chemiluminescent signal in a macrophage hybridoma cell line

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INFECTION AND IMMUNITY, Sept. 1991, p. 3303-3308 Vol. 59, No. 9 0019-9567/91/093303-06$02.00/0 Trypanosoma cruzi but Not Trypanosoma brucei Fails To Induce a Chemiluminescent Signal in a Macrophage Hybridoma Cell Line B. VRAY,l* P. DE BAETSELIER,2 A. OUAISSI, AND Y. CARLIER4 Laboratoire de Parasitologie Experimentale, Faculte des Sciences,' and Laboratoire de Parasitologie, Faculte de Medecine,4 Universite Libre de Bruxelles, Brussels, and Unit of Cellular Immunology, Institute of Molecular Biology, Vrije Universiteit Brussel, Sint Genesius Rode,2 Belgium, and Centre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, F-59000 Lille, France3 Received 2 January 1991/Accepted 27 June 1991 Macrophage-Trypanosoma cruzi interactions were studied by using a newly generated macrophage hybrid- oma cell line (2C11-12) that was selected for its capacity to produce high levels of reactive oxygen intermediates. This cell line was found to be a suitable host cell for T. cruzi, and intracellular parasitic development could be inhibited by activation with gamma interferon. When exposed to opsonized Trypanosoma brucei, Micrococcus lysodeikticus, or Legionella pneumophila, the activated macrophage cell line produces a high chemiluminescent signal, indicating the release of reactive oxygen intermediates. Alternatively, when opsonized T. cruzi was added to these activated macrophages, this parasite failed to stimulate a chemiluminescent response, suggesting an impairment in the triggering of the respiratory burst. When activated, macrophages are engaged in the respira- tory burst. Following their interaction with microorganisms, activated macrophages release reactive oxygen intermedi- ates (ROTs), which are thought to be involved in the killing of various parasites (19, 25) and yeasts (40). For example, gamma interferon (IFN--y)-activated macrophages are capa- ble of impairing the intracellular multiplication of Trypano- soma cruzi (36, 37, 49), and hydrogen peroxide is capable of directly killing T. cruzi (15, 31, 32). However, there is a lack of direct evidence for ROI production during macrophage-T. cruzi interaction. Since the activated 2C11-12 macrophage hybridoma cell line produces high levels of chemilumines- cence (CL) after treatment with phorbol myristate acetate or opsonized pathogens (9), we investigated the susceptibility of this cell line to infection by T. cruzi and its ability to produce a CL signal when interacting with this parasite. This response was compared with that generated by Trypano- soma brucei and the bacteria Micrococcus lysodeikticus and Legionella pneumophila. T. cruzi (Y strain) was maintained in male BALB/c mice by weekly intraperitoneal inoculations. Irradiated (700-rad X rays) Fischer rats were used to produce large quantities of trypomastigote forms (17). T. cruzi epimastigotes were grown in GLSH medium at 28°C (20). T. brucei (strain ANTAT, l.l.E clone) was maintained by serial passage in mice and harvested as described previously (14). Lyophi- lized cells of M. lysodeikticus (ATCC 4698) were purchased from Sigma Chemical Co., St. Louis, Mo. L. pneumophila (serogroup 1, Philadelphia strain 1, ATCC 33152) was culti- vated and harvested as described previously (35). The 2C11-12 cell line (10) was cultivated in RPMI 1640 medium (GIBCO, Grand Island, N.Y.) supplemented with N- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 25 mM), glutamine (2 mM), fetal calf serum (10%), NCTC- 135 medium (10%) (11), penicillin (100 IU/ml), and strepto- mycin (100 ,ug/ml; GIBCO) and incubated at 37°C in a 5% CO2 atmosphere. For infection experiments, 2C11-12 cells were seeded (105 cells per ml per well) in 24-well plates * Corresponding author. (Nunc, Roskilde, Denmark) with sterile round coverslips (Thermanox, 13-mm diameter; Miles Scientific, Naperville, Ill.). Trypomastigotes were added to the cells in various parasite-to-cell ratios. After 16 h, the cultures were washed with prewarmed medium to remove all free parasites. At different time intervals (24, 48, 72, and 96 h), the cells were fixed with methanol and stained with Giemsa stain. At least 200 cells per coverslip were microscopically counted to determine the level of infection. The parasitic index was calculated by multiplying the percentage of infected cells by the mean number of amastigotes per infected cell. The statistical significance of these results was determined by the nonparametric Mann-Whitney Wilcoxon test (39). 2C11-12 cells were activated by adding mouse recombi- nant IFN--y (1, 10, or 50 U/ml) to the culture medium 24 h before the cells were infected with parasites. The culture medium was removed 24 and 48 h after the initiation of the infection and replaced with fresh medium containing IFN-y. Polymyxin B sulfate (10 U/ml; Sigma) was added to inhibit the effect of lipopolysaccharide (LPS) traces which could be present in the culture medium (12, 21). For CL assays, 104 2C11-12 cells, previously activated for 48 h with mouse recombinant IFN--y (50 U/ml) or LPS B from Salmonella enteritidis (10 ,ug/ml; Difco, Detroit, Mich.), were grown in sterile polypropylene vials (Nunc). When IFN--y was used for cell activation, polymyxin B sulfate (10 U/ml) was added (see above). The opsonized or nonopsonized T. cruzi and T. brucei were added at a ratio of 20 parasites per cell unless otherwise indicated. When added to activated 2C11-12 cells, opsonized M. lysodeikticus cells induced a high CL signal and were routinely used as the positive control (we assumed that they induce 100% of the CL capacity of activated 2C11-12 cells). A ratio of 2,000 opsonized M. lysodeikticus cells per 2C11-12 cell was used (11). L. pneumophila was added at the same ratio. Each CL assay was repeated, in duplicate, at least three times. The CL signal was recorded over a 20-min period with a Biolumat (Berthold Co., Wildbab, Germany). The maxi- mum CL emission curve (cpm x 1,000) was used to calculate the CL percentage value. An anti-T. cruzi serum (S-1) was produced by inoculating 3303

