| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B Family Member RelB Is Required for Innate and Adaptive Immunity to Toxoplasma gondii1
*
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104;
Department of Immunology, The Strathclyde Institute of Biomedical Sciences, University of Strathclyde, Glasgow, Scotland; and
Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B family of transcription factors are associated with the
regulation of innate and adaptive immunity to infection. Infection of
C57BL/6 mice with Toxoplasma gondii resulted in
up-regulation of NF-B activity that included the NF-B family
member RelB. To assess the role of RelB in the regulation of the immune
response to this infection, we challenged RelB-deficient mice
(RelB-/-) and wild-type (WT) littermate controls with
T. gondii. Although WT controls were resistant to
T. gondii, RelB-/- mice succumbed 10–15
days after infection. Examination of accessory cell functions
associated with resistance to T. gondii revealed that
RelB-/- macrophages stimulated with IFN-
plus LPS or
TNF-
produced IL-12 as well as reactive nitrogen intermediates and
inhibited parasite replication similar to WT macrophages. Analysis of
the systemic responses of RelB-/- and WT mice revealed
that infected mice had similar serum levels of IL-12. However,
RelB-/- mice challenged with T. gondii
produced negligible levels of IFN- and had reduced NK cell activity
compared with WT mice. Similarly, splenocytes from uninfected
RelB-/- mice stimulated with polyclonal stimuli were
deficient in their ability to produce IFN-. Together, our results
demonstrate that RelB is essential for the development of innate NK and
adaptive T cell responses that lead to the production of IFN- and
resistance to T. gondii. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, which mediates
innate resistance to many viral, bacterial, and parasitic infections
(3, 4, 5). However, the innate production of IL-12 is also
required for the development of TH1 responses, which are critical for
long term immunity to many intracellular pathogens (6).
Similarly, other cytokines (IL-1 and TNF-) and costimulatory events
(CD28/B7) that can regulate NK cell responses (7, 8, 9, 10) are
also involved in the development of T cell responses (11, 12). Thus, many of the same events that mediate innate NK cell
production of IFN- are responsible for the development of TH1 cells
that produce IFN-.
To understand the molecular events that regulate the development of
innate and adaptive immunity, studies were initiated to determine the
role of NF-B in the immune response to Toxoplasma gondii.
In mammalian cells the NF-B family of transcription factors is
composed of several members (NF-B1,
NF-B2, RelA, RelB, and c-Rel) that belong to
an ancient family associated with the regulation of immune response
genes (13, 14, 15). In unstimulated cells, NF-B dimers are
retained in the cytoplasm in an inactive form as a consequence of their
association with the inhibitory family of IB molecules. In response
to an external signal, IB is phosphorylated, ubiquinated, and
degraded leading to the release of NF-B, which can then translocate
to the nucleus, bind to B DNA motifs present in specific gene
promoters, and activate transcription (16, 17). These
events are independent of de novo protein synthesis and represent a
system that allows a rapid response to appropriate stimuli. 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 in
macrophages, as well as other cell types (18, 19, 20, 21, 22). In
addition, cytokines such as TNF-, IL-1, and IL-18 can also activate
NF-B (16, 23). 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 adhesion molecule-1 or E selectin (ELAM), VCAM, and ICAM),
cytokines (IL-1, TNF-, and IL-12), as well as inducible NO synthase
(16). 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.
The role of the NF-B family of transcription factors in immunity is
not restricted to regulation of innate immunity. Several NF-B
members are expressed predominantly in lymphoid tissues (24, 25) and are important in the regulation of T and B cell
responses (25, 26, 27, 28). Whereas RelA and c-Rel complexes are
part of the inducible activity, RelB-containing complexes are part of
the constitutive B activity present in lymphoid tissues (25, 29). RelB is also expressed by dendritic cells (24, 30) and is required for the development of the myeloid-related
CD8- dendritic cells (31). The
importance of this transcription factor in immune homeostasis is
illustrated by studies that characterized
RelB-/- mice (32, 33). Lymphoid
development in the absence of RelB appears to be normal, but these mice
develop a multifocal, mixed inflammatory cell infiltration in several
organs, myeloid hyperplasia, and splenomegaly due to extramedullary
hematopoiesis (33). The basis for this inflammatory
response is unclear but is dependent on the presence of T cells
(34) and has been linked to overexpression of chemokine
genes by fibroblasts (35).
Given the important role for RelB in the regulation of the immune
response and studies that suggested that dendritic cells may be the
initial source of IL-12 after infection with T. gondii
(36), the role of RelB in resistance to this parasite was
assessed. The studies presented here reveal that RelB activity is
elevated during toxoplasmosis and that this transcription factor has an
important role in resistance to T. gondii. Comparison of
wild-type (WT)3 and
RelB-/- mice demonstrates that RelB is not
required for accessory cell production of IL-12 or the ability of
macrophages to respond to IFN- and control parasite replication.
