Parasitol Latinoam 59: 3 - 7, 2004

ARTÍCULO ORIGINAL

Effect of Trypanosoma lewisi infection on the Toxoplasma gondii multiplication in white rat peritoneal macrophages

 

MISAEL CHINCHILLA *, OLGA M. GUERRERO * y ALFREDO CASTRO *

* Centro de Investigación en Enfermedades Tropicales, CIET, Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica. San José, Costa Rica, América Central.

Dirección para correspondencia

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ABSTRACT

Peritoneal macrophages (PM) from normal Wistar rats were treated in vitro with peritoneal supernatant or sera, obtained from rats infected with 10⁶ Trypanosoma lewisi trypomastigotes before the infection with Toxoplasma gondii tachyzoites. In this experimental model, Toxoplasma multiplication in PM was increased, as compared to macrophages treated with supernatant or sera from control rats not infected with T. lewisi. This effect was observed only if the active supernatant or sera came from rats infected with the T. lewisi 3 to 6 d before Toxoplasma inoculation. Furthermore, immunosuppressive activity was only detectable after at least 24 h incubation with the supernatant or sera. These results are in accordance with our in vivo previous studies which demonstrated a clear immunosuppressive effect of T. lewisi during T.gondii infection of the remarkably resistant Wistar rats.

Key words: Immunosuppression, Toxoplasma gondii, Trypanosoma lewisi, peritoneal macrophages.

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INTRODUCTION

Immunosuppressive effects in parasitic infections allow greater parasite multiplication in the final host, causing important pathology development. That is the case of filariosis, schistosomosis, trichinosis and others diseases^1,2. More specifically in Protozoa, such as malaria infection³ and trypanosomosis⁴ immunosuppression has been reported. It has been demonstrated that the "brucei" group species exert a significant immune inhibition effect in animals and human beings^5-8. A similar phenomenon has been observed in Trypanosoma musculi infections⁹.

Immunosuppression can be achieved by treatment with inflammatory inhibitors such as corticosteroids¹⁰, but this process is usually very slow. However in a recent model we have demonstrated that T. lewisi infections make the white rat, an animal remarkably resistant to T. gondii¹¹, as susceptible as the white mouse¹². In this case the effect is produced only after 4 days of T. lewisi infection¹³. On the other hand another study showed that macrophages from T. lewisi infected rats do not present any increased multiplication of T. gondii in vitro¹⁴.

The immunosuppresive effect on rat normal macrophages caused by immune supernatant and sera from T. lewisi infected rats is the aim of this study.

MATERIAL AND METHODS

Wistar white rats (100-150 g body weight) were obtained from Animal Care Laboratories, Hospital México, and San José, Costa Rica. The animals were infected with T. lewisi and served as a source of peritoneal macrophages. NGP white mice (20-25 g) were infected with T. gondii to obtain tachyzoites.

Tachyzoites of the RH strain of T. gondii obtained from infected mice and T. lewisi blood forms from a strain isolated from a costarican Rattus rattus were used throughout these studies.

Experimental model

Non-infected white rats were injected intraperitoneally with 3 ml of 0.5% sodium caseinate in 0.85% saline solution. Five days later, peritoneal macrophages were withdrawn with Minimal Essential Medium plus 10% heat-inactivated fetal calf serum and antibiotics (MEM-FCS). The cells suspended in 0.3 ml were cultured in cover slips (22 x 22 mm) incubated in 5% CO₂ at 37°C for 24 h. The cells were washed with MEM-FCS and treated with peritoneal supernatant or sera coming from T. lewisi infected rats. After 24 h incubation, the cells were infected with 10⁶ tachyzoites of T. gondii. After one and 24 h incubation periods, the cells were washed, air dried, fixed in methanol and stained with Giemsa dye for 8 min. To determine the infection rate the number of intracellular tachyzoites were counted as previously described¹⁵.

Before collection of the intraperitoneal components used in this model, rats were inoculated with 10⁶ T. lewisi trypomastigotes. Depending on the experiment, five to twelve hours later peritoneal exudate was withdrawn as before, and centrifuged to separate the sedimented cells from the supernatant which were used together with the immune serum from T. lewisi infected animals.

Statistical significance was determined by using the paired T test.

RESULTS

Macrophages from non-infected rats (normal macrophages: NM) treated with supernatant from T. lewisi infected animals (TS) showed an increased multiplication of intracellular Toxoplasma tachyzoites, compared to that of NM treated with supernatant from non infected rats (normal supernatant: NS). None of the sedimented cells increased the number of tachyzoites in the phagocytes (Figure 1).

