By
From the * Departments of Medicine and Pathology, University of Chicago, Chicago, Illinois 60637;
and the Department of Pharmacology, University of California San Diego, San Diego,
California 92093
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Abstract |
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To examine the role of nuclear factor (NF)-B in T cell development and activation in vivo,
we produced transgenic mice that express a superinhibitory mutant form of inhibitor
B-
(I
B-
A32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant
[m]I
B-
mice). Thymocyte development proceeded normally in the mI
B-
mice. However, the numbers of peripheral CD8+ T cells were significantly reduced in these animals. The
mI
B-
thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after
cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4+CD8+; DP) thymocytes from the mI
B-
mice were resistant to
-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by
-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of
-CD3 mAb was preceded
by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mI
B-
thymocytes maintained high level expression of bcl-xL after
-CD3 treatment. Taken together, these results demonstrated important roles for NF-
B in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-
B is required for the
-CD3-mediated apoptosis of DP thymocytes through a pathway that involves
the regulation of the antiapoptotic gene, bcl-xL.
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Introduction |
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The mammalian nuclear factor (NF)-B1 transcription
factors, NF-
B1 (p50/p105), NF-
B2 (p52/p100),
RelA (p65), c-Rel, and RelB are involved in the regulation of immune and inflammatory responses, cellular proliferation, and cell death (for review see references 1). NF-
B proteins modulate these diverse biological processes by
regulating the expression of a wide variety of genes encoding cytokines and cytokine receptors, chemokines, cell
adhesion molecules, cell-surface receptors, hematopoietic
growth factors, acute phase proteins, and other transcription factors. Transcriptional activation or repression of NF-
B target genes requires binding of NF-
B dimers to
B
DNA binding sites. Although p50/p65 heterodimers are
considered the prototypical NF-
B dimer, many different
combinations of NF-
B homo- and heterodimers exist (5)
and there is evidence to suggest that these different NF-
B
dimers regulate the expression of different genes (6).
In most cells, the transcriptional activity of NF-B proteins is controlled at the posttranslational level by regulated
associations with members of the inhibitor (I)
B family of
inhibitory proteins. NF-
B proteins are retained in the cytoplasm of unstimulated cells and thus rendered inactive via
interactions with one or more of the seven known I
B
proteins: I
B-
, I
B-
, I
B-
, Bcl-3, I
B-
, p105, and
p100 (3, 7). In response to a wide variety of stimuli, including antigen receptor cross-linking, and exposure to cytokines (TNF-
, IL-1), bacterial components (LPS), viruses, ionizing radiation, or oxidative stress, I
B-
and -
proteins are phosphorylated and degraded and cytoplasmic
NF-
B dimers released from their inhibitory proteins are
rapidly translocated to the nucleus where they regulate the
transcription of genes containing
B binding sites (8).
Recent studies have demonstrated that phosphorylation of
Ser32 and Ser36 and subsequent polyubiquitination of I
B-
are essential for the release and nuclear translocation of NF-
B dimers (12). Substitution of I
B-
Ser32 and Ser36 by Ala residues prevents phosphorylation and degradation
of I
B-
, thereby retaining NF-
B dimers in an inactive
form in the cytoplasm.
NF-B proteins are thought to play important roles in T
cell activation and development (19, 20). Functionally important
B binding sites have been identified in a large
number of T cell transcriptional regulatory elements, including the IL-2, IL-2R
, G-CSF, and macrophage inflammatory protein (MIP)-2 promoters (1). Preformed
NF-
B proteins are present in the cytoplasm of thymocytes and resting peripheral T cells (21). TCR engagement results in the rapid phosphorylation of I
B-
and the concomitant nuclear migration of active NF-
B dimers (19,
22, 23). Despite these findings, it has been difficult to precisely elucidate the role of NF-
B in regulating T cell development, activation, and apoptosis in vivo. During the
last several years, gene targeting experiments have demonstrated that RelB, c-Rel, and NF-
B1 each play important but distinct roles in regulating the development and function of the mammalian immune system. NF-
B1-deficient
mice displayed defects in B cell proliferation in response to
the mitogen LPS but not to IgM cross-linking (24, 25).
