Decreased Resistance of B Cell-Deficient Mice

to Infection with Toxoplasma gondii Despite

This information is current as
of April 1, 2020.
Unimpaired Expression of IFN- γ, TNF-α,
and Inducible Nitric Oxide Synthase
Hoil Kang, Jack S. Remington and Yasuhiro Suzuki
J Immunol 2000; 164:2629-2634; ;
doi: 10.4049/jimmunol.164.5.2629
http://www.jimmunol.org/content/164/5/2629

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Decreased Resistance of B Cell-Deficient Mice to Infection with
Toxoplasma gondii Despite Unimpaired Expression of IFN-␥,
TNF-␣, and Inducible Nitric Oxide Synthase1

Hoil Kang, Jack S. Remington, and Yasuhiro Suzuki2

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (␮MT) mice. Following peroral
infection with 10 cysts of the ME49 strain, all ␮MT mice survived the acute stage of the infection but died between 3 and 4 wk
after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which ␮MT animals succumbed
to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also
detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites
were observed in brains of ␮MT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed
only in brains of ␮MT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-␥
were detected in sera of both strains of mice. Amounts of mRNA for IFN-␥, IL-10, and inducible NO synthase in the brain did

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not differ between infected ␮MT and control mice. Expression of mRNA for TNF-␣ was increased in brains of ␮MT mice.
Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in
the brain in the infected ␮MT mice. These results indicate that B cells play an important role, most likely through their production
of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung. The
Journal of Immunology, 2000, 164: 2629 –2634.

C ell-mediated immunity is critical for host resistance
against acute acquired infection with T. gondii (1– 4) and
against development of toxoplasmic encephalitis during
the chronic stage of the infection (5–9). Although infection with T.
gondii also stimulates humoral immunity, the role of Abs in resis-
tance against T. gondii remains unclear. Previous studies have
demonstrated that administration of anti-T. gondii polyclonal (10,
11) or mAbs (12, 13) administered before infection resulted in
reduction in mortality or prolonged time to death in mice. How-
ever, these studies did not address whether the Ab response during
the natural course of the immune response following infection
plays a protective role against this organism.
Frenkel and Taylor (14) previously examined the effects of B
cell depletion on toxoplasmosis in mice by treatment with anti-␮
Ab. From their results, they suggested that Ab may not be decisive
in resistance against the acute infection, but may be important in
controlling the latent infection. Because of the potential side ef-
fects of injection of large quantities of xenogenic Ab (15), these
studies did not provide conclusive results. Chronic anti-␮ Ab treat-
ment involves delivery of an extraordinary amount of Ab, which
may alter the microenvironment and concomitant cell-cell interac-
Department of Immunology and Infectious Diseases, Research Institute, Palo Alto
Medical Foundation, Palo Alto, CA 94301; and Division of Infectious Diseases and
Geographic Medicine, Department of Medicine, Stanford University School of Med-
icine, Stanford, CA 94305
Received for publication August 23, 1999. Accepted for publication December
16, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by U.S. Public Health Service Grants AI04717 and
AI38260.
2
Address correspondence and reprint requests to Dr. Yasuhiro Suzuki, Department of
Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Founda-
tion, AMES Building, 795 El Camino Real, Palo Alto, CA 94301. E-mail address:
ysuzuki@leleand.stanford.edu
tions that occur during infection with T. gondii (15–17). In addi-
tion, despite the extraordinary amount of Ab used, the anti-␮ Ab
may not completely deplete B cell populations in vivo, and al-
though serum IgM is reduced to undetectable levels, IgG is gen-
erally readily detectable, albeit at levels 10- to 1000-fold lower
than normal (17). To elucidate whether B cells play a protective
role in resistance against infection with T. gondii, additional stud-
ies using alternative approaches to deplete B lymphocytes are
needed.
B cell-deficient (␮MT) mice have recently been generated by
disruption of one of the membrane exons of the ␮-chain gene (18).
These animals have no detectable B cells or circulating Ab, yet
display normal development of the T lymphocyte compartment
(18). Moreover, previous studies have been shown that ␮MT mice
have normal Ag-presenting function for priming of CD4⫹ T cells
to most soluble Ags as well as unimpaired CD8⫹ T lymphocyte
response (19 –21). Thus, ␮MT mice appear to be a suitable model
to analyze the role of B cells in host resistance to T. gondii infec-
tion. In the present study, we examined susceptibility of ␮MT
mice to peroral infection with T. gondii. These mice suffered early
mortality associated with continuous proliferation of tachyzoites in
their brains and lungs in the absence of Ab responses to the par-
asite but with unimpaired expression of IFN-␥, TNF-␣, and induc-
ible NO synthase (iNOS).3
Materials and Methods
Mice
Female Swiss-Webster (Taconic Farms, Germantown, NY), C57BL/6-
background B cell-deficient (␮MT) (The Jackson Laboratory, Bar Harbor,
ME), and control C57BL/6 mice (The Jackson Laboratory) were 6 – 8 wk
old when used.
3
Abbreviation used in this paper: iNOS, inducible NO synthase.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

