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Induction of toxoplasmostasis in a human glioblastoma by interferon γ
Journal of Neuroimmunology, 43 (1993) 31-38 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00
31
JNI 02305
Induction of toxoplasmostasis in a human glioblastoma by interferon y Walter D a u b e n e r a Korinna Pilz a Samira Seghrouchni Z e n n a t i a, Thomas Bilzer b, Hans-Georg Fischer a and Ulrich Hadding b a Institut fiir Medische Mikrobiologie und Virologie, and oAbteilung fiir Neuropathologie, Heinrich-Heine- Universitdt, Diisseldorf, Germany (Received 8 June 1992) (Revision received 16 September 1992) (Accepted 16 September 1992)
Key words: Gliobtastoma cells; Toxoplasma; Interferon 3'
Summary In the course of human toxoplasmosis central nervous system involvement often occurs. As a model for toxoplasma growth within human brain cells the proliferation of Toxoplasma gondii strain BK within the human glioblastoma cell line 86HG39 was analysed. We found that 86HG39 cells support the growth of toxoplasma similar to human monocyte derived macrophages and in contrast to human monocytes. The growth of Toxoplasma gondii within interferon 3' (IFNT) treated 86HG39 cells is reduced due to toxoplasmostasis and not due to toxoplasmocide effects. The mechanism of IFNy induced toxoplasmostasis was also investigated. It was found that IFNy did not induce 0 2 production a n d / o r nitrite oxide production, and inhibitors of O f and NO 2 did not influence IFNy induced toxoplasmostasis. In contrast, the supplementation of L-tryptophan to the culture medium completely abolished the IFNy effect. We therefore conclude that the induction of L-tryptophan degradation in 86HG39 cells by IFNy, possibly by activation of the indoleamine-2,3-dioxygenase, is responsible for the IFNy induced toxoplasmostasis within the glioblastoma cell line.
Introduction
Toxoplasma gondii is an obligate intracellular parasite that is gaining wider recognition as an opportunistic pathogen producing fulminant infection in compromised hosts. Central nervous system (CNS) involvement by acquired toxoplasmosis is often seen, but neurologic symptoms are variable. In healthy human adults Toxoplasma gondii proliferation within the CNS is restricted, resulting in the formation of brain cysts containing large amounts of bradyzoites. These cysts persist for the remainder of the life of the host. In the case of immunosuppression, for example by infection with the human immunodeficiency virus, a reactivation of Toxoplasma gondii occurs, resulting in a life-threatening encephalitis.
Correspondence to: W. D~iubener, Institut ffir Medische Mikrobiologie und Virologie, Moorenstrasse 5, D-4000 Diisseldorf, Germany.
The knowledge of the immune mechanisms that participate in the host defense against toxoplasma in vivo derives primarily from studies in animal models. It was shown that despite the production of antibodies which after complement activation are effective in the killing of extracellular toxoplasma (Schreiber and Feldmann, 1980) cellular immune reactions are of importance. In this cellular immune response, NK cells (Hauser et al., 1983; Hauser and Tsai, 1986) and T cells (Khan et al., 1988; Kasper et al., 1992) were involved but the principal part in the control of toxoplasma growth is played by mononuclear phagocytes as shown by in vitro data. Different studies have shown remarkable differences of toxoplasma growth between cells of human or murine origin. Human monocytes (Murray et al., 1985) and different types of human macrophage (Catterall et al., 1987, Israelski et al., 1990) were able to restrict intracellular toxoplasma growth without prior activation by lymphokines. In contrast, all types of murine macrophages were only able to restrict toxoplasma growth after activation by
32 lymphokines, in particular IFNT (Remington et al.. 1975; Adams et al., 1990). Cellular immune reactions within the central nervous system, which is always involved in toxoplasmosis, were strictly controlled by the blood-brain-barrier consisting of endothelial ceils and astrocytes. Furthermore, only few cells expressing MHC antigens necessary for the initiation of cellular immune response were found within normal brain parenchyma. Within the CNS two types of accessory cells endowed with macrophage-like capacity exist, microglia cells and astrocytes (Fontana et al., 1987b). Despite immunhistological studies concerning the fate of Toxoplasma gondii within the CNS (Kittas et al., 1984, Schltiter et al., 1991) only few in vitro data are available. For example, it is described that IFNT is involved in the control of toxoplasma growth within murine astrocytes (Jones et al., 1986). To our knowledge no data about toxoplasma growth within human astrocytes are available. Most of our knowledge about human astrocyte cell function is obtained by the use of glioblastoma cells of astrocytic origin, because, due to ethic reasons, human native brain cells are difficult to obtain. Human glioblastoma cells have been described to exhibit many macrophage-like functions, for example monokine secretion, the capacity for phagocytosis (Fontana et al., 1987a), IFNT responsible class I1 antigen expression (Carell et al., 1982) and target function for human cytotoxic T cells (Dhib-Jalbut et al., 1990). In this study the growth of Toxoplasma gondii strain BK within the glioblastoma cell line 86HG39 is investigated. This cell line is described to express the astrocyte marker protein G F A P (Bilzer et al., 1991). The accessory cell capacity of 86HG39 cells involving MHC antigen expression, monokine secretion and the capacity to activate human T ceils has been described recently (D~iubener et al., 1992).
Materials and M e t h o d s
Toxoplasma gondii Toxoplasma gondii strain BK was a kind gift of Drs. Seitz and Saathoff (Institut ftir Med. Parasitologie, Bonn, FRG). Toxoplasma gondii was expanded by the use of the murine fibroblast cell line L929 (ATCC, Rockville, MA) as feeder cells. In brief, 1.2 × 10 6 L929 cells were cultured in culture flasks (Costar, Cambridge, MA) and 10 × 106 T. gondii were added in 5-ml Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY) supplemented with 2% glutamine (Gibco, Grand Island, NY) and 10% heat inactivated FCS (Seromed, Biochrom, Berlin, FRG). After 3-5 days of cultivation the L929 cells were completely lysed by T. gondii. The parasites were harvested and cen-
trifuged at 500 rpm for 5 rain to pellet the few remainig L929 cells and cell debris. The supernatant was removed and centrifuged at 2000 rpm for 10 rain. Thereafter 7~ gondii were resuspended in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 5 ~ FCS, and used for infection of different cells.
Culti~ation of 86HG39 cells The glioblastoma cell line 86HG39 which is defined by means of immunocytochemical and morphological criteria (Bilzer et al., 1991) was used in this study between the 70th and 150th in vitro passage. Cells were grown in tissue culture flasks and divided weekly in ratios between 1 : 2 and 1 : 10 depending on the proliferation rate. To simplify harvesting, the adherent glioblastoma cells were detached with t r y p s i n / E D T A (Seromed, Biochrom, Berlin, FRG) and viability was tested after harvesting by Trypan blue exclusion. For IFNT treatment 86HG39 cells were harvested, washed three times with PBS and resuspended in RPMI 1640 medium supplemented with 5% FCS. Then 1-1.5 M 10 6 cells were cultured in tissue culture flasks and IFNT (Genzyme, Cambridge, MA) was added. In preliminary experiments it was found that treatment of 86HG39 cells with 5 0 0 U / m l IFNT for 72 h was optimal for the induction of toxoplasmostatic effects, and therefore these conditions were used in all experiments.
Preparation of human monocytes and monocyte derh'ed macrophage Human PBL were obtained from buffy coates from healthy donors (Dr. P. Wernet, Institut fiir Blutgerinnungswesen und Transfusionsmedizin, Heinrich-Heinc Universit~it, Diisseldorf, FRG) by Ficoll Hypaque separation. The PBL were allowed to adhere on plastic Petri dishes (Greiner, Ntirtingen, FRG) for 3 h. The non-adherent cells were then removed and the remaining cells were used as monocytes or cultured for 7 days in Iscove's medium 5% FCS to allow differentiation into monocyte-derived macrophage (MDM) as described (Murray et al., 1985). IFNy treatment of MDM was done exactly as described for 86HG39 cells.