RelB is required for the differentiation and/or survival of
dendritic cells and thymic medullary epithelial cells and
RelB
/
mice displayed severely defective cellular immune responses (26, 27). The immune dysfunction observed in RelB
/
mice was worsened in NF-
B1/RelB
double knockout mice, suggesting that the lack of RelB is
compensated for by other NF-
B1-containing dimers (28).
c-Rel-deficient mice displayed defective B and T cell proliferation in response to mitogen stimulation as well as
markedly decreased IL-2 production after TCR engagement (29). In contrast, mice lacking RelA died on embryonic day 15 from massive hepatocyte apoptosis (30, 31).
However, progenitor cells derived from RelA
/
mice
were able to give rise to normal T cells, suggesting that RelA is not required for T cell development (30, 32).
Recent studies have suggested that, in addition to regulating lymphocyte function, NF-B proteins might play a
critical role in protecting cells against apoptosis. Support for
this model came both from the finding of hepatocyte apoptosis in the RelA
/
mice and from experiments demonstrating that overexpression of a dominant-negative form of
the I
B-
protein in transgenic mice (33) and several cell
lines promoted apoptosis in vitro (34). However, other
studies have suggested that NF-
B can also function in a
proapoptotic fashion. For example, induction of apoptosis in 293 cells after serum withdrawal requires RelA activation (40). Similarly, radiation-induced apoptosis in ataxia
telangiectasia cells is suppressed by dominant-negative
I
B-
proteins (41) and inhibition of NF-
B activation
prevents apoptosis in cultured human thymocytes (42). Finally, NF-
B activation promotes apoptosis in neural cells
(43) and T cell hybridomas (44), and high levels of c-Rel
induce apoptosis in avian embryos and in bone marrow
cells in vitro (45).
Although gene targeting experiments have been useful
for identifying the essential nonredundant roles of individual NF-B transcription factors in T cell development and
function, the interpretation of these experiments can be
obscured by functional redundancies of related gene products in the mutant animals and by embryonic lethal phenotypes that preclude a complete analysis of lymphoid development and function. Moreover, in some cases it can be
difficult to distinguish cell autonomous and non-cell autonomous phenotypes because mutant animals lack expression of the targeted gene in all cell lineages. We and others
(33, 46) have used a complementary approach that circumvents these potential problems to study the role of NF-
B
in T cell development, activation, and apoptosis in vivo.
Specifically, we have generated transgenic mice that express
a mutant superinhibitory form of the I
B-
protein under
the control of the T cell-specific CD2 promoter and enhancer. This superinhibitory form of I
B-
cannot be
phosphorylated on Ser32 and Ser36 and degraded in response
to TCR cross-linking and would therefore be expected to
constitutively inhibit the nuclear translocation of NF-
B
proteins after T cell activation. Studies of the mI
B-
mice
revealed several important functions for NF-
B in T cells
in vivo. First, NF-
B is required to develop or maintain
normal numbers of peripheral CD8+ T cells. Second,
NF-
B activation is necessary for both cytokine production and thymocyte proliferation after TCR engagement.
Finally and somewhat unexpectedly, NF-
B is required for
-CD3-mediated apoptosis of double positive (DP) thymocytes in vivo via a pathway that involves downregulated
expression of the antiapoptotic gene bcl-xL in DP thymocytes.
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Materials and Methods |
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Transgene Construction and Generation of Transgenic Mice.
The construction of the hemagglutinin (HA)-tagged ICell Culture.
Single cell suspensions of thymocytes and splenocytes were cultured at 37°C, 5% CO2 in RPMI 1640 medium containing 10% heat inactivated FCS, 100 U/ml penicillin/streptomycin, 2 mM glutamine, 0.1 mM nonessential amino acids, 5.5 × 102 µMFetal Thymic Organ Culture.
Fetuses of CD1 and mIELISA Assays.
For ELISA assays, 96-well plates (Dynatech Labs.) were coated overnight at 4°C withWestern Blot Analysis.
