parasite immunology

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

The presence and phenotype of apoptotic lymphocytes was

studied in spleen cell suspensions taken from CB6F1 mice

infected with Plasmodium chabaudi chabaudi AS. High

levels of apoptotic cells were found, associated with high

parasitaemias and splenomegaly. This was also accompa-

nied by expansion and disarray of spleen white pulp.

Apoptosis levels lowered when parasitaemia was cleared,

but were still higher than in normal mice. At this time, the

spleen was diminishing in size and the white pulp was

contracting and rearranging. When parasitaemia was

patent, the cells most affected by apoptosis were CD41 T

cells followed by CD81 T cells, and to a lesser extent

B2201 B cells. When parasitaemia was cleared, CD81 T

cells and B2201 B cells returned to basal levels of

apoptosis, while CD41 T cells still had higher apoptosis

levels than normal mice. A similar pattern of lymphocyte

subpopulation apoptosis was found in infected BALB/c

mice, despite the fact that, for this mouse model, it has been

reported that B cells are the cells that are most affected by

apoptosis. We consider that the high levels of apoptosis in

CD41 T cells when parasitaemias are still high are not

easily explained by a normal mechanism of down regulation

of the immune response.

Keywords apoptosis, mouse malaria, CD41 T cells,

CD81 T cells, B cells, Plasmodium chabaudi

INTRODUCTION

Apoptosis is a natural mechanism of cell death, which

involves membrane cell blebbing, cell shrinkage, chromatin

condensation, nuclear fragmentation and DNA degradation

(1). This mechanism, which has been also called pro-

grammed cell death, is essential for the homeostasis of the

whole organism and has a central role from development to

the maintenance of body shape and function. There has

been major advances in the understanding of the biochem-

ical events that underlie the apoptotic process (2).

Apoptosis is triggered by a variety of both internal and

external signals. Most of the morphological changes

observed in a cell that is undergoing apoptosis are caused

by a family of cystein proteases which are activated in

cascade and are known as caspases. These enzymes have

restricted protein targets which are normally inactivated

after the caspase exerts its action. In some cases, the target

of a caspase is an enzyme inhibitor so the final action of the

caspases is the activation of the enzyme.

Inside the cell, there are proteins which facilitates the

apoptotic process (pro-apoptotic) and others which suppress

it (anti-apoptotic). The relative amount of pro- and anti-

apoptotic proteins determines if a cell is sensitive or

resistant to apoptotic signals. One of the first identified

anti-apoptotic proteins was called Bcl-2, which was

discovered in a B-cell lymphoma. Now we know that

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

responding to the antigen undergo extensive proliferation.

When the antigen is eliminated, the number of T-cells must

be down regulated and this is achieved by inducing

apoptosis in the responding T-cells, a process termed

q 2001 Blackwell Science Ltd 617

Correspondence: Luis Favila-Castillo, Department of Immunology,

National School of Biological Sciences, IPN, Carpio y Plan de Ayala,

Colonia Santo TomaÂs, MeÂxico DF 11340, MeÂxico.

Received: 4 January 2001

Accepted for publication: 29 June 2001

activation-induced cell death. The apoptotic signal is

received by the T-cell through a membrane receptor called

CD95 or Fas, which is a type I transmembrane receptor.

The death signal is provided by the ligand of CD95 (CD95L

or FasL), which is expressed on lymphoid cells in the late

phases of the immune response. The elimination of

responding cells is considered a normal mechanism to

turn off an ongoing immune response and to avoid self

damage (3). There are other signals which contribute to

activation-induced cell death, such as a receptor for tumour

necrosis factor (TNF)R2 which transmits apoptotic signals

when bound to its ligand TNF-a. Finally, apoptotic cells are

engulfed by phagocytic cells, eliminating them without

causing tissue inflammation or alterations in tissue

morphology or function.

The idea that parasitic microorganisms might tamper with

apoptotic signals or biochemical processes and hence use

them to their advantage is attractive as it could help explain,

at least in part, how parasites can become established in

their host and how infection becomes chronic.

An increase in apoptosis of lymphoid cells has been

described during mouse infection with microorganisms

such as viruses (4,5), ricketssia (6), bacteria (7,8), protozoa

(9,10) and helminths (11). The relevant questions in these

systems are: (i) does the parasitic microorganism use

apoptotic signals to interfere with the immune response and

become established in its host or (ii) is the increase in

apoptosis during the infection associated with the normal

silencing of the immune response against the parasite? A

third possibility is that the increase in apoptosis is an

epiphenomenon, which is not relevant to the host±parasite

interaction.

In mice infected with the lymphocytic choriomeningitis

virus (4) or with Listeria monocytogenes (7), lymphocyte

apoptosis has been associated with silencing of the immune

response. Whereas, in mice infected with Trypanosoma

cruzi, lymphocyte apoptosis has been interpreted as a way

to limit host tissue damage by the immune response, but

which collaterally promotes the establishment of a chronic

infection (10). On the other hand, a protein that induces

apoptosis in macrophages has been identified in Yersinia,

and it has been shown that when Yersinia is inoculated into

mice the expression of this protein is an important virulent

factor (12).

