Effect of cytokines on growth of Toxoplasma gondii in murine astrocytes.

Abstract

Cytokines play a significant role in the regulation of Toxoplasma gondii in the central nervous system. Cytokine-activated microglia are important host defense cells in central nervous system infections. Recent evidence indicates that astrocytes can also be activated by cytokines to inhibit intracellular pathogens. In this study, we examined the effect of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 on the growth of T. gondii in a primary murine astrocyte culture. Pretreatment of astrocytes with IFN-gamma resulted in 65% inhibition of T. gondii growth. Neither TNF-alpha, IL-1, nor IL-6 alone had any effect on T. gondii growth. IFN-gamma in combination with either TNF-alpha, IL-1, or IL-6 caused a 75 to 80% inhibition of growth. While nitric oxide was produced by astrocytes treated with these cytokines, inhibition of T. gondii growth was not reversed by the addition of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Furthermore, IFN-gamma in combination with IL-1, IL-6, or TNF-alpha also induced inhibition in astrocytes derived from syngeneic mice deficient in the enzyme inducible nitric oxide synthase. This finding suggests that the mechanism of cytokine inhibition is not nitric oxide mediated. Similarly, the addition of tryptophan had no effect on inhibition, indicating that the mechanism was not mediated via induction of the enzyme indoleamine 2, 3-dioxygenase. The mechanism of inhibition remains to be elucidated. Results from this study demonstrate that cytokine-activated astrocytes are capable of significantly inhibiting the growth of T. gondii. These data indicate that astrocytes may be important host defense cells in controlling toxoplasmosis in the brain.

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INFECTION AND IMMUNITY,
0019-9567/98/$04.00?0
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Oct. 1998, p. 4989–4993Vol. 66, No. 10
Effect of Cytokines on Growth of Toxoplasma gondii
in Murine Astrocytes
S. K. HALONEN,1F.-C. CHIU,1,2AND L. M. WEISS3,4*
Departments of Neurology,1Anesthesiology,2Pathology,3and Medicine,4
Albert Einstein College of Medicine, Bronx, New York 10461
Received 12 January 1998/Returned for modification 19 February 1998/Accepted 6 July 1998
Cytokines play a significant role in the regulation of Toxoplasma gondii in the central nervous system.
Cytokine-activated microglia are important host defense cells in central nervous system infections. Recent
evidence indicates that astrocytes can also be activated by cytokines to inhibit intracellular pathogens. In this
study, we examined the effect of gamma interferon (IFN-?), tumor necrosis factor alpha (TNF-?), interleukin-6
(IL-6), and IL-1 on the growth of T. gondii in a primary murine astrocyte culture. Pretreatment of astrocytes
with IFN-? resulted in 65% inhibition of T. gondii growth. Neither TNF-?, IL-1, nor IL-6 alone had any effect
on T. gondii growth. IFN-? in combination with either TNF-?, IL-1, or IL-6 caused a 75 to 80% inhibition of
growth. While nitric oxide was produced by astrocytes treated with these cytokines, inhibition of T. gondii
growth was not reversed by the addition of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine.
Furthermore, IFN-? in combination with IL-1, IL-6, or TNF-? also induced inhibition in astrocytes derived
from syngeneic mice deficient in the enzyme inducible nitric oxide synthase. This finding suggests that the
mechanism of cytokine inhibition is not nitric oxide mediated. Similarly, the addition of tryptophan had no
effect on inhibition, indicating that the mechanism was not mediated via induction of the enzyme indoleamine
2,3-dioxygenase. The mechanism of inhibition remains to be elucidated. Results from this study demonstrate
that cytokine-activated astrocytes are capable of significantly inhibiting the growth of T. gondii. These data
indicate that astrocytes may be important host defense cells in controlling toxoplasmosis in the brain.
Toxoplasma gondii is an important pathogen in the central
nervous system and causes a severe encephalitis in patients
with AIDS. Cytokines play an important role in the regulation
of T. gondii replication in the central nervous system (17).
