Replication of Toxoplasma gondii, but Not Trypanosoma cruzi, IsRegulated in Human Fibroblasts Activated with Gamma
Interferon: Requirement of a FunctionalJAK/STAT Pathway
ISABELA PENNA CERAVOLO,1 ANDREA C. L. CHAVES,1 CLAUDIO A. BONJARDIM,2
DAVID SIBLEY,3 ALVARO J. ROMANHA,1 AND RICARDO T. GAZZINELLI4,5*
Cellular and Molecular Parasitology Laboratory1 and Chagas’ Disease Laboratory,5 Centro de Pesquisas Rene Rachou,FIOCRUZ, and Viruses Laboratory, Microbiology Department,2 and Biochemistry and Immunology Department,4 ICB,UFMG, Belo Horizonte, Brazil, and Molecular Microbiology Department, Washington University, St. Louis, Missouri3
Received 28 September 1998/Returned for modification 18 November 1998/Accepted 12 February 1999
To study the role of tryptophan degradation by indoleamine 2,3-dioxygenase (INDO) in the control ofTrypanosoma cruzi or Toxoplasma gondii replication, we used human fibroblasts and a fibrosarcoma cell line(2C4). The cells were cultured in the presence or absence of recombinant gamma interferon (rIFN-g) and/orrecombinant tumor necrosis factor alpha (rTNF-a) for 24 h and were then infected with either T. cruzi or T.gondii. Intracellular parasite replication was evaluated 24 or 48 h after infection. Treatment with rIFN-g and/orrTNF-a had no inhibitory effect on T. cruzi replication. In contrast, 54, 73, or 30% inhibition of T. gondiireplication was observed in the cells treated with rIFN-g alone, rIFN-g plus rTNF-a, or TNF-a alone,respectively. The replication of T. gondii tachyzoites in cytokine-activated cells was restored by the addition ofextra tryptophan to the culture medium. Similarly, T. gondii tachyzoites transfected with bacterial tryptophansynthase were not sensitive to the microbiostatic effect of rIFN-g. We also investigated the basis of the cytokineeffect on parasite replication by using the three mutant cell lines B3, B9, and B10 derived from 2C4 andexpressing defective STAT1a (signal transducer and activator of transcription), JAK2 (Janus family ofcytoplasmic tyrosine kinases), or JAK1, respectively, three important elements of a signaling pathway triggeredby rIFN-g. We found that rTNF-a was able to induce low levels expression of INDO mRNA in the parental cellline, as well as the cell line lacking functional JAK2. In contrast to the parental cell line (2C4), rIFN-g was notable to induce the expression of INDO mRNA or microbiostatic activity in any of the mutant cell lines. Thesefindings indicate the essential requirement of the JAK/STAT pathway for the induction of high levels of INDOmRNA, tryptophan degradation, and the anti-Toxoplasma activity inside human nonprofessional phagocyticcells.
Several of the gene products and functions induced bygamma interferon (IFN-g) in either professional or nonpro-fessional phagocytic cells (PPC and NPPC) have been impli-cated in resistance to microbial infection. In macrophages orPPC the activation by IFN-g leads to the production of reactiveoxygen intermediates (31) and reactive nitrogen intermediates(1, 16), the generation of leukotrine derivatives (54), and tryp-tophan degradation (8, 35), all of which are involved in thecontrol of intracellular pathogens. Thus, the growth of differ-ent intracellular protozoa, such as Leishmania spp., Toxo-plasma gondii, and Trypanosoma cruzi (28, 29, 30, 38, 55), istightly regulated inside macrophages activated by IFN-g. Manyof these microbiostatic-microbicidal functions induced byIFN-g have also been shown to be potentiated by tumor ne-crosis factor alpha (TNF-a) (16, 23).
In human NPPC, indoleamine 2,3-dioxygenase (INDO) ap-pears to be the main enzyme induced by IFN-g and is impli-cated in the control of the intracellular replication of T. gondiitachyzoites (35). INDO is the first enzyme in a major pathway
responsible for the degradation of tryptophan. More specifi-cally, this enzyme catalyzes the oxidative decyclization of L-tryptophan to N-formylkynurenine (36, 48). In both PPC andNPPC, inhibition of intracellular parasite replication inducedby IFN-g can be reversed by the addition of tryptophan to thetissue culture medium (17, 35). These results indicate that theinduction of INDO by IFN-g leads to tryptophan depletionand to the interruption of parasite replication inside the ver-tebrate host cells (9, 10, 49).
Since, like T. gondii tachyzoites, the T. cruzi amastigotes canalso replicate inside most nucleated cells from their vertebratehosts, it is tempting to speculate that not only macrophages butalso NPPC may display mechanisms that will lead to resistanceto infection with this latter parasite. In this study, we proposedto investigate the regulation of T. cruzi replication inside NPPCof human origin activated with rIFN-g and/or rTNF-a. T. gon-dii parasites were used as a control since their replication isknown to be controlled inside human NPPC after activationwith IFN-g. In addition, mutant cell lines were used to test theeffect of different elements from the signaling pathway(s) trig-gered by recombinant IFN-g [rIFN-g] on the induction ofINDO and the control of intracellular protozoa inside NPPC.Our results, show that (i) in contrast to T. gondii, T. cruziparasites can grow even in the presence of low levels of tryp-tophan inside NPPC cells activated with rIFN-g alone or incombination with rTNF-a; (ii) rTNF-a triggers low levels of
* Corresponding author. Mailing address: Laboratory of Chagas’Disease, CPqRR-FIOCRUZ, CEP 30190-002, Cx. Postal 1743, Av.Augusto de Lima 1715, Belo Horizonte, MG 30190-002, Brazil. Phone:031-295-3566. Fax: 031-295-3115. E-mail: [email protected].