Figure 1. Multiplication rte of T. gondii in peritoneal macrophages treated with different peritoneal exudates componenents. N sed = sediment from non infected rats. T sed = sediment from T. lewisi infected rats. N S = supernatant from non infected rats. T S = supernatant from T. lewisi infected rats.

When NM were treated with supernatant from rats infected with T. lewisi 12 days before, there was no evidence of increasing T. gondii multiplication as compared with the results observed in the corresponding control (Figure 2).

Figure 2. Multiplication rats of T. gondii in peritoneal macrophages treated 5 or 12 d before with different supernatant or sediments. N sed = sediment from non infected rats. T sed 5 = sediment from non infected rats 5 d before with T. lewisi. T sed 12 = sediment from non infected rats 12 d before with T. lewisi. N S = supernatant from non infected rats. NS 5 = supernatant from rats infected 5 d before with T. lewisi. NS 12 = supernatant from rats infected 12 d before with T. lewisi.

Based on these results, the time of T. lewisi infection necessary to produce the immuno-suppressive activity was investigated. Our results showed that the immunosuppressive effect started 3 days after the infection and disappeared 9 days later ( Figure 3). These data also demonstrate that the best effect is found in rats that have been infected with T. lewisi for 3 to 6 days before.

Figure 3. Multiplication rate of T. gondii in peritoneal macrophages treated with "active" supernatant at different periods of time. N = normal. d= days

In order to determine the optimal time of contact necessary for the immunosuppressive manifestation in vitro, NM were treated with TS for 1, 2, 4 or 24 h and then infected with T. gondii. A higher multiplication rate was found after 24 h contact (Figure 4).

Figure 4. Multiplication rate of T. gondii in peritoneal macrophages treated with supernatant for different period of time (h). N= normal h= hours of contact

To determine whether the sera from T. lewisi infected rats produced the same effect or it was exclusively found in peritoneal supernatant, NM were treated for 24 h with sera from rats collected after 1, 2, 3, 4, 5 and 6 days of infection and then inoculated with Toxoplasma tachyzoites. An increased multiplication was observed in phagocytic cells treated with sera from animals infected with T. lewisi 3 to 6 days before (Figure 5). NM treated with sera from non-infected animals showed a normal T. gondii multiplication.

Figure 5. Multiplication rate of T.gondii in peritoneal macrophages treated with sera from T.lewisi infected rats. N= normal d= days of infection

DISCUSSION

Immunosuppressive activity in intracelullar organisms is a process where lymphocytes, their products, as well as phagocytic cells play an important role^16,17. In T. musculi, for example, suppression of the immune defence probably depends on a direct interference on B cells². These studies show, partially, an effect that could be considered similar, since the sera from T. lewisi infected rats induced an increase of Toxoplasma multiplication inside the macrophages (Figure 5). Suppression of immunity by IL-4 and IL-10 produced by spleen cells has been observed in Trypanosoma congolense¹⁸ and there are similar studies with lymphokines for other african trypanosome infections^8,19,20 and for T. cruzi ⁴. In addition, studies dealing with gamma interferon show an apparently increase of T. gondii multiplication in macrophages²¹. These data agree with the results (Figures 1 and 2) because the suppressive effect was found in the peritoneal exudate supernatant where the lymphocyte products are elicited.

As in previous studies with kidney cells infected with T. gondii¹⁵ the optimal contact time to demonstrate the immune effect was 24 h (Figure 4).

Gomes et al²² reported that the glycoinositol phospholipid blocked TCD4+ y CD8+ T cells proliferation in T. cruzi infections which could affect the immunity in human infections with Chagas disease. Furthermore, a glycoprotein (AGG10) of this parasite inhibits the expression of IL-2 receptors chains, indicating a role for this glycoprotein in the immunosuppression as observed in the acute phase of this disease²³. Studies in progress dealing with T. lewisi lysates attempt to find out if trypanosome membrane components are important in the model. In this respect we have observed that the immune effect is found in supernatants from rats infected as early as 3 days before (Figure 3). Since the animals are inoculated with 10⁶ parasites, and the multiplication rate is very fast, membrane components could play an important role in the immunosuppression model reported in this paper.

 

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Acknowledgements: This study was supported by a grant Project 803-A2-045 of the Vicerrectoría de Investigación of the Universidad de Costa Rica.

Address for correspondence: Dr. Misael Chinchilla. Facultad de Microbiología. Universidad de Costa Rica. San José, Costa Rica. América Central. Phone 207-42-77; FAX: 506-225-23-74.