Parasite Immunology, 2001: 23: 617±626Mouse splenic CD41 and CD81 T cells undergo extensive apoptosisduring a Plasmodium c...
L.Sanchez-Torres et al.                                                                                      Parasite Immu...
Volume 23, Number 12, December 2001                                                        T cell apoptosis in P. chabaudi...
L.Sanchez-Torres et al.                                                                                    Parasite Immuno...
Volume 23, Number 12, December 2001                                                                                       ...
L.Sanchez-Torres et al.                                             Parasite Immunology622                       q 2001 Bl...
Volume 23, Number 12, December 2001                                                                                       ...
L.Sanchez-Torres et al.                                                                                                   ...
Volume 23, Number 12, December 2001                                                           T cell apoptosis in P. chaba...
L.Sanchez-Torres et al.                                                                                                   ...
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  1. 1. Parasite Immunology, 2001: 23: 617±626Mouse splenic CD41 and CD81 T cells undergo extensive apoptosisduring a Plasmodium chabaudi chabaudi AS infectionLUVIA SANCHEZ-TORRES, ANDREA RODRIGUEZ-ROPON, MARIBEL AGUILAR-MEDINA& LUIS FAVILA-CASTILLODepartment of Immunology, National School of Biological Sciences, IPN, Mexico City , MexicoSUMMARY INTRODUCTIONThe presence and phenotype of apoptotic lymphocytes was Apoptosis is a natural mechanism of cell death, whichstudied in spleen cell suspensions taken from CB6F1 mice involves membrane cell blebbing, cell shrinkage, chromatininfected with Plasmodium chabaudi chabaudi AS. High condensation, nuclear fragmentation and DNA degradationlevels of apoptotic cells were found, associated with high (1). This mechanism, which has been also called pro-parasitaemias and splenomegaly. This was also accompa- grammed cell death, is essential for the homeostasis of thenied by expansion and disarray of spleen white pulp. whole organism and has a central role from development toApoptosis levels lowered when parasitaemia was cleared, the maintenance of body shape and function. There hasbut were still higher than in normal mice. At this time, the been major advances in the understanding of the biochem-spleen was diminishing in size and the white pulp was ical events that underlie the apoptotic process (2).contracting and rearranging. When parasitaemia was Apoptosis is triggered by a variety of both internal andpatent, the cells most affected by apoptosis were CD41 T external signals. Most of the morphological changescells followed by CD81 T cells, and to a lesser extent observed in a cell that is undergoing apoptosis are causedB2201 B cells. When parasitaemia was cleared, CD81 T by a family of cystein proteases which are activated incells and B2201 B cells returned to basal levels of cascade and are known as caspases. These enzymes haveapoptosis, while CD41 T cells still had higher apoptosis restricted protein targets which are normally inactivatedlevels than normal mice. A similar pattern of lymphocyte after the caspase exerts its action. In some cases, the targetsubpopulation apoptosis was found in infected BALB/c of a caspase is an enzyme inhibitor so the final action of themice, despite the fact that, for this mouse model, it has been caspases is the activation of the enzyme.reported that B cells are the cells that are most affected by Inside the cell, there are proteins which facilitates theapoptosis. We consider that the high levels of apoptosis in apoptotic process (pro-apoptotic) and others which suppressCD41 T cells when parasitaemias are still high are not it (anti-apoptotic). The relative amount of pro- and anti-easily explained by a normal mechanism of down regulation apoptotic proteins determines if a cell is sensitive orof the immune response. resistant to apoptotic signals. One of the first identified anti-apoptotic proteins was called Bcl-2, which wasKeywords apoptosis, mouse malaria, CD41 T cells, discovered in a B-cell lymphoma. Now we know thatCD81 T cells, B cells, Plasmodium chabaudi there is a family of these proteins and some of them, such as Bax and Bak, are in fact pro-apoptotic. Many proteins of the Bcl-2 family work at the mitochondria membrane level. If pro-apoptotic proteins predominate, the mitochondria releases several molecules, including cytochome C, which promote the apoptotic process. During the course of an immune response, T-cell clonesCorrespondence: Luis Favila-Castillo, Department of Immunology,National School of Biological Sciences, IPN, Carpio y Plan de Ayala, responding to the antigen undergo extensive proliferation. Â Â ÂColonia Santo Tomas, Mexico DF 11340, Mexico. When the antigen is eliminated, the number of T-cells mustReceived: 4 January 2001 be down regulated and this is achieved by inducingAccepted for publication: 29 June 2001 apoptosis in the responding T-cells, a process termedq 2001 Blackwell Science Ltd 617