Cytoplasmic and nuclear thymocyte extracts were prepared as previously described (49). In brief, thymocytes were washed with PBS, incubated in buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) for 10 min at 4°C and centrifuged to separate nuclei (pellet) from cytoplasmic proteins (supernatant). Nuclei were lysed by incubation with buffer C (20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) at 4°C and lysates were cleared by centrifugation. Nuclear and cytoplasmic extracts were frozen atElectrophoretic Mobility Shift Assay.
A double-stranded oligonucleotide, spanning theFlow Cytometry.
Single cell suspensions of lymphocytes (106 cells) were washed in PBS and incubated with PE-Ribonuclease Protection Assays.
Ribonuclease protection assays were performed using a commercially available kit as described by the manufacturer (PharMingen) using the mAPO-2 multiprobe template set, 10 µg thymic RNA, and 0.1 mCiTUNEL Assays.
TUNEL (Tdt-mediated dUTP nick end labeling) assays were performed on paraffin-embedded sections of thymi according to Gavrieli et al. (51). Photomicrographs were obtained with a Zeiss Axioskop. ![]() |
Results |
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To inhibit inducible NF-B activity in thymocytes in vivo, we produced
transgenic mice that expressed a "superinhibitory" form of
I
B-
under the control of the T cell-specific CD2 promoter and enhancer (mI
B-
mice). We chose to use an
I
B-
transgene because previous studies have demonstrated that I
B-
plays a critical role in regulating inducible NF-
B expression (for review see references 2,
52). The superinhibitory mI
B-
A32/36 transgene containing
Ser32 and Ser36 to Ala substitutions cannot be phosphorylated and degraded in response to TCR engagement and
would therefore be expected to constituitively inhibit NF-
B activity in both resting and activated thymocytes and T
cells. The CD2 promoter and enhancer (48, 53; Fig. 1 A)
were used in these studies because (a) they program T cell-
specific transgene expression and (b) they are expressed at
high levels in all thymocyte subsets including double negative (CD4
CD8
; DN), DP (CD4+CD8+), and single
positive (CD4+ or CD8+; SP) cells (54). The transgene
construct also contained three copies of an 11-amino acid
influenza virus HA epitope tag (55) that allowed us to distinguish the transgene-encoded protein from endogenous
murine I
B-
. A line of mI
B-
mice containing ~12 copies of the transgene was produced by injection of this
construct into fertilized CD1 mouse embryos (Fig. 1 B).
Homozygous transgenic mice were generated by breeding
heterozygous littermates in order to obtain maximal levels
of I
B-
A32/36 protein expression, an important consideration in producing a dominant-negative phenotype. A second line of transgenic mice expressing the I
B-
A32/36
cDNA under the control of the proximal lck promoter and
the CD2 enhancer was also produced (data not shown).
Similar phenotypes were observed in both lines of transgenic mice and are therefore not distinguished in the results
described below.
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Endogenous IB-
is phosphorylated and degraded after
treatment of T cells with the NF-
B inducer, TNF-
(47).
To analyze the relative levels of endogenous and transgene-encoded I
B-
and to study the differential regulation of
the two proteins in response to TNF-
, we performed
Western blot analyses using whole cell extracts from mI
B-
and control thymocytes that had been treated for different
times with TNF-
(Fig. 2 A). Basal levels of the I
B-
A32/36
protein as detected with both the
-HA and
-I
B-
(
-MAD) antibodies slightly exceeded levels of the endogenous I
B-
protein in the mI
B-
thymocytes. In both
wild-type and mI
B-
thymocytes, endogenous I
B-
was almost completely degraded after 15 min of TNF-
treatment and remained undetectable until ~30 min after
treatment. As previously reported (9, 15), endogenous
I
B-
was reexpressed between 30 and 60 min after TNF-
treatment (data not shown). This reexpression reflects NF-
B-mediated activation of the I
B-
promoter. In marked
contrast, levels of the transgene-encoded I
B-
A32/36 were
unchanged by TNF-
treatment at all time points (Fig. 2
A). Activation of mI
B-
thymocytes with PMA plus ionomycin or TCR cross-linking resulted in a similar degradation of endogenous I
B-
, whereas the levels of I
B-
A32/36 remain unaffected (data not shown).