In malaria, there are several phenomena in which

apoptosis of lymphoid cells could be involved. There is a

very intense proliferation of lymphoid cells (13,14) and

severe splenomegaly, and these events are subdued when

parasitaemia is controlled. In both cases, apoptosis could be

part of a normal mechanism to reduce the number of

lymphoid cells and, as a consequence, spleen size. On the

other hand, general immunosuppression has been described

in different malaria mouse models, in humans and in other

animal models, where the infection becomes chronic (15±

17). In these latter two situations, apoptosis of lymphoid

cells might help explain the development of immunosup-

pression and chronicity.

The organ most affected in malaria is the spleen. Besides

suffering changes in size and function, the spleen is also

closely related to the development and maintenance of a

protective immune response (18). Using a spleen transplant

technique in a malaria rat model, it has been shown that,

after primary infection, the spleen develops all the required

elements to protect itself against reinfection (19).

One way to understand the role of lymphoid cell

apoptosis in malaria is to study the presence of apoptotic

lymphoid cells during different stages of infection, and to

relate any changes to other physiological alterations in the

immune system. In this respect, it is also useful to

determine how different lymphoid cell populations are

affected by apoptosis. A recent study (20) investigated

apoptosis in mouse spleen cells infected with P. chabaudi

chabaudi AS. It was found that the frequency and absolute

numbers of apoptotic cells in the spleen increased during

primary infection, reached a peak around 4 days following

peak parasitaemia and then descended to almost normal

levels after the parasite was cleared. The findings also

demonstrated that apoptosis involves T cells (CD41 and

CD81), B cells and macrophages and that the majority of

apoptotic cells are B cells.

Here, we carried out a similar study using the same

parasite, and, in general, confirmed the previous results

(20). We also describe changes in spleen size and histology

during the infection, and correlate these changes with

apoptosis of spleen cells. Finally, it should be noted that we

studied apoptosis in lymphocyte subpopulations using a

different experimental approach to the previous study (20).

We therefore conclude that when apoptosis is expressed as

the percentage of apoptotic cells within a particular

lymphocyte subpopulation, the cells most affected by

apoptosis are CD41 T cells, followed by CD81 T cells,

while B2201 B cells are proportionally less affected. With

this information, the possible role of apoptosis at the

different stages of infection is discussed.

MATERIALS AND METHODS

Mice

Female (BALB/cXC57Bl/6) F1 hybrids or BALB/c 14±22-

week-old mice were used for all experiments. The parents

were originally purchased from the Jackson Laboratory

(Bar Harbor, ME, USA) and hybrids or BALB/c mice were

bred in our facilities at the Department of Immunology, in

L.Sanchez-Torres et al. Parasite Immunology

618 q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626

the National School of Biological Sciences, IPN, Mexico

City. Experimental mice were maintained in a reversed 12-h

light-dark cycle from 07.00 h to 19.00 h.

Parasite and infection

Plasmodium chabaudi chabaudi AS was kindly donated by

Dr David Walliker (University of Edinburgh, Edinburgh,

UK), and is a cloned and mosquito-transmitted line. The

parasite has been syringe passaged from the original

material no more than eight times and reference populations

are cryopreserved in liquid nitrogen. For all experiments,

parasites from frozen material were inoculated into young

mice (8±10 weeks old) and when parasitaemias were

patent, experimental mice were inoculated intraperitoneally

with 5 � 104 parasitized red blood cells. Control mice were

inoculated with the same amount of normal red blood cells

as the total amount of red cells received by the infected

mice. The parasite causes a synchronical infection and

undergoes schizogony every 24 h between 09.00 h and

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 splenomegaly

A group of mice was infected with P. chabaudi, as

described above, and was only used for the determination

of splenomegaly. At different times following infection,

groups of three mice were sacrificed, their spleens extracted

and weighed. At each experimental point, one normal, age

matched mouse was also killed and its spleen weight

recorded. A total of eight normal mice were sacrificed

during the experiment and all resulting data were pooled.

Processing of spleens from infected and control mice

A group of mice was infected with the parasite, and at

different times following infection, groups of infected or

control mice were sacrificed, their spleens extracted

(always between 13.00 h and 14.00 h) and cut in half.

From one half, a cell suspension was prepared by

expressing the spleen through nylon mesh with the help

of a plastic syringe plunger. The spleen cells were collected

in cold phosphate-buffered saline (PBS), centrifuged,

resuspended in PBS, counted and used for the determina-

tion of apoptotic cells by cytofluorometry (see below). The

other spleen half was fixed in 10% formalin in PBS,

embedded in paraffin-wax and processed by standard

histological techniques used to prepare and stain 4 mm

sections with haematoxilin and eosin (H&E).

