In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma gondii.
ABSTRACT Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii.
Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination.
Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation.
Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.
In Vitro Effect of TNF-? and IFN-? in Retinal Cell
Infection with Toxoplasma gondii
Emmanuelle Delair,1Claudine Creuzet,2,3Jean Dupouy-Camet,2,3and
PURPOSE. Toxoplasma gondii is an intracellular protozoan par-
asite and the most common cause of infectious uveitis. This
study was conducted to evaluate the in vitro effect of tumor
necrosis factor (TNF)-? and interferon (IFN)-? in rat retinal
cells infected with T. gondii.
METHODS. Rat retinal cells, retinal pigment epithelial (RPE)
cells, and retinal Mu ¨ller glial (RMG) cells were in vitro infected
with T. gondii RH strain tachyzoites. Cultured cells were stim-
ulated with various concentrations of TNF-? and IFN-?. The
effect of TNF-? and IFN-? in T. gondii invasion and replication
between retinal cells was determined through two different
methods: measuring [3H]-uracil incorporation and counting
infected cells by microscopic examination.
RESULTS. Infection by T. gondii was lesser within RPE cells than
within RMG cells. IFN-? significantly inhibits [3H]-uracil incor-
poration in RMG and RPE cells (respectively, 35%, 83%, and
87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%,
30%, and 75% for RPE cells). TNF-? significantly inhibits [3H]-
uracil incorporation in RPE cells (23% and 38% inhibition at 1
and 10 ng/mL), but not in RMG cells. These results were
confirmed by confocal microscopic data. The percentage of
infected cells decreased from 20% to 7% after IFN-? stimula-
CONCLUSIONS. Both cytokines IFN-? and TNF-? inhibited T. gon-
dii replication in the RPE cells, whereas only IFN-? had an
anti-Toxoplasma activity within the RMG cells. The differences
in cytokine response may be the reason that RPE cells are less
efficiently infected by T. gondii than are RMG cells. (Invest
fests itself through exacerbations of chorioretinitis resulting
from the rupture of quiescent cysts in the retina. These cysts
contain bradyzoites, a form of the parasite with low levels of
metabolic activity. The cysts persist in the host tissues for years
without causing any local inflammatory reaction.2Some sur-
face antigens of the parasite are expressed and pass though the
oxoplasmosis is the most common cause of infectious
posterior uveitis.1Ocular toxoplasmosis typically mani-
cyst wall, maintaining full protective immunity and thus pre-
venting further infestation. However, the wall of a cyst some-
times breaks releasing the bradyzoites which, through an as yet
poorly understood mechanism, transform into tachyzoites, the
form of the parasite with high levels of metabolic activity, and
reactivate infection locally.3In response to the infection with
Toxoplasma gondii, the host organism sets up an immune
reaction, mainly of cellular type, via T lymphocytes—essen-
tially T lymphocytes CD8?—and cytokines, including IFN-?.4
The protective role of IFN-? and TNF-? on the reduction of the
toxoplasmic retinochoroiditis is clearly demonstrated.5
Some histologic analyses performed on the eyes of animals
affected by ocular toxoplasmosis made it possible to locate the
T. gondii cysts, mainly in the pigment epithelium and the inner
layers of the neurosensory retina.6The cell types in which the
cysts develop have not been clearly identified, but the cysts are
most often found close to a Mu ¨ller cell. Mu ¨ller cells support
glial cells whose extensions spread over the whole thickness of
the retina. These cells can present the antigen, and in vitro
express class II molecules of the major histocompatibility com-
plex after stimulation with IFN-?.7The retinal pigment epithe-
lium (RPE) is a single layer of cells overlying the photorecep-
tors and forming a selectively permeable barrier between the
neural retina and the highly permeable choroidal vessels. RPE
plays several roles in local immune response, expressing the
major histocompatibility complex (MHC) class II, expressing
the antigen to sensitized T cells, and producing various cyto-
kines and NO. The toxoplasmic retinochoroiditis is partly due
to the lysis of the retinal cells infected with the parasite and
partly to the adjacent inflammatory response in the choroid
and retina.8The role of cytokines in toxoplasmic retinocho-
roiditis has been studied in vivo in animal models, where it has
been posited that cytokine IFN-? plays an important role in
controlling the disease.5,9IFN-? is one of the major cytokines
of the adaptive immune response produced by activated T cells
on infection with various intracellular infectious agents (myco-
bacteria, virus, T. gondii). TNF-? has considerable importance
in the innate immune response to any infection and is as well
produced by activated T cells at levels that can also influence
intraocular immunity. It is accepted that host resistance to
toxoplasmic infection is due to IFN-? generated by innate
natural killer cells and adaptive CD4 and CD8 T lympho-
cytes.4,10Our work was designed to assess the ability of T.