2630
Infection with T. gondii
Cysts of the ME49 strain were obtained from brains of chronically infected
Swiss-Webster mice as described previously (22). ␮MT and control mice
were infected with 10 cysts perorally by gavage.
Histopathology
At 25 days after infection, mice were euthanized by asphyxiation with
CO2. Their brains, lungs, hearts, livers, spleens, kidneys, and small and
large intestines were removed and immediately fixed in a solution contain-
ing 10% Formalin, 70% ethanol, and 5% acetic acid. Two to four 5-␮m-
thick sections (50- or 100-␮m distance between sections) of the organs
from each mouse were stained with hematoxylin and eosin. Immunoper-
oxidase staining using rabbit IgG Ab against either tachyzoite-specific
SAG2 or bradyzoite-specific BAG1 were used for detection of tachyzoites
and cysts, respectively (23, 24). The specificity of these Abs were de-
scribed previously (25, 26). Sections stained with hematoxylin and eosin
were evaluated for inflammatory changes, and sections stained by the im-
munoperoxidase method were evaluated for the number of T. gondii cysts
and areas associated with tachyzoites.
Detection of mRNA for cytokines and tachyzoite-specific SAG1
RNA was isolated from brains of infected ␮MT and control mice by using
RNA Stat60 (Tel-Test, Friendswood, TX) by following the commercial
insert. cDNA was synthesized using the RNA as described previously (22,
27). PCR for cytokines was performed with 5 ␮l of either a 1:10 dilution
or 1:50 dilution of the original cDNA reaction mixture with a Geneamp
9700 thermocycler (Perkin-Elmer, Emeryville, CA) using 30 cycles to pro-
duce the amount of DNA within a linear range as described previously (22,
27). This number of cycles was determined in preliminary studies using
different amounts of cDNA of the sample. Specific primers for ␤-actin,
IFN-␥, TNF-␣, IL-10 (Clontech, Palo Alto, CA), and iNOS (28) designed
to span at least one intron allowed differentiation of amplified target DNA
derived from either cDNA or genomic DNA in the PCR. PCR for SAG1
were performed using 1 ␮l of the original cDNA reaction mixture, as
described by Gazzinelli et al. (28). For each gene product, the number of
cycles was determined experimentally and was defined as that number of
cycles that would achieve a detectable concentration that was well below
the saturating conditions. In these conditions, 3-fold differences in cDNA
concentrations showed clear differences in the amounts of PCR products.
Detection of PCR products
Ten-microliter aliquots of the final PCR mixtures were electrophoresed at
100 V for 1 h on a 1.5% agarose gel and denatured (22, 27). The DNA was
then transferred to a Duralon-UV membrane (Stratagene, La Jolla, CA) by
standard blotting procedures (29) and UV cross-linked. Oligonucleotide
probes for ␤-actin, IFN-␥, TNF-␣, IL-10 (Clontech), and iNOS (26), which
hybridize to the PCR products wholly within the region amplified by the
primers, were end labeled as described for the 3⬘-end labeling and signal
amplification system for a FluorImager (Amersham, Little Chalfont, En-
gland), and hybridization was detected by scanning of the membranes with
a FluorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA) as de-
scribed previously (30). Quantification of mRNA was performed by den-
sitometry analysis with the FluorImager and normalized to the ␤-actin
level.
Serum levels of IFN-␥
Mice were bled at 25 days after infection and the concentrations of IFN-␥
in their sera were measured by ELISA using mAbs against IFN-␥ (R4 –
6A2 as capture, XMG 1.2 as secondary) obtained from PharMingen (San
Diego, CA).
Toxoplasma Ab
