INTRODUCTION

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Toxoplasma gondii encephalitis is a common opportunistic infection of the central nervous system in AIDS patients that is a consequence of the reactivation of the cyst stage of the organism (27). Cysts cause little to no pathology and are thought to persist for the lifetime of the host in muscle and brain (13). Cysts in the brain are thought to periodically rupture, releasing bradyzoites into the brain, but in immunocompetent hosts parasite replication is limited (12). In immune-suppressed patients, however, when cysts rupture, the bradyzoites differentiate into the rapidly replicating tachyzoite stage, resulting in a necrotizing and often fatal encephalitis.

Cytokines play an important role in the regulation of T. gondii in the central nervous system (10, 22, 23). Gamma interferon (IFN-) has been shown to be the main cytokine controlling replication of the parasite in the brain (35). IFN--activated microglia have been demonstrated to be important effector cells in the brain, controlling T. gondii via a nitric oxide-mediated mechanism (7-9, 14). Astrocytes are the predominant host cell for T. gondii in the brain and support prolific growth of the tachyzoite stage (18). IFN- has recently been demonstrated to inhibit parasite replication in astrocytes (19). This IFN--induced inhibition in astrocytes was found to be via a nitric oxide- and tryptophan starvation-independent mechanism (19).

IFN- inhibits the replication of T. gondii in fibroblasts (32), retinal pigment cells (29), endothelial cells (37), and enterocytes (11). In fibroblasts and retinal pigment cells, the mechanism of inhibition is via tryptophan starvation, while in enterocytes the mechanism of inhibition was found to be due to limiting the availability of intracellular iron (11, 29, 32). The mechanism in endothelial cells is unknown but appears to be independent of reactive oxygen intermediates and the tryptophan starvation pathway (37).

We investigate here the role of reactive oxygen intermediates and iron in IFN--induced inhibition of T. gondii in murine astrocytes. Astrocytes genetically deficient in reactive oxygen intermediates and the reactive oxygen scavengers catalase, mannitol, and thiourea were used to investigate the role of reactive oxygen intermediates in IFN--induced inhibition in murine astrocytes. Iron salts, ferric chloride, ferric nitrate, ferric transferrin, and the iron chelator desferrioxamine were used to investigate the role of iron in IFN--induced inhibition.

	MATERIALS AND METHODS

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Primary astrocyte culture. Murine astrocytes from C57BL/6 × SV129 mice or syngeneic mice deficient in phagocyte oxidase (gp91^phox/ [33]) were cultured from the brains of neonatal mice. Murine pups were sacrificed, the brains were removed from the cranium, and then 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-µm (pore-size) Nitex mesh, centrifuged at 200 × g, suspended in growth medium at a concentration of 10⁶ cells/ml and then plated onto poly-L-lysine-coated dishes. Astrocytes were maintained in endotoxin-free minimal essential medium (MEM; GIBCO-BRL, Gaithersburg, Md.) supplemented with 20% fetal bovine serum (FBS; GIBCO-BRL), 5% glucose, and 100 U of penicillin and streptomycin (GIBCO-BRL) per ml. The growth medium was changed every 3 days. After 7 days in vitro a confluent layer of 1 × 10⁴ to 2 × 10⁴ cells/cm² of astrocytes is achieved. 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 (Sigma L-2895). Astrocytes were dissociated in trypsin-EDTA, replated onto poly-L-lysine-coated coverslips at 10⁴ cells/cm in a 24-well plate, 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. The ME49 strain of T. gondii was utilized. Tachyzoites were obtained by in vitro culture in human foreskin fibroblast cells. Parasites were harvested after 2 to 3 days in culture. Parasites were resuspended in MEM supplemented with 10% FBS and then incubated with murine astrocyte cultures infected with 5 × 10⁴ parasites per well, a target ratio of 5:1 (parasites/host cells), for 2 h to allow the parasites to invade. The astrocyte cultures were then washed to remove any extracellular parasites and incubated with medium alone or in the presence of reactive oxygen scavengers or iron salts as described below.