gondii to invade rat retinal cells—pigment epithelium and
Mu ¨ller cells—and to quantify the proliferation of T. gondii
within these cells. The purpose of our study was also to analyze
the effect of the cytokines IFN-? and TNF-? on retinal cell
infection. We attempted to demonstrate that cytokines have an
effect on infection prevention and also on a possible defense
mechanism after infection. Nitric oxide (NO) production was
also measured during the infection.
MATERIALS AND METHODS
The cell cultures used in these experiments were primary cultures of
retinal cells from Lewis rats. These rats were killed by cervical dislo-
From the1Universite ´ Paris Descartes, Faculte ´ de Medicine, Ho ˆpital
Cochin, Service d’ophtalmologie, Paris, France;2Institut Cochin, De ´-
partement des Maladies Infectieuses, Biologie Comparative des Api-
complexes, Universite ´ Paris Descartes, CNRS, (UMR 8104), Paris,
France; and3INSERM U567, Paris, France.
Submitted for publication October 24, 2007; revised March 24,
June 26, and August 1, 2008; accepted February 27, 2009.
Disclosure: E. Delair, None; C. Creuzet, None; J. Dupouy-Ca-
met, None; M.-P. Roisin, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Emmanuelle Delair, Universite ´ Paris Des-
cartes, Faculte ´ de Me ´decine, Ho ˆpital Cochin, Service d’ophtalmologie,
27 rue du Faubourg Saint-Jacques, 75679 Paris Cedex 14, France;
Investigative Ophthalmology & Visual Science, April 2009, Vol. 50, No. 4
Copyright © Association for Research in Vision and Ophthalmology
cation between their 8th and 12th days of life. Their eyes were swiftly
enucleated in sterile conditions; immediately put in Dulbecco’s modi-
fied Eagle’s medium (DMEM) containing 4.5% glucose, 1% glutamine,
1% penicillin/streptomycin, 1% amphotericin B (complete DMEM); and
left overnight in the dark at room temperature.
The eyeballs were then incubated in 0.5 mL of complete DMEM
containing 0.1% trypsin and 70 U/mL collagenase for 1 hour at 37°C.
Next, they were put in a dish containing DMEM supplemented with
10% fetal calf serum (FCS) to inhibit the action of trypsin. Each eyeball
was then dissected under a microscope to isolate the pigment epithe-
lium and the neurosensory retina separately.
The neurosensory retinas were placed in 10-cm dishes (four to five
per dish) containing 5 mL DMEM with 10% FCS and then were broken
down with forceps into small fragments that were incubated at 37°C
with 5% CO2for 5 to 6 days in the same medium. The fragments were
then carefully removed from the dish and the cells grew to confluence.
RMG cell purity was checked by immunocytochemistry. Briefly, RMG
cells (passages 2 to 3, approximately 4 days of culture subsequent to
the last passage) appeared slightly positive for the marker GFAP (glial
fibrillary acidic protein) and highly positive for the marker vimentin.
The pigment epithelia in suspension were centrifuged at 1300 rpm
for 10 minutes. The tissue was dissociated by adding 0.1 mL trypsin per
eye to the pellet assisted mechanically by passing the contents of the
tube 10 times through a Pasteur pipette. The action of trypsin was then
inhibited by 5 mL DMEM with 10% FCS. The pellet was rinsed and
diluted in DMEM with 10% serum (2 mL for four to five retinas). This
solution was placed in 60-cm dishes containing 2 mL DMEM?10% FCS
(four to five retinas per dish) and incubated 4 to 5 days at 37°C in 5%
Preparation of T. gondii
The RH strain tachyzoites of T. gondii were propagated by intraperi-
toneal injection of IOPS/OF1 female mice. After 3 or 4 days of infec-
tion, the mice were killed and the tachyzoites recovered under sterile
conditions by injecting 10 mL of 9% NaCl solution in the peritoneal
cavity and removing the contents with the same syringe. The
tachyzoites were washed in PBS and centrifuged for 10 minutes at 2500
rpm. The pellet was then resuspended in culture medium and the
tachyzoites counted with a hemocytometer. Viability was assessed via
trypan blue exclusion. Parasites were 95% viable and samples con-
tained less than one host cell per 300 parasites.