  2. 2. L.Sanchez-Torres et al. Parasite Immunologyactivation-induced cell death. The apoptotic signal is in different malaria mouse models, in humans and in otherreceived by the T-cell through a membrane receptor called animal models, where the infection becomes chronic (15±CD95 or Fas, which is a type I transmembrane receptor. 17). In these latter two situations, apoptosis of lymphoidThe death signal is provided by the ligand of CD95 (CD95L cells might help explain the development of immunosup-or FasL), which is expressed on lymphoid cells in the late pression and chronicity.phases of the immune response. The elimination of The organ most affected in malaria is the spleen. Besidesresponding cells is considered a normal mechanism to suffering changes in size and function, the spleen is alsoturn off an ongoing immune response and to avoid self closely related to the development and maintenance of adamage (3). There are other signals which contribute to protective immune response (18). Using a spleen transplantactivation-induced cell death, such as a receptor for tumour technique in a malaria rat model, it has been shown that,necrosis factor (TNF)R2 which transmits apoptotic signals after primary infection, the spleen develops all the requiredwhen bound to its ligand TNF-a. Finally, apoptotic cells are elements to protect itself against reinfection (19).engulfed by phagocytic cells, eliminating them without One way to understand the role of lymphoid cellcausing tissue inflammation or alterations in tissue apoptosis in malaria is to study the presence of apoptoticmorphology or function. lymphoid cells during different stages of infection, and to The idea that parasitic microorganisms might tamper with relate any changes to other physiological alterations in theapoptotic signals or biochemical processes and hence use immune system. In this respect, it is also useful tothem to their advantage is attractive as it could help explain, determine how different lymphoid cell populations areat least in part, how parasites can become established in affected by apoptosis. A recent study (20) investigatedtheir host and how infection becomes chronic. apoptosis in mouse spleen cells infected with P. chabaudi An increase in apoptosis of lymphoid cells has been chabaudi AS. It was found that the frequency and absolutedescribed during mouse infection with microorganisms numbers of apoptotic cells in the spleen increased duringsuch as viruses (4,5), ricketssia (6), bacteria (7,8), protozoa primary infection, reached a peak around 4 days following(9,10) and helminths (11). The relevant questions in these peak parasitaemia and then descended to almost normalsystems are: (i) does the parasitic microorganism use levels after the parasite was cleared. The findings alsoapoptotic signals to interfere with the immune response and demonstrated that apoptosis involves T cells (CD41 andbecome established in its host or (ii) is the increase in CD81), B cells and macrophages and that the majority ofapoptosis during the infection associated with the normal apoptotic cells are B cells.silencing of the immune response against the parasite? A Here, we carried out a similar study using the samethird possibility is that the increase in apoptosis is an parasite, and, in general, confirmed the previous resultsepiphenomenon, which is not relevant to the host±parasite (20). We also describe changes in spleen size and histologyinteraction. during the infection, and correlate these changes with In mice infected with the lymphocytic choriomeningitis apoptosis of spleen cells. Finally, it should be noted that wevirus (4) or with Listeria monocytogenes (7), lymphocyte studied apoptosis in lymphocyte subpopulations using aapoptosis has been associated with silencing of the immune different experimental approach to the previous study (20).response. Whereas, in mice infected with Trypanosoma We therefore conclude that when apoptosis is expressed ascruzi, lymphocyte apoptosis has been interpreted as a way the percentage of apoptotic cells within a particularto limit host tissue damage by the immune response, but lymphocyte subpopulation, the cells most affected bywhich collaterally promotes the establishment of a chronic apoptosis are CD41 T cells, followed by CD81 T cells,infection (10). On the other hand, a protein that induces while B2201 B cells are proportionally less affected. Withapoptosis in macrophages has been identified in Yersinia, this information, the possible role of apoptosis at theand it has been shown that when Yersinia