The effect of cytokines on the replication of T. gondii within rat retinal vascular endothelial cells

Journal of Neuroimmunology 102 Ž2000. 182–188 www.elsevier.comrlocaterjneuroim

The effect of cytokines on the replication of T. gondii within rat retinal vascular endothelial cells Catriona L. Brunton, Graham R. Wallace, Elizabeth Graham, Miles R. Stanford

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Department of Opthalmology, Rayne Institute, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK Received 14 June 1999; received in revised form 23 August 1999; accepted 23 August 1999

Abstract Toxoplasma gondii infection of the eye can result in a recurrent necrotising retinochoroiditis ŽTR. which may lead to a permanent loss of visual acuity. The mechanisms responsible for the control of TR within the retina are unknown. The aim of this study was to examine the effects of cytokines on the replication of T. gondii RH strain tachyzoites within rat retinal vascular endothelial ŽrRVE. cells. Pretreatment of rRVE with IFNg, TNF or IL-1b resulted in a significant decrease in T. gondii replication from day 2 onwards. There was no significant difference in nitric oxide ŽNO. production by IFNg, TNF or IL-1b treated rRVE as compared to controls at any time point. By comparison, the addition of L-tryptophan to IFNg treated cultures significantly restored T. gondii replication from 48 h post inoculation. Thus, IFNg, TNF and IL-1b can significantly inhibit the replication of T. gondii within rRVE. However, this inhibition appears to be independent of NO production. L-tryptophan catabolism may have a role in IFNg mediated inhibition of T. gondii replication in rRVE cells. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Toxoplasma gondii; Retinochoroiditis; Cytokines; Retinal vascular endothelial cells

1. Introduction Toxoplasmosis is a widespread protozoan parasitic infection of humans. Approximately 500 million people world-wide have antibodies against the causative organism Toxoplasma gondii ŽKean, 1972.. In the immunocompetent individual the disease is usually asymptomatic and selflimiting ŽMiller, 1995.. However, in those who are immunosuppressed as a consequence of chemotherapy or AIDS, latent infection can become reactivated resulting in acute disseminating disease with neurological symptoms the most common feature. Infection during pregnancy can result in congenital toxoplasmosis as tachyzoites of T. gondii can cross the placenta and infect foetal tissues. The risks of infection and morbidity are dependent on the stage of gestation at which the mother becomes infected; infection during the first trimester results in the most severe morbidity ŽDesmonts and Couvrer, 1974.. Toxoplasmic retinochoroiditis ŽTR. is most commonly associated with congenital infection although it has been found that 1%–3% of individuals with acquired infection ) Corresponding author. Tel.: q44-171-928-9292 ext. 3393; fax: q44171-401-9062; e-mail: [email protected]

develop ocular disease ŽOlle et al., 1996.. TR is characterised by an acute necrotising retinochoroiditis which accounts for 28%–50% of all cases of posterior uveitis world-wide ŽPavesio and Lightman, 1996.. Although TR is self-limiting in immunocompetent individuals, lesions can often reoccur and may lead to permanent loss of visual acuity ŽRothova, 1993.. The pathogenesis of TR is still a matter of debate as it is not clear whether tissue damage is caused by the parasite itself or by the host response. Moreover, the immune mechanisms involved in the control of ocular disease have not yet been elucidated. In vivo murine studies have provided some evidence for the importance of the cytokine IFNg and treatment with antiIFNg antibodies results in an increase in ocular lesions and higher parasite load within the eye ŽGazzinelli et al., 1994; Olle et al., 1996.. Several mechanisms have been demonstrated by which this cytokine can inhibit toxoplasmosis. IFNg stimulation of macrophages leads to the production of nitric oxide ŽNO. which results in the destruction of T. gondii tachyzoites ŽMayer, 1997.. Treatment of chronically infected mice with the NO inhibitor aminoguanidine results in a significant increase in inflammation in the choroid, retina and vitreous which suggests a role for NO in the control of