INDO mRNA, which was associated with low microbiostaticactivity against T. gondii; (iii) rTNF-a also increased, in anadditive manner, the effect of IFN-g on the induction of INDOmRNA as well as on tachyzoite growth regulation inside cellsfrom the fibroblast lineage; and (iv) JAK1 (Janus family of
cytoplasmic tyrosine kinases), JAK2, and STAT1a (signaltransducer and activator of transcription) are all required forthe maximal induction of INDO mRNA, tryptophan degrada-tion, and the control of parasite replication by rIFN-g alone orwhen added with rTNF-a.
MATERIALS AND METHODS
Cell lines. The human fibrosarcoma cell lines 2C4, B3, B9, and B10 and thehuman foreskin fibroblast line CRL1634 (a gift from Alan Sher, National Insti-tute of Allergy and Infectious Diseases, National Institutes of Health) were used.
The mutants (B3, B9, and B10) were selected from the 2C4 human cell line,which was derived from a human fibrosarcoma HT1080 (5, 24, 57). The mutantcell lines were mutagenized with ICR 191 (acridine mutagen; Sigma ChemicalCo., St. Louis, Mo.). After five rounds of mutagenesis, the cells were fluorescenceactivator cell sorted and then were grown and clonally selected in the presenceof IFN-g (51). Thus, the mutants termed B3, B9, and B10 were independentisolates derived from 2C4 (5). B3 was defective in STAT1a and B10 and B9 weredefective in JAK1 and JAK2, respectively, three important elements of a signal-ing pathway triggered by IFN-g (6, 11, 32, 53). The genotypes of these cells (genedeletions) were all confirmed by genetic crosses involving mutants from the sameand from different complementation groups, as well as by biochemical analysis(5, 49). Further, by measuring the cytokine-induced expression of membranesurface proteins (e.g., transfected CD2 and major histocompatibility complexclass I and class II), the steady-state levels of 9-27,29,59-oligoadenylate syn-thetase, and the guanylate binding protein mRNAs, we confirmed the expectedphenotype of the B3, B9, and B10 cell lines. Thus, we showed that B3 and B10are unresponsive to both IFN type 1 (IFN-a/b) and type 2 (IFN-g), whereas B9conserved intact the IFN type 1 signaling pathway (5).
The cells were grown in Dulbecco modified Eagle medium (DMEM; GIBCOLaboratories, Grand Island, N.Y.) or RPMI 1640 medium (GIBCO) and sup-plemented with 10% heat-inactivated fetal bovine serum, 5 mM L-glutamine, 100IU of penicillin and 100 mg of streptomycin per ml, and 25 mM of HEPES buffer(pH 7.3). The tryptophan concentrations in the DMEM and RPMI media were16 mg (78.4 mM) and 5 mg (24.5 mM) per liter, respectively. All cell lines were
FIG. 1. (A) Effect of rIFN-g (900 IU/ml) and/or rTNF-a (60 IU/ml) onintracellular parasite replication in human foreskin fibroblasts. The replicationwas evaluated 48 h postinfection. An asterisk indicates a result statisticallydifferent from the control group (P , 0.05). Results indicate the means 6 thestandard errors of the means from two independent experiments done in dupli-cate. (B) Effect of rIFN-g (900 IU/ml) and/or rTNF-a (60 IU/ml) on intracellularparasite replication in 2C4 cells. The replication was evaluated at 48 h postin-fection. An asterisk indicates a result statistically different from the control group(P , 0.05). Results indicate the means 6 the standard errors of the means fromthree independent experiments done in duplicate. (C) Illustration of rIFN-geffect on intracellular replication of T. gondii tachyzoites and T. cruzi amasti-gotes. Note that, in contrast to T. gondii, T. cruzi growth was observed in thepresence or absence of rIFN-g.
FIG. 2. Effect of the absence of L-tryptophan (L-Trp) in 2C4 cells stimulatedwith rIFN-g (900 IU/ml) and/or rTNF-a (60 IU/ml) and infected with (A) T.gondii or (B) T. cruzi. The cells and parasites were maintained without L-tryp-tophan as described in Materials and Methods. The replication was evaluated24 h postinfection. An asterisk indicates a result statistically different from thecontrol group (P , 0.05). Results indicate the means 6 the standard errors of themeans from three independent experiments done in duplicate.
cultured at 37°C in humidified air containing 5% CO2. The cells were counted ina Neubauer hemocytometer after trypsin treatment, and their viability was as-sessed by trypan blue exclusion.
Parasites. The Y strain of T. cruzi (44) and the RH strain of T. gondii wereused in the parasite growth assays. An RH strain transfected with the tryptophansynthase (TS) gene from Escherichia coli (43) was used in some experiments.Both parasites were maintained by serial passages in 2C4 human fibroblastmonolayer cultures. The T. cruzi trypomastigote and T. gondii tachyzoite formswere obtained on days 5 and 2 of cell culture, respectively.
T. gondii and T. cruzi cell infection. Confluent cells were trypsinized (0.034%trypsin in 0.1% EDTA; both from Sigma), added to eight-well tissue culturechamber slides (Nunc, Inc.) in duplicates at a final concentration of 3 3 104
cells/well in 430 ml of DMEM or RPMI in the presence or absence of cytokines,and incubated at 37°C in 5% CO2 at 95% humidity for 24 h. The medium waschanged after 24 h, and T. gondii or T. cruzi was added at a ratio of 3/1 and 10/1parasites per cell, respectively, in a volume of 200 ml, with or without thecytokines. After 3 h of infection for T. gondii and 6 h for T. cruzi, the parasitesthat had not infected the cells were removed by washing, replaced with 430 ml offresh medium, and further incubated with or without the cytokines for 24 or 48 hat 37°C and 5% CO2. In the case of the T. cruzi, the infected cells were incubatedat 33°C in 5% CO2 at 95% humidity (4). After 24 or 48 h of incubation, thechambers were washed with phosphate-buffered saline (PBS), fixed with meth-anol, stained with May-Grunwald-Giemsa, and mounted on slides. The resultsevaluated under light microscopy were expressed as an infection index. Theinfection index is the average number of parasites per 100 cells obtained fromthree independent experiments performed in duplicate.
