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Inhibitory effect of interferon-gamma activated ovine

umbilical vein endothelial cells on the intracellular

replication of Toxoplasma gondii

I.H. Dimier, D.T. Bout

To cite this version:

(2)

Original

article

Inhibitory

effect of

interferon-γ

activated ovine

umbilical vein endothelial cells

on

the intracellular

replication

of

Toxoplasma

gondii

IH Dimier*

DT Bout

CJF INSERM 93-09

Immunologie

des maladies infectieuses,

Équipe

associée Inra

immunologie

parasitaire,

UFR des sciences

pharmaceutiques,

31, avenue

Monge,

37200 Tours, France

(Received

21 December 1995; accepted 25

April 1996)

Summary ― Toxoplasma gondii

is an

obligate

intracellular parasite that is a

major

cause of abortion

and neonatal

mortality

in sheep. In

congenital toxoplasmosis, Tgondiifirst

invades the umbilical vein

endothelial cells and are then disseminated

throughout

the fetus. Treatment of ovine umbilical vein

endothelial cells with bovine recombinant

y-interferon (IFN-y)

blocked the

growth

of

T gondii.

Growth of the parasite was measured

by

3H-uracil

incorporation

18 h after the onset of the infection and

by

micro-scopic

enumeration of

parallel

cultures. This assay revealed that when the cells were

pretreated

with

IFN-y in

concentrations

ranging

from 0.15-1 250 U/mL, a

high degree

of inhibition of

T gondii

replica-tion was observed with the effect

being dose-dependent.

Maximum activation was achieved

by

incu-bating

with 625 U/mL

IFN-y and

no

activity

was present at 0.15 U/mL. This

technique

could be of

rel-evance as a first line of defense

against congenital

ovine

Toxoplasma

infection. Inhibition of

T gondii

replication

is due to a different mechanism from that

existing

in mouse

macrophages

and human

fibroblasts.

L

-Arginine-dependent

production of reactive

nitrogen

and oxygen intermediates was not

responsible

for the inhibition of

T gondii replication. Supplements

of five amino acids were able to

overcome the inhibition

partially

but

significantly.

The mechanism of the inhibition remains to be

elu-cidated.

ovine umbilical vein endothelial cell

/ Toxoplasma gondii

l IFN-y

Résumé ― Les cellules endothéliales de la veine ombilicale ovine activées par l’interféron y

inhibent la

multiplication

intracellulaire de

Toxoplasma gondü. Toxoplasma gondii

est un parasite

intracellulaire

obligatoire

à

l’origine

de nombreux avortements et de mortalité néonatale chez le

mou-ton. Dans la

toxoplasmose congénitale,

T

gondü

infecte dans un

premier

temps les cellules endothéliales

*

(3)

de la veine ombilicale avant de se disséminer à l’ensemble du foetus. Les cellules endothéliales de la veine ombilicale ovine activées par l’interféron gamma

(IFN-y)

recombinant bovin inhibent la

multipli-cation de T

gondü.

La

multiplication

du

parasite

est estimée par

incorporation d

3

H-uracile,

18 heures

après

l’infection et par numération

microscopique

de cultures

parallèles.

Les cellules

pré-incubées

avec de

l’IFN-yselon

une gamme de concentrations allant de 0.15 à 1250 Ulml montrent un fort

degré

d’inhibition de

réplication

de T

gondü

avec un effet

dose-dependant.

L’activation maximale est obtenue

avec 625 Ulmi

d’IFN-y,

aucune activité n’est décelée à 0,15 Ulml. L’inhibition de

replication

est due à

un mécanisme différent de ceux rencontrés dans les

macrophages

murins et les fibroblastes humains.

La

production

de dérivés nitrés et

oxygénés n’est pas responsable

de l’inhibition de

réplication

de T

gondii.

L’addition de 5 acides aminés lève

partiellement

mais

significativement

l’inhibition. Le mécan-isme d’inhibition reste inconnu.

cellules endothéliales de la veine ombilicale

ovine/Toxoplasma gondii lIFN-y

INTRODUCTION

Toxoplasma

gondii

is a

protozoan

parasite

that infects all warm-blooded

animals,

including

man

(Werk, 1985).

In the

farming

industry,

ovine

toxoplasmosis

is of

great

economic

importance,

because it causes

sheep

to abort

leading

to neonatal loss

(Bux-ton,

1990).