Cell Culture Infection with T. gondii
Cell cultures were grown in DMEM containing 10% SVF. One hour
before infection, the medium was replaced by DMEM containing 5%
SVF. When they grew to confluence, the cells were infected with the
tachyzoites on 24-well dishes at a 5:1 ratio (250,000 tachyzoites per
50,000 cells). After 2 hours of infection, the supernatant was removed,
the medium changed, and the cultures rinsed and reincubated for a
variable period. In the last series of experiments, the cells were mas-
sively infected with a 15:1 ratio (750,000 tachyzoites for 50,000 cells)
to assess tachyzoite penetration 6 hours after infection. The control
experiments involved infection of NIH-3T3 human fibroblasts with T.
The cells were fixed in 4% paraformaldehyde, saturated with 10% FCS,
permeated with a 0.5% Triton solution, and stained with rabbit poly-
clonal anti-GFAP antibodies (glia-specific, dilution 1/100; Dako,
Trappes, France) for Mu ¨ller cells, with mouse monoclonal anti-pan-
cytokeratin antibodies for the pigment epithelial cells (dilution 1/50),
and with mouse monoclonal anti-Toxoplasma or rabbit polyclonal
anti-Toxoplasma antibodies. The second matching antibodies used
(anti-rabbit or anti-mouse; dilution 1/100) were stained with FITC or
TRITC. After they were rinsed, the preparations were mounted
(Vectashield; Vector Laboratories, Compie `gne, France) and examined
under epifluorescence and confocal microscopes. The invasion of the
different cellular types was quantified by counting the number of
infected cells and the number of intracellular T. gondii in 200 cells
randomly selected in each preparation. The purity of the cultures was
checked by simultaneously using the anti-GFAP and anti-pan-cytokera-
Radioactive Uracil Incorporation
The radioactivity emitted by [3H]-uracil incorporated in the T. gondii
DNA synthesis was measured to quantify tachyzoite proliferation
within the infected cells, as described by Creuzet et al.11The cultures
were infected with 250,000 tachyzoites per well (tachyzoite-to-host
cell ratio, 5:1) for 2 hours, washed with PBS to eliminate extracellular
tachyzoites, and incubated for various periods in DMEM containing 3%
dialyzed calf serum and 4 ?Ci/mL (5,6)-[3H] uracil. At the end of the
incubation period, the supernatant was eliminated, the cells were lysed
for 15 minutes in 1 mL PBS containing 1% sodium dodecyl sulfate (SDS)
and 1 mM uracil, and the nucleic acids were precipitated by adding
10% trichloroacetic acid (TCA). The contents of the wells were placed
on fiberglass filters (GF/C; Whatman, Sarl, France) and washed three
times with 5% TCA. The radioactivity was then determined with a
scintillation counter. For each experiment, wells without T. gondii
were included to check that uracil incorporation was specific to the
The cell cultures were stimulated with LPS (10 and 100 ng/mL), rat
recombinant IFN-? (0.1, 1, and 10 ng/mL), rat recombinant TNF-? (0.1,
1, and 10 ng/mL), alone or in combination, 72 hours before the
infection. After infection with T. gondii, the cultures were incubated in
DMEM containing 3% and 5% SVF (for uracil incorporation and immu-
nocytofluorescence, respectively) for 48 hours, without adding cyto-
kines. The effect of cytokines on the infection was assessed according
to two parameters: the penetration of tachyzoites within the host cells,
and the proliferation of tachyzoites within the infected cells.
Tachyzoite proliferation within the infected cells was quantified by
using two methods: uracil incorporation and analysis of the prepara-
tions by immunocytofluorescence. The preparations were analyzed 24
hours after infection for Mu ¨ller cells and 48 hours after infection for
pigment epithelial cells. The penetration into the host cells was studied
only in the Mu ¨ller cells, by analyzing the preparations 6 hours after
massive infection by immunocytofluorescence.
Quantification of NO Production
After 72 hours of cell culture stimulation with different cytokines, the
supernatants were collected to quantify nitrite production by a color-
imetric method based on the Griess reaction.12Put briefly, 100 ?L of
supernatant was added to 100 ?L of Griess reagent (1% sulfanilamide
and 0.1% naphthyl-ethylenediamine), and 10 minutes later the absor-
bance was measured at 540 nm. The nitrite level was quantified from
a standard curve constructed with different concentrations of sodium
nitrite. NO production was also measured 48 hours after infection.
