The study examines apoptosis in mouse splenic T cell and B cell populations during infection with Plasmodium chabaudi chabaudi AS malaria. High levels of apoptosis were found to correlate with high parasitemia and splenomegaly, particularly in CD4+ T cells. Apoptosis levels decreased as parasitemia was cleared but remained elevated compared to normal mice, with CD8+ T cells and B cells returning to basal apoptosis levels while CD4+ T cells remained higher.

1. Parasite Immunology, 2001: 23: 617±626
Mouse splenic CD41 and CD81 T cells undergo extensive apoptosis
during a Plasmodium chabaudi chabaudi AS infection
LUVIA SANCHEZ-TORRES, ANDREA RODRIGUEZ-ROPON, MARIBEL AGUILAR-MEDINA
& LUIS FAVILA-CASTILLO
Department of Immunology, National School of Biological Sciences, IPN, Mexico City , Mexico
SUMMARY INTRODUCTION
The presence and phenotype of apoptotic lymphocytes was Apoptosis is a natural mechanism of cell death, which
studied in spleen cell suspensions taken from CB6F1 mice involves membrane cell blebbing, cell shrinkage, chromatin
infected with Plasmodium chabaudi chabaudi AS. High condensation, nuclear fragmentation and DNA degradation
levels 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 the
nied by expansion and disarray of spleen white pulp. whole organism and has a central role from development to
Apoptosis levels lowered when parasitaemia was cleared, the maintenance of body shape and function. There has
but 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 and
patent, the cells most affected by apoptosis were CD41 T external signals. Most of the morphological changes
cells followed by CD81 T cells, and to a lesser extent observed in a cell that is undergoing apoptosis are caused
B2201 B cells. When parasitaemia was cleared, CD81 T by a family of cystein proteases which are activated in
cells and B2201 B cells returned to basal levels of cascade and are known as caspases. These enzymes have
apoptosis, while CD41 T cells still had higher apoptosis restricted protein targets which are normally inactivated
levels than normal mice. A similar pattern of lymphocyte after the caspase exerts its action. In some cases, the target
subpopulation apoptosis was found in infected BALB/c of a caspase is an enzyme inhibitor so the final action of the
mice, 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 the
apoptosis. We consider that the high levels of apoptosis in apoptotic process (pro-apoptotic) and others which suppress
CD41 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 or
of the immune response. resistant to apoptotic signals. One of the first identified
anti-apoptotic proteins was called Bcl-2, which was
Keywords apoptosis, mouse malaria, CD41 T cells, discovered in a B-cell lymphoma. Now we know that
CD81 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 clones
Correspondence: 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
Colonia Santo Tomas, Mexico DF 11340, Mexico. When the antigen is eliminated, the number of T-cells must
Received: 4 January 2001 be down regulated and this is achieved by inducing
Accepted for publication: 29 June 2001 apoptosis in the responding T-cells, a process termed
q 2001 Blackwell Science Ltd 617

2. L.Sanchez-Torres et al. Parasite Immunology
activation-induced cell death. The apoptotic signal is in different malaria mouse models, in humans and in other
received 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 lymphoid
The 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. Besides
responding cells is considered a normal mechanism to suffering changes in size and function, the spleen is also
turn off an ongoing immune response and to avoid self closely related to the development and maintenance of a
damage (3). There are other signals which contribute to protective immune response (18). Using a spleen transplant
activation-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 required
when 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 cell
causing tissue inflammation or alterations in tissue apoptosis in malaria is to study the presence of apoptotic
morphology 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 the
apoptotic signals or biochemical processes and hence use immune system. In this respect, it is also useful to
them to their advantage is attractive as it could help explain, determine how different lymphoid cell populations are
at least in part, how parasites can become established in affected by apoptosis. A recent study (20) investigated
their 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 absolute
described during mouse infection with microorganisms numbers of apoptotic cells in the spleen increased during
such 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 normal
systems are: (i) does the parasitic microorganism use levels after the parasite was cleared. The findings also
apoptotic signals to interfere with the immune response and demonstrated that apoptosis involves T cells (CD41 and
become established in its host or (ii) is the increase in CD81), B cells and macrophages and that the majority of
apoptosis 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 same
third possibility is that the increase in apoptosis is an parasite, and, in general, confirmed the previous results
epiphenomenon, which is not relevant to the host±parasite (20). We also describe changes in spleen size and histology
interaction. 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 we
virus (4) or with Listeria monocytogenes (7), lymphocyte studied apoptosis in lymphocyte subpopulations using a
apoptosis 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 as
cruzi, lymphocyte apoptosis has been interpreted as a way the percentage of apoptotic cells within a particular
to limit host tissue damage by the immune response, but lymphocyte subpopulation, the cells most affected by
which 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. With
apoptosis in macrophages has been identified in Yersinia, this information, the possible role of apoptosis at the
and 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 virulent
factor (12).