Rather, RelB is required for the infection-induced activation of NK
cells and production of IFN- by T cells. Thus, our studies identify
RelB as a common link between NK and T cell responses to infection in
which resistance is mediated by IFN-.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female Swiss Webster, CBA/CaJ, and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). RelB-/- mice (33) were bred on the 129/B6 background and maintained within Thoren caging units within the animal facilities at the University of Pennsylvania. Mice were between 4 and 6 wk of age when used for experiments. The RelB-/- mice originated on a 129/B6 background and had been back-crossed for five generations on a C57BL/6 background. Heterozygote mice that lack a copy of the relB gene were used to generate mice that lack both copies of the relB gene (RelB-/-) as well as WT controls. Mice were typed using a PCR based methodology that distinguishes the WT relB gene from the targeted relB-neo allele as previously described (37). Approximately 50% of RelB-/- mice die between 8 and 12 wk after birth due to a multifocal, mixed inflammatory cell infiltration in several organs (33). However, RelB-/- mice were healthy at the time they were used in these studies, and deaths in uninfected mice during the course of these experiments was rare. All experiments performed were in accordance with the Institutional Review Board of the University of Pennsylvania. Because of the difficulties associated with breeding and maintenance of these mice, male and female mice were used for the studies reported here.
Parasites
Soluble Ags of T. gondii (TLA) were prepared from RH strain tachyzoites as previously described (38). RH strain tachyzoites were routinely maintained in the laboratory in human foreskin fibroblasts. TLA was titrated to determine the optimal concentration for splenocyte proliferation and was used at 25–40 µg/ml for these experiments. 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.
Electrophoretic mobility shift assays (EMSAs)
Whole cell extracts from spleens of C57BL/6 mice were prepared
as previously described (25). The double-stranded
oligodeoxynucleotides corresponding to the palindromic B site
(5'-GGGAATTCCC-3') were used for these assays. The double-stranded
oligodeoxynucleotide (1 pmol) was labeled by filling the overlapping
ends with the Klenow fragment of DNA polymerase I and
[-32P]dCTP. Unincorporated nucleotides were
removed and 10 fmol of labeled oligonucleotide (50,000 cpm) were
incubated with 10 µg of protein extracts, 2 µg of poly(dI-dC), in
buffer containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 5 mM
MgCl2, 1 mM DTT, 0.7 mM PMSF, and 17% glycerol
in a final volume of 22 µl for 15 min at 20°C. The different
antisera used on these assays were anti-RelA (SC 372-G),
anti-RelB (SC 226X), and anti-p50 (SC 114-G) from Santa Cruz
Biotechnology (Santa Cruz, CA). Complexes were separated on 5.5%
polyacrylamide gels run on 0.25% Tris-borate-EDTA buffer, dried, and
exposed to Kodak X-Omat AR film (Rochester, NY) at -70°C.
Histology
At different times postinfection, samples of lung, liver, heart, spleen, and brain were removed from each mouse, fixed in 4% formaldehyde/70% ethanol/0.8 N acetic acid, 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 (PECs) were prepared as previously described and used to estimate the percentage of cells infected with T. gondii (8). The percentage of cells infected was <0.1%, but parasites could still be detected a value of 0.1% was assigned.
Reagents
Anti-mouse CD3
(145-2C11) was prepared from hybridoma
supernatants. Hamster anti-murine CD40 (4C11) was provided by Dr.
Bob Coffman (DNAX, Palo Alto, CA). IFN-, TNF-, IL-2, IL-4, IL-6,
and IL-10 levels were measured using two site ELISAs as previously
described (40, 41). 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 was purchased from Genzyme (Cambridge, MA).
PMA, ionomycin, LPS, and Con A were purchased from Sigma (St. Louis,
MO). Levels of reactive nitrogen intermediates (RNI) were measured
using the Greiss assay as previously described (42).
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, MD)) into single cell suspension as
previously described (42). 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-1, TNF-, IL-4, and IFN-.
CD3+ T cells were purified using columns (R&D
Systems, Minneapolis, MN) and routinely produced populations of T cells
that were >95% CD3+. To analyze levels of
cytokine mRNA, total RNA was isolated from the spleens of mice by the
guanidine isothiocyante method and assayed for cytokine mRNA levels
using the Riboquant Mutiprobe Protection Assay System (PharMingen, San
Diego, CA). In brief, 10–20 µg of RNA from each sample was
hybridized in solution with the appropriate radiolabeled antisense RNA.
The probe mCK-2b was used for the detection of cytokine mRNA as
recommended by the manufacturers. After hybridization, free probe and
remaining ssRNA were digested with RNAases, 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 PhosphorImager and data analyzed
using MultiAnalyst software (Bio-Rad, Hercules, CA). Cytolysis of
51Cr-labeled YAC-1 cells (American Type Tissue
Culture Collection, Manassas, VA) was used to measure NK cell cytolytic
activity as described previously (43).
Analysis of macrophage functions
Bone marrow macrophages (BMM
) from
RelB-/-, WT, and heterozygous 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
(p/s). After at least 6 days incubation at 37°C and 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, BMM were resuspended in complete
RPMI and plated onto 96-well plates, 100 µl/well at 2 x
106 BMM/ml. Medium alone, 250 ng/ml LPS, or
1000 U of 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 IL-1ß, TNF-, IL-6, IL-12, and NO
production as above. For antitoxoplasma activity, 4 x
105 BMM in complete RPMI were plated on 15-mm
glass coverslips in 24-well plates. Cells were then incubated at 37°C
and 5% CO2 in medium alone or medium containing
100 U/ml IFN-, or in 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 1:1,
parasite/macrophage. At 2 and 16 h postinfection, cultures were
fixed in formalin and baseline infections and parasite growth assessed
microscopically after staining of coverslip cultures using Diff-Quik
(Dade Diagnostics, Aguada, PR).
