INTRODUCTION

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Trypanosoma cruzi is an obligate intracellular protozoan parasite of mammals and the etiologic agent of Chagas' disease. The parasite infects a variety of host cell types, including macrophages; intracellular replication as amastigotes is followed by the release of trypomastigotes that can reach the bloodstream before infecting other host cells. The acute phase is characterized by a large increase in parasite replication, and trypomastigotes are observed in the blood of infected mice. After control of the acute phase in immunocompetent mice, the infection turns into a chronic phase (starting around day 21 postinfection [p.i.]) where parasites are no longer detectable by light microscopy in the bloodstream but form inflammatory nests in various tissues, a process associated with chagasic pathology, in which antiparasite cytotoxic T lymphocytes or autoimmune mechanisms may play a role (14, 21, 29).

Several cell subsets of both the innate and the specific immune system were reported to be required for survival during the acute phase of the infection in murine T. cruzi infection, such as NK cells (4), CD4⁺ (20, 27) and CD8⁺ T cells (20, 26, 28), and B cells (11). Two essential mediators of resistance to T. cruzi have been found to be gamma interferon (IFN-) (1, 10, 12, 18, 23, 31) and nitric oxide (NO) (10, 17, 33), which has direct strong cytotoxic effects on T. cruzi (6, 33). IFN- is thought to be the most important inducer of the inducible NO synthase (iNOS) for increased NO production by macrophages (2, 8, 9) and thus essential for mediation of NO-dependent parasite control during acute infection.

There have been several reports that effector cell pathways essential for T. cruzi control during acute infection are dispensable during chronic infection (4, 16, 26). In this study, we found that this also applies for NO production during the chronic and also during the late acute phase; both phases are characterized by control through the adaptive immune system and not through NK cells (4). We show that there is only a narrow time window during acute infection where NO is indispensable.

(This study formed part of a Ph.D. thesis by M.S. at the Faculty of Biology, University of Hamburg.)

	MATERIALS AND METHODS

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Mice and parasites. Six- to eight-week-old IFN- knockout (KO) BALB/c mice and wild-type BALB/c littermates as well as C57BL/6 mice maintained under specific-pathogen-free conditions were used for the experiments.

Treatment with L-NIL and AG and determination of nitrite. L-N⁶-(1-iminoethyl)-lysine (L-NIL) (Alexis, Grünwald, Federal Republic of Germany) and aminoguanidine (AG) (Sigma, Munich, Federal Republic of Germany) were dissolved at 3 mM and 90 mM, respectively, in drinking water, which was the only source for fluid intake of mice during the duration of blockade experiments. That the mice actually continued drinking normally was verified by recording of their weight twice a week. Production of NO in serum was assessed by determination of NO₂ and NO₃ in mouse sera (Griess reaction) as described elsewhere (19).

Determination of IFN- in serum. IFN- concentrations in sera of mice were determined by specific two-site enzyme-linked immunosorbent assays using standard protocols. The antibody pairs for capture and detection (biotinylated) were purchased from Pharmingen (Hamburg, Federal Republic of Germany) in the combination recommended. Recombinant IFN- (Pharmingen) was used as a standard. Enzyme-linked immunosorbent assays were developed after incubation with streptavidin-peroxidase complex (1:10,000; Boehringer, Mannheim, Federal Republic of Germany), using 3,5,3',5'-tetramethylbenzidine as substrate (dissolved [6 mg/ml] in dimethyl sulfoxide; Roth, Karlsruhe, Federal Republic of Germany). Sensitivity was 5 pg/ml.

PCR. DNA was purified from blood samples using the QIAamp blood mini DNA kit from Qiagen (Hilden, Federal Republic of Germany). Two oligonucleotides (T1, 5' GAC GGC AAG AAC GCC AAG GCA 3'; T2, 5' TCA CGC GCT CTC CGG CAC GTT GTC 3') derived from T. cruzi cDNA sequence coding for a 24-kDa protein were used as described elsewhere (15).