MATERIALS AND METHODS
In malaria, there are several phenomena in which
apoptosis of lymphoid cells could be involved. There is a
Mice
very intense proliferation of lymphoid cells (13,14) and
severe 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 parents
part of a normal mechanism to reduce the number of were originally purchased from the Jackson Laboratory
lymphoid cells and, as a consequence, spleen size. On the (Bar Harbor, ME, USA) and hybrids or BALB/c mice were
other hand, general immunosuppression has been described bred in our facilities at the Department of Immunology, in
618 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626

3. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malaria
the National School of Biological Sciences, IPN, Mexico Determination of apoptotic cells by cytofluorometry
City. Experimental mice were maintained in a reversed 12-h
The presence of apoptotic cells was determined in cell
light-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) kit
Parasite and infection (Zymed Laboratories, San Francisco, CA, USA) was used,
according to the manufacturer's instructions. Briefly: spleen
Plasmodium chabaudi chabaudi AS was kindly donated by cells, obtained as described above, were adjusted to
Dr 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 cell
parasite has been syringe passaged from the original suspension. The mixture was incubated for 10 min at room
material no more than eight times and reference populations temperature. After centrifugation, cells were resuspended in
are cryopreserved in liquid nitrogen. For all experiments, 190 ml of binding buffer and 10 ml of propidium iodide
parasites from frozen material were inoculated into young (20 mg/ml) were added. Cells were analysed in a
mice (8±10 weeks old) and when parasitaemias were FACScalibur cytofluorometer (Becton Dickinson, San
patent, experimental mice were inoculated intraperitoneally Jose, CA, USA) using the Cell Quest program (version
with 5 Â 104 parasitized red blood cells. Control mice were 2´0), and 10 000 events per sample were recorded. Cells
inoculated with the same amount of normal red blood cells that were positive for both propidium iodide and Annexin V
as the total amount of red cells received by the infected were considered as necrotic cells, and were excluded from
mice. The parasite causes a synchronical infection and the analysis. The results are expressed as the percentage of
undergoes schizogony every 24 h between 09.00 h and apoptotic cells (^ SD).