is inoculated into different stages of infection is discussed.mice the expression of this protein is an important virulentfactor (12). MATERIALS AND METHODS In malaria, there are several phenomena in whichapoptosis of lymphoid cells could be involved. There is a Micevery intense proliferation of lymphoid cells (13,14) andsevere splenomegaly, and these events are subdued when Female (BALB/cXC57Bl/6) F1 hybrids or BALB/c 14±22-parasitaemia is controlled. In both cases, apoptosis could be week-old mice were used for all experiments. The parentspart of a normal mechanism to reduce the number of were originally purchased from the Jackson Laboratorylymphoid cells and, as a consequence, spleen size. On the (Bar Harbor, ME, USA) and hybrids or BALB/c mice wereother hand, general immunosuppression has been described bred in our facilities at the Department of Immunology, in618 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626
  3. 3. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malariathe National School of Biological Sciences, IPN, Mexico Determination of apoptotic cells by cytofluorometryCity. Experimental mice were maintained in a reversed 12-h The presence of apoptotic cells was determined in celllight-dark cycle from 07.00 h to 19.00 h. suspensions by their ability to bind Annexin V (21). The Apodetect Annexin V-fluorescein-isothiocyanate (FITC) kitParasite and infection (Zymed Laboratories, San Francisco, CA, USA) was used, according to the manufacturers instructions. Briefly: spleenPlasmodium chabaudi chabaudi AS was kindly donated by cells, obtained as described above, were adjusted toDr David Walliker (University of Edinburgh, Edinburgh, 5 Â 105 per ml in binding buffer and 10 ml of FITC-UK), and is a cloned and mosquito-transmitted line. The Annexin V were added to a 190-ml sample of the cellparasite has been syringe passaged from the original suspension. The mixture was incubated for 10 min at roommaterial no more than eight times and reference populations temperature. After centrifugation, cells were resuspended inare cryopreserved in liquid nitrogen. For all experiments, 190 ml of binding buffer and 10 ml of propidium iodideparasites from frozen material were inoculated into young (20 mg/ml) were added. Cells were analysed in amice (8±10 weeks old) and when parasitaemias were FACScalibur cytofluorometer (Becton Dickinson, Sanpatent, experimental mice were inoculated intraperitoneally Jose, CA, USA) using the Cell Quest program (versionwith 5 Â 104 parasitized red blood cells. Control mice were 2´0), and 10 000 events per sample were recorded. Cellsinoculated with the same amount of normal red blood cells that were positive for both propidium iodide and Annexin Vas the total amount of red cells received by the infected were considered as necrotic cells, and were excluded frommice. The parasite causes a synchronical infection and the analysis. The results are expressed as the percentage ofundergoes schizogony every 24 h between 09.00 h and apoptotic cells (^ SD).11.00 h. Parasitaemia was determined by light microscopyof methanol-fixed, Giemsa-stained thin blood films, andwas expressed as percent parasitaemia. Determination of apoptotic cells in sections Apoptotic cells were detected by the TUNEL reaction (22). An in situ cell death detection kit (Boehringer MannheimDetermination of splenomegaly GmbH, Mannheim, Germany) was used according to theA group of mice was infected with P. chabaudi, as manufacturers instructions. Briefly, wax was removeddescribed above, and was only used for the determination from sections, they were rehydrated and treated withof splenomegaly. At different times following infection, proteinase K. The sections were then incubated withgroups of three mice were sacrificed, their spleens extracted terminal deoxinucleotidyl transferase and FITC-labelledand weighed. At each experimental point, one normal, age nucleotides, which were detected using an alkalinematched mouse was also killed and its spleen weight phosphatase conjugated anti-FITC antibody, using nitrobluerecorded. A total of eight normal mice were sacrificed tetrazolium as the substrate. Apoptotic cells were stainedduring the experiment and all resulting data were pooled. blue and sections were then counterstained with nuclear red, dehydrated and mounted in synthetic resin.Processing of spleens from infected and control mice Purification of lymphocyte subpopulations by magneticA group of mice was infected with the parasite, and at beadsdifferent times following infection, groups of infected orcontrol mice were sacrificed, their spleens extracted CD41 or CD81 T cells or B2201 B cells were purified(always between 13.00 h and 14.00 h) and cut in half. from total spleen cell suspensions, prepared as describedFrom one half, a cell suspension was prepared by above. Spleen cells (107) were resuspended in 90 ml of PBSexpressing the spleen through