In order to verify the effect of exogenous tryptophan in restoring the parasitereplication, the 2C4 cells activated with cytokines were cultured as describedabove, in the presence or absence of exogenous 1 mM L-tryptophan (Sigma), andthen infected with either tachyzoite or trypomastigote forms. The intracellularparasites were counted at 24 h postinfection. To further certify a tryptophanrequirement for the intracellular growth of T. gondii or T. cruzi, parasite repli-cation was evaluated in a culture medium prepared from a kit supplied byGIBCO that had all the components of a complete medium except tryptophan.The tryptophan-free medium was supplemented with 3% dialyzed heat-inacti-vated fetal bovine serum and 100 mM indole. Parasite intracellular growth wasevaluated at 24 and 48 h postinfection.
Cytophatic assay. Vaccinia virus strain WR was a gift from C. Jungwirth(University of Wurzburg, Wurzburg, Germany). It was propagated in Vero cellsand purified as previously described by Joklik (19). The 2C4 parental and mutantcell lines were cultured as described above at a density of 1.5 3 104/well on a24-well plate and, when 90% of confluence was reached, rIFN-g (500 IU/ml) wasadded overnight. Fresh medium supplemented with rIFN-g was added to thecultures, which were then infected with vaccinia virus at a 0.01 multiplicity ofinfection for 2 h. The infection was stopped by aspirating and replacing themedium with 2 ml of fresh medium. After 30 h, the virus plaques were visualizedby adding 1 ml of 0.3% crystal violet in formalin solution (7).
Cytokines. Human rIFN-g and rTNF-a, with a specific activity of 3 3 107
U/mg of protein, were provided by Genentech, Inc. (San Francisco, Calif.) andwere maintained at 4°C. The concentrations used in the assays were 900 IU/mlfor rIFN-g and 60 IU/ml for rTNF-a. These concentrations were chosen after
previous titrations of the cytophatic effect of the vesicular stomatitis virus in 2C4cells.
INDO mRNA expression assay. In order to verify the induction of INDOmRNA expression, 75-cm2 culture flasks containing 2 3 106 to 5 3 106 cells wereused. Briefly, the cells were incubated in the presence or absence of cytokines for14 to 16 h. The medium was discarded, and the cells were washed with PBS. TotalRNA was extracted with TRIzol (GIBCO) according to the manufacturer’sinstructions. The cDNA synthesis was obtained in a final volume of 100 mlcontaining 200 U of reverse transcriptase (RT) obtained from Moloney murineleukemia virus (Pharmacia), 200 mM concentrations of each deoxynucleosidetriphosphate (Promega), 2.5 ml of buffer (0.1 M MgCl2, 0.5 M Tris-HCl, 1 mMDTT, 2 mg of bovine serum albumin per ml; pH 7.2 at 20°C) (BoehringerMannheim), 240 pmol of oligo-dT10 (Boehringer Mannheim) per ml, 0.1 Mdithiothreitol (Bio-Rad), 1 U of the RNase inhibitor RNasin (Promega) per ml,and 0.4 mg of total RNA. The cDNA samples were stored at 220°C.
The INDO mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)were detected in the host cells by RT-PCR assays. The primers 59-AGT TGA
FIG. 3. Effect of the addition of L-tryptophan (L-Trp) on the intracellularreplication of T. gondii tachyzoites in 2C4 cells activated with rIFN-g (900 IU/ml)and/or rTNF-a (60 IU/ml). Tryptophan was added to the culture medium at afinal concentration of 1 mM immediately after the parasite infection, and thereplication was evaluated 24 h later. An asterisk indicates a result statisticallydifferent from the control group (P , 0.05). Results indicate the means 6 thestandard errors of the means from three independent experiments done induplicate.
FIG. 4. Comparison of effects of rIFN-g (900 IU/ml) and/or rTNF-a (60IU/ml) on 2C4 human fibroblasts infected with wild-type or transfected strains ofT. gondii and maintained in the presence (A) or in the absence (B) of L-tryptophan (L-Trp). Both strains were maintained in tryptophan-free medium.RPMI was supplemented with 3% dialyzed heat-inactivated fetal bovine serumand 100 mM indole. The replication was evaluated 24 h after infection. Anasterisk indicates a result statistically different from the control group (P , 0.05).Results indicate the means 6 the standard errors of the means from threeindependent experiments done in duplicate. (C) PCR products of TS from E.coli. PCR was performed as described in Materials and Methods. The PCRproduct (3 ml) was electrophoresed in 6% polyacrylamide gel and silver stained.DNA of bacteriophage fX digested by endonuclease HaeIII was used as amolecular size marker. Lanes: 1 and 2, DNA from T. gondii RH wild-type strain,1 and 10 ng, respectively; 3 and 4, T. gondii TS-transfected strain, 1 and 10 ng,respectively; 5 and 6, E. coli, 1 and 10 ng, respectively; 7, negative control (noDNA added). An 1,155-bp fragment was expected.
VOL. 67, 1999 MICROBIOSTATIC EFFECT OF IFN-g IN HUMAN FIBROBLASTS 2235
GAA GTT AAA CAT GC-39 and 59-CAT GAT CGT GGA TTT GGT GA-39were used to detect INDO mRNA. The expected fragment size was 487 bp. ThemRNA expression of the human housekeeping gene GAPDH was used as areference for the inducible INDO. The primers 59-GTG GTG AAG CAG GCGTCG-39 and 59-GAC TGA GTG TGG CAG GGA-39 were used to detectGAPDH mRNA expression. The expected fragment size was 311 bp. The RT-PCR program consisted of 30 cycles with an initial denaturation at 95°C for 5min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and a finalannealing of 1 min at 55°C and an extension at 72°C for 5 min. The amplificationwas carried out in a thermocycler (PTC 100; MJR Research) in a final volume of10 ml containing 0.5 U of Taq DNA polymerase (CENBIOT, RS, Porto Alegre,Brazil), 200 mM concentrations of each deoxynucleoside triphosphate, 1.5 mMMgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 8.5), together with 10.0 and 1.0pmol of the INDO and GAPDH primers, respectively. The reaction mixture wasoverlaid with 20 ml of mineral oil. After amplification, 3 ml of the products wasmixed with 3 ml of a 23 sample buffer (0.25% bromophenol blue, 0.25% xylenecyanol, 30% glycerol) and subjected to electrophoresis through a 6% nondena-turing polyacrylamide gel. Gels were fixed with 10% ethanol–0.5% acetic acid for10 min, and the bands were revealed by staining with 0.2% silver nitrate for 10min with 0.75 M NaOH–0.1 M formaldehyde for 10 min as previously described(40).