Gamma interferon (IFN-?) has been shown to prevent reacti-
vation of Toxoplasma encephalitis in mice (30). Tumor necro-
sis factor alpha (TNF-?), interleukin-1 (IL-1), and interleu-
kin-6 (IL-6) are up-regulated in the brains of mice with chronic
toxoplasmosis (9, 15, 16). Studies indicate that IFN-?, TNF-?,
IL-1, and IL-6 may control the growth of T. gondii in the brain
via activation of microglia (4, 5). Studies of mice indicate that
cytokines induce anti-Toxoplasma activity in microglia via a
nitric oxide (NO)-mediated mechanism (10).
Recent evidence indicates that cytokines can also activate
astrocytes to inhibit growth of T. gondii (6, 8, 25, 27). For
example, IFN-? has been shown to inhibit growth of T. gondii
in the glioblastoma cell line 86HG39 (6). Inhibition was shown
to be via induction of indoleamine 2,3-dioxygenase (IDO),
resulting in the degradation of intracellular tryptophan (8).
Pelloux et al. found that in the astrocytoma cell line GHE,
TNF-? inhibited, IL-1 stimulated, and IFN-? and IL-6 had no
effect on growth of T. gondii (25). Finally, in primary human
astrocytes, IFN-? and IL-1 in combination have been shown to
inhibit growth of T. gondii via the production of NO (27).
Interpretation of these results is difficult due to variability
found in astrocyte cell lines and differences between tumor
cells and primary astrocytes.
For this reason, we have chosen to study the effects of cyto-
kines on growth of T. gondii in a primary astrocyte culture. In
this study, we evaluated the effects of IFN-?, TNF-?, IL-1, and
IL-6 on the replication of T. gondii ME49 in a primary murine
astrocyte culture. The effects of these cytokines individually,
and the effects of IFN-? in combination with IL-1, TNF-?, and
IL-6, on the growth of T. gondii were examined. The ability of
each of these cytokines and cytokine combinations to induce
nitric oxide production was assessed by using the Griess re-
agent. A nitric oxide-mediated mechanism of cytokine inhibi-
tion of T. gondii growth was addressed by using NG-monometh-
yl-L-arginine (NMMA), a nitric oxide inhibitor, and by using
astrocytes derived from syngeneic mice deficient in the enzyme
inducible nitric oxide synthase (iNOS). The IDO mechanism of
inhibition was investigated via the addition of exogenous tryp-
tophan. The aim of this study was to clarify the effect of cyto-
kines on T. gondii replication in astrocytes and increase our
understanding of the role of astrocytes in the host defense
against T. gondii in the central nervous system.
MATERIALS AND METHODS
Primary astrocyte culture. Murine astrocytes from C57BL/6 ? SV129 mice or
syngeneic mice, deficient in iNOS (iNOS?/?; gift of C. Nathan) (22), were
cultivated from the brains of neonatal (less than 24 h old) mice. Murine pups
were sacrificed, the brain was removed from the cranium, the forebrain was
dissected and the meninges were removed. The tissue was minced and incubated
in 0.25% trypsin for 5 min at 37°C. After 5 min, the trypsin was inactivated with
a solution containing DNase and soybean trypsinase inhibitors, and the tissue
was further disrupted by trituration in a 20-ml pipette. The dissociated cells were
filtered through a 74-?m-pore-size Nitex mesh, centrifuged at 200 ? g, sus-
pended in growth medium at a concentration of 106cells/ml, and plated onto
poly-L-lysine-coated dishes. Astrocytes were maintained in endotoxin-free min-
imal essential medium (BRL-GIBCO, Gaithersburg, Md.) supplemented with
20% fetal bovine serum (BRL-GIBCO), 5% glucose, and 100 U of penicillin and
streptomycin (BRL-GIBCO) per ml. The growth medium was changed every 3
days. After 7 days in vitro, a confluent layer of 1 ? 104to 2 ? 104cells/cm2of
astrocytes is reached. By this method, cells were found to be ?95% astrocytes, as
judged by positive staining for glial fibrillary acidic protein. Cultures contained
?5% microglia, as identified by staining with the lectin BS1-B4 (catalog no.