Assay for toxoplasma proliferation It has been shown that intracellular Toxoplasma gondii incorporate large amounts of 3H-uracil into nucleic acid because they have substantially more uridine phosphorylase (which converts uracil into uridine which is then incorporated into nucleic acid) than mammalian cells (Pfefferkorn and Pfefferkorn, 1977). This 3H-uracil incorporation method is used in this study for monitoring of toxoplasma growth with minor modifications. In brief, 1.5 × 10 4 86HG39 cells or 3 × 10 4 freshly prepared monocytes or monocyte derived macrophage were cultured in 96-well flat bottom culture plates
33 (Greiner, Niirtingen, FRG) in a total volume of 100/zl per well. Then 1 × 104 freshly prepared toxoplasma were added in 50 ~1. In some experiments L-tryptophan, D-tryptophan, superoxide dismutase (obtained from Sigma, Deisenhofen, F R G ) or NG-monomethyl-c arginine (Calbiochem, Bad Soden, FRG) was added. After 24, 48, 72 or 96 h 0.4/xCi 3H-uracil (Amersham Buchler, Braunschweig, F R G ) was added to the culture and left for 14-18 h. The cultures were then processed for liquid scintillation counting. For monitoring of T. gondii growth within the first 30 h after infection another method similar to those described by several other authors (Murray et al., 1985; Catterall et al., 1987), was used. 2 X 104 86HG39 cells in 100 /xl were allowed to adhere on glass cover slips placed in a 24-well culture plate (Costar, Cambridge, MA). Medium was then removed and 3 x 104 viable T. gondii were added in 100 /~1. After 3 h, extracellular T. gondii were removed by extensive washing and 0.5 ml RPMI 1640 5% FCS was added. At this time point (zero) two cover slips were removed and fixed with Diff-Quick fixative (Merz + Dade, Diidingen, Switzerland). The other two cover slips per group were handled in the same way after 30 h. After fixation 200 /xl of a 1:500 diluted serum sample obtained from a 32-year-old person which had an acute but asymptomatic toxoplasmosis was added for 30 rain. After two washing steps a FITC labelled anti human Ig antibody (BioMerieux, Marcy, France), diluted 1 : 300 in PBS containing Evans blue was added for 30 min. After two additional washing steps 200 cells per cover slip were counted using a fluorescense microscope (Zeiss, K61n, FRG). The number of infected cells and the number of T. gondii per infected cell were determined. In each group two cover slips were counted. The data are given as the mean _+SE of four independent experiments.
Determination of NO~The determination of NO 2 was performed by the use of Griess-reagent (0.3% naphthylethylenediamide-dihydrochloride, 1.0% sulfanylamide obtained from Sigma (Deisenhofen, F R G ) as described (Ding et al., 1988). 2 x 10 4 86HG39 cells IFNy treated or not were cultured in a 24-well plate in 2 ml Iscove's 5% FCS. After 24, 48, 72 and 96 h the supernatant was removed and 100/zl were mixed with an equal volume of Griess reagent. Absorbance at 543 nm was measured by the use of a microplate reader (Nunc, Wiesbaden, FRG). As positive control supernatants of murine bone marrow derived macrophages, obtained from A. Bielinsky from our institute, were used. The amount of NO~- produced was estimated by comparison with a standard consisting of a two-fold diluted sodium nitrate solution. The detection limit of the test was 2 - 4 n m o l / m l NO 2.
Measurement of Oj- production To 3 X 104 86HG39 cells/well of a 96-well flat bottom culture plate in RPMI 1640 free of phenol red, (Gibco, Grand Island, NY) phorbol-myrestat-acetate (PMA, Sigma Deisenhofen FRG) was added between 20 n g / m l and 0.2 n g / m l . 0 2 production was measured by the reduction of cytochrome c (Sigma, Deisenhofen, F R G ) (Pick and Mizel, 1981). As positive control, human granulocytes obtained from Ficoll gradient after NH4CI lysis of erythrocytes were used.
Determination of IL-6 production 2 x l0 s 86HG39 cells IFNy treated or not were cultured in a 24-well culture plate and stimulated with 1 / z g / m l LPS (Sigma, Deisenhofen, F R G ) for 24 h. The supernatant was then removed and added in three-fold dilutions to 2.5 x 103 B9 cells. The proliferation of B9 cells was monitored by 3H-thymidine incorporation after 42-72 h in vitro culture. In each experiment rhu IL-6 (Pharma Biotechnologie, Hannover, F R G ) was used as positive control and one unit of IL-6 gives raise to half maximal proliferation. The B9 bioassay is described to be specific for IL-6 and no cross-reactions with other lymphokines, especially with IL-1, are known (Helle et al., 1988). B9 cells in our laboratory were maintained in Iscove's 5% FCS containing 1% of supernatant of LPS stimulated PBL.
Results
Growth of Toxoplasma gondii strain BK in human peripheral blood accessory cells Toxoplasma gondii strain R H did not proliferate within freshly prepared human monocytes, but monocyte-derived macrophage after 5 - 7 days of in vitro incubation support the growth of this parasite (Murray et al., 1985). We first tested whether Toxoplasma gondii strain BK used by us, which is similar to strain R H with respect to its virulence, shows the same growth behavior in human accessory cells. Therefore human monocytes were infected with Toxoplasma gondii strain BK and parasite growth was monitored by 3H-uracil incorporation. In parallel, monocyte-derived macrophages (MDM) and IFNy treated MDM were used. It was found that Toxoplasma gondii strain BK did not or only marginally replicated within fresh monocytes whilst a remarkable parasite growth occurs within MDM. The treatment of MDM with r I F N y (500 U / m l ) 72 h before toxoplasma infection led to an activation of MDM and these cells were then able to inhibit parasite growth. From these data we conclude that Toxoplasma gondii strain BK is similar to strain R H with respect to its growth within human peripheral blood accessory cells.
34 40000
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2
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4
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days after infection
Z "--"~
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,~= SH-tnymidine cam 86HG39 + T o x o p a s m a i = SH-Lraci~ cpr~ 86HG39 ~ Toxoplasma
Fig. l. 1.5 X 104 86HG39 cells were cultured with or without 10 x 104 Toxoplasma gondii. The proliferation of the glioblastoma cells was monitored by 3H-thymidine incorporation, while toxoplasma growth was measured by 3H-uracil uptake in parallel. Data are given as mean c p m + S E of triplicate cultures. 3H-thymidine and 3H-uracil incorporation in Toxoplasma gondii cultured without 86HG39 cells was less than 500 cpm. Also 3H-uracil incorporation of uninfected 86HG39 cells was less than 500 cpm.
Growth of Toxoplasma gondii within the human glioblastoma cell line 86HG39 Firstly, experiments were performed to assay whether glioblastoma cells can support the growth of Toxoplasma gondii. Therefore 1.5 x 10 4 86HG39 cells per well were infected with 1 x 104 T. gondii and parasite growth was monitored by 3H-uracil incorporation on different days after infection. The growth of 86HG39 cells infected or not was determined in parallel by 3H-thymidine uptake (Fig. 1). It was found that Toxoplasma gondii strain BK is able to proliferate within 86HG39 cells measured by 3H-uracil incorporation and visualized by microscopic examination of infected cultures. Due to the fact that T. gondii is an obligate intracellular parasite the growth of infected
Fig. 2. 1.5 X 105 86HG39 cells, IFNT-treated or not, were cultured with I X 104 viable T. gondii. T. gondff growth was monitored by 3H-uracil uptake on different days after infection. Data are given as mean cpm _+SE of triplicate cultures.
86HG39 cells is reduced by killing of the cells by T.
gondii. Thereafter, also the growth of the parasite declines because of the lack of viable feeder cells. Similar growth data were obtained by the infection of 86HG39 monolayers with T. gondii for 3 h and counting the number of infected cells and the number of T. gondii per infected cell at different times after infection. The data obtained (not shown) revealed that the number of infected cells remained stable for up to 30 h, and therefore we conclude that 86HG39 ceils were not able to kill intracellular T. gondii. In contrast, the number of T. gondii per infected cell increased, indicating the growth of the parasite.