11.00 h. Parasitaemia was determined by light microscopy
of methanol-fixed, Giemsa-stained thin blood films, and
was 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 Mannheim
Determination of splenomegaly
GmbH, Mannheim, Germany) was used according to the
A group of mice was infected with P. chabaudi, as manufacturer's instructions. Briefly, wax was removed
described above, and was only used for the determination from sections, they were rehydrated and treated with
of splenomegaly. At different times following infection, proteinase K. The sections were then incubated with
groups of three mice were sacrificed, their spleens extracted terminal deoxinucleotidyl transferase and FITC-labelled
and weighed. At each experimental point, one normal, age nucleotides, which were detected using an alkaline
matched mouse was also killed and its spleen weight phosphatase conjugated anti-FITC antibody, using nitroblue
recorded. A total of eight normal mice were sacrificed tetrazolium as the substrate. Apoptotic cells were stained
during 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 magnetic
A group of mice was infected with the parasite, and at
beads
different times following infection, groups of infected or
control 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 described
From one half, a cell suspension was prepared by above. Spleen cells (107) were resuspended in 90 ml of PBS
expressing the spleen through nylon mesh with the help (containing 2 mm ethylenediaminetetraacetic acid and
of a plastic syringe plunger. The spleen cells were collected 0´5% bovine seric albumin) and 10 ml of the respective
in 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 on
tion of apoptotic cells by cytofluorometry (see below). The ice for 15 min, washed twice, resuspended in 500 ml of
other 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 then
histological 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 were
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4. L.Sanchez-Torres et al. Parasite Immunology
collected. The column was removed from the magnetic 21±38 p.i., when parasitaemia had been cleared. During
field and positive cells were eluted with 1´5 ml of PBS and this time, the level of apoptosis, although low, was still
collected. The purification procedure was repeated using significantly higher than in normal mice (16´7% apoptotic
the 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 different
CD8 monoclonal antibodies and PE-conjugated anti-B220 group of infected mice, which gave a parasitaemia curve
monoclonal antibody. The percentage of apoptotic cells was very similar to the one shown in Figure 1(a) (data not
determined in the purified cells by cytofluorometry, as shown). The spleen started to increase in size early in
described 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 time
Statistical analysis
and at peak parasitaemia it was around 12-fold higher than
Student's t-test was used to assess statistical significance. a normal spleen (12X, P , 0´001). The spleen size reached
P , 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 was
RESULTS
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 a
during P. chabaudi primary infection
size of 2´5X at day 31 p.i. (Figure 1c, P , 0´001). The
The curve of parasitaemia caused by P. chabaudi infection spleen stays around this size for several months after
in 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. chabaudi
mia) to reach a maximum at day 8 p.i. (peak parasitaemia).
primary infection
Then parasitaemia starts to rapidly decline (descending
parasitaemia) and the parasite becomes undetectable by H&E stained sections were prepared from the spleens of
light microscopy around days 17±19 p.i., and stays this mice used to generate the parasitaemia curve shown in
way up to day 38 p.i. (parasitaemia cleared) (Figure 1a). In Figure 1(a). A section from a normal spleen is shown in
this study, parasitaemia has been considered to be cleared at Figure 2(a), where normal distribution and aspect of red
this time; however, the presence of the parasite can be and white pulp is observed. There were a few lymphoid
demonstrated by subinoculation of blood into normal mice cells with picnotic nuclei (0´2±0´5 per field, using a  40
up to days 60 or 70 p.i. After this time, parasites cannot be objective) which were considered to be apoptotic cells since
demonstrated 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 was
determined during P. chabaudi primary infection in the organized and did not show obvious proliferative activity
same 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 the
of 13 normal mice, a background of 7´6% (^ 0´6) apoptotic amount of apoptotic cells. At day 8 p.i., the time of peak
cells was detected. In infected mice, the percentage of parasitaemia, cells in the white pulp had proliferated
apoptotic 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 disappear
When mice were approaching, or had reached peak (Figure 2c). At this time, the spleen had increased
parasitaemia (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) and
apoptotic 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 apoptotic
peak at day 10 p.i., when 53´1% of the spleen cells were cells was detected in cell suspensions, total tissue
found to be apoptotic (P , 0´01). At this time, descending disorganization was observed. This disorganization was
parasitaemia had already started, although parasitaemia was due mainly to hiperplasia of the lymphoid tissue, the red
still high (Figure 1a). After this peak of apoptotic cells, pulp had almost disappeared (Figure 2d) and apoptotic cells
apoptosis 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. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malaria
but it was still not at its peak. The time of peak of apoptotic cells and clusters, although still high (around
splenomegaly 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 splenomegaly
reorganize although it is still hyperplasic (Figure 2e). The was subdued, white and red pulp are clearly separated again
red 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.).
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6. L.Sanchez-Torres et al. Parasite Immunology
622 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626

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 stages
Figure 3 Apoptotic cells detected by Annexin V binding in of parasitaemia (Figure 4a,b,d,e). In CB6F1 mice, the
subpopulations of spleen lymphocytes purified by magnetic beads and percentage of both CD41 and CD81 T cells tended to
analysed by citofluorometry. The cells were determined in 3±6 CB6F1 return to normal levels when the parasitaemia has been
or 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 stayed
18% desending parasitaemia (DESC) or mice 3 days after parasitaemia
had 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.
