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B2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii1
*
Medical Research Council Centre for Immune Regulation, School of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom;
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and
Department of Immunology, The Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B family of transcription factors are involved in the
regulation of innate and adaptive immune functions associated with
resistance to infection. To assess the role of NF-B2 in
the regulation of cell-mediated immunity, mice deficient in the
NF-B2 gene (NF-B2-/-) were
challenged with the intracellular parasite Toxoplasma
gondii. Resistance to this opportunistic pathogen is dependent
on the production of IL-12, which is required for the development of
innate NK cell and adaptive T cell responses dominated by the
production of IFN-
necessary to control replication of this
parasite. Although wild-type controls were resistant to T.
gondii, NF-B2-/- mice developed
severe toxoplasmic encephalitis and succumbed to disease between 3 and
10 wk following infection. However, NF-B2 was not
required for the ability of macrophages to produce IL-12 or to inhibit
parasite replication and during the acute stage of infection,
NF-B2-/- mice had no defect in their
ability to produce IL-12 or IFN- and infection-induced NK cell
responses appeared normal. In contrast, during the chronic phase of the
infection, susceptibility of NF-B2-/- mice
to toxoplasmic encephalitis was associated with a reduced capacity of
their splenocytes to produce IFN- associated with a loss of
CD4+ and CD8+ T cells. This loss of T cells
correlated with increased levels of apoptosis and with elevated
expression of the pro-apoptotic molecule Fas by T cells from infected
NF-B2-/- mice. Together, these results
suggest a role for NF-B2 in the regulation of lymphocyte
apoptosis and a unique role for this transcription factor in
maintenance of T cell responses required for long-term resistance to
T. gondii. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B family of transcription factors, composed of
NF-B1, NF-B2, RelA,
RelB, and c-Rel, is induced in response to signals that lead to cell
growth, differentiation, inflammatory responses, and apoptosis
(1, 2, 3, 4). The types of signals commonly associated with
activation of NF-B are those which are a consequence of inflammation
and infection. Bacteria and their products are some of the best
activators of NF-B (5, 6, 7, 8) and cytokines such as TNF-
and IL-1 can also activate NF-B (2). The activation and
nuclear translocation of NF-B leads to increased transcription of a
number of different genes including chemokines (IL-8), adhesion
molecules (endothelial leukocyte activation molecule, VCAM, ICAM),
cytokines (IL-1, TNF-, IL-12), and inducible NO synthase (9, 10). Thus, the activation of NF-B is strongly associated with
the production of cytokines and activation of effector molecules
associated with innate immunity to infection. However, the role of
NF-B in immunity is not restricted to regulation of innate immunity
and several NF-B members are expressed in T and B cells and are
important in the regulation of their responses
(11, 12, 13, 14).
The importance of NF-B in the development of immune responses and
associated effector functions is illustrated by gene deletion studies
(15, 16, 17, 18, 19, 20, 21, 22). Thus, mice deficient in different NF-B family
members are more susceptible to various viral, bacterial, and parasitic
infections, but in many cases the basis for the increased
susceptibility to these pathogens is uncertain. To understand the role
of NF-B in the development of innate and adaptive immunity to
infection, studies were initiated to determine the role of different
NF-B family members in the immune response to T. gondii.
This parasite is an opportunistic pathogen in patients with acquired
and primary deficiencies in cell-mediated immunity
(23, 24, 25). Resistance to T. gondii is dependent
on the ability of accessory cells to produce IL-12, which stimulates
the production of IFN- by NK and T cells, which is required to
control replication of this parasite (26). Several
molecular studies indicate that NF-B is likely to be involved in
many of these steps. For example, the IL-12 p40 promoter has two
NF-B binding sites that are involved in the production of IL-12
(27), and NF-B is important in the development,
differentiation, and function of dendritic cells (20, 21, 28, 29, 30), which can produce IL-12 in response to T.
gondii (31). Evidence that NF-B is involved in the
regulation of the IFN- gene is provided by the identification of
functional NF-B sites in the promoter (32), studies in
which inhibition of NF-B activation in T cells resulted in reduced
production of IFN- following primary TCR stimulation (33, 34) and reports that mice deficient in c-Rel or RelB have
defects in their ability to produce IFN- (13, 35).
