Central Role of Endogenous Gamma Interferon in Protective Immunity against Blood-Stage Plasmodium chabaudi AS Infection

Abstract

The role of endogenous gamma interferon (IFN-γ) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-γ gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 106 parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had
severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and
IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide
(NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated
spleen cells and B220+ and Mac-1+spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80+, NK1.1+, and F4/80+Ia+ spleen cells than infected GKO mice; male WT had more CD3+ cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher
proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels
of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a,
and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality,
phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection.
These results thus provide unequivocal evidence for the central role of endogenous IFN-γ in the development of protective
immunity against blood-stage P. chabaudi AS.

Full text

INFECTION AND IMMUNITY,
0019-9567/00/$04.00⫹0Aug. 2000, p. 4399–4406 Vol. 68, No. 8
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Central Role of Endogenous Gamma Interferon in Protective
Immunity against Blood-Stage Plasmodium chabaudi
AS Infection
ZHONG SU AND MARY M. STEVENSON*
Centre for the Study of Host Resistance, Montreal General Hospital Research Institute
and McGill University, Montreal, Quebec, Canada
Received 12 January 2000/Returned for modification 25 February 2000/Accepted 1 May 2000
The role of endogenous gamma interferon (IFN-␥) in protective immunity against blood-stage Plasmodium
chabaudi AS malaria was studied using IFN-␥gene knockout (GKO) and wild-type (WT) C57BL/6 mice.
Following infection with 10
6
parasitized erythrocytes, GKO mice developed significantly higher parasitemia
during acute infection than WT mice and had severe mortality. In infected GKO mice, production of inter-
leukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages
was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production
observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total
nucleated spleen cells and B220
ⴙ
and Mac-1
ⴙ
spleen cells both before and after infection. Infected WT mice,
however, had significantly more F4/80
ⴙ
, NK1.1
ⴙ
, and F4/80
ⴙ
Ia
ⴙ
spleen cells than infected GKO mice; male
WT had more CD3
ⴙ
cells than male GKO mice. In comparison with those from WT mice, splenocytes from
infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanava-
lin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT
mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO
mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, pheno-
types of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during
infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-␥in the
development of protective immunity against blood-stage P. chabaudi AS.
Studies of experimental murine models as well as humans
suggest an important role for gamma interferon (IFN-␥)in
protective immune responses to blood-stage malaria (19, 22).
Treatment of mice with exogenous IFN-␥delays the onset of
parasitemia and decreases the number of infected erythrocytes
during Plasmodium chabaudi adami infection (4). Shear and
her colleagues (31) demonstrated that daily treatment with
recombinant IFN-␥resulted in a less severe course of infection
and increased survival in mice infected with the lethal strain of
Plasmodium yoelii 17x. Furthermore, these investigators found
a correlation between the timing and level of IFN-␥production
in vitro by spleen cells and the outcome of infection with lethal
versus nonlethal strains of P. yoelii 17x. These observations
were confirmed in a recent study demonstrating that endoge-
nous levels of IFN-␥in the spleen during blood-stage malaria
infection differ between nonlethal and lethal Plasmodium spe-
cies at 24 h after infection (5). Studies in our laboratory of
resistant C57BL/6 and susceptible A/J mice demonstrated a
correlation between the level of resistance to blood-stage P.
chabaudi AS infection and IFN-␥mRNA expression and pro-
tein production by spleen cells (15, 27, 34). In addition, treat-
ment of P. chabaudi AS-infected C57BL/6 mice with neutral-
izing monoclonal antibodies (MAbs) to IFN-␥exacerbates the
course of infection, but there is no effect on survival (21, 35).
Recent studies by van der Heyde et al. (42) using IFN-␥
knockout (GKO) mice on the 129 background and Favre et al.
(6) using IFN-␥receptor knockout (KO) mice on a mixed
genetic background demonstrated a role for endogenous
IFN-␥in the development of protective immunity to infection
with P. chabaudi adami and P. chabaudi AS, respectively. In
contrast, Tsuji et al. (41) failed to observe significant differ-
ences in parasitemia levels between IFN-␥receptor KO and
wild-type (WT) mice on a mixed genetic background during
blood-stage infection with P. chabaudi adami although protec-
tion induced by immunization with attenuated sporozoites
against liver-stage P. yoelii 17XNL was impaired in the IFN-␥
receptor KO mice. None of these studies, however, addressed
the issue of the effects of background genes on the immune
responses against blood-stage malaria in KO mice lacking
IFN-␥responses.
The major cell types producing IFN-␥during blood-stage
malaria are NK cells and T cells, primarily CD4
⫹
Th cells.
Studies of nude mice or NK cell-depleted mice demonstrated
that early production of IFN-␥during infection with nonlethal
P. yoelii is dependent on both NK and T cells (5). Using the
model of P. chabaudi AS infection in resistant C57BL/6 and
susceptible A/J mice, we demonstrated that NK cells produce
IFN-␥during early infection and that the ability of these cells
to produce IFN-␥correlates with resistance (23). However,
during the acute phase of P. chabaudi AS infection, just before
peak parasitemia, CD4
⫹
Th cells are the major source of
IFN-␥(17, 21, 34). Taken together, these observations dem-
onstrate that IFN-␥produced during innate as well as acquired
immune responses plays a central role in protective immunity
during blood-stage malaria.
This study was performed to determine the role of endoge-
nous IFN-␥in the development of protective immunity against
blood-stage P. chabaudi AS infection. We used GKO mice on
the resistant C57BL/6 background to investigate the protective
* Corresponding author. Mailing address: Montreal General Hos-
pital Research Institute, 1650 Cedar Ave., Montreal, Quebec H3G
1A4, Canada. Phone: (514) 937-6011, ext. 4507. Fax: (514) 934-8332.
E-mail: mcev@musica.mcgill.ca.