0165-5728r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 6 7 - 8

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ocular disease ŽHayashi et al., 1996.. The treatment of human retinal pigment epithelial cells ŽRPE. in vitro with IFNg has been shown to result in a dose dependent inhibition of parasite growth within these cells. However, NO production was not demonstrated in these cultures and the addition of an NO inhibitor did not affect parasite replication. Instead, IFNg inhibited parasite growth in RPE cells by starving T. gondii of L-tryptophan, an amino acid essential for growth. IFNg induces the upregulation of indoleamine-2,3-dioxygenase ŽIDO. which converts Ltryptophan to kynurenine ŽNagineni et al., 1996.. It is not yet clear how T. gondii invades the retina, whether the parasite enters the retina via the bloodstream or within infected macrophages. Clinical evidence strongly supports the concept that the parasite initially causes inflammation of the retina, with very little evidence of choroidal disease being present in the early stages. Available pathological evidence, taken from eyes with fulminant endophthalmitis, has shown choroiditis with infection of RPE cells but it is unlikely that these cells are infected during the initial parasite invasion ŽNagineni et al., 1996.. The retinal vascular endothelium ŽRVE. which appears to have a critical role in recruiting leukocytes from the circulation during immune mediated disease such as posterior uveitis may also be the primary site for parasite invasion in TR but little is known about this process or the effects of cytokines upon it ŽGreenwood and Calder, 1993; Greenwood et al., 1996.. We therefore examined the effects of various cytokines on the growth of T. gondii within rRVE cells to try and determine the mechanisms involved in this process.

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2. Materials and methods 2.1. Rat retinal Õascular endothelial (rRVE) cell cultures A SV40 large T immortalised cell line ŽJG2.1. was generously donated by Dr. John Greenwood, University College, London. rRVE cells were grown to confluence on collagen coated plastic tissue culture flasks. Collagen ŽSigma, UK. was diluted 1:20 in Hanks balanced salt solution ŽHanks BSS, Sigma. and used to coat the bottom of the flask for 30 min, the flasks were then removed to an ammonia atmosphere for a further 30 min. The collagen mixture was rinsed from the flasks three times using Hanks BSS. rRVE cells were cultured in Hams F10 ŽSigma. supplemented with 2 mM L-glutamine ŽSigma., 20 mgrml gentamycin ŽSigma. and 20 mgrml penicillin–streptomycin ŽGibco BRL, UK., 20% plasma derived serum ŽPDS, First Link, UK., 83 mlr10 ml endothelial cell growth factor ŽECGF, Sigma., 50 mlr10 ml of 1 mgrml ascorbic acid ŽSigma. solution and 10 Urml heparin at 378C in a humidified CO 2 incubator. 2.2. Maintenance of T. gondii T. gondii RH, a virulent non-cyst forming strain, was obtained from the Toxoplasma Reference Laboratory at St. George’s Hospital, Tooting, London. T. gondii RH strain were first passaged in mice by intraperitoneal injection of tachyzoites, animals were killed after 3 days and parasites were collected by washing the peritoneum.

Fig. 1. Effect of recombinant rat IFNg on T. gondii replication in rRVE cells. Confluent cultures of rRVE cells were treated with 100 Urml IFNg for 24 h prior to parasite inoculation. The cultures were washed 6 h after parasite inoculation to remove non-adherent parasites. Culture supernatants were collected on days 1–4 after parasite inoculation and T. gondii replication quantified by counting the number of extracellular tachyzoites. The results are the means " the standard errors of nine experiments.

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Fig. 2. Effects of recombinant rat IFNg, TNF and IL-1b on T. gondii replication within rRVE cells. Confluent cultures of rRVE cells were treated with 100 Urml IFNg, 1 ngrml TNF, 1 ngrml IL-1b or combinations of these cytokines for 24 h prior to parasite inoculation. For each cytokine treatment a further rRVE culture was treated with the iNOS inhibitor N G MMA Ž400 mM.. The cultures were washed 6 h after parasite inoculation to remove non-adherent parasites. Culture supernatants were collected on days 1–4 after parasite inoculation and T. gondii replication quantified by counting the number of extracellular tachyzoites. The results are the means" the standard errors of six experiments.