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Page 1: Chemiluminescent Signal in a Macrophage Hybridoma Cell Line

INFECTION AND IMMUNITY, Sept. 1991, p. 3303-3308 Vol. 59, No. 90019-9567/91/093303-06$02.00/0

Trypanosoma cruzi but Not Trypanosoma brucei Fails To Induce aChemiluminescent Signal in a Macrophage Hybridoma Cell Line

B. VRAY,l* P. DE BAETSELIER,2 A. OUAISSI, AND Y. CARLIER4

Laboratoire de Parasitologie Experimentale, Faculte des Sciences,' and Laboratoire de Parasitologie,Faculte de Medecine,4 Universite Libre de Bruxelles, Brussels, and Unit of Cellular Immunology,

Institute of Molecular Biology, Vrije Universiteit Brussel, Sint Genesius Rode,2 Belgium, andCentre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, F-59000 Lille, France3

Received 2 January 1991/Accepted 27 June 1991

Macrophage-Trypanosoma cruzi interactions were studied by using a newly generated macrophage hybrid-oma cell line (2C11-12) that was selected for its capacity to produce high levels of reactive oxygen intermediates.This cell line was found to be a suitable host cell for T. cruzi, and intracellular parasitic development could beinhibited by activation with gamma interferon. When exposed to opsonized Trypanosoma brucei, Micrococcuslysodeikticus, or Legionella pneumophila, the activated macrophage cell line produces a high chemiluminescentsignal, indicating the release of reactive oxygen intermediates. Alternatively, when opsonized T. cruzi wasadded to these activated macrophages, this parasite failed to stimulate a chemiluminescent response, suggestingan impairment in the triggering of the respiratory burst.