4399

effect of this cytokine and, more importantly, to elucidate its
immunoregulatory role in the development of protection
against blood-stage infection with this parasite. We previously
demonstrated that male mice are more susceptible to infection
with this parasite than female mice (33). Since male GKO mice
were also found to be more susceptible to infection than fe-
male GKO mice, we performed separate analyses of male and
female GKO as well as WT mice. Our results demonstrate the
pivotal role of endogenous IFN-␥in the development of pro-
tective immune responses and survival during blood-stage P.
chabaudi AS infection. Furthermore, we identified important
gender differences in host responses to this infection.
MATERIALS AND METHODS
Mice, parasite, and experimental infections. Breeding pairs of GKO mice,
provided by Genentech, Inc. (South San Francisco, Calif.), and backcrossed onto
the C57BL/6 strain for eight generations, were a kind gift from F. P. Heinzel
(Case Western Reserve University School of Medicine, Cleveland, Ohio) (12).
GKO mice were bred in the animal facility of the Montreal General Hospital
Research Institute under specific-pathogen-free conditions. Spleen cells from
these mice stimulated in vitro with either concanavalin A (ConA) or parasite
antigen failed to produce detectable levels of IFN-␥. WT control C57BL/6 mice
were obtained from Charles River (St. Constant, Quebec, Canada) and main-
tained in the same facility. WT and GKO mice, 8 to 12 weeks old, were age and
sex matched in all experiments. P. chabaudi AS was maintained as previously
described (26). Infections were initiated by intraperitoneal injection of 10
6
P.
chabaudi AS parasitized erythrocytes (PRBC). Parasitemia and mortality were
monitored daily as previously described (26). Mice were sacrificed at various
times, and blood was obtained by cardiac puncture, allowed to clot for 30 min at
4°C, and centrifuged at 3,000 ⫻gfor 3 min. Sera were collected and stored at 4°C
for measurement of interleukin 12 (IL-12) p70 or at ⫺20°C for determination of
the levels of other cytokines.
Spleen cell culture and proliferation assay. Spleens from normal and infected
mice were removed aseptically and pressed through a sterile fine-wire mesh with
10 ml of RPMI 1640 (Life Technologies, Burlington, Ontario, Canada) supple-
mented with 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Lo-
gan, Utah), 25 mM HEPES (Life Technologies), 0.12% gentamicin (Schering,
Montreal, Quebec, Canada), and 2 mM glutamine (Life Technologies). Cell
suspensions were centrifuged at 350 ⫻gfor 10 min. Erythrocytes were lysed with
0.175 M NH
4
Cl, and the cells were washed twice in fresh medium. Membrane
debris was removed by filtering the cell suspensions through sterile gauze. The
viability of the cells was determined by trypan blue exclusion and was always
⬎90%. Total cell counts were performed on individual samples, and differential
counts were performed on cytospin preparations stained with Diff-Quik (Amer-
ican Scientific Products, McGaw Park, Ill.). For proliferation assays, spleen cells
were adjusted to 2.5 ⫻10
6
cells/ml and aliquots of 0.1 ml were plated in triplicate
in 96-well flat-bottom plates, stimulated with 10
6
washed PRBC/ml as the ma-
laria parasite antigen, 5 ␮g of ConA (Calbiochem, La Jolla, Calif.)/ml, or me-
dium as the control, and incubated for 72 h at 37°C in a humidified CO
2
incubator. During the last 16 h of culture, 1 ␮Ci of [
3
H]thymidine (specific
activity, 6.7 Ci/mmol) was added to each well, the cells were harvested with an
automatic cell harvester, and the incorporated radioactivity was measured in a
liquid scintillation counter. For determination of cytokine production, spleen
cells were adjusted to 5 ⫻10
6
cells/ml and aliquots of 1 ml were plated in
triplicate in 24-well tissue culture plates in the presence or absence of PRBC, as
described above, and incubated for 48 h at 37°C in a humidified CO
2
incubator.
Supernatants were collected, centrifuged at 350 ⫻gfor 5 min, and stored at
⫺20°C until assayed for cytokine levels. For culture of splenic macrophages, the
percentages of macrophages in spleen cells were determined on Diff-Quik-
stained cytospin slides and the cell suspensions were adjusted to 10
6
macro-
phages/ml. Aliquots of 0.1 ml/well in triplicate were incubated in flat-bottom
96-well plates at 37°C for 2 h. Nonadherent cells were removed. Adherent cells,
which were ⬎95% macrophages based on morphology, phagocytosis of inert
latex beads, and nonspecific esterase staining (28), were washed twice with warm
medium and incubated with 0.2 ml of medium alone or medium containing 1 ␮g
of Escherichia coli 0127:B8 lipopolysaccharide (LPS) (Difco, Detroit, Mich.)/ml.
Supernatants were collected 20 h later and assayed for IL-12 p70 and nitric oxide
(NO).
Cytokine ELISAs. Cytokine levels in sera and spleen cell or macrophage
supernatants were measured using two-site sandwich enzyme-linked immunosor-
bent assays (ELISAs) for IFN-␥, tumor necrosis factor alpha (TNF-␣), and IL-12
p70 as previously described (27, 36). For IL-4, the capturing and detecting
antibodies were BVD4-1D11 MAb and biotinylated BVD6-24G2 MAb, respec-
tively. For IL-10, JES5.2A5 MAb (American Type Culture Collection, Manassas,
Va.) and biotinylated SXC-1 MAb (PharMingen Canada, Mississauga, Ontario,
Canada) were used as capturing and detecting antibodies, respectively. Standard
curves for each cytokine were generated using recombinant cytokines (Phar-
Mingen Canada). Reactivity was revealed using ABTS [2,2⬘-azinobis(3-ethylben-
zthiazolinesulfonic acid)] substrate (Boehringer Mannheim, Laval, Quebec, Can-
ada), and optical density (OD) values were read in a microplate reader at 405 nm
with a reference wavelength of 492 nm.
