Immunology 1992 77 95-98
Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection M. C. LEITE-DE-MORAES,*t M. HONTEBEYRIE-JOSKOWICZ,* M. DARDENNEI & W. SAVINOt Unite d'Immunoparasitologie, Institut Pasteur, Paris, France, tDepartment of Immunology, Institute Oswaldo Cruz, Rio de Janeiro, Brazil and jHopital Necker, CNRS URA 1461, Paris, France
Acceptedfor publication 20 April 1992
SUMMARY Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcRafl and TcRyb cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4IOwCD8- and CD4-CD8IOw cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection. The data presented here demonstrate that total thymocyte numbers as well as the percentages of thymic lymphocyte subsets, as CD4+CD8+, CD4+CD8-, CD4-CD8+, CD4-CD8-, CD3IOW, CD3high, TcRo4# and yb, severely altered in the acute phase of infection, returned to normal values in chronically infected animals, probably due to the increase in the percentage of thymocyte precursors, such as CD4IOWCD8- or CD4-CD8Iow cells, together with the increase in the IL-2R+ and cycling cells that occurred by the late phase of acute infection.
INTRODUCTION Chagas' disease is a parasitic affection caused by the flagellate protozoan Trypanosoma cruzi. During the course of the disease, one can distinguish an acute phase, with easy detection of the parasite in the blood and target tissues, that contrasts with the chronic phase, when parasitaemia is no longer detectable and T. cruzi is virtually absent from the tissues, in spite of severe associated pathologies (reviewed in ref. 1). In the last few years cumulative experimental data, essentially obtained in the mouse model of T. cruzi infection, have clearly demonstrated immunological imbalances in acutely infected animals. Recently, a series of experiments definitely demonstrated that T lymphocytes, and more particularly CD4+ cells, are involved in the pathophysiology of the disease.2A In fact, these recent findings are in keeping with pioneer works showing that neonatally thymectomized as well as athymic (nu/ nu) mice exhibit enhanced severity of the disease.5 6 Considering the importance of T-cell-mediated mechanisms in Chagas' disease, we recently performed a number of experiments in order to better understand whether the thymus is also affected in T. cruzi infection. These studies, carried out in acutely infected animals, revealed a severe thymic atrophy with significant changes in the lymphoid compartment of the organ.7'8 It thus seemed worthwhile to investigate whether these alterations persisted during the course of a chronic infection.
Mice and parasites Six- to 8-week-old male C3H/HeJ mice were obtained from Pasteur Institute facilities (Paris, France). Each experimental group consisted of four to eight animals. The CL strain of T. cruzi was maintained by passage in C3H/HeJ mice. Animals were infected with 105 bloodstream-derived trypomastigote forms. They were killed at different times post-infection. The acute phase was determined following the parasitaemia as described by Brener,9 while the chronic phase was defined by negative parasitaemia and classical increased levels of circulating anti- T. cruzi immunoglobulins (IgG) detected by ELISA (i.e. 100:1 chronic versus acute titre ratio, not shown), as described elsewhere. '°
Correspondence: M. C. Leite-de-Moraes, Unite d'Immunoparasitologie, Institut Pasteur, 25, rue du Dr. Roux, 75724, Paris, Cedex 15, France.
Antibodies Fluoresceinated rat monoclonal antibodies (mAb) anti-Thy-1.2 (clone 30.H.12), anti-CD8 (clone YTS 169.4), as well as
MATERIALS AND METHODS
95
96
M. C. Leite-de-Moraes et al.
phycoerythrin-coupled anti-CD4 mAb (clone GK 1.5), were purchased from Becton-Dickinson (Grenoble, France). The hamster anti-CD3 mAb (clone 145-2C1 1), the anti-TcRai mAb (clone H57-597), the anti-TcRyb mAb (clone GT3) and the rat anti-IL-2R (clone PC61) are currently being expanded in our laboratory, and were used as culture supernatants or ascitic fluids.
O T. cruzi
0
E
30-
8 20-T
Immunofluorescence Thymi were removed and gently homogenized to yield a singlecell suspension in Hanks' solution containing 2% foetal calf serum (FCS) and 0 1% NaN3. Cell viability was determined by the Trypan blue exclusion test. Viable thymocytes (106 cells/ tube) were incubated for 30 min at 4C with optimal concentrations of each specific antibody. Washing was performed twice with Hanks' solution. For indirect immunofluorescence cells were subjected to the second antibody. They were then washed and fluorescence analysis were carried out using a flow cytometry analyser (FACScan; Becton-Dickinson & Co., Mountain View, CA). Detection of cellular DNA content Thymic cell suspensions from normal or infected mice were analysed by flow cytometry for their cellular DNA content after staining with the Vindelov lowsalt propidium-iodide (PI) solution, as described elsewhere." The number of cycling cells was calculated on the basis of a DNA content higher than that of resting diploid cells.
