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(Journal of Leukocyte Biology. 2002;72:727-734.)
© 2002 by Society for Leukocyte Biology

Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen

Cinthia Stempin, Laura Giordanengo, Susana Gea and Fabio Cerbán

Inmunología, Departamento Bioquímica Clínica, Facultad Ciencias Químicas, Universidad Nacional Córdoba, Argentina

Correspondence: Fabio M. Cerban, Inmunología, Departamento Bioquímica Clínica, Facultad Ciencias Químicas, Universidad Nacional Córdoba, Pabellón, Argentina, Ala 1, Subsuelo, Ciudad Universitaria s/n, 5000, Córdoba, Argentina. E-mail: fcerban{at}bioclin.fcq.unc.edu.ar


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ABSTRACT
 
We studied the macrophage (Mo) activation pathways through Mo interaction with immunogenic Trypanosoma cruzi antigens as cruzipain (Cz) and R13. J774 cells, peritoneal and spleen Mo from normal mice, were used. Although Mo classic activation was observed in the presence of lipopolysaccharide, evaluated through nitric oxide (NO) and interleukin (IL)-12 production, Cz and R13 did not activate Mo in this way. To study the alternative pathway, we examined the arginase activity in Mo cultured with Cz. An increase of arginase activity was detected in all Mo sources assayed. An increase of IL-10 and transforming growth factor-ß in culture supernatants from Mo stimulated with Cz was observed. The study of expression of B7.1 and B7.2 in spleen Mo revealed that Cz induces preferential expression of B7.2. In vitro studies revealed that Cz stimulated J774 cells and then, infected with trypomastigotes of T. cruzi, developed a higher number of intracellular parasites than unstimulated infected Mo. Thus, Cz favors the perpetuation of T. cruzi infection. In addition, a down-regulation of inducible NO synthase was observed in J774 cells stimulated with Cz. These results suggest that Cz interaction with Mo could modulate the immune response generated against T. cruzi through the induction of a preferential metabolic pathway in Mo.

Key Words: arginase • iNOS • cytokines • immune deviation


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INTRODUCTION
 
Trypanosoma cruzi is an obligate, intracellular, protozoan parasite of mammalians and the ethiologic agent of Chagas disease, a widely distributed, debilitating infection, which constitutes a major health problem in many Latin American countries. The parasite infects a variety of host cell types, including macrophages (Mo). Intracellular replication of amastigotes is followed by the release of trypomastigotes (Tp) that can reach the bloodstream before infecting other host cells [1 ].

Cytokines play a key role in regulating parasite replication and immune response in infected hosts. The resistance of mice to infection with T. cruzi has been associated with the production of the proinflammatory cytokine as interleukin-12 (IL-12), which triggers the production of interferon-{gamma} (IFN-{gamma}) by natural killer (NK) and T cells [2 ]. IFN-{gamma} and bacterial lipopolysaccharides (LPS) have been identified as the major mediators of this classical Mo activation pathway [3 ]. The IFN-{gamma} produced in turn activates Mo to release nitric oxide (NO) by up-regulation of inducible NO synthase (iNOS) and kills the obligate intracellular amastigote forms of the parasite [4 ]. Tumor necrosis factor {alpha}, another cytokine associated with resistance to infection [5 ], acts synergistically with IFN-{gamma} to induce microbicidal activity in Mo by NO production. This metabolic pathway has a fundamental importance in murine Mo as a key defense element in various infectious diseases. In contrast, the susceptibility of mice with T. cruzi infection has been associated with the production of the anti-inflammatory cytokines such as IL-10 and transforming growth factor-ß (TGF-ß) [6 ].

Anti-inflammatory agents such as IL-4 and glucocorticoids were shown to inhibit secretion of proinflammatory cytokines by Mo. Moreover, this agent enhances the capacity for endocytosis of Mo, introducing the concept of alternative activation of Mo. Alternatively activated Mo preferentially express innate immunity receptors with broad specificity for foreign antigens such as the Mo mannose receptor, scavenger receptor, and CD163 [3 ].

Despite this enhanced capacity for phagocytosis, alternatively activated Mo do not exert enhanced killing functions toward microbes. NO production is counteracted in alternatively activated Mo by enhanced expression of arginase enzyme, competing with iNOS for L-arginine as its substrate, and leads to the products L-ornithine and urea [7 ]. The arginase activity is up-regulated by the T cell type 2-type cytokines IL-4, IL-10, and IL-13 [8 , 9 ].