Determination of nitrite (NO
2
ⴚ
) and nitrate (NO
3
ⴚ
) concentrations. For NO,
the concentration of NO
2⫺
in cell culture supernatants was measured by the
Griess reaction (11). NO
2⫺
concentrations were calculated using NaNO
2
as a
standard. Serum NO levels were determined based on NO
3⫺
concentrations
using a previously described method (14). NaNO
3
, diluted in serum from unin-
fected WT mice and dialyzed against phosphate-buffered saline (PBS) for 24 h,
was used as a standard to calculate serum NO
3⫺
levels.
Flow cytometry. Spleen cells were adjusted to 2 ⫻10
7
cells/ml in staining buffer
(PBS with 1% bovine serum albumin and 0.2% sodium azide). Aliquots of 50 ␮l
of cells were incubated with anti-mouse CD16/CD32 MAb (clone 2.4G2; Phar-
Mingen) to block FcR. Blocked spleen cells were then labeled with a fluorescein
isothiocyanate (FITC)-conjugated MAb against mouse CD3 (clone 145-2C11;
PharMingen), B220 (clone RA3-6B2; PharMingen), Mac-1 (clone MI/70; Sero-
tec, Oxford, United Kingdom), macrophage activation marker F4/80 (clone CL:
A3-1; Serotec), or a phycoerythrin (PE)-conjugated MAb to mouse NK1.1 (clone
PK136; PharMingen). To determine Ia antigen expression on macrophages,
two-color flow cytometry was performed by labeling with a PE-conjugated MAb
against F4/80 followed, after washing with staining buffer, by staining with a
FITC-conjugated MAb to I-A
b
(A␣
b
) (clone AF6-120.1; PharMingen). Isotype-
matched MAbs conjugated to either FITC or PE were used as negative controls
for all experiments. Acquisition of cells and analysis of data were performed
immediately after staining using a FACscan equipped with CellQuest software
(Becton Dickinson, Mountain View, Calif.).
Serum P. chabaudi AS-specific antibody titers. Levels of P. chabaudi AS-
specific antibody isotypes in serum were determined by ELISA. P. chabaudi AS
antigen was prepared as described previously (47). Immulon II plates (Dynatech,
Chantilly, Va.) were coated with parasite antigen overnight at 4°C and subse-
quently blocked with 1% bovine serum albumin in PBS for 1 h. Individual serum
samples were serially diluted twofold, and 50 ␮l of each dilution was added to
each plate and incubated for2hatroom temperature. After extensive washing,
horseradish peroxidase-conjugated goat anti-mouse isotype antibodies (SBA,
Birmingham, Ala.) were added and incubated at room temperature for another
2 h. Reactivity was visualized using ABTS substrate, and OD values were read in
a microplate reader at 405 nm with a reference wavelength of 492 nm. Antibody
isotype levels in serum are expressed as ELISA titers, the reciprocal of the lowest
dilution that yields the background OD.
Statistical analysis. Data are presented as means ⫾standard errors of the
means (SEM). Statistical significance of differences in means between WT and
GKO mice, between normal and infected mice, and between sexes was analyzed
by Student’s ttest using Mystat (Systat, Evanston, Ill.). A Pvalue of ⬍0.05 was
considered significant.
RESULTS
Course of P. chabaudi AS infection in WT and GKO mice.
First, we examined the course of parasitemia and monitored
mortality in WT and GKO mice on the resistant C57BL/6
background. WT mice, either male or female, developed pri-
mary parasitemia which peaked on day 7 postinfection (Fig. 1A
and B). The peak parasitemia level in male WT mice (38% ⫾
3.4% PRBC) was significantly higher than that in female WT
mice (24% ⫾4.2%; P⬍0.05). Parasitemia levels declined in
female WT mice, and infection was cleared by day 23. Male
WT mice also showed a reduction in parasitemia after the first
peak and cleared the parasite by day 35 postinfection. All male
and female WT mice survived primary infection (Fig. 1C and
D). In comparison, GKO mice experienced a more severe
course of P. chabaudi AS infection. Male GKO mice developed
significantly higher parasitemia during days 7 to 9 postinfection
than male WT mice (Fig. 1A), and 100% of male GKO mice
died by day 14 after infection (Fig. 1C). Female GKO mice had
significantly higher parasitemia between days 8 and 10 than
female WT mice (Fig. 1B), and 40% died between days 12 and
20 postinfection (Fig. 1D). Surviving female GKO mice expe-
rienced two recrudescent parasitemias of 45 and 15% PRBC
on days 16 and 25 postinfection, respectively. These results
demonstrate that GKO mice are susceptible to P. chabaudi AS
infection in terms of both increased parasitemia and decreased
survival. Furthermore, male mice, either GKO or WT, devel-
oped significantly higher peak parasitemias than their female
counterparts and male GKO mice suffered more severe mor-
tality than female GKO mice.
4400 SU AND STEVENSON INFECT.IMMUN.

Serum cytokine levels in P. chabaudi AS-infected WT and
GKO mice. The immunoregulatory role of IFN-␥in the devel-
opment of resistance to acute P. chabaudi AS infection was
analyzed by determining levels of IL-12 p70, TNF-␣, and NO
in serum from WT and GKO mice during infection. Basal
serum IL-12 p70 and NO levels in uninfected WT and GKO
mice were not significantly different, while basal levels of
TNF-␣were significantly higher in uninfected WT mice (Table
1). Consistent with our previous findings (27), P. chabaudi AS
infection in WT mice induced increased IL-12 p70 production
in vivo, which peaked on day 2 postinfection (Table 1) and then
declined thereafter (data not shown). GKO mice also had
significant increases (P⬍0.05 for both sexes) in serum IL-12
p70 on day 2 postinfection, but the levels were significantly
lower than those observed in WT mice (P⬍0.01 for both
sexes). On day 7 postinfection, there were significant increases
in serum TNF-␣levels in WT (P⬍0.01) as well as GKO (P⬍
0.05) mice compared to those in their uninfected controls.