When activated, macrophages are engaged in the respira-tory burst. Following their interaction with microorganisms,activated macrophages release reactive oxygen intermedi-ates (ROTs), which are thought to be involved in the killing ofvarious parasites (19, 25) and yeasts (40). For example,gamma interferon (IFN--y)-activated macrophages are capa-ble of impairing the intracellular multiplication of Trypano-soma cruzi (36, 37, 49), and hydrogen peroxide is capable ofdirectly killing T. cruzi (15, 31, 32). However, there is a lackof direct evidence for ROI production during macrophage-T.cruzi interaction. Since the activated 2C11-12 macrophagehybridoma cell line produces high levels of chemilumines-cence (CL) after treatment with phorbol myristate acetate oropsonized pathogens (9), we investigated the susceptibilityof this cell line to infection by T. cruzi and its ability toproduce a CL signal when interacting with this parasite. Thisresponse was compared with that generated by Trypano-soma brucei and the bacteria Micrococcus lysodeikticus andLegionella pneumophila.

T. cruzi (Y strain) was maintained in male BALB/c miceby weekly intraperitoneal inoculations. Irradiated (700-rad Xrays) Fischer rats were used to produce large quantities oftrypomastigote forms (17). T. cruzi epimastigotes weregrown in GLSH medium at 28°C (20). T. brucei (strainANTAT, l.l.E clone) was maintained by serial passage inmice and harvested as described previously (14). Lyophi-lized cells of M. lysodeikticus (ATCC 4698) were purchasedfrom Sigma Chemical Co., St. Louis, Mo. L. pneumophila(serogroup 1, Philadelphia strain 1, ATCC 33152) was culti-vated and harvested as described previously (35).The 2C11-12 cell line (10) was cultivated in RPMI 1640

medium (GIBCO, Grand Island, N.Y.) supplemented with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES;25 mM), glutamine (2 mM), fetal calf serum (10%), NCTC-135 medium (10%) (11), penicillin (100 IU/ml), and strepto-mycin (100 ,ug/ml; GIBCO) and incubated at 37°C in a 5%CO2 atmosphere. For infection experiments, 2C11-12 cellswere seeded (105 cells per ml per well) in 24-well plates

* Corresponding author.

(Nunc, Roskilde, Denmark) with sterile round coverslips(Thermanox, 13-mm diameter; Miles Scientific, Naperville,Ill.). Trypomastigotes were added to the cells in variousparasite-to-cell ratios. After 16 h, the cultures were washedwith prewarmed medium to remove all free parasites. Atdifferent time intervals (24, 48, 72, and 96 h), the cells werefixed with methanol and stained with Giemsa stain. At least200 cells per coverslip were microscopically counted todetermine the level of infection. The parasitic index wascalculated by multiplying the percentage of infected cells bythe mean number of amastigotes per infected cell. Thestatistical significance of these results was determined by thenonparametric Mann-Whitney Wilcoxon test (39).

2C11-12 cells were activated by adding mouse recombi-nant IFN--y (1, 10, or 50 U/ml) to the culture medium 24 hbefore the cells were infected with parasites. The culturemedium was removed 24 and 48 h after the initiation of theinfection and replaced with fresh medium containing IFN-y.Polymyxin B sulfate (10 U/ml; Sigma) was added to inhibitthe effect of lipopolysaccharide (LPS) traces which could bepresent in the culture medium (12, 21). For CL assays, 1042C11-12 cells, previously activated for 48 h with mouserecombinant IFN--y (50 U/ml) or LPS B from Salmonellaenteritidis (10 ,ug/ml; Difco, Detroit, Mich.), were grown insterile polypropylene vials (Nunc). When IFN--y was usedfor cell activation, polymyxin B sulfate (10 U/ml) was added(see above). The opsonized or nonopsonized T. cruzi and T.brucei were added at a ratio of 20 parasites per cell unlessotherwise indicated. When added to activated 2C11-12 cells,opsonized M. lysodeikticus cells induced a high CL signaland were routinely used as the positive control (we assumedthat they induce 100% of the CL capacity of activated2C11-12 cells). A ratio of 2,000 opsonized M. lysodeikticuscells per 2C11-12 cell was used (11). L. pneumophila wasadded at the same ratio.Each CL assay was repeated, in duplicate, at least three

times. The CL signal was recorded over a 20-min period witha Biolumat (Berthold Co., Wildbab, Germany). The maxi-mum CL emission curve (cpm x 1,000) was used to calculatethe CL percentage value.An anti-T. cruzi serum (S-1) was produced by inoculating