Determination of apoptotic cells by cytofluorometry

The presence of apoptotic cells was determined in cell

suspensions by their ability to bind Annexin V (21). The

Apodetect Annexin V-fluorescein-isothiocyanate (FITC) kit

(Zymed Laboratories, San Francisco, CA, USA) was used,

according to the manufacturer's instructions. Briefly: spleen

cells, obtained as described above, were adjusted to

5 � 105 per ml in binding buffer and 10 ml of FITC-

Annexin V were added to a 190-ml sample of the cell

suspension. The mixture was incubated for 10 min at room

temperature. After centrifugation, cells were resuspended in

190 ml of binding buffer and 10 ml of propidium iodide

(20 mg/ml) were added. Cells were analysed in a

FACScalibur cytofluorometer (Becton Dickinson, San

Jose, CA, USA) using the Cell Quest program (version

2´0), and 10 000 events per sample were recorded. Cells

that were positive for both propidium iodide and Annexin V

were considered as necrotic cells, and were excluded from

the analysis. The results are expressed as the percentage of

apoptotic cells (^ SD).

Determination of apoptotic cells in sections

Apoptotic cells were detected by the TUNEL reaction (22).

An in situ cell death detection kit (Boehringer Mannheim

GmbH, Mannheim, Germany) was used according to the

manufacturer's instructions. Briefly, wax was removed

from sections, they were rehydrated and treated with

proteinase K. The sections were then incubated with

terminal deoxinucleotidyl transferase and FITC-labelled

nucleotides, which were detected using an alkaline

phosphatase conjugated anti-FITC antibody, using nitroblue

tetrazolium as the substrate. Apoptotic cells were stained

blue and sections were then counterstained with nuclear

red, dehydrated and mounted in synthetic resin.

Purification of lymphocyte subpopulations by magnetic

beads

CD41 or CD81 T cells or B2201 B cells were purified

from total spleen cell suspensions, prepared as described

above. Spleen cells (107) were resuspended in 90 ml of PBS

(containing 2 mm ethylenediaminetetraacetic acid and

0´5% bovine seric albumin) and 10 ml of the respective

paramagnetic bead bound antibody (Miltenyi Biotec,

Aarhus, Denmark) were added. Cells were incubated on

ice for 15 min, washed twice, resuspended in 500 ml of

PBS and loaded onto a positive selection column (MS1/

RS1 MACS column, Miltenyi). The column was then

exposed to a strong magnetic field (VarioMacs, Miltenyi).

Negative cells were eluted with 1´5 ml of PBS and were

Volume 23, Number 12, December 2001 T cell apoptosis in P. chabaudi malaria

q 2001 Blackwell Science Ltd, Parasite Immunology, 23, 617±626 619

collected. The column was removed from the magnetic

field and positive cells were eluted with 1´5 ml of PBS and

collected. The purification procedure was repeated using

the positive cells. The purity of the cells was assessed by

cytofluorometry using FITC-conjugated anti-CD4 or anti-

CD8 monoclonal antibodies and PE-conjugated anti-B220

monoclonal antibody. The percentage of apoptotic cells was

determined in the purified cells by cytofluorometry, as

described above.

Statistical analysis

Student's t-test was used to assess statistical significance.

P , 0´05 was considered statistically significant.

RESULTS

Apoptosis of spleen cells and changes in spleen size

during P. chabaudi primary infection

The curve of parasitaemia caused by P. chabaudi infection

in CB6F1 mice has four distinct zones. Four days after

inoculation, parasites start to be detected by light micro-

scopy and numbers sharply increase (ascending parasitae-

mia) to reach a maximum at day 8 p.i. (peak parasitaemia).

Then parasitaemia starts to rapidly decline (descending

parasitaemia) and the parasite becomes undetectable by

light microscopy around days 17±19 p.i., and stays this

way up to day 38 p.i. (parasitaemia cleared) (Figure 1a). In

this study, parasitaemia has been considered to be cleared at

this time; however, the presence of the parasite can be

demonstrated by subinoculation of blood into normal mice

up to days 60 or 70 p.i. After this time, parasites cannot be

demonstrated in blood by subinoculation (data not shown).

The percentage of apoptotic cells in the spleen was

determined during P. chabaudi primary infection in the

same mice used to generate the parasitaemia curve

(Figure 1a). Results are shown in Figure 1(b). In a group

of 13 normal mice, a background of 7´6% (^ 0´6) apoptotic

cells was detected. In infected mice, the percentage of

apoptotic cells at the beginning of ascending parasitaemia

(days 2 and 4 p.i.) was not different from normal controls.