When

T gondii is

acquired by

a

previously unexposed

individual

during

preg-nancy, it can cross the

placenta

and may cause an acute infection in the

immunolog-ically-incompetent

fetus.

Cell-mediated

immunity

to

T gondii is

believed to be the

major

mechanism of resistance to

toxoplasmosis

and is medi-ated

through IFN-y

activation

(Suzuki

and

Orellana, 1988). Understanding

the mech-anisms of host resistance and

identifying

the

components

of

protective immunity

is an

important prerequisite

for the

develop-ment of novel and safe ways of

controlling

toxoplasmosis.

During congenital

toxoplasmosis,

infec-tion of the umbilical cord blood vessels is the

major

route of

potential

transmission to

the fetus.

Experiments

with human umbilical vein endothelial cells have revealed that the

anti-toxoplasma activity

of these cells can be stimulated

by

human

IFN-y (Suzuki

and

Remington, 1988).

It was considered

impor-tant to extend these results to the ovine model. In this

report

we observed that

bovine recombinant

IFN-y significantly

enhanced the

ability

of ovine umbilical vein endothelial cells

(OUVEC)

to inhibit the

growth

of

T gondii

and we tested the effect of oxygen intermediates and

tryptophan

on this inhibition.

MATERIALS AND METHODS

Cell-culture

Ovine endothelial cells were obtained from

umbil-ical cord veins

by digestion

with 0.2%

collagen-ase, as was

previously

carried out in the human model

(Wautier and

Wautier,

1984).

OUVEC were

cultured in RPMI 1640 medium

(GIBCO)

con-taining

10% fetal calf serum

(Flow),

2 mM

L

-glu-tamin

(GIBCO),

20 mM

Hepes (GIBCO),

100

U/mL

penicillin (Sigma),

100

N

g/mL streptomycin

(Sigma)

in 25-cm3flasks

(Nunclon).

The cultures

were maintained at 37 °C in 5%

C0

2

at 95%

humidity.

When confluence was reached, the pri-mary OUVEC cultures were

trypsinized (0.2%

trypsin

in 0.02%

EDTA)

and seeded into 96-well

plates (Falcon)

at a concentration of 2.5 x 104 cells/well for the

[

3

H]uracil

test or in 24-well

plates

(Falcon)

at a concentration of 5 x 104in 200

pL

for

microscopic

examination.

Production ofT

gondii tachyzoites

(4)

female Swiss

(OF1)

mice. For the in vitro infection of OUVEC,

tachyzoites

were harvested

by

peri-toneal

lavage

2 or 3

days

after

ip

infection into

mice, counted with an

haemocytometer

and added to the OUVEC culture within 3 h of

being

harvested at a concentration of 5 x 104protozoa

per well.

Assay ofT gondii

intracellular

replication

and effect of

IFN-y

When the OUVEC cultures were confluent, the culture medium was

replaced

with various

con-centrations of

IFN-y ranging

from 1 250-0.15 5

U/mL.

The cultures were incubated for 22 h and then washed twice.

Tgondiitachyzoites

were added to the OUVEC in 200

pL

of

seeding

medium

(5

x

10

4

tachyzoites/well)

and the

plates

were returned

to normal culture conditions. After 2 h, 2.5

N

Ci

5,6-[

3

H]uracil (Amersham, specific

activity 50 Ci

mmol-1)

were added to each well in 50

pL

medium and the cultures were incubated for a

further 16 h. This precursor is

incorporated

by

T

gondii

but not

by

the host cells because

only

the

parasite contains

significant quantities

of the

required salvage

enzyme uracil

phosphoribosyl-transferase. The incorporation of this

compound

is correlated with intracellular

growth.

Uninfected cultures were also labeled in every

experiment

and

consistently

incorporated <1% of the

radioac-tivity

found in the control cultures that were

infected but not treated with interferon. Prior to cell

harvesting,

the culture appearance was

checked to

verify

whether

T gondii-induced

cell

lysis

had started.

Non-incorporated [

3

H]uracil

and

tachyzoites

which could have failed to infect the

OUVEC were removed by two washes with

phos-phate-buffered

saline. The OUVEC were then

lysed by

the addition of 0.1 % sodium

dodecyl

sul-phate (Serva,

100

pL/well)

and, after 30 min at

room temperature, the nucleic acids were

pre-cipitated by the addition of 3 M trichloroacetic

acid

(UCB,

50

pL/ well).