In the present study, we investigated the Mo activation pathways when these cells interact with T. cruzi antigens characterized at an immunological level as cruzipain (Cz) and R13 [10 , 11 ]. We demonstrate that only Cz induces the alternative activation of Mo. This antigen up-regulated the arginase activity and the B7.2 expression and enhances the IL-10 and TGF-ß production. Thus, this alternatively activated Mo could generate a T2-type response. It is noteworthy that an increase of parasite survival was observed when Mo, previously activated with Cz, were cultured with T. cruzi Tp. These results suggest that Cz may be involved in the parasite persistence in the host.


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MATERIALS AND METHODS
 
Antigens
Cz purification
Epimastigote forms of T. cruzi Tulahuen strain were grown at 28°C in brain heart infusion medium (Becton Dickinson, France), supplemented with 0.5% tryptose, 10% fetal bovine serum (FBS), 200 mg/ml hemin, 100 U/ml penicillin, and 100 mg/ml streptomycin. Parasites were harvested at the exponential growth phase, centrifuged at 5000 g for 10 min at 4°C, and washed with phosphate-buffered saline (PBS). The parasites were resuspended in 3 vol 0.25 M sucrose and 5 mM KCl, and the irreversible protease inhibitors, 1 mM TLCK and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co., St. Louis, MO), were added. The epimastigotes were disrupted by three cycles of freezing (-20°C) and thawing (4°C), and the homogenates were centrifuged at 7000 g for 15 min at 4°C. Saturated ammonium sulfate solution, adjusted to pH 7 with NH4OH, was added at 50% saturation to the supernatant with stirring in an ice bath [12 ]. The precipitate obtained after centrifugation of this suspension was carefully dissolved and dialyzed in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4. CaCl2, MgCl2, and MnCl2 were added to a final concentration of 5 mM each. Subsequently, the samples were submitted to affinity chromatography as previously described [13 ]. The absence of enzyme activity was controlled by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing 0.1% gelatin as substrate. After the electrophoresis performed at 120 V, the gel was incubated with buffer, 50 mM sodium phosphate, pH 5.7, at 37°C overnight, and it was stained with Coomassie brillant blue R250. The samples were neither reduced nor boiled [14 ].

R13: peptide carboxy-terminal of T. cruzi ribosomal P1 or P2 proteins
This peptide (EEEDDDMGFGLFD) was purchased from Neosystem S. A. (France).

LPS
LPS from Escherichia coli serotype 0111:B4 was purchased from Sigma Chemical Co. Cz, R13, and LPS were used at a concentration of 10 µg/ml in all experiments assayed.

Cell line culture
The J774 cell line was cultured in RPMI 1640 containing 10% FBS, 4 mM L-glutamine, and 40 µg/ml gentamycin at 37°C in 95% air, and 5% CO2. J774 cells (5x105 cells/well) were stimulated with LPS, Cz, R13, or IL-4 (10 U/ml).

Spleen Mo culture
For Mo purification, normal spleen mononuclear cells obtained from BALB/c mice were incubated with 3 ml lysing buffer and then were incubated with RPMI 20% FBS in plastic dishes for 2 h at 37°C in 5% of CO2 (1x107 cells/Petri dish). Nonadherent cells were removed. The adherent cells were obtained by incubation in an ice bath and were resuspended in RPMI complete medium. These cells (1x106 cells) were stimulated with LPS, Cz, R13, or IL-4. This procedure yields >85% of Mac-1 (CO11b) + cells, as determined by fluorescein-activated cell sorter (FACS) analysis.

Resident peritoneal Mo culture
Normal resident peritoneal cells from BALB/c mice were obtained by various peritoneal washings with complete RPMI medium. Peritoneal cells (2.5x105 cells/well) were washed and cultured in complete RPMI medium with LPS, Cz, R13, or IL-4. The percentage of Mac-1+ cells was >60%.

Adherent peritoneal cells were obtained in the same form of adherent spleen cells. The adherent peritoneal cells (5x105 cells/well) were used for IL-10 determination. This procedure yields >85% of Mac-1+ cells, as determined by FACS analysis.

