INFECTION AND IMMUNITY, May 2002, p. 2368–2374 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.5.2368–2374.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 5
Dendritic Cells as Effector Cells: Gamma Interferon Activation of Murine Dendritic Cells Triggers Oxygen-Dependent Inhibition of Toxoplasma gondii Replication F. Aline, D. Bout, and I. Dimier-Poisson* UMR Université-INRA d’Immunologie Parasitaire, UFR des Sciences Pharmaceutiques, 37200 Tours, France Received 15 October 2001/Returned for modification 20 November 2001/Accepted 1 February 2002
Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts, including humans. Much interest has focused on the ability of gamma interferon (IFN-␥)-activated macrophages to prevent intracellular replication, but some other cells (e.g., fibroblasts, endothelial cells, microglial cells, astrocytes, enterocytes and retinal pigment cells) can also be activated to induce this inhibition of proliferation. Dendritic cells are generally known to be involved in the induction of immune responses, but no previous study had investigated the possibility that dendritic cells may act as effector cells of this system. Our results show that IFN-␥-activation inhibits the replication of T. gondii in dendritic cells, with the inhibition being dose dependent. Neither nitrogen derivatives nor tryptophan starvation appears to be involved in the inhibition of parasite replication by IFN-␥. Experiments with oxygen scavengers indicate that intracellular T. gondii replication is oxygen dependent. Our findings suggest that, in addition to their essential role in stimulating the immune system, dendritic cells probably act as effector cells in the first line of defense against pathogen invasion. Toxoplasma gondii causes one of the most common parasitic infections, affecting a wide range of hosts, including humans beings and domestic animals, throughout the world. This parasite has an unusually wide host spectrum, since almost all mammalian cells, including red blood cells, can be infected by this parasite in vitro (39), although intracellular growth usually occurs within nucleated cells. Data from studies performed in vitro and in vivo have shown that gamma interferon (IFN-␥) is one of the main mediators of resistance to T. gondii (36), possibly through activation of the IFN-␥-inducible gene IGTP (7, 18, 38). Administration of IFN-␥ in a murine model gives significant protection against T. gondii (24). Furthermore, mice injected with a monoclonal antibody to IFN-␥ failed to survive to an intraperitoneal infection with T. gondii, whereas infected mice given no antibody survived and went on to develop chronic T. gondii infection (36). Much interest has centered on the ability of activated macrophages to prevent the intracellular replication of T. gondii (40), but some other cells, such as fibroblasts (30), endothelial cells (41), microglial cells (6, 14), astrocytes (29), retinal pigment cells (28), enterocytes (12), and a human uroepithelial carcinoma cell line (9) can also be activated to induce this inhibition of proliferation. Studies have identified several antiparasitic mechanisms, including the production of nitrogen monoxide (23), which is responsible for the lysis of the intracellular parasite Leishmania major in murine macrophages (22). This mechanism seems to be responsible for the inhibition of T. gondii replication in mouse peritoneal macrophages preincubated with IFN-␥ (1)
but not in preincubated astrocytes (16, 17), endothelial cells, or enterocytes (10, 11, 12). The second mechanism of inhibition related to IFN-␥-activated enterocytes has been shown to be due to a limitation of the availability of intracellular iron (12), an element essential for metabolic processes. A third potential mechanism we investigated involves the synthesis of toxic oxygen radicals. These intermediates seem to be responsible for the inhibition of T. gondii replication in immune murine peritoneal macrophages (26) but not in in vivo-activated peritoneal macrophages (34), endothelial cells (41), or astrocytes (17). The final anti-T. gondii mechanism is related to the production of an enzyme, 2,3-dioxygenase-indoleamine, which is synthesized in IFN-␥-activated human macrophages, retinal pigment cells, and fibroblasts (27, 28, 30). This enzyme breaks down intracellular tryptophan, depriving the parasite of this amino acid, which is essential for its proliferation. A recent study (5) reports that dendritic cells (DC), unlike neutrophils and lymphocytes, are highly permissive toward T. gondii invasion and replication. For this reason, it was interesting to test their ability to act as effector cells in the immunological response. DC are generally known to be involved in the induction of immune responses (35). They are found in most tissues and, in particular, in tissues that interface with the external environment (for example, the mucosal surfaces through which microorganisms, such as T. gondii, enter the host). These professional antigen-presenting cells act as “sentinels” in the immunological response to various infecting agents, such as bacteria (37), parasites (I. Dimier-Poisson, F. Aline, M.-N. Mévélec, D. Buzoni-Gatel, and D. Bout, unpublished data), or viruses (3). Indeed, when they encounter an immunological stimulus, most DC use macropinocytosis to concentrate the antigen in the major histocompatibility complex (MHC) class II compartment (32) before they mature and move into the lymphoid tissues (31) in search of antigen-spe-