Toxoplasma Ab titers in sera of animals obtained at 25 days after infection
were measured by the Sabin-Feldman dye test (31).
Flow cytometry
Spleen cells were obtained from ␮MT and control mice before and 23 days
after infection. A total of 1 ⫻ 106 of spleen cells was pretreated on ice for
10 min with 10 ␮l of a predetermined optimal concentration of anti-Fc␥II/
III receptors (2.4G2) to block non-Ag-specific binding of Abs to the Fc␥II/
III receptors. Thereafter, the cells were incubated on ice for 30 min with 10
␮l of optimal concentrations of FITC-conjugated anti-CD8 mAb (53-6.7)
and PE-conjugated anti-CD4 mAb (RM4-5), or PE-conjugated anti-NK1.1
mAb (PK136). The mAbs were obtained from PharMingen. Analysis of
ROLE OF B CELLS IN RESISTANCE TO T. gondii
FIGURE 1. Mortality in ␮MT and control mice following infection
with T. gondii. ␮MT and control mice were infected perorally with 10 cysts
of the ME49 strain. The data are representative of two separate experiments
performed.
stained cells was performed with a FACScan (Becton Dickinson, Mountain
View, CA). Dead cells were gated out on the basis of propidium iodide
staining.
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Administration of anti-T. gondii IgG Ab
T. gondii-specific IgG Abs were purified by using a protein A column from
sera of rabbits that had been infected i.p. with 50 cysts of the ME49 strain
5 wk earlier. ␮MT mice were injected i.p. with 0.12 ml (0.8 mg of protein)
of the purified IgG solution (dye test tier, ⱖ1:16,000) every 3 days begin-
ning at 2 days after infection for 40 days. Control mice were injected with
the same amounts of normal rabbit IgG purified from seronegative rabbits
in the same manner.
Statistical analysis
Levels of significance for differences between groups of mice were deter-
mined by Student’s t or Mann-Whitney U test. Mann-Whitney U test was
applied when SDs were significantly different between groups tested. Dif-
ferences which provided p ⬍ 0.05 were considered to be significant.
Results
Effect of T. gondii infection on mortality in B cell-deficient and
control mice
Following infection with 10 cysts of the ME49 strain, ␮MT mice
all died between 21 and 28 days after infection (Fig. 1). In contrast,
all control mice were alive at 59 days after infection, and thereafter
began dying; all died by 85 days after infection ( p ⫽ 0.0007,
Fig. 1).
Histological changes in the organs of infected ␮MT mice
Because of the remarkable difference in mortality between infected
␮MT and control mice, we examined their brains, hearts, lungs,
livers, kidneys, and small and large intestines at 25 days after
infection (when some ␮MT mice had already died). The most re-
markable histological changes were observed in brains of ␮MT
mice. Significantly greater numbers of areas associated with T.
gondii tachyzoites were observed in brains of ␮MT than in control
mice ( p ⫽ 0.0001, Fig. 2B). In ␮MT mice, there were many small
areas of acute focal inflammation with the parasites (Fig. 3A).
Large areas of necrosis of brain tissue associated with numerous
tachyzoites were observed in ␮MT but not in control mice (Fig.
3B). Numbers of cysts were also significantly greater in brains of
␮MT than in control mice ( p ⫽ 0.019, Fig. 2A). These facts in-
dicate that continuous proliferation of tachyzoites and formation of
greater numbers of cysts occur in brains of ␮MT mice following
infection.
Small numbers of tachyzoites were observed in the lungs in 50%
of the ␮MT mice examined (n ⫽ 10). T. gondii cysts were also
observed in lungs of two of these mice (Fig. 3C). Neither
The Journal of Immunology 2631