Histology. Previously described protocols for histology were followed (5). Complete hearts and complete femoral muscles from each animal were fixed in 70% ethanol and embedded in paraffin using standard methods. Sections (4 µm thick) were prepared from each tissue. Tissue sections were first stained in hematoxylin (Merck, Darmstadt, Germany) for 20 min and rinsed in tap water for 30 min, and this was followed by counterstaining with 1% (wt/vol) eosin (Merck) for 3 min. The tissue sections were analyzed with a light microscope at a magnification of ×1,000. For quantification of amastigote pseudocysts, two sections from each tissue of each animal were examined by an investigator who was not aware if the mice had received NO-blocking treatment before.

	RESULTS

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NO is produced early in infection during trypomastigote parasitemia. When infected with sublethal doses of 10 or 100 trypomastigotes, BALB/c mice developed peak parasitemia within 15 to 20 days p.i. (Fig. 1), consistent with the acute phase of T. cruzi infection. A high amount of IFN- was observed in the serum from 10 days p.i., and it was followed by the appearance of NO (Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Parasitemia, serum NO, and serum IFN- of BALB/c mice infected with the T. cruzi Tulahuen strain. The mice were infected with 100 blood-derived trypomastigotes. The data are representative of five independent experiments using four animals per group and experiment. The arithmetic means ± standard deviations (error bars) are given. Interestingly, IFN- remains highly elevated during the chronic phase of infection. Consistent results were obtained when the inoculation dose was 10 trypomastigotes per mouse.

View larger version (93K): [in this window] [in a new window]   FIG. 2.   Detection of T. cruzi DNA in the blood of acute and chronically infected mice. Lanes 1 and 2, 10 and 100 µl of blood from uninfected mice, respectively; lanes 3 and 4, 10 and 100 µl of blood from chronically infected mice at day 90 p.i., respectively; lanes 5 and 6, 10 and 100 µl of blood from acutely infected mice at day 14 p.i. The data are representative of four independent experiments. HMW and LMW, high- and low-molecular-weight markers, respectively.

NO is essential for control of parasitemia and for survival in the early acute phase of infection. Administration of AG or L-NIL starting 1 day before the infection led to a high parasitemia (Fig. 3) and to 100% mortality of mice by day 17 p.i. (Fig. 4). The efficacies of AG and L-NIL were equivalent in this trial. When NO production was blocked by AG from day 7 p.i., the development of high parasitemia was delayed by 8 days (i.e., for the time span treatment was delayed; i.e., day 1 versus day 7 [Fig. 3A]), but mortality was still 100% (Fig. 4A). NO synthase inhibition from day 13 p.i. by AG resulted in only 50% mortality (Fig. 4A); in this group, those animals that died developed high parasitemia before death while those animals that survived eventually controlled their parasitemia (Fig. 3A).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Parasitemia of BALB/c mice infected with 100 blood-derived trypomastigotes of the Tulahuen strain. The treatment with AG (A) or L-NIL (B) was done as indicated in the figure. (A) For each time point, the arithmetic means ± standard deviations (error bars) for groups of four to six animals are given. The data are representative of three independent experiments (except for treatment from day 20 to 30 p.i., which was done two times). The values for the group receiving AG from days 13 to 30 p.i. are split at day 20 into the resistant (solid lines) and susceptible (dotted lines) subgroups. (B) For each time point, the means ± standard deviations (error bars) for groups of eight animals are given.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   NO mediates survival of mice only during a small window in the early phase of the acute infection with T. cruzi Tulahuen. BALB/c mice were infected with 100 blood-derived trypomastigotes, and the treatment with AG (A) or L-NIL (B) in the drinking water was started as indicated. For each time point, the arithmetic means for four to six (A) or eight (B) animals are given. The data in panel A are representative of three independent experiments (except for treatment from day 20 to 30 p.i., which was done two times); in panel B, one experiment per group was performed. Note that in all animal groups, baseline survival at the beginning of the experiment was 100%. The lines are drawn not overlapping for clarity.