3303

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3304 NOTES

BALB/c mice with irradiated trypomastigotes and then with500 nonirradiated parasites (50). The absence of parasitemiaindicated that the specific immune response was protective.Another anti-T. cruzi serum (S-2) was obtained by theintraperitoneal inoculation of BALB/c mice with 40 livetrypanosomes. A very low parasitemia was observed during25 days. The mice were then killed, and the sera wereharvested, filtered, and pooled. A pool of sera (S-3) fromhealthy mice served as the control. The production of rabbitanti-T. cruzi epimastigote serum (S-4), anti-T. brucei serum(IRSb), anti-M. Iysodeikticus serum (IRSm), and anti-L.pneumophila serum (IRSp) has been previously described(9, 11, 18, 44).

T. cruzi trypomastigotes (106 per ml) were opsonized withS-1, S-2, or S-3 (final dilutions, 1/10, 1/100, and 1/1,000,respectively) for 30 min at 37°C and then washed in RPMImedium. Fibronectin (FN) opsonization was performed byincubating T. cruzi parasites (106 per ml) with human FN atvarious concentrations (10, 50, 100, 200, and 400 ,ug/ml) for45 min. Antibody opsonization was also performed withrabbit anti-FN immunoglobulin G, obtained as previouslyreported (33, 34), and used at a 1/40 final dilution in a secondlayer on previously FN-opsonized parasites. T. cruzi epi-mastigotes (106 per ml), T. brucei (107 per ml), M. lysodeik-ticus (1010 per ml), and L. pneumophila (1010 per ml) were

opsonized by incubating them with their respective rabbitspecific immune sera (final dilution, 1/100) for 30 min at 37°Cand then washing them in RPMI medium.

In some experiments, T. cruzi trypomastigotes were op-sonized by incubating them in fresh guinea pig serum (asource of complement; final dilution, 1/40) at 37°C for 30 minbefore being washed.Our results show that when parasites were incubated with

2C11-12 cells at different parasite-to-cell ratios (3/1, 10/1,50/1, 100/1, and 200/1), the parasitic index increased from243 to 2,190, in parallel with the greater number of parasites.A kinetic study of 2C11-12 cell infection by T. cruzi indicateda steady elevation of the parasitic index as a function of timeafter infection (data not shown). Microscopic observationsof stained cells showed intracellular amastigotes progres-sively enlarging. After 72 h, most of them had differentiatedinto trypomastigotes, and numerous motile trypomastigotesin the cytoplasm, free-swimming trypomastigotes, and someamastigotes were clearly observed by phase-contrast mi-croscopy. Additional kinetic studies with opsonized para-sites were also performed (using the S-1 and S-2 anti-T. cruziantisera; see above), and higher parasitic indexes wererecorded (data not shown). There was a more statisticallysignificant difference (P < 0.01) between nonopsonized andcontrol serum (S-3)-treated parasites than between nonop-

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FIG. 1. Parasitic index in relation to IFN--y activation. 2C11-12 cells were activated with various doses of IFN-,y and infected with

parasites (parasite/cell ratio, 1:3). The parasitic indexes were recorded for nonopsonized parasites (A) or for parasites opsonized with S1 (0)or S2 (A) or treated with S3 (0).

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NOTES 3305

sonized and S-1- or S-2-opsonized parasites. Parasites har-vested from the infected 2C11-12 cell culture medium wereinoculated in male BALB/c mice (4 x 105 parasites permouse), and the animals developed a normal pattern ofparasitemia, indicating that passage through the 2C11-12cells did not alter the virulence of the parasites. When2C11-12 cells were maintained in an activated state by IFN-yfor three days after infection, a dose-dependent inhibitoryeffect on parasite multiplication was observed. As shown inFig. 1, an extremely low parasitic index was recorded when50 U of IFN--y per ml was used to activate host cells. Whenantibody-opsonized parasites were used to infect the2C11-12 cells, IFN--y activation also induced a strong dose-dependent inhibitory effect on parasite multiplication. Toassess the capacity of T. cruzi to induce an oxidative burst in2C11-12 cells, CL assays using T. cruzi and other, unrelatedmicroorganisms were performed. No CL signal was re-corded when nonactivated 2C11-12 cells were exposed toopsonized or nonopsonized T. cruzi, T. brucei, M. lysodeik-ticus, or L. pneumophila. When cells activated with IFN--ywere exposed to nonopsonized microorganisms, a low andinsignificant CL signal was recorded. Similar results wereobtained with LPS (data not shown).However, a high CL response (2,320 x 103 + 1,042 x 103