Determination of arginase activity
Arginase activity was measured in cell lysates as previously described [15 ]. Briefly, cells were lysed with 50 µl 0.1% Triton X-100 containing 5 µg pepstatin, 5 µg aprotinin, and 5 µg antipain as protease inhibitors. This mixture was stirred for 30 min at room temperature. To lysed cells were added 50 µl 10 mM MnCl2 and 50 mM Tris-HCl to activate the enzyme by heating for 10 min at 56°C. Arginine hydrolysis was initiated by the addition of 25 µl 0.5 M L-arginine, pH 9.7, at 37°C for 45 min. The reaction was stopped with 400 µl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 540 nm after the addition of 25 µl {alpha}-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 45 min. IL-4 (10 U/ml; PeproTech, Inc., Rocky Hill, NJ) was used as a positive control for arginase activity. The results are expressed as µg of urea.

NO assay
The production of NO was measured indirectly by assaying nitrites in the culture supernatant using the Griess reaction. Supernatants were collected at 48 h and mixed with an equal volume of Griess reagent [16 ]. Optical density measurements were averaged and converted to micromoles of nitrites per well using a standard curve of sodium nitrite.

Flow cytometric analysis
To examine the expression of B7.1, B7.2, and CD23, adherent spleen cells (2x106) were activated for 24 h with LPS, Cz, or R13. The cells were collected after 24 h and washed twice with Hanks’ balanced salt solution (HBSS) and preincubated with anti-mouse CD32/CD16 antibody for 1 h at 4°C to block immunoglobulin (Ig) nonspecific trapping through Fc receptors. Following Fc blocking, cells were incubated with labeled monoclonal antibodies (mAb) against mouse B7.1-fluorescein isothiocyanate (FITC; hamster), mouse B7.2-FITC (rat), or mouse CD23-phycoerythrin (PE; rat), conjugated for 30 min at 4°C using 1 µg antibody/106 cells. FITC- and PE-labeled antibodies were purchased from PharMingen (San Diego, CA). The cells were washed three times with HBSS, fixed in 2% formaldehyde, and stored at 4°C in the dark until analysis. This analysis was carried out in a FACS flow cytometer (Ortho Diagnostic System, Raritan, NJ) within the large population. Large cells were defined by analysis of forward-scatter. Results were analyzed using WinMDI software.

The percentage of Mac-1+ cells in adherent spleen, total or adherent peritoneal cells, was determined by FACS analysis using rat mAb against mouse Mac-1-PE (PharMingen).

Cytokine determination
Macrophagic cells were washed twice and resuspended in complete RPMI medium. Cells were cultured separately in the presence of LPS, Cz, or R13 in 24-well plates (Becton Dickinson). Culture supernatants were collected after 48 h and assayed for the presence of IL-12 p40, IL-10, and TGF-ß by a capture enzyme-linked immunosorbent assay (ELISA) using mAb pairs purchased from PharMingen. Briefly, ELISA plates (Becton Dickinson) were coated with anticytokine antibodies overnight at 4°C. Plates were washed and blocked with 10% bovine serum albumin (BSA) for 2 h at room temperature. Supernatants (100 µl) from different groups were added to the plates and incubated overnight at 4°C. Plates were washed and incubated further with biotinylated anticytokine antibody for 1 h at room temperature. After washing, avidin-peroxidase was added to the wells and incubated for an additional 30 min. Plates were washed and developed using o-phenylendiamine and H2O2 as substrate. Plates were read at 490 nm in an ELISA plate-reader (BioRad, Hercules, CA). Standard curves were generated with recombinant cytokines (PharMingen).

Trypanocidal activity
J774 cells (1.25x105 cells/well) were cultured previously with LPS, Cz, R13, IL-4, or medium alone for 24 h and then were infected with 1 x 106 T. cruzi Tp forms per well. For avoiding the Mo reinvasion of Tp, noninternalized parasites were removed by six washes with RPMI 24 h later. The growth of parasites in Mo was evaluated by counting intracellular amastigotes by immunofluorescence assays.