Furthermore, activation of NF-B is involved in the regulation of NO
production (9) associated with the control of parasite
replication (36, 37). Thus, the association of NF-B
activation with production of IL-12 and IFN- as well as macrophage
effector cell functions highlight the possible role that this family of
transcription factors plays in resistance to T. gondii.
Direct evidence that NF-B is involved in resistance to T.
gondii is provided by studies in which mice deficient in
NF-B2 or RelB were shown to be susceptible to
toxoplasmosis (20, 35). However, the mechanism that
underlies the increased susceptibility of these mice to toxoplasmosis
is unclear and these findings raise several questions about the
function of NF-B in resistance to infection. The studies presented
in this study confirm the important role of
NF-B2 in resistance to T. gondii
and reveal that NF-B2 is not required for the
induction of the innate or adaptive responses associated with
resistance to T. gondii. Rather, these studies identify a
unique role for NF-B2 in the regulation of
lymphocyte apoptosis in chronically infected mice and demonstrate that
it is required for the long-term maintenance of adaptive immunity to
this parasite.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female CBA/CaJ mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). The NF-B2-/- mice originated on a 129/B6
background and had been backcrossed for four to five generations on a
C57BL/6 background. Mice were typed using a PCR-based methodology which
distinguishes the wild-type
(WT)3
NF-B2 gene from the targeted
NF-B2-neo allele as previously
described (35). NF-B2-/- mice are healthy and do not display
any signs of autoimmunity or developmental defects (21).
The NF-B2-/- and
NF-B2+/+ mice used in these
experiments were littermates generated by crosses of heterozygous mice.
As a consequence it was frequently difficult to obtain large numbers of
sex-matched controls, thus male and female mice were used for the
studies reported here. Direct comparisons within experimental groups
used single sexes.
Parasites
Toxoplasma lysate Ags (TLA) were prepared from RH strain tachyzoites as previously described (38). RH strain tachyzoites were routinely maintained in the laboratory in human fibroblasts. Cysts of the ME49 strain of T. gondii were harvested from brains of CBA/CaJ mice infected for 1–2 mo. For experimental infections, mice were given 20 ME49 cysts i.p. in a volume of 0.2 ml.
Histology
At different times postinfection, samples of lung, liver, heart, spleen, and brain were removed from each mouse, fixed in 10% neutral buffered formalin, and embedded in paraffin. Organs were sectioned and stained with hematoxylin and eosin for evaluation of pathological changes. T. gondii parasites and Ags were detected in livers of infected mice by peroxidase-antiperoxidase staining using polyclonal rabbit Abs against T. gondii (39). Cytospin preparations of peritoneal exudate cells were prepared as previously described and used to estimate the percentage of cells infected with T. gondii (40). A value of 0.1% was assigned where the percentage of cells infected was <0.1% but parasites could still be detected. In situ detection of apoptotic cells in histological sections was performed using a TUNEL assay protocol (Boehringer Mannheim, Indianapolis, IN). Briefly, a TdT was used to incorporate fluorescein-dUTP onto the 3' ends of DNA strand breaks. Positively labeled cells were visualized using an anti-fluorescein-peroxidase conjugate in combination with diaminobenzadine. Sections were counterstained with hematoxylin and analyzed using light microscopy.
Reagents
Anti-mouse CD3
mAb (145-2C11) was prepared from hybridoma
supernatants. IFN-, TNF-, IL-2, IL-4, IL-6, and IL-10 levels were
measured using two-site ELISAs as previously described (41, 42). IL-12 p40 levels were measured using mAb C17.8
and biotinylated mAb C15.6 (grown from hybridomas provided by Giorgio
Trinchieri (Wistar Institute, Philadelphia, PA)). Recombinant murine
IL-2, IL-4, IL-6, IL-10, TNF-, and IFN- were purchased from
Genzyme (Cambridge, MA). LPS was purchased from Sigma (St. Louis, MO).
Levels of reactive nitrogen intermediates were measured using the
Greiss assay as previously described (43).
Analysis of T and NK cell responses
Spleens from uninfected or infected animals were harvested and
dissociated in complete RPMI 1640 (10% heat-inactivated FCS (Sigma),
1000 U/ml penicillin, 10 mg/ml streptomycin, 0.25 mg/ml fungizone
(BioWhittaker, Walkersville, MA)) into single cell suspension as
previously described (43). Cells were plated at a cell
density of 4 x 105 cells/well in a final
volume of 200 µl in 96-well plates and incubated with various
stimuli, and supernatants were harvested after 48 h and assayed
for the production of IL-2, IL-12, IL-4, and IFN-. Cytolysis of
51Cr-labeled YAC-1 cells (American Type Culture
Collection, Manassas, VA) was used to measure NK cell cytolytic
activity as described previously (44).