* Corresponding author. Mailing address: UFR des Sciences Pharmaceutiques, 31 Ave. Monge, 37200 Tours, France. Phone: 33-247-3671-84. Fax: 33-247-36-72-52. E-mail:
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cific T cells (15, 19, 25) and B cells (13) to initiate an antigenspecific immune response. In the present study, we demonstrate for the first time that DC do indeed act as effector cells and can be activated by IFN-␥ to inhibit the intracellular proliferation of T. gondii. We also demonstrate that one of the mechanisms for this inhibition is mediated by the production of an oxygen intermediate, since incubation with oxygen scavengers restored the intracellular replication of T. gondii. MATERIALS AND METHODS Animals. DC were isolated from the spleens of female CBA/J (H-2k) and C57BL/6 (H-2b) mice aged 6 to 10 weeks (Janvier). Female Swiss (OF1) mice were used to maintain the T. gondii cycle. Parasites. T. gondii tachyzoites of the highly virulent RH strain (21), maintained by twice-weekly intraperitoneal passage of 2 ⫻ 106 tachyzoites in female Swiss (OF1) mice, were harvested from the peritoneal fluid of mice that had been infected by intraperitoneal route 3 to 4 days earlier. For the in vitro infection of cells, tachyzoites were counted on a Malassez hemocytometer, and added to the DC. Purification of spleen DC. DC were prepared from the spleens of naive 6- to 10-week-old C57BL/6 or CBA/J mice. The DC were purified by a procedure described by Iwasaki and Kelsall for DC from Peyer’s patches (20). The spleens were dissected and digested with collagenase D (400 Mandl U/ml; Boehringer, Mannheim, Germany) plus DNase I (15 g/ml; Boehringer) for 30 min at 37°C. A single-cell suspension was prepared, and cells were incubated with anti-mouse CD11c-coated magnetic beads (Miltenyi Biotech) and selected on MACS separation columns (Miltenyi Biotech). Sorted spleen DC from CBA/J mice or C57BL/6 mice were routinely 98 to 100% positive for CD11c. Spleen DC were cultured in IMDM medium (Gibco-BRL) containing 10% heat-inactivated fetal calf serum (FCS; Gibco-BRL), 1% L-glutamine (Sigma), and 1% penicillinstreptomycin (Sigma). DC2.4 cultures. The DC2.4 cell line was kindly provided by K. L. Rock (Dana Farber Cancer Institute, Boston, Mass.). DC2.4 cells were obtained from bone marrow cells infected with a retrovirus encoding myc and raf by using supernatant from NIH J2 Leuk cells, as previously described (33). DC2.4 cells were resuspended in complete RPMI medium (RPMI 1640 medium [Gibco-BRL] containing 10% heat-inactivated FCS [Gibco-BRL], 1% Lglutamine [Sigma], and 1% penicillin-streptomycin [Sigma]) and then placed in 25-cm2 flasks (Corning). The cultures were maintained at 37°C in 5% CO2 at 95% humidity, and the medium was changed every 3 days. FACScan analysis. Before the inhibition assay, the purity of isolated spleen DC was determined on a FACScan apparatus (Becton Dickinson). Cells were incubated with 2.4G2 (rat anti-mouse Fc receptor monoclonal antibody [MAb]; Pharmingen) for 10 min to prevent antibody binding to FcR; they were then stained and incubated with the fluorescein isothiocyanate-coupled MAbs RA36B2 (Rat immunoglobulin G2a [IgG2a] and IgG2k anti-CD45R/B220; Pharmingen), 53-2.1 (rat IgG2a and -k anti-CD90; Pharmingen), and Ly-2 (rat IgG2a and -k anti-CD8␣). In order to determine their presentation and costimulation activity, spleen DC from CBA/J mice were also stained and incubated with 14.4.4 (murine IgG2a anti-I-Ek,d), 16-10A1 (rat IgG2a anti-CD80), and GL1 (hamster MAb antiCD86). In order to confirm that the DC2.4 line was maintained properly, the cells were stained and incubated with the same antibodies. Labeled spleen DC and DC2.4 cell cultures were analyzed on a FACScan apparatus (Becton Dickinson). Assay of T. gondii intracellular replication in IFN-␥ DC. (i) Uracil assay. DC2.4 and spleen DC cultures were trypsinized and seeded into 96-well plates (Falcon) at a concentration of 5 ⫻ 104 cells/well in 50 l of complete Iscove modified Dulbecco medium (IMDM). The plates were incubated at 37°C in 5% CO2 at 95% humidity. One day after seeding, 50-l volumes of various concentrations of IFN-␥ (R&D Systems, Abingdon, United Kingdom) diluted in complete IMDM were added. The cultures were incubated for 18 h and then washed twice with prewarmed complete IMDM. T. gondii tachyzoites were added according to a ratio of three tachyzoites per DC to the DC in 50 l of complete IMDM, and the plates were incubated. After 1 h, 1 Ci of [5,6-3H]uracil (NEN, Boston, Mass.), which is specifically incorpo-