TS-transfected T. gondii. The expression of the TS in the transgenic strain ofT. gondii (43) was confirmed by PCR. The reaction components were the sameas those described for INDO and GAPDH except for the use of 1.0 pmol ofprimers 59-CCC CTA TTT TGG TGA GTT TG-39 and 59-CCC CTA TTT TGGTGA GTT TG-39 and either 1.0 or 10.0 ng of template DNA. The expectedfragment size was 1,155 bp. The PCR program consisted of 30 cycles, with aninitial denaturation at 95°C for 5 min, annealing at 50°C for 1 min, and extensionat 72°C for 1 min, followed by annealing at 50°C for 1 min and a final extensionof 5 min.
Statistical analysis. Differences between groups were assessed with the Stu-dent t test. P values of ,0.05 were considered statistically significant.
RESULTS
Differential regulation of T. cruzi and T. gondii growth inrIFN-g activated NPPC cells through tryptophan degradation.In order to study the regulation of parasite growth insideNPPC, we used the fibrosarcoma cell line (2C4). Initially, wecompared the ability of human foreskin fibroblasts (Fig. 1A)and the 2C4 cell line (Fig. 1B), exposed to rIFN-g and/orrTNF-a, to control T. gondii and T. cruzi replication. Thehuman fibroblasts or 2C4 cells were cultured in the absence orpresence of the cytokines rIFN-g (900 IU/ml) and/or rTNF-a(60 IU/ml) for 24 h and then infected with either T. cruzitrypomastigotes or T. gondii tachyzoites. The cell culture wasinterrupted at 48 h postinfection, and the intracellular para-sites were counted. The results presented on Fig. 1A (humanforeskin fibroblasts) and 1B (2C4 cell line) and illustrated inFig. 1C (2C4 cell line), show that activation of either humanfibroblasts or 2C4 cells with rIFN-g resulted in a strong inhib-itory effect of intracellular tachyzoite growth. The rTNF-aalone had a persistent, small inhibitory effect on T. gondiigrowth, which was observed in different experiments. However,this difference was not statistically significant within a singleexperiment. The addition of rIFN-g and rTNF-a resulted in anadditive inhibitory effect on the tachyzoite growth. Interest-ingly, activation of either human foreskin fibroblasts or the 2C4cell line with rIFN-g and/or rTNF-a had no effect on theintracellular growth of T. cruzi amastigotes.
Because previous studies have demonstrated that the acti-vation of human NPPC with IFN-g results in degradation ofthe intracellular pool of tryptophan (10, 35, 36), which is re-sponsible for the inhibition of parasite replication, we decidedto repeat the above experiment without tryptophan. The 2C4cell line was cultured in regular medium or in medium lackingtryptophan in the presence or absence of rIFN-g and/orrTNF-a for 24 h and was then infected with either T. gondii(Fig. 2A) or T. cruzi (Fig. 2B). In agreement with early studies(35), tachyzoite growth was largely inhibited in the tryptophan-free medium even in absence of cytokines. A small but notstatistically significant effect on T. cruzi growth was observed in
cells cultured in the absence of tryptophan. Thus, our dataindicate that T. cruzi amastigotes presented an equal ratio ofparasite replication at both low and high levels of tryptophaninside the host cells. Consistent with the hypothesis that tryp-tophan degradation mediates the inhibition of tachyzoitegrowth induced by rIFN-g, we observed that in the presence ofan excess amount of tryptophan, the inhibitory effect of intra-cellular tachyzoite growth induced by either rIFN-g or rIFN-gplus rTNF-a was reversed (Fig. 3).
We also tested the ability of T. gondii parasites transfectedwith the enzyme TS from E. coli to survive inside 2C4 cellsunder rIFN-g stimulation. The results presented in Fig. 4 showthat, in contrast to the wild type, the replication of transgenicparasites was not sensitive to NPPC exposure to rIFN-g and/orrTNF-a in the presence (Fig. 4A) or in the absence of trypto-phan (Fig. 4B). Figure 4C shows the detection of the TS geneby PCR by using DNA extracted from wild-type tachyzoites(lanes 1 and 2), transfected parasites (lanes 3 and 4), E. coli(lanes 5 and 6), and a negative control of the PCR reaction(lane 7). In contrast to transfected parasites and E. coli, PCRwith DNA from wild-type parasites did not yield a PCR prod-uct of the predicted size (1,155 bp).
Study of the involvement of the JAK/STAT pathway in theinduction of both INDO mRNA expression and antiparasiteeffector function by IFN-g. To further investigate the ability ofrIFN-g to induce INDO expression and the regulation of par-asite expression in NPPC, we used three mutant cell lines,derived from 2C4 line, that have specific defects on the IFN-gsignaling pathway. As shown in Fig. 5, the mutant cell lines B3,
FIG. 5. IFN-g signaling pathway showing the defects in the human fibroblastmutants B3, B9, and B10.
