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INFECTION AND IMMUNITY, June 2003, p. 3648–3651 Vol. 71, No. 6
0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.6.3648–3651.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Chemokine Receptor CCR2 Is Not Essential for the Development of
Experimental Cerebral Malaria
Elodie Belnoue,
1
Fabio T. M. Costa,
1
AnaM.Viga´rio,
1
Tatiana Voza,
2
Franc¸oise Gonnet,
1,2
Ire`ne Landau,
2
Nico van Rooijen,
3
Matthias Mack,
4
William A. Kuziel,
5
and Laurent Re´nia
1
*
De´partement d’Immunologie, Institut Cochin, INSERM U567, CNRS UMR 8104, Universite´ Rene´ Descartes, Hoˆpital Cochin,
1
and Museum National d’Histoire Naturelle,
2
Paris, France; Department of Cell Biology and Immunology, Faculty of
Medicine, Vrije Universiteit, Amsterdam 1081 BT, The Netherlands
3
; Medical Polyclinic, University of Munich,
80336 Munich, Germany
4
; and Department of Microbiology and Institute for Cellular and
Molecular Biology, University of Texas, Austin, Texas 78712-1095
5
Received 13 January 2003/Returned for modification 14 February 2003/Accepted 6 March 2003
Infection with Plasmodium berghei ANKA induces cerebral malaria in susceptible mice. Brain-sequestered
CD8
ⴙ
T cells are responsible for this pathology. We have evaluated the role of CCR2, a chemokine receptor
expressed on CD8
ⴙ
T cells. Infected CCR2-deficient mice were as susceptible to cerebral malaria as wild-type
mice were, and CD8
ⴙ
T-cell migration to the brain was not abolished.
Cerebral malaria (CM) contributes to around 2 million deaths
annually, mainly in African children. Brain sequestration of par-
asitized erythrocytes (PE) is thought to be responsible for this
pathology (4, 18). However, though necessary, PE sequestration
cannot account alone for CM, since this phenomenon has been
observed in non-CM cases (25). Leukocyte sequestration has of-
ten been described within brain postcapillary venules from pa-
tients who died of CM (9, 21); however, ethical considerations
limit investigation of the role of these cells in pathogenesis. In a
mouse model of CM with Plasmodium berghei ANKA, character-
ized by paralysis, deviation of the head, ataxia, convulsions, and
coma, histological studies have shown that PE and leukocytes are
sequestered in brain capillaries (10, 12, 20, 22). We have recently
demonstrated that recruitment of macrophages, neutrophils, and
T lymphocytes to the brain is associated with the onset of the
disease and that the recruited CD8
⫹
T-cell subset is responsible
for the neurological symptoms and the ensuing death (2). We
postulated that a chemokine receptor(s) must be necessary for
the migration of these pathogenic CD8
⫹
T cells to the brain. We
focused on one of these chemokine receptors, CCR2, since it has
been shown previously to be expressed on CD8
⫹
T cells migrating
to the brain after a viral infection (19). CCR2 is a member of the
seven-transmembrane G protein-coupled receptor superfamily
and binds ligands such as CCL2 (MCP-1), CCL7 (MCP-3), and
CCL12 (MCP-5) (29). In the mouse, CCR2 is expressed on
monocytes; T cells, in particular CD8
⫹
T cells (17); endothelial
cells; and brain cells like astrocytes and microglial cells (5, 11).
CCR2 has been shown elsewhere to be implicated in leukocyte
adhesion, monocyte recruitment (13, 26), and dendritic cell traf-
ficking (23).