FIGURE 2. Number of T. gondii cysts (A) and areas of inflammation

associated with tachyzoites (B) in brains of infected ␮MT and control mice.
Mice were infected perorally with 10 cysts of the ME49 strain and 25 days
later, histological study was performed on their brains. Four sagittal sec-
tions (distance between section of 50 ␮m) from each mouse were stained
with an immunoperoxidase stain using Ab against bradyzoite-specific
BAG1 (A) or tachyzoite-specific SAG2 (B) and evaluated for counting. The
mean value from these sections for each mouse was calculated as the num-
ber per section. These values were shown in the figure and were used for
statistical analysis to compare differences between groups. The data reflect
results of two separate experiments. Each experimental group in each ex-

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periment contains three to six mice.

tachyzoites nor cysts were detectable in lungs of control mice (n ⫽

7). Despite these differences in the presence of the parasite be-
tween ␮MT and control mice, infiltration of inflammatory cells in
the lungs was similar in both groups.
Although the tachyzoites were not detected, mild infiltration of
inflammatory cells was observed in the liver and heart of both
␮MT and control mice. Neither the parasite nor inflammatory re-
sponses were observed in the kidneys or intestines of ␮MT mice or
controls.

Tachyzoite-specific SAG1 mRNA in brains of infected mice

At 25 days after infection, amounts of tachyzoite-specific mRNA
encoding SAG1 in the total RNA fractions were measured by re-
verse transcripting the RNA, followed by amplification of SAG1-
specific cDNA using PCR (RT-PCR). Significantly greater
amounts of SAG1 mRNA were detected in total RNA obtained
from brains of infected ␮MT mice than control mice ( p ⫽ 0.0002),
although there were variations between individual ␮MT mice
(Fig. 4).