Toxoplasmostasis induced in 86HG39 cells by IFNy IFNy is the major lymphokine involved in the control of the growth of intracellular pathogens (Suzuki et al., 1988). Furthermore, IFN7 is described to inhibit tumor cell growth (Ozaki et al., 1988). The addition of IFN7 to 86HG39 cells results in a dose-dependent 25
30
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-~ 10 0 x 0 I"-
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86HG39 cells treated with IFN'/
Fig. 3, IFNy-treated or -untreated 86HG39 cells were infected with Toxoplasma gondii for 3 h and the number of infected cells and the number of T. gondii per infected cell was determined by indirect immunofluorescence as described in Materials and Methods. The data are given as mean _+ SE of four independent experiments.
35 growth inhibition and similar data were obtained when 86HG39 cells were only pretreated with IFN 7. The reduced proliferation rate is not due to a complete killing of 86HG39 cells by IFNy because IFN 7 treated cells were found to be viable measured by the Trypan blue exclusion method. Furthermore, viability of the glioblastoma cells was proven by the investigation of LPS induced IL-6 production of IFNy-treated and -untreated cells by the B9 bioassay. IFNy-treated 86HG39 cells release, in contrast to untreated cells, small amounts of IL-6. After addition of 1/xg/ml LPS IFNy-treated and -untreated cells produce comparable amounts of IL-6. This and the recently published data on IFNy-induced enhancement of MHC class II antigen expression (D~iubener et al., 1992) led us to the conclusion that IFNy is active on the glioblastoma cell line 86HG39 and that treated cells are still viable. The following experiments revealed that in contrast to normal cells, IFNy-activated cells were capable of restricting the intracellular growth of Toxoplasma gondii measured by 3H-uracil incorporation on different days after infection. For example T. gondii proliferation within IFNy-treated cells on day 3 after infection was less than 15% of positive control (data shown in Fig. 2). This effect was also seen by microscopic examination of infected cultures. While in the culture with untreated cells, 3 days after infection large amounts of extracellular T. gondii were seen, in cultures with IFNy-treated cells only few extracellular a n d / o r intracellular parasites were visible. Subsequent experiments were done to clarify whether this growth inhibition of T. gondii within IFNy-treated 86HG39 cells is due to toxoplasmostatic or toxoplasmocidal effects. Therefore, as described in Materials and Methods, IFNy-treated and -untreated cells were infected with Toxoplasma gondii and extracellular T. gondii were removed after 3 h. T. gondii growth was then monitored by counting the amount of infected cells and the number of T. gondii per infected cell on different time points after infection. The result of four independent experiments are shown in Fig. 3. At timepoint zero, directly after infection, the percentage of infected cells is not significantly different between IFNy-treated and -untreated 86HG39 cells. The small reduction of the number of infected cells after 30 h in treated and untreated is more likely explained by the growth of tumor cells than due to toxoplasmocidal effects. In contrast, the number of T. gondii per infected cell is reduced in IFNy-treated vs. -untreated cells (7 vs. 20 T. gondii) These results show that IFNy treatment of 86HG39 cells induces no toxoplasmocidal but toxoplasmostatic effects.
Mechanism of IFNy induced toxoplasmostasis Several cellular mechanisms involved in the defense against Toxoplasma gondii were stimulated in different
40000 -
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~
NGMMA
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Fig. 4. 86HG39 cells, IFNy-treated or not, were cultured with l x 104 Toxoplasma gondii in culture medium with or without supplemental L-tryptophan (100/xg/ml), u-tryptophan (100 p,g/ml), NGMMA (0.1 mmol), or SOD (300 U/ml). Toxoplasma growth was determined by 3H-uracil incorporation 72 h after infection. Data are given as mean cpm _+SE of triplicate cultures.
cells. The main mechanisms are: induction of the oxidative burst (Murray et al., 1985), induction of Larginine dependent production of NO[ (Adams et al., 1990) and the induction of a tryptophan-degrading enzyme (indoleamine-2,3-dioxygenase) (Pfefferkorn, 1984). The measurement of Oy production of IFNytreated and -untreated cells was done as described in Materials and Methods according to the method established by Mizell et al. (1981). It was found that 86HG39 cells were unable to produce Oy, while human granulocytes used as control produce up to 13 nmol O [ / m l . Similar negative results were obtained by the measurement of NO~- production by IFNy-treated and -untreated 86HG39 cells. In this case murine bone marrow cells were used as positive control and 35 nmol N O [ / m l was found in the supernatant of these cells. In the next experiments we analyzed whether degradation of L-tryptophan might be the IFNy-induced toxoplasmostatic mechanism. Therefore, L-tryptophan was added to T. gondii-infected IFNy-treated and -untreated cells. T. gondii growth was monitored on different days after infection. The data shown in Fig. 4 indicate that L-tryptophan supplementation to IFNytreated 86HG39 cells restores the growth of Toxoplasma gondii. The magnitude of T. gondii proliferation in IFNy-treated 86HG39 cells after L-tryptophan supplementation is similar to the growth in untreated cells. In contrast, T. gondii growth without supplemental L-tryptophan is reduced. This IFNy antagonistic effect is specific for L-tryptophan because addition of D-tryptophan to IFNy-treated 86HG39 cells did not restore T. gondii proliferation. Furthermore, neither
36
SOD, a scavenger of 0 2 , nor NCLmonomethyl-Larginine, an inhibitor of N O 2 production, were able to influence IFNy induced toxoplasmostasis. We conclude therefore that IFNT-induced tryptophan degradation accounts for the toxoplasmostatic effect exhibited by IFNy-activated 86HG39 cells.