B9, and B10 are defective in STAT1a, JAK2, and JAK1, re-spectively. Recent studies have demonstrated that cells lackingfunctional JAK1 can exhibit substantial expression of genesinduced by IFN-g. However, the antiviral activity is only ex-hibited in cells which have functional JAK1, JAK2, and
STAT1a (15, 18, 45). We also tested cells lacking differentcomponents from the JAK/STAT pathway (Fig. 5) in theirability to control viral replication (Fig. 6A), T. gondii tachyzoitegrowth (Fig. 6B), and INDO mRNA expression (Fig. 6C).Interestingly, we found that JAK2-deficient cell line (B9) can
FIG. 6. (A) Cytophatic effect of vaccinia virus infectivity assay in parental 2C4 and mutant B3 and B10 cells. Row a shows the controls, in duplicate, with the threecell lines without vaccinia virus and rIFN-g. b1, c1, and d1 show the protective effect of rIFN-g (500 IU/ml) against vaccinia virus in 2C4 cells. b2 and c2 show the viraleffect on 2C4 cells without rIFN-g. d2 shows rIFN-g plus 2C4 cells. b3, c3, and d3 show the viral effect on the B3 mutant cell line plus rIFN-g. b4 and c4 show the effectof the virus on the mutant B3 cells. d4 shows rIFN-g plus B3 cells. b5, c5, and d5 show the viral effect on the B10 mutant cell line plus rIFN-g. b6 and c6 show theviral effect on the B10 cells. d6 shows rIFN-g plus B10 cells. (B) Effect of rIFN-g (900 IU/ml) and/or rTNF-a (60 IU/ml) on the parental (2C4) and mutant (B3, B9,and B10) cells infected with T. gondii. The replication was evaluated 48 h after infection. An asterisk indicates a result statistically different from control group (P ,0.05). Results indicate the means 6 the standard errors of the means from three independent experiments done in duplicate. (C) Effect of rIFN-g (900 IU/ml) and/orrTNF-a (60 IU/ml) on the induction of INDO mRNA. The parental (2C4) and mutant (B3, B9, and B10) cells were incubated with rIFN-g and/or rTNF-a for 14 to16 h. Lanes 1, 5, 9, and 13 are controls without cytokine; lanes 2, 6, 10, and 14 are rIFN-g; lanes 3, 7, 11, and 15 are rIFN-g plus rTNF-a; lanes 4, 8, 12, and 16 arerTNF-a; and lane 17 is the negative control (no cDNA added). Expression of INDO and GAPDH mRNA was detected by RT-PCR. PCR products (3 ml) wereelectrophoresed in 6% polyacrylamide gel and silver stained. The expected fragment sizes were 487 bp for the INDO and 311 bp for the GAPDH genes.
VOL. 67, 1999 MICROBIOSTATIC EFFECT OF IFN-g IN HUMAN FIBROBLASTS 2237
express low levels of INDO mRNA in response to rTNF-a butnot in response to IFN-g. As with the antiviral activity, maxi-mal induction of INDO mRNA and control of tachyzoite rep-lication was only observed in cells with functional JAK1 andJAK2 (and STAT1).
In addition, we observed a smaller ratio of infectivity whenwe compared the mutant cells to the parental cell line. How-ever, these differences were observed in some but not all of theexperiments performed.
DISCUSSION
Different studies indicate that the activation of human mac-rophages and NPPC with IFN-g leads to the induction ofINDO, a key enzyme involved in the degradation of the essen-tial amino acid tryptophan. In NPPC and macrophages, tryp-tophan starvation has been shown to be an important IFN-g-induced mechanism involved in the regulation of intracellularpathogen replication (3, 10, 35, 55). Thus, the addition of extratryptophan completely restores replication of the intracellularprotozoan T. gondii and the bacteria Chlamydia psittaci andChlamydia pneumoniae inside human fibroblast and epithelialcells, respectively (8, 35, 46). In contrast, additional tryptophanhas only a partial effect in restoring the replication of T. gondii,Leishmania donovani, and C. psittaci inside human macro-phages activated with IFN-g (10, 30). These latter results sug-gest that mechanisms other than tryptophan degradation maybe triggered in PPC by activation with IFN-g (12, 34, 47).
Because T. cruzi can infect any nucleated cell from its ver-tebrate host, it is tempting to speculate that cytokine activationof NPPC may also result in the induction of microbiostaticfunction, which is responsible for the control of parasite rep-lication during the chronic stage of infection. Furthermore,IFN-g and/or TNF-a have been shown to play an importantrole in resistance to T. cruzi during the activation of bothhuman and mouse macrophages (13, 26, 27); we thereforedecided to study the capacity of these cytokines to induceanti-T. cruzi activity in human fibroblasts. As a control we usedT. gondii tachyzoites, whose replication inside human fibro-blasts has been shown to be sensitive to activation by IFN-g.Interestingly, our results showed that in contrast to T. gondiitachyzoites, replication of T. cruzi inside human fibrosarcomacells or fibroblasts was not affected by cell activation withrIFN-g. Consistent with these observations, T. cruzi replicationwas not affected by tryptophan starvation, i.e., when parasiteswere cultured in tissue culture and medium lacking trypto-phan. In contrast, tachyzoite replication was almost abolishedin the same culture conditions. In agreement with our findingsare the studies showing that T. cruzi replication is not affectedin NPPC originating from humans or other mammals activatedwith human lymphoblastoid IFN and IFN obtained by infectingmonolayers of human amniotic cells with inactivated New-castle disease virus (14, 33). However, different studies suggestthat IFN-g, as well as IFN-a/b, may inhibit T. cruzi replicationinside rat or murine NPPC (31, 37). The reasons for thesediscrepancies are still unclear.