RESULTS Thymocyte recovery in T. cruzi chronically infected mice In order to evaluate the thymocyte phenotypic alterations in T. cruzi-infected mice, we carried out a longer kinetic analysis starting at the acute and continuing until the chronic phase. Confirming our previous results,7'8 mice infected with 105 parasites and analysed at the acute phase (0-5 and 1 month after infection) underwent severe thymic atrophy. Conversely, in chronically infected animals, at a stage where parasitaemia was absent and the level of circulating immunoglobulins increased, there was a progressive recovery of thymus weight and thymocyte numbers (Fig. 1) compared to age-matched controls. Besides the recovery of thymocyte numbers in the chronic infection, the percentages of CD4/CD8-defined thymocyte subsets also returned to normal values (Fig. 2). It is interesting to note that the ratio of single positive cells bearing low densities of CD4 or CD8 molecules was higher in the late phase of acute infection. Since these cells are precluded as intrathymic precursors of double positive thymocytes, they might be related to the thymus recovery observed in the chronic phase. Actually, the analysis of cycling cells as well as of the thymocytes expressing the IL-2 receptor revealed a significant increase in both parameters, during the acute phase (Fig. 3). Lastly, we evaluated the CD3-Ti complex expression in acute and chronically infected mice. Again, the decrease of CD3lOw, paralleled by an increase in the CD3high thymocyte percentage, associated with an enhancement of the percentage of cells bearing the TcRcxa or yb observed during the acute phase, was restored to normal values in chronically infected animals (Fig. 4).
E
0 05
3 Months after infection
5
Figure 1. Number of viable thymocytes in C3H/HeJ mice infected by T. cruzi with the dose of 105 parasites/mouse at different times after infection. The data shown represent the mean + SEM of five to eight mice individually tested. **P
Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection M. C. LEITE-DE-MORAES,*t M. HONTEBEYRIE-JOSKOWICZ,* M. DARDENNEI & W. SAVINOt Unite d'Immunoparasitologie, Institut Pasteur, Paris, France, tDepartment of Immunology, Institute Oswaldo Cruz, Rio de Janeiro, Brazil and jHopital Necker, CNRS URA 1461, Paris, France
Acceptedfor publication 20 April 1992
SUMMARY Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcRafl and TcRyb cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4IOwCD8- and CD4-CD8IOw cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection. The data presented here demonstrate that total thymocyte numbers as well as the percentages of thymic lymphocyte subsets, as CD4+CD8+, CD4+CD8-, CD4-CD8+, CD4-CD8-, CD3IOW, CD3high, TcRo4# and yb, severely altered in the acute phase of infection, returned to normal values in chronically infected animals, probably due to the increase in the percentage of thymocyte precursors, such as CD4IOWCD8- or CD4-CD8Iow cells, together with the increase in the IL-2R+ and cycling cells that occurred by the late phase of acute infection.
INTRODUCTION Chagas' disease is a parasitic affection caused by the flagellate protozoan Trypanosoma cruzi. During the course of the disease, one can distinguish an acute phase, with easy detection of the parasite in the blood and target tissues, that contrasts with the chronic phase, when parasitaemia is no longer detectable and T. cruzi is virtually absent from the tissues, in spite of severe associated pathologies (reviewed in ref. 1). In the last few years cumulative experimental data, essentially obtained in the mouse model of T. cruzi infection, have clearly demonstrated immunological imbalances in acutely infected animals. Recently, a series of experiments definitely demonstrated that T lymphocytes, and more particularly CD4+ cells, are involved in the pathophysiology of the disease.2A In fact, these recent findings are in keeping with pioneer works showing that neonatally thymectomized as well as athymic (nu/ nu) mice exhibit enhanced severity of the disease.5 6 Considering the importance of T-cell-mediated mechanisms in Chagas' disease, we recently performed a number of experiments in order to better understand whether the thymus is also affected in T. cruzi infection. These studies, carried out in acutely infected animals, revealed a severe thymic atrophy with significant changes in the lymphoid compartment of the organ.7'8 It thus seemed worthwhile to investigate whether these alterations persisted during the course of a chronic infection.