Within each experiment, all conditions were repeated in triplicate
wells, and each experiment was performed three times. Data were
analyzed by nonparametric (Wilcoxon signed-rank test) and/or para-
metric methods (Student’s t-test, analysis of variance; StatView; SAS
Institute, Cary, NC).
Infection According to Cellular Type
The proliferation of tachyzoites within the host cells 24 hours
after infection was assessed by Uracil incorporation. The par-
IOVS, April 2009, Vol. 50, No. 4
Retinal Cell Infection by Toxoplasma gondii
asites grew within both cell types studied, retinal pigment
epithelium and retinal Mu ¨ller cells (Fig. 1). Uracil incorpo-
ration was not significantly different between Mu ¨ller cells
and NIH-3T3. However, this incorporation was significantly
less in the retinal pigment epithelial cells than in NIH-3T3
(25.4% ? 2.5%).
Cytokine Modification of the Infection
Proliferation of Tachyzoites within the Host Cell.
Analysis by [3H]-Uracil Incorporation. The pigment epithelial
cells were significantly less sensitive to infection with T. gondii
when stimulated beforehand with IFN-? or TNF-? (Fig. 2A).
These cytokines triggered a dose-dependent inhibition reaction
on tachyzoite proliferation. Inhibition was observed to in-
crease with cytokine concentration in the culture medium.
At 0.1 ng/mL, neither IFN-? nor TNF-? had an effect. The
effect appeared at concentrations of 1 ng/mL (30% inhibi-
tion with IFN-? and 23% inhibition with TNF-?), and was
highest at 10 ng/mL (75% inhibition with IFN-? and 38%
inhibition with TNF-?). IFN-? at 10 ng/mL concentration had
a higher inhibiting effect than did TNF-? at the same con-
centration. We observed a synergic effect between these
two cytokines, wherein adding TNF-? significantly increased
the inhibiting effect of IFN-?. LPS had no effect on
tachyzoite proliferation; however, it significantly increased
the action of IFN-?.
With regard to Mu ¨ller cells, IFN-?, but not TNF-?, had an
inhibiting effect on tachyzoite proliferation (Fig. 2B). IFN-?
significantly inhibited [3H]-uracil incorporation by RMG cells
(respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10
ng/mL). Neither TNF-? nor LPS showed any synergic effect
with IFN-? in Mu ¨ller cells. Thus, Mu ¨ller cells appeared to be
more sensitive to the inhibiting action of IFN-? than did pig-
ment epithelial cells.
Analysis by Immunocytofluorescence. The inhibiting ef-
fect of IFN-? on T. gondii infection was also confirmed by
microscopic confocal analysis (Fig. 3). The percentage of in-
fected cells was 20% in the absence of stimulation and 7% after
IFN-? stimulation (Fig. 4A). The number of intracellular
tachyzoites for 200 cells was significantly reduced when the
cells were stimulated beforehand with IFN-? (Fig. 4B).
Penetration of Host Cells by Tachyzoites. In the ab-
sence of cytokine stimulation, 39% of RMG cells were in-
fected and several tachyzoites entered the cell cytoplasm.
Tachyzoites’ capacity to penetrate Mu ¨ller cells is reduced by
infection. Different types of cells (human fibroblasts NIH-3T3 [3T3]
and RPE cells and RMG cells from Lewis rats] were incubated in DMEM
supplemented with 3% FCS and infected for 2 hours with tachyzoites
(ratio 1:5). [3H]-uracil incorporation (expressed in DPM) was assayed
24 hours after infection.
[3H]-uracil incorporation in retinal cells 24 hours after
tachyzoite multiplication among in-
fected retinal cells: RPE (A) and RMG
(B). Culture cells were stimulated 72
hours before infection with various
cytokines (IFN-? and/or TNF-? 0.1, 1,
or 10 ng/mL) and/or with LPS at 100
ng/mL. IFN-? and TNF-? were used at
a concentration of 10 ng/mL when
mixed with other cytokines. Cell cul-
ture without cytokine stimulation
served as the control. [3H]-uracil in-
corporation was assayed 48 hours af-
ter infection (ratio 1:5). *P ? 0.05;
**P ? 0.001.
Influence of cytokines on
1756Delair et al.
IOVS, April 2009, Vol. 50, No. 4
IFN-?. The percentage of infected cells and the number of
intracellular tachyzoites for 200 cells dropped from 39% to
19.5% and from 93 to 46, respectively, after the addition of
IFN-? (Fig. 5).
The NO production of the studied cells was very small in the
basal state when the cells were not stimulated (some 2 ?M).