Parasites were passaged in vitro in human foetal foreskin fibroblasts ŽHFFF, European Collection of Animal Cell Cultures, 86031405.. HFFF cells were grown to confluence in minimal essential medium ŽMEM, Gibco. supplemented with 5 ml L-glutamine and 10% HIFBS in a 75-cm2 tissue culture flask. Nineteen milliliters of fresh medium were added and HFFF cells were infected with a 1-ml inoculum of tachyzoites Ž5 = 10 6 per ml.. Parasite concentration was determined by placing the parasite suspension on a haemocytometer ŽNeubauer. and parasites were counted by viewing under the light microscope. The infected cells were maintained at 378C in a humidified CO 2 incubator. 2.3. Cytokine treatment rRVE cells were grown to confluence on collagen coated glass coverslips and washed twice with phosphate buffered saline ŽPBS. to remove heparin ŽDaubener et al., 1995.. IFNg Ž100 Urml, R & D systems, UK., TNF Ž1 ngrml, Serotec, UK. and IL-1b Ž1 ngrml, R & D Systems. were added for 24 h prior to inoculation with the parasite. The cultures were washed twice with PBS and T. gondii RH strain tachyzoites were added at a parasite to cell ratio of 20:1. After 6 h the cultures were washed in PBS Ž=2. to remove any non-adherent parasites. On days 1, 2, 3 and 4 post inoculation culture supernatants were collected and T. gondii replication assessed by counting the number of tachyzoites released into the supernatant fluid. In separate experiments following cytokine treatment, N G-monomethyl-L-arginine Ž N G MMA, 400 mM, Calbiochem, UK, protocol as reported in Halonen et al., 1998., or L-tryptophan Ž20–100 mgrml, Sigma, protocol as reported in

Nagineni et al., 1996. were added at the same time as T. gondii. L-tryptophan was further added at the same dose on each day of the experiment to replace any L-tryptophan degraded by IFNg activated IDO. 2.4. Quantification of NO secretion Measurement of NO metabolites was carried out according to the following protocol. Eight nitrite standards were made up in 0 to 35 mM concentrations in order to construct a standard curve, 100 ml of each was then pipetted in duplicate into the wells of a 96-well plate ŽCostar, UK.. Supernatants to be tested were added in duplicate to the 96-well plate as 100 ml aliquots. One hundred microliter of Griess reagent ŽICN, UK. was added to each test well and the plate incubated at room temperature for 10 min. The absorbance was then measured photometrically at 550 nm on a plate reader. 2.5. Statistics Statistical evaluation of differences in parasite replication and NO production between the experimental samples

Table 1 Effect of recombinant rat IFNg on NO production by rRVE cells. The results are the means"the standard errors of nine experiments

Day 1 Day 2 Day 3 Day 4

IFNgqT. gondii IFNg

ControlqT. gondii Control

3.80"1.09 4.18"1.06 4.59"1.43 5.33"1.43

3.58"1.14 3.41"0.76 4.05"0.92 4.48"1.11

2.74"0.47 3.55"0.48 3.87"0.58 5.34"0.58

2.5"0.52 2.63"0.47 3.26"0.61 5.24"0.92

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Table 2 Effect of recombinant rat IFNg, TNF and IL-1b on NO production by rRVE cells. The results are the means" the standard errors of six experiments