DISCUSSION
subpopulation in the unseparated cell suspension was also
determined. 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, and
increase in apoptotic cells in all three stages of para- related our findings to parasitaemia and changes in spleen
sitaemia, both for CB6F1 and BALB/c mice. In CB6F1 size and histology. When parasites just started to be
mice, the level of CD41 apoptotic T cells approached detectable by light microscopy there were no noticeable
normal levels when the parasitaemia was cleared (Fig- changes in spleen histology (Figure 2b), size (Figure 1c) or
ure 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) which
CB6F1 mice. For BALB/c mice, 43´3% of CD41 T cells correlates with an increase in spleen size (Figure 1c) and
were apoptotic at descending parasitaemia (parasitaemia the number of apoptotic cells had started to increase
Figure 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 and
red 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 to
disappear; (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 have
invaded 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. The
white and red pulp are again clearly separated but the white pulp shows clusters of material sorrounded by a clear area (arrows); (G) Magnification of
the 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 apoptotic
cell, and other clusters, which are larger, include apoptotic cells and cell debris (seven such clusters are shown); (H) TUNEL reaction of the clusters
shown 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. 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, these
PERCENT 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 takes
Figure 4 Percent of lymphocyte subpopulations in spleen cells from
approximately only 1 h to complete and the beads do not
normal mice (NL), infected mice with 5±15% of ascending
parasitaemia (ASC), peak parasitaemia (PEAK), 2±18% desending interfere with cytofluorometric analysis. At all the studied
parasitaemia (DESC) or mice 3 days after parasitaemia had become stages of patent parasitaemia, CD41 and CD81 T cells had
undetectable by light microscopy (cleared, CLD). Bars are 1 SD; ND, high percentages of apoptotic cells (Figure 3a,b). When
not 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 apoptotic
moderately (Figure 1b). The peak of apoptotic cells was CD41 T cells, although diminished, were still significantly
found at day 10 p.i., when there was more than 50% greater in number compared to normal mice. The
apoptosis. Although parasite growth was already seen to be percentage of total CD41 and CD81 T cells was found to
under 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 apoptotic
apoptotic 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 total
fact 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 apoptosis
possible explanation for the presence of apoptotic cells at were T lymphocytes, particularly CD41 T cells, from
this time of the infection is that they are induced, at least in which approximately 50% were apoptotic at peak and
part, by parasite products, as has been described for descending parasitaemia, compared to approximately 2% in
P. falciparum malaria (23). Spleen size reached a peak at normal mice, while the least affected population comprised
day 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 was
The percentage of apoptotic cells had diminished, although also studied in mice infected with the same Plasmodium
it was still high (approximately 30%, Figure 1b). We strain that we used, except that in that study BALB/c mice
believe that at this stage apoptosis is related to the turning were used instead of CB6F1 mice. Using Annexin V to
off of the antiparasite immune response, since after this detect apoptotic cells by cytofluorometry, these authors
time the spleen size diminished rapidly, red and white pulp reported results very similar to ours in terms of percentage
became 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 of
reduced to a near normal size (day 38 p.i.), the white and apoptotic cells during patent parasitaemia are B cells, while
624 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626

9. Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malaria
apoptosis in CD41 and CD81 T cells is only moderately Listeria monocytogenes (7) and Yersinia pseudotuberculosis
increased. 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 of
difference lies in the different experimental approach that the lymphoid cells, is a general phenomenon in a variety of