nylon mesh with the help (containing 2 mm ethylenediaminetetraacetic acid andof a plastic syringe plunger. The spleen cells were collected 0´5% bovine seric albumin) and 10 ml of the respectivein cold phosphate-buffered saline (PBS), centrifuged, paramagnetic bead bound antibody (Miltenyi Biotec,resuspended in PBS, counted and used for the determina- Aarhus, Denmark) were added. Cells were incubated ontion of apoptotic cells by cytofluorometry (see below). The ice for 15 min, washed twice, resuspended in 500 ml ofother spleen half was fixed in 10% formalin in PBS, PBS and loaded onto a positive selection column (MS1/embedded in paraffin-wax and processed by standard RS1 MACS column, Miltenyi). The column was thenhistological techniques used to prepare and stain 4 mm exposed to a strong magnetic field (VarioMacs, Miltenyi).sections with haematoxilin and eosin (H&E). Negative cells were eluted with 1´5 ml of PBS and wereq 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626 619
  4. 4. L.Sanchez-Torres et al. Parasite Immunologycollected. The column was removed from the magnetic 21±38 p.i., when parasitaemia had been cleared. Duringfield and positive cells were eluted with 1´5 ml of PBS and this time, the level of apoptosis, although low, was stillcollected. The purification procedure was repeated using significantly higher than in normal mice (16´7% apoptoticthe positive cells. The purity of the cells was assessed by cells, day 38 p.i., P , 0´001).cytofluorometry using FITC-conjugated anti-CD4 or anti- Changes in spleen size were determined in a differentCD8 monoclonal antibodies and PE-conjugated anti-B220 group of infected mice, which gave a parasitaemia curvemonoclonal antibody. The percentage of apoptotic cells was very similar to the one shown in Figure 1(a) (data notdetermined in the purified cells by cytofluorometry, as shown). The spleen started to increase in size early indescribed above. ascending parasitaemia. At day 4 p.i., the spleen was already significantly bigger than in normal mice (Figure 1c, P , 0´05). The spleen size increased sharply after this timeStatistical analysis and at peak parasitaemia it was around 12-fold higher thanStudents t-test was used to assess statistical significance. a normal spleen (12X, P , 0´001). The spleen size reachedP , 0´05 was considered statistically significant. a peak at day 14 p.i. (17X, P , 0´001) which was 4 days later than the peak of apoptosis (Figure 1b) and 6 days later than peak parasitaemia, when descending parasitaemia wasRESULTS around 0´01% (Figure 1a). After this time, the size of the spleen decreased sharply to become 6´5X at day 16 p.i.Apoptosis of spleen cells and changes in spleen size (parasitaemia cleared) and continued to decrease to reach aduring P. chabaudi primary infection size of 2´5X at day 31 p.i. (Figure 1c, P , 0´001). TheThe curve of parasitaemia caused by P. chabaudi infection spleen stays around this size for several months afterin CB6F1 mice has four distinct zones. Four days after infection (data not shown).inoculation, parasites start to be detected by light micro-scopy and numbers sharply increase (ascending parasitae- Histological changes in the spleen during P. chabaudimia) to reach a maximum at day 8 p.i. (peak parasitaemia). primary infectionThen parasitaemia starts to rapidly decline (descendingparasitaemia) and the parasite becomes undetectable by H&E stained sections were prepared from the spleens oflight microscopy around days 17±19 p.i., and stays this mice used to generate the parasitaemia curve shown inway up to day 38 p.i. (parasitaemia cleared) (Figure 1a). In Figure 1(a). A section from a normal spleen is shown inthis study, parasitaemia has been considered to be cleared at Figure 2(a), where normal distribution and aspect of redthis time; however, the presence of the parasite can be and white pulp is observed. There were a few lymphoiddemonstrated by subinoculation of blood into normal mice cells with picnotic nuclei (0´2±0´5 per field, using a  40up to days 60 or 70 p.i. After this time, parasites cannot be objective) which were considered to be apoptotic cells sincedemonstrated in blood by subinoculation (data not shown). they were TUNEL positive (see below). At day 4 p.i., when The percentage of apoptotic cells in the spleen was parasites just started to be detected, the white pulp wasdetermined during P. chabaudi primary infection in the organized and did not show obvious proliferative activitysame mice used to generate the parasitaemia curve (Figure 2b). In general, no differences in histology(Figure 1a). Results are shown in Figure 1(b). In a group compared to normal spleens were detected, including theof 13 normal mice, a background of 7´6% (^ 0´6) apoptotic amount of apoptotic cells. At day 8 p.i., the time of peakcells was detected. In infected mice, the percentage of parasitaemia, cells in the white pulp had proliferatedapoptotic