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rated by the parasite, were added to each well in 50 l of complete IMDM, and the cultures were incubated overnight. The parasite contained a significant amount of uracil phophoribosyltransferase, an enzyme responsible for the conversion of uracil into UMP, which is not normally present in the host cell. Once synthesized, the uridine was incorporated in the mRNA of T. gondii. Nonincorporated [3H]uracil and any tachyzoites that had failed to infect DC were removed by two successive washes in 200 l of complete RPMI. DC2.4 cells and spleen DC were lysed by freezing overnight followed by thawing. The contents of the well were deposited on glass fiber filters by using an automated cell harvester (Tomtec), and the radioactivity level was determined by using a liquid scintillation counter (Wallac Microbeta). The results are expressed in counts per minute (cpm). For each experiment, the controls included DC, which had or had not been preincubated with IFN-␥, that were cultured without T. gondii. The radioactivity for these samples was always near the background levels, confirming that [3H]uracil incorporation occurred specifically in the presence of the parasite. (ii) Fixing, staining, and photomicrography. Thermanox plastic coverslips (13 mm in diameter) treated on one side were placed in a 24-well plate (Falcon), and 1 ml of FCS (Gibco-BRL) was added for 30 min and then removed. DC2.4 cultures were trypsinized and seeded into 24-well plates at a concentration of 1 ⫻ 105 cells/well in 1 ml of complete IMDM. The plates were incubated at 37°C in 5% CO2 at 95% humidity. At 2 days after seeding, 200 l of IFN-␥ (500 pg/ml) diluted in complete IMDM was added. The cultures were incubated for 12 h, and T. gondii tachyzoı¨tes were added according a ratio of three tachyzoites per DC for 24 h. After incubation, the DC were washed in 1 ml of 1⫻ phosphate-buffered saline. The plastic coverslips were subjected to May-Grunwald-Giemsa fixing and staining with a Hemacolor (Merck, Darmstadt, Germany) microscopy staining kit and then washed in distilled water. The cells were then mounted between a slide (LMR; Labo-Moderne, Paris, France) and a cover glass in a drop of aqueous mounting medium (Immu-Mount; Shandon, Pittsburgh, Pa.). Photomicrographs of the various preparations were made by using a microscope Axioskop MC80 (Carl Zeiss) on a Fujichrome Sensia 400 ASA film and used to carry out a microscopic count of the various tachyzoite division stages. Effect of oxygen scavengers on the IFN-␥-induced inhibition of T. gondii replication. DC2.4 cells and spleen DC were cultured, activated, infected, and harvested as described above, but this time oxygen intermediate scavengers were added to the cultures during their incubation with IFN-␥ (100 pg/ml) and then added again during their infection with T. gondii. The oxygen scavengers used and their final concentrations in the cultures were as follows: 50 mM D-mannitol (OH⫺ scavenger), 10 mM benzoic acid (OH scavenger), 2.23 mg of superoxide dismutase (O2⫺ scavenger)/ml, and 1 mM DABCO (O2⫺ quencher). All were purchased from Sigma and were made up in complete RPMI medium immediately before use. Murray and Cohn (26) had previously used these concentrations and experimental conditions to block the oxygen-dependent inhibition of T. gondii replication. The results are expressed in percent counts per minute, taking the values for the infected DC as 100% cpm. Effect of L-tryptophan on IFN-␥-induced inhibition of T. gondii replication. DC2.4 cells and spleen DC were cultured, activated, and infected as described above, except that exogenous L-tryptophan (Sigma) in complete IMDM was added, along with the IFN-␥ (100 pg/ml), and added again during T. gondii infection. The concentration of L-tryptophan in the wells ranged from 0.5 to 1,000 g/ml. The results are expressed in percent counts per minute, taking the values for unactivated DC as 100% cpm. Test for nitrite production by DC. The Griess reaction was used to test for the presence of nitrite, an indicator of the production of nitrogenous intermediates from L-arginine, in a 24-h-old supernatant of IFN-␥-activated DC, cultured as described above. Cell culture supernatant (150 l) was added to 700 l of a freshly prepared mixture of 1% sulfanilamide (Sigma) in 1.2 N hydrochloric acid and 0.3% N-1-naphtylethylenediamine dihydrochloride (Sigma). The absorbance at 540 nm was determined after incubation for 30 min in the dark. Nitrite concentrations were deduced by reference to a calibration curve prepared by using standard solutions of sodium nitrite (Merck) diluted in the same medium as that used for the cell culture. Statistical analysis. The significance of the differences between the groups was determined by using InStat 2.01 software. Comparisons between experimental groups were made by using analysis of variance.