L-2895; Sigma, St. Louis, Mo.). Astrocytes were dissociated in trypsin-EDTA,
replated onto poly-L-lysine-coated coverslips or 24-well plates at 104cells/cm,
* Corresponding author. Mailing address: Dept. of Pathology, Rm.
F-504, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-
2142. Fax: (718) 430-8543. E-mail: lmweiss@aecom.yu.edu.
4989

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and cultured for 7 to 10 days after replating. These astrocytes were then infected
with T. gondii ME49 as described below.
Culture of T. gondii. Tachyzoites from T. gondii ME49 were obtained by in
vitro culture in Vero cells. Parasites were harvested after 4 to 5 days in culture,
resuspended in minimal essential medium supplemented with 10% fetal bovine
serum, and used for infection of murine astrocyte cultures.
Chemicals and cytokines. Murine recombinant IFN-?, IL-1?, TNF-?, and IL-6
were purchased from Genzyme (Cambridge, Mass.). NMMA and L-tryptophan
were obtained from Sigma. [3H]uracil and [3H]tryptophan were purchased from
Amersham Pharmacia Biotech (Arlington Heights, Ill.). The Griess reagent kit
(catalog no. G-7921) was obtained from Molecular Probes (Eugene, Oreg.).
Cytokine treatments. Murine astrocytes were stimulated with IFN-?, TNF-?,
IL-1? (each at 100 U/ml), or IL-6 (1 ng/ml), alone or in combination, for 72 h
prior to infection, and supernatants were removed for determination of nitric
oxide production. Cultures were then infected with T. gondii and incubated for
48 h without cytokines, and growth was determined by the [3H]uracil method
described below. The percentage of infected astrocytes for each condition was
determined by counting the number of infected cells per 500 cells under both
phase and immunofluorescence microscopy. Testing for each condition was per-
formed in triplicate. Immunofluorescence microscopy was performed with a 1:50
dilution of a commercial polyclonal rabbit anti-Toxoplasma antibody (DAKO,
Carpenteria, Calif.) followed by detection with anti-rabbit fluorescein immuno-
globulin G (Boehringer Mannheim, Indianapolis, Ind.) as previously described
(13). All cultures were incubated in endotoxin-free medium, and no endotoxin
contamination was detected in any of the experimental cultures.
Determination of Toxoplasma growth. Cultures were infected with 105T. gondii
tachyzoites per well (a 5:1 tachyzoite/host cell ratio) for 2 h. The monolayer was
then extensively washed to remove extracellular tachyzoites and [3H]uracil (2.5
?Ci per well) was added. T. gondii growth was determined 48 h later by the
[3H]uracil incorporation method described below. Before cell harvesting, the
appearance of the culture was checked to verify that T. gondii-induced cell lysis
had not begun. The monolayer was washed three times with phosphate-buffered
saline to remove any nonincorporated [3H]uracil. The astrocyte monolayer was
then lysed by incubation in 0.1% sodium dodecyl sulfate for 15 min at room
temperature. Nucleic acids were precipitated by the addition of 3 M trichloro-
acetic acid. The contents of the wells were deposited on Whatman glass filters
and washed extensively with 0.1 M trichloroacetic acid, and then radioactivity was
determined with a liquid scintillation counter (21). For each experiment, controls
included murine astrocytes cultured in the absence of T. gondii. The radioactivity
of these samples was always near background levels, thus confirming that [3H]u-
racil incorporation was specific to the parasite.
Measurement of nitric oxide. Supernatants from astrocyte cultures were col-
lected after 72 h of incubation in cytokines, and nitrite was assayed with the
Griess reagent kit (Molecular Probes). Briefly, a 150-?l aliquot of culture su-
pernatant was mixed with 50 ?l of the Griess reagent [0.05% N-(1-naphthyl)
ethylenediamine dihydrochloride–0.5% sulfanilic acid in phosphoric acid] and
diluted with 1.3 ml of water, and absorbance was measured at 548 nm in a
spectrophotometer. The amount of nitrite in the sample was calculated from a
sodium nitrite standard curve.