Levels of mRNA for cytokines and iNOS in brains of infected

mice
It was recently reported that spleen cells from Chlamydia tracho-
matis-infected B cell-deficient mice failed to produce Th1-related
(IFN-␥) or Th2-related (IL-6 and IL-10) cytokines after C. tracho-
matis-specific in vitro restimulation (16). Since an IFN-␥-mediated
immune response has been shown to be critical for resistance
against development of toxoplasmic encephalitis (5–9, 32), we ex-
amined whether expression of IFN-␥, TNF-␣, and iNOS is im-
paired in brains of ␮MT mice infected with T. gondii. We also
examined expression of IL-10 in brains of these mice because this
cytokine has been reported to inhibit T. gondii microbicidal activ- FIGURE 3. Histological changes and detection of T. gondii in the brain
and lung of infected ␮MT mice. Mice were infected perorally with 10 cysts
ity of macrophages activated by IFN-␥ (33). Amount of mRNA for
of the ME49 strain, and histological study was performed 25 days later. A,
the cytokines and iNOS were measured by RT-PCR in the total
An area of acute focal inflammation associated with tachyzoites in the
RNA fractions obtained from brains of mice at 25 days after in- brain. B, A large area of necrosis of brain tissue associated with
fection. The amounts of mRNA for IFN-␥, IL-10, and iNOS did tachyzoites. C, A cyst observed in the lung. Sections were stained by im-
not differ between these mice (Fig. 4). The amounts of mRNA for munoperoxidase stain using rabbit IgG Abs against (A and B) tachyzoite-
TNF-␣ were significantly greater in ␮MT than in control mice specific SAG2 or (C) bradyzoite-specific BAG1 (arrowheads indicate A
(TNF-␣:␤-actin ratio, 0.191 ⫾ 0.081 vs 0.052 ⫾ 0.013, p ⫽ and B T. gondii tachyzoites and C a cyst). The experiment was performed
0.0021) (Fig. 4). In uninfected ␮MT or control mice, the amounts twice, and there were three to six mice in each group in each experiment.
2632
FIGURE 4. Detection of cytokine mRNAs using PCR-associated am-
plification of RNA in brains of ␮MT and control mice infected with T.
gondii. Groups of three to five mice were killed 25 days after infection with
the ME49 strain, and their brains were analyzed for the presence of cyto-
kine mRNAs (see Materials and Methods). Lanes 1-3, control mice; lanes
4 – 8, ␮MT mice. The data are representative of two separate experiments
performed.
of mRNA for each of these molecules were very low and mostly
undetectable (ratios to ␤-actin were below 0.001).
IFN-␥ levels in sera of infected ␮MT and control mice
To compare systemic IFN-␥ production between ␮MT and control
mice, we measured serum levels of this cytokine at 25 days after
infection. Levels of IFN-␥ did not differ between these mice
(1.95 ⫾ 0.54 ng/ml in controls (n ⫽ 7) vs 2.35 ⫾ 0.53 ng/ml in
␮MT (n ⫽ 9)). The results were reproducible in two separate
experiments.
Toxoplasma Ab titers
Toxoplasma dye test Ab titers were examined in sera obtained at
25 days after infection. Whereas high Ab titers (dye test titers
ranged from 1:1024 to 1:4096) were observed in sera of control
mice, they were undetectable in sera of ␮MT animals.
Changes in relative percentages of T cell subsets and NK cells
in the spleen in ␮MT and control mice following infection
We examined relative percentages of T cell subsets and NK cells
in the spleen of ␮MT and control mice by flow cytometry before
and 23 days after infection. Relative percentages of CD8⫹ T cells
significantly increased following infection in both ␮MT (from
37.6 ⫾ 4.0% to 50.1 ⫾ 4.9%; p ⫽ 0.014) and control mice (from
12.0 ⫾ 0.5% to 18.3 ⫾ 4.1%; p ⫽ 0.032). In contrast, relative
percentages of NK cells significantly decreased following infec-
tion in both ␮MT (from 8.3 ⫾ 1.7% to 2.9 ⫾ 0.8%; p ⫽ 0.0015)
and control mice (from 3.3 ⫾ 0.4% to 1.1 ⫾ 0.4%; p ⫽ 0.0006).
Relative percentages of CD4⫹ T cells did not differ before and
after infection in both groups of animals (data not shown). Because
of the absence of B cells in ␮MT mice, relative percentages of T
cells and NK cells were higher in ␮MT than in control mice.
Passive transfer of anti-T. gondii IgG to infected ␮MT mice
To examine whether the cause of early death in infected ␮MT
mice was due to the absence of Ab responses, we treated these
mice with rabbit anti-T. gondii IgG Abs every 3 days beginning 2
days after infection. Control ␮MT mice were treated with normal
rabbit IgG in the same manner. All control ␮MT mice died from
21 to 25 days after infection (Fig. 5). ␮MT mice treated with
anti-T. gondii IgG Abs were all alive at the end of observation
ROLE OF B CELLS IN RESISTANCE TO T. gondii
FIGURE 5. Effect of treatment with anti-T. gondii IgG Ab on mortality