Cytofluorometric analysis
After dissociation and lysis of erythrocytes, cells 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, CD8, or the NK cell-specific marker DX5 for 20 min on ice in the presence of saturating amounts of Fc Block (PharMingen). Cells were then washed and analyzed using a FACScalibur Flow cytometer (Becton Dickinson). For biotinylated Abs, cells were stained and washed as described above and then incubated with FITC or PE-conjugated streptavidin (PharMingen) for 20 min on ice. Cells were then washed with FACS buffer and analyzed. The Abs and streptavidin reagents were used at dilutions empirically determined to give optimal staining for flow cytometric analyses. Results were analyzed using CellQuest software (Becton Dickinson).
Statistics
INSTAT software (GraphPad, San Diego, CA) was used for unpaired two-tailed Student’s t test, paired t test evaluations, or Mann-Whitney nonparametric test. A p value of <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To analyze whether infection with T. gondii was able to
activate NF-B binding activity in the spleen, C57BL/6 mice were
infected with the ME49 strain of T. gondii, and whole cell
extracts were prepared from spleens at different time points after
infection and used to analyze B-binding activity by EMSA (Fig. 1
). Uninfected animals showed a faster
migrating band corresponding to p50 homodimers (line
B) and a slower migrating activity that corresponded to RelA- and
RelB-containing complexes (line A) as demonstrated by
the addition of antiserum specific for these proteins. Ab specific for
RelB did not induce an obvious shift in the NF-B complexes (data not
shown), whereas Abs specific for RelA resulted in a decrease in the
RelA complexes (Fig. 1, middle panel). Only when the RelA
complexes were removed was the supershift caused by the RelB-specific
Abs revealed (Fig. 1, right panel). The binding
corresponding to the RelA- and RelB-containing complexes is weak in
uninfected animals but strongly up-regulated by 2 days after infection.
The specificity of these binding complexes was analyzed by the addition
of an 100-fold excess of cold B oligonucleotide that completely
abolished the binding (data not shown). These results indicate that
infection with T. gondii induces a B-binding activity in
the spleen that is composed of RelA- and RelB-containing complexes.
|
To address the role of the NF-B family member, RelB in the
immune response to T. gondii RelB-/-
and WT littermate controls were challenged i.p. with the ME49 strain of
T. gondii. In contrast to WT mice,
RelB-/- mice were highly susceptible to
infection with 100% mortality within 16 days of infection (Fig. 2A). To assess levels of
parasite replication, we compared the numbers of infected cells present
in cytospin preparations of PECs at day 5 postinfection. This analysis
revealed that RelB-/- mice had a higher
percentage of cells infected than WT mice (Fig. 2B). The
cellular composition of PECs from WT and
RelB-/- mice was different (Fig. 2, C and D). WT mice contained predominantly
macrophages, monocytes, and lymphocytes with few neutrophils. In
contrast, PECs from infected RelB-/- mice
consisted mostly of hypersegmented neutrophils and macrophages.
Examination of moribund infected RelB-/- mice
revealed that there were large numbers of free parasites present in the
peritoneum of these mice with few host cells. Histopathological
analysis of mice infected for 10 days revealed that WT mice developed a
severe interstitial pneumonia with rare organisms, whereas the liver
displayed scattered lymphocytic infiltrates with few organisms apparent
(Fig. 3, A, D, and
G). Analysis of uninfected RelB-/- mice (Fig. 3, C, F, and I) revealed the presence
of inflammatory infiltrates in the lungs and liver but no inflammation
in the heart similar to previous reports (32, 33).
However, infected RelB-/- mice developed a
severe necrotizing myocarditis (Fig. 3B) characterized by
the presence of parasites and the lung had a necrotizing interstitial
pneumonia associated with tachyzoites (Fig. 3H). Severe
hepatocellular necrosis associated with the presence of organisms was
observed in the liver (Fig. 3E). Numerous parasites were
present in the spleen, associated with mild necrosis, and small numbers
of parasites were also present in the brain without any evidence of an
inflammatory response directed against them (data not shown). Together,
our findings demonstrate that infection of
RelB-/- mice with T. gondii results
in high levels of parasite replication which results in the death of
these mice during the acute phase of the infection.
|
|
In an attempt to identify the basis for the increased
susceptibility of RelB-/- mice to
toxoplasmosism, macrophage functions associated with resistance to
T. gondii were examined. BMM from WT and
RelB-/- mice stimulated with IFN- and LPS
produced similar levels of RNI and were able to control replication of
T. gondii (Fig. 4,
A and B). Similar results were observed when
cells were stimulated with IFN- plus TNF- (data not shown).