Day 1 Day 2 Day 3 Day 4 Day 4 q N G MMA

Control

IFNg

TNF

IFNg q TNF

IL-1b

IFNg q IL-1b

2.30 " 0.26 2.29 " 0.22 2.22 " 0.29 2.45 " 0.26 2.39 " 0.15

2.89 " 0.41 2.65 " 0.37 2.47 " 0.43 3.13 " 0.65 2.43 " 0.34

2.64 " 0.37 2.76 " 0.33 2.38 " 0.31 2.59 " 0.28 2.76 " 0.25

3.49 " 0.45 3.12 " 0.33 3.13 " 0.38 3.87 " 0.49 3.98 " 0.18

2.18 " 0.29 2.10 " 0.21 2.97 " 0.99 2.27 " 0.41 2.58 " 0.08

2.82 " 0.35 2.54 " 0.23 2.69 " 0.20 2.92 " 0.38 2.75 " 0.26

were determined by the Students’ paired t-test. p - 0.05 was considered significant. 3. Results 3.1. Effect of IFNg , TNF and IL-1b on T. gondii replication within rRVE It has previously been shown that IFNg, TNF and IL-1b can affect T. gondii replication within human RPE cells and astrocytes ŽLee and Brosnan, 1996; Nagineni et al., 1996.. Pre-treatment with IFNg inhibited, but did not completely halt, the growth of T. gondii tachyzoites by rRVE cells from 24 h post inoculation. Parasite replication was significantly inhibited at 48, 72 and 96 h post inoculation as compared to controls Ž p - 0.005, Fig. 1..

We investigated whether TNF or IL-1b could affect parasite growth within rRVE, either alone or in conjunction with IFNg. From 72 h post infection there was a significant inhibition of parasite growth between cultures treated with IFNg, TNF or IL-1b alone as compared to controls Ž p - 0.05, Fig. 2.. Cultures treated with a combination of IFNg and TNF or IFNg and IL-1b also displayed a significant inhibition of parasite growth as compared to controls Ž p - 0.05, Fig. 2.. However, the cytokine combination did not have a synergistic effect on parasite replication as compared to cultures treated with a single cytokine. N G MMA treated cultures demonstrated a significant inhibition of parasite growth as compared to controls Ž p - 0.05, Fig. 2.; therefore the addition of N G MMA did not restore the release of tachyzoites by cytokine treated rRVE.

Fig. 3. Effect of L-tryptophan on parasite growth within IFNg treated, T. gondii infected rRVE cells. rRVE cells were treated with 100 Urml IFNg for 24 h prior to parasite inoculation. L-tryptophan was added at concentrations of 20 mgrml, 50 mgrml or 100 mgrml at the point of parasite inoculation. L-tryptophan was added at the same concentrations on each day of the experiment in order to replace any L-tryptophan degraded by the action of IFNg. Culture supernatants were collected on days 1–3 after parasite inoculation and T. gondii replication quantified by counting the number of extracellular tachyzoites. The results are the means " the standard errors of four experiments comparing L-tryptophan treated and IFNg treated cultures.

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Table 3 Effect of L-tryptophan on NO production by IFNg treated, T. gondii infected rRVE cells. The results are the means" the standard errors of six experiments