NO production by both pigment epithelial cells and Mu ¨ller
cells increased significantly after stimulation with IFN-
??TNF-? or IFN-??TNF-??LPS combinations (Table 1). Nei-
ther IFN-? alone nor TNF-? significantly increased NO produc-
tion. The presence of T. gondii did not stimulate production
(no significant difference between control wells without T.
gondii and infected wells). In the second series of measure-
gondii. The proliferation of tachyzoites among RPE cells was assayed
by immunocytofluorescence 48 hours after infection (ratio 5:1), with-
out cytokine stimulation (A), and after stimulation with IFN-? at 10
ng/mL 72 hours before infection (B). After stimulation with IFN-?, the
number of infected cells and the proliferation of tachyzoites within the
cells were lower. The ability of tachyzoites to penetrate RMG cells was
evaluated 6 hours after massive infection (ratio 15:1), without cytokine
stimulation (C, D) and after stimulation with IFN-? at 10 ng/mL 72
hours before infection (E). Tachyzoite proliferation among RMG cells
was assayed 24 hours after infection (ratio 5:1) without cytokine
stimulation (F). In the absence of prior stimulation with cytokines, the
tachyzoites grew within the parasitophorous vacuoles and formed
rosettes containing 10 to 20 tachyzoites each. After stimulation with
IFN-?, tachyzoite proliferation was lower, in most cases with only one
parasitophorous vacuole per cell, and the vacuoles contained only 5 to
10 tachyzoites (data not shown).
Confocal images of RPE and RMG cells infected with T.
immunocytofluorescence 24 hours after infection. Cultures of RMG cells
were stimulated 72 hours before infection with IFN-? at 10 ng/mL, more
or less associated with LPS at 100 ng/mL, or with TNF-? at 10 ng/mL. Cell
culture without cytokine stimulation served as the control. The cells were
fixed 24 hours after infection (ratio 5:1), stained with antibodies, and
examined with epifluorescence and confocal microscopes. The percent-
age of infected cells (A) and the number of tachyzoites (B) were counted
among 200 cells randomly selected from each preparation.
Influence of cytokines on RMG cell infection determined by
IOVS, April 2009, Vol. 50, No. 4
Retinal Cell Infection by Toxoplasma gondii
ments performed 48 hours after infection, since the cells were
no longer under the effects of cytokines, they were less stim-
ulated and produced less NO.
Ocular toxoplasmosis, whether acquired or congenital, typi-
cally manifests itself in the form of a chorioretinitis reflecting
the inflammation and necrosis of an area including the neuro-
retina, the pigment epithelium, and the choroid in response to
the release of tachyzoites. Cysts and free forms of tachyzoites
were found in the neuroretina and the pigment epithelium.
The two types of retinal cells we have chosen to study, Mu ¨ller
cells (RMG) and pigment epithelial cells (RPE), play an impor-
tant role in the local immune response by producing certain
cytokines (TNF, IL-1) and acting as antigen-presenting
cells.13,14We therefore chose to study the penetration and the
proliferation of T. gondii in these cells and to assess variations
in response to the action of IFN-? and TNF-?. In our experi-
ments T. gondii infected the Mu ¨ller cells and the pigment
epithelial cells, and the infection was lesser within the RPE
cells than within the RMG cells. T. gondii proliferation was also
inhibited by IFN-?, TNF-?, and the IFN-??TNF-? combination
in the RPE cells, but only by IFN-? or the IFN-??TNF-? com-
bination in the RMG cells. Pretreatment of RMG cells with
IFN-? prevented infection (decreasing the percentage of in-
fected cells) and also induced a postinfection defense mecha-
nism (decreasing the number of parasites per infected cell 6
hours after massive infection, as well as decreasing parasite
multiplication among cells 48 hours after infection). It is pos-
sible that the infection’s reduction could be partly due to a
consequence of TNF- and IFN-?-mediated apoptosis of RPE or
RMG cells. Nevertheless, we showed that the number of RMG
cells infected and the number of tachyzoites per cell decreased
significantly when the cells were pretreated with IFN-? (Fig. 4),
and this effect was not influenced by the mediated apoptosis.
The literature does not mention any study on the penetration
and proliferation of T. gondii in RMG cells, and only one team
has studied the interactions between T. gondii and RPE cells.