When mice were approaching, or had reached peak

parasitaemia (days 7 and 8 p.i.), there was a small but

statistically significant increase in apoptotic cells (15´2%

apoptotic cells, day 7 p.i., P , 0´001). After this time, the

percentage of apoptotic cells increased steadily to reach a

peak at day 10 p.i., when 53´1% of the spleen cells were

found to be apoptotic (P , 0´01). At this time, descending

parasitaemia had already started, although parasitaemia was

still high (Figure 1a). After this peak of apoptotic cells,

apoptosis started to decline and reached a plateau from days

21±38 p.i., when parasitaemia had been cleared. During

this time, the level of apoptosis, although low, was still

significantly higher than in normal mice (16´7% apoptotic

cells, day 38 p.i., P , 0´001).

Changes in spleen size were determined in a different

group of infected mice, which gave a parasitaemia curve

very similar to the one shown in Figure 1(a) (data not

shown). The spleen started to increase in size early in

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

and at peak parasitaemia it was around 12-fold higher than

a normal spleen (12X, P , 0´001). The spleen size reached

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

around 0´01% (Figure 1a). After this time, the size of the

spleen decreased sharply to become 6´5X at day 16 p.i.

(parasitaemia cleared) and continued to decrease to reach a

size of 2´5X at day 31 p.i. (Figure 1c, P , 0´001). The

spleen stays around this size for several months after

infection (data not shown).

Histological changes in the spleen during P. chabaudi

primary infection

H&E stained sections were prepared from the spleens of

mice used to generate the parasitaemia curve shown in

Figure 1(a). A section from a normal spleen is shown in

Figure 2(a), where normal distribution and aspect of red

and white pulp is observed. There were a few lymphoid

cells with picnotic nuclei (0´2±0´5 per field, using a � 40

objective) which were considered to be apoptotic cells since

they were TUNEL positive (see below). At day 4 p.i., when

parasites just started to be detected, the white pulp was

organized and did not show obvious proliferative activity

(Figure 2b). In general, no differences in histology

compared to normal spleens were detected, including the

amount of apoptotic cells. At day 8 p.i., the time of peak

parasitaemia, cells in the white pulp had proliferated

considerably, such that white pulp had enlarged and the

limits between white and red pulp started to disappear

(Figure 2c). At this time, the spleen had increased

considerably in size, although it had not reached its peak.

The amount of apoptotic cells increased (2±3 per field) and

clusters of apoptotic cells started to appear. At day 10 p.i.,

which is the time when the peak in numbers of apoptotic

cells was detected in cell suspensions, total tissue

disorganization was observed. This disorganization was

due mainly to hiperplasia of the lymphoid tissue, the red

pulp had almost disappeared (Figure 2d) and apoptotic cells

were abundant (10±20 per field). Spleen size was increased,



but it was still not at its peak. The time of peak

splenomegaly is day 14 p.i., which is when parasitaemia

is being resolved, and the white pulp has started to

reorganize although it is still hyperplasic (Figure 2e). The

red pulp started to be distinguishable again and the amount

of apoptotic cells and clusters, although still high (around

10 per field) was lower than at day 10 p.i. At day 38 p.i.,

when parasitaemia has been controlled and splenomegaly

was subdued, white and red pulp are clearly separated again

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

spleens and started to appear in the spleens of infected mice

until day 19 p.i. (not shown) but were much more abundant

at day 38 p.i. At higher magnification (� 100 objective),

these structures were conglomerates of apoptotic cells,

bodies and cell debris, apparently inside a macrophage

(Figure 2g). All these structures were TUNEL positive, as

were individual cells with condensed chromatin which were

probably those recognized as apoptotic in H&E stained

sections (Figure 2h).

Apoptosis in lymphocytes subpopulations

Our results show that there is an increase in apoptotic cells

in the spleen during P. chabaudi primary infection. We next

studied apoptosis at the level of lymphocyte subpopulation

at different stages of infection. For this, we determined the

percentage of apoptotic cells in CD41 and CD81 T cells

and B2001 B cells, purified by antibody coated magnetic

beads, as described in Materials and Methods.

We prepared a purified spleen cell subpopulation from

normal and infected CB6F1 or BALB/c mice at high

ascending parasitaemias, when the number of apoptotic

cells started to rise significantly (Figure 1b), from peak

parasitaemia or from descending parasitaemia. For CB6F1

mice, samples were also studied a few days following

parasitaemia clearance. For all experiments, the percentage

of apoptotic cells in the unseparated whole spleen cell

suspension was also determined, and it was found to be

within the range predicted by the curve shown in

Figure 1(b) (data not shown). The percentage of each cell

0 5 10 15 20 25 30 35 400·001

0·01

0·1

1

10

100

% P

ara

site

mia

0 5 10 15 20 25 30 35 400

60

70

80

50

40

30

20

10

% A

po

pto

tic

cells

Sp

leen

weig

ht

(g)

0 5 10 15 20 25 30 35 400·0

0·2

0·4

0·6

0·8

1·0

1·2

1·4

1·6

Days postinfection

(a)

(b)

(c)

Figure 1 CB6F1 mice were infected with P. chabaudi. (a) Resulting

parasitaemias were followed in six mice and expressed as percent

parasitaemia (mean ^ SD). (b) Apoptosis in spleen cell suspensions of

mice infected with P. chabaudi, determined by Annexin V binding. The

percentage of apoptotic cells was determined in mice from the same

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),

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

indicated times, in a different group of mice than in a (mean ^SD). The

open triangle in day 1 is the mean weight of eight normal spleens.