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To analyze the effects of IB-
A32/36 expression on the
nuclear translocation of NF-
B proteins after T cell activation, we performed electrophoretic mobility shift assays
(EMSAs) using nuclear extracts prepared from both resting
thymocytes and thymocytes activated in vivo by intraperitoneal injection of an
-CD3 mAb (Fig. 2 B). As reported
previously, nuclear extracts from unstimulated wild-type
and mI
B-
thymocytes both contained p50 homodimers that bound to the radiolabeled
B probe (22, 56). The
presence of such p50 homodimers in uninduced thymic
nuclear extracts probably reflected the fact that p50 binds to
I
B proteins with lower affinity and therefore is not quantitatively retained in the cytoplasm of either wild-type or
mI
B-
thymocytes (57). Because p50 homodimers do
not contain a transcriptional activation domain they are
thought to repress
B-dependent gene expression (58).
After activation with
-CD3 mAb, the level of p50
homodimers increased equivalently in the wild-type and
mI
B-
thymocyte nuclear extracts. In addition, an inducible low mobility
B binding complex appeared in the
nuclear extracts of wild-type thymocytes. Antibody supershift experiments demonstrated that this complex contained
predominantly p50/p65 heterodimers as well as smaller
amounts of c-Rel- and RelB-containing heterodimers
(Fig. 2 B). The induction of this low mobility
B binding
activity was markedly inhibited after
-CD3-mediated activation of the mI
B-
thymocytes (Fig. 2 B). Taken together, these Western blotting and EMSA experiments
demonstrated that thymocytes from the mI
B-
transgenic
animals expressed high levels of cytoplasmic I
B-
A32/36
protein that was not degraded after TCR engagement in
vivo. Moreover, constitutive expression of the I
B-
A32/36 protein significantly reduced the induction of nuclear p50/
p65, c-Rel/p50, and RelB/p50 NF-
B heterodimers after either TNF-
or
-CD3-mediated activation of the
mI
B-
thymocytes.
To investigate T cell development in the mIB-
mice, thymocytes and peripheral T cells from transgenic
and wild-type animals were analyzed by flow cytometry
using antibodies specific for a variety of developmentally
regulated T cell surface antigens (Fig. 3). Thymi from the
mI
B-
animals contained normal populations of DN, DP,
and SP cells. Levels of CD3 and TCR-
/
expression were
also indistinguishable on wild-type and mI
B-
thymocytes (Fig. 3 B), as were expression of TCR-
/
and
heat-shock antigen (data not shown). Thus, expression of
I
B-
A32/36 did not appear to perturb thymocyte ontogeny.
Total numbers of SP peripheral T cells in both the spleen
and the lymph nodes were normal in mI
B-
mice as were
the numbers of splenic CD4+ T cells. However, as described previously by Boothby at al. and Esslinger et al. (33,
46), the numbers of peripheral CD8+ T cells were reduced
by ~50% in mI
B-
mice (Fig. 3 C).
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To assess the effects of IB-
A32/36
expression on cytokine production and proliferation, thymocytes from mI
B-
and wild-type control littermates were activated in vitro for 72 h with PMA plus ionomycin,
-CD3
mAb, or
-CD3 plus
-CD28 mAbs. When compared with
wild-type thymocytes, proliferation of the mI
B-
thymocytes was reduced by 63% in response to PMA plus ionomycin and by 78% after activation with
-CD3 mAb (Fig. 4).
Previous studies have suggested that CD28 costimulation may
function, at least in part, through an NF-
B-dependent pathway (59). Therefore, we tested the ability of CD28 costimulation to rescue the defect in
-CD3-mediated thymocyte proliferation seen in the mI
B-
thymocytes. Interestingly, the
observed reductions in thymocyte proliferation were partially, but not completely, rescued by
-CD28 costimulation (Fig.