NO is dispensable for survival as well as blood and tissue parasite burden in the late acute phase of infection. The above results suggested that NO is indispensable at a time during early T. cruzi infection (day 1 to 20) when the adaptive immune system is not yet effective but that it may become dispensable at later times. To gain further evidence for the existence of such a time window where NO is essential, NO synthase activity was blocked during the time when the adaptive immune system in this model is known to be involved in the control of the parasites (26) but the innate, NK-derived IFN--dependent defense mechanisms are no longer essential (4), i.e., from day 20 p.i. on. Note that at this time, parasitemia is not yet under control since blood trypomastigote levels do not drop before 22 days p.i. (Fig. 1). NO in mouse serum has similar kinetics (Fig. 5).

View larger version (24K): [in this window] [in a new window]   FIG. 5.   NO in the serum of BALB/c mice infected with 100 blood-derived trypomastigotes of the Tulahuen strain. The treatment with AG or L-NIL was started as indicated in the figure. For each time point, the arithmetic mean ± standard deviation (error bar) for four, six, or eight animals is given. The data are representative of three independent experiments (except for treatment from days 20 to 30 p.i. which was done two times, and with L-NIL, which was done once).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Quantification of amastigote pseudocysts from hearts and skeletal muscles of BALB/c mice infected with 100 blood-derived trypomastigotes. Tissues were subjected to histological analysis as described in Materials and Methods. Amastigote pseudocysts were counted with a light microscope at a magnification of ×1,000. Two sections of different parts of each tissue from each animal were completely analyzed. Thirty-two animals were used for this experiment. At each time point from day 7 until day 42 p.i., four animals were sacrificed and tissues were analyzed. The arithmetic means + standard deviations (error bars) are given. *, eight animals which had been infected in the same experimental series were given L-NIL from days 20 to 42 p.i. and were sacrificed at day 42 p.i.

NO inhibition does not affect the chronic phase of T. cruzi infection. Inhibition of NO production by either AG or L-NIL during the chronic phase (days 60 to 120 p.i.) did not lead to mortality or reappearance of parasitemia (Table 1; Fig. 3B).

	DISCUSSION

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The results generated in this study allow a more precise view of the trypanocidal role of NO in the control of murine T. cruzi infection. The essentiality of iNOS induction for parasite control has been demonstrated using enzyme blockers (17) or iNOS KO mice (10); the latter mice had much higher parasite loads in blood and tissue, which were associated with extreme susceptibility (100% mortality after application of only 15 blood forms/mouse [10]). NO activity was linked to trypanocidal action of macrophages given that IFN- rendered infected macrophages capable of inhibiting intracellular replication of T. cruzi in vitro by a mechanism that involves the production of NO (8). Recently, more molecular pathways of NO action on the parasites have been elucidated (30, 32). By depletion studies as well as gene KO (10, 17), IFN- as well as a variety of cells that produce this cytokine (NK [4], [22], CD4⁺ [20, 27] and CD8⁺ T cells [26, 28]) was shown to be essential for protection against T. cruzi. However, the consensus of these studies is that depletion of any of these cell subsets affects only the early, acute phase of infection while it does not lead to a reversion of parasitemia if depletion is initiated after blood stage trypomastigotes have disappeared from the blood (3, 12, 26).

We show here that this also applies to NO-mediated parasite control: while there was a strong exacerbation of parasitemia, with 100% mortality in animals treated from the beginning of infection with NO-synthase inhibitor L-NIL or AG (Table 1; Fig. 3), it was impossible to render animals parasitemic by long-term (60 days) AG or L-NIL application if started at day 60 p.i. (Table 1; Fig. 3). In addition, it was impossible to increase parasite loads both in blood and in tissue by NO blockade after day 20 p.i.; in contrast, tissue and blood parasite levels declined in the presence of either NOS inhibitor (Fig. 3).