Because resistance to T. gondii is dependent on the ability
of IL-12 to stimulate the production of IFN-, we determined if the
absence of RelB would alter accessory cell production of IL-12. BMM
from uninfected WT and RelB-/- mice stimulated
with IFN- and LPS produced similar levels of IL-12, and challenge of
mice with 20 cysts of T. gondii resulted in increased serum
levels of IL-12 that were similar in WT and
RelB-/- mice (Fig. 4C). Stimulation
of splenocytes from uninfected mice with TLA resulted in the production
of similar levels of IL-12 by WT and RelB-/-
mice. Moreover, splenocytes from WT mice infected for 5 days and
stimulated in vitro with TLA produced increased levels of IL-12
compared with uninfected controls, similar to previous reports
(44, 45). However, splenocytes from
RelB-/- mice infected for 5 days and stimulated
in vitro with TLA did not produce increased levels of IL-12 compared
with uninfected RelB-/- mice (Fig. 4C).
|
in response to IFN-
plus LPS. Similar to IL-12, there was no difference in the levels of
IL-10 or IL-6 produced (Fig. 5,
A and B). However,
RelB-/- macrophages produced lower levels of
TNF- (Fig. 5C, although this failed to reach statistical
significance) in accord with previous studies (37), but
increased (p < 0.05) levels of IL-1ß (Fig. 5D).
|
and
activation of NK cells
To determine whether a defect in the production of IFN- was
responsible for the increased susceptibility to toxoplasmosis of
RelB-/- mice, we measured the production of
IFN- by splenocytes isolated from mice infected for 5 days and
stimulated with TLA. FACS analysis revealed that there was no
difference in the percentage of CD4+ and
CD8+ T cells in the spleens of infected WT and
RelB-/- mice. However, splenocytes from
infected WT mice produced high levels of IFN-, but splenocytes from
infected RelB-/- mice produced minimal levels
of IFN- (Fig. 6A). Analysis
of serum levels of IFN- 5 days after infection revealed that
RelB-/- mice produced significantly less
IFN- than WT mice (Fig. 6B). To analyze the production of
IFN- in RelB-/- mice after infection, we
isolated RNA from spleens of uninfected mice and mice infected for 5
days and performed ribonuclease protection assay (RPA) analysis (Fig. 6C). This analysis showed that after infection WT mice
up-regulated the levels of IFN- mRNA whereas the
RelB-/- mice showed low levels of IFN- mRNA
in uninfected and infected mice. Together with the recall responses and
serum levels of IFN-, these results indicate that the absence of
RelB leads to reduced production of IFN- and this occurs at the
level of transcription.
|
A). Because this
infection-induced activation of NK cells is dependent on IFN-
(43), the defect in NK cell responses observed after
infection of RelB-/- mice with T.
gondii could be a consequence of the reduced production of IFN-
in these mice. Alternatively, the defective NK cell activity could be
due to a lack of NK cells or a defect in their cytolytic function. FACS
analysis revealed the presence of similar numbers of NK cells in WT and
RelB-/- mice (data not shown) and overnight
stimulation of RelB-/- splenocytes with IL-2
plus IL-18 resulted in increased NK cell cytolytic activity of
splenocytes from RelB-/- mice (Fig. 7B). Similar results were observed with the combination of
IL-12 plus IL-18 or IL-2 plus IL-12. The in vitro NK cell activity
observed with WT mice stimulated with IL-2 plus IL-18 was 2- to 3-fold
increased over the activity observed with
RelB-/- mice (data not shown). Because IFN-
is required for the optimal activation of NK cell cytolytic activity,
this difference in NK cell activity likely reflects the levels of
endogenous IFN- in these cultures. Nevertheless, these results
suggest that during toxoplasmosis the absence of RelB results in a
defect in NK cell activation that is not due to an intrinsic defect in
cytolytic function of NK cells but is associated with decreased
production of IFN-.
|
in response to
nonspecific T cell stimuli uncovered a major defect in their ability to
respond to anti-CD3 alone or in combination with IL-12, IL-18, or
IL-2 (Fig. 8A). Additional
stimuli such as Con A, anti-CD40, or LPS also revealed defects in
IFN- production (Fig. 8B). However, stimulation with PMA
(a protein kinase C inducer) plus ionomycin (a
Ca2+ ionophore) restored the production of
IFN- although it was still reduced compared with WT responses. The
combination of IL-12 and IL-18 has been used to directly stimulate NK
cell production of IFN- by splenocytes (46, 47, 48), and
when splenocytes from RelB-/- mice were
stimulated with this combination, they produced 80–90% less IFN-
compared with WT mice (data not shown).
|
To determine whether there were any other defects in the
production of T cell associated cytokines splenocytes from uninfected
WT and RelB-/-, mice were stimulated with
either anti-CD3, Con A, anti-CD40, LPS, or PMA plus ionomycin
and the levels of IL-2 and IL-4 in these cultures measured by ELISA.
The absence of RelB led to a reduction in the levels of IL-2 produced
by splenocytes incubated with anti-CD3, Con A, anti-CD40, or
LPS used (data not shown), but the data was most striking when cells
were stimulated with PMA and ionomycin (Fig. 8
C). However,
the reduced production of IL-2 by splenocytes from
RelB-/- mice did not contribute to the lack of
IFN- as exogenous IL-2 failed to restore production of IFN- by
these cells (Fig. 8A).
Stimulation with anti-CD3 alone or in combination with IL-12,
IL-18, or IL-2 or the polyclonal stimuli Con A, anti-CD40, or LPS
did not result in a significant difference in IL-4 production by
splenocytes from WT or RelB-/- mice (data not
shown). However, stimulation with PMA and ionomycin revealed that
splenocytes from RelB-/- mice produced higher
levels of IL-4 than splenocytes from WT mice (Fig. 8D).