4). Thus, the CD28 signaling pathway can at least partially
bypass the requirement for NF-
B activation in thymocyte
activation by TCR engagement.
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Many cytokine genes including IL-2, IL-3, and GM-CSF are potential targets for NF-B (60). However, the
role of NF-
B in regulating activation-specific cytokine
expression in vivo, particularly the expression of the IL-2
gene, remains controversial. Therefore, we compared cytokine production by the wild-type and mI
B-
thymocytes after activation with
-CD3 plus
-CD28 mAbs
(Fig. 5). As compared with wild-type thymocytes, the
mI
B-
thymocytes displayed significantly decreased production of IL-2, IL-3, and GM-CSF after
-CD3 plus
-CD28 activation (Fig. 5). IL-2 production was most
significantly reduced (28% of wild-type levels), whereas
GM-CSF and IL-3 production were less dramatically inhibited (Fig. 5). Taken together, these experiments demonstrated that NF-
B is required both for the induction of
multiple cytokines and for normal thymocyte proliferation
after TCR cross-linking.
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Systemic administration of -CD3
mAb has been shown to result in the rapid depletion of
>90% of DP thymocytes by apoptosis (61). NF-
B positively regulates the expression of death-rescuing genes,
thereby preventing TNF-
-induced apoptosis at least in certain cell types (34). Based upon these findings, it was reasonable to hypothesize that DP thymocytes from the mI
B-
mice that failed to activate NF-
B normally would display
increased apoptosis after systemic administration of
-CD3
mAb. To directly test the role of NF-
B in mediating
the
-CD3-mediated apoptotic death of DP thymocytes,
mI
B-
and wild-type animals were injected intraperitoneally with
-CD3 mAb and thymocyte subsets were assessed by flow cytometry 48 h after injection. As shown in
Fig. 6 and consistent with previous reports (62, 63), treatment of wild-type animals with
-CD3 mAb resulted in
dose-dependent reductions in the size of the DP thymocyte
population. Wild-type animals treated with 40 µg of
-CD3
mAb displayed a 98% reduction in DP thymocytes within
48 h after
-CD3 administration. Somewhat surprisingly, this
-CD3-mediated loss of DP thymocytes was completely
and reproducibly prevented in the mI
B-
mice even after
administration of 40 µg of
-CD3 mAb (Fig. 6). Similar reductions in DP thymocyte apoptosis were observed at both
24 and 48 h after injection and after administration of either
20 or 40 µg of
-CD3 mAb (Fig. 6 and data not shown).
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To confirm the protective effects of IB-
A32/36 expression on DP thymocyte apoptosis in vivo, we performed
TUNEL assays on thymi from wild-type and mI
B-
mice
after intraperitoneal injection of
-CD3 mAb (Fig. 7).
Large numbers of apoptotic cells were seen in wild-type
thymi after treatment with
-CD3 mAb as compared with
littermates treated with an isotype-matched control antibody. In contrast, the frequency of apoptotic cells in the
mI
B-
thymi was dramatically reduced as compared with
that observed in wild-type thymi after treatment with similar amounts of
-CD3 mAb (Fig. 7).
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Like -CD3 treatment,
irradiation of the thymus is known to cause DP thymocyte
apoptosis in vivo (64). However, recent studies have suggested that these different apoptotic stimuli may use distinct
signaling pathways to regulate programmed cell death.
Given the resistance of mI
B-
DP thymocytes to
-CD3-mediated apoptosis, it was of interest to determine
the sensitivity of these cells to
irradiation in vivo. As
shown in Fig. 6 C, DP thymocytes from the mI
B-
mice
remained fully sensitive to
irradiation-induced apoptosis
in vivo. Thus, expression of the I
B-
A32/36 transgene protected DP thymocytes from apoptosis in response to
-CD3
but failed to protect these cells against
irradiation-
induced cell death.