Coverslips were taken at different times of T. cruzi infection of Mo for immunofluorescence staining. Coverslips were washed in PBS, and the cells were fixed for 40 min in 4% buffered formalin. Coverslips were washed in PBS, and the cells were permeabilized with Triton X-100 1% for 15 min. Coverslips were washed again in PBS, and the cells were blocked with BSA 1% for 15 min. After that, cells were stained overnight with Chagas disease patient serum diluted 1:100. Coverslips were washed, and the secondary staining was then performing with FITC-anti-human IgG diluted 1:100 in BSA 1%. After 1 h, coverslips were washed and then mounted in PBS-glycerin.

Moreover, the growth of parasites in Mo was also evaluated by counting Tp released from culture supernatant at the fifth day after infection in a Newbauer chamber [5 , 17 , 18 ].

Immunoblot analysis of iNOS (NOS 2)
J774 cells (1x106) were stimulated with LPS or Cz. After 48 h, cells were washed, pelleted, and lysed for 30 min on ice in lysis buffer [1% Triton X-100, 0.5% sodium deoxicolate, 9% SDS, 0.5% dithiothreitol, 1 mM sodium ortovanadate, 10 µg PMSF, 30 µg aprotinin in PBS]. Cell debris was spun down at 13,000 g for 15 min. Aliquots of cell lysates were diluted in SDS sample buffer, boiled at 100°C for 3 min, spun down, and applied to precast 7.5% acrylamide Tris-glycine gels at 40 µg protein/lane and run at 150 V for 1 h. Prestained protein molecular mass standards (14.3–200 kDa) were run in parallel. Samples were transferred to nitrocellulose membrane (BioRad) at 100 V for 1 h. Membranes were probed using the rabbit polyclonal antibody anti-NOS 2 (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution followed by anti-rabbit peroxidase conjugated (Sigma Chemical Co.) at a 1:6000 dilution. Bands were visualized using chemiluminescence reaction.

Statistical analysis
Mann-Whitney U test was used to compare data results between nonstimulated and stimulated groups. A P value < 0.05 was considered statistically significant.


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RESULTS
 
Cz does not induce nitrite release in J774 cells
We first analyzed the NO production, as it is involved in the resistance against parasite infection. When J774 cells were cultured with LPS, an increase in NO production was observed, according to previous data [19 ] but not in the presence of T. cruzi antigens, Cz, or R13 (Fig. 1A ). We found that pretreatment with Cz but not R13 was able to decrease the NO production by Mo in response to LPS (Fig. 1B) . When J774 cells were cultured with Cz plus LPS and R13 plus LPS, neither Cz nor R13 decreased NO production induced by LPS alone (data not shown).



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  		Figure 1. (A) Production of NO by J774 cells. J774 cells were incubated with LPS, Cz, R13, or without antigens (-). After 48 h, nitrites were measured. The bars represent the mean of triplicates ± SD. Data represent one of three independent experiments with similar results. (B) J774 cells were activated with Cz or R13. After 24 h, the cells were washed, and then, LPS was added. After 24 h, nitrites were measured. Data represent one of two independent experiments with similar results (Cz plus LPS vs. LPS alone; *, P<0.05).

Induction of arginase activity in Cz-activated Mo
Most studies about Mo activation have focused on the expression or activity of iNOS. However, the substrate L-arginine is used not only by iNOS but also by arginase. In addition, T2-type cytokines shift arginine metabolism in Mo, decreasing NO and inducing ornithine and urea production by arginase and subsequent polyamine biosynthesis, leading to spermine, spermidine, and putrescine production. To investigate the possibility that Cz shifts arginine metabolism in Mo, increasing arginase activity, we incubated Mo (J774, total peritoneal or adherent spleen cells) in the presence of LPS, Cz, or R13 for 48 h. In all assays with Cz, the arginase activity was higher than control cell cultures with medium alone, which suggests that Cz modifies the Mo metabolism (Fig. 2A 2B 2C ). R13 only induce arginase activity in J774 cells (Fig. 2A) . An increase of urea levels was even observed when Mo were stimulated with LPS (Fig. 2A 2B 2C) .



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  		Figure 2. Induction of arginase activity from different sources of Mo. J774 cell line (A), total peritoneal cells (B), and adherent spleen cells (C) were stimulated for 48 h with the indicated antigen. Then, these cells were lysed, and arginase activity was determined. Data represent the mean ± SD of four independent experiments (*, P<0.05).