Mice and parasites Six- to 8-week-old male C3H/HeJ mice were obtained from Pasteur Institute facilities (Paris, France). Each experimental group consisted of four to eight animals. The CL strain of T. cruzi was maintained by passage in C3H/HeJ mice. Animals were infected with 105 bloodstream-derived trypomastigote forms. They were killed at different times post-infection. The acute phase was determined following the parasitaemia as described by Brener,9 while the chronic phase was defined by negative parasitaemia and classical increased levels of circulating anti- T. cruzi immunoglobulins (IgG) detected by ELISA (i.e. 100:1 chronic versus acute titre ratio, not shown), as described elsewhere. '°
Correspondence: M. C. Leite-de-Moraes, Unite d'Immunoparasitologie, Institut Pasteur, 25, rue du Dr. Roux, 75724, Paris, Cedex 15, France.
Antibodies Fluoresceinated rat monoclonal antibodies (mAb) anti-Thy-1.2 (clone 30.H.12), anti-CD8 (clone YTS 169.4), as well as
MATERIALS AND METHODS
95
96
M. C. Leite-de-Moraes et al.
phycoerythrin-coupled anti-CD4 mAb (clone GK 1.5), were purchased from Becton-Dickinson (Grenoble, France). The hamster anti-CD3 mAb (clone 145-2C1 1), the anti-TcRai mAb (clone H57-597), the anti-TcRyb mAb (clone GT3) and the rat anti-IL-2R (clone PC61) are currently being expanded in our laboratory, and were used as culture supernatants or ascitic fluids.
O T. cruzi
0
E
30-
8 20-T
Immunofluorescence Thymi were removed and gently homogenized to yield a singlecell suspension in Hanks' solution containing 2% foetal calf serum (FCS) and 0 1% NaN3. Cell viability was determined by the Trypan blue exclusion test. Viable thymocytes (106 cells/ tube) were incubated for 30 min at 4C with optimal concentrations of each specific antibody. Washing was performed twice with Hanks' solution. For indirect immunofluorescence cells were subjected to the second antibody. They were then washed and fluorescence analysis were carried out using a flow cytometry analyser (FACScan; Becton-Dickinson & Co., Mountain View, CA). Detection of cellular DNA content Thymic cell suspensions from normal or infected mice were analysed by flow cytometry for their cellular DNA content after staining with the Vindelov lowsalt propidium-iodide (PI) solution, as described elsewhere." The number of cycling cells was calculated on the basis of a DNA content higher than that of resting diploid cells.
RESULTS Thymocyte recovery in T. cruzi chronically infected mice In order to evaluate the thymocyte phenotypic alterations in T. cruzi-infected mice, we carried out a longer kinetic analysis starting at the acute and continuing until the chronic phase. Confirming our previous results,7'8 mice infected with 105 parasites and analysed at the acute phase (0-5 and 1 month after infection) underwent severe thymic atrophy. Conversely, in chronically infected animals, at a stage where parasitaemia was absent and the level of circulating immunoglobulins increased, there was a progressive recovery of thymus weight and thymocyte numbers (Fig. 1) compared to age-matched controls. Besides the recovery of thymocyte numbers in the chronic infection, the percentages of CD4/CD8-defined thymocyte subsets also returned to normal values (Fig. 2). It is interesting to note that the ratio of single positive cells bearing low densities of CD4 or CD8 molecules was higher in the late phase of acute infection. Since these cells are precluded as intrathymic precursors of double positive thymocytes, they might be related to the thymus recovery observed in the chronic phase. Actually, the analysis of cycling cells as well as of the thymocytes expressing the IL-2 receptor revealed a significant increase in both parameters, during the acute phase (Fig. 3). Lastly, we evaluated the CD3-Ti complex expression in acute and chronically infected mice. Again, the decrease of CD3lOw, paralleled by an increase in the CD3high thymocyte percentage, associated with an enhancement of the percentage of cells bearing the TcRcxa or yb observed during the acute phase, was restored to normal values in chronically infected animals (Fig. 4).
E
0 05
3 Months after infection
5
Figure 1. Number of viable thymocytes in C3H/HeJ mice infected by T. cruzi with the dose of 105 parasites/mouse at different times after infection. The data shown represent the mean + SEM of five to eight mice individually tested. **P