Nagineni et al.15studied the capacity of T. gondii to infect
human RPE cells, but the percentage of infected cells was not
specified, the authors having used the size of the lysis plaques
to assess the infection intensity. They observed that IFN-? and
TNF-? reduced the intensity of infection in RPE cells in a
dose-dependent way. When they studied the infection of the
astrocytes of human cerebral tissue, which are glial cells just
like RMG cells, Pelloux et al.16found 16% of infected cells 24
hours after infection, which is close to the 20% we obtained
with RMG cells. With regard to the action of IFN-?, their results
were different from ours, as they noted no inhibiting effect on
tachyzoite proliferation. Two hypotheses can explain these
differences. First, the incubation time of the cells with the
cytokines was shorter, the cells having been treated with IFN-?
24 hours before infection (compared with 72 hours in our
experiments). Second, the astrocytes used by the authors were
of human origin, whereas we worked on rat cells. Another
team, using murine astrocytes preincubated 72 hours before
infection with different cytokines, found an inhibiting effect
for IFN-? and the IFN-??TNF-? combination on T. gondii
proliferation, as we did with RMG cells.17However, no inhib-
iting effect was observed when TNF was used alone, a result
similar to ours with RMG cells. In our study, we used dissoci-
ated RPE cells, retaining no cells of the original single layer.
The barrier function played by RPE cells is mostly dependent
on the integrity of tight junctions. It is known that IFN-?
induces alterations of RPE tight junctions.18Studies have
shown that IFN-? stimulates expression of intercellular adhe-
sion molecule (ICAM)-1 in human RPE cells.19ICAM-1 secre-
tion by RPE cells might actively participate in immune reac-
tions in the retina by recruiting and activating lymphocytes,
contributing to the immunopathologic process in inflamma-
termined by immunocytofluorescence 6 hours after massive infection.
Cultures of RMG cells were stimulated 72 hours before infection with
IFN-? at 10 ng/mL, more or less associated with LPS at 100 ng/mL, or
with TNF-? at 10 ng/mL. Cell cultures without cytokine stimulation
served as the control. The cells were fixed 6 hours after massive
infection (ratio 15:1), stained with antibodies, and examined under
epifluorescence and confocal microscope. The percentage of infected
cells (A) and the number of tachyzoites (B) were counted among 200
cells randomly selected on each preparation.
Influence of cytokines on massive RMG cell infection de-
1758Delair et al.
IOVS, April 2009, Vol. 50, No. 4
We also confirmed the capability of RPE and RMG cells to
produce NO under certain conditions (for instance, when
simultaneously stimulated with IFN-? and TNF-? more or less
associated with LPS). Goureau et al.20showed that NO syn-
thetase, which transforms L-arginine into NO in RMG cells,
corresponds to the inducible form of the enzyme (as in the
macrophages). This NO production by the activated cells could
be a defense mechanism against T. gondii but also has a toxic
effect on the retina’s neuronal cells.21In vivo, an increase in
NO production is observed in the aqueous humor and the
vitreous body of rats with experimentally induced uveitis.12
The responsibility of NO in the development of uveitis is
demonstrated by the reduction in the intraocular inflammation
when the rats are treated with an inhibitor of NO synthetase
(type L-NMMA). During a generalized toxoplasmic infection,
NO production, in particular by splenic cells, is a defense
mechanism that limits the proliferation of tachyzoites. When
mice are treated with an inhibitor of NO synthetase from the
start of infection, a clear aggravation of the ophthalmologic
injuries is observed compared with mice in which NO produc-
tion is not inhibited.22Thus, NO production appears to be a
“systemic” defense against toxoplasmic infection, but it has not
been demonstrated that this mechanism is valid for “local”
defense in the retina. The mechanism used by IFN-? to inhibit
T. gondii proliferation in RPE and RMG cells is not yet known.
This inhibiting effect is not due to a direct action of IFN-? on
T. gondii that might reduce the latter’s virulence, since in our
experiments cultures were rinsed before being reincubated
with T. gondii without adding cytokines. However, several
hypotheses can be contemplated. The first hypothesis would
be to consider that this inhibition depends on NO production,
for instance as has been demonstrated for macrophages.23Our
results seem to reject this hypothesis, both for RPE and RMG
cells, since under certain conditions, for instance in the pres-
ence of 1 ng/mL IFN-? alone, the inhibiting effect was evident
without any increase in NO production by the cells. In their
study on human RPE cells, Nagineni et al.18demonstrated that
the inhibition of T. gondii proliferation by IFN-? depended on
mechanisms independent of NO but dependent on indoleam-
ine 2,3-dioxygenase (IDO). This second hypothesis considers
that the IDO enzyme could play a part in the IFN-?–dependent
inhibition phenomenon. Because this enzyme degrades trypto-
phan, its action can be shown if the inhibiting effect due to
IFN-? disappears when adding tryptophan to the medium.