Analysis of macrophage functions
Bone marrow-derived macrophages (BMMø) from
NF-B2-/- and WT littermates
were derived from bone marrow cells grown on petri dishes (150 x
15-mm Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in DMEM
containing 20% (v/v) heat-inactivated FCS, 20% L-cell-conditioned
medium, 100 U/ml penicillin, and 100 µg/ml streptomycin. After at
least 6 days of incubation at 37°C with 5% CO2
in a humidified incubator, adherent cells were harvested using ice-cold
buffered saline without calcium or magnesium and washed three times in
complete RPMI 1640. For measurement of cytokines and NO production,
BMMø were resuspended in complete RPMI 1640 and plated onto 96-well
plates, 100 µl/well at 2 x 106 BMMø/ml.
Medium alone, 250 ng/ml LPS, or 1000 U rTNF- were added to cultures
with or without 100 U/ml IFN- to a final volume of 200 µl/well.
Supernatants were collected at 48 h and used to measure TNF-,
IL-6, IL-12, and NO production as above. For anti-Toxoplasmaactivity, 4 x 105 BMMø in complete
RPMI 1640 were plated on 15-mm glass coverslips in 24-well plates.
Cells were then incubated at 37°C with 5% CO2
in medium alone, medium containing 100 U/ml IFN-, or medium
containing 250 ng/ml LPS or 1000 U/ml TNF- with or without the
addition of 100 U/ml IFN-. After 4 h, cultures were infected
with RH tachyzoites at a ratio of one parasite/macrophage, and at 2 and
16 h postinfection, cultures were fixed in formalin and baseline
infections and parasite growth was assessed microscopically following
staining of coverslip cultures using Diff-Quik (Dade Diagnostics,
Aguada, PR).
Cytofluorometric analysis
After dissociation of the spleen and lysis of erythrocytes, splenocytes were resuspended at a final concentration of 1 x 107 cells/ml in FACS buffer composed of 1x PBS (BioWhittaker, Walkersville, MD), 0.2% BSA Fraction V (Sigma), and 4 mM sodium azide. For FACS analysis, 1 x 106 cells were stained with various conjugated mAbs specific for CD4 or CD8 for 20 min on ice in the presence of saturating amounts of Fc Block (PharMingen, San Diego, CA). Cells were then washed and analyzed using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). For biotinylated mAbs, cells were stained and washed as described above, then incubated with FITC- or PE-conjugated streptavidin (PharMingen) for 20 min on ice. Cells were then washed with FACS buffer and analyzed. Each mAb and streptavidin reagent was used at dilutions empirically determined to give optimal staining for flow cytometric analyses. Results were analyzed using CellQuest software (Becton Dickinson).
RNase protection assay (RPA) analysis
Total RNA was isolated from the spleens of mice by the guanidine isothiocyanate method and was assayed for cytokine mRNA levels using the Riboquant Mutiprobe Protection Assay System (PharMingen). Briefly, 10 µg RNA from each sample was hybridized in solution with the appropriate radiolabeled antisense RNA probe set. mAPO-1, mAPO-2 and mAPO-3 were employed for the detection of mRNA for genes involved in the regulation of apoptosis as recommended by the manufacturers. Following hybridization, free probe and remaining ssRNA were digested with RNases, and the protected probes were purified and resolved on 5% denaturing polyacrylamide gels using Ultra Pure Sequagel reagents (National Diagnostics, Atlanta, GA). Dried gels were then exposed to a phosphorimaging screen (Bio-Rad, Richmond, CA) and visualized using a Bio-Rad Molecular Imager System.