Effect of NMMA or tryptophan on cytokine inhibition. Murine astrocytes were
cultured as described above except that in some experiments, either NMMA
(final concentration of 400 ?M) or tryptophan (final concentration of 100 ?g/ml)
was added to the culture at the time of cytokine addition. Tryptophan uptake by
astrocytes was measured by monitoring [3H]tryptophan uptake as described by
Pfefferkorn (28). IDO activity was measured photometrically at 490 nm, by using
the Ehrlich reagent to monitor the degradation of tryptophan to kynurenine as
described by Da ¨ubener et al. (7).
Statistics. Within each experiment, all conditions were repeated in triplicate,
and each experiment was replicated two to three times. Data were analyzed by
nonparametric (Wilcoxon signed-rank test) and/or parametric (Student t test and
analysis of variance) methods, using Sigma Stat version 1.0 (Jandel Scientific, San
Rafael, Calif.).
RESULTS
Effect of IFN-?, TNF-?, IL-1, and IL-6 on growth of T. gondii
in murine astrocytes. Astrocytes were pretreated with cyto-
kines and then infected with T. gondii, and growth was mea-
sured 48 h after infection. The effect of treating astrocytes with
IFN-?, TNF-?, IL-1, and IL-6 alone and in combination is
presented in Fig. 1. IFN-? inhibited the growth of T. gondii by
approximately 65% (P ? 0.05), but there was no effect from
IL-6, TNF-?, or IL-1 alone. IFN-? in combination with either
IL-6, IL-1, or TNF-? inhibited the growth of T. gondii in
murine astrocytes by approximately 75 to 80% (P ? 0.05), a
level 10 to 15% greater than that observed after treatment with
IFN-? alone (P ? 0.05). The addition of IL-6 did not reverse
the inhibition of growth induced by either IFN-?–IL-1 or IFN-
?–TNF-? in astrocytes. No inhibition of T. gondii growth was
seen with either IFN-? alone or any of the IFN-? cytokine
combinations when cytokines were added at the time of infec-
tion or 24 h prior to infection (data not shown).
Cultures pretreated with IFN-? alone and in combination
with each of TNF-?, IL-1, and IL-6 were also assessed micro-
scopically. The level of infection of cultures treated with cyto-
kines was found to be ?5%, compared to 30 to 35% in the
controls (Fig. 1, inset), which correlated with the results of the
uracil uptake assay. The percent infected cells approximately
doubled when IL-6 was added to other cytokines (i.e., 1.2%
infected with IFN-? and 2.8% infected with IFN-?–IL-6).
While not statistically significant, this finding raises the possi-
bility that in astrocytes, as found for macrophages (2), IL-6
may reverse part of the activation due to other cytokines.
NO production in cytokine-treated astrocytes. NO produc-
tion after treatment with each of the cytokine combinations is
presented in Table 1. NO levels in the controls (i.e., no cyto-
kines added) were between 2 and 3 ?M, and no increase in NO
was seen with IFN-?, TNF-?, IL-1, or IL-6 treatment alone. All
cytokine combinations resulted in a statistically significant el-
evation in NO above the control level (P ? 0.05): IFN-?–IL-1,
9.9 ?M; IFN-?–TNF-?, 15.2 ?M; and IFN-?–IL-6, 5 ?M. The
addition of IL-6 to IFN-?–IL-1 decreased the NO levels in-
duced by IFN-?–IL-1 slightly, while the addition of IL-6 to
IFN-?–TNF-? had no effect on NO production.
Effect of NMMA on cytokine inhibition of T. gondii growth in
murine astrocytes. Addition of NMMA decreased nitrite levels
in all cytokine combinations to background levels (?2 ?M) in
all cultures (Table 1). The presence or absence of NMMA
during the 72-h pretreatment with cytokines prior to infection
of astrocytes with T. gondii did not affect the cytokine-medi-
ated inhibition of T. gondii growth due to IFN-?–IL-1, IFN-?–
IL-6, IFN-?–IL-1–IL-6, IFN-?–TNF-?, or IFN-?–TNF-?–IL-6
(Table 2) or to the individual cytokines (data not shown).