cpm) was obtained when antibody-opsonized M. lysodeikti-cus interacted with IFN--y-activated 2C11-12 cells, con-firming that these cells are capable of releasing high levels ofROIs. This bacterium-induced high CL level was obtainedregularly in all experiments. Similarly, a high CL signal wasalso obtained with opsonized T. brucei and L. pneumophila.In contrast, T. cruzi opsonized with mouse anti-T. cruziserum S-1 or S-2 induced a low CL emission (Table 1 andFig. 2). FN favors the binding of T. cruzi parasites tomacrophages (34, 48), including the 2C11-12 cell line (46a).Experiments using FN-opsonized parasites were performed,and they also gave weak CL signals (4.5% + 1.1%), similarto those observed with nonopsonized parasites. This indi-cates that antibody-independent as well as antibody-depen-dent receptor/ligand systems that promote the binding of T.cruzi to 2C11-12 cells were not capable of triggering asignificant respiratory burst in 2C11-12 cells.

Since the species specificity of the Fc receptor could playa role in the induction of antibody-dependent CL signals(T. brucei, M. lysodeikticus, and L. pneumophila wereopsonized with rabbit antibodies, whereas T. cruzi wasopsonized with mouse antibodies), T. cruzi was also op-sonized with rabbit anti-FN immunoglobulin G after FNcoating. However, the CL signal remained low (Table 1),indicating that neither the rabbit nor the mouse antibodyopsonization was capable of triggering the respiratory burst.Similarly, only a very weak CL signal could be found byusing complement-opsonized T. cruzi parasites.

Since the high CL signal induced by antibody-opsonizedbacteria was obtained with a high bacterium/macrophageratio (2,000:1), antibody-opsonized parasite/macrophage ra-tios higher than the 20:1 ratio previously used were alsotested. However, even at higher ratios, the CL signal re-mained low (Table 1). Triggering macrophages with LPSalone or with IFN--y and then with LPS has no enhancing CLeffect (data not shown).

Epimastigotes of T. cruzi were also unable to trigger therespiratory burst of IFN--y-activated 2C11-12 cells, exceptwhen S-4-opsonized parasites and a high parasite/cell ratio(300:1) were used; however, the CL signal was only 30% ofthe bacterial effect (Table 1).Our results indicate that 2C11-12 cells, similar to other

TABLE 1. Percentage of CL recorded from IFN-y-activated2C11-12 cells interacting with M. lysodeikticus, L. pneumophila,

T. brucei, or T. cruzi

Triggering agent and Source of opsonin % CL + SDaparasite/cell ratio

M. lysodeikticus2,000:1 IRSm 1002,000:1 Normal rabbit serum 3.5 0.92,000: 1 b 3.8 ± 1.9

L. pneumophila2,000:1 IRSp 113.4 ± 10.12,000:1 1.2 1.2

T. brucei20:1 IRSb 106.8 ± 43,Oc20:1 Normal rabbit serum 5.5 ± 1.620:1 6.7 ± 1.4

T. cruzi (trypomastigote)20:1 4.9 ± 3.220:1 5.3 ± 3.220:1 S-i 9.2 ± 4.520:1 S-1 9.6 ± 2.920:1 S-2 8.3 ± 5.220:1 S-2 8.2 ± 3.620:1 S-3 9.9 ± 4.920:1 S-3 5.8 ± 4.620:1 FN 4.5 ± 1.120:1 Anti-FN IgGd 5.7 ± 0.620:1 Complement 2.3 ± 0.950:1 S-1 5.9 ± 2.3100:1 S-1 11.1 ± 3.4200:1 S-1 13.0 ± 5.4700:1 S-i 10.0 ± 4.9

T. cruzi (epimastigote)10:1 0.6 ± 0.4100:1 0.4 ± 0.5300:1 4.3 ± 3.110:1 S-4 1.7 ± 0.8100:1 S-4 3.7 ± 0.5300:1 S-4 30.8 ± 8.8e10:1 Normal rabbit serum 1.2 ± 0.4100:1 Normal rabbit serum 0.5 ± 0.3300:1 Normal rabbit serum 6.1 ± 0.9

a Mean ± standard deviation of at least three experiments performed induplicate. Unless otherwise indicated, P < 0.01 versus control as determinedby the Mann-Whitney Wilcoxon test.