Statistics
Instat software (GraphPad, San Diego, CA) was used for unpaired two-tailed student t test, paired t test evaluations, or Mann-Whitney nonparametric test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B2-/- mice succumb to TE
To address the role of NF-B2 in the
immune response to T. gondii,
NF-B2-/- and WT littermate
controls were challenged i.p. with 20 cysts of the ME49 strain of
T. gondii. In contrast to WT mice,
NF-B2-/- mice were
susceptible to infection and mice succumbed to infection beginning on
day 25 with 100% mortality by day 90 of infection (Fig. 1
A). Histopathological
analysis of chronically infected mice revealed that WT mice developed a
mild meningoencephalitis (Fig. 1B), whereas
NF-B2-/- mice developed a
severe meningoencephalitis characterized by the presence of remarkable
numbers of parasites, large numbers of inflammatory cells, and areas of
necrosis (Fig. 1C). The lungs of infected WT and
NF-B2-/- mice had a mild to
moderate interstitial pneumonia with no organisms readily detected.
Similarly, the livers of these mice had a mild mutifocal random and
perivascular hepatitis with no obvious differences between WT and
NF-B2-/- mice (data not
shown). These findings confirm previous studies that
NF-B2-/- mice die earlier
than WT mice following infection with T. gondii
(20) but demonstrate for the first time that the
susceptibility of these mice is associated with increased numbers
of parasites and the development of severe TE.
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B2 in macrophage functions
Because activation of NF-B is associated with the effector and
regulatory functions necessary to control replication of T.
gondii, the lack of NF-B2 in macrophages
could result in increased susceptibility to TE. Therefore, the ability
of BMMø from WT and
NF-B2-/- mice to produce
cytokines or control parasite replication was assessed. BMMø from WT
and NF-B2-/- mice
stimulated with IFN- and LPS produced similar levels of reactive
nitrogen intermediates and were able to control replication of T.
gondii (Fig. 2, A and
B). Analysis of the cytokines produced by BMMø from WT and
NF-B2-/- mice stimulated
with IFN- and LPS revealed that they produced similar levels of IL-6
and TNF- (data not shown). However, BMMø from
NF-B2-/- mice produced
increased levels of IL-12 but decreased levels of IL-10 (Fig. 2, C and D). Because endogenous IL-10 is an
inhibitor of macrophage production of IL-12, it is possible that the
increased levels of IL-12 are a consequence of a reduced capacity to
produce IL-10. Nevertheless, based on the parameters examined, no
obvious defects were identified in macrophage function that would
provide an explanation for the increased susceptibility of
NF-B2-/- mice to
TE.
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B2 is not required for infection-induced
production of IL-12, IFN-, or activation of NK cells
Because NF-B is associated with the regulation of innate as
well as adaptive immunity to infection, the early events associated
with resistance to T. gondii were characterized to determine
whether they contributed to the susceptibility of these mice to TE. WT
and NF-B2-/- mice were
infected with T. gondii and the systemic levels of IL-12,
IFN-, and NK cell activity were used as a measure of the early
protective responses to this infection (40, 45). By day 5
postinfection there were elevated serum levels of IL-12 and IFN-
that were similar in WT and
NF-B2-/- mice (Fig. 3, A and B).
Interestingly, similar to the results observed with BMMø, there was a
trend to higher levels of IL-12 in the
NF-B2-/- mice compared with
WT mice, but this was not statistically significant. In addition,
infection of WT and
NF-B2-/- mice with T.
gondii resulted in a similar increase in NK cell cytolytic
activity (Fig. 3C). Together, these data indicate that the
absence of NF-B2 does not inhibit the
infection-induced increases in IL-12, IFN-, and NK cell
activation.
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B2 in the
development of the early immune response the capacity of splenocytes
from uninfected and infected WT and
NF-B2-/- mice to produce
IL-12 and IFN- were compared. Stimulation of splenocytes from
uninfected mice with TLA resulted in the production of comparable
levels of IL-12 by WT and
NF-B2-/- mice (Fig. 4). Stimulation of splenocytes from
infected mice with TLA resulted in increased production of IL-12
compared with splenocytes from uninfected mice, but there was no
significant difference between WT and
NF-B2-/- mice (Fig. 4A). The ability of splenocytes from uninfected
NF-B2-/- mice to produce
IFN- in response to soluble anti-CD3 revealed that equivalent
levels of IFN- were produced under these conditions and similar
results were observed with splenocytes from acutely infected mice (Fig. 4B). Splenocytes from uninfected WT and
NF-B2-/- mice produced
minimal levels of IFN- in response to TLA, whereas cells from
infected WT and NF-B2-/-
produced increased levels of IFN-, but no significant difference was
observed between these experimental groups. These results need to be
interpreted with care as
NF-B2-/- mice actually have
increased numbers of CD4+ and
CD8+ T cells compared with littermate controls
(21). Nevertheless, together with the studies which
examined NK cell cytolytic activity and serum levels of IFN- and
IL-12, it appears that in the absence of
NF-B2, the generation of protective immune
responses appear to be intact and these mice are able to survive the
acute phase of this infection.