TNF-␣levels were, however, significantly higher in WT mice
than the levels detected in their GKO counterparts (P⬍0.01
for both sexes). These observations suggest that optimum pro-
duction of IL-12 p70 as well as TNF-␣during blood-stage
malaria is IFN-␥dependent. Infection with blood-stage P.
chabaudi AS in WT mice resulted in significant and substantial
increases in serum NO on day 7 postinfection. However, only
basal levels of NO were detected in the sera of infected GKO
mice, suggesting that NO production in vivo during blood-
stage malaria is totally IFN-␥dependent. Serum IFN-␥levels
increased significantly in both male and female WT mice fol-
lowing blood-stage P. chabaudi AS infection; in vivo IFN-␥
production was significantly higher in female than in male WT
mice (Table 1). No gender differences in serum IL-12 p70,
TNF-␣, or NO levels in WT and GKO mice were detected
before or after infection.
Spleen cell phenotypes in P. chabaudi AS-infected WT and
GKO mice. Next, we analyzed the numbers and phenotypes of
leukocytes in the spleens of WT and GKO mice during P.
chabaudi AS infection. There were no significant differences in
the total numbers of nucleated spleen cells or in the numbers
of T cells, B cells, macrophages, and NK cells between unin-
fected WT and GKO mice (Table 2). There were significant
and similar increases in the total numbers of nucleated cells, B
cells, and Mac-1
⫹
cells in the spleens of infected mice com-
pared to corresponding values for uninfected WT and GKO
mice (P⬍0.01). Among infected male mice, the number of
CD3
⫹
cells in WT mice was significantly higher than the num-
ber in GKO mice (P⬍0.05). Compared to infected GKO
mice, infected WT mice had significantly higher numbers of
F4/80
⫹
macrophages (P⬍0.01 for male and P⬍0.05 for
female mice) and significantly higher numbers of NK 1.1
⫹
cells
(P⬍0.01 for male and P⬍0.05 for female mice) in their
spleens. There were significant increases in the number of
F4/80
⫹
Ia
⫹
cells following P. chabaudi AS infection compared
to corresponding values for uninfected WT (P⬍0.01 for both
sexes) and GKO (P⬍0.05 for both sexes) mice (data not
shown). The number of F4/80
⫹
Ia
⫹
spleen cells from GKO
mice was, however, significantly lower than that from WT mice
(P⬍0.05 for males and P⬍0.01 for females). There were
significantly higher numbers of F4/80
⫹
macrophages in in-
fected female WT mice than in male WT mice (P⬍0.05) and
higher numbers of F4/80
⫹
Ia
⫹
spleen cells in uninfected and
infected female WT mice than in their male counterparts (P⬍
0.05). These results suggest that, in the absence of IFN-␥, the
recruitment and/or local proliferation of F4/80
⫹
macrophages
and NK 1.1
⫹
cells is impaired during blood-stage malaria.
These results also suggest that higher numbers of F4/80
⫹
and
F4/80
⫹
Ia
⫹
cells in female WT mice may be linked to signifi-
cantly lower their peak parasitemia levels being than those in
WT male mice.
In vitro proliferation and cytokine production by spleen
cells from P. chabaudi AS-infected WT and GKO mice. We also
compared the in vitro function of spleen cells and splenic
macrophages from WT and GKO mice. As shown in Table 3,
the in vitro proliferation of spleen cells from uninfected mice
was low in the presence of medium or PRBC and there were
FIG. 1. Parasitemia and mortality of WT and GKO mice following infection
with P. chabaudi AS. Male (A and C) and female (B and D) WT and GKO mice
were infected intraperitoneally with 10
6
PRBC, and the course of parasitemia (A
and B) was determined. Data are means ⫾SEM of six mice per group from one
of three experiments. Cumulative survival (C and D) of infected WT and GKO
mice was determined from 16 to 19 mice pooled from three experiments. ⴱ,
statistically significant differences in mean parasitemia between WT and GKO
mice (P⬍0.05).
TABLE 1. Serum cytokine and NO levels in uninfected and
P. chabaudi AS-infected WT and GKO mice
a
Cytokine or
NO and
mouse type
Level
b
in mice that were:
Uninfected Infected
Male Female Male Female
IL-12p70
WT 0.27 ⫾0.04 0.27 ⫾0.06 2.97 ⫾0.3* 3.32 ⫾0.19*
GKO 0.22 ⫾0.03 0.16 ⫾0.08 0.71 ⫾0.20 0.78 ⫾0.14
IFN-␥
WT 1.3 ⫾0.2 1.1 ⫾0.2 32.3 ⫾3.6 50.8 ⫾4.3#
GKO ND
c
ND ND ND
TNF-␣
WT 0.32 ⫾0.05* 0.31 ⫾0.02* 1.25 ⫾0.10* 1.01 ⫾0.10*
GKO 0.15 ⫾0.02 0.22 ⫾0.01 0.39 ⫾0.03 0.40 ⫾0.02
NO
WT 9.8 ⫾2.0 5.1 ⫾1.3 59.6 ⫾6.5* 76.2 ⫾5.8*
GKO 9.9 ⫾3.0 4.8 ⫾0.9 11.1 ⫾1.2 9.2 ⫾1.6
a
Levels of IL-12 p70 in serum of infected mice were analyzed on day 2, and
levels of IFN-␥, TNF-␣, and NO were analyzed on day 7, following P. chabaudi
AS infection. Data are means ⫾SEM for three to five mice per group. Results
from one of three replicate experiments are shown.
b
Units are nanograms per milliliter for cytokines and micromolar for NO. ⴱ,
P⬍0.01 for WT versus GKO mice; #, P⬍0.01 for WT female versus male mice.
c
ND, not detectable.