, nonopsonized.'P < 0.05 versus control.d IgG, immunoglobulin G.e p < 0.01 versus control and versus all other values.

types of macrophages (3, 24, 27, 42), are suitable hosts for T.cruzi. 2C11-12 cell-derived amastigotes and trypomastigoteswere readily identified and found to be as infectious as thebloodstream forms. The parasitic index was shown to bedependent upon (i) parasite/cell ratio, (ii) incubation time,and (iii) opsonization. This is in agreement with other studiesusing conventional peritoneal macrophages as the targetcells (1, 5, 8, 28, 29). Without opsonization, the parasite isable to bind to and invade 2C11-12 cells. This observationsuggests that various ligand/receptor systems are involved inthe process. The recognition entities used by the parasitesare probably similar to those involved in their interactionwith normal mouse peritoneal macrophages (3). The bindingprocess can be reinforced by opsonizing T. cruzi withspecific antibodies which enhance phagocytosis of the para-site by 2C11-12 cells, as reported for other macrophages (1,22, 23, 45). The possibility of obtaining nearly 100% infectionof 2C11-12 cells with a high parasite/cell ratio (100:1 or 200:1)

VOL. 59, 1991

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FIG. 2. Representative data obtained from a CL assay. IFN--y-activated 2C11-12 cells were incubated with T. cruzi (parasite/cell ratio,20:1). Parasites either were not treated ([1) or were opsonized with S-2 at the following dilutions: 1/10 (O), 1/100 (0), or 1/1,000 (0). Opsonized(A) and nonopsonized (A) M. lysodeikticus were included as controls.

confirms the homogeneous character of 2C11-12 cells andtheir sensitivity to infection by the parasite.

Activation of 2C11-12 cells by IFN-y inhibits the develop-ment of T. cruzi in a dose-dependent manner. Similar resultswere reported by other investigators using various types ofeffector cells (2, 36, 37, 49).Taken together, our results indicate that 2C11-12 cells can

harbor the complete cycle of intracellular multiplication anddifferentiation by the parasite T. cruzi and, therefore, thatthis cell line can be adopted as an additional in vitro modelfor T. cruzi infection. This has the further advantage ofavoiding the heterogeneity of conventional peritoneal mac-rophages (7).

It has been claimed that the production of ROIs byactivated macrophages is involved in the killing of T. cruzi,(31, 32, 41). Our results clearly demonstrate the failure of T.cruzi to trigger the respiratory burst of 2C11-12 macro-phages. This finding supports the idea that ROIs are notinvolved in T. cruzi killing and that an oxygen-independentmechanism might be implicated in parasite control (reviewedin reference 30). Indeed, the cell line we used was selectedfor its ability to produce high levels of ROIs, resulting in ahigh CL signal (9-11). Accordingly, antibody-opsonized T.brucei, M. lysodeikticus, or L. pneumophila induced a veryhigh CL signal in IFN--y-activated 2C11-12 cells. Theseresults indicate that both extracellular organisms (T. bruceiand M. lysodeikticus) and intracellular organisms (L. pneu-mophila) are perfectly capable of triggering a CL emission inthe 2C11-12 cell line.