Pfefferkorn24demonstrated that this mechanism was respon-
sible for the inhibition of T. gondii proliferation in response to
IFN-? in human fibroblasts.
Others have studied the influence of cytokines on RPE
infection with different infectious agents, in particular the
inhibiting role of IFN-? on RPE infection with cytomegalovirus
according to an IDO-dependent mechanism.25
TNF-? inhibited tachyzoite proliferation in pigment epithe-
lial cells but not in Mu ¨ller cells. The possible presence of TNF-?
receptors on the surface of pigment epithelial cells could
explain the difference in sensitivity. Two types of TNF-? re-
ceptors have been revealed on the surface of MRC5 human
fibroblasts and have been shown to be involved in defense
mechanisms against T. gondii26,27
In conclusion, we have demonstrated the inhibiting role of
IFN-? in the penetration and proliferation of RH strain
tachyzoites in retinal cells, pigment epithelial cells, and Mu ¨ller
cells, in a dose-dependent way and according to a mechanism
that seems independent of NO production. Inhibition by TNF-?
applies only in RPE cells (not RMG cells), also in a dose-
dependent way and also according to a mechanism that ap-
pears to be independent of NO production. Both cytokines
IFN-? and TNF-? inhibited T. gondii replication in RPE cells,
whereas only IFN-? had an anti-Toxoplasma activity within
RMG cells. The differences in cytokine response may be the
reason that RPE cells are less efficiently infected by T. gondii
than RMG cells.
The authors thank Yvonne de Kozak (CNRS [Centre National de la
Recherche Scientifique], Unite ´ 450 INSERM [Institut National de la
Sante ´ et de la Recherche Me ´dicale], Universite ´ Pierre et Marie Curie,
Paris VI, France) for excellent instruction in retinal cell culture, and
Isabelle Tardieux (UMR [Unite ´ Mixte de Recherche] 8104-U567, IFR
[Institut Fe ´de ´ratif de Recherche] 116, Institut Cochin, Universite ´ Paris
Descartes) for help in obtaining T. gondii tachyzoites.
1. Smit RL, Baarsma GS, de Vries J. Classification of 750 consecutive
uveitis patients in the Rotterdam Eye Hospital. Int Ophthalmol.
TABLE 1. Influence of Cytokines on NO Production by RPE and RMG Cells
Before InfectionAfter InfectionBefore InfectionAfter Infection
IFN ? TNF
LPS ? IFN
LPS ? TNF
LPS ? IFN ? TNF
5 ? 1
93.75 ? 2.2
10.97 ? 0.7
3.66 ? 0.3
88.9 ? 1.2
7.8 ? 0.4
11 ? 0.4
25 ? 1
37.1 ? 0.75 ? 0.4
NO production was measured in the culture supernatant 72 hours after cell culture stimulation with
various cytokines (before infection) and 48 hours after infection with tachyzoites. IFN-? and TNF-? were
used alone at different concentrations (0.1, 1, or 10 ng/mL). IFN-? and TNF-? were used at a concentration
of 10 ng/mL in combination or with LPS at 100 ng/mL. Cell culture with no cytokine stimulation served
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Retinal Cell Infection by Toxoplasma gondii
2. Jacobs L, Remington JS, Melton ML. The resistance of the encysted
form of Toxoplasma gondii. J Parasitol. 1960;46:11–21.
3. Ferguson JP, Hutchison WM, Pettersen E. Tissue cyst rupture in
mice chronically infected with Toxoplasma gondii. Parasitol Res.
4. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-
gamma: the major mediator of resistance against Toxoplasma gon-
dii. Science. 1988;240:516–518.
5. Gazzinelli R, Bre ´zin A, Li Q, Nussenblatt RB, Chan CC. Toxoplasma
gondii: acquired ocular toxoplasmosis in the murine model, pro-
tective role of TNF-alpha and IFN-gamma. Exp Parasitol. 1994;78:
6. McMenamin PG, Dutton GN, Hay J, Cameron S. The ultrastructural
pathology of congenital murine toxoplasmic retinochoroiditis.
Part I: The localization and morphology of Toxoplasma cysts in the
retina. Exp Eye Res. 1986;43(4):529–543.
7. Mano T, Puro DG. Phagocytosis by human retinal glial cells in
culture. Invest Ophthalmol Vis Sci. 1990;31(6):1047–1055.