The contents of the wells

were

deposited

on

glass

fiber filters with an auto-mated cell harvester

(Skatron)

and

radioactivity

was determined in an LKB liquid scintillation

counter using ACS II scintillation fluid

(Amer-sham).

The results were

expressed

as counts

per minute

(CPM).

For each

experiment,

controls included OUVEC, either

pre-incubated

or not with

IFN-y,

cultured in the absence of

T gondii.

The

radioactivity

level for these samples was always

around

background

level, thus

confirming

that

[

3

H]uracil incorporation

was

specific

to the para-site.

Effect of

N

G

MMA

on the

IFN-y induced

inhibition ofT

gondii replication

The

protocol

for the culture, activation and

infec-tion of OUVEC was as described above except

that various concentrations of NGMMA

(p-hydroxy-azobenzene-p’-sulfonate

salt; molecular

weight,

484.3;

Sigma), ranging

from 15-500

pM

and

freshly

made in

complete

RPMI, were added

along

with the

IFN-y (each

in 100

pL).

The final

concentration of

IFN-y

in the cultures was

100 U/mL. After incubation and

washing,

NGMMA

was

again

added to the cultures

immediately

before the addition of T

gondii tachyzoites (each

in 100

pL).

Measurement of OUVEC nitrite

production

The presence of nitrite in 24-h supernatants of

IFN-y-activated

OUVEC was determined

by

the

Griess reaction. Cell culture supernatant

(150 uL)

was added to 700

pL

of a

freshly-prepared

mixture

(50/50)

of 1°/ sulfanilamide

(Sigma)

in 1.2 N

hydrochloric

acid and 0.3%

N-1-naph-thylethylenediamine dihydrochloride (Sigma).

The

absorbance at 450 nm was determined after

incu-bation for 30 min in the dark. Nitrite

concentra-tions were calculated

by

reference to a calibration

curve

prepared by using

standard solutions of

sodium nitrite

(Prolabo)

made up in the same

medium as for the cell culture.

Effect of oxygen scavengers on the

IFN-yinduced

inhibition ofT

gondii

replication

OUVEC were cultured, activated and infected as

previously described, except that

oxygen-inter-mediate scavengers were added to the cultures

together

with

IFN-y (100 U/mL) during

the last 3 h of the incubation

period

and the scavengers

(5)

final concentrations were as follows: 2.23

mg/mL

superoxide

dismutase

(3

250

U/mg

0

2

-scav-enger),

1.91

mg/mL

catalase

(42

000

U/mg,

64

mg/ml, H

2

0

2

scavenger),

50 mM D-mannitol

(OH-scavenger),

1 mM DABCO

(0

2

-quencher),

10 mM benzoic acid

(sodium

salt,

OH-scavenger),

and 10 mM L-histidine

hydrochloride

(0

2

-quencher).

All were

purchased

from

Sigma

and

were made up

immediately

before use in

com-plete

RPMI medium. These concentrations and

conditions

successfully

blocked the

oxygen-dependent

inhibition of

Tgondii replication

in the

study

performed by Murray

et al

(1979).

Effect of

!-tryptophan

and other amino acids on the

IFN-y-induced

inhibition ofT

gondii

replication

OUVEC were cultured, activated and infected

as

previously

described, except that exogenous

L

-tryptophan (Sigma)

in

complete

RPMI was

added

along

with the

IFN-y (100 U/mL)

and was

then added

again immediately

before addition of the T

gondii tachyzoites.

The concentration

of the amino acids in the wells

ranged

from 0.5-1 000

pg/mL.

Statistical

analysis

Values shown are the means of

triplicate

sam-ples

with SE bars and are

representative

of at least four independent

experiments.

Statistical differences between the various groups were

assessed

using

the Student’s t test. The level of

significance

was set at 0.05.

RESULTS

Effect

of IFN-y on

T

gondii replication

in OUVEC

The in vitro treatment of OUVEC with IFN-y stimulates these cells to inhibit the

growth

of the intracellular

parasite

T gondii.

The treated cells become able to

prevent

the otherwise unchecked intracellular

replica-tion of

T gondii.

We demonstrated this

activity by

measuring

the

incorporation

of

[

3

H]uracil

by

the

proliferating parasites

and

through microscopic

observation of the infected cultures. In normal unactivated

OUVEC,

T

gondii incorporated

large

amounts of

[

3

H]uracil,

suggesting

a

rapid

and marked

proliferation

(fig

1).