With the use of a recently described monoclonal antibody
(MAb) to mouse CCR2 (17), expression of this molecule was
investigated by cytofluorometry on total brain-sequestered leuko-
cytes (BSL) and on the cell populations (macrophages and T
lymphocytes) which are known to express CCR2 (17), isolated
from 129/Ola ⫻C57BL/6J F
2
wild-type (WT) naive mice or P.
berghei ANKA-infected WT mice with or without CM. BSL were
isolated as previously described (2), and leukocyte subsets were
identified with the following antibodies: biotinylated rat immuno-
globulin G2b (IgG2b) MAb anti-mouse F4/80 (Tebu, Le Perray-
en-Yvelines, France), hamster IgG MAb anti-mouse CD3 conju-
gated to phycoerythrin (clone 17A2; PharMingen), rat IgG2a
antibody anti-mouse CD8␣conjugated to quantum red (clone
53-6.7; Sigma), rat IgG2a MAb anti-mouse CD4 conjugated to
quantum red (clone H129-19; Sigma), and purified rat antibody
anti-mouse CCR2 (17). Ultravidin conjugated to phycoerythrin
(Leinco Technologies Inc., St. Louis, Mo.) and goat IgG anti-rat
IgG conjugated to fluorescein isothiocyanate (Polysciences, Inc.,
Warrington, Pa.) were used as secondary reagents. For each sam-
ple, 5,000 cells were analyzed. CCR2
⫹
BSL were more numerous
in WT mice with CM than in those without CM (NCM) or in
naive mice (Fig. 1). BSL from WT mice with CM also expressed
more CCR2 on their surface (mean fluorescence intensity [MFI],
57.1 ⫾10.4) than did BSL from mice without CM (MFI, 30.1 ⫾
2.9; one-factor analysis of variance and Tukey test, P⬍0.05; five
mice per group) or BSL from naive mice (MFI, 25.15 ⫾2; P⬍
0.01). Moreover, a strong and significant accumulation of CD8
⫹
T cells expressing CCR2 was observed in the brains of CM mice
but not in those from NCM or naive mice (Fig. 1B).
Since CCR2 is expressed on pathogenic CD8
⫹
T cells, we
next investigated susceptibility in CCR2-knockout (KO) mice
(14). These mice display severe deficits in macrophage (7, 14),
neutrophil (6), and T-cell (8) migration in response to either
antigenic or nonantigenic challenge and an impaired type 1
cytokine response (7). CCR2-KO and WT mice were infected
with 10
6
PE, and their parasitemia and anemia (hemoglobin
levels) were determined every other day as previously de-
scribed (28). All the KO mice but only 60 to 80% of WT mice
developed CM and died between days 6 and 10 after infection
(Fig. 2A and B). Though parasite levels were not significantly
different between the two groups during the first week, the
remaining WT mice died 2 weeks later (Fig. 2B) of hyperpara-
sitemia (Fig. 2C) and anemia (Fig. 2D).
* Corresponding author. Mailing address: De´partement d’Immuno-
logie, Institut Cochin, Hoˆpital Cochin, Baˆtiment Gustave Roussy, 27
rue du Fbg St Jacques, 75014 Paris, France. Phone: 33 1 40 51 65 11.
Fax: 33 1 40 51 65 35. E-mail: renia@cochin.inserm.fr.
3648
Histopathological analysis of the midbrain region of infected
mice was performed as described previously (1) and revealed
petechial hemorrhages and leukocyte accumulation in the cap-
illaries of WT mice with CM, whereas these changes were not
observed in infected WT mice without CM (data not shown).
Brains of infected KO mice with CM showed ring hemorrhages
with apparently fewer leukocytes in the capillaries than in
those of WT mice with CM (data not shown). We thus quan-
tified the total number of BSL from WT and KO mice. As
shown in Fig. 3, BSL from KO mice with CM were less nu-
merous than BSL from WT mice with CM. Nevertheless, there
was a significant threefold increase in BSL number in infected
KO mice compared to naive KO mice. There were eight times
more BSL from WT mice with CM than from naive WT mice.