Discussion
In the course of human toxoplasmosis, an interaction between brain cells and the tachyzoite stage of Toxoplasma gondii takes place in the acute infection as well as by the reactivation of toxoplasma cysts. In this report the human GFAP-positive glioblastoma cell line 86HG39 was used to study the growth of T. gondii. We found that T. gondii strain BK is able to proliferate within 86HG39 cells. The growth of the parasite within the glioblastoma cell line up to 30 h after infection was monitored by the determination of infected glioblastoma ceils and by the enumeration of T. gondii per infected cell using the indirect immunofluorescence technique. We found, in agreement with Derouin et al. (1987), that the staining of intracellular T. gondii with antibodies leads to a clear indentification of the parasite and is superior to the staining of parasites with Diff-Quick or Giemsa solution used by other groups. The magnitude of T. gondii growth within 86HG39 cells is similar to that described for human monocyte derived macrophage and human and murine fibroblasts. The growth of Toxoplasma gondii within the glioblastoma cell line 86HG39 was also monitored by the 3H-uracil incorporation method described by McLeod et al. (1979). The basis of this method is that T. gondii is able to incorporate uracil via uridine phosphorylase in the DNA while human cells are not. The growth data of T. gondii within 86HG39 cells measured by 3H-uracil incorporation are similar to those reported for human umbilical cord vein endothelial cells (Woodman et al., 1991) and for human fibroblasts (Pfefferkorn and Guyre, 1984). Furthermore, in this report the growth of the T. gondii feeder cells was determined in parallel to the proliferation of the parasite. In respect to T. gondii growth all cell types tested up to now can be divided into three groups. Human monocytes are the prototype for the first group: these cells can be infected by Toxoplasma gondii but they restrict intracellular parasite growth without prior activation. This inhibition of T. gondii growth by human monocytes is due to toxoplasmocidal effects, shown by the reduced number of infected cells few hours after infection (Murray et al., 1985). The second group includes cells which can not inhibit T. gondii growth even after I F N y treatment, such as human EBV-trans-
formed B cells (Canessa et al., 1988). The third group of cells consists of those which are able to restrict intracellular T. gondii growth only after activation, especially by IFN7. This group includes all types of murine macrophages and human monocyte derived macrophages (Murray et al., 1985; Adams et al., 1990). We showed that 86HG39 cells are unable to restrict intracellular 7". gondii growth without stimulation, but IFN7 treatment induces a remarkable toxoplasmostatic effect. Therefore, 86HG39 cells belong to the third group described. Only few data about the fate of Toxoplasrna gondii within brain cells are available. For example, it was described by Jones et al. (1986) that Toxoplasrna gondii is able to replicate within murine astrocytes. That study was performed by the use of the low virulent Toxoplasma gondii strain Pe which is able to form pseudocysts containing large amounts of bradyzoites. IFN7 treatment of murine astrocytes led to an enhanced development of pseudocysts. In our experiments in IFNy-treated and -untreated 86HG39 cells no extracellular T. gondii cysts were found. This discrepancy might be explained by the use of the highly virulent Toxoplasma gondii strain BK by us which, in contrast to strain Pe, is unable to form pseudocysts even in vivo. The mechanism of IFNy-induced control of T. gondii growth within various cells seems to be different. In macrophage the enhanced activation of an oxidative burst reaction is described (Murray et al., 1985), as well as an induction of NO 2 production by IFN7 (Adams et al., 1990). We found that the cell line 86HG39 is unable to produce 0 2 and NO 2 and therefore these anti-parasitic reactions are not involved in the induction of toxoplasmostasis within 86HG39 cells. Furthermore, inhibitors of the oxidative burst or of NO 2 production did not influence the IFNy-induced toxoplasmostasis. As shown in different reports another IFNy-induced effect is the induction of tryptophan degradation in several cell types, including the glioblastoma cell line U137MG (Werner-Felmayer et al., 1989). The IFNTinduced tryptophan degradation is described to be responsible for the reduced proliferation of tumor cells after administration of IFN7 and cell proliferation is restored when L-tryptophan is added to IFNy-treated cells (Ozaki et al., 1988). Similar results were obtained using IFNy-treated 86HG39 cells. Tryptophan degradation after IFN7 treatment was shown by different authors to be dependent on the activation of the enzyme indoleamine-2,3-dioxygenase (Taylor and Feng, 1991). In addition to this anti-tumor effect tryptophan degradation was found to be the IFNy-induced antichlamydial effect within T 24 cells (Byrne et al., 1986). Furthermore, it was found that k-tryptophan degradation is responsible for the restricted Toxoplasrna gondii
37
growth within human fibroblasts (Pfefferkorn, 1984). Whether the inhibition of T. gondii growth in human fibroblasts is due to toxoplasmostatic or toxoplasmocidal effects remains unclear. We found that the toxoplasmostatic effect of IFN3,treated 86HG39 cells could be reversed by the addition of L-tryptophan to the culture medium. In contrast, the same amount of D-tryptophan added to infected IFN~/-treated 86HG39 cells did not influence the IFN3,-induced toxoplasmostatic effect within 86HG39 cells. Therefore it is assumed that the IFN3,-induced toxoplasmostatic effect is due to an activation of indoleamine-2,3-dioxygenase, the only known tryptophan degrading enzyme which is induced by IFNy (Taylor and Feng, 1991). We are now analysing whether other inducers of indoleamine-2,3-dioxygenase like IFNa and IFN/3 (Carlin et al., 1987) are also able to induce toxoplasmostasis within 86HG39 cells. Furthermore, because of the fact that the cell line 86HG39 is able to induce T cell proliferation of histocompatible T cells specific for soluble antigen Toxoplasma gondii as shown recently (D~iubener et al., 1992) we intend to establish a culture system consisting of 86HG39 cells, T. gondii-specific T ceils and viable T. gondii. In summary, this study describes the growth of Toxoplasma gondii within cells of the glioblastoma cell line 86HG39. We found that IFN~/ treatment of these cells leads to the induction of a toxoplasmostatic effect by the induction of L-tryptophan degradation. The capacity of 86HG39 cells to control T. gondii growth after activation, and the capacity of these cells to interact with antigen-specific T cells will allow the establishment of an in vitro model for cellular immune reactions within the CNS in the course of human toxoplasmosis.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft SFB 194, Diisseldorf. We thank Dr. Seitz and Dr. Saathoff, Institut fiir Med. Parasitology, Bonn, FRG for the kind gift of Toxoplasma gondii strain BK, and Dr. C. MacKenzie for critical reading of the manuscript.
References Adams, L.B., Hibbs, J.B., Taintor, R.R. and Krahenbuhl, J.L. (1990) Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii: Role for synthesis of inorganic nitrogen oxides from L-arginine. J. Immunol. 144, 2725-2729. Bilzer, T., Stavrou, D., Dahme, E., Keiditsch, E., Biirring, K.F., Anzil, A.P. and Wechsler, W. (1991) Morphological, immunocytochemical and growth characteristics of three human glioblastomas established in vitro. Virchows Arch. A Pathol. Anat. 418, 281-293.
Byrne, G.I., Lehmann, L.K. and Landry, G.J. (1986) Induction of tryptophan catabolism is the mechanism for gamma interferon mediated inhibition of intracellular Chlamydia psittaci replication in T 24 cells. Infect. Immun. 53, 347-351. Canessa, A., Pistoia, V., Roncella, S., Merti, A., Melioli, G., Terragna, A. and Ferrarini, M. (1988) An in vitro model for toxoplasma infection in man: Interaction between CD4 + monoclonal T-cells and macrophages result in killing of trophozoites. J. Immunol. 140, 3580-3588. Carlin, J.M., Borden, E.C., Sondel, P.M. and Byrne, G.I. (1987) Biological response modifier-induced indoleamine-2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures. J. Immunol. 139, 2414-2418. Carrel, S., DeTribolet, N. and Gross, N. (1982) Expression of HLADR and common acute lymphoblastic leukemia antigens on glioma cells. Eur. J. Immunol. 12, 354-357. Catterall, J.R., Black, C.M., Leventhal, J.P., Rizk, N.W., Wachtel, J.S. and Remington, J.S. (1987) Non-oxidative microbicidal activity in normal human alveolar and peritoneal macrophages. Infect. Immun. 55, 1635-1640. D~iubener, W., Seghrouchni-Zennati, S., Wernet, P., Bilzer, T., Fischer, H.G. and Hadding, U. (1992) Human glioblastoma cell line 86HG39 activates T cells in an antigen specific MHC class II dependent manner. J. Neuroimmunol., in press. Derouin, F., Mazero, M.C. and Garin, Y.J.F. (1987) Comparative study of tissue culture and mouse inoculation methods for demonstration of Toxoplasma gondii. J. Clin. Microb. 25, 15971600. Dhib-Jalbut, S., Kutta, C.V., Flerlage, M., Shimojo, N. and McFarland, H.F. (1990) Adult human glial cells can present target antigen to HLA-restricted cytotoxic T cells. J. Neuroimmunol. 29, 203-211. Ding, A.H., Nathan, C.F. and Stuehr, D.J. (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. Immunol. 