Infected mice had significantly bigger spleens from days 4±31 p.i.

(P , 0´05 at day 4 p.i. and P , 0´001 from day 7 p.i. to day 31 p.i.).



subpopulation in the unseparated cell suspension was also

determined.

For CD41 T cells, we found an important and significant

increase in apoptotic cells in all three stages of para-

sitaemia, both for CB6F1 and BALB/c mice. In CB6F1

mice, the level of CD41 apoptotic T cells approached

normal levels when the parasitaemia was cleared (Fig-

ure 3a). At peak parasitaemia, 48´3% of CD41 T cells were

apoptotic in CB6F1 mice compared to 2´3% in normal

CB6F1 mice. For BALB/c mice, 43´3% of CD41 T cells

were apoptotic at descending parasitaemia (parasitaemia

was 7±11% in the sampled mice) compared to 2´7% in

normal BALB/c mice.

For CD81 T cells, there was also a significant increase in

apoptotic cells in all the three stages of parasitaemia, both

for CB6F1 and BALB/c mice. In CB6F1 mice, the level of

CD81 apoptotic T cells approached normal levels when the

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,

19´0% of CD81 T cells were apoptotic and, for BALB/c

mice, 23´9% were apoptotic, both at peak parasitaemia,

compared to 2´6% in normal mice for both mouse strains.

For B2201 B cells, although there was a tendency for the

percentage of apoptotic cells to increase during patent

parasitaemia for both CB6F1 and BALB/c mice; the

difference was significant only at descending parasitaemia.

For CB6F1 mice, the percentage of apoptotic B2201 B

cells returned to normal levels when parasitaemia was

cleared (Figure 3c,f).

The percentage of CD41 and CD81 T cells in the spleen

diminished during infection, both in CB6F1 and in BALB/c

mice, following a similar pattern during the different stages

of parasitaemia (Figure 4a,b,d,e). In CB6F1 mice, the

percentage of both CD41 and CD81 T cells tended to

return to normal levels when the parasitaemia has been

cleared (Figure 4a,b).

The percentage of B2201 B cells increased or stayed

within normal range during the different stages of para-

sitaemia, both in CB6F1 and BALB/c mice (Figure 4c,f).

DISCUSSION

In this study, we investigated apoptosis of lymphocytes at

different stages of P. chabaudi infection in mice, and

related our findings to parasitaemia and changes in spleen

size and histology. When parasites just started to be

detectable by light microscopy there were no noticeable

changes in spleen histology (Figure 2b), size (Figure 1c) or

percentage of apoptotic cells (Figure 1b). At peak para-

sitaemia, the white pulp had expanded (Figure 2c) which

correlates with an increase in spleen size (Figure 1c) and

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) � 200; (F) � 100; (G±H) � 1000.

0NL

CB6F1 mice

CD4+ T cells

ASC PEAK DESC CLD

10

20

30

40

50

60

(a)

∗∗

∗∗

∗∗∗∗

0NL

BALB/c mice

CD4+ T cells

ASC PEAK DESC CLD

ND10

20

30

40

50

60

(d)

∗∗

∗

∗∗

0NL

CD8+ T cells

ASC PEAK DESC CLD

ND10

20

30

40

50

60

(e)

∗∗∗ ∗∗

0NL

B220+ B cells

ASC PEAK DESC CLD

ND10

20

30

40

50

60

(f )

∗

0NL

CD8+ T cells

ASC PEAK DESC CLD

10

20

30

% A

po

pto

tic

cells

40

50

60

(b)

∗∗∗∗ ∗

0NL

B220+ B cells

ASC PEAK DESC CLD

10

20

30

40

50

60

(c)

∗

Figure 3 Apoptotic cells detected by Annexin V binding in

subpopulations of spleen lymphocytes purified by magnetic beads and

analysed by citofluorometry. The cells were determined in 3±6 CB6F1

or BALB/c mice per group of normal mice (NL), infected mice with 5±

15% of ascending parasitaemia (ASC), peak parasitaemia (PEAK), 2±

18% desending parasitaemia (DESC) or mice 3 days after parasitaemia

had become undetectable by light microscopy (cleared, CLD). Bars are

1 SD; ND, not determined, *P , 0´05, **P , 0´01, compared to

normal mice.



moderately (Figure 1b). The peak of apoptotic cells was

found at day 10 p.i., when there was more than 50%

apoptosis. Although parasite growth was already seen to be

under control, parasitaemia was still high (approximately

20%), we therefore do not believe that this peak of

apoptotic cells can be interpreted as a mechanism for

turning off the immune response. This is supported by the

fact that splenomegaly had not reached its peak and the

spleen histology was in total disarray (Figure 2d). One

possible explanation for the presence of apoptotic cells at

this time of the infection is that they are induced, at least in

part, by parasite products, as has been described for

P. falciparum malaria (23). Spleen size reached a peak at

day 14 p.i. and at this time parasitaemia was low, and the

white and red pulp had started to reorganize (Figure 2e).