FIG. 1. Effect of cytokines on growth of T. gondii in mouse astrocytes. Cells
were incubated with the cytokines IFN-? (100 U/ml), IL1 (1 ng/ml), IL-6 (100
U/ml), and TNF-? (100 U/ml) for 72 h before infection. Control cultures were
incubated in medium alone. [3H]uracil (2.5 ?Ci/ml) was added 2 h after infec-
tion, and cells were harvested 48 h later. Results are averages of three separate
experiments. Bars indicate ? standard error of the mean. ?, significance at the
P ? 0.05 level versus control as calculated by Student’s t test. (Insert) Percent-
ages of cells (mean ? standard deviation) infected with T. gondii ME49 as
determined by microscopy (multiplicity of infection, 5:1). The difference between
each cytokine treatment and the control was significant at P ? 0.05; there was no
significant difference between any of the various cytokine treatments.
4990 HALONEN ET AL.INFECT. IMMUN.

Page 3

Effect of cytokines on growth of T. gondii in iNOS?/?murine
astrocytes. Treatment of iNOS?/?astrocytes with IFN-?–IL-1,
IFN-?–IL-6, IFN-?–IL-1 plus IL-6, IFN-?–TNF-?, or IFN-?–
TNF-? plus IL-6 inhibited the growth of T. gondii significantly
(75 to 80%) (Table 3), as was seen with syngeneic control
murine astrocytes. In addition, as found for control astrocytes,
the addition of IL-6 had little or no effect on the degree of
inhibition caused by either IFN-?–IL-1 or IFN-?–TNF-?. As
expected, no nitric oxide was detected in the supernatants from
the iNOS?/?astrocytes after treatment with any of the cyto-
kine combinations (Table 3).
Effect of tryptophan on cytokine inhibition of T. gondii
growth. Tryptophan (100 ?g/ml) added to cultures stimulated
with cytokines did not reverse inhibition of T. gondii growth
caused by any of the cytokine treatments in normal or in
iNOS?/?astrocytes (Table 4). Tryptophan uptake by astro-
cytes was verified by [3H]tryptophan incorporation (28). No
IDO activity was detected in astrocytes with or without cyto-
kine stimulation.
DISCUSSION
The cytokines IFN-?, TNF-?, IL-1, and IL-6 are known to be
important in controlling the replication of T. gondii in the
brain. The importance of cytokine activation of microglia in
regulating T. gondii infection in the brain is well established,
but the role of astrocytes is less well understood. Variable
effects on the growth of T. gondii have been demonstrated in
different astrocyte tumor lines with IFN-?, TNF-?, IL-1, and/or
IL-6 treatment. In the glioblastoma cell line 86HG39, IFN-?