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TABLE 1. Phenotype analysis of isolated DCa Cell type
% Splenic DC
% DC2.4 cells
MHC class II CD80 CD86 CD8␣
94 55 58 65
97 90 90 ND
a DC were incubated with a rat anti-mouse Fc receptor antibody and then incubated with the fluorescein isothiocyanate-coupled MAbs Ly-2 (anti-CD8␣), 14.4.4S (anti-MHC class II), 16-10A1 (anti-CD80), and GL1 (anti-CD86). Labeled spleen DC and DC2.4 cells were analyzed on a FACScan apparatus. The values shown are representative of at least three experiments. ND, not determined.
RESULTS Phenotype analysis of isolated DC. We analyzed the expression of T- and B-cell markers on DC-enriched populations positively selected from spleens on the basis of their expression of CD11c. We found that a nonsignificant proportion of antiCD11c magnetic bead-selected cells expressed B220 (⬍5%), whereas CD90 staining was undetectable (data not shown). Spleen CD11c⫹ DC from CBA/J mice or C57BL/6 mice were 94% positive for MHC class II (Table 1) and 55 to 58% positive for the costimulatory molecules CD80 and CD86, as described by Bourguin et al. (4).
Spleen CD11c⫹ DC from C57BL/6 mice were 65% positive for CD8␣, as described by Anjuere et al. (2). DC2.4 were routinely ⱖ90% positive for CD80, CD86, and MHC II, as shown by Rock and coworkers (33). Effect of IFN-␥ on T. gondii replication in DC2.4 cells and spleen DC investigated by the uracil technique. Unactivated, T. gondii-infected DC2.4 cells, spleen DC from CBA/6 mice, and spleen DC from C57BL/6 mice all incorporated large amounts of [3H]uracil (22,000, 18,000, and 21,000 cpm, respectively) (Fig. 1), suggesting that high levels of proliferation of tachyzoites are found in DC. Uninfected cells were also radiolabeled in every experiment but consistently incorporated ⬍5% of the radioactivity found in the control culture. Furthermore, the level of [3H]uracil was dramatically reduced in DC that had been preincubated with IFN-␥ (Fig. 1). In DC2.4 cells (Fig. 1A), the IFN-␥-dependent inhibition was dose dependent from 100 pg/ml (the basal cpm rate) to 12 pg/ml, the lowest dose to induce inhibition in DC2.4 cells (15,000 cpm). The IFN-␥-dependent inhibition in spleen DC from CBA/J or C57BL/6 mice was observed from 500 pg/ml (the basal cpm rate) to 100 pg/ml (12,000 or 16,000 cpm, respectively) (Fig. 1B and C), the lowest dose to induce inhibition. Replication was abolished at doses of IFN-␥ of 500 pg/ml (spleen DC) and 100 pg/ml (DC2.4 cells) (P ⬎ 0.05), respectively. Concentrations of
FIG. 1. Effect of IFN-␥ on T. gondii replication in DC ([3H]uracil assay) observed 18 h after infection with three tachyzoites per cell. The values shown are the means of triplicate samples, with standard error bars. Infected DC, unactivated, infected DC; untreated DC, unactivated, noninfected DC. (A) DC2.4 cells. (B) Spleen DC from CBA/J mice. (C) Spleen DC from C57BL/6 mice.