The mechanism by which T. cruzi escapes tryptophan star-vation is completely unknown. One possibility would be theexpression of an enzyme which is involved in the synthesis oftryptophan. In measuring the amount of indole consumed, wedetected only trace amounts of TS activity when we used T.cruzi epimastigote or trypomastigote extracts compared to ourfindings with E. coli. Moreover, by using primers specific forthe conserved E. coli TS sequence, we were unable to amplifya fragment that showed homology with the sequence of E. coliTS (data not shown). Another explanation for this effect would
be the generation of free tryptophan in the host due to theparasite protease activity. In fact, early studies demonstratedthat parasite treatment with specific protease inhibitors re-duced the amount of T. cruzi replication inside host cells (25).It is also noteworthy that all of the parasites sensitive to tryp-tophan starvation (i.e., T. gondii, L. donovani, and Chlamydiasp.) reside within a parasitophorous vacuole, in contrast to T.cruzi amastigotes, which replicate in the host cell cytoplasm(2). Thus, one could speculate that the residual pool of tryp-tophan in cells activated with IFN-g would be less available inthe parasitophorous vacuole. It is also possible that the tryp-tophan starvation only reduces the speed of parasite replica-tion inside NPPC. Since the T. cruzi replication is slower thanthe T. gondii tachyzoite replication, the proliferation of theformer parasite is not affected by the decreased levels of tryp-tophan inside host cells.
Finally, we also studied the ability of IFN-g to induce mi-crobiostatic activity in fibroblasts, as well as expression ofINDO mRNA in the 2C4 parental cell lines and in derivativecells lacking functional elements (i.e., STAT1a, JAK1, andJAK2) of the IFN-g signaling pathway. As expected, our re-sults show that 2C4 cells were responsive to IFN-g, whereasnone of mutant cell lines showed antiviral, antiparasitic activityor INDO mRNA expression when stimulated with rIFN-g.Thus, these results indicate the involvement of the JAK/STATpathway on the induction of INDO mRNA expression andmicrobiostatic function in NPPC exposed to IFN-g.
Interestingly, we found that rTNF-a was able to trigger theexpression of low levels of INDO mRNA in the mutant B9 cellline, which has an intact signaling pathway to respond to type1 IFNs. Previous studies have also shown that TNF-a andIFN-b can trigger the expression of INDO mRNA (41, 42, 52)and that TNF-a is able to induce mRNA synthesis of IFN-b inhuman fibroblasts (21, 39, 50). In light of our findings, wesuggest that TNF-a may be triggering INDO mRNA expres-sion through the induction IFN-b synthesis in human fibro-blasts. However, the levels of INDO mRNA induced by TNF-aand IFN-b are apparently not high enough to efficiently controlT. gondii replication inside fibroblasts.
These results clearly establish the involvement of the JAK/STAT signaling pathway in the INDO-mediated antimicrobialactivity of IFN-g, as suggested earlier by nucleic acid sequenceanalysis (22) and functional studies with plasmid constructscontaining INDO gene promoter (11, 22). However, in con-trast to previously reported studies, our results demonstratefor the first time the involvement of individual components(i.e., JAK1, JAK2, and STAT1) of the IFN-g signaling cascadein the induction of both INDO mRNA expression and micro-biostatic activity in NPPC.
ACKNOWLEDGMENTS
This work was supported in part by the Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq-522.056-95/4), theFundacao de Amparo a Pesquisa do Estado de Minas Gerais(FAPEMIG-CBS 1221/95), and PAPES/FIOCRUZ. R.T.G., A.J.R.,and C.A.B. are research fellows from CNPq. I.P.C. and A.C.L.C. aregraduate fellows from Coordenacao de Aperfeicoamento de Pessoalde Nıvel Superior (CAPES) and CNPq, respectively.
We are grateful to J. C. Magalhaes for technical assistance on thevirus infectivity assays.
REFERENCES
1. Adams, L. B., J. B. Hibbs, R. R. Taintor, and J. L. Krahenbuhl. 1990.Microbiostatic effect of murine activated macrophages for Toxoplasma gon-dii: role for synthesis of inorganic nitrogen oxides from L-arginine. J. Immu-nol. 144:2725–2729.
2. Andrews, N. W. 1993. Living dangerously: how Trypanosoma cruzi uses lyso-
somes to get inside host cells, and then escapes into the cytoplasm. Biol. Res.26:65–67.
3. Beatty, W. L., T. A. Belanger, A. A. Desai, R. P. Morrison, and G. I. Byrne.1994. Tryptophan depletion as a mechanism of gamma interferon-mediatedchlamydial persistence. Infect. Immun. 62:3705–3711.
4. Bertelli, M. S. M., R. R. Golgher, and Z. Brener. 1977. Intraspecific variationin Trypanosoma cruzi: effect of temperature on the intracellular differentia-tion in tissue culture. J. Parasitol. 63:434–437.
5. Bonjardim, C. A. 1998. JAK/STAT deficient cell lines. Braz. J. Med. Biol.Res. 31:1389–1395.
6. Briscoe, J., N. C. Rogers, B. A. Witthuhn, D. Watling, A. G. Harpur, A. F.Wilks, G. R. Starck, J. N. Ihle, and I. M. Kerr. 1996. Kinase-negativemutants of JAK1 can sustain interferon-g-inducible gene expression but notan antiviral state. EMBO J. 15:799–809.
7. Buller, R. M. L., S. Chakrabarti, B. Moss, and T. Fredrickson. 1988. Cellproliferative response to Vaccinia virus is mediates by VGF. Virology 164:182–192.
8. Byrne, G. J., L. K. Lehmann, and G. J. Landry. 1986. Induction of trypto-phan catabolism is the mechanism for gamma-interferon-mediated inhibi-tion of intracellular Chlamydia psittaci replication in T24 cells. Infect. Im-mun. 53:347–351.
9. Caplen, H. S., and S. L. Gupta. 1988. Differential regulation of a cellulargene by human interferon-g and interferon-a. J. Biol. Chem. 263:332–339.
10. Carlin, J. M., E. C. Borden, and G. I. Byrne. 1989. Interferon-inducedindoleamine 2,3-dioxygenase activity inhibits Chlamydia psittaci replicationin human macrophages. J. Interferon Res. 9:329–337.