These findings suggested that the overproduction of IL-4 could account
for the reduced levels of IFN- produced by splenocytes from
RelB-/- mice. However, the addition of
neutralizing Ab specific for IL-4 (or mAbs specific for IL-10 or
TGF-ß) to cultures of RelB-/- splenocytes
stimulated with anti-CD3 alone or in combination with IL-18, IL-12,
or IL-2 did not restore the production of IFN- (data not shown). It
should be noted that RPA analysis of spleens from infected WT and
RelB-/- mice revealed minimal levels of mRNA
for IL-2 and IL-4 in these samples (data not shown), suggesting that
the decreased production of IFN- by RelB-/-
mice was not due to dysregulated production of these cytokines during
infection.
Purified T cells from RelB-/- mice are
defective in production of IL-2 and IFN- but not IL-4
To assess the contribution of non-T cell populations to the
defective production of IFN-, IL-2, and IL-4 observed with
splenocytes from RelB-/- mice,
CD3+ T cells were purified from spleens of
uninfected WT and RelB-/- mice and stimulated
with plate-bound anti-CD3 alone or in combination with IL-12,
IL-18, or IL-2. Whereas T cells from WT mice produced high levels of
IFN-, T cells from RelB-/- mice produced 5-
to 10-fold lower levels of IFN- (Fig. 9A). Stimulation of purified T
cells from WT mice with PMA plus ionomycin resulted in the production
of high levels of IL-2, but the levels of IL-2 produced by purified T
cells from RelB-/- mice was reduced
significantly in comparison (Fig. 9B). In contrast, levels
of IL-4 produced by these cells were not significantly different (Fig. 9C). Similar results were observed with purified T cells
stimulated with anti-CD3 (data not shown). Comparison of the
responses of splenocytes and purified T cells reveal that the loss of
RelB results in reduced production of IFN- and IL-2 by both
populations. However, the increased production of IL-4 observed with
splenocytes from RelB-/- mice was not observed
with purified T cells. Together, these results demonstrate that T cells
deficient in RelB have an intrinsic defect in their ability to produce
IFN- and IL-2 but that a non-T cell population is responsible for
the increased production of IL-4 observed with splenocytes from
RelB-/- mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Several studies have correlated the nuclear
translocation of RelA/NF-B1 with the ability of T cells to produce
IFN- (23, 49), and there are several putative NF-B
sites in the IFN- promoter that are involved in transcription of
this gene (50). Thus, the most likely explanation for the
defect in the production of IFN- is that RelB is involved in the
regulation of the IFN- gene. An alternative explanation may be that
the absence of RelB results in an immunological environment that
indirectly inhibits T and NK cell production of IFN-. Further
studies will be required to understand the molecular basis of the role
of RelB in the regulation of the production of IFN-.
Although the absence of RelB results in several immune defects, we
believe the principal reason that RelB-/- mice
show increased susceptibility to toxoplasmosis is due to the lack of
IFN-. Thus, the phenotype we observe in
RelB-/- mice is similar to mice deficient in
the production of IFN- (43, 45, 51, 52). However,
previous studies with RelB-/- mice concluded
that production of IFN- by these mice was normal (37, 53). In one study (53), this conclusion was based
on intracellular staining for IFN- in T cells stimulated with PMA
and ionomycin. When we used these stimuli, we found that we could
induce the production of significant levels of IFN- by splenocytes
from RelB-/- mice. However, these compounds,
which bypass receptor-mediated events, are associated with activation
of protein kinase C and the calmodulin calcium-dependent protein
phosphatase calcineurin pathways and synergize to activate many
transcription factors including NF-B (54, 55). When we
used more physiological stimuli (i.e., infection or stimulation through
the TCR alone or in combination with cytokines), we identified a major
defect in the production of IFN-. In other studies
(37), it was found that RelB-/-
mice infected with L. monocytogenes had serum levels of
IFN- that were greater than infected WT mice. The reason for this
discrepancy with our studies is unclear but the presence of large
numbers of these bacteria (4 logs greater than WT mice) may stimulate a
pathway for the production of IFN- that is independent of
RelB.
These studies have also shown that the absence of RelB can affect the
production of IL-1ß, IL-2, and IL-4 and are in agreement with
previous studies which reported that RelB was required for maximal
production of TNF- (37). Based on current literature,
it is unlikely that these defects would contribute to the early death
of RelB-/- mice infected with T.
gondii. Although TNF- is associated with resistance to T.
gondii, mice deficient in the TNF receptors survive acute
toxoplasmosis and die as a consequence of a severe encephalitis
(56, 57). Because IL-1 is involved in mediating resistance
to T. gondii (7, 58), the increased production
of IL-1ß would more likely contribute to resistance to T.
gondii rather than to enhance parasite replication. The reduced
production of IL-2 by T cells from RelB-/- mice
could contribute to the defect in IFN- production and susceptibility
to infection. However, exogenous IL-2 did not restore the production of
IFN- by T cells from RelB-/- mice and mice
deficient in the common chain, which is used for IL-2 signaling,
have an IFN--dependent mechanism of resistance to acute
toxoplasmosis (59). Moreover, RPA analysis of spleens from
infected mice did not reveal any differences in the levels of IL-2 mRNA
between WT and RelB-/- mice. Thus, we conclude
that the defect in IL-2 production would be unlikely to contribute to
the enhanced susceptibility of RelB-/- mice to
toxoplasmosis.