141, 2407-2412. Fontana, A., Bodmer, S. and Rei, K. (1987) Immunoregulatory factors secreted by astrocytes and glioblastoma cells. Lymphokines 14, 91-121. Fontana, A., Frei, K., Bodmer, S. and Hofer, E. (1987) Immunemediated encephalitis: On the role of antigen presenting cells in brain tissue. !mmunol. Rev. 100, 185-201. Hauser, W.E. and Tsai, V. (1986) Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for Toxoplasma gondii in vitro. J. Immunol. 136, 313-319. Hauser, W.E., Sharma, S.D. and Remington, J.S. (1983) Augmentation of NK cell activity by soluble and particulate fractions of Toxoplasma gondii. J. Immunol. 131,458-463. Helle, M., Boerje, L. and Aarden, L.A. (1988) Functional discrimination between interleukin 6 and interleukin 1. Eur. J. Immunol. 18, 1535-1540. Israelski, D.M., Araujo, F.G., Wachtel, J.S., Heinrichs, L. and Remington, J.S. (1990) Differences in microbicidal activities of human macrophages against Toxoplasma gondii and Trypanosoma cruzi. Infect. Immun. 58, 263-265. Jones, T.C., Bienz, K.A. and Erb, P. (1986) In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gammainterferon. Infect. Immun. 51, 147-156. Kasper, L.H., Khan, I.A., Ely, K.H., Buelow, R. and Boothroyd, J.C. (1992) Antigen specific (p30) mouse CD8 T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages. J. Immunol. 148, 1493-1498. Khan, I.A., Smith, K.A. and Kasper, L.H. (1988) Induction of antigen specific parasitocidal cytotoxic T cell splenocytes by a major membrane protein (p30) of Toxoplasma gondii. J. Immunol. 141, 3600-3605. Kittas, S., Kittas, C., Paizi-Biza, P. and Henry, L. (1984) A histological and immunohistochemical study of the changes induced in the
38 brain of white mice by infection with Toxoplasma gondii. Br. J. Exp. Pathol. 65, 67-74. McLeod, R. and Remington, J.S. (1979) A method to evaluate the capacity of monocytes and macrophages to inhibit multiplication of an intracellular pathogen. J. Immunol. Methods 27, 19-29. Murray, H.W., Rubin, B.Y., Carriero, S.M., Harris, A.M. and Jaffee, E.A. (1985) Human monocuclear phagocyte antiprotozoal mechanism: Oxygen-dependent, vs. oxygen-independent activity against intracellular Toxoplasma gondii. J. Immunol. 134, 1982-1988. Ozaki, Y., Edelstein, M.P. and Duch, D.S. (1988) Induction of indoleamine-2,3-dioxygenase: A mechanism of the antitumor activity of interferon 3'. Proc. Natl. Acad. Sci. USA 85, 1242-1246. Pfefferkorn, E.R. (1984) Interferon y blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc. Natl. Acad. Sci. USA 81,908-912. Pfefferkorn, E.R. and Guyre, P. (1984) Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon. Infect. Immun. 44, 211-216. Pfefferkorn, E.R. and Pfefferkorn, L.C. (1977) Specific labeling of intracellular Toxoplasma gondii with uracil. J. Protozool. 24, 449-453. Pick, E. and Mizel, D. (1981) Rapid microassay for the measurement of superoxide and hydrogen peroxide production by maerophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46, 211-226.
Remington, J.S., Krahenbuhl. J.L. and Mendenhall, J.W. (1972) A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6, 829-834. Schlfiter, D., L6hler, J., Deckert, M., Hof, H. and Schwendemann, G. (1991) Toxoplasma encephalitis of immunocompetent and nude mice: immunohistological characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products. J. Neuroimmunol. 31, 185-198. Schreiber, A.B. and Feldmann, H.A. (1980) Identification of the activator system for antibody to Toxoplasma in the classical complement pathway. J. Infect. Dis. 141, 366-369. Suzuki, Y., Orellana, M.A., Schreiber, R.D. and Remington, J.S. (1988) Interferon 3'. The major mediator of resistence against Toxoplasma gondii. Science 240, 516-518. Taylor, M.W. and Feng, G. (1991) Relationship between interferon y, indoleamine-2-3-dioxygenase, and tryptophan catabolism. FASEB 5, 2516-2522. Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A., Reibnegger, G. and Wachter, H. (1989) Characteristics of interferon induced tryptophan metabolism in human cells in vitro. Biochem. Biophys. Acta 1012, 140-147. Woodman, J.P., Dimier, I.H. and Bout, D.T. (1991) Human endothelial cells are activated by IFN-y to inhibit Toxoplasma gondii replication. J. Immunol. 147, 2019-2023.
31
JNI 02305
Induction of toxoplasmostasis in a human glioblastoma by interferon y Walter D a u b e n e r a Korinna Pilz a Samira Seghrouchni Z e n n a t i a, Thomas Bilzer b, Hans-Georg Fischer a and Ulrich Hadding b a Institut fiir Medische Mikrobiologie und Virologie, and oAbteilung fiir Neuropathologie, Heinrich-Heine- Universitdt, Diisseldorf, Germany (Received 8 June 1992) (Revision received 16 September 1992) (Accepted 16 September 1992)
Key words: Gliobtastoma cells; Toxoplasma; Interferon 3'
Summary In the course of human toxoplasmosis central nervous system involvement often occurs. As a model for toxoplasma growth within human brain cells the proliferation of Toxoplasma gondii strain BK within the human glioblastoma cell line 86HG39 was analysed. We found that 86HG39 cells support the growth of toxoplasma similar to human monocyte derived macrophages and in contrast to human monocytes. The growth of Toxoplasma gondii within interferon 3' (IFNT) treated 86HG39 cells is reduced due to toxoplasmostasis and not due to toxoplasmocide effects. The mechanism of IFNy induced toxoplasmostasis was also investigated. It was found that IFNy did not induce 0 2 production a n d / o r nitrite oxide production, and inhibitors of O f and NO 2 did not influence IFNy induced toxoplasmostasis. In contrast, the supplementation of L-tryptophan to the culture medium completely abolished the IFNy effect. We therefore conclude that the induction of L-tryptophan degradation in 86HG39 cells by IFNy, possibly by activation of the indoleamine-2,3-dioxygenase, is responsible for the IFNy induced toxoplasmostasis within the glioblastoma cell line.
Introduction
Toxoplasma gondii is an obligate intracellular parasite that is gaining wider recognition as an opportunistic pathogen producing fulminant infection in compromised hosts. Central nervous system (CNS) involvement by acquired toxoplasmosis is often seen, but neurologic symptoms are variable. In healthy human adults Toxoplasma gondii proliferation within the CNS is restricted, resulting in the formation of brain cysts containing large amounts of bradyzoites. These cysts persist for the remainder of the life of the host. In the case of immunosuppression, for example by infection with the human immunodeficiency virus, a reactivation of Toxoplasma gondii occurs, resulting in a life-threatening encephalitis.
Correspondence to: W. D~iubener, Institut ffir Medische Mikrobiologie und Virologie, Moorenstrasse 5, D-4000 Diisseldorf, Germany.
The knowledge of the immune mechanisms that participate in the host defense against toxoplasma in vivo derives primarily from studies in animal models. It was shown that despite the production of antibodies which after complement activation are effective in the killing of extracellular toxoplasma (Schreiber and Feldmann, 1980) cellular immune reactions are of importance. In this cellular immune response, NK cells (Hauser et al., 1983; Hauser and Tsai, 1986) and T cells (Khan et al., 1988; Kasper et al., 1992) were involved but the principal part in the control of toxoplasma growth is played by mononuclear phagocytes as shown by in vitro data. Different studies have shown remarkable differences of toxoplasma growth between cells of human or murine origin. Human monocytes (Murray et al., 1985) and different types of human macrophage (Catterall et al., 1987, Israelski et al., 1990) were able to restrict intracellular toxoplasma growth without prior activation by lymphokines. In contrast, all types of murine macrophages were only able to restrict toxoplasma growth after activation by
32 lymphokines, in particular IFNT (Remington et al.. 1975; Adams et al., 1990). Cellular immune reactions within the central nervous system, which is always involved in toxoplasmosis, were strictly controlled by the blood-brain-barrier consisting of endothelial ceils and astrocytes. Furthermore, only few cells expressing MHC antigens necessary for the initiation of cellular immune response were found within normal brain parenchyma. Within the CNS two types of accessory cells endowed with macrophage-like capacity exist, microglia cells and astrocytes (Fontana et al., 1987b). Despite immunhistological studies concerning the fate of Toxoplasma gondii within the CNS (Kittas et al., 1984, Schltiter et al., 1991) only few in vitro data are available. For example, it is described that IFNT is involved in the control of toxoplasma growth within murine astrocytes (Jones et al., 1986). To our knowledge no data about toxoplasma growth within human astrocytes are available. Most of our knowledge about human astrocyte cell function is obtained by the use of glioblastoma cells of astrocytic origin, because, due to ethic reasons, human native brain cells are difficult to obtain. Human glioblastoma cells have been described to exhibit many macrophage-like functions, for example monokine secretion, the capacity for phagocytosis (Fontana et al., 1987a), IFNT responsible class I1 antigen expression (Carell et al., 1982) and target function for human cytotoxic T cells (Dhib-Jalbut et al., 1990). In this study the growth of Toxoplasma gondii strain BK within the glioblastoma cell line 86HG39 is investigated. This cell line is described to express the astrocyte marker protein G F A P (Bilzer et al., 1991). The accessory cell capacity of 86HG39 cells involving MHC antigen expression, monokine secretion and the capacity to activate human T ceils has been described recently (D~iubener et al., 1992).