VOL. 68, 2000 IMMUNE RESPONSES TO BLOOD-STAGE MALARIA IN GKO MICE 4401

no significant differences between WT and GKO mice. In cul-
tures stimulated with ConA, there was a significantly higher
response in cells from uninfected GKO than in cells from WT
mice (P⬍0.01 for both sexes). In comparison to those of
spleen cells from uninfected mice, the responses of spleen cells
from P. chabaudi AS-infected WT or GKO mice to medium
control or PRBC were significantly higher (P⬍0.001). Com-
pared to infected WT mice, infected GKO mice had signifi-
cantly higher spontaneous proliferation in cultures containing
medium (P⬍0.01 for both sexes) and significantly higher
responses to parasite antigen (P⬍0.01 for both sexes). The
responses of cells from infected mice of either genotype or
gender to ConA were significantly lower than those of cells
from their uninfected counterparts (P⬍0.001), but infected
GKO mice had significantly higher responses than infected
WT mice (P⬍0.01 for both sexes). Notable gender differences
were observed in cultures stimulated with parasite antigen.
Infected male WT mice had a significantly lower response to
PRBC than their female counterparts (P⬍0.05), while male
GKO mice had significantly higher responses to PRBC than
female GKO mice (P⬍0.01).
Spleen cells harvested from WT mice during acute P.
chabaudi AS infection produced high levels of IFN-␥in vitro
following stimulation with parasite antigen (Fig. 2A). Interest-
ingly, PRBC-stimulated spleen cells from infected female WT
mice produced significantly higher levels of IFN-␥than simi-
larly stimulated cells from infected male WT mice (P⬍0.05).
As expected, there was no detectable IFN-␥in spleen cell
supernatants from GKO mice stimulated with specific antigen
regardless of infection. Production of the proinflammatory cy-
tokine TNF-␣was markedly increased in cultures of spleen
cells from both infected WT and GKO mice stimulated with
PRBC, and there were no significant differences between WT
and GKO mice (Fig. 2B). As shown in Fig. 2C, spleen cells
from infected WT and GKO mice produced comparable levels
of IL-4 following in vitro stimulation with PRBC. However,
PRBC-stimulated spleen cells from infected GKO mice pro-
duced significantly higher levels of IL-10 than their WT coun-
terparts (P⬍0.05 for both sexes) (Fig. 2D). Production of
IL-10 and TNF-␣was not detectable in nonstimulated, me-
dium control cultures regardless of infection or the genotype of
the mice. Spontaneous production of IFN-␥was detected only
in spleen cell cultures from infected WT mice (male, 10.9 ⫾1.0
ng/ml; female, 13.5 ⫾0.3 ng/ml), but the difference was not
significant. Supernatants from medium control cultures from
infected WT and GKO mice had low levels of IL-4, and no
significant difference between these two groups of mice was
observed (data not shown). Gender differences similar to those
in IFN-␥production were observed in IL-10 production.
Spleen cells from infected female GKO mice, compared to
their male counterparts, produced significantly higher levels of
IL-10 in response to PRBC (P⬍0.05). Together, these results
demonstrate that, in the absence of IFN-␥, IL-10 production in
response to parasite antigen is significantly increased in male
and female GKO mice.
To determine if macrophage effector functions are impaired
in GKO mice during acute P. chabaudi AS infection, splenic
macrophages were analyzed for their ability to produce IL-12
p70 and NO in vitro in response to LPS. In contrast to the
marked and significant increases in IL-12 p70 production by
macrophages from infected mice compared to that by macro-
phages from uninfected WT mice (P⬍0.01 for both sexes),
IL-12 p70 production by macrophages from infected GKO
mice was only modestly increased over basal levels (P⬍0.05
for female mice only) and the levels were significantly lower
than those in infected WT mice (P⬍0.05 for both sexes) (Fig.
3A). In addition, macrophages from infected WT mice, com-
pared to their uninfected counterparts, produced significantly
higher levels of NO in vitro in response to LPS (P⬍0.001)
(Fig. 3B). Consistent with in vivo observations described
above, splenic macrophages from infected GKO mice pro-
duced only basal levels of NO. No gender differences in pro-
duction of IL-12 p70 or NO in vitro were observed.
Parasite-specific antibody responses in P. chabaudi AS-in-
fected WT and GKO mice. To investigate the effect of IFN-␥
deficiency on malaria-specific antibody production, specific an-
tibody isotype levels were determined in the sera of infected
WT and GKO mice. Total parasite-specific immunoglobulin
(Ig) levels among P. chabaudi AS-infected male and female
WT mice and infected female GKO mice were similar (Table
4). However, marked differences in antibody isotypes between
infected WT and GKO mice were evident. Infected female WT
mice had significantly higher levels of parasite-specific IgM
(P⬍0.05), IgG2a (P⬍0.01), and IgG3 (P⬍0.005) than their
GKO counterparts, while infected female GKO mice had sig-
nificantly higher levels of IgG1 than their WT counterparts
(P⬍0.01). There were no significant differences between male
and female WT mice. P. chabaudi AS-infected male GKO mice
were unavailable for this analysis since 100% of these animals
succumbed to infection.
TABLE 2. Spleen cellularity and phenotypes in uninfected and P. chabaudi AS-infected WT and GKO mice
a
Cell
type
No. of nucleated cells (10
7
)/spleen (%) for mice that were
b
:
Uninfected
WT GKO
Male Female Male Female
Total 8.0 ⫾1.0 9.7 ⫾1.0 6.6 ⫾0.6 8.2 ⫾0.9
CD3
⫹
2.80 ⫾0.4 (31.8 ⫾0.6) 3.50 ⫾0.3 (36.0 ⫾1.3) 2.10 ⫾0.2 (31.5 ⫾1.2) 2.70 ⫾0.3 (33.5 ⫾1.5)
B220
⫹
5.30 ⫾0.7 (61.0 ⫾1.1) 5.50 ⫾0.6 (57.0 ⫾0.8) 4.30 ⫾0.4 (66.1 ⫾0.5) 4.80 ⫾0.7 (57.9 ⫾2.0)
Mac-1
⫹
0.34 ⫾0.03 (4.0 ⫾0.2) 0.44 ⫾0.05 (4.5 ⫾0.1) 0.23 ⫾0.03 (3.5 ⫾0.3) 0.37 ⫾0.08 (4.5 ⫾0.7)
F4/80
⫹
0.28 ⫾0.04 (3.2 ⫾0.1) 0.50 ⫾0.04 (5.2 ⫾0.3) 0.21 ⫾0.04 (3.1 ⫾0.4) 0.42 ⫾0.07 (5.3 ⫾0.5)
NK1.1 0.35 ⫾0.05 (4.0 ⫾0.1) 0.37 ⫾0.04 (3.8 ⫾0.2) 0.23 ⫾0.04 (3.5 ⫾0.2) 0.36 ⫾0.03 (4.4 ⫾0.2)
a
Percentages of spleen cell phenotypes from uninfected and day-7 P. chabaudi AS-infected mice were determined by fluorescence-activated cell sorter analysis. The
number of cells of each phenotype was calculated from the total number of spleen cells and the percentage of cells with the phenotype (in parentheses). Data are
means ⫾SEM of three individual mice per group.