cells at the beginning of ascending parasitaemia considerably, such that white pulp had enlarged and the(days 2 and 4 p.i.) was not different from normal controls. limits between white and red pulp started to disappearWhen mice were approaching, or had reached peak (Figure 2c). At this time, the spleen had increasedparasitaemia (days 7 and 8 p.i.), there was a small but considerably in size, although it had not reached its peak.statistically significant increase in apoptotic cells (15´2% The amount of apoptotic cells increased (2±3 per field) andapoptotic cells, day 7 p.i., P , 0´001). After this time, the clusters of apoptotic cells started to appear. At day 10 p.i.,percentage of apoptotic cells increased steadily to reach a which is the time when the peak in numbers of apoptoticpeak at day 10 p.i., when 53´1% of the spleen cells were cells was detected in cell suspensions, total tissuefound to be apoptotic (P , 0´01). At this time, descending disorganization was observed. This disorganization wasparasitaemia had already started, although parasitaemia was due mainly to hiperplasia of the lymphoid tissue, the redstill high (Figure 1a). After this peak of apoptotic cells, pulp had almost disappeared (Figure 2d) and apoptotic cellsapoptosis started to decline and reached a plateau from days were abundant (10±20 per field). Spleen size was increased,620 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626
  5. 5. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malariabut it was still not at its peak. The time of peak of apoptotic cells and clusters, although still high (aroundsplenomegaly is day 14 p.i., which is when parasitaemia 10 per field) was lower than at day 10 p.i. At day 38 p.i.,is being resolved, and the white pulp has started to when parasitaemia has been controlled and splenomegalyreorganize although it is still hyperplasic (Figure 2e). The was subdued, white and red pulp are clearly separated againred pulp started to be distinguishable again and the amount and in the white pulp few individual apoptotic cells were observed (2±3 per field). In addition, many discrete less dense areas were easily recognizable at low magnification (Figure 2f). These structures were not seen in normal (a) spleens and started to appear in the spleens of infected mice 100 until day 19 p.i. (not shown) but were much more abundant at day 38 p.i. At higher magnification (Â 100 objective), 10 these structures were conglomerates of apoptotic cells, bodies and cell debris, apparently inside a macrophage % Parasitemia 1 (Figure 2g). All these structures were TUNEL positive, as were individual cells with condensed chromatin which were 0·1 probably those recognized as apoptotic in H&E stained sections (Figure 2h). 0·01 0·001 Apoptosis in lymphocytes subpopulations 0 5 10 15 20 25 30 35 40 Our results show that there is an increase in apoptotic cells (b) in the spleen during P. chabaudi primary infection. We next 80 studied apoptosis at the level of lymphocyte subpopulation at different stages of infection. For this, we determined the 70 percentage of apoptotic cells in CD41 and CD81 T cells 60 and B2001 B cells, purified by antibody coated magnetic % Apoptotic cells beads, as described in Materials and Methods. 50 We prepared a purified spleen cell subpopulation from 40 normal and infected CB6F1 or BALB/c mice at high 30 ascending parasitaemias, when the number of apoptotic cells started to rise significantly (Figure 1b), from peak 20 parasitaemia or from descending parasitaemia. For CB6F1 10 mice, samples were also studied a few days following parasitaemia clearance. For all experiments, the percentage 0 0 5 10 15 20 25 30 35 40 of apoptotic cells in the unseparated whole spleen cell suspension was also determined, and it was found to be (c) within the range predicted by the curve shown in 1·6 Figure 1(b) (data not shown). The percentage of each cell 1·4 1·2 Figure 1 CB6F1 mice were infected with P. chabaudi. (a) Resulting Spleen weight (g) parasitaemias were followed in six mice and expressed as percent 1·0 parasitaemia (mean ^ SD). (b) Apoptosis in spleen cell suspensions of 0·8 mice infected with P. chabaudi, determined by Annexin V binding. The percentage of apoptotic cells was determined in mice from the same 0·6 group as in a at the indicated times (3±6 mice per experimental point). The open triangle in day 1 is the result of 13 normal mice (mean ^ SD), 0·4 P , 0´01 from days 7±38 p.i. compared to normal mice. (c) The weight of the spleen of three mice per experimental point was recorded at the 0·2 indicated times, in a different group of mice than in a (mean ^ SD). The 0·0 open triangle in day 1 is the mean weight of eight normal spleens. 0 5 10 15 20 25 30 35 40 Infected mice had significantly bigger spleens from days 4±31 p.i. Days postinfection (P , 0´05 at day 4 p.i. and P , 0´001 from day 7 p.i. to day 31 p.i.).q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626 621