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B2 is required for the maintenance of T cell
responses
Because NF-B2-/- mice
succumbed to TE during the chronic stage of the infection the T cell
responses of chronically infected mice was analyzed. Interestingly,
when chronically infected
NF-B2-/- mice developed
clinical signs of disease, there was a rapid progression and death of
mice within a week, whereas other infected
NF-B2-/- mice in the same
group appeared healthy. Therefore, experiments were designed in which
groups of WT and NF-B2-/-
mice were infected, and as
NF-B2-/- mice became
moribund they were sacrificed and their immune responses compared with
chronically infected, but healthy, WT and
NF-B2-/- littermates. These
studies revealed that following stimulation of splenocytes with
anti-CD3 or TLA, healthy
NF-B2-/- mice produced
lower levels of IFN- than chronically infected WT mice and that
moribund NF-B2-/- mice had
a major defect in their ability to produce IFN- (Fig. 5A). In contrast, IL-12 levels
were essentially intact and only reduced in mice that were moribund
(Fig. 5B). Because endogenous production of IFN- enhances
TLA-induced IL-12 production (45), the reduced levels of
IL-12 detected are likely due to the almost complete lack of IFN- in
these cultures. Together, these data associate the susceptibility of
NF-B2-/- mice to TE with a
reduced capacity to produce IFN- required for resistance to
TE.
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B2-/- mice had a major
defect in their capacity to produce IFN-, they produced higher basal
levels of IL-2 than WT mice. However, the levels of IL-2 produced by
splenocytes from healthy infected
NF-B2-/- mice stimulated
with TLA were similar to WT mice (Fig. 5
C) and moribund mice
produced negligible levels of IL-2 (data not shown). These findings led
us to compare the ability of T cells from uninfected WT and
NF-B2-/- mice to produce
IL-2 in response to polyclonal stimuli. Splenocytes from
NF-B2-/- mice produced
almost 10-fold more IL-2 than splenocytes from WT mice in response to
ConA (Fig. 5D) and similar difference was also observed with
anti-CD3 although the absolute levels of IL-2 produced were much
reduced (data not shown). These results demonstrate that in the absence
of NF-B2 T cells overproduce IL-2.
Previous studies have shown that uninfected
NF-B2-/- mice have similar
numbers of splenocytes compared with WT mice but have an approximate
3-fold increase in the percentage of CD4+ and
CD8+ T cells and a reduced number of B cells
compared with WT mice (20, 21), and our studies confirmed
these findings (data not shown). Analysis of the spleens from
chronically infected mice revealed that healthy
NF-B2-/- mice had similar
numbers of CD4+ and CD8+ T
cells compared with WT mice (Fig. 6) and
that moribund NF-B2-/- mice
displayed a profound loss of CD4+ and
CD8+ T cells. In addition, although uninfected
NF-B2-/- mice have a
reduced number of B cells (21), this was further reduced
following infection and moribund mice had an almost complete loss of B
cells (Fig. 6). These data suggest that the reduced levels of IFN-
produced in recall responses by chronically infected
NF-B2-/- mice is associated
with the loss of T cells from these spleens. The loss of T cells also
provides a likely explanation why splenocytes from uninfected
NF-B2-/- mice produce
higher levels of IL-2 than splenocytes from WT mice, but during the
chronic stage of infection the levels of IL-2 produced are
comparable.
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B2-/- mice results in
increased levels of apoptosis and T cell expression of Fas
Gross pathological analysis of infected
NF-B2-/- mice that were
moribund revealed that their spleens were 
25% of the size of
chronically infected WT mice, and histological analysis suggested that
although there were no readily detectable areas of parasite replication
in the spleen there were high levels of apoptosis compared with
chronically infected WT mice. Importantly, based on histological
analysis, there were no discernable differences between acutely
infected WT and NF-B2-/-
mice and the use of TUNEL analysis as a measure of the levels of
apoptosis in the spleens of WT and
NF-B2-/- mice revealed
that, similar to previous reports (21), uninfected WT and
NF-B2-/- mice had similar
levels of apoptosis (data not shown). However, TUNEL analysis confirmed
the preliminary histological analysis and revealed remarkable numbers
of apoptotic cells in the spleens of chronically infected
NF-B2-/- mice, which was
not observed in chronically infected WT mice (Fig. 6).