Day 1 Day 2 Day 3

IFNg qT. gondii

IFNg q T. gondii q20 mgrml L-tryptophan

IFNg q T. gondii q50 mgrml L-tryptophan

IFNg q T. gondii q100 mgrml L-tryptophan

T. gondii q100 mgrml L-tryptophan

4.78 " 0.27 5.70 " 0.79 4.83 " 1.34

5.27 " 0.14 5.57 " 1.19 5.85 " 1.49

4.25 " 0.71 7.89 " 1.93 5.21 " 0.84

4.21 " 1.26 4.77 " 0.45 5.57 " 1.73

2.74 " 0.76 4.02 " 1.25 3.26 " 0.72

3.2. Effect of IFNg , TNF and IL-1b on NO production by rRVE The relationship between IFNg, TNF and IL-1b induced parasite growth inhibition and NO production by rRVE cells was investigated by assaying nitrite levels in the culture supernatants using Griess Reagent. There was no significant difference in concentration of NO metabolites between IFNg treated cultures and control cultures at any time point ŽTable 1.. Although there was an increase in NO metabolites from Day 1 to Day 4 there was no significant difference between cultures and the increase may have been accounted for by accumulation of metabolites in the supernatants. There were also no significant differences between T. gondii infected cultures and controls nor between IFNg treated T. gondii infected cultures and controls. There were no significant differences in the release of NO metabolites from TNF or IL-1b treated cultures and controls ŽTable 2.. In addition, combinations of IFNg and TNF or IFNg and IL-1b did not produce significantly different levels of NO metabolites as compared to controls. In order to test further whether IFNg was stimulating the production of NO metabolites by rRVE, the iNOS inhibitor N G MMA was added to cultures treated with cytokines as described above. There was no significant differences in levels of NO metabolites between cultures treated with N G MMA and those which were not, regardless of cytokine treatment ŽTable 2.. 3.3. Effect of L-tryptophan on T. gondii replication within IFNg treated rRVE We investigated whether the addition of L-tryptophan to IFNg treated T. gondii infected rRVE cells would restore parasite replication in IFNg treated cultures. There was a significant increase in tachyzoite numbers between L-tryptophan treated cultures and IFNg only treated cultures at a concentration of 50 mgrml L-tryptophan and above, from 48 h post inoculation ŽFig. 3.. Addition of L-tryptophan at the point of parasite inoculation significantly restores parasite growth in IFNg treated cultures. Therefore, IFNg appears to inhibit parasite replication in rRVE cells by starving the parasites of this amino acid. L-tryptophan had

no effect on NO production by IFNg treated rRVE or on T. gondii infected rRVE alone ŽTable 3..

4. Discussion RVE cells form a barrier between the retina and invading organisms from the bloodstream and are involved in the recruitment of leukocytes from the circulation during immune mediated disease ŽGreenwood et al., 1996.. The effect of cytokines on the replication of T. gondii in rRVE cells and the mechanisms that may be involved have not been previously investigated. In this study we show that T. gondii will readily infect and replicate within rat rRVE cells. Pre-treatment of rRVE with IFNg, TNF or IL-1b inhibited the growth of the parasite within these cells. These cytokines appeared to restrict T. gondii replication by starving the parasites of the amino acid L-tryptophan. The rRVE cells used in this study were a SV40 large T immortalised cell line ŽJG2.1. and we recognise that there may be differences in responses between transformed and primary cells. However, it has been demonstrated that the cell line used has similar morphology to primary cultures and retains expression of characteristic surface markers, including von Willebrand factor, the transferrin receptor and RECA-1 antigen ŽGreenwood et al., 1996.. These shared characteristics determined the use of the SV40 transformed JG2.1 rRVE cell line for all the experiments reported in this investigation. The role of cytokines in TR has been studied in vivo in animal models where it has been proposed that the cytokine IFNg has an important role to play in the control of this disease ŽGazzinelli et al., 1994; Olle et al., 1996.. In vitro studies have demonstrated that T. gondii can readily survive and replicate within unstimulated resident murine peritoneal macrophages where growth within the parasitophorus vacuole fails to stimulate the oxidative burst and fusion with lysosomes ŽAdams et al., 1990.. It has since been shown that the production of IFNg by natural killer cells activates killing of T. gondii within infected macrophages ŽMcCabe and Remington, 1986.. Available evidence suggests that stimulation of macrophages by invading T. gondii leads to the production of IL-1b, IL-12