  6. 6. L.Sanchez-Torres et al. Parasite Immunology622 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626
  7. 7. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malaria CB6F1 mice BALB/c mice was 7±11% in the sampled mice) compared to 2´7% in (a) (d) 60 + 60 + normal BALB/c mice. For CD81 T cells, there was also a significant increase in CD4 T cells CD4 T cells 50 50 ∗∗ 40 ∗∗ 40 ∗∗ apoptotic cells in all the three stages of parasitaemia, both 30 30 ∗ for CB6F1 and BALB/c mice. In CB6F1 mice, the level of 20 ∗∗ 20 10 10 ∗∗ CD81 apoptotic T cells approached normal levels when the ∗∗ 0 NL ASC PEAK DESC CLD 0 NL ASC PEAK DESC ND CLD parasite was cleared (Figure 3b,e). The increase in apoptotic CD81 T cells was approximately one-half the increase in apoptotic CD41 T cells. For CB6F1 mice, (b) (e) 60 60 19´0% of CD81 T cells were apoptotic and, for BALB/c + + CD8 T cells CD8 T cells 50 50 % Apoptotic cells 40 40 mice, 23´9% were apoptotic, both at peak parasitaemia, 30 30 compared to 2´6% in normal mice for both mouse strains. ∗ For B2201 B cells, although there was a tendency for the 20 20 ∗∗ ∗∗ ∗ ∗∗ 10 ∗∗ 10 0 0 ND percentage of apoptotic cells to increase during patent NL ASC PEAK DESC CLD parasitaemia for both CB6F1 and BALB/c mice; the NL ASC PEAK DESC CLD (c) (f ) difference was significant only at descending parasitaemia. For CB6F1 mice, the percentage of apoptotic B2201 B 60 60 + + B220 B cells B220 B cells 50 50 40 40 cells returned to normal levels when parasitaemia was 30 30 cleared (Figure 3c,f). 20 20 The percentage of CD41 and CD81 T cells in the spleen 10 ∗ 10 ∗ ND diminished during infection, both in CB6F1 and in BALB/c 0 0 NL ASC PEAK DESC CLD NL ASC PEAK DESC CLD mice, following a similar pattern during the different stagesFigure 3 Apoptotic cells detected by Annexin V binding in of parasitaemia (Figure 4a,b,d,e). In CB6F1 mice, thesubpopulations of spleen lymphocytes purified by magnetic beads and percentage of both CD41 and CD81 T cells tended toanalysed by citofluorometry. The cells were determined in 3±6 CB6F1 return to normal levels when the parasitaemia has beenor BALB/c mice per group of normal mice (NL), infected mice with 5± cleared (Figure 4a,b).15% of ascending parasitaemia (ASC), peak parasitaemia (PEAK), 2± The percentage of B2201 B cells increased or stayed18% desending parasitaemia (DESC) or mice 3 days after parasitaemiahad become undetectable by light microscopy (cleared, CLD). Bars are within normal range during the different stages of para-1 SD; ND, not determined, *P , 0´05, **P , 0´01, compared to sitaemia, both in CB6F1 and BALB/c mice (Figure 4c,f).normal mice. DISCUSSIONsubpopulation in the unseparated cell suspension was alsodetermined. In this study, we investigated apoptosis of lymphocytes at For CD41 T cells, we found an important and significant different stages of P. chabaudi infection in mice, andincrease in apoptotic cells in all three stages of para- related our findings to parasitaemia and changes in spleensitaemia, both for CB6F1 and BALB/c mice. In CB6F1 size and histology. When parasites just started to bemice, the level of CD41 apoptotic T cells approached detectable by light microscopy there were no noticeablenormal levels when the parasitaemia was cleared (Fig- changes in spleen histology (Figure 2b), size (Figure 1c) orure 3a). At peak parasitaemia, 48´3% of CD41 T cells were percentage of apoptotic cells (Figure 1b). At peak para-apoptotic in CB6F1 mice compared to 2´3% in normal sitaemia, the white pulp had expanded (Figure 2c) whichCB6F1 mice. For BALB/c mice, 43´3% of CD41 T cells correlates with an increase in spleen size (Figure 1c) andwere apoptotic at descending parasitaemia (parasitaemia the number of apoptotic cells had started to increaseFigure 2 Haematoxilin and eosin stained sections from: (A) normal spleen; (B) spleen from a mouse at day 4 p.i. with P. chabaudi where white andred pulp are clearly separated; (C) spleen from a mouse at day 8 p.i. with P. chabaudi. Note that the separation between white and red pulp starts todisappear; (D) spleen from a mouse at day 10 p.i. Note that the white pulp is not longer distinguished as cells from the white pulp appear to haveinvaded most of the organ and there are only a few red blood cells; (E) spleen from a mouse at day 14 p.i. The white pulp is becoming rearrenged,although it is larger than in a normal spleen. Groups of red blood cells can be seen again in the red pulp; (F) spleen from a mouse at day 38 p.i. Thewhite and red pulp are again clearly separated but the white pulp shows clusters of material sorrounded by a clear area (arrows); (G) Magnification ofthe clusters seen in F, there are two kind of clusters, some are in fact formed by a single cell with a condensed nucleus which looks like an apoptoticcell, and other clusters, which are larger, include apoptotic cells and cell debris (seven such clusters are shown); (H) TUNEL reaction of the clustersshown in (G), both single cells and clusters are positive. Original magnification: (A) to (E) Â 200; (F) Â 100; (G±H) Â 1000.q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626 623