alone has been shown to inhibit the growth of T. gondii,
while in the glioblastoma cell line 87HG31, the combination
of TNF-? and IFN-? was required to inhibit growth (6, 8); in
GHE astrocytoma cells, TNF-? but not IFN-? inhibited the
growth of T. gondii (25). Additionally, in astrocytoma cells,
IL-1 was found to stimulate growth and IL-6 had no effect on
T. gondii growth (25).
In the present study using primary murine astrocytes, we
have demonstrated that pretreatment with IFN-? alone or in
combination with IL-1, IL-6, or TNF-? but not IL-1, IL-6, or
TNF-? alone significantly inhibited the growth of T. gondii in
these cells. This result is consistent with findings for glioblas-
toma cell line 86HG39 (6) but in contrast to those for astro-
TABLE 1. Effect of cytokines on NO production
in mouse astrocytes
Treatment
Nitrite concn
(?M)a
Control...................................................................................... 2.0 ? 1.2
IFN-? ........................................................................................ 1.9 ? 0.8
TNF-?....................................................................................... 1.3 ? 0.5
IL-1............................................................................................ 1.4 ? 0.6
IL-6............................................................................................ 0.7 ? 0.7
IFN-?, IL-1............................................................................... 9.9 ? 2.3b
IFN-?, IL-6............................................................................... 5.0 ? 0.8b
IFN-?, IL-1, IL-6..................................................................... 6.3 ? 1.7b
IFN-?, TNF-?..........................................................................15.2 ? 1.0b
IFN-?, TNF-?, IL-6 ................................................................13.8 ? 0.7b
IFN-?, IL-1, NMMA............................................................... 2.3 ? 1.7
IFN-?, IL-6, NMMA............................................................... 2.1 ? 1.7
IFN-?, IL-1, IL-6, NMMA..................................................... 1.8 ? 1.1
IFN-?, TNF-?, NMMA.......................................................... 2.2 ? 1.6
IFN-?, TNF-?, IL-6, NMMA ................................................ 2.6 ? 1.6
aMean ? standard deviation of three separate experiments for NO in the
supernatants of astrocyte cultures treated with IFN-? (100 U/ml), IL-1 (1 ng/ml),
IL-6 (100 U/ml), and TNF-? (100 U/ml), in the presence or absence of NMMA
(400 ?M); control cultures were incubated in medium alone. Cytokines were
added to cultures 72 h prior to infection, and NMMA was added at the time of
cytokine addition. 0 ? level of detection (?1.0 ?M).
bP ? 0.05 from the value of the control group as calculated by Student’s t test.
TABLE 2. Effect of NMMA on cytokine inhibition
of T. gondii growth in murine astrocytes
Treatment
Inhibition of T. gondii growth
(% of control)a
Medium only Cytokines ? NMMA
IFN-?, IL-1
IFN-?, IL-6
IFN-?, IL-1, IL-6
IFN-?, TNF-?
IFN-?, TNF-?, IL-6
22.4 ? 3.1
20.4 ? 2.3
18.2 ? 1.5
23.3 ? 3.7
14.3 ? 2.9
24.7 ? 1.1
21.0 ? 0.8
15.2 ? 4.1
16.6 ? 2.5
16.8 ? 2.5
aMean ? standard deviation of three separate experiments for astrocyte
cultures treated with IFN-? (100 U/ml), IL-1 (1 ng/ml), IL-6 (100 U/ml), and
TNF-? (100 U/ml), in the presence or absence of NMMA (400 ?M), and for
control cultures incubated in medium alone. Cells were incubated with cytokines
for 72 h prior to infection, and NMMA was added at the time of cytokine
addition; [3H]uracil (2.5 ?Ci/ml) was added 2 h after infection, and cells were
harvested 48 h later. No endotoxin contamination was detected in any of the
cultures.
TABLE 3. Effect of cytokines on growth of T. gondii
in iNOS?/?murine astrocytes
Treatment
Inhibition of
T. gondii growth
(% of control)a
IFN-?, IL-1 ............................................................................ 22.8 ? 2.3
IFN-?, IL-6 ............................................................................ 21.1 ? 0.8
IFN-?, IL-1, IL-6................................................................... 23.3 ? 3.1
IFN-?, TNF-?........................................................................ 22.2 ? 1.8
IFN-?, TNF-?, IL-6.............................................................. 19.2 ? 5.9
aMean ? standard deviation of two separate experiments for astrocyte cul-
tures treated with IFN-? (100 U/ml), IL-1 (1 ng/ml), IL-6 (100 U/ml), and TNF-?
(100 U/ml). Cells were incubated with cytokines for 72 h prior to infection;
[3H]uracil (2.5 ?Ci/ml) was added 2 h after infection, and cells were harvested
48 h later. Control cultures were incubated in medium alone. All values are
significantly different (P ? 0.05) from the control group as calculated by Stu-
dent’s t test. Nitrite was not detectable (i.e., ?1.0 ?M) in any sample.
TABLE 4. Effect of tryptophan on cytokine inhibition
of T. gondii growth
Treatment
Inhibition of T. gondii growth (% of control)a
Control astrocytesiNOS?/?astrocytes
Medium
Cytokines ?
tryptophan
Medium
Cytokines ?
tryptophan
IFN-?