8. Roberts F, Mets MB, Ferguson DJ, et al. Histopathological features
of ocular toxoplasmosis in the fetus and infant. Arch Ophthalmol.
9. Olle P, Bessieres MH, Malecaze F, Seguela JP. The evolution of
ocular toxoplasmosis in anti-interferon gamma treated mice. Curr
Eye Res. 1996;15(7):701–707.
10. Denkers EY, Gazzinelli RT. Regulation and function of T-cell-me-
diated immunity during Toxoplasma gondii infection. Clin Micro-
biol Rev. 1998;11(4):569–588.
11. Creuzet C, Robert F, Roisin MP, et al. Neurons in primary culture
are less efficiently infected with Toxoplasma gondii than glial cells.
Parasitol Res. 1998;84(1):25–30.
12. Goureau O, Bellot J, Thillaye B, Courtois Y, de Kozak Y. Increased
nitric oxide production in endotoxin-induced uveitis. J Immunol.
13. Roberge FG, Caspi R, Nussenblatt RB. Glial retinal Mu ¨ller cells
produce Il-1 activity and have a dual effect on autoimmune T
helper lymphocytes. J Immunol. 1988;140:2193–2196.
14. De Kozak Y, Hicks D, Chatenoud L, Bellot J, Thillaye B, Faure P.
Intracellular TNF in endotoxin- and S-antigen-induced uveitis: in
vitro synthesis of TNF by retinal Mu ¨ller glial and pigment epithelial
cells. Region Immunol. 1994;6:76–80.
15. Nagineni C, Pardhasaradhi K, Martins M, Detrick B, Hooks J.
Mechanisms of interferon-induced inhibition of Toxoplasma gon-
dii replication in human pigment epithelial cells. Infect Immun.
16. Pelloux H, Pernod G, Polack B, Ambroise-Thomas P. Influence of
cytokines on Toxoplasma gondii growth in human astrocytoma-
derived cells. Parasitol Res. 1996;82:598–603.
17. Halonen S, Chiu F, Weiss L. Effect of cytokines on growth of
Toxoplasma gondii in murine astrocytes. Infect Immun. 1998;66:
18. Zech JC, Pouvreau I, Cotinet A, Goureau O, Le Varlet B, de Kozak
Y. Effect of cytokines and nitric oxide on tight junctions in cul-
tured rat retinal pigment epithelium. Invest Ophthalmol Vis Sci.
19. Nagineni CN, Kutty RK, Detrick B, Hooks JJ. Inflammatory cyto-
kines induce intercellular adhesion molecule-1 (ICAM-1) mRNA
synthesis and protein secretion by human retinal pigment epithe-
lial cell cultures. Cytokine. 1996;8(8):622–630.
20. Goureau O, Hicks D, Courtois Y, de Kozak Y. Induction and
regulation of nitric oxide synthetase in retinal Mu ¨ller glial cells.
J Neurochem. 1994;63:310–317.
21. Goureau O, Regnier-Ricard F, Courtois Y. Requirement for nitric
oxide in retinal neuronal cell death induced by activated Mu ¨ller
glial cells. J Neurochem. 1999;72:2506–2515.
22. Hayashi S, Chan C, Gazzinelli R, Pham N, Cheung M, Roberge F.
Protective role of nitric oxide in ocular toxoplasmosis. Br J Oph-
23. Remington JS, Krahenbuhl J, Mendenhall HJ. A role for activated
macrophages in resistance to infection with Toxoplasma gondii.
Infect Immun. 1972;6:829–834.
24. Pfefferkorn ER. Interferon ? blocks the growth of Toxoplasma
gondii in human fibroblasts by inducing the host cells to degrade
tryptophan. Proc Natl Acad Sci U S A. 1984;81:908–912.
25. Bodaghi B, Goureau O, Zipeto D, Laurent L, Virelizier JL, Michel-
son S. Role of IFN-?-induced indoleamine 2,3-dioxygenase and
inducible nitric oxide synthetase in the replication of human
cytomegalovirus in retinal pigment epithelial cells. J Immunol.
26. Derouich-Guergour D, Pelloux H, Aldebert D, Demenge P, Am-
broise-Thomas P. Evidence for tumor necrosis factor receptors
(TNFRs) in human MRC5 fibroblast cells. Eur Cytokine Netw.
27. Derouich-Guergour D, Brenier-Pinchart MP, Ambroise-Thomas P,
Pelloux H. Tumor necrosis factor ?: role in the physiopathology of
protozoan parasite infections. Int J Parasitol. 2001;31:763–769.
1760Delair et al.
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