11. Chon, S. Y., H. H. Hassanain, and S. L. Gupta. 1996. Cooperative role ofinterferon regulatory factor-1 and p91 (STAT1) response elements in inter-feron-g-inducible expression of human indoleamine 2,3-dioxygenase gene.J. Biol. Chem. 271:17247–17252.
12. de la Maza, L. M., and E. M. Peterson. 1988. Dependence of the in vitroantiproliferative activity of recombinant human g-interferon on the concen-tration of tryptophan in culture media. Cancer Res. 48:346–350.
13. Gazzinelli, R. T., I. P. Oswald, S. Hieny, S. L. James, and A. Sher. 1992. Themicrobicidal activity of interferon-gamma-treated macrophages againstTrypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-medi-ated mechanism inhibitable by interleukin-10 and transforming growth fac-tor-beta. Eur. J. Immunol. 22:2501–2506.
14. Golgher, R. R., M. S. M. Bertelli, M. L. Petrillo-Peixoto, and Z. Brener. 1980.Effect of interferon on the development of Trypanosoma cruzi in tissueculture “Vero cells.” Mem. Inst. Oswaldo Cruz 75:157–160.
15. Greelund, A. C., M. A. Farrar, B. L. Viviano, and R. D. Schreiber. 1994.Ligand-induced IFN gamma receptor tyrosine phosphorylation couples thereceptor to its signal transduction system (p91). EMBO J. 13:1591–1600.
16. Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A.Nacy. 1990. Leishmania major amastigotes initiate the L-arginine-dependentkilling mechanism in IFN-g-stimulated macrophages by induction of tumornecrosis factor-a. J. Immunol. 145:4290–4297.
17. Gupta, S. L., J. M. Carlin, P. Pyati, W. Daı, E. R. Pfefferkorn, and M. J.Murphy, Jr. 1994. Antiparasitic and antiproliferative effects of indoleamine2,3-dioxygense enzyme expression in human fibroblasts. Infect. Immun. 62:2277–2284.
18. Heim, M. H., I. M. Kerr, G. R. Starck, and J. E. Darnell. 1995. Contributionof STAT SH2 groups to specific interferon signalling by the JAK-STATpathway. Science 267:1347–1349.
19. Joklik, W. R. 1962. The purification of four strains of poxvirus. Virology18:9–18.
20. Kierszenbaum, F., and G. Sonnenfeld. 1984. b-Interferon inhibits cell infec-tion by Trypanosoma cruzi. J. Immunol. 132:905–908.
21. Kohase, M., D. Henriksen-DeStefano, L. T. May, J. Vilcek, and P. B. Sehgal.1986. Induction of beta 2-interferon by tumor necrosis factor: a homeostaticmechanism in the control of cell proliferation. Cell 45:659–666.
22. Konan, K. V., and M. W. Taylor. 1996. Importance of the two interferon-stimulated response element (ISRE) sequences in the regulation of thehuman indoleamine 2,3-dioxygenase gene. J. Biol. Chem. 271:19140–19145.
23. Liew, F. Y., Y. Li, and S. Millott. 1990. Tumor necrosis factor-alpha syner-gizes with IFN-gamma in mediating killing of Leishmania major through theinduction of nitric oxide. J. Immunol. 15:4306–4310.
24. McKendry, R., J. John, D. Flavell, M. Muller, I. M. Kerr, and G. R. Stark.1991. High-frequency mutagenesis of human cells and characterization of amutant unresponsive to both a and g interferons. Proc. Natl. Acad. Sci. USA88:11455–11459.
25. Meirelles, M. N. L., L. Juliano, E. Carmona, S. G. Silva, E. M. Costa,A. C. M. Murta, and J. Scharfstein. 1992. Inhibitors of the major cysteinylproteinase (GP57/51) impair host cell invasion and arrest the intracellulardevelopment of Trypanosoma cruzi in vitro. Mol. Biochem. Parasitol. 52:175–184.
26. Munoz-Fernandez, M. A., M. A. Fernandez, and M. Fresno. 1992. Activationof human macrophages for the killing of intracellular Trypanosoma cruzi byTNF-a and IFN-g through a nitric oxide-dependent mechanism. Immunol.Lett. 33:35–40.
27. Munoz-Fernandez, M. A., M. A. Fernandez, and M. Fresno. 1992. Synergism
between tumor-necrosis factor-alpha and interferon-gamma on macrophageactivation for the killing of intracellular Trypanosoma cruzi through a nitricoxide-dependent mechanism. Eur. J. Immunol. 22:301–307.
28. Murray, H. W., B. Y. Rubin, and C. D. Rothermel. 1983. Killing of intracel-lular Leishmania donovani by human mononuclear phagocytes: evidence thatinterferon-g is the activating lymphokine. J. Clin. Invest. 72:1506–1510.
29. Murray, H. W., B. Y. Rubin, S. M. Carriero, A. T. Denn, A. M. Harris, andE. A. Jaffee. 1985. Human mononuclear phagocyte antiprotozoal mecha-nisms: oxygen-dependent versus oxygen-independent activity against intra-cellular Toxoplasma gondii. J. Immunol. 134:1982–1988.
30. Murray, H. W., A. Szuro-Sudol, D. Wellner, M. J. Oca, A. M. Granger, D. M.Libby, C. D. Rothermel, and B. Y. Rubin. 1989. Role of tryptophan degra-dation in respiratory burst-independent antimicrobial activity of gamma in-terferon-stimulated human macrophages. Infect. Immun. 57:845–849.
31. Nathan, C. F., H. W. Murray, M. E. Wiebe, and B. Y. Rubin. 1983. Identi-fication of interferon-g as the lymphokine that activates human macrophageoxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670–689.
32. O’Shea, J. J. 1997. JAKs, STATs, cytokine signal transduction, and immu-noregulation: are we there yet? Immunity 7:1–11.
33. Osuna, A., G. Ortega, F. Gamarro, S. Castanys, and L. M. Ruiz-Perez. 1985.Effect of interferon on the infectivity of Trypanosoma cruzi in cultured HeLacells. Int. J. Parasitol. 15:167–170.