Chemicals and cytokines. Murine recombinant IFN-, interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-), and IL-6 were purchased from Genzyme (Cambridge, Mass.). All other reagents were purchased from Sigma (St. Louis, Mo.). Ferric citrate was prepared by mixing a 1:1 ratio of trisodium citrate with ferric chloride and then adjusting the pH to neutrality with 1 M NaOH.

Cytokine and chemical treatments. Murine astrocytes were stimulated with IFN-, TNF-, or IL-6, alone or in various combinations. Cytokines were used at the following concentrations: IFN- at 100 U/ml, TNF- at 100 U/ml, and IL-6 at 100 U/ml. Astrocytes were incubated with cytokines for 72 h prior to infection. Cultures were washed to remove the cytokines, infected with T. gondii as described above, and then incubated for the next 48 h without cytokines. In some experiments, reactive oxygen scavengers (catalase, thiourea, or mannitol) or iron salts (ferric citrate or ferric transferrin) were added following infection with T. gondii.

Microscopic analysis of T. gondii intracellular replication. The percentage of infected astrocytes was determined by counting the number of infected cells per 500 cells under both phase and immunofluorescent microscopy. In some experiments, growth of parasites was determined by counting the number of tachyzoites in 100 vacuoles. Each condition was tested in triplicate. Immunofluorescence was assessed using a 1:50 dilution of a commercial polyclonal rabbit anti-toxoplasma antibody (Dako, Carpinteria, Calif.), followed by detection with anti-rabbit fluorescein immunoglobulin G (Boehringer-Mannheim, Indianapolis, Ind.) as previously described (18). All cultures were incubated in endotoxin-free media, and no endotoxin contamination was detected in any of the experimental cultures.

Statistics. Within each experiment all conditions were repeated in triplicate wells, and each experiment was replicated two to three times (as indicated in the tables). Data were analyzed by nonparametric (Wilcoxon signed-rank test) and/or parametric (Student t test-analysis of variance) methods using Sigma Stat version 1.0 (Jandel Scientific, San Rafael, Calif.).

	RESULTS

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Effect of cytokines on T. gondii in phox^/ murine astrocytes. Astrocyte cultures were generated from mice genetically deficient in generating reactive oxygen intermediates, stimulated with IFN-, either alone or in combination with TNF-, IL-1, and IL-6 for 72 h, and then infected with T. gondii; the parasite growth was assessed 48 h later. All cytokine combinations significantly (P < 0.05) inhibited the growth of T. gondii in phox^/ murine astrocytes (Table 1). IFN- alone resulted in a 53.6% inhibition of growth compared to control cells, while IFN- in combination with TNF- or IL-6 caused a slightly greater inhibition (34 to 43% of control; Table 1). The IFN--induced inhibition in phox^/ astrocytes was comparable to the cytokine inhibition seen in normal murine astrocytes, whereas IFN- alone induced inhibition of 35 to 40% and a slight synergism was seen with IFN- in combination with other cytokines (18).

View this table: [in this window] [in a new window]   TABLE 1.   Effect of cytokine treatments in phox/ murine astrocytesa

Effect of oxygen scavengers on IFN--induced inhibition of T. gondii. To further test the role of an oxygen-dependent mechanism in the anti-Toxoplasma activity of IFN- in astrocytes, various inhibitors or scavengers were added to murine astrocyte cultures. Neither catalase, which converts hydrogen peroxide to water and oxygen, nor mannitol or thiourea, which are scavengers of hydroxyl radicals, reversed the inhibitory effect induced by IFN- (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Effect of reactive oxygen intermediate on IFN- in murine astrocytesa

Effect of iron(III) on the IFN--induced inhibition of T. gondii. To test whether the IFN--induced inhibition of T. gondii in astrocytes is iron dependent, cells were incubated with the siderophore desferrioxamine (DFO). DFO (50 µM) did not induce inhibition of T. gondii in astrocytes; the addition of the iron salts, ferric citrate, or ferric transferrin to DFO did, however, cause a significant increase (two- to threefold) in the growth of T. gondii in astrocytes (Table 3). The role of iron in the IFN--induced anti-Toxoplasma effect was further tested by the addition of ferric citrate at 5, 50, and 100 µM to IFN--treated cultures. Ferric citrate did not reverse the IFN--induced inhibition of T. gondii in astrocytes at any of the concentrations used (Table 4). These results indicate that the IFN--induced anti-Toxoplasma effect is iron independent in astrocytes.