IFN-?, IL-1
IFN-?, IL-6
IFN-?, IL-1, IL-6
IFN-?, TNF-?
IFN-?, TNF-?, IL-6
35.5 ? 2.3
28.4 ? 3.9
21.5 ? 2.9
20.6 ? 2.1
23.5 ? 3.5
21.0 ? 2.2
38.7 ? 3.4
33.3 ? 3.5
29.6 ? 1.4
22.8 ? 5.4
22.5 ? 5.1
23.7 ? 1.1
32.3 ? 2.3
21.1 ? 1.1
20.5 ? 1.7
25.5 ? 4.9
23.4 ? 0.7
15.0 ? 3.8
31.0 ? 2.1
15.6 ? 2.0
17.0 ? 4.1
19.5 ? 1.7
16.8 ? 5.6
12.5 ? 1.6
aMean ? SD of two separate experiments for astrocyte cultures treated with
IFN-? (100 U/ml), IL-1 (1 ng/ml), IL-6 (100 U/ml), and TNF-? (100 U/ml) in the
presence and absence of tryptophan (100 ?g/ml) and for control cultures incu-
bated with medium alone. Cells were incubated with cytokines for 72 h prior to
infection, and tryptophan was added at the time of cytokine addition; [3H]uracil
(2.5 ?Ci/ml) was added 2 h after infection, and cells were harvested 48 h later.
No statistically significant difference was detected between any of the cultures as
determined by Student’s t test and analysis of variance.
VOL. 66, 1998CYTOKINE INHIBITION OF T. GONDII IN MURINE ASTROCYTES4991

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cytoma cell line GHE (25). The absence of IFN-?-induced in-
hibition in GHE astrocytoma cells may be due to the fact that
in this study, cells were pretreated with IFN-? for only 24 h.
Peterson et al. (26) found that pretreatment with IFN-? for
24 h did not activate murine astrocytes to inhibit T. gondii,
which is consistent with our observations. We found that mu-
rine astrocytes needed to be pretreated with IFN-? for 48 to
72 h to induce inhibition. Similarly, Da ¨ubener et al. (6, 8)
found optimal inhibition with IFN-? when glioblastoma cells
were pretreated for 72 h. Treatment of astrocytes with cyto-
kines after infection did not induce inhibition. Thus, priming of
astrocytes by IFN-? is required for anti-Toxoplasma activity.
This phenomenon has also been reported for monocytes (3).
IFN-? in combination with either IL-1, TNF-?, or IL-6 in-
hibited T. gondii growth in murine astrocytes. The effect of
adding either IL-1, IL-6, or TNF-? to IFN-? significantly (by 15
to 20%) increased inhibition of growth induced by IFN-?
alone. Similarly, in microglia, IFN-? activation is enhanced by
TNF-?, IL-1, or IL-6 (4, 5). In macrophages, IL-6 has been
reported to reverse the inhibition caused by IFN-?–IL-1 (2). In
our study, IL-6 did not reverse the inhibition caused by either
IFN-?–IL-1 or IFN-?–TNF-? in murine astrocytes. The effect
of IL-6 is of interest due to the implication that IL-6 plays an
important role in the immunopathogenesis of Toxoplasma en-
cephalitis (31).
IFN-? in combination with IL-1 and other cytokines has
been shown to stimulate nitric oxide production via the enzyme
iNOS in both human and murine astrocytes (14, 19). Treat-
ment of primary murine astrocytes with IFN-? in combination
with other cytokines also resulted in the production of nitric
oxide. However, inhibition of T. gondii growth was found to be
nitric oxide independent, as demonstrated by the inability of
NMMA to reverse the inhibition and the ability of cytokines to
inhibit T. gondii growth in iNOS?/?astrocytes.