The overproduction of IL-4 by splenocytes from
RelB-/- mice stimulated with PMA and ionomycin
is in accord with previous studies which showed increased levels of
IL-4 mRNA in uninfected RelB-/- mice
(37). Because purified T cells from WT and
RelB-/- mice stimulated with PMA and ionomycin
produced similar amounts of IL-4 our findings suggest that non-T cells
may be the source of the increased levels of IL-4. Granulocytes have
been reported as a source of IL-4 (60, 61, 62), and the
increased numbers of granulocytes present in
RelB-/- mice supports the idea that these cells
represent an endogenous source of IL-4 in the spleen. However, it is
unlikely that overproduction of IL-4 contributes to the decreased
ability of RelB-/- T cells to produce IFN-
because the inclusion of a neutralizing Ab specific for IL-4 did not
increase the production of IFN- by splenocytes from
RelB-/- mice stimulated with anti-CD3 alone
or in combination with IL-2, IL-12, or IL-18. In addition, based on the
production of IL-4 by splenocytes from infected mice and RPA analysis,
we did not observe the development of a TH2 type response in WT or
RelB-/- mice.
Comparison of the ability of BMM from WT or
RelB-/- mice to produce IL-12 revealed no
significant differences between these populations. Moreover,
splenocytes from uninfected RelB-/- mice
produced normal levels of IL-12 in response to TLA. Recent studies have
suggested that, in uninfected mice, the major source of IL-12 produced
in response to parasite Ags in the spleen is
CD8+ dendritic cells (36).
Although RelB-/- lack the myeloid-related
CD8- dendritic cells, they do possess
CD8+ lymphoid dendritic cells
(31) and this correlates with the production of IL-12 in
response to TLA by splenocytes from uninfected
RelB-/- mice. However, splenocytes from
infected RelB-/- mice did not display the
infection-induced increase in production of IL-12 that was observed in
WT mice. Interestingly, there were no significant differences in the
serum levels of IL-12 between infected WT and
RelB-/- mice, in accord with our studies with
BMM. This discrepancy between systemic levels of IL-12 and levels
produced by splenocytes in recall responses was also reported in
studies with IFN--/- mice infected with
T. gondii (45). Because IFN- is important in
priming accessory cells to produce IL-12, the lack of an
infection-induced increase in IL-12 production by splenocytes from
infected RelB-/- mice is probably a consequence
of the reduced production of IFN- in these mice. However, the
infection-induced increase in serum IL-12 appears to be IFN-
independent and may reflect the contribution of IL-12 production by
macrophages at local sites of inflammation like the peritoneal
cavity.
There are two published studies on the role of the NF-B system in
the immune response to T. gondii. Mice deficient in Bcl-3
(an IB family member with transcriptional activity) survive the
acute phase of infection but die within 3–5 wk after infection
(63). These mice appear to have a normal initial IFN-
response, likely from NK cells, but fail to mount a protective T cell
response to T. gondii (63).
NF-B2-/- mice are also resistant during the
acute phase of toxoplasmosis but display a protracted pattern of
mortality during the chronic phase of infection (64) (J.
Caamaño and C. Hunter, unpublished observations). These results
suggest a requirement for Bcl-3 and NF-B2 in the development or
maintenance of appropriate T cell responses. In addition, we have
infected NF-B1-/- mice with the ME49 strain
T. gondii, and these mice are not more susceptible to
toxoplasmosis (J. Caamaño and C. Hunter, unpublished
observations). The results obtained with mice deficient in Bcl-3,
NF-B1, or NF-B2 contrast with the phenotype we observed from
studies with RelB-/- mice. Although RelB is not
required for macrophage functions associated with resistance to
T. gondii, it is essential for the development of
innate NK and adaptive T cell responses that lead to the production of
IFN- and resistance to intracellular pathogens. Thus, these studies
identify a role for RelB in the regulation of innate and adaptive
immunity to infection that is distinct from NF-B1, NF-B2, and
Bcl-3.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address:
3 Abbreviations used in this paper: WT, wild-type; TLA, soluble Ags of T. gondii; PEC, peritoneal exudate cell; RNI, reactive nitrogen intermediate; BMM, bone marrow macrophages; RH, virulent strain of gondii; RPA, ribonuclease protection assay.
Received for publication May 26, 1999. Accepted for publication August 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
by NK cells: A role for IL-1ß in the T cell independent mechanism of resistance against intracellular pathogens. J. Immunol. 155:4347.[Abstract]
are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiological antagonist. Proc. Natl. Acad. Sci. USA 90:3725. response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-. J. Immunol. 150:3982.[Abstract]
B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B. Immunol. Today 19:80.[Medline]
B and its inhibitor IB-/MAD-3 in monocytes by Mycobacterium tuberculosis and during human tuberculosis. J. Immunol. 159:4109.[Abstract]
B and is a potent inducer of chemokine and adhesion molecule gene expression in endothelial cells and fibroblasts. J. Immunol. 158:3285.[Abstract]
B and
activatory protein 1 and the induction of inflammatory cytokines.
J. Exp. Med. 186:247.
B. Infect. Immun. 65:3337.[Abstract]
production and activates IRAK and NF-B. Immunity 7:571.[Medline]
B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Nature 365:767.