Materials and M e t h o d s
Toxoplasma gondii Toxoplasma gondii strain BK was a kind gift of Drs. Seitz and Saathoff (Institut ftir Med. Parasitologie, Bonn, FRG). Toxoplasma gondii was expanded by the use of the murine fibroblast cell line L929 (ATCC, Rockville, MA) as feeder cells. In brief, 1.2 × 10 6 L929 cells were cultured in culture flasks (Costar, Cambridge, MA) and 10 × 106 T. gondii were added in 5-ml Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY) supplemented with 2% glutamine (Gibco, Grand Island, NY) and 10% heat inactivated FCS (Seromed, Biochrom, Berlin, FRG). After 3-5 days of cultivation the L929 cells were completely lysed by T. gondii. The parasites were harvested and cen-
trifuged at 500 rpm for 5 rain to pellet the few remainig L929 cells and cell debris. The supernatant was removed and centrifuged at 2000 rpm for 10 rain. Thereafter 7~ gondii were resuspended in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 5 ~ FCS, and used for infection of different cells.
Culti~ation of 86HG39 cells The glioblastoma cell line 86HG39 which is defined by means of immunocytochemical and morphological criteria (Bilzer et al., 1991) was used in this study between the 70th and 150th in vitro passage. Cells were grown in tissue culture flasks and divided weekly in ratios between 1 : 2 and 1 : 10 depending on the proliferation rate. To simplify harvesting, the adherent glioblastoma cells were detached with t r y p s i n / E D T A (Seromed, Biochrom, Berlin, FRG) and viability was tested after harvesting by Trypan blue exclusion. For IFNT treatment 86HG39 cells were harvested, washed three times with PBS and resuspended in RPMI 1640 medium supplemented with 5% FCS. Then 1-1.5 M 10 6 cells were cultured in tissue culture flasks and IFNT (Genzyme, Cambridge, MA) was added. In preliminary experiments it was found that treatment of 86HG39 cells with 5 0 0 U / m l IFNT for 72 h was optimal for the induction of toxoplasmostatic effects, and therefore these conditions were used in all experiments.
Preparation of human monocytes and monocyte derh'ed macrophage Human PBL were obtained from buffy coates from healthy donors (Dr. P. Wernet, Institut fiir Blutgerinnungswesen und Transfusionsmedizin, Heinrich-Heinc Universit~it, Diisseldorf, FRG) by Ficoll Hypaque separation. The PBL were allowed to adhere on plastic Petri dishes (Greiner, Ntirtingen, FRG) for 3 h. The non-adherent cells were then removed and the remaining cells were used as monocytes or cultured for 7 days in Iscove's medium 5% FCS to allow differentiation into monocyte-derived macrophage (MDM) as described (Murray et al., 1985). IFNy treatment of MDM was done exactly as described for 86HG39 cells.
Assay for toxoplasma proliferation It has been shown that intracellular Toxoplasma gondii incorporate large amounts of 3H-uracil into nucleic acid because they have substantially more uridine phosphorylase (which converts uracil into uridine which is then incorporated into nucleic acid) than mammalian cells (Pfefferkorn and Pfefferkorn, 1977). This 3H-uracil incorporation method is used in this study for monitoring of toxoplasma growth with minor modifications. In brief, 1.5 × 10 4 86HG39 cells or 3 × 10 4 freshly prepared monocytes or monocyte derived macrophage were cultured in 96-well flat bottom culture plates
33 (Greiner, Niirtingen, FRG) in a total volume of 100/zl per well. Then 1 × 104 freshly prepared toxoplasma were added in 50 ~1. In some experiments L-tryptophan, D-tryptophan, superoxide dismutase (obtained from Sigma, Deisenhofen, F R G ) or NG-monomethyl-c arginine (Calbiochem, Bad Soden, FRG) was added. After 24, 48, 72 or 96 h 0.4/xCi 3H-uracil (Amersham Buchler, Braunschweig, F R G ) was added to the culture and left for 14-18 h. The cultures were then processed for liquid scintillation counting. For monitoring of T. gondii growth within the first 30 h after infection another method similar to those described by several other authors (Murray et al., 1985; Catterall et al., 1987), was used. 2 X 104 86HG39 cells in 100 /xl were allowed to adhere on glass cover slips placed in a 24-well culture plate (Costar, Cambridge, MA). Medium was then removed and 3 x 104 viable T. gondii were added in 100 /~1. After 3 h, extracellular T. gondii were removed by extensive washing and 0.5 ml RPMI 1640 5% FCS was added. At this time point (zero) two cover slips were removed and fixed with Diff-Quick fixative (Merz + Dade, Diidingen, Switzerland). The other two cover slips per group were handled in the same way after 30 h. After fixation 200 /xl of a 1:500 diluted serum sample obtained from a 32-year-old person which had an acute but asymptomatic toxoplasmosis was added for 30 rain. After two washing steps a FITC labelled anti human Ig antibody (BioMerieux, Marcy, France), diluted 1 : 300 in PBS containing Evans blue was added for 30 min. After two additional washing steps 200 cells per cover slip were counted using a fluorescense microscope (Zeiss, K61n, FRG). The number of infected cells and the number of T. gondii per infected cell were determined. In each group two cover slips were counted. The data are given as the mean _+SE of four independent experiments.
Determination of NO~The determination of NO 2 was performed by the use of Griess-reagent (0.3% naphthylethylenediamide-dihydrochloride, 1.0% sulfanylamide obtained from Sigma (Deisenhofen, F R G ) as described (Ding et al., 1988). 2 x 10 4 86HG39 cells IFNy treated or not were cultured in a 24-well plate in 2 ml Iscove's 5% FCS. After 24, 48, 72 and 96 h the supernatant was removed and 100/zl were mixed with an equal volume of Griess reagent. Absorbance at 543 nm was measured by the use of a microplate reader (Nunc, Wiesbaden, FRG). As positive control supernatants of murine bone marrow derived macrophages, obtained from A. Bielinsky from our institute, were used. The amount of NO~- produced was estimated by comparison with a standard consisting of a two-fold diluted sodium nitrate solution. The detection limit of the test was 2 - 4 n m o l / m l NO 2.
Measurement of Oj- production To 3 X 104 86HG39 cells/well of a 96-well flat bottom culture plate in RPMI 1640 free of phenol red, (Gibco, Grand Island, NY) phorbol-myrestat-acetate (PMA, Sigma Deisenhofen FRG) was added between 20 n g / m l and 0.2 n g / m l . 0 2 production was measured by the reduction of cytochrome c (Sigma, Deisenhofen, F R G ) (Pick and Mizel, 1981). As positive control, human granulocytes obtained from Ficoll gradient after NH4CI lysis of erythrocytes were used.