Our data indicate that a coinduction of iNOS and arginase was observed in J774 cells using 10 µg/ml LPS. Accordingly, LPS at a concentration lower than 10 ng/ml only induces iNOS and not arginase, and at a higher dose, it induces both enzyme activities [20 ]. IL-4 was used as a positive control for arginase activity.

Cz induces IL-10 and TGF-ß but not IL-12 secretions
Previously, it was reported that the IL-12 is produced by Mo in response to live Tp [2 ] and induces cytokine production, primarily of IFN-{gamma}, from NK and T cells. Moreover, IFN-{gamma} contributes to classic Mo activation. To investigate whether Cz is able to induce Mo IL-12 secretion, adherent spleen cells or J774 cells were cultured with LPS, Cz, or R13. A significant increment of IL-12 secretion was observed in the presence of LPS but not in the presence of Cz or R13 (Fig. 3A and 3B ). Moreover, adherent spleen cells activated with Cz plus LPS produce lower IL-12 levels than cells activated with LPS alone. However, adherent spleen cells activated with R13 plus LPS produce similar IL-12 levels to cells activated with LPS alone (data not shown).



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  		Figure 3. Induction of IL-12 p40 levels by J774 cells (A) and adherent spleen cells (B). Cells were incubated with the indicated antigen. The bars represent the mean of triplicates ± SD. Data represent one of two independent experiments with similar results (*, P<0.05).

To investigate the IL-10 production, total and adherent peritoneal cells and adherent spleen cells were stimulated with Cz, LPS, or R13. A clear increase of IL-10 secretion was observed in total (Fig. 4A ) and adherent peritoneal cell (Fig. 4C) cultures in the presence of Cz or LPS. The increment of IL-10 production induced by Cz in adherent spleen cells was lower than peritoneal cell cultures (Fig. 4B) .



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  		Figure 4. Induction of IL-10 levels in total peritoneal cells (A), adherent spleen cells (B), and adherent peritoneal cells (C). Cells were incubated with LPS, Cz, R13, or without antigens (-). The bars represent the mean of triplicates ± SD. Data represent one of two independent experiments with similar results (*, P<0.05).

In addition, TGF-ß has been shown to increase the severity of T. cruzi infection in mice [21 ]. Therefore, we studied TGF-ß production in J774 cells stimulated with Cz, LPS, or R13. A significant increase of TGF-ß secretion was observed in J774 cells only in the presence of Cz (Fig. 5 ).



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  		Figure 5. Induction of TGF-ß levels in J774 cells. These cells were incubated with LPS, Cz, R13, or without antigens (-). The bars represent the mean of triplicates ± SD. Data represent one of two independent experiments with similar results (*, P<0.05).

B7.2 is better induced than B7.1 on adherent spleen cells in the presence of Cz
To study the influence of the interaction between Cz and normal adherent spleen cells on the regulation of immunologically important surface molecules, the expression of B7.1, B7.2, and CD23 was investigated by FACS analysis. As shown in Figure 6 , adherent spleen cells incubated with Cz showed an up-regulated B7.1 and B7.2 expression, the expression of B7.2 being higher than B7.1 as compared with nonstimulated cells. No changes in the CD23 expression were observed when adherent spleen cells were cultured with Cz or R13 (data not shown). These cells stimulated with LPS expressed high levels of B7.1 and B7.2, and CD23 expression was absent (data not shown). R13 induced a lower expression of the B7.2 than LPS and Cz. These data clearly indicate that B7.1 is better induced by LPS, and B7.2 is induced to the same extent by LPS and Cz.



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  		Figure 6. Expression of B7.1 and B7.2 in adherent spleen cells. Cells were cultured in the presence of medium alone (-), LPS, Cz, or R13. After 24 h, the cells were collected, and B7.1 and B7.2 expression was determined by FACS.

Increase of T. cruzi survival in J774 cells previously cultured with Cz
To investigate the biological role of Cz within the Mo compartment in T. cruzi infection, J774 cells were previously incubated with LPS, Cz, R13, or IL-4 for 24 h, and then the cells were infected by exposure to T. cruzi Tp for 24 h. The cells were subsequently washed, fixed, and stained, and intracellular amastigotes were counted at 24, 48, 72, and 96 h by immunofluorescence assays. In addition, extracellular Tp released from cells were determined at 120 h after infection.