In contrast,

[

3

H]uracil

incorporation

was

dramatically

reduced in OUVEC that had been

prein-cubated with

IFN-y (fig

1

). Maximal

activa-tion of

anti-Toxoplasma activity

was obtained with 625 U/mL of

IFN-y.

The

IFN-y-induced

inhibition was

dose-dependent,

with a minimum effective dose of about 0.15 U/mL

(fig 1

Microscopic

observation of the infected endothelial cells confirmed the results of the

isotope uptake

assay. In a

typical experiment

where the cultures were treated with 150 U/mL IFN for 18

h,

the number of

Toxoplasma

per endothe-lial cell was 8-16 per

parasitophorous

vac-uole in the controls and 1-2 per

para-sitophorous

vacuole in the treated cultures

(fig

2).

There was no

significant

difference however between the average number of infected cells in the untreated cultures

(25

±

1%)

and in the

IFN-!y-treated

cultures

(150 U/mL)

2 h

(27

± 2%)

and 18 h

(25

±

3%)

after the onset of

infection,

suggesting

that

IFN-y

had no direct effect on the T

gondii

infection. The effect of

IFN-y

treat-ment was to

markedly

decrease the num-ber of intracellular

Toxoplasma

as

com-pared

to the control cultures.

Effect

of N

G

MMA

on the

IFN!y-induced

inhibition ofT

gondii replication

and measurement of nitrite

production

For all tested concentrations

(15-500 pM),

N

G

MMA

had no effect on the

IFN-y induced

inhibition of

T gondii

replication by

OUVEC.

(6)

Effect of oxygen scavengers on the

IFN-!induced

inhibition ofT

gondii

i i

replication

None of the six oxygen scavengers tested

(Superoxide

dismutase, catalase,

D

-manni-tol,

DABCO,

benzoic acid and L-histidine

hydrochloride)

had any effect on the inhibi-tion of

Tgondii replication by

IFN-y activated

cells.

Effect of

L

-tryptophan

and other amino acids on the

IFN-y-induced

inhibition of

T gondii

replication

Supplementation

of the culture medium with

L

-tryptophan

did not overcome the

IFN-y-induced inhibition of

Tgondii replication.

In

additional

experiments,

concentrations of up to 1

mg/mL

were

tested,

with no

lessen-ing

of the inhibition observed

(fig 3).

In order to determine if the

inhibitory

mechanism of

Tgondii replication

in

IFN-y-treated OUVEC was

dependent

on another amino acid in the culture

medium,

the

remaining

19 amino acids were tested

sep-arately

as with the

protocol

used for

trypto-phan.

The results showed that five amino acids:

tyrosin, cystein, glutamic

acid,

arginin

and

aspartic

acid at concentrations >10

0 pg/mL

partially

but

significantly (P

<

0.05)

overcame the

IFN-y-induced

inhibi-tion of

T gondii replication (fig 3).

DISCUSSION

(7)
(8)

OUVEC possess a microbiostatic

capacity

and are able to

prevent

the otherwise unchecked intracellular

replication

of T

gondii after IFN-yactivation. Significant

inhi-bition of

T gondii replication

was obtained

at concentrations of 0.15-1 250 U/mL. Above a concentration of 20 U/mL

replica-tion was

completely

inhibited

(P< 0.05)

as

compared

with the control

cells, ie,

the par-asite infected the cell but did not

multiply.

It is

unlikely

that

IFN-y affected

the

T gondii

directly

because the cells had been washed before the

parasite

was added and there was no

significant

difference between the number of infected cells in the untreated cultures and in the

IFN-y-treated

cultures.

IFN-y

is a multifunctional

cytokine

with

species-specific immunomodulatory,

antivi-ral and

antiprotozoal

activities,

all of which

probably

function

by

different mechanisms.

Toxoplasma

is

capable

of

invading

and

repli-cating

in

nearly

all mammalian cells. Studies of

IFN-y-activated macrophages

and fibro-blasts have identified several

anti-Toxo-plasma

mechanisms. There are several

potential

mechanisms

by

which the inhibition of

replication

of

T gondii

may have been effected. One such mechanism was found in

activated

macrophages. By

means of

block-ing experiments

with a

competitive

inhibitor of

L

-arginine,

N

G

MMA

(Adams

et

al,

1990)

it was shown to involve

L

-arginine-dependent

production

of nitric

oxide,

with

subsequent

conversion to nitrite and nitrate

In contrast to mouse

macrophages,

the

L

-arginine-dependent production

of

nitro-gen intermediates does not seem to be a

significant

mechanism in the inhibition of T

gondii replication by

IFN-y-activated

OUVEC.