b
ⴱ, and ⴱⴱ,P⬍0.05 and P⬍0.01, respectively, for WT versus GKO mice; #, P⬍0.05 for female versus male mice.
4402 SU AND STEVENSON INFECT.IMMUN.

DISCUSSION
GKO mice on the resistant C57BL/6 background were used
in the present study to directly assess the role of endogenous
IFN-␥in the development of protective immunity against P.
chabaudi AS infection. GKO mice, both male and female,
developed significantly higher levels of parasitemia than WT
mice during acute infection, and surviving female GKO mice
had several prominent recrudescent parasitemias during the
chronic stage of infection. The infection was lethal in 100% of
male and 40% of female GKO mice. These results provide
unequivocal evidence for the protective effect of this Th1 cy-
tokine against blood-stage P. chabaudi AS infection. Unlike
what was found for P. chabaudi AS-infected GKO mice, which
had severe mortality, mortality was not observed in studies
using neutralizing MAbs to block IFN-␥activity during infec-
tion in WT mice (21, 35) or in studies of GKO mice infected
with P. yoelii 17XNL and P. chabaudi adami (42). This is likely
due to the inability of neutralizing antibodies to completely
block the activity of IFN-␥in vivo and, for P. yoelii and P.
chabaudi adami infections, to differences in virulence among
various rodent malaria species.
To elucidate the immunological mechanisms underlying the
dramatic difference between WT and GKO mice, we analyzed
the effects of IFN-␥deficiency on the in vivo and in vitro
production of several key molecules known to modulate pro-
tective immunity against P. chabaudi AS infection. Early IL-12
production during infection with a number of intracellular
pathogens, including protozoan parasites (8, 20), has been
recognized as important for stimulating IFN-␥production by
NK cells and inducing the differentiation of CD4
⫹
Th0 cells to
a Th1 phenotype (9, 40). Previously, we demonstrated that an
early IL-12 response is critical for development of IFN-␥-
dependent protection against P. chabaudi AS infection (27,
36). It is not known, however, if the early IL-12 response
during P. chabaudi AS infection requires IFN-␥. The depen-
dency of IL-12 production on IFN-␥varies with different in-
tracellular pathogens (7, 8, 30). Results in the present study
show that, although IL-12 production was increased in GKO
mice following P. chabaudi AS infection, the level at day 2
postinfection was significantly lower than the response ob-
served in WT mice. This observation suggests that production
of optimum levels of IL-12 early during infection is dependent
on IFN-␥, which is produced as early as 24 h after blood-stage
malaria infection (5). Analysis of IL-12 production in vitro by
splenic macrophages from infected WT and GKO mice further
demonstrated the dependence of IL-12 production on IFN-␥.
These observations indicate that there may be a positive-feed-
back loop between IL-12 and IFN-␥which is important for the
host to rapidly mount IFN-␥-dependent protective immunity
against P. chabaudi AS infection. This is supported by our
earlier observation that recombinant IL-12 (rIL-12) treatment
of susceptible A/J mice against P. chabaudi AS infection was
most effective when treatment was started on the day of infec-
tion (36; unpublished observation). Furthermore, treatment
with rIL-12 did not alter the lethal course of P. chabaudi AS
infection in GKO mice (data not shown). Taken together,
these results demonstrate that IFN-␥is required for optimum
IL-12 production during early infection and for expression of
IL-12-induced protection.
TNF-␣is both protective and pathogenic during malaria
infection in humans and in mice depending on the quantity,
timing, and tissue site of its production as well as the malaria
TABLE 3. Proliferative responses of spleen cells from uninfected and P. chabaudi AS-infected WT and GKO mice
a
Mouse
type
[
3
H]thymidine uptake (cpm) by spleen cells from mice that were
b
:
Uninfected Infected
Medium PRBC ConA Medium PRBC ConA
Female
WT 396 ⫾40 270 ⫾24 10,995 ⫾890 767 ⫾33† 1,456 ⫾221†# 3,331 ⫾603†
GKO 406 ⫾58 221 ⫾6 22,451 ⫾1,229* 1,743 ⫾426*† 3,298 ⫾568*† 7,767 ⫾1,225*†
Male
WT 250 ⫾19 190 ⫾6 8,691 ⫾635 695 ⫾74† 837 ⫾70† 2,273 ⫾241†
GKO 301 ⫾31 215 ⫾8 19,261 ⫾1,503* 1,314 ⫾125*† 6,250 ⫾474*†## 7,599 ⫾1,129*†
a
Spleen cells from uninfected or day-7 P. chabaudi AS-infected WT and GKO mice were cultured with PRBC (10
6
/ml), ConA (5 ␮g/ml), or medium as the control.
[
3
H]thymidine uptake was measured, and data are means ⫾SEM for three mice per group.
b
†, P⬍0.001 for uninfected versus infected mice; ⴱ,P⬍0.01 for WT versus GKO mice; # and ##, P⬍0.05 and P⬍0.01, respectively, for male versus female
mice.