and time to death of ␮MT mice infected with T. gondii. Mice were infected
perorally with 10 cysts of the ME49 strain and injected i.p. with 0.8 mg of
either rabbit anti-T. gondii (‚) or normal IgG Ab (F) every 3 days begin-
ning at 2 days after infection for 40 days. The data shown involved four to
five mice per group.
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period (42 days after infection) (Fig. 5). Histological studies were
performed on brains and lungs of ␮MT animals in both groups at
23 days after infection. Significantly fewer numbers of T. gondii
cysts and inflammatory areas associated with tachyzoites were ob-
served in brains of mice treated with anti-T. gondii IgG Abs than
those treated with normal IgG ( p ⫽ 0.017 for either the number of
cysts or inflammatory areas) (Fig. 6). Small numbers of
tachyzoites were detected in lungs of animals only treated with
normal IgG (data not shown). Toxoplasma Abs were detected in
sera of mice treated with anti-T. gondii IgG Abs (dye test titer at
1:2048) but not in those treated with normal IgG.
Discussion
The results described above reveal that mice deficient in B cells
were able to survive the acute stage of infection with T. gondii but
succumbed to the parasite 3– 4 wk after infection. In contrast, all
control mice were still alive 8 wk after infection. As expected,
FIGURE 6. Effect of treatment with anti-T. gondii IgG Ab on number of
T. gondii cysts (A) and areas of inflammation associated with tachyzoites
(B) in the brain in infected ␮MT mice. Mice were infected perorally with
10 cysts of the ME49 strain and injected i.p. with 0.8 mg of either rabbit
anti-T. gondii or normal IgG Ab every 3 days beginning at 2 days after
infection. Twenty-three days after infection, histological study was per-
formed on their brains. Four sagittal sections (distance between section of
50 ␮m) from each mouse were stained with an immunoperoxidase stain
using Ab against bradyzoite-specific BAG1 (A) or tachyzoite-specific
SAG2 (B) and evaluated for counting. The mean value from these sections
for each mouse was calculated as the number per section. These values
were shown in the figure and were used for statistical analysis to compare
differences between groups. Each experimental group in each experiment
contains three to seven mice.
The Journal of Immunology
Toxoplasma Abs were detectable in sera of infected control but not
of ␮MT mice. Adoptive transfer of anti-T. gondii IgG Abs to in-
fected ␮MT mice prevented their early mortality. Thus, production
of Abs against T. gondii by B cells was critical for prevention of
mortality in mice during the chronic stage of infection. This is the
first evidence for the importance of B cells in host resistance to T.
gondii during the natural course of infection.
Histological study revealed large numbers of tachyzoites in
brains of ␮MT mice but not of control mice at the time when ␮MT
mice had begun to die. Tachyzoites were also demonstrable in the
lung of only ␮MT mice but the numbers of the organisms in their
lungs were markedly fewer than in their brains. These results in-
dicate that B cells, most likely Ab production by these cells, are
important for preventing persistence of tachyzoite replication in
the brain and lung in mice during the late stage of the infection.
This is further supported by the evidence that treatment with anti-
T. gondii IgG Abs markedly decreased the parasite load in these
organs. Since tachyzoites were not detectable in hearts, livers, kid-
neys, or intestines of the ␮MT mice when they had begun to die,
the mechanism of host defense against T. gondii appears to differ
in different organs.
In addition to greater tachyzoite load in brains and lungs of
␮MT mice, we observed differences in the numbers of T. gondii
cysts in these organs. Markedly and significantly greater numbers
of cysts were detected in brains of ␮MT than in control mice. In
addition, cysts were detectable only in lungs of ␮MT mice al-
though the numbers of cysts were small. In our previous experi-
ence, formation of T. gondii cysts in the lung is unusual. Persistent
proliferation of tachyzoites in the brain and lung of ␮MT mice
may have contributed to formation of greater numbers of cysts in
these organs.
Frenkel and Taylor (14) previously addressed the role of B cells
in resistance against T. gondii by examining the occurrence of
reactivation of latent infection in anti-␮ Ab-treated mice infected
with a virulent strain and treated with sulfadiazine. They observed
mortality associated with toxoplasmic pneumonia and/or enceph-
alitis in chronically infected anti-␮-treated animals after discon-
tinuation of sulfadiazine treatment. In the present study, persis-
tence of tachyzoite proliferation was observed in brains and lungs