34. Ozaki, Y., M. P. Edelstein, and D. S. Duch. 1988. Induction of indoleamine2,3-dioxygenase: a mechanism of the antitumor activity of interferon-g. Proc.Natl. Acad. Sci. USA 85:1242–1246.
35. Pfefferkorn, E. R. 1984. Interferon-g blocks the growth of Toxoplasma gondiiin human fibroblasts by inducing the host cells to degrade tryptophan. Proc.Natl. Acad. Sci. USA 81:908–912.
36. Pfefferkorn, E. R., M. Eckel, and S. Rebhun. 1986. Interferon-g suppressesthe growth of Toxoplasma gondii in human fibroblasts through starvation fortryptophan. Mol. Biochem. Parasitol. 20:215–224.
37. Plata, F., J. Wietzerbin, F. G. Pons, E. Falcoff, and H. Eisen. 1984. Syner-gistic protection by specific antibodies and interferon against infection byTrypanosoma cruzi in vitro. Eur. J. Immunol. 14:930–935.
38. Reed, S. G. 1988. In vivo administration of recombinant IFN-g inducesmacrophage activation and prevents acute disease, immune suppression, anddeath in experimental Trypanosoma cruzi infection. J. Immunol. 140:4342–4347.
39. Reis, L. F., T. Ho Lee, and J. Vilcek. 1989. Tumor necrosis factor actssynergistically with autocrine interferon-beta and increases interferon-betamRNA levels in human fibroblasts. J. Biol. Chem. 264:16351–16354.
40. Santos, F. R., S. D. J. Pena, and J. T. Epplen. 1993. Genetic and populationstudy of a Y-linked tetranucleotide repeat DNA polymorphism. Hum.Genet. 90:655–656.
41. Schemer-Avni, Y., D. Wallach, and I. Sarov. 1988. Inhibition of Chlamydiatrachomatis growth by recombinant tumor necrosis factor. Infect. Immun.56:2503–2506.
42. Schmitz, J. L., J. M. Carlin, E. C. Borden, and G. I. Byrne. 1989. Betainterferon inhibits Toxoplasma gondii growth in human monocyte-derivedmacrophages. Infect. Immun. 57:3254–3256.
43. Sibley, L. D., M. Messina, and I. R. Niesman. 1994. Stable DNA transfor-mation in the obligate intracellular parasite Toxoplasma gondii by comple-mentation of tryptophan auxotrophy. Proc. Natl. Acad. Sci. USA 91:5508–5512.
44. Silva, L. H. P., and V. Nussensweig. 1953. Sobre uma cepa de Trypanosomacruzi altamente virulenta para o camundongo branco. Fol. Clin. Biol. 20:191–208.
45. Stahl, N., T. J. Farruggella, T. G. Boulton, Z. Zhong, J. E. Darnell, Jr., andG. D. Yancopoulos. 1995. Choice of STATs and other substrates specified bymodular tyrosine based motifs in cytokine receptors. Science 267:1349–1353.
46. Summersgill, J. T., N. N. Sahney, A. G. Charlotte, T. C. Quinn, and J. A.Ramirez. 1995. Inhibition of Chlamydia pneumoniae growth in Hep-2 pre-treated with gamma interferon and tumor necrosis factor alpha. Infect.Immun. 63:2801–2803.
47. Takikawa, O., T. Kuroiwa, F. Yamasaki, and R. Kido. 1988. Mechanism ofinterferon-g action. Characterization of indoleamine 2,3-dioxygenase in cul-tured human cells induced by interferon-g and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J. Biol. Chem.263:2041–2048.
48. Taylor, M. W., and F. Grensheng. 1991. Relationship between interferongamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J.5:2516–2522.
49. Thomas, S. M., L. F. Garrity, C. R. Brandt, C. S. Schobert, G.-S. Feng, M. W.Taylor, J. M. Carlin, and G. J. Byrne. 1993. IFN-g-mediated antimicrobialresponse. J. Immunol. 150:5529–5534.
50. Van Damme, J., M. de Ley, J. Van Snick, C. A. Dinarello, and A. Billiau.1987. The role of interferon-b1 and the 26-kDa protein (interferon-b2) asmediators of the antiviral effect of interleukin 1 and tumor necrosis factor.J. Immunol. 139:1867–1872.
51. Watling, D., D. Guschin, M. Muller, O. Silvennoinen, B. A. Witthuhn, F. W.Quelle, N. C. Rodgers, G. R. Starck, J. N. Ihle, and I. M. Kerr. 1993.Complementation by the protein tyrosine kinase JAK2 of a mutant cell line
VOL. 67, 1999 MICROBIOSTATIC EFFECT OF IFN-g IN HUMAN FIBROBLASTS 2239
defective in the interferon gamma signal transduction pathway. Nature 366:166–170.
52. Werner-Felmayer, G., E. R. Werner, D. Fuchs, A. Hausen, G. Reibnegger,and H. Wachter. 1989. Tumor necrosis factor-alpha and lipopolysaccharideenhance interferon-induced tryptophan degradation and pteridine synthesisin human cells. Biol. Chem. Hoppe-Seyler 370:1063–1069.
53. Will, A., U. Hemmann, H. Friedmann, M. Rollinghoff, and A. Gessner. 1996.Intracellular murine IFN-g mediates virus resistance, expression of oligoad-
enylate synthetase, and activation of STAT transcription factors. J. Immunol.157:4576–4583.
54. Wirth, J. J., and F. Kierszembaum. 1985. Effects of leukotriene C4 onmacrophage association with and intracellular fate of Trypanosoma cruzi.Mol. Biochem. Parasitol. 15:1–10.
55. Wirth, J. J., F. Kierszenbaum, G. Sonnenfeld, and A. Zlotnik. 1985. Enhanc-ing effects of gamma interferon on phagocytic cell association with andkilling of Trypanosoma cruzi. Infect. Immun. 49:61–66.