TABLE 2—Continued
No. of nucleated cells (10
7
)/spleen (%) for mice that were
b
:
Infected
WT GKO
Male Female Male Female
30.5 ⫾2.2 31.6 ⫾4.5 24.2 ⫾2.9 28.2 ⫾1.6
8.7 ⫾0.3* (28.8 ⫾1.1) 10.6 ⫾1.8 (33.2 ⫾1.3) 6.5 ⫾0.6 (26.5 ⫾1.8) 9.5 ⫾0.3 (33.9 ⫾2.8)
18.4 ⫾2.8 (59.6 ⫾4.9) 19.7 ⫾2.8 (62.4 ⫾0.8) 15.3 ⫾1.7 (63.0 ⫾3.5) 14.1 ⫾0.2 (50.4 ⫾3.6)
1.76 ⫾0.3 (5.8 ⫾0.9) 1.81 ⫾0.32 (5.7 ⫾0.3) 1.30 ⫾0.30 (5.4 ⫾0.5) 1.43 ⫾0.14 (4.9 ⫾0.4)
1.53 ⫾0.14** (5.0 ⫾0.3)* 3.82 ⫾0.35*# (12.4 ⫾1.2)** 0.53 ⫾0.08 (2.3 ⫾0.8) 0.99 ⫾0.14 (3.5 ⫾0.6)
0.84 ⫾0.11** (2.7 ⫾0.2) 1.34 ⫾0.29* (4.1 ⫾0.4)* 0.39 ⫾0.04 (1.7 ⫾0.3) 0.66 ⫾0.10 (2.3 ⫾0.2)
VOL. 68, 2000 IMMUNE RESPONSES TO BLOOD-STAGE MALARIA IN GKO MICE 4403

parasite species involved (10, 15, 37). Treatment of P. chabaudi
AS-susceptible A/J mice with human recombinant TNF-␣sup-
presses parasitemia and reduces mortality. Furthermore, a
Th1-associated increase in TNF-␣expression in the spleen
correlates with resistance to this infection (15, 32). However,
mice deficient in TNF-␣(unpublished observation) or in the
TNF-␣p55 and p75 receptors (29) develop similar levels of
peak parasitemia and clear P. chabaudi AS infection at the
same time as WT control mice, suggesting that the absence of
TNF-␣activity does not impair protective Th1 responses
against blood-stage malaria. Indeed, TNF-␣receptor-deficient
mice have normal serum IL-12 p70, IFN-␥, and NO levels
during infection (29). TNF-␣production was increased in vivo
in both WT and GKO mice during infection, but serum TNF-␣
levels were significantly lower in GKO mice. These observa-
tions are consistent with findings in experimental models of
sepsis in mice lacking either IFN-␥(12) or the IFN-␥receptor
(16) and demonstrate that IFN-␥has an important role in
up-regulating TNF-␣production during malaria.
High levels of NO in the sera of P. chabaudi AS-infected WT
mice and in supernatants of LPS-stimulated splenic macro-
phage from these mice were detected, but this response was
completely abolished in infected GKO mice. These results
demonstrate the critical role of IFN-␥in inducing NO produc-
tion during blood-stage malaria and, consistent with our pre-
vious findings (14), suggest a correlation between NO produc-
tion and resistance to P. chabaudi AS infection. Previously, we
reported that treatment of P. chabaudi AS-infected mice with
the selective inducible NO synthase (iNOS) inhibitor, amino-
guanidine, results in high mortality but does not alter the
course of parasitemia (14, 36). Based on these observations, we
proposed that NO plays a role in protecting the host by regu-
lating the immune response rather than direct parasite killing
(14). Indeed, we showed previously that NO suppresses the in
vitro proliferative responses of spleen cells from P. chabaudi
AS-infected WT B6 mice to specific antigens and the mitogen
ConA (1). The proliferation of spleen cells from infected WT
mice in response to ConA and malaria antigen was significantly
lower than the response of cells from infected GKO mice, and
suppression was coincident with high levels of NO in spleen
cell cultures from WT but not GKO mice (data not shown).
Recent studies of P. chabaudi AS-infected mice treated with
FIG. 2. Cytokine production in vitro by spleen cells from uninfected and P.
chabaudi AS-infected WT and GKO mice. Single cell suspensions were prepared
from spleens recovered from uninfected and day-7-infected male and female WT
and GKO mice and cultured for 48 h in the presence of PRBC (10
6
/ml). Super-
natants were collected, and the levels of IFN-␥(A), TNF-␣(B), IL-4 (C), and
IL-10 (D) were analyzed by ELISA. Data are means ⫾SEM of four or five
individual mice from one of three experiments. ⴱand #, statistically significant
differences (P⬍0.05) between GKO and WT mice and between female and
male mice of the same genotype, respectively; ND, not detectable.
FIG. 3. Production of IL-12 p70 and NO in vitro by splenic macrophages
from uninfected and P. chabaudi AS-infected WT and GKO mice. Splenic
macrophages from uninfected and infected (day 2 for IL-12 p70 and day 7 for
NO) mice were cultured for 20 h in the presence of 1 ␮g of LPS/ml. Supernatants
were collected, and the levels of IL-12 p70 (A) and NO
2⫺
(B) were determined.
Data are means ⫾SEM of three individual mice per group from one of two
experiments. Statistically significant differences are indicated as follows: †, P⬍
0.05; ‡, P⬍0.01; §, P⬍0.001 (uninfected versus infected mice); ⴱ,P⬍0.05; ⴱⴱ,
P⬍0.01 (WT versus GKO mice).
TABLE 4. Malaria-specific antibody isotype levels in serum of P. chabaudi AS infected WT and GKO mice
a
Mouse
type
Level of P. chabaudi AS-specific antibody isotype
b
:
Total Ig IgM IgG1 IgG2a IgG2b IgG3
Female
WT 1,610 ⫾119 3,069 ⫾578* 113 ⫾27 755 ⫾84** 72 ⫾13 604 ⫾92*
GKO 1,611 ⫾144 1,428 ⫾169 612 ⫾83** 153 ⫾29 91 ⫾23 158 ⫾77
Male
c
WT 1,807 ⫾165 2,228 ⫾187 138 ⫾21 665 ⫾40 153 ⫾34 624 ⫾40
a
Parasite-specific antibody titers were determined by ELISA in serum collected 14 days following infection with P. chabaudi AS. Data are means ⫾SEM for four
mice per group.
b
ⴱand ⴱⴱ,P⬍0.05 and P⬍0.01, respectively, for WT versus GKO mice of the same sex.
c
All male GKO mice died by day 14.