B. J. Exp. Med. 186:999.B transcription factors during lymphocyte activation. Curr. Opin. Immunol. 10:353.[Medline]
B/IB function in mice. Semin. Cancer Biol. 8:93.[Medline]
B activities in mouse thymus and spleen. Oncogene 9:3289.[Medline]
B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia. Proc. Natl. Acad. Sci. USA 92:10944.- dendritic cells but not of lymphoid-related CD8+ dendritic cells. Immunity 9:839.[Medline]
B/Rel family. Cell 80:331.[Medline]
interferon and other cytokines in suppression of the spleen cell proliferative response to concanavlin A and Toxoplasma antigen during acute toxoplasmosis. Infect. Immun. 63:751.[Abstract]
interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha. Infect. Immun. 62:2818. synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
interferon by natural killer cells. Infect. Immun. 65:3594.[Abstract]
(IL-1), IL-1ß and interferon--inducing factor on the production of interferon- by NK cells. Eur. J. Immunol. 27:2787.[Medline]
gene expression. J. Interferon Cytokine Res. 16:563.[Medline]
B and NFAT with the interferon-promoter. J. Biol. Chem. 272:30412.: the major mediator of resistance against Toxoplasma gondii. Science 240:516. promoter in the murine dendritic cell line 18 and the murine mast cell line CPII is differentially regulated. J. Immunol. 157:2645.[Abstract]
B/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 7:175.[Medline]
-chain (c) reveals an unexpected role for CD4+ T lymphocytes in early IFN--dependent resistance to Toxoplasma gondii. J. Immunol. 160:2565.B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.This article has been cited by other articles:
|
|
 |
|
  L. A. Lieberman and D. E. Higgins A Small-Molecule Screen Identifies the Antipsychotic Drug Pimozide as an Inhibitor of Listeria monocytogenes Infection Antimicrob. Agents Chemother., February 1, 2009; 53(2): 756 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Roth-Cross, S. J. Bender, and S. R. Weiss Murine Coronavirus Mouse Hepatitis Virus Is Recognized by MDA5 and Induces Type I Interferon in Brain Macrophages/Microglia J. Virol., October 15, 2008; 82(20): 9829 - 9838. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Dzierszinski, M. Pepper, J. S. Stumhofer, D. F. LaRosa, E. H. Wilson, L. A. Turka, S. K. Halonen, C. A. Hunter, and D. S. Roos Presentation of Toxoplasma gondii Antigens via the Endogenous Major Histocompatibility Complex Class I Pathway in Nonprofessional and Professional Antigen-Presenting Cells Infect. Immun., November 1, 2007; 75(11): 5200 - 5209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Martucci, S. Franchi, D. Lattuada, A. E. Panerai, and P. Sacerdote Differential involvement of RelB in morphine-induced modulation of chemotaxis, NO, and cytokine production in murine macrophages and lymphocytes J. Leukoc. Biol., January 1, 2007; 81(1): 344 - 354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Masek, J. Fiore, M. Leitges, S.-F. Yan, B. D. Freedman, and C. A. Hunter Host cell Ca2+ and protein kinase C regulate innate recognition of Toxoplasma gondii J. Cell Sci., November 1, 2006; 119(21): 4565 - 4573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cannarile, F. Fallarino, M. Agostini, S. Cuzzocrea, E. Mazzon, C. Vacca, T. Genovese, G. Migliorati, E. Ayroldi, and C. Riccardi Increased GILZ expression in transgenic mice up-regulates Th-2 lymphokines Blood, February 1, 2006; 107(3): 1039 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Molestina and A. P. Sinai Host and parasite-derived IKK activities direct distinct temporal phases of NF-{kappa}B activation and target gene expression following Toxoplasma gondii infection J. Cell Sci., December 15, 2005; 118(24): 5785 - 5796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Corn, C. Hunter, H.-C. Liou, U. Siebenlist, and M. R. Boothby Opposing Roles for RelB and Bcl-3 in Regulation of T-Box Expressed in T Cells, GATA-3, and Th Effector Differentiation J. Immunol., August 15, 2005; 175(4): 2102 - 2110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shapira, O. S. Harb, J. Margarit, M. Matrajt, J. Han, A. Hoffmann, B. Freedman, M. J. May, D. S. Roos, and C. A. Hunter Initiation and termination of NF-{kappa}B signaling by the intracellular protozoan parasite Toxoplasma gondii J. Cell Sci., August 1, 2005; 118(15): 3501 - 3508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-L. C. Jin, L. Lan, M. Zoudilova, and M. Conti Specific Role of Phosphodiesterase 4B in Lipopolysaccharide-Induced Signaling in Mouse Macrophages J. Immunol., August 1, 2005; 175(3): 1523 - 1531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Kane, L. Cervi, J. Sun, A. S. McKee, K. S. Masek, S. Shapira, C. A. Hunter, and E. J. Pearce Helminth Antigens Modulate TLR-Initiated Dendritic Cell Activation J. Immunol., December 15, 2004; 173(12): 7454 - 7461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Mason, J. Fiore, T. Kobayashi, K. S. Masek, Y. Choi, and C. A. Hunter TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii Infect. Immun., October 1, 2004; 72(10): 5662 - 5667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schmidt-Supprian, J. Tian, H. Ji, C. Terhorst, A. K. Bhan, E. P. Grant, M. Pasparakis, S. Casola, A. J. Coyle, and K. Rajewsky I{kappa}B Kinase 2 Deficiency in T Cells Leads to Defects in Priming, B Cell Help, Germinal Center Reactions, and Homeostatic Expansion J. Immunol., August 1, 2004; 173(3): 1612 - 1619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. I. Samson, S. Memet, C. A. J. Vosshenrich, F. Colucci, O. Richard, D. Ndiaye, A. Israel, and J. P. Di Santo Combined deficiency in I{kappa}B{alpha} and I{kappa}B{epsilon} reveals a critical window of NF-{kappa}B activity in natural killer cell differentiation Blood, June 15, 2004; 103(12): 4573 - 4580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ichikawa, S. Hida, Y. Omatsu, S. Shimoyama, K. Takahara, S. Miyagawa, K. Inaba, and S. Taki Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2 PNAS, March 16, 2004; 101(11): 3909 - 3914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Mason, H.