NCM WT mice contained the same number of BSL as did
naive WT mice (Fig. 3). BSL from the different mouse groups
were further phenotyped by cytofluorometry. Macrophages
were identified as F4/80
⫹
, neutrophils were identified as F4/
80
⫺
and Gr-1
⫹
(rat IgG2b MAb anti-mouse Gr-1 conjugated
to fluorescein isothiocyanate, clone RB6-8C5; PharMingen),
and T cells were identified as described above. We observed a
significant increase in the numbers of macrophages, neutro-
phils, and CD4
⫹
and CD8
⫹
T lymphocytes (but not of other
cell types) in CM WT mice compared with naive or NCM WT
mice. Macrophages and CD8
⫹
T cells, but no other cell types,
increased in infected KO mice with CM compared with naive
KO mice. However, the number of macrophages in KO mice
with CM was significantly lower than in CM WT mice (Fig. 3).
In contrast, similar numbers of CD8
⫹
T cells, the subset re-
sponsible for CM in WT mice, were found in CM WT and CM
KO mice. Depletion experiments were carried out to investi-
gate the role of brain-sequestered CD8
⫹
T cells in CCR2-KO
mice with CM. Depletion of BSL subsets was performed at day
6, just before the onset of CM, by injecting intraperitoneally 1
mg of the following MAbs: rat IgG anti-mouse CD8 (clone
2.43; ATCC TIB 210), rat IgG anti-mouse CD4 (clone GK1.5;
ATCC TIB 207), or antipolymorphonuclear cells (15). More
than 98% of blood CD8
⫹
or CD4
⫹
T cells were depleted as
verified by fluorescence-activated cell sorting (FACS) analysis.
Depletion of blood neutrophils was more than 80% as verified
FIG. 1. CCR2 is expressed on BSL. (A) Representative dot plot of BSL from CCR2 WT mice (naive, NCM, and CM) stained with an
anti-CCR2 MAb versus size (forward size scatter [FSC]). Data are representative of five animals per group. (B) Number of total sequestered
leukocyte subsets (white bars) and CCR2
⫹
leukocyte subsets (black bars) from the whole brain of WT mice (naive, NCM, and CM). Samples of
brain leukocyte suspension from mice infected with 10
6
PE and healthy mice were stained with MAbs specific for neutrophils, macrophages, and
CD4
⫹
and CD8
⫹
T cells and for CCR2 and analyzed by flow cytometry. Absolute numbers of a given subset were calculated by multiplying the
percentage of positive cells for this subset by the total number of BSL. CCR2
⫹
cell numbers were determined by using the percentage of CCR2
positive cells within each subset multiplied by the total number of this subset. *, P⬍0.05 versus CM mice (one-factor analysis of variance followed
by Tukey test). #, P⬍0.05 versus NCM mice. This experiment is representative of three.
VOL. 71, 2003 NOTES 3649
by FACS analysis with anti-Gr-1 MAb. Purified rat IgG (Sig-
ma) was used as a negative control. Macrophages were de-
pleted at day 5 after P.berghei ANKA injection by intravenous
injection of 0.2 ml of phosphate-buffered saline containing
approximately 1 mg of dichloromethylenediphosphonate (Cl
2
-
MDP) encapsulated in liposomes (27). More than 90% of
blood F4/80
⫹
cells were depleted as verified by FACS analysis
2 days later. All CCR2-KO mice depleted of CD4
⫹
T cells,
neutrophils, or macrophages died of CM (Fig. 4 and data not
shown), whereas none of the anti-CD8-treated KO mice de-
FIG. 2. CM incidence, survival, parasite load, and hemoglobin lev-
els after P.berghei ANKA (PbA) infection of CCR2 WT and KO mice.
(A) CM incidence occurring between day 6 and day 10 in WT (n⫽32)
and KO (n⫽27) mice infected with 10
6
PE. On day 10, as calculated
by Fisher’s exact test, Pwas 0.0049 between WT and KO mice. (B) Sur-
vival of WT (n⫽17) and KO (n⫽17) mice infected with 10
6
PE.
Neurological signs first appear late on days 6 to 10 (shaded area), with
death occurring in ⬍24 h after their onset. (C) PE per milliliter of
blood ⫾standard errors of the means. WT (n⫽5) and KO (n⫽5)
mice were infected with 10
6
PE. Mortality is indicated at the top (KO
mice) and at the bottom (WT mice) as the number of dead mice (d) on
that day. The difference between WT and KO mice on day 6 was not
significant. (D) Hemoglobin levels (means ⫾standard errors of the
means) in WT (n⫽5) and KO (n⫽5) mice infected with 10
6
PE.