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and TNF ŽSher et al., 1993; Gazzinelli et al., 1994; Hunter et al., 1995.. These cytokines induce the production of IFNg by NK cells and in turn IFNg induces transcription of the enzyme inducible NO synthase ŽiNOS. leading to the production of NO by macrophages resulting in destruction of the parasite ŽMayer, 1997.. In the central nervous system, microglia and astrocytes have IFNg dependent anti-toxoplasma mechanisms. IFNg stimulated human microglia are able to restrict parasite entry but do not appear to affect intracellular survival of T. gondii ŽChao et al., 1994.. IFNg and IL-1b have a synergistic effect on the upregulation of iNOS in human astrocytes and in vitro studies have indicated that these cells can induce toxoplasmostasis using a NO-dependent mechanism ŽPetersen, 1995; Lee and Brosnan, 1996.. Recent studies, however, indicate that NO may have a lesser role than previously considered in long term latent T. gondii infections ŽSchluter ¨ et al., 1999.. Treatment with iNOS inhibitors of the susceptible C57rBL mouse strain, infected with a cyst-forming strain of T. gondii, resulted in an increased cyst burden and more rapid mortality. However, the resistant, Balbrc mouse strain was unaffected. These findings indicate that a long term chronic infection in resistant animals is maintained by a NO-independent mechanism. In this investigation we demonstrated that stimulation of rRVE with IFNg, TNF or IL-1b did not result in a significant increase in NO metabolites as compared to controls. In addition, treatment of cytokine stimulated cultures with the iNOS inhibitor N G MMA did not result in a significant reduction of NO metabolites. Therefore, IFNg treated rRVE cells appear to be capable of restricting T. gondii replication by a NO-independent mechanism. Addition of L-tryptophan to IFNg stimulated rRVE significantly restored the growth of T. gondii within these cells at concentrations of 50 mgrml and above, from 48 h post infection. This indicates that IFNg induced toxoplasmostasis may be mediated by L-tryptophan starvation. IFNg induces the upregulation of IDO which converts L-tryptophan to kynurenine, thus depriving the parasite of an amino acid which is essential for replication ŽNagineni et al., 1996.. The catabolism of L-tryptophan by IDO has been shown to lead to toxoplasmostasis in fibroblasts, microglial cells and RPE ŽPfefferkorn, 1984; Daubener et al., 1993; Nagineni et al., 1996.. Accumulation of NO in retinal tissue is potentially harmful; it has been reported that NO can interfere with RPE phagocytosis leading to photoreceptor degradation and has also been implicated in endotoxin induced uveitis in rats ŽBequet et al., 1994; Parks et al., 1994.. It is possible that L-tryptophan starvation is a mechanism by which non-phagocytic cells can restrict parasite growth whilst minimising immune mediated damage to surrounding host tissue. Other possible mechanisms of control of T. gondii infection by cytokines have been described. Studies using human macrophages showed that there was no role for

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IFNg mediated iron depletion in the inhibition of T. gondii replication ŽMurray et al., 1991.. Conversely, rat gut enterocytes are capable of inhibiting the growth of T. gondii on the addition of IFNg by limiting the amount of intracellular iron ŽDimier and Bout, 1998.. Both DFO and IFNg mediated inhibition were reversed on the addition of ferrous sulphate or holotransferrin to enterocyte cultures. We are currently investigating iron depletion as a potential mechanism by which IFNg restricts parasite replication within rRVE. It is not known whether T. gondii enters the retina within infected macrophages or directly from the bloodstream. RVE cells could act as a first line of defence against invading parasites, as these cells form an important part of the blood–retinal barrier and may be the point of entry of T. gondii into the retina. We have shown that RH strain T. gondii tachyzoites may actively invade and replicate within rRVE cells and that rRVE cannot inhibit the parasite without cytokine stimulation in vitro. IFNg, TNF and IL-1b can induce toxoplasmostasis in rRVE cells by a mechanism which is not dependent on NO production but may be controlled by L-tryptophan starvation. Regardless of the mechanism, even in conjunction with a combination of cytokines, rRVE cannot completely halt the replication of T. gondii and it is possible that these cells have a greater role in recruiting leukocytes to the site of infection rather than actively challenging the parasite. Once through the blood–retinal barrier T. gondii may stimulate resident macrophages or other retinal cells to produce cytokines such as TNF. This would lead to the upregulation of adhesion molecules and chemokines on the RVE and subsequent leukocyte infiltration. Further work to characterise the effect of toxoplasma infection on other resident retinal cells is now required.

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