Determination of IL-6 production 2 x l0 s 86HG39 cells IFNy treated or not were cultured in a 24-well culture plate and stimulated with 1 / z g / m l LPS (Sigma, Deisenhofen, F R G ) for 24 h. The supernatant was then removed and added in three-fold dilutions to 2.5 x 103 B9 cells. The proliferation of B9 cells was monitored by 3H-thymidine incorporation after 42-72 h in vitro culture. In each experiment rhu IL-6 (Pharma Biotechnologie, Hannover, F R G ) was used as positive control and one unit of IL-6 gives raise to half maximal proliferation. The B9 bioassay is described to be specific for IL-6 and no cross-reactions with other lymphokines, especially with IL-1, are known (Helle et al., 1988). B9 cells in our laboratory were maintained in Iscove's 5% FCS containing 1% of supernatant of LPS stimulated PBL.
Results
Growth of Toxoplasma gondii strain BK in human peripheral blood accessory cells Toxoplasma gondii strain R H did not proliferate within freshly prepared human monocytes, but monocyte-derived macrophage after 5 - 7 days of in vitro incubation support the growth of this parasite (Murray et al., 1985). We first tested whether Toxoplasma gondii strain BK used by us, which is similar to strain R H with respect to its virulence, shows the same growth behavior in human accessory cells. Therefore human monocytes were infected with Toxoplasma gondii strain BK and parasite growth was monitored by 3H-uracil incorporation. In parallel, monocyte-derived macrophages (MDM) and IFNy treated MDM were used. It was found that Toxoplasma gondii strain BK did not or only marginally replicated within fresh monocytes whilst a remarkable parasite growth occurs within MDM. The treatment of MDM with r I F N y (500 U / m l ) 72 h before toxoplasma infection led to an activation of MDM and these cells were then able to inhibit parasite growth. From these data we conclude that Toxoplasma gondii strain BK is similar to strain R H with respect to its growth within human peripheral blood accessory cells.
34 40000
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,~= SH-tnymidine cam 86HG39 + T o x o p a s m a i = SH-Lraci~ cpr~ 86HG39 ~ Toxoplasma
Fig. l. 1.5 X 104 86HG39 cells were cultured with or without 10 x 104 Toxoplasma gondii. The proliferation of the glioblastoma cells was monitored by 3H-thymidine incorporation, while toxoplasma growth was measured by 3H-uracil uptake in parallel. Data are given as mean c p m + S E of triplicate cultures. 3H-thymidine and 3H-uracil incorporation in Toxoplasma gondii cultured without 86HG39 cells was less than 500 cpm. Also 3H-uracil incorporation of uninfected 86HG39 cells was less than 500 cpm.
Growth of Toxoplasma gondii within the human glioblastoma cell line 86HG39 Firstly, experiments were performed to assay whether glioblastoma cells can support the growth of Toxoplasma gondii. Therefore 1.5 x 10 4 86HG39 cells per well were infected with 1 x 104 T. gondii and parasite growth was monitored by 3H-uracil incorporation on different days after infection. The growth of 86HG39 cells infected or not was determined in parallel by 3H-thymidine uptake (Fig. 1). It was found that Toxoplasma gondii strain BK is able to proliferate within 86HG39 cells measured by 3H-uracil incorporation and visualized by microscopic examination of infected cultures. Due to the fact that T. gondii is an obligate intracellular parasite the growth of infected
Fig. 2. 1.5 X 105 86HG39 cells, IFNT-treated or not, were cultured with I X 104 viable T. gondii. T. gondff growth was monitored by 3H-uracil uptake on different days after infection. Data are given as mean cpm _+SE of triplicate cultures.
86HG39 cells is reduced by killing of the cells by T.
gondii. Thereafter, also the growth of the parasite declines because of the lack of viable feeder cells. Similar growth data were obtained by the infection of 86HG39 monolayers with T. gondii for 3 h and counting the number of infected cells and the number of T. gondii per infected cell at different times after infection. The data obtained (not shown) revealed that the number of infected cells remained stable for up to 30 h, and therefore we conclude that 86HG39 ceils were not able to kill intracellular T. gondii. In contrast, the number of T. gondii per infected cell increased, indicating the growth of the parasite.
Toxoplasmostasis induced in 86HG39 cells by IFNy IFNy is the major lymphokine involved in the control of the growth of intracellular pathogens (Suzuki et al., 1988). Furthermore, IFN7 is described to inhibit tumor cell growth (Ozaki et al., 1988). The addition of IFN7 to 86HG39 cells results in a dose-dependent 25
30
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-~ 10 0 x 0 I"-
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Fig. 3, IFNy-treated or -untreated 86HG39 cells were infected with Toxoplasma gondii for 3 h and the number of infected cells and the number of T. gondii per infected cell was determined by indirect immunofluorescence as described in Materials and Methods. The data are given as mean _+ SE of four independent experiments.
35 growth inhibition and similar data were obtained when 86HG39 cells were only pretreated with IFN 7. The reduced proliferation rate is not due to a complete killing of 86HG39 cells by IFNy because IFN 7 treated cells were found to be viable measured by the Trypan blue exclusion method. Furthermore, viability of the glioblastoma cells was proven by the investigation of LPS induced IL-6 production of IFNy-treated and -untreated cells by the B9 bioassay. IFNy-treated 86HG39 cells release, in contrast to untreated cells, small amounts of IL-6. After addition of 1/xg/ml LPS IFNy-treated and -untreated cells produce comparable amounts of IL-6. This and the recently published data on IFNy-induced enhancement of MHC class II antigen expression (D~iubener et al., 1992) led us to the conclusion that IFNy is active on the glioblastoma cell line 86HG39 and that treated cells are still viable. The following experiments revealed that in contrast to normal cells, IFNy-activated cells were capable of restricting the intracellular growth of Toxoplasma gondii measured by 3H-uracil incorporation on different days after infection. For example T. gondii proliferation within IFNy-treated cells on day 3 after infection was less than 15% of positive control (data shown in Fig. 2). This effect was also seen by microscopic examination of infected cultures. While in the culture with untreated cells, 3 days after infection large amounts of extracellular T. gondii were seen, in cultures with IFNy-treated cells only few extracellular a n d / o r intracellular parasites were visible. Subsequent experiments were done to clarify whether this growth inhibition of T. gondii within IFNy-treated 86HG39 cells is due to toxoplasmostatic or toxoplasmocidal effects. Therefore, as described in Materials and Methods, IFNy-treated and -untreated cells were infected with Toxoplasma gondii and extracellular T. gondii were removed after 3 h. T. gondii growth was then monitored by counting the amount of infected cells and the number of T. gondii per infected cell on different time points after infection. The result of four independent experiments are shown in Fig. 3. At timepoint zero, directly after infection, the percentage of infected cells is not significantly different between IFNy-treated and -untreated 86HG39 cells. The small reduction of the number of infected cells after 30 h in treated and untreated is more likely explained by the growth of tumor cells than due to toxoplasmocidal effects. In contrast, the number of T. gondii per infected cell is reduced in IFNy-treated vs. -untreated cells (7 vs. 20 T. gondii) These results show that IFNy treatment of 86HG39 cells induces no toxoplasmocidal but toxoplasmostatic effects.