As expected, untreated cells were unable to restrict parasite growth and allowed survival of amastigotes. In contrast, Mo activated in vitro with LPS were able to restrict the growth of intracellular amastigotes (Fig. 7A ) and the release of extracellular Tp (Fig. 7C) . However, pretreatment of Mo with Cz resulted in uncontrolled parasite replication, as the number of parasites is higher than untreated cells. Moreover, IL-4, used as a positive control of alternative pathway activation, also promotes T. cruzi growth in a similar form to Cz (Fig. 7A and 7C) . In Figure 7B , a representative field of stimulated Mo was shown for each group. A clear increase of intracellular amastigotes in Cz- and IL-4-activated Mo was observed. Thus, these data strongly suggest that Cz favors T. cruzi survival in Mo.



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  		Figure 7. Trypanocidal activity. J774 cells (1.25x105 cells/well) were plated on 12-mm round-glass coverslips and incubated for 24 h with LPS, Cz, R13, IL-4, or medium alone (-). J774-stimulated cells were infected with 1 x 106 Tp per well. After 24 h, the cells were washed for removing the noninternalized parasites. Coverslips were taken at different times of T. cruzi infection of Mo, and the number of amastigotes/100 cells was counting for immunofluorescence staining (A). Cells were observed at a magnification of 200x. We showed a representative field for each group (B). The number of Tp was counted at the fifth day after infection in the culture supernatant (C). The bars represent the mean of triplicates ± SD. Data represent one of two independent experiments with similar results (*, P<0.05).

Cz failed to induce iNOS protein in J774 cells
To ascertain that Cz down-regulate the iNOS expression in J774 cells and thus favor the parasite intracellular replication observed in Figure 7 , the presence of iNOS protein in J774 cells cultured with LPS, Cz, or medium alone was analyzed by Western blot with anti-iNOS (NOS 2) polyclonal antiserum. Whereas the expression of iNOS was undetectable in J774 cells previously incubated with Cz, a major band was detected after incubation of these cells with LPS. The presence of iNOS, although in minor intensity, was also revealed in unstimulated, J774 Mo cells (Fig. 8 ). These results clearly show that Cz induce down-regulation of iNOS in J774 cells.



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  		Figure 8. Analysis of iNOS expression in J774 cells. These cells were treated with medium alone (-), LPS, or Cz. After 48 h, the cells were lysed and analyzed by Western blot with a polyclonal rabbit anti-mouse iNOS (NOS 2).


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DISCUSSION
 
The Mo is a pivotal cell in the host immune response to T. cruzi. During the very early stages of infection, the parasites are found within Mo, which may inhibit their replication or provide a favorable environment in which the parasites can multiply and be disseminated. Then, the interaction of Mo with T. cruzi, intracellular products or exoantigens, could be relevant in the control or persistence of infection.

In the present study, we provide experimental evidence of the influence of Cz on Mo activation pathways, showing that Cz-activated Mo trigger an alternative activation profile. In our system, we observed that Cz-activated Mo produce urea (by arginase activation), IL-10, and TGF-ß but not NO and IL-12. We also found that R13 is mainly inactive, inducing arginase activity in J774 cells but not in spleen and peritoneal Mo. This phenomenon could be explained by a different umbral of activation of J774 cells with respect to primary Mo.

The survival of parasites increases in Mo, alternatively activated by Cz. One candidate that favors the parasite replication in Mo culture is TGF-ß, induced by Cz. TGF-ß shifts arginine metabolism in Mo, decreasing NO and inducing ornithine production (by arginase) and subsequent polyamine biosynthesis [5 , 21 ]. Moreover, TGF-ß also exerts potent regulatory effects on Mo function, including suppression of IL-12 production [22 ]. Accordingly, we demonstrated that Cz decrease NO and IL-12 production from Mo stimulated by LPS.

IL-10, induced by Cz in peritoneal cells, is another cytokine that could enhance the alternative activation of Mo and favor parasite replication. It is possible that IL-10 released by Mo could deviate the immune system to the T2 response. However, we cannot discard the possible participation of B-1 cells in IL-10 production, as these cells could be Mac-1+.