FIG. 3. Levels of whole-brain-sequestered leukocytes in CCR2-KO and WT mice after P.berghei ANKA infection. Enumeration of BSL was
performed on perfused brains from KO (n⫽15) and WT (n⫽8) mice at the time when CM is diagnosable (days 6 to 10), NCM WT mice (days
9 to 10) (n⫽6), and naive KO (n⫽10) or WT (n⫽11) mice. Cell numbers were determined as described for Fig. 1B. Values are expressed as
means ⫾standard errors of the means. *, P⬍0.05 (one-factor analysis of variance followed by Tukey test), significantly different from naive WT
mice; #, P⬍0.05, Tukey test, significantly different from NCM WT mice; and , P⬍0.05, Tukey test, significantly different from CM KO mice.
This experiment is representative of three.
FIG. 4. Role of CD8
⫹
T cells in CM in CCR2 KO mice. The
effector role of CD8
⫹
T cells was demonstrated through a series of
depletion experiments with infected CCR2-KO and WT mice. The
figure shows survival (A) and CM incidence (B) in infected WT or KO
mice injected with the following rat antibodies: control IgG (n⫽5),
anti-CD8 (n⫽5), anti-CD4 (n⫽5), and anti-polymorphonuclear cell
(PMN) (n⫽5) on day 6. *, P⬍0.05 (Fisher test) versus P.berghei
ANKA-infected WT mice treated with control rat IgG; #, P⬍0.0001
(Fisher test) versus rat IgG-treated KO mice. This experiment is rep-
resentative of two.
3650 NOTES INFECT.IMMUN.
veloped CM. Identical results were observed in infected and
similarly depleted WT mice (Fig. 4 and data not shown).
Finally the role of cytokines was investigated, since a type 1
response, which is altered in CCR2-KO mice (7, 23, 24), has
been associated elsewhere with CM development (1, 16). Both
CM WT and KO mice, however, developed similar serological
and cellular type 1 responses overall (data not shown).
Our results clearly show that CCR2 is not necessary for CM to
occur. CCR2 deficiency was associated with a reduction in num-
bers of macrophages, neutrophils, and CD4
⫹
T cells but not of
CD8
⫹
T cells. Our results further confirm that CD8
⫹
T cells are
responsible for CM death (2). It is remarkable that the pathology
in WT and CCR2-KO mice was due to the sequestration of less
than 10
5
CD8
⫹
T cells in the vasculature of a whole brain. CCR2
has also been shown previously to be expressed on brain cells like
endothelial cells, astrocytes, and microglial cells (5, 11), and sig-
naling through this receptor may activate these cell types for
chemokine and cytokine production. However, our results indi-
cate that CCR2 signaling in these cells is not required for the
development of CM. Since migration of CD8
⫹
T cells to the brain
occurred normally in CCR2 KO mice, this implies that another
chemokine receptor(s) is involved in this process. We have shown
recently that CCR5 deficiency results in the decrease in CM
susceptibility in mice of the same genetic background (3). Prelim-
inary results indicate that more than 80% of brain-sequestered
CD8
⫹
T cells from infected WT or CCR2 KO mice express
CCR5 (data not shown). More studies are needed to determine if
other chemokine receptors are involved in rodent and eventually
in human CM.
We thank Georges Snounou for critical reading of the manuscript.
This work was supported in part by a grant from Junta Nacional de
Investigac¸a˜o Cientifica e Tecnologica (JNICT) and Fondation de La
Recherche Me´dicale to Laurent Re´nia. Elodie Belnoue held a fellow-
ship from MENRT. Fabio T. M Costa was supported by a fellowship
from the CAPES foundation, Brazil. Ana Margarida Viga´rio held a
fellowship from Junta Nacional de Investigac¸a˜o Cientifica e Tecno-
logica (JNICT), Portugal.
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Editor: W. A. Petri, Jr.
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