Mechanism of IFNy induced toxoplasmostasis Several cellular mechanisms involved in the defense against Toxoplasma gondii were stimulated in different
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Fig. 4. 86HG39 cells, IFNy-treated or not, were cultured with l x 104 Toxoplasma gondii in culture medium with or without supplemental L-tryptophan (100/xg/ml), u-tryptophan (100 p,g/ml), NGMMA (0.1 mmol), or SOD (300 U/ml). Toxoplasma growth was determined by 3H-uracil incorporation 72 h after infection. Data are given as mean cpm _+SE of triplicate cultures.
cells. The main mechanisms are: induction of the oxidative burst (Murray et al., 1985), induction of Larginine dependent production of NO[ (Adams et al., 1990) and the induction of a tryptophan-degrading enzyme (indoleamine-2,3-dioxygenase) (Pfefferkorn, 1984). The measurement of Oy production of IFNytreated and -untreated cells was done as described in Materials and Methods according to the method established by Mizell et al. (1981). It was found that 86HG39 cells were unable to produce Oy, while human granulocytes used as control produce up to 13 nmol O [ / m l . Similar negative results were obtained by the measurement of NO~- production by IFNy-treated and -untreated 86HG39 cells. In this case murine bone marrow cells were used as positive control and 35 nmol N O [ / m l was found in the supernatant of these cells. In the next experiments we analyzed whether degradation of L-tryptophan might be the IFNy-induced toxoplasmostatic mechanism. Therefore, L-tryptophan was added to T. gondii-infected IFNy-treated and -untreated cells. T. gondii growth was monitored on different days after infection. The data shown in Fig. 4 indicate that L-tryptophan supplementation to IFNytreated 86HG39 cells restores the growth of Toxoplasma gondii. The magnitude of T. gondii proliferation in IFNy-treated 86HG39 cells after L-tryptophan supplementation is similar to the growth in untreated cells. In contrast, T. gondii growth without supplemental L-tryptophan is reduced. This IFNy antagonistic effect is specific for L-tryptophan because addition of D-tryptophan to IFNy-treated 86HG39 cells did not restore T. gondii proliferation. Furthermore, neither
36
SOD, a scavenger of 0 2 , nor NCLmonomethyl-Larginine, an inhibitor of N O 2 production, were able to influence IFNy induced toxoplasmostasis. We conclude therefore that IFNT-induced tryptophan degradation accounts for the toxoplasmostatic effect exhibited by IFNy-activated 86HG39 cells.
Discussion
In the course of human toxoplasmosis, an interaction between brain cells and the tachyzoite stage of Toxoplasma gondii takes place in the acute infection as well as by the reactivation of toxoplasma cysts. In this report the human GFAP-positive glioblastoma cell line 86HG39 was used to study the growth of T. gondii. We found that T. gondii strain BK is able to proliferate within 86HG39 cells. The growth of the parasite within the glioblastoma cell line up to 30 h after infection was monitored by the determination of infected glioblastoma ceils and by the enumeration of T. gondii per infected cell using the indirect immunofluorescence technique. We found, in agreement with Derouin et al. (1987), that the staining of intracellular T. gondii with antibodies leads to a clear indentification of the parasite and is superior to the staining of parasites with Diff-Quick or Giemsa solution used by other groups. The magnitude of T. gondii growth within 86HG39 cells is similar to that described for human monocyte derived macrophage and human and murine fibroblasts. The growth of Toxoplasma gondii within the glioblastoma cell line 86HG39 was also monitored by the 3H-uracil incorporation method described by McLeod et al. (1979). The basis of this method is that T. gondii is able to incorporate uracil via uridine phosphorylase in the DNA while human cells are not. The growth data of T. gondii within 86HG39 cells measured by 3H-uracil incorporation are similar to those reported for human umbilical cord vein endothelial cells (Woodman et al., 1991) and for human fibroblasts (Pfefferkorn and Guyre, 1984). Furthermore, in this report the growth of the T. gondii feeder cells was determined in parallel to the proliferation of the parasite. In respect to T. gondii growth all cell types tested up to now can be divided into three groups. Human monocytes are the prototype for the first group: these cells can be infected by Toxoplasma gondii but they restrict intracellular parasite growth without prior activation. This inhibition of T. gondii growth by human monocytes is due to toxoplasmocidal effects, shown by the reduced number of infected cells few hours after infection (Murray et al., 1985). The second group includes cells which can not inhibit T. gondii growth even after I F N y treatment, such as human EBV-trans-
formed B cells (Canessa et al., 1988). The third group of cells consists of those which are able to restrict intracellular T. gondii growth only after activation, especially by IFN7. This group includes all types of murine macrophages and human monocyte derived macrophages (Murray et al., 1985; Adams et al., 1990). We showed that 86HG39 cells are unable to restrict intracellular 7". gondii growth without stimulation, but IFN7 treatment induces a remarkable toxoplasmostatic effect. Therefore, 86HG39 cells belong to the third group described. Only few data about the fate of Toxoplasrna gondii within brain cells are available. For example, it was described by Jones et al. (1986) that Toxoplasrna gondii is able to replicate within murine astrocytes. That study was performed by the use of the low virulent Toxoplasma gondii strain Pe which is able to form pseudocysts containing large amounts of bradyzoites. IFN7 treatment of murine astrocytes led to an enhanced development of pseudocysts. In our experiments in IFNy-treated and -untreated 86HG39 cells no extracellular T. gondii cysts were found. This discrepancy might be explained by the use of the highly virulent Toxoplasma gondii strain BK by us which, in contrast to strain Pe, is unable to form pseudocysts even in vivo. The mechanism of IFNy-induced control of T. gondii growth within various cells seems to be different. In macrophage the enhanced activation of an oxidative burst reaction is described (Murray et al., 1985), as well as an induction of NO 2 production by IFN7 (Adams et al., 1990). We found that the cell line 86HG39 is unable to produce 0 2 and NO 2 and therefore these anti-parasitic reactions are not involved in the induction of toxoplasmostasis within 86HG39 cells. Furthermore, inhibitors of the oxidative burst or of NO 2 production did not influence the IFNy-induced toxoplasmostasis. As shown in different reports another IFNy-induced effect is the induction of tryptophan degradation in several cell types, including the glioblastoma cell line U137MG (Werner-Felmayer et al., 1989). The IFNTinduced tryptophan degradation is described to be responsible for the reduced proliferation of tumor cells after administration of IFN7 and cell proliferation is restored when L-tryptophan is added to IFNy-treated cells (Ozaki et al., 1988). Similar results were obtained using IFNy-treated 86HG39 cells. Tryptophan degradation after IFN7 treatment was shown by different authors to be dependent on the activation of the enzyme indoleamine-2,3-dioxygenase (Taylor and Feng, 1991). In addition to this anti-tumor effect tryptophan degradation was found to be the IFNy-induced antichlamydial effect within T 24 cells (Byrne et al., 1986). Furthermore, it was found that k-tryptophan degradation is responsible for the restricted Toxoplasrna gondii
37
growth within human fibroblasts (Pfefferkorn, 1984). Whether the inhibition of T. gondii growth in human fibroblasts is due to toxoplasmostatic or toxoplasmocidal effects remains unclear. We found that the toxoplasmostatic effect of IFN3,treated 86HG39 cells could be reversed by the addition of L-tryptophan to the culture medium. In contrast, the same amount of D-tryptophan added to infected IFN~/-treated 86HG39 cells did not influence the IFN3,-induced toxoplasmostatic effect within 86HG39 cells. Therefore it is assumed that the IFN3,-induced toxoplasmostatic effect is due to an activation of indoleamine-2,3-dioxygenase, the only known tryptophan degrading enzyme which is induced by IFNy (Taylor and Feng, 1991). We are now analysing whether other inducers of indoleamine-2,3-dioxygenase like IFNa and IFN/3 (Carlin et al., 1987) are also able to induce toxoplasmostasis within 86HG39 cells. Furthermore, because of the fact that the cell line 86HG39 is able to induce T cell proliferation of histocompatible T cells specific for soluble antigen Toxoplasma gondii as shown recently (D~iubener et al., 1992) we intend to establish a culture system consisting of 86HG39 cells, T. gondii-specific T ceils and viable T. gondii. In summary, this study describes the growth of Toxoplasma gondii within cells of the glioblastoma cell line 86HG39. We found that IFN~/ treatment of these cells leads to the induction of a toxoplasmostatic effect by the induction of L-tryptophan degradation. The capacity of 86HG39 cells to control T. gondii growth after activation, and the capacity of these cells to interact with antigen-specific T cells will allow the establishment of an in vitro model for cellular immune reactions within the CNS in the course of human toxoplasmosis.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft SFB 194, Diisseldorf. We thank Dr. Seitz and Dr. Saathoff, Institut fiir Med. Parasitology, Bonn, FRG for the kind gift of Toxoplasma gondii strain BK, and Dr. C. MacKenzie for critical reading of the manuscript.
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