  8. 8. L.Sanchez-Torres et al. Parasite Immunology (a) CB6F1 mice (d) BALB/c mice red pulp were seen to be clearly separated. However, we 60 CD4+ T cells 60 CD4+ T cells found clusters of apoptotic cells and bodies in the white 50 50 pulp, which were easily recognized since they were less 40 40 dense that the surrounding tissue. These structures, which 30 30 ∗ are shown in more detail in Figure 2(g,h), have been 20 20 ∗∗ 10 ∗∗ ∗∗ 10 ∗∗ described in other pathologies as tingible body macro- ∗∗ ∗∗ 0 0 ND phages, which are macrophages that have engulfed NL ASC PEAK DESC CLD NL ASC PEAK DESC CLD apoptotic cells and bodies (24). In this study, thesePERCENT OF LYMPHOCYTE SUBPOPULATION structures were detected as being clearly TUNEL positive (b) (e) 60 CD8+ T cells 60 CD8+ T cells 50 50 (Figure 2h). We do not know their significance, although it 40 40 is interesting that they started to appear rather late in the 30 30 infection (day 19 p.i.) and were very abundant at day 38 p.i. 20 ∗∗ 20 ∗ (Figure 2f). We have observed the same structures in pig 10 ∗∗ 10 ∗∗ 0 0 ND lymph nodes undergoing a viral infection; however, the NL ASC PEAK DESC CLD NL ASC PEAK DESC CLD structures appeared much earlier (day 4 p.i) (25). 60 (c) B220+ B cells 60 (f) B220+ B cells On the other hand, the identification of apoptotic 50 ∗∗ 50 cells by Annexin V binding, which detects an early change ∗∗ 40 40 ∗∗ in membrane structure preceding DNA fragmentation, 30 30 correlated well with the observation of picnotic nuclei, a 20 20 late stage of apoptosis, in H&E stained spleen sections. 10 10 ND We also investigated apoptosis in different lymphocyte subpopulations. Cells were purified by antibody coated 0 0 NL ASC PEAK DESC CLD NL ASC PEAK DESC CLD magnetic beads. This is an efficient procedure that takesFigure 4 Percent of lymphocyte subpopulations in spleen cells from approximately only 1 h to complete and the beads do notnormal mice (NL), infected mice with 5±15% of ascendingparasitaemia (ASC), peak parasitaemia (PEAK), 2±18% desending interfere with cytofluorometric analysis. At all the studiedparasitaemia (DESC) or mice 3 days after parasitaemia had become stages of patent parasitaemia, CD41 and CD81 T cells hadundetectable by light microscopy (cleared, CLD). Bars are 1 SD; ND, high percentages of apoptotic cells (Figure 3a,b). Whennot determined, *P , 0´05, **P , 0´01, compared to normal mice. parasitaemia was cleared, the percentage of apoptotic CD81 T cells returned to normal levels while apoptoticmoderately (Figure 1b). The peak of apoptotic cells was CD41 T cells, although diminished, were still significantlyfound at day 10 p.i., when there was more than 50% greater in number compared to normal mice. Theapoptosis. Although parasite growth was already seen to be percentage of total CD41 and CD81 T cells was found tounder control, parasitaemia was still high (approximately diminish during patent parasitaemia (Figure 4a,b,d,e).20%), we therefore do not believe that this peak of During patent parasitaemia, the percentage of apoptoticapoptotic cells can be interpreted as a mechanism for B2201 B cells was not greatly modified (Figure 3c,f),turning off the immune response. This is supported by the although there was an increase in the percentage of totalfact that splenomegaly had not reached its peak and the B2201 B cells (Figure 4c,f).spleen histology was in total disarray (Figure 2d). One In conclusion, the population most affected by apoptosispossible explanation for the presence of apoptotic cells at were T lymphocytes, particularly CD41 T cells, fromthis time of the infection is that they are induced, at least in which approximately 50% were apoptotic at peak andpart, by parasite products, as has been described for descending parasitaemia, compared to approximately 2% inP. falciparum malaria (23). Spleen size reached a peak at normal mice, while the least affected population comprisedday 14 p.i. and at this time parasitaemia was low, and the the B2201 B cells.white and red pulp had started to reorganize (Figure 2e). In a previous study (20), apoptosis of spleen cells wasThe percentage of apoptotic cells had diminished, although also studied in mice infected with the same Plasmodiumit was still high (approximately 30%, Figure 1b). We strain that we used, except that in that study BALB/c micebelieve that at this stage apoptosis is related to the turning were used instead of CB6F1 mice. Using Annexin V tooff of the antiparasite immune response, since after this detect apoptotic cells by cytofluorometry, these authorstime the spleen size diminished rapidly, red and white pulp reported results very similar to ours in terms of percentagebecame clearly separated again and parasites became and kinetics of apoptotic cells during primary infection.undetectable by light microscopy. When the spleen had However, they reach the conclusion that the majority ofreduced to a near normal size (day 38 p.i.), the white and apoptotic cells during patent parasitaemia are B cells, while624 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626