The high levels of apoptosis observed in the spleens of chronically
infected NF-B2-/- mice led
us to examine the expression of genes associated with regulation of
apoptosis. RPA analysis was used to quantitate the levels of mRNA for
Fas ligand (FasL), Fas, Fas-associated death domain (FADD),
Fas-associated phosphatase, Fas-associated protein factor, TNF-related
apoptosis-inducing ligand, TNFR-assocated death domain protein,
receptor interacting protein, Bcl2, bak, bax, bad, and various caspases
in the spleens of WT and
NF-B2-/- mice. This
approach did not reveal any difference in the levels of mRNA specific
for these genes between uninfected WT and
NF-B2-/- mice and the only
marked difference obtained when infected WT and
NF-B2-/- mice were compared
was in the levels of Fas and FasL. The results presented in Fig. 7 show the data obtained for Fas, FasL,
and FADD using uninfected WT and
NF-B2-/- mice or WT and
NF-B2-/- mice infected for
38 days. This analysis revealed that although there were no differences
in the levels of Fas, FasL, and FADD between uninfected WT and
NF-B2-/- mice, infected
NF-B2-/- mice had increased
levels of mRNA for Fas and FasL compared with infected WT mice but
there was no significant difference in the levels of FADD (Fig. 7). In
similar experiments which compared levels of mRNA for Fas and FasL
at days 11, 59, and 68 postinfection, similar results were also
observed (data not shown). Based on these results, FACS analysis was
used to examine T cell expression of Fas. As shown in Fig. 8, T cells directly isolated from
chronically infected
NF-B2-/- mice expressed
elevated levels of Fas, but this was not observed in uninfected mice or
infected WT mice (Fig. 8). FACS analysis also revealed a small increase
in the expression of FasL (Fig. 8). Together, the TUNEL, RPA, and FACS
analysis indicate that infection of
NF-B2-/- mice with T.
gondii leads to increased expression of Fas and this correlates
with the increased levels of apoptosis observed in these mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B2 is not required for the innate NK and
adaptive T cell responses that lead to the production of IFN-
required for resistance to T. gondii, nor is
NF-B2 required for the macrophage effector
functions associated with control of this intracellular pathogen.
However, our studies indicate that in chronically infected mice
NF-B2 has a role in the maintenance of
lymphocyte responses necessary to control the chronic stage of this
infection. Thus, there is a remarkable loss of T cells in
chronically infected
NF-B2-/- mice which
correlates with a reduced capacity to produce IFN- and
susceptibility to TE. In addition, it is noteworthy that while
uninfected NF-B2-/- mice
have reduced numbers of B cells compared with WT mice (20, 21) there was a further reduction in this population of cells
during the chronic stage of infection and this may contribute to the
increased susceptibility of these mice to TE (46, 47).
Nevertheless, like the patients with AIDS, certain cancers, or who are
being treated with immunosuppressive drug regimens (23, 24, 48), the remarkable loss of lymphocytes and/or lymphocyte
functions in chronically infected
NF-B2-/- mice provides a
mechanism that underlies the development of TE in these mice.
Although our studies have demonstrated a novel role for
NF-B2 in the regulation of IL-2, IL-10, and
IL-12 production, the most striking observation was the high levels of
apoptosis and loss of lymphocytes observed in chronically infected
NF-B2-/- mice. Importantly,
the increased numbers of apoptotic cells was only observed in
NF-B2-/- mice infected with
T. gondii, suggesting that these events are directly related
to the regulation of the immune response to this persistent parasite.
There are several possible explanations for the loss of T cells during
chronic toxoplasmosis. Previous studies have identified a clear role
for NF-B proteins in the regulation of anti-apoptotic genes,
such as TNFR-associated factor, cellular inhibitor of apoptosis, and
IEX-1L (49, 50, 51, 52, 53) although, recent studies indicate that
NF-B can also have a pro-apoptotic role (54). Perhaps
the most direct explanation for the increased apoptosis observed in the
infected NF-B2-/- mice is
that NF-B2-/- lymphocytes
may have reduced expression of anti-apoptotic factors and/or
increased levels of proapoptotic proteins such as Fas, rendering these
cells more susceptible to apoptosis (55, 56, 57, 58). However, to
the best of our knowledge, NF-B2 has not been
associated with the regulation of apoptosis (20, 21), and
further studies will be required to determine whether
NF-B2 can regulate downstream targets that
protect against apoptosis.