4404 SU AND STEVENSON INFECT.IMMUN.

aminoguanidine demonstrated that NO also suppresses IFN-␥,
TNF-␣, and IL-2 production during acute infection (39). NO-
mediated suppression of spleen cell proliferation and IFN-␥
production has also been demonstrated in iNOS-deficient mice
infected with Leishmania major (46) and Trypanosoma brucei
rhodesiense (13). Together, these results suggest that NO pro-
duction during P. chabaudi AS infection regulates the intensity
and duration of Th1-associated immune responses and main-
tains the balance between the protective and pathologic effects
of IFN-␥and TNF-␣.
While increased mRNA expression and production of the
Th1 cytokines IL-12, IFN-␥, and TNF-␣by spleen cells corre-
late with resistance to P. chabaudi AS infection, the exquisite
susceptibility of A/J mice to this parasite correlates with spleen
cell production of Th2 cytokines (15, 23, 27, 34, 36). Interest-
ingly, the absence of IFN-␥coincided with significantly in-
creased production of the Th2 cytokine IL-10 by spleen cells
from P. chabaudi AS-infected GKO compared to that by
spleen cells from WT mice. We also investigated whether the
abnormalities in systemic and in vitro IL-12 p70, TNF-␣, NO,
and IL-10 production in GKO mice were related to imbalances
in splenic leukocyte populations. Fluorescence-activated cell
sorter analysis of nucleated spleen cells showed that the re-
cruitment and/or local proliferation of NK cells and F4/80
⫹
macrophages expressing Ia antigen were deficient in infected
male and female GKO mice compared to their WT counter-
parts. Studies of P. chabaudi AS-infected IL-10 deficient mice
showed that increased mortality among these mice is accom-
panied by an enhanced Th1 IFN-␥response during acute in-
fection which is retained in the chronic phase of infection (18).
Th1 cytokine responses are also sustained during P. chabaudi
AS infection in IL-4-deficient mice compared to WT litter-
mates (44). We observed that IL-10 production was signifi-
cantly higher in spleen cells from TNF-␣receptor-deficient
mice than in those from WT mice, but comparable levels of
IFN-␥and IL-4 were produced by cells from the two genotypes
(29). These observations suggest that there is coordinate and
tight counterregulation by cytokines during blood-stage P.
chabaudi AS infection. Cytokine-deficient mice should, there-
fore, be very useful in understanding the interactions of cyto-
kines and their balance during blood-stage malaria.
The requirement for antibodies in the resolution of blood-
stage P. chabaudi AS infection was clearly demonstrated in
B-cell-depleted mice, which are unable to resolve P. chabaudi
AS infection efficiently (38, 43, 45). Intact control mice, which
control the infection and clear the parasites, initially mount a
strong Th1-associated IgG2a response followed by a Th2-as-
sociated IgG1 response during the chronic stage of infection
(38). The IgG fraction of immune sera from P. chabaudi AS-
infected mice binds to the surfaces of PRBC and facilitates
their phagocytosis by macrophages (24). We observed that
female GKO mice produced significantly lower levels of para-
site-specific IgM, IgG2a, and IgG3 but more IgG1 than their
WT counterparts. This alteration in antibody isotypes may, in
part, account for the persisting parasitemia in female GKO
mice, the majority of which survive primary blood-stage P.
chabaudi AS infection.
Gender differences among humans as well as experimental
animals in resistance to parasitic diseases, including malaria,
are apparent. We observed that male mice, either WT or
GKO, developed higher peak parasitemias than their female
counterparts, and 100% of male GKO mice succumbed to the
infection. These results confirm and extend our previous ob-
servation of a gender difference in resistance to P. chabaudi AS
infection (33). The increased susceptibility of male mice to this
parasite is due to the immunosuppressive effects of testoster-
one, which is known to modulate the production of and re-
sponse to cytokines (2, 3, 25). Here, we provide evidence of
gender-associated immunologic differences in response to P.
chabaudi AS infection. In comparison with infected female WT
mice, male WT mice had significantly lower levels of serum
IFN-␥and reduced in vitro production of this cytokine by
spleen cells in response to PRBC. Significantly lower levels of
IL-10 were also apparent in supernatants of spleen cells from
male versus female GKO mice stimulated with parasite antigen
or ConA (data not shown). A difference between male and
female WT mice in the numbers of F4/80
⫹
Ia
⫹
macrophages in
the spleens of infected as well as uninfected mice was also
apparent. Intriguingly, as observed here and previously, the
gender difference in response to P. chabaudi AS infection is
more prominent in cytokine- or cytokine receptor-deficient
mice (18, 29). Further studies are required to understand the
interactions between sex hormones and the immune response
during blood-stage malaria.
In conclusion, the findings presented in this study unequiv-
ocally demonstrate the critical and central role of endogenous
IFN-␥in regulating protective immune responses against
blood-stage P. chabaudi AS infection. In the absence of IFN-␥,
production of important counterregulatory molecules is dra-
matically altered. Production of protective Th1 mediators, in-
cluding IL-12, TNF-␣, and NO, is deficient, while there is
increased production of the Th2 cytokine IL-10. Antibody pro-
duction in GKO mice is also altered. Furthermore, this study
highlights the additive effects of the immunosuppressive male
sex hormone testosterone and the lack of IFN-␥, which render
male GKO mice extremely susceptible to P. chabaudi AS.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research
Council of Canada (MT14663) to M.M.S.
We gratefully acknowledge the technical assistance of Mi Fong Tam
for breeding GKO mice, maintaining the parasite, and performing the
infection studies. The secretarial assistance of Marlene Salhany is also
gratefully appreciated.
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Editor: J. M. Mansfield
4406 SU AND STEVENSON INFECT.IMMUN.