The administration
of -CD3 mAbs to mice in vivo can effect thymocytes either
directly or indirectly via the activation of peripheral blood
CD3+ T cells. To attempt to distinguish these possibilities, we
tested the susceptibility of mI
B-
thymocytes to
-CD3-
mediated apoptosis in fetal thymic organ culture (FTOC)
(Fig. 8). Wild-type thymocytes were killed efficiently by 3 d
of FTOC treatment with an
-CD3 mAb. In contrast, DP
thymocytes from the mI
B-
mice were relatively resistant
to
-CD3-mediated killing in FTOC. These results suggested that at least some of the resistance to
-CD3-mediated DP
thymocyte apoptosis seen in the mI
B-
mice reflects a
thymus-specific effect of transgene expression.
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The Bcl-2 family of proteins is
comprised of multiple members that function either as
death agonists (Bax, Bak, Bcl-XS, and Bad) or death antagonists (Bcl-2, Bcl-xL, Bcl-x, Mcl-1, A1, and Bcl-w) (65).
Two antiapoptotic members of this family, Bcl-xL and Bcl-2,
are expressed in a reciprocal pattern during thymocyte development. Bcl-2 is expressed at high levels in immature
DN thymocytes. Its expression is downregulated in DP
cells and it is then reexpressed as these DP cells mature to
SP thymocytes and peripheral T cells (66, 67). Conversely,
Bcl-xL is expressed at low or undetectable levels in immature DN cells. Its expression is significantly upregulated in
DP thymocytes, and it is then downregulated as these cells
progress to the SP stage of thymocyte ontogeny (68, 69).
Interestingly, DP thymocytes from transgenic mice that
overexpress Bcl-2 or Bcl-xL are protected from multiple proapoptotic stimuli, including
-CD3 treatment in vivo
(69, 70). Given these findings, it was logical to postulate
that altered expression of Bcl-2-family genes in the mI
B-
thymocytes might account for their decreased susceptibility
to proapoptotic stimuli. Accordingly, we used an RNAse
protection assay to directly monitor the expression of Bcl-2-related genes in thymocytes from wild-type and mI
B-
transgenic mice. As shown in Fig. 9 A, both basal and
-CD3-treated levels of bfl-1, bak, bax, bcl-2, and bad
mRNAs were equivalent in wild-type and mI
B-
thymocytes. Similarly, we failed to detect differential expression
of mRNAs encoding Fas, Fas-L, TRAF, TRADD, Fadd,
RIP, and FLICE in either unstimulated or
-CD3-treated
wild-type and mI
B-
thymocytes (data not shown). Basal
levels of bcl-xL were equivalent in wild-type and mI
B-
thymocytes. However, after
-CD3 treatment, the expression of bcl-xL was significantly decreased in the wild-type
cells but was maintained at pretreatment levels in the
mI
B-
thymocytes (Fig. 9 A).
|
Because Bcl-xL is the predominant antiapoptotic protein
expressed in DP thymocytes, the preferential loss of Bcl-xL
from wild-type thymocytes after -CD3 administration
might have simply reflected the death of these DP cells. To
confirm that the pattern of Bcl-xL protein expression paralleled that of the mRNA and to assess the possibility that the
observed changes in bcl-xL mRNA expression reflected the
preferential death of the DP thymocytes in the wild-type animals, we performed Western blot analyses on thymocyte
extracts 18 and 39 h after
-CD3 treatment in vivo. As
shown in Fig. 9 B, levels of Bcl-xL protein were significantly reduced in wild-type thymocytes at both 18 and
39 h after administration of
-CD3 mAb. In contrast, levels
of Bcl-xL protein were maintained at unstimulated levels in
the mI
B-
thymocytes at both time points. Because there is not a significant loss of DP thymocytes 18 h after
-CD3
treatment, these results indicated that the loss of Bcl-xL
protein precedes thymocyte apoptosis and is prevented in
the mI
B-
DP thymocytes. We confirmed this result by
performing intracellular staining of viable DP thymocytes
using Bcl-xL-specific antibodies (Fig. 9 C). 24 h after
-CD3 administration, viable DP thymocytes displayed a
significant decrease in intracellular Bcl-xL as measured by flow cytometry. In contrast, DP thymocytes from the
mI