of ␮MT mice during the late stage of the primary infection. Thus,
Ab appears to play a critical role in prevention of proliferation of
tachyzoites in these organs in both primary infection and reacti-
vation of latent infection. In addition to the brain and lung, myo-
carditis was also reported in the anti-␮-treated mice (14). How-
ever, this pathology was not evident in ␮MT mice in the present
study. This difference may be due to the side effects of treatment
with large amounts of xenogenic anti-␮ Ab on the immune system
in the previous study, the presence or absence of sulfadiazine treat-
ment, differences in virulence of the parasite and mouse strains,
and/or different infection conditions (primary vs reactivation).
Despite the absence of humoral immunity, we observed signif-
icant increases in relative percentages of CD8⫹ T cells and de-
creases in relative percentages of NK cells in spleens of both ␮MT
and control mice following infection. We also observed unim-
paired IFN-␥ expression in T. gondii-infected ␮MT mice. Cellular
immunity mediated by IFN-␥ (5, 6, 32), TNF-␣ (7, 8, 27, 28), and
iNOS (9, 34) have been reported to be critical for resistance against
development of toxoplasmic encephalitis in mice. Although in-
fected ␮MT mice developed severe encephalitis, expression of
these molecules in their brains were similar to or higher than those
in control mice. Serum levels of IFN-␥ were also similar in both
groups of mice. In addition, expression of IL-10 in the brain did
not differ between ␮MT and control mice. These results further
2633
support the likelihood that absence of Ab production is a major
factor in the reduced resistance to T. gondii in these mice.
Eperon et al. (35) recently reported that following infection with
Neospora caninum, ␮MT mice developed focal necrotic cerebral
lesions which were absent in control mice. In their studies, IFN-␥
production by spleen cells was lower in ␮MT than in control mice
during the early stage of infection; however, it did not differ be-
tween these animals during the later stage when cerebral pathology
was observed in ␮MT mice (35). Yang and Brunham (16) recently
reported that production of both Th1- and Th2-type cytokines were
suppressed in ␮MT mice following C. trachomotis infection. In
infection with lymphocytic choriomeningitis virus, ␮MT mice
were found to have a CD4 helper defect (36). A regulatory role of
B cells on T cell responses may differ depending on infectious
agents which stimulate the immune system, the stages of infection,
and subsets of T cells.
In the presence of specific Abs, T. gondii tachyzoites are rapidly
lysed by activation of complement through the classical pathway
in vitro (37, 38), although the parasites are resistant to complement
in the absence of Abs (39). This Ab-dependent complement-me-
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diated killing may play an important role in resistance of mice to
the infection. Abs also appear to play a protective role in collab-
oration with macrophages. Macrophages have been shown to kill
Ab-coated tachyzoites in vitro (40, 41). Since the present study
revealed persistent proliferation of tachyzoites in brains and lungs
of ␮MT mice, Ab coating of the parasite to allow for phagocytosis
and killing by resident macrophages (microglia and alveolar mac-
rophages) in the brain and lung may be an important protective
mechanism. In addition, Ab may inhibit intracellular proliferation
of tachyzoites. Mineo and Kasper (42) reported that treatment of
tachyzoites with a mAb to T. gondii in the absence of complement
did not inhibit either attachment or invasion of the parasite into
human fibroblasts but inhibited their intracellular proliferation.
It has been well documented that IFN-␥-mediated cellular im-
munity is required for survival of mice during both the acute and
chronic stages of the infection with T. gondii (1–9). The results of
the present study indicate the importance of the B cell Ab response
for prevention of persistent proliferation of tachyzoites in the brain
and lung during the chronic stage of infection. Thus, humoral and
cellular immunity act in concert in host resistance against T. gon-
dii. The role of each immune response appears to differ depending
on the stage of the infection and its anatomical location.
Acknowledgments
We thank Dr. Fausto Araujo for providing rabbit anti-T. gondii serum and
Edgar K. Gufwoli, Hanna Lee, and Pauline Chu for excellent technical
assistance with histological studies. We also thank the serology laboratory
of Palo Alto Medical Foundation for performing the Sabin-Feldman
dye test.
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