The second

putative anti-Toxoplasma

mechanism

investigated

was the effect of toxic oxygen radical metabolites

(Murray

et

al,

1979).

Oxidative metabolites have been

implicated

in the destruction of

T gondii on

the basis of studies

demonstrating

that the

capacity

of activated mouse

peritoneal

(9)

perox-ide is correlated with their

capacity

to kill this

parasite (Murray

et

al,

1979).

This

poten-tial antimicrobial mechanism seems

appli-cable to situations where intracellular

para-sites are killed but not to situations where the

parasites

are

only

inhibited from

multi-plying.

Reactive oxygen intermediates do not,

therefore,

appear to constitute the mechanism of inhibition for T

gondii

repli-cation in

IFN-y-activated

OUVEC.

Finally,

we tested the mechanism

by

which

IFN-y-activated

human fibroblasts inhibit T

gondii proliferation, namely

that of

tryptophan

degradation.

Pfefferkorn

(1984)

showed that

IFN-ystimulates

human fibrob-lasts to

produce

an enzyme,

indoleamine-2,3-dioxygenase,

that

degrades tryptophan,

thus

starving

T gondii of

this essential amino acid. We found that

surplus

tryptophan

had no effect on the

IFN-y-induced

inhibition of

T gondii by

cells,

despite testing

it up to the

physiologically-enormous

concentration of 1

mg/mL. Among

the 19 amino acids

tested,

five

(tyrosin, cystein, glutamic

acid,

aspartic

acid and

asparagin)

were observed to

par-tially

decrease the amount of inhibition. Induction of other amino acid starvation

by

synthesis

of

degradation

enzymes was

hypothesized

like

tryptophan degradation

mechanism.

However,

this mechanism does

not seem to be very efficient insofar as very

high

amino acid concentrations

(1

mg/mL)

only partially

affect the inhibition of

parasite

replication.

Thus,

amino acid

degradation

may not feature in the

repertoire

of mecha-nisms

by

which activated OUVEC exert an antimicrobiostatic effect.

The mechanism

by

which

IFN-y-activated

OUVEC inhibit

replication

remains to be elu-cidated. The fact that endothelial cells from

the umbilical cord can be stimulated to

exhibit

anti-Toxoplasma activity

invites addi-tional studies to determine the in vivo rele-vance of this

phenomenon.

This,

in turn, may lead to studies aimed at

blocking

the transmission of

T gondii from

mother to

fetus,

thus

contributing

to the

prevention

of ovine

congenital toxoplasmosis.

REFERENCES

Adams LB, Hibbs JB, Taintor RR, Krahenbuhl JL (1990)

Microbiostatic effect of murine activated

macrophages for Toxoplasma gondii. Role for syn-thesis of inorganic nitrogen oxides from L-arginine. J

Immunol 144, 2725-2730

Buxton D (1990) Ovine toxoplasmosis: a review. J R

Soc Med 83, 509-514 4

Murray HW. Juangbhanich CW, Nathan CF, Cohn ZA

(1979) Macrophage oxygen-dependent antimicro-bial activity. 11. The role of oxygen intermediates.

J Exp Med 150, 950-956

Pfefferkorn ER (1984) interferon-y blocks the growth of T gondii in human fibroblasts by inducing the host

cells to degrade tryptophan. Proc Natl Acad Sci USA 81, 908-912 2

Suzuki Y, Orellana M (1988) Interferon-y the major medi-ator of resistance against Toxoplasma gondii.

Sci-ence 240, 51-517 7

Suzuki Y, Remington J (1988) Dual regulation of

resis-tance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+T cells in mice. J Immunol 140,

3943-3949

Wautier MP, Wautier JL (1984) Culture de cellules endotheliales. In: Nouvelles techniques d’6tude d’hemostase et de thrombose (S Levy-Toledano,

ed), Les editions Inserm, Paris, 117-120 Werk R (1985) How does Toxoplasma gondii enter host

cells? Rev Infect Dis 7, 449-453

Woodman JP, Dimier IH, Bout DT (1991) Human

endothelial cells are activated by IFN-yto inhibit

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