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IFN-␥ ranging from 100 down to 25 pg/ml inhibited T. gondii proliferation in DC2.4 cells (Fig. 1A) but did not significantly inhibit T. gondii replication in spleen DC (Fig. 1B and C). Doses of IFN-␥ ranging from 12 to 0.2 pg/ml did not significantly inhibit T. gondii replication in either DC2.4 cells or spleen DC. These findings suggest that the intracellular proliferation of T. gondii is inhibited by IFN-␥. The trypan blue assay did not reveal any toxic effects of IFN-␥ on the DC, even at higher doses. Investigation of the effect of IFN-␥ on T. gondii replication by the microscopic counting of tachyzoites at various multiplication stages. To determine whether parasite division was impaired or whether the parasite could have been lysed by IFN-␥-treated cells, DC from the DC2.4 cell line infected overnight were scored for the numbers of parasites per vacuole. Photomicrographs of these infected cells are shown in Fig. 2A and B. At day 1 postinfection, at a DC-to-parasite ratio of 1:5, large vacuoles containing parasites at higher multiplications of infection were found in untreated, infected DC (Fig. 2A), with 38% of the infected cells showing tachyzoites at stage 8 and 41% showing tachyzoites by at least stage 16 (Fig. 2C). These findings confirm that DC do allow rapid tachyzoite division (5). In IFN-␥-treated DC, the infected DC mainly harbored division stages 2 and 4 (Fig. 2B), showing that IFN-␥-treated DC are less permissive toward normal tachyzoite division. Effect of L-tryptophan on IFN-␥-induced inhibition of T. gondii replication. Supplementation of the culture medium with 1 mg of L-tryptophan/ml never restored the radioactivity level to that observed in the DC culture (Fig. 3). This result showed that L-tryptophan had no effect on the IFN-␥-induced inhibition of T. gondii replication in either the DC2.4 cell line or spleen DC. Effect of oxygen scavengers on IFN-␥-induced inhibition of T. gondii replication. In order to find out whether the mechanism responsible for the inhibition of multiplication observed after IFN-␥ activation of DC is oxygen dependent, four oxygen scavengers were tested. The results showed that adding D-mannitol, DABCO, benzoic acid, or superoxide dismutase restored the initial replication of T. gondii in IFN-␥-activated DC (Fig. 4). Very little uracil incorporation occurred when the infected DC had been activated with IFN-␥, whereas preincubation with one of these four oxygen scavengers totally or partially restored the high level of radioactivity observed in infected but inactivated DC. The mechanisms responsible for the inhibition of replication of T. gondii therefore seem to be connected to an oxygen pathway. Effect of IFN-␥ activation on nitrite production by DC. We examined the relationship between the ability of IFN-␥stimulated cells to produce NO and their anti-Toxoplasma activities. No detectable amounts of nitrite were found in the culture supernatants of IFN-␥-stimulated DC. The IFN-␥ treatment did not increase the production of NO by spleen DC or DC2.4 cells compared to that of control cultures (data not shown). These findings imply that the inhibition of T. gondii replication in DC by IFN-␥ is not dependent on NO production.
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FIG. 2. Photomicrographs of May-Grunwald-Giemsa preparations of tachyzoite-infected DC 2.4 cells. Host cells were incubated for 24 h with tachyzoites at a cell-to-parasite ratio of 1:5. Arrowheads indicate parasites. Magnification, ⫻850. (A) Tachyzoite-infected DC showing rapid tachyzoite division, with up to eight tachyzoites per vacuole. (B) Tachyzoite-infected DC treated with IFN-␥ (500 pg/ml), showing slower tachyzoite division, as evidenced by the presence of ⬍4 tachyzoites per vacuole. Photomicrographs were made with Fujichrome Sensia 400 ASA film, and the slides were scanned into Adobe Photoshop files by using a UMAX scanner (Astra 1220S). (C) Microscopic counts of tachyzoites at various division stages (division stage: numbers of tachyzoites per infected cell) made at a magnification of ⫻200. The values are the means of three experiments, and 10 to 20 microscope fields were counted.
DISCUSSION Our findings show that IFN-␥ activation inhibits the replication of T. gondii in an immortalized DC line, as well as in primary spleen DC. These cells are known to be among the first cells to encounter antigens, partly because they are widely distributed in most tissues, notably those in contact with the external environment, thus explaining their importance in studies investigating interactions between host cells and infectious agents, such as T. gondii, for which the oral route is the usual route of entry. These professional antigen-presenting cells are the “sentinels” of the immune system, but no previous study had investigated the possibility that DC may act as effector cells in this system. For the first time, we demonstrate here that DC do act as effectors, and this constitutes a microbiostatic capacity.