Although NF-B2 may be directly involved in the
regulation of apoptosis, there are alternative explanations for the
loss of lymphocytes in the chronically infected
NF-B2-/- mice. It is
possible that the phenotype presented by the
NF-B2-/- mice during the
chronic phase of infection with T. gondii is a result of an
immunological environment that promotes apoptosis. For example,
NF-B2-/- mice lack
follicular dendritic cells (21) and NF-B is also
involved in the generation of metallophilic marginal zone macrophages
(59) and the role of these cell types in the maintenance
of T cell responses is unknown. In addition, the increased capacity of
NF-B2-/- T cells to produce
IL-2 and elevated levels of expression of Fas by T cells from
NF-B2-/- mice suggest that
activation-induced cell death (AICD) may be involved in the loss of
lymphocytes. AICD is a process in which IL-2 primes activated T cells
for apoptosis (60) mediated by the interaction of Fas with
FasL (61, 62). Of relevance to our studies, the activation
of NF-B is involved in the expression of Fas, FasL, and IL-2, and
several studies have shown that blockade of NF-B antagonizes AICD
(55, 56, 63, 64, 65, 66). It is not clear why there is only an
increase in the number of apoptotic cells and loss of lymphocytes in
chronically infected mice, but long-term activated T cells are more
sensitive to Fas-induced apoptosis than resting or short-term activated
T cells (67, 68). Furthermore, it has been reported that
Th1 cells (which dominate during infection with T. gondii)
are more susceptible than TH2 cells to AICD (69).
Nevertheless, the events that lead to the loss of T cells in infected
NF-B2-/- mice remain
unclear and only by integrating studies which examine how the absence
of NF-B2 affects susceptibility to apoptosis,
production of IL-2 and expression of Fas can we determine the molecular
basis for these events in
NF-B2-/- mice.
An important challenge to understanding the role of NF-B in
regulation of immunity is to distinguish the role of specific NF-B
members in the different regulatory and effector functions essential to
coordinate the development of protective immunity. The response of
NF-B2-/- mice to infection
with T. gondii contrasts with what is known about the role
of other NF-B members in resistance to T. gondii.
RelB-/- mice are highly susceptible to acute
toxoplasmosis associated with a defect in the ability of their NK and T
cells to produce IFN- (35). In contrast,
NF-B1-/- mice infected with
T. gondii are not more susceptible to toxoplasmosis and can
generate and maintain normal T cell responses (J.C. and C.A.H.,
unpublished observations). Because many of the NF-B family members
are thought of as being functionally able to compensate for each other,
the identification of a role for NF-B2 in the
regulation of adaptive immunity to infection that is distinct from
NF-B1 and RelB indicates a unique role for
NF-B2 in immunity to this infection. It is
also important to recognize the parallels between the
NF-B2-/- mice and the
situation in AIDS patients who are chronically infected with T.
gondii and who, as their T cell numbers fall, can no longer
control this infection and develop TE. T cells from individuals with
AIDS have an increased susceptibility to apoptosis and there are high
levels of apoptosis of activated T cells in these patients
(70, 71, 72, 73), and it has been proposed that AICD contributes
to the loss of T cells that allows opportunistic infections to cause
disease. Understanding the role of NF-B2 in
the regulation of T cell survival and maintenance of protective T cell
responses may be helpful in the design of strategies to prevent the
loss of T cells that renders these patients susceptible to
opportunistic pathogens.
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Acknowledgments |
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B2-/- mice. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008.
3 Abbreviations used in this paper: WT, wild type; TLA, Toxoplasma lysate Ag; TE, toxoplasmic encephalitis; BMMø, bone marrow-derived macrophages; RPA, RNase protection assay; FasL, Fas ligand; FADD, Fas-associated death domain; AICD, activation-induced cell death.
Received for publication June 14, 2000. Accepted for publication August 23, 2000.
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References |
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)
or stimulated with IFN-
).
B, The levels of nitrite produced by BMMø stimulated
with LPS plus IFN-
