages obtained from resistant and

susceptible strains ofmouse and also to determine

the effect of these lipid mediators on the course of

in vivo infection. References

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2. GreenSJ, MeltzerMS,Hibbs_JrJB, NacyCA. Activatedmacrophages destroy

intracellular Leishmania major amastigotes by t-arginine-dependent killing

mechanism._JImmunol 1990;144: 278-283.

3. MauelJ, RansijnA, Buchmiller-RouillerY. Killingof Leishmania parasites in

activatedmurinemacrophagesisbased t-arginine-dependentprocessthat

producesnitrogen derivatives.JLeuk Biol 1991;49:73-82.

4. ScottP,JamesS,Sher A. The respiratory burstis notrequired for killing intracellular and extracellular parasitesby lymphokine-activated macrophagecellline._EurJ

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5.CrockerPR,DavisEV,BlackwellJM.Variable expression of themurinenatural

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cutaneousleishmaniasis.AmJPathol1984; 114:137-148.

7. Barcinski MA, SchechtmanD, Quinta, etal. Granulocyte-macrophage

colony-stimulating factorincreasesthe infectivity of Leishmaniaamazonensisbyprotecting promastigotes from heat-induced death.InfectImmun 1992;60: 3523-3527.

8. FarrellJP,Kirkipatrick CE. Experimentalcutaneousleishmaniasis.II. Apossible

role for prostaglandinsinexacerbation of diseaseinLeishmaniamajor-infected

BALB/cmice.Jlmmunol1987;138: 902-907.

9. ReinerNE, Malemud_CJ.Arachidonic acid metabolismin murineLeishmaniasis (donovam): ex-vivoevidence for increasedcyclooxygenaseand 5-1ipoxygenase activityinspleencells. Cell lmmunol1984;$8:501-510.

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macrophagesinfected with Leishmania donovani:in vitroevidence for parasite-induced alterationsin cyclooxygenaseand lipoxygenase pathways.JImmunol 1985;134: 556-563.

11. GoodwinJS, CeuppensJ.Regulationof theimmuneresponse by prostaglandins. Jlmmunol1983;3: 295-297.

12. RinconM,Tugores A,Lopez-RivasA,etaL ProstaglandinE and theincreaseof

intracellular cAMP inhibitthe expression of interleukin-2 receptorsinhumanT

cells.EurJImmunol1988;18: 1791-1794.

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OKT3-inducedincrease incytoplasmatic free calciumintheJukartT cell line. Eur JImmunol1989;19: 1953-1958.

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inhibition ofhuman T-cellactivationbyPGE Jlmmunol1990;145: 2616-2625.

15. GehardBM,BazanHEP, BazanNG.BN52021 blocks PAF-mediated suppression of cellular immunity. In:Braquet P,ed. Ginkgolides--chemistry, biology, pharma-cologyand clinicalperspectives. Barcelona: Prous.Sci.Publish.,1988;703-729. 16. PignolB,HenaneS,Mencia-HuertaJM,Rola-PleszczynskiM,BraquetP. Effectof

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ACKNOWLEDGEMENTS.The authors thankMariadeFtima FariasP.Carvalhoand

DuniaRodriguez for technicalassistance.Thiswork supported byFundaqo de

Amparo Pesquisa do Estado de So Paulo (FAPESP) and Conselho Nacional de DesenvolvimentoCientifico Tecnol6gico(CNPq)from Brazil.

Received 22 November 1993;

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