we took. They analysed unseparated spleen cell suspensions infections. In principle, this can be explained as a normal
by double staining. Apoptotic cells were stained with homeostatic response involved in the termination of the
Annexin 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 that
surface marker antibody labelled with a second fluoro- the peak of apoptotic cells is reached when there are still
chrome. They performed a cytofluorometric analysis of the relatively high parasite loads, which, from our point of
sample using one label to identify and quantify the view, is still too early a time to terminate the immune
apoptotic cells and then, using the second label, determined response. On the other hand, it is well established that
within the apoptotic population the percentage of cells CD41 T cells are important in the control P. chabaudi
carrying a particular marker (i.e. CD41). In this way, they primary infection (26,27). Other authors working with
did not determine the percentage of cells with that another malaria mouse model (P. berghei) have described
particular marker that are not part of the nonapoptotic P. berghei specific CD41 T cells that are specifically
population. 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 this
subpopulation and then determine the percentage of deletion is mediated by apoptosis (28). This, together with
apoptotic cells within that purified subpopulation. This our observation that approximately 50% of CD41 T cells
gave us an idea of how apoptosis affects a particular undergo apoptosis during P. chabaudi infection, provides
lymphocyte subpopulation. In this way, we could see that sufficient data to stimulate the study of lymphocyte
CD41 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 possibility
that the difference between ours and the previous study (20) ACKNOWLEDGEMENTS
is explained by the use of different mouse strains. We Â
This work was supported by Coordinacion General de
therefore infected BALB/c mice with P. chabaudi, and Â
Posgrado e Investigacion del IPN. Luis Favila-Castillo is
determined the percentage of apoptotic cells in purified fellow from COFAA-IPN, EDD-IPN and SNI-SEP. Luvia
lymphocyte subpopulations in the same way as we did for   Â
Sanchez-Torres, Andrea Rodrõguez-Ropon and Maribel
CB6F1 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 a
CB6F1 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 the
CD81 T cells. The variations in the percentage of spleen use of the English language.
CD41, CD81 T cells and B2201 B cells during
parasitaemia were also similar between BALB/c and
CB6F1 mice (Figure 4a±f). We therefore conclude that, REFERENCES
during P. chabaudi infection, the cells most affected by 1 Kerr JFR, Willie AH, Currie AR. Apoptosis: a basic biological
apoptosis 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 in
described in mice infected with Cytomegalovirus (5), the immune system: turning lymphocytes off. Science 1998;
lymphocytic choriomeningitis virus (4), T. cruzi (10), 280: 243±248.
Toxoplasma gondii (9), Schistosoma mansoni (11) and 4 Razvi ES, Jiang Z, Woda BA, Welsh RM. Lymphocyte apoptosis
Mycobacterium avium (8). An increase in apoptosis of B during the silencing of the immune response to acute viral
cells has been reported in mice infected with cytomegalo- infections in normal, lpr and Bcl-2-transgenic mice. Am J Pathol
1995; 147: 79±91.
virus (5) and lymphocytic choriomeningitis virus (4). In
5 Yoshida H, Sumichika H, Hamano S, He X, Minamishima Y,
other infections, the phenotype of the apoptotic cells has not Kimura G, Nomoto K. Induction of apoptosis of T cells by
been studied, but apoptosis of lymphoid cells has been infecting mice with murine cytomegalovirus. J Virol 1995; 69:
reported in mice infected with Rickettsia tsutsugamushi (6), 4769±4775.
q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626 625

10. L.Sanchez-Torres et al. Parasite Immunology
6 Kasuya S, Nagano I, Ikeda T, Chitoshi G, Shimokawa K, Takahashi Series No. 1. Role of the Spleen in the Immunology of Parasitic
Y. Apoptosis of lymphocytes in mice induced by infection with Diseases. Basel: Schwabe; 1979 183±204.
Rickettsia tsutsugamushi. Infect Immun 1996; 64: 3937±3941. 19 Favila-Castillo L, Monroy-Ostria A, Kobayashi E, Hirunpe-
7 Fuse Y, Nishimura H, Maeda K, Yoshikai Y. CD95 (Fas) charat C, Kamada N, Good MF. Protection of rats against
may control the expansion of activated T cells after malaria by a transplanted immune spleen. Parasite Immunol
elimination of bacteria in murine listeriosis. Int Immunol 1997; 1996; 18: 325±331.