View this table: [in this window] [in a new window]   TABLE 4.   Role of iron in IFN--treated murine astrocytesa

Effect of IFN- on invasion and growth of T. gondii. The effect of IFN- pretreatment of astrocytes on invasion and growth of T. gondii was also tested by counting the percent infected cells and the number of tachyzoites per vacuole at 2 and 24 h postinvasion, respectively. No significant difference was seen in the percent infected cells at 2 h between control and IFN--treated cells (Table 5), indicating that IFN- pretreatment of astrocytes has no effect on the invasion of host cells. By 24 h, however, both the percent infected cells and the number of tachyzoites per vacuole were significantly less in IFN--treated cells versus control cells (Table 5). The decrease in the percentage of infected cells indicates that IFN- induces a microbicidal effect, while the decrease in the number of parasites per vacuole suggests that a microbiostatic effect occurs by 24 h postinvasion.

View this table: [in this window] [in a new window]   TABLE 5.   Effect of IFN- on growth of T. gondii in murine astrocytesa

	DISCUSSION

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IFN- is the main cytokine controlling T. gondii in the brain (35). Previous studies demonstrated that IFN- significantly inhibits T. gondii in astrocytes via a nitric oxide- and tryptophan-independent mechanism (19). In this study the mechanism of IFN--induced inhibition of T. gondii in astrocytes was further investigated. IFN--induced inhibition was found to be independent of reactive oxygen intermediates, as evidenced by the inability of oxygen radical scavengers to reverse the inhibition and the fact that IFN- could also induce inhibition in astrocytes incapable of producing the reactive oxygen intermediates. The role of iron deprivation in IFN--induced inhibition was also addressed. The inability of DFO to induce the inhibition of the growth of T. gondii and the inability of ferric salts to reverse the IFN--mediated growth inhibition indicate that the IFN--induced inhibition of T. gondii in murine astrocytes is independent of iron deprivation. IFN- was found not to affect invasion by T. gondii of astrocytes but was found to have a microbiostatic and microbicidal effect that was evident by 24 h after invasion.

The mechanisms of IFN--induced inhibition of T. gondii which have been demonstrated in other cell types include reactive oxygen intermediates, induction of nitric oxide production, tryptophan starvation, and iron deprivation. In human mononuclear phagocytes, IFN- induces toxoplasmacidal activity via reactive oxygen intermediates (28). In murine macrophages and microglia, IFN- activates inhibition of T. gondii via L-arginine-dependent production of nitric oxide (1, 5). In nonmyeloid cells, IFN--induced inhibition of T. gondii was found to occur via tryptophan degradation in human fibroblasts and retinal pigment cells (29, 32), while in enterocytes inhibition occurred via iron deprivation (37).

In murine astrocytes, we have previously shown that IFN--induced inhibition of T. gondii was independent of nitric oxide intermediates and tryptophan degradation (19). We found in the present study that IFN--induced inhibition of T. gondii in astrocytes was also independent of reactive oxygen derivatives and iron deprivation. Astrocytes have been shown to produce superoxide via a neutrophil-type NADPH oxidase during recovery from hypoxia (21, 36). The respiratory burst as an antitoxoplasmic mechanism in astrocytes has not previously been investigated. The finding that reactive oxygen intermediates do not play a role in the antitoxoplasmic activity of astrocytes is consistent with studies that have found that p47 phox^/ mice, which lack an inducible oxidative burst, are able to control both the acute and chronic stages of T. gondii infection (2). Iron deprivation, a common antimicrobial mechanism, was also not found to be the mechanism of IFN--induced inhibition of T. gondii in astrocytes. These data indicate that the IFN--induced inhibition of T. gondii in astrocytes occurs via an unknown mechanism.