This is in contrast to murine infection with another kinetoplastid, Leishmania major. Here it was shown that in chronic infection (123 days p.i.), L-NIL application led to a reactivation of cutaneous lesions in C57BL/6 mice which became significant after only 17 days of treatment (24). In our own laboratory, we extended these findings when treating C57BL/6 mice with L-NIL towards the end of the acute phase of L. major infection, i.e., at a time when the footpad swelling is already declining: we observed a fast reactivation of cutaneous lesions in these mice which became significant as early as 10 days following the initiation of L-NIL administration (data not shown). This is in strong contrast to the further decline of T. cruzi parasite loads in blood and tissue at the end of the acute phase under L-NIL treatment (days 20 to 42 p.i.).

Thus, comparison of these two infections strongly suggests that fundamental differences in the control of chronic T. cruzi and L. major infections exist. A potential reason for this discrepancy is that L. major resides in macrophages which control infection by NO whereas T. cruzi also invades other types of cells which do not express iNOS.

A main novel finding of our study is that NO is not even essential for the whole period of acute infection: blood parasite levels, in our experimental system, did not decline before day 22 p.i. In untreated mice, the levels of NO reaction products in the serum fell concomitantly with blood parasite loads (Fig. 1), and NO synthase inhibition during that period did not block clearance of parasites from blood (Fig. 3). Consistently, NO synthase inhibition, if initiated from day 20 p.i., did not affect survival, in contrast to earlier inhibition, i.e., from day 1 or day 7 p.i. (Fig. 4). When treatment was started at day 13 p.i., mortality was only 50% (Fig. 4). This reflects a decreasing need for NO in parasitemia control from day 7 p.i. (essential) to day 20 p.i. (dispensable). Importantly, tissue amastigotes, after day 20 p.i., are also controlled by mechanisms other than NO, since L-NIL treatment did not prevent the reduction of pseudocysts in hearts and skeletal muscles, so that equivalent low levels of amastigotes were found both in the presence and in the absence of L-NIL at day 42 p.i. (Fig. 6).

The question of whether AG is as potent an NO synthesis blocker as is L-NIL may be raised. AG was shown to be indeed inferior to L-NIL on a molar ratio in vitro (25). However, these authors did not compare the two inhibitors in vivo, in contrast to us, and we adapted the molarity of the inhibitors for our in vivo study to equipotent levels. We always got consistent results with AG and L-NIL (Table 1; Fig. 3 and 4).

Our data fit very well to earlier observations that IFN- itself is not needed during the whole acute phase of T. cruzi infection: IFN- neutralization did not alter the parasite load of mice if applied later than day 11 p.i. (4). It was also shown that this early IFN- production is dependent on the presence of NK cells in T. cruzi infection (4). In contrast, if T. cruzi-infected mice are depleted of CD8⁺ T cells, parasitemia is unaltered compared to that in control infected mice until day 21 p.i., but thereafter, blood parasite loads rise quickly and mice die from infection (26). Mice lacking CD8⁺ T cells through gene KO also die around day 28 (i.e., later than in our study when NO production was blocked) despite elevated IFN- (28).

Taking all these results into account, it appears that NO is essential for the parasite control during the first 2 weeks of infection, i.e., a time when NK cells are known to control the parasite load through IFN- production (4). It is, however, dispensable thereafter in late acute infection, i.e., during the period when blood parasite levels decrease under the control of CD8⁺ T cells (26). Thus, the synopsis of the results suggests a cascade of events where NK cells, by their production of IFN-, activate effector cells, most likely macrophages, to induce NOS2 and to exert control of early parasite replication. After 2 weeks p.i., this mechanism is gradually replaced by the action of the adaptive immune system in which CD8⁺ T cells (20, 26, 28), and apparently CD4⁺ T cells (20, 27) and B cells (11) producing neutralizing antibodies, act in concert to further limit parasite replication in an NO-independent way.