  9. 9. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malariaapoptosis in CD41 and CD81 T cells is only moderately Listeria monocytogenes (7) and Yersinia pseudotuberculosisincreased. At first sight, it seems that we reached opposite (12).conclusions to their study; however, we believe that the It seems that an increase in apoptosis, in some or most ofdifference lies in the different experimental approach that the lymphoid cells, is a general phenomenon in a variety ofwe took. They analysed unseparated spleen cell suspensions infections. In principle, this can be explained as a normalby double staining. Apoptotic cells were stained with homeostatic response involved in the termination of theAnnexin V labelled with one fluorochrome and the immune response induced by the microorganism (3).lymphocyte subpopulation was marked with an anti-cell Nevertheless, in our mouse malaria model, we found thatsurface marker antibody labelled with a second fluoro- the peak of apoptotic cells is reached when there are stillchrome. They performed a cytofluorometric analysis of the relatively high parasite loads, which, from our point ofsample using one label to identify and quantify the view, is still too early a time to terminate the immuneapoptotic cells and then, using the second label, determined response. On the other hand, it is well established thatwithin the apoptotic population the percentage of cells CD41 T cells are important in the control P. chabaudicarrying a particular marker (i.e. CD41). In this way, they primary infection (26,27). Other authors working withdid not determine the percentage of cells with that another malaria mouse model (P. berghei) have describedparticular marker that are not part of the nonapoptotic P. berghei specific CD41 T cells that are specificallypopulation. deleted when adoptively transferred into recipient mice Our approach was to first purify a particular lymphocyte challenged with the same parasite. It is suggested that thissubpopulation and then determine the percentage of deletion is mediated by apoptosis (28). This, together withapoptotic cells within that purified subpopulation. This our observation that approximately 50% of CD41 T cellsgave us an idea of how apoptosis affects a particular undergo apoptosis during P. chabaudi infection, provideslymphocyte subpopulation. In this way, we could see that sufficient data to stimulate the study of lymphocyteCD41 T cells comprise the lymphocyte subpopulation most apoptosis in malaria.affected by apoptosis, and not B2201 B cells. Furthermore, it is important to exclude the possibilitythat the difference between ours and the previous study (20) ACKNOWLEDGEMENTSis explained by the use of different mouse strains. We  This work was supported by Coordinacion General detherefore infected BALB/c mice with P. chabaudi, and  Posgrado e Investigacion del IPN. Luis Favila-Castillo isdetermined the percentage of apoptotic cells in purified fellow from COFAA-IPN, EDD-IPN and SNI-SEP. Luvialymphocyte subpopulations in the same way as we did for    Sanchez-Torres, Andrea Rodrõguez-Ropon and MaribelCB6F1 mice, as shown in Figure 3(d±f). Although there are Aguilar-Medina were supported by CONACYT Mexico.some minor differences in the results between BALB/c and Luvia Sanchez-Torres received during part of this study aCB6F1 mice, the conclusion is that in BALB/c mice the scholarship from the Von Behring and Kitasato Foundation,cells most affected by apoptosis are also CD41 and Mexico City. We thank Dr Edith Ormerod for reviewing theCD81 T cells. The variations in the percentage of spleen use of the English language.CD41, CD81 T cells and B2201 B cells duringparasitaemia were also similar between BALB/c andCB6F1 mice (Figure 4a±f). We therefore conclude that, REFERENCESduring P. chabaudi infection, the cells most affected by 1 Kerr JFR, Willie AH, Currie AR. Apoptosis: a basic biologicalapoptosis are CD41 and CD81 T cells. phenomenon with wide ranging implications in tissue kinetics. An increase in apoptosis of lymphoid cells has been Br J Cancer 1972; 26: 239±257.described for several kinds of infection. Important increases 2 Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770±776.in apoptosis of CD41 or CD41 plus CD81 T cells have been 3 Van Parijs LP, Abbas AK. Homeostasis and self-tolerance indescribed in mice infected with Cytomegalovirus (5), the immune system: turning lymphocytes off. 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