IFN- is known to induce a diverse array of effects on cells (5, 6). IFN- is a 34-kDa glycoprotein that binds to a membrane receptor. The IFN- receptor is ubiquitously expressed on all nucleated cells at modest levels (6). Binding of IFN- to the membrane receptor transmits signals to the cytoplasm and nucleus by the Jak-STAT pathway which mediate the transcription of IFN--specific genes (5, 6). Several primary response genes are themselves transcription factors and are required for the induction of other secondary components of the cellular response to IFN-. More than 200 IFN--regulated genes have been identified (6). The function of many of these genes is known, and they have been identified as being involved in a diverse range of distinct cellular programs which collectively orchestrate the immune response. For example, IFN- induces the expression of major histocompatibility complex (MHC) I and II molecules, which are involved in antigen presentation; the induction of enzymes, resulting in the respiratory burst; nitric oxide and tryptophan degradation, which have antimicrobial effects; and the induction of expression of ICAM molecules and chemokines, which are involved in leukocyte-endothelium interactions. The function of many of the other known IFN- response genes, however, is not understood.

While the mechanism of IFN--induced inhibition of T. gondii in astrocytes is not understood, it was found that IFN- resulted in a microbiostatic and microbicidal effect that was evident by 24 h after invasion. IFN- has a wide variety of effects on the physiology of cells, including cell shape changes, an antiproliferative effect, and the induction of mitogen-activated protein kinases, which may regulate some of these effects (4, 5, 26). One possible mode of action of IFN- in T. gondii may be through disruption of the intracellular organization of the cytoskeleton or other host cell organelles, which may in turn affect the parasitophorous vacuole, an organelle essential for the intracellular survival of T. gondii. The acquisition of host cell cytoskeleton, endoplasmic reticulum, and mitochondria around the parasitophorous vacuole of T. gondii is well documented, and inhibition of lysosomal fusion with the parasitophorous vacuole is also known to be essential for intracellular survival (24, 34). In support of this, IFN- was found to interfere with the intracellular development and survival of the parasite in astrocytes, and it is possible that this effect of IFN- is due to the disruption of interactions of the parasitophorous vacuole with the host cell organelles.

Whatever the mechanism of IFN--induced inhibition of T. gondii in astrocytes, these studies suggest that astrocytes are an important effector cell in the brain. IFN- induces upregulation of MHC class I and II molecules in astrocytes, and it has been suggested that astrocytes may serve as important antigen-presenting cells in the brain (3). IFN--activated astrocytes, for example, could serve to stimulate MHC class I-restricted CD8⁺ cells, which are cytolytic for infected cells and thought to play a major role in host immunity against T. gondii (17). Our studies indicate that IFN--activated astrocytes also have direct antimicrobial effects on T. gondii.

It is well established that IFN--activated macrophages and microglia, cells of hemopoietic origin, have direct antimicrobial effects in T. gondii and other intracellular pathogens through toxic reactive nitrogen and oxygen intermediates (7-9, 14). Until recently, the role of IFN--activated microbicidal mechanisms in nonhemopoietic cells has been unclear. Yap and Sher (38) addressed this question recently in a study in which susceptibility to T. gondii infection was tested in chimeric mice in which IFN- receptors were expressed on both hemopoietic and nonhemopoietic cells or on hemopoietic cells only. Yap and Sher found that resistance to both acute and chronic infections by T. gondii required the expression of IFN- receptors in both the hemopoietic and nonhemopoietic compartments (38). These results indicate that nonhemopoietic cells are necessary for host resistance to T. gondii. Since T. gondii infects a number of nonhemopoietic cells, including cells of epithelial, mesodermal, and neuronal origin, IFN--activated nonhemopoietic cells may be of particular importance to host resistance to T. gondii. For instance, it has been suggested that IFN--activated enterocytes and endothelial cells are important effector cells controlling parasite dissemination during an acute infection and in congenital toxoplasmosis, respectively (11, 37). Likewise, our studies indicate that IFN--activated astrocytes may be important effector cells controlling replication of T. gondii in the central nervous system and are possibly involved in the prevention of reactivated toxoplasmic encephalitis. Further studies investigating the role of these nonhemopoietic cells in acute infection, congenital toxoplasmosis, and toxoplasmic encephalitis may yield important insights into the pathogenesis of T. gondii.