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FIG. 3. Effect of L-tryptophan on the induction of inhibition of T. gondii replication in DC by IFN-␥. Infected DC, unactivated, infected DC; untreated DC, unactivated, noninfected DC; activated and infected DC, infected DC treated with IFN-␥ at the lowest concentration required to inhibit T. gondii replication (100 pg/ml for DC2.4; 500 pg/ml for spleen DC), cultured with or without L-tryptophan (Trp). The values shown are the means of triplicate samples. These results are representative of three different experiments. (A) DC2.4; (B) spleen DC from CBA/J mice; (C) spleen DC from C57BL/6 mice.
Thus, DC should be considered as cells involved in both induction and effector mechanisms of a specific immune response. Here we report significant inhibition of T. gondii replication in DC activated by IFN-␥ at concentrations of from 100 to 12 pg/ml in the DC2.4 cell line and from 500 to 100 pg/ml in spleen DC. At concentrations from 100 pg/ml (DC 2.4) and from 500 pg/ml (spleen DC), replication was completely abolished (P ⬍ 0.05). Microscopic examination indicated that the parasite still infected the DC but that its division was subsequently markedly impaired in the IFN-␥-activated DC. This enables the IFN-␥-activated DC to prevent the intracellular replication of T. gondii, and this inhibition is dose dependent. The blue trypan assay showed that IFN-␥ itself had no toxic effect even at the higher dose. Moreover, any direct impact of IFN-␥ itself can be excluded since the DC were washed after activation with IFN-␥ for 18 h. It was necessary to be certain about this, since cell-mediated immunity is the main mechanism by which the immune-competent host controls Toxoplasma infection, especially by inducing IFN-␥ production by CD8⫹ and CD4⫹ T lymphocytes. Indeed, IFN-␥ clearly plays a crucial role in resistance to T. gondii (36), since in a murine model the administration of IFN-␥ leads to a significant protection against T. gondii (24). There are several possible mechanisms for the inhibition of replication. One such mechanism, known to occur in activated macrophages, is the L-arginine-dependent production of nitric oxide, with its subsequent conversion into nitrite and nitrate. Unlike mouse macrophages, but like human endothelial cells, the L-arginine-dependent production of nitrogen intermediates
does not seem to be a significant mechanism in the inhibition of T. gondii replication by IFN-␥-activated DC. Another possible anti-Toxoplasma mechanism that we investigated was the breakdown of tryptophan. IFN-␥ stimulates human fibroblasts to produce an enzyme, indoleamine-2.3dioxygenase, which breaks down tryptophan, thus starving T. gondii of this essential amino acid. We found that the presence of tryptophan did not prevent the IFN-␥-induced inhibition of T. gondii by DC and that this mechanism does not seem to be involved in any cell type in the murine model. Finally, we found that the mechanism by which IFN-␥-activated DC inhibit T. gondii replication involves the production of toxic oxygen metabolites. Reactive oxygen intermediates appear to be the mechanism of inhibition of T. gondii replication by IFN-␥-activated DC, since this inhibition was entirely or partially reversed when the cells were cultured in a medium supplemented with oxygen scavengers. A mechanism of this type has been observed in the inhibition of T. gondii replication in murine macrophages (26) and in the elimination of the bacterium Rhodococcus equi (8) by IFN-␥-activated murine macrophages. Our findings suggest that, in addition to their essential role in stimulating the immune system, DC probably act as effector cells in the first line of defense against pathogen invasion. The ability of DC to inhibit T. gondii replication after IFN-␥ activation through an oxygen-dependent mechanism may have important implications for the defense against invasion by T. gondii and other infectious agents and their subsequent dissemination.
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FIG. 4. Effect of oxygen scavengers on IFN-␥-induced inhibition of T. gondii replication in DC. Infected DC, unactivated, infected DC; untreated DC, unactivated, noninfected DC; activated and infected DC, infected DC treated with IFN-␥ at the lowest concentration required to inhibit T. gondii replication (100 pg/ml for DC2.4; 500 pg/ml for spleen DC), cultured with or without various oxygen scavengers. The values shown are the means of triplicate samples. The results are representative of three different experiments. (A) DC2.4; (B) spleen DC from CBA/J mice; (C) spleen DC from C57BL/6 mice.
ACKNOWLEDGMENTS We thank D. Tabareau and R. Magné for excellent technical assistance. We thank F. Velge-Roussel for help in histological analysis.
8.
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