The percentage of apoptotic cells had diminished, although

it was still high (approximately 30%, Figure 1b). We

believe that at this stage apoptosis is related to the turning

off of the antiparasite immune response, since after this

time the spleen size diminished rapidly, red and white pulp

became clearly separated again and parasites became

undetectable by light microscopy. When the spleen had

reduced to a near normal size (day 38 p.i.), the white and

red pulp were seen to be clearly separated. However, we

found clusters of apoptotic cells and bodies in the white

pulp, which were easily recognized since they were less

dense that the surrounding tissue. These structures, which

are shown in more detail in Figure 2(g,h), have been

described in other pathologies as tingible body macro-

phages, which are macrophages that have engulfed

apoptotic cells and bodies (24). In this study, these

structures were detected as being clearly TUNEL positive

(Figure 2h). We do not know their significance, although it

is interesting that they started to appear rather late in the

infection (day 19 p.i.) and were very abundant at day 38 p.i.

(Figure 2f). We have observed the same structures in pig

lymph nodes undergoing a viral infection; however, the

structures appeared much earlier (day 4 p.i) (25).

On the other hand, the identification of apoptotic

cells by Annexin V binding, which detects an early change

in membrane structure preceding DNA fragmentation,

correlated well with the observation of picnotic nuclei, a

late stage of apoptosis, in H&E stained spleen sections.

We also investigated apoptosis in different lymphocyte

subpopulations. Cells were purified by antibody coated

magnetic beads. This is an efficient procedure that takes

approximately only 1 h to complete and the beads do not

interfere with cytofluorometric analysis. At all the studied

stages of patent parasitaemia, CD41 and CD81 T cells had

high percentages of apoptotic cells (Figure 3a,b). When

parasitaemia was cleared, the percentage of apoptotic

CD81 T cells returned to normal levels while apoptotic

CD41 T cells, although diminished, were still significantly

greater in number compared to normal mice. The

percentage of total CD41 and CD81 T cells was found to

diminish during patent parasitaemia (Figure 4a,b,d,e).

During patent parasitaemia, the percentage of apoptotic

B2201 B cells was not greatly modified (Figure 3c,f),

although there was an increase in the percentage of total

B2201 B cells (Figure 4c,f).

In conclusion, the population most affected by apoptosis

were T lymphocytes, particularly CD41 T cells, from

which approximately 50% were apoptotic at peak and

descending parasitaemia, compared to approximately 2% in

normal mice, while the least affected population comprised

the B2201 B cells.

In a previous study (20), apoptosis of spleen cells was

also studied in mice infected with the same Plasmodium

strain that we used, except that in that study BALB/c mice

were used instead of CB6F1 mice. Using Annexin V to

detect apoptotic cells by cytofluorometry, these authors

reported results very similar to ours in terms of percentage

and kinetics of apoptotic cells during primary infection.

However, they reach the conclusion that the majority of

apoptotic cells during patent parasitaemia are B cells, while

CB6F1 mice

0

10

20

30

40

50

60

∗∗

∗∗∗∗

∗

CD4+ T cells

NL ASC PEAK DESC CLD

(a)

0

10

20

30

40

50

60

∗∗∗∗

CD8+ T cells

NL ASC

PE

RC

EN

T O

F LY

MP

HO

CY

TE

SU

BP

OP

ULA

TIO

N

PEAK DESC CLD

(b)

0

10

20

30

40

50

60

∗∗

B220+ B cells


(c)

BALB/c mice

0

10

20

30

40

50

60

∗∗

∗∗∗∗

CD4+ T cells


ND

ND

ND

(d)

0

10

20

30

40

50

60

∗∗∗

CD8+ T cells


(e)

0

10

20

30

40

50

60

∗∗ ∗∗

B220+ B cells


(f)

Figure 4 Percent of lymphocyte subpopulations in spleen cells from

normal mice (NL), infected mice with 5±15% of ascending

parasitaemia (ASC), peak parasitaemia (PEAK), 2±18% desending

parasitaemia (DESC) or mice 3 days after parasitaemia had become

undetectable by light microscopy (cleared, CLD). Bars are 1 SD; ND,

not determined, *P , 0´05, **P , 0´01, compared to normal mice.



apoptosis in CD41 and CD81 T cells is only moderately

increased. At first sight, it seems that we reached opposite

conclusions to their study; however, we believe that the

difference lies in the different experimental approach that

we took. They analysed unseparated spleen cell suspensions

by double staining. Apoptotic cells were stained with

Annexin V labelled with one fluorochrome and the

lymphocyte subpopulation was marked with an anti-cell

surface marker antibody labelled with a second fluoro-

chrome. They performed a cytofluorometric analysis of the

sample using one label to identify and quantify the

apoptotic cells and then, using the second label, determined

within the apoptotic population the percentage of cells

carrying a particular marker (i.e. CD41). In this way, they

did not determine the percentage of cells with that

particular marker that are not part of the nonapoptotic

population.