Conversely, Cz promotes T. cruzi intracellular growth in a similar form to IL-4. This cytokine is a very strong inducer of arginase and the alternative pathway of Mo activation. The opposite effect is produced by IL-12, as this cytokine has been previously shown to inhibit T. cruzi replication [2 ] by inducing iNOS and elevated NO synthesis. We observed an important decrease in the number of parasites in activated Mo with LPS, which secreted high levels of IL-12. Then, the balance between anti-inflammatory (IL-10 and TGF-ß) and proinflammatory (IL-12) cytokines at the beginning of T. cruzi infection could be crucial for the installation of the parasite. IL-10 and some microbial antigens are endogenous inhibitors of regulation of IL-12 [23 , 24 ]. Our data suggest that Cz could be involved in the down-regulation of IL-12.

Most studies about the regulation of Mo NO release have focused on the expression of iNOS [25 26 27 28 ]. However, the substrate L-arginine is used not only by iNOS but also by arginase. Therefore, the regulation of L-arginine available to iNOS depends on arginase activity modulating NO production from Mo as well as the parasite survival.

Our results also indicated that Cz induces iNOS down-regulation in J774 cells, suggesting that this antigen is important to interact with antigen-presenting cells or effector-macrophagic cells by its negative consequence in some mechanisms for parasite clearance. In fact, the results showed an increment in arginase activity in Mo, previously cultured with Cz. Furthermore, our results clearly demonstrate that J774 cells incubated with Cz possess minor ability to eliminate intracellular parasites than those incubated with LPS. This phenomenon would also be associated with the down-regulation of iNOS and no induction of NO by Cz, and these two parameters were clearly induced by LPS. Therefore, Cz-activated Mo are functionally different from LPS-activated Mo.

The surface molecules of Mo may also influence the profile of immune response. The alternative activation produces the expression in Mo cell surface of important molecules that regulate the immune response. For this reason, we investigated the B7.1 and B7.2 expression in adherent spleen cells stimulated with Cz. An up-regulation mainly of B7.2 in Cz-activated, adherent spleen cells was demonstrated by FACS analysis. Some studies have suggested that B7.1 and B7.2 costimulation leads to T cell differentiation along different pathways [29 ]. It was proposed that B7.2-mediated costimulation induces IL-4 production in naive T cells by pushing them toward T helper cell type 2 (Th2) development, whereas B7.1 provides a more neutral signal, resulting in an elevated, IL-2 production [30 ]. However, other studies using B7.1 and B7.2 transfectants have not found any differences in the cytokine production following T cell costimulation [31 ]. Thus, the differences in B7.1 and B7.2 activity may reflect intrinsic difference in the molecules or differential expression on individual cell types at distinct times during an immune response [32 ]. Recently, the B7.1/B7.2 costimulation concept has been revisited, as the B7 superfamily is expanding, and there is an increment of complexity in costimulation signals regulating the induction of Th1 or Th2 cells [33 ]. However, our results may be indicating that a higher increment of B7.2 than B7.1 in adherent spleen cells would be associated with the persistence of T. cruzi in Mo.

On the other hand, arginase activity induced by parasites such as Schistosoma, Leishmania, and T. brucei has been reported to favor their survival in the host and to influence the pathogenesis of the disease [34 35 36 37 38 39 ]. In leishmaniasis, it was reported that the arginase activity is essential for parasite growth through polyamine synthesis from L-ornithine [35 ]. Moreover, arginase expression and increase in circulating polyamine levels were found in mice infected with Schistosoma mansoni [36 ]. In Brugia malayi infection, alternatively activated Mo and Th2 cell differentiation were also induced [38 , 39 ].

The present work extends these observations to T. cruzi infection. Our results suggest that agents that alternatively activate Mo could promote T. cruzi intracellular growth. It is interesting that this work is the first to identify a parasite antigen able to trigger alternative Mo activation in vitro. This Mo-activation profile associated with a functional ability of these cells to promote the intracellular growth of the parasite might be an interesting evasion mechanism of the innate immune response used by T. cruzi to favor its installation in the host.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), SECYT-UNC, Fundación Banco Nación, and Agencia Córdoba Ciencia. C. S. and L. G. thank Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECYT-UNC) and CONICET, respectively, for the fellowships granted. F. C. and S. G. are Research Career Investigators from CONICET.

We thank M. P. Aoki for help with immunofluorescence assays.

Received December 28, 2001; revised April 15, 2002; accepted May 21, 2002.


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