IFN-? has been shown to inhibit Toxoplasma growth via
induction of the enzyme IDO, which results in the degradation
of tryptophan in human fibroblasts, glioblastoma cells, retinal
epithelial cells, and macrophages (8, 12, 23, 24, 29). Addition-
ally, the IDO pathway has been shown to be activated by IFN-?
and TNF-? in some glioblastoma cells and native human as-
trocytes (7). In our study, the addition of tryptophan did not
reverse the inhibition caused by the IFN-? alone or IFN-? in
combination with either TNF-?, IL-1, or IL-6, and no increase
in IDO activity could be detected in astrocytes following cyto-
kine treatment. These data suggest that in murine astrocytes,
cytokines inhibit T. gondii via an IDO-independent pathway.
IFN-?-induced inhibition of T. gondii in endothelial cells has
been shown to be mediated by an IDO-independent mecha-
nism and was also demonstrated not to be mediated via nitric
oxide or reactive oxygen intermediates (32). The presence of
some other mechanism(s) induced by cytokines is not surpris-
ing given the many diverse effects that cytokines have on as-
trocyte functions (1, 20). For example, IL-1 induces a reactive
astrocyte phenotype characterized by the expression of TNF-?,
IL-6, and colony-stimulating factor in astrocytes (20). IFN-?
also induces many changes in cell physiology, including meta-
bolic and cytoskeletal changes. The cytokine-induced inhibi-
tion of T. gondii in astrocytes may be due to some of these
generalized effects on host cell function. It is possible that iron
starvation (11) or other reactive oxygen intermediates can be
induced by IFN-? alone or in combination with other cytokines
and that these mechanisms, which are the focus of our current
investigations, are responsible for the cytokine-mediated inhi-
bition of T. gondii growth in murine astrocytes. Whatever the
mechanism(s) involved, this study clearly demonstrates that cy-
tokine-activated astrocytes induce significant anti-Toxoplasma
activity and that IFN-? is the primary cytokine mediating this
inhibition.
The ability of cytokines to activate astrocytes to inhibit rep-
lication of T. gondii may also be involved in the mechanism of
reactivated Toxoplasma infections in AIDS. For instance, evi-
dence suggests that in patients with AIDS, a shift from a Th1
to a Th2 cytokine profile occurs in the brain (18). The presence
of the Th1 cytokines IFN-?, TNF-?, and IL-1 in the brain
would presumably activate astrocytes to exert anti-Toxoplasma
activity. Concomitantly, a shift to a Th2 cytokine profile, which
is accompanied by a decrease in IFN-? and subsequent de-
creases in TNF-? and IL-1, might then promote growth of
T. gondii in astrocytes. The effect of Th2 cytokines on T. gondii
in astrocytes is not known, but data from our previous study
(13) showed that astrocytes, when unstimulated by IFN-? or
other cytokines, serve as excellent host cells for T. gondii,
supporting extensive replication resulting in the lysis and con-
tinual reinfection of astrocytes. This finding, in conjunction
with the Th1/Th2 shift hypothesis, suggests that astrocytes may
play a pivotal role in the pathophysiology of Toxoplasma en-
cephalitis in the brains of patients with AIDS.
In conclusion, we found in murine astrocytes, IFN-? alone
or in combination with IL-1, TNF-?, or IL-6 significantly in-
hibited growth of T. gondii. Although IFN-?–IL-1 and IFN-?–
TNF-? induced NO production, inhibition was not found to be
via an NO-mediated mechanism. Cytokine-mediated inhibition
was also not due to induction of IDO. The NO/IDO-indepen-
dent pathway responsible for inhibition of T. gondii growth
is currently under investigation in our laboratory. Given that
IFN-? has been shown to be the main cytokine controlling
growth of T. gondii in the brain and that TNF-?, IL-1, and IL-6
are up-regulated in the brains of mice with chronic toxoplas-
mosis, results from the present study indicate astrocytes are
most likely an important effector cell in limiting T. gondii
replication in the brain.
ACKNOWLEDGMENTS
Sandra K. Halonen is an Aaron Diamond Foundation Fellow. This
work was supported in part by a grant from The Aaron Diamond
Foundation and in part by PHS grant AI 39454.
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Editor: J. M. Mansfield
VOL. 66, 1998CYTOKINE INHIBITION OF T. GONDII IN MURINE ASTROCYTES4993