65: 1883±1891. 20 Helmby H, Jonsson G, Troye-Blomberg M. Cellular changes and
8 Gilbertson B, Zhong J, Cheers C. Anergy, IFN-g production and apoptosis in the spleens and peripheral blood of mice infected with
apoptosis in terminal infection of mice with Mycobacterium blood-stage Plasmodium chabaudi chabaudi AS. Infect Immun
avium. J Immunol 1999; 163: 2073±2080. 2000; 68: 1485±1490.
9 Liesenfeld O, Kosek JC, Suzuki Y. Gamma interferon induces 21 Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A
Fas-dependant apoptosis of Peyer's patch T cells in mice novel assay for apoptosis. Flow cytometric detection of phospha-
following peroral infection with Toxoplasma gondii. Infect Immun tidylserine expression on early apoptotic cells using fluorescein
1997; 65: 4682±4689. labeled annexin V. J Immunol Meth 1995; 184: 39±51.
10 Lopes MF, Veiga VF, Santos AR, Fonseca MEF, DosReis GA. 22 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of
Activation-induced CD41 T cell death by apoptosis in experi- programmed cell death in situ via specific labeling of nuclear
mental Chagas disease. J Immunol 1995; 154: 744±752. DNA fragmentation. J Cell Biol 1980; 119: 493±501.
11 Fallon PG, Smith P, Dunne DW. Type 1 and type 2 cytokine- 23 Â Â
Toure-Balde A, Sarthou JL, Aribot G et al. Plasmodium
producing mouse CD41 and CD81 T cells in acute Schistosoma falciparum induces apoptosis in human mononuclear cells. Infect
mansoni infection. Eur J Immunol 1998; 28: 1408±1416. Immun 1996; 64: 744±750.
12 Monack DM, Mecsas J, Bouley JD, Falkow S. Yersinia-induced 24 Theerapol S, Zhang Y, Kluge JP, Halbur PG, Paul PS. A pneumo-
apoptosis in vivo aids in the establishment of a systemic infection virulent United States strain of porcine reproductive and respiratory
of mice. J Exp Med 1998; 188: 2127±2137. virus induces apoptosis in bystander cells both in vitro and in vivo. J
13 Freeman RR, Parish R. Polyclonal B cell activation during rodent Gen Virol 1998; 71: 2989±2995.
malarial infections. Clin Exp Immunol 1978; 32: 41±45. 25 Rodriguez-Ropon A, Sanchez-Torres L, Favila-Castillo L,
14 Jayawardena AN, Targett GAT, Leuchars E, Carter RL, Doenhoff Estrada-Parra S, Hernandez-Jauregui P. Porcine rubulavirus causes
MJ, Davies AJS. T cell activation in murine malaria. Nature 1975; apoptosis in lymph nodes and increases CD81 peripheral
258: 149±151. lymphocytes in piglets. Submitted.
15 Greenwood BM, Playfair JHL, Torrigiani G. Immunosuppression 26 Langhorne J, Simon-Haarhaus B, Meding SJ. The role of CD41 T
in murine malaria: I. General characteristics. Clin Exp Immunol cells in the protective immune response to Plasmodium chabaudi
1971; 8: 467±478. in vivo. Immunol Lett 1990; 25: 101±107.
16 Rockett KA, Awburn MM, Rockett EJ, Cowden WB, Clark IA. 27 Taylor-Robinson AW, Phillips RS, Severn A, Moncada S, Liew
Possible role of nitric oxide in malarial immunosuppression. FY. The role of TH1 and TH2 cells in a rodent malaria infection.
Parasite Immunol 1994; 16: 243±249. Science 1993; 260: 1931±1934.
17 Russo DM, Weidanz WP. Activation of antigen-specific sup- 28 Hirunpetcharat C, Good MF. Deletion of Plasmodium berghei-
pressor T cell by the intravenous injection of soluble blood-stage specific CD41 T cells adoptively transferred into recipient mice
malarial antigens. Cell Immunol 1988; 115: 437±446. after challenge with homologous parasite. Proc Natl Acad Sci USA
18 Wyler DJ, Oster CN, Quinn TC. In Tropical Diseases Research 1998; 95: 1715±1720.
626 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626