-C. Liou, and C. A. Hunter T Cell-Intrinsic Expression of c-Rel Regulates Th1 Cell Responses Essential for Resistance to Toxoplasma gondii J. Immunol., March 15, 2004; 172(6): 3704 - 3711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Speirs, L. Lieberman, J. Caamano, C. A. Hunter, and P. Scott Cutting Edge: NF-{kappa}B2 Is a Negative Regulator of Dendritic Cell Function J. Immunol., January 15, 2004; 172(2): 752 - 756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Lieberman, M. Banica, S. L. Reiner, and C. A. Hunter STAT1 Plays a Critical Role in the Regulation of Antimicrobial Effector Mechanisms, but Not in the Development of Th1-Type Responses during Toxoplasmosis J. Immunol., January 1, 2004; 172(1): 457 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Molestina, T. M. Payne, I. Coppens, and A. P. Sinai Activation of NF-{kappa}B by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated I{kappa}B to the parasitophorous vacuole membrane J. Cell Sci., November 1, 2003; 116(21): 4359 - 4371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Tato, A. Villarino, J. H. Caamano, M. Boothby, and C. A. Hunter Inhibition of NF-{kappa}B Activity in T and NK Cells Results in Defective Effector Cell Expansion and Production of IFN-{gamma} Required for Resistance to Toxoplasma gondii J. Immunol., March 15, 2003; 170(6): 3139 - 3146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Artis, K. Speirs, K. Joyce, M. Goldschmidt, J. Caamano, C. A. Hunter, and P. Scott NF-{kappa}B1 Is Required for Optimal CD4+ Th1 Cell Development and Resistance to Leishmania major J. Immunol., February 15, 2003; 170(4): 1995 - 2003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhou, J. Zhang, M. G. Lichtenheld, and G. G. Meadows A Role for NF-{kappa}B Activation in Perforin Expression of NK Cells Upon IL-2 Receptor Signaling J. Immunol., August 1, 2002; 169(3): 1319 - 1325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Dobbin, N. C. Smith, and A. M. Johnson Heat Shock Protein 70 Is a Potential Virulence Factor in Murine Toxoplasma Infection Via Immunomodulation of Host NF-{kappa}B and Nitric Oxide J. Immunol., July 15, 2002; 169(2): 958 - 965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Tato and C. A. Hunter Host-Pathogen Interactions: Subversion and Utilization of the NF-{kappa}B Pathway during Infection Infect. Immun., July 1, 2002; 70(7): 3311 - 3317. [Full Text] [PDF]
|
|
|
|
|
|
K. Speirs, J. Caamano, M. H. Goldschmidt, C. A. Hunter, and P. Scott NF-{kappa}B2 Is Required for Optimal CD40-Induced IL-12 Production but Dispensable for Th1 Cell Differentiation J. Immunol., May 1, 2002; 168(9): 4406 - 4413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mason, J. Aliberti, J. C. Caamano, H.-C. Liou, and C. A. Hunter Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli J. Immunol., March 15, 2002; 168(6): 2590 - 2594. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Uhl, S. Giguere, T. J. Jack, and T. Hodge Increased Pulmonary Activation of Nuclear Factor-{kappa}B (NF-{kappa}B) in Foals Inoculated with Rhodococcus equi is Associated with Increased Expression of Inflammatory Cytokines Vet. Pathol., January 1, 2002; 39(1): 132 - 136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wysocka, S. Robertson, H. Riemann, J. Caamano, C. Hunter, A. Mackiewicz, L. J. Montaner, G. Trinchieri, and C. L. Karp IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness J. Immunol., June 15, 2001; 166(12): 7504 - 7513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Blass, E. Pure, and C. A. Hunter A Role for CD44 in the Production of IFN-{{gamma}} and Immunopathology During Infection with Toxoplasma gondii J. Immunol., May 1, 2001; 166(9): 5726 - 5732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Caamano, C. Tato, G. Cai, E. N. Villegas, K. Speirs, L. Craig, J. Alexander, and C. A. Hunter Identification of a Role for NF-{kappa}B2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii J. Immunol., November 15, 2000; 165(10): 5720 - 5728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Cai, T. Radzanowski, E. N. Villegas, R. Kastelein, and C. A. Hunter Identification of STAT4-Dependent and Independent Mechanisms of Resistance to Toxoplasma gondii J. Immunol., September 1, 2000; 165(5): 2619 - 2627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. J. Blader, I. D. Manger, and J. C. Boothroyd Microarray Analysis Reveals Previously Unknown Changes in Toxoplasma gondii-infected Human Cells J. Biol. Chem., June 22, 2001; 276(26): 24223 - 24231. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