In agreement with previous reports (9, 16), optimal CL

emission by 2C11-12 cells required preincubation of the cellswith IFN-y and opsonization of the microorganisms. Appar-ently, the 2C11-12 Fc receptor needs to be occupied by theFc portion of the opsonizing antibodies for efficient stimula-tion of the respiratory burst and CL emission. Surprisingly,antibody-opsonized T. cruzi trypomastigotes are not able toprovide this Fc-mediated CL inducing signal. Even a highparasite/cell ratio for opsonization failed to induce a signifi-cant CL response. Moreover, the combination of differentactivating agents (LPS and IFN--y) previously shown to exerta synergistic influence on macrophage killing activity againstT. cruzi epimastigotes did not increase the low CL response(2). These results indicate that (i) interactions between T.cruzi and 2C11-12 cells do not result in a significant release ofROIs in spite of the capacity of this cell line to produce highlevels of ROIs and (ii) the inhibition of intracellular T. cruziparasite development observed in IFN--y-activated 2C11-12cells is therefore likely to be independent of the respiratoryburst.

In addition, the positive CL signal was obtained eitherwith extracellular (T. brucei and M. lysodeikticus) or intra-cellular (L. pneumophila) organisms, indicating that theabsence of CL signal in response to the addition of T. cruziwas specific and not linked to a defect of the cell line indealing with intracellular pathogens.These present findings corroborate a recently published

work describing the failure of opsonized or nonopsonizedtrypomastigotes, amastigotes, and epimastigotes of T. cruzito stimulate superoxide anions and hydrogen peroxide byactivated mouse peritoneal and spleen macrophages (26).

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VOL. 59, 1991

Similarly, inhibition of CL response was reported for Leish-mania major promastigotes (4, 13), Leishmania enrietti (4),and Toxoplasma gondii (47). However, in sharp contrastwith other protozoa, such as T. brucei (discussed here), thenonpathogenic Trypanosoma dionisii (43) and Trypanosomamusculi (46) are perfectly capable of triggering a respiratoryburst. This observation highlights the differences betweenthese protozoa in their interactions with the host immuneresponse based on the presence (or absence) of an intracel-lular replication phase.

Finally, the fact that IFN--y-activated 2C11-12 cells inhibitthe intracellular multiplication of T. cruzi detectable ROIproduction points to the possible role of oxygen-independentmetabolites in the cellular control of T. cruzi infection.Oxygen-independent metabolic pathways were shown to beimplicated in host control of infection by L. major (13),Leishmania donovani (38), and T. gondii (6), and an L-argi-nine-dependent mechanism could be a good candidate toexplain these results (15).

Recombinant mouse IFN-,y was kindly supplied by A. Billiau,Rega Institute, Leuven, Belgium, and T. brucei was kindly providedby N. Van Meirvenne, Institute of Tropical Medicine PrinceLeopold, Antwerp, Belgium. Human FN was a generous gift ofA. B. Schreiber, Fort Washington, Pa. We thank C. Truyens whoprovided us with anti-epimastigote immune serum. The technicalassistance of L. Bris, B. Frih, and E. Vercauteren was appreciated.

This work was supported by grant 3.9003.87 from F.R.S.M. and a

grant "Action de Recherche Concertee-U.L.B.-1991."

REFERENCES1. Alcantara, A., and Z. Brener. 1978. The in vitro interaction of

Trypanosoma cruzi bloodstream forms and mouse peritonealmacrophages. Acta Trop. 35:209-219.

2. Alcina, A., and M. Fresno. 1987. Activation by synergismbetween endotoxin and lymphokines of the mouse macrophagecell line J774 against infection by Trypanosoma cruzi. ParasiteImmunol. 9:175-186.

3. Araujo-Jorge, T. C. 1989. The biology of Trypanosoma cruzi-macrophage interaction. Mem. Inst. Oswaldo Cruz Rio 84:441-462.

4. Buchmuller-Rouiller, Y., and J. Mauel. 1987. Impairment of theoxidative metabolism of mouse peritoneal macrophages byintracellular Leishmania spp. Infect. Immun. 55:587-593.

5. Carvalho, T. U., and W. De Souza. 1986. Infectivity of amasti-gotes of Trypanosoma cruzi. Rev. Inst. Med. Trop. Sao Paulo28:205-212.

6. Catterall, J. R., C. M. Black, J. P. Leventhal, N. W. Rizk, J. S.Wachtel, and J. S. Remington. 1987. Nonoxidative microbicidalactivity in normal humal alveolar and peritoneal macrophages.Infect. Immun. 55:1635-1640.

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INFECT. IMMUN.