Our approach was to first purify a particular lymphocyte

subpopulation and then determine the percentage of

apoptotic cells within that purified subpopulation. This

gave us an idea of how apoptosis affects a particular

lymphocyte subpopulation. In this way, we could see that

CD41 T cells comprise the lymphocyte subpopulation most

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)

is explained by the use of different mouse strains. We

therefore infected BALB/c mice with P. chabaudi, and

determined the percentage of apoptotic cells in purified

lymphocyte subpopulations in the same way as we did for

CB6F1 mice, as shown in Figure 3(d±f). Although there are

some minor differences in the results between BALB/c and

CB6F1 mice, the conclusion is that in BALB/c mice the

cells most affected by apoptosis are also CD41 and

CD81 T cells. The variations in the percentage of spleen

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,

during P. chabaudi infection, the cells most affected by

apoptosis are CD41 and CD81 T cells.

An increase in apoptosis of lymphoid cells has been

described for several kinds of infection. Important increases

in apoptosis of CD41 or CD41 plus CD81 T cells have been

described in mice infected with Cytomegalovirus (5),

lymphocytic choriomeningitis virus (4), T. cruzi (10),

Toxoplasma gondii (9), Schistosoma mansoni (11) and

Mycobacterium avium (8). An increase in apoptosis of B

cells has been reported in mice infected with cytomegalo-

virus (5) and lymphocytic choriomeningitis virus (4). In

other infections, the phenotype of the apoptotic cells has not

been studied, but apoptosis of lymphoid cells has been

reported in mice infected with Rickettsia tsutsugamushi (6),

Listeria monocytogenes (7) and Yersinia pseudotuberculosis

(12).

It seems that an increase in apoptosis, in some or most of

the lymphoid cells, is a general phenomenon in a variety of

infections. In principle, this can be explained as a normal

homeostatic response involved in the termination of the

immune response induced by the microorganism (3).

Nevertheless, in our mouse malaria model, we found that

the peak of apoptotic cells is reached when there are still

relatively high parasite loads, which, from our point of

view, is still too early a time to terminate the immune

response. On the other hand, it is well established that

CD41 T cells are important in the control P. chabaudi

primary infection (26,27). Other authors working with

another malaria mouse model (P. berghei) have described

P. berghei specific CD41 T cells that are specifically

deleted when adoptively transferred into recipient mice

challenged with the same parasite. It is suggested that this

deletion is mediated by apoptosis (28). This, together with

our observation that approximately 50% of CD41 T cells

undergo apoptosis during P. chabaudi infection, provides

sufficient data to stimulate the study of lymphocyte

apoptosis in malaria.

ACKNOWLEDGEMENTS

This work was supported by CoordinacioÂn General de

Posgrado e InvestigacioÂn del IPN. Luis Favila-Castillo is

fellow from COFAA-IPN, EDD-IPN and SNI-SEP. Luvia

SaÂnchez-Torres, Andrea RodrõÂguez-RopoÂn and Maribel

Aguilar-Medina were supported by CONACYT Mexico.

Luvia Sanchez-Torres received during part of this study a

scholarship from the Von Behring and Kitasato Foundation,

Mexico City. We thank Dr Edith Ormerod for reviewing the

use of the English language.

REFERENCES

1 Kerr JFR, Willie AH, Currie AR. Apoptosis: a basic biological

phenomenon with wide ranging implications in tissue kinetics.

Br J Cancer 1972; 26: 239±257.

2 Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407:

770±776.

3 Van Parijs LP, Abbas AK. Homeostasis and self-tolerance in

the immune system: turning lymphocytes off. Science 1998;

280: 243±248.

4 Razvi ES, Jiang Z, Woda BA, Welsh RM. Lymphocyte apoptosis

during the silencing of the immune response to acute viral

infections in normal, lpr and Bcl-2-transgenic mice. Am J Pathol

1995; 147: 79±91.

5 Yoshida H, Sumichika H, Hamano S, He X, Minamishima Y,

Kimura G, Nomoto K. Induction of apoptosis of T cells by

infecting mice with murine cytomegalovirus. J Virol 1995; 69:

4769±4775.



6 Kasuya S, Nagano I, Ikeda T, Chitoshi G, Shimokawa K, Takahashi

Y. Apoptosis of lymphocytes in mice induced by infection with

Rickettsia tsutsugamushi. Infect Immun 1996; 64: 3937±3941.

7 Fuse Y, Nishimura H, Maeda K, Yoshikai Y. CD95 (Fas)

may control the expansion of activated T cells after

elimination of bacteria in murine listeriosis. Int Immunol 1997;

65: 1883±1891.

8 Gilbertson B, Zhong J, Cheers C. Anergy, IFN-g production and

apoptosis in terminal infection of mice with Mycobacterium

avium. J Immunol 1999; 163: 2073±2080.

9 Liesenfeld O, Kosek JC, Suzuki Y. Gamma interferon induces

Fas-dependant apoptosis of Peyer's patch T cells in mice

following peroral infection with Toxoplasma gondii. Infect Immun

1997; 65: 4682±4689.

10 Lopes MF, Veiga VF, Santos AR, Fonseca MEF, DosReis GA.

Activation-induced CD41 T cell death by apoptosis in experi-

mental Chagas disease. J Immunol 1995; 154: 744±752.

11 Fallon PG, Smith P, Dunne DW. Type 1 and type 2 cytokine-

producing mouse CD41 and CD81 T cells in acute Schistosoma

mansoni infection. Eur J Immunol 1998; 28: 1408±1416.

12 Monack DM, Mecsas J, Bouley JD, Falkow S. Yersinia-induced

apoptosis in vivo aids in the establishment of a systemic infection

of mice. J Exp Med 1998; 188: 2127±2137.

13 Freeman RR, Parish R. Polyclonal B cell activation during rodent

malarial infections. Clin Exp Immunol 1978; 32: 41±45.

14 Jayawardena AN, Targett GAT, Leuchars E, Carter RL, Doenhoff

MJ, Davies AJS. T cell activation in murine malaria. Nature 1975;

258: 149±151.

15 Greenwood BM, Playfair JHL, Torrigiani G. Immunosuppression

in murine malaria: I. General characteristics. Clin Exp Immunol

1971; 8: 467±478.

16 Rockett KA, Awburn MM, Rockett EJ, Cowden WB, Clark IA.

Possible role of nitric oxide in malarial immunosuppression.

Parasite Immunol 1994; 16: 243±249.

17 Russo DM, Weidanz WP. Activation of antigen-specific sup-

pressor T cell by the intravenous injection of soluble blood-stage

malarial antigens. Cell Immunol 1988; 115: 437±446.

18 Wyler DJ, Oster CN, Quinn TC. In Tropical Diseases Research

Series No. 1. Role of the Spleen in the Immunology of Parasitic

Diseases. Basel: Schwabe; 1979 183±204.

19 Favila-Castillo L, Monroy-Ostria A, Kobayashi E, Hirunpe-

charat C, Kamada N, Good MF. Protection of rats against

malaria by a transplanted immune spleen. Parasite Immunol

1996; 18: 325±331.

20 Helmby H, Jonsson G, Troye-Blomberg M. Cellular changes and

apoptosis in the spleens and peripheral blood of mice infected with

blood-stage Plasmodium chabaudi chabaudi AS. Infect Immun

2000; 68: 1485±1490.

21 Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A

novel assay for apoptosis. Flow cytometric detection of phospha-

tidylserine expression on early apoptotic cells using fluorescein

labeled annexin V. J Immunol Meth 1995; 184: 39±51.

22 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of

programmed cell death in situ via specific labeling of nuclear

DNA fragmentation. J Cell Biol 1980; 119: 493±501.

23 ToureÂ-BaldeÂ A, Sarthou JL, Aribot G et al. Plasmodium

falciparum induces apoptosis in human mononuclear cells. Infect

Immun 1996; 64: 744±750.

24 Theerapol S, Zhang Y, Kluge JP, Halbur PG, Paul PS. A pneumo-

virulent United States strain of porcine reproductive and respiratory

virus induces apoptosis in bystander cells both in vitro and in vivo. J

Gen Virol 1998; 71: 2989±2995.

25 Rodriguez-Ropon A, Sanchez-Torres L, Favila-Castillo L,

Estrada-Parra S, Hernandez-Jauregui P. Porcine rubulavirus causes

apoptosis in lymph nodes and increases CD81 peripheral

lymphocytes in piglets. Submitted.

26 Langhorne J, Simon-Haarhaus B, Meding SJ. The role of CD41 T

cells in the protective immune response to Plasmodium chabaudi

in vivo. Immunol Lett 1990; 25: 101±107.

27 Taylor-Robinson AW, Phillips RS, Severn A, Moncada S, Liew

FY. The role of TH1 and TH2 cells in a rodent malaria infection.

Science 1993; 260: 1931±1934.

28 Hirunpetcharat C, Good MF. Deletion of Plasmodium berghei-

specific CD41 T cells adoptively transferred into recipient mice

after challenge with homologous parasite. Proc Natl Acad Sci USA

1998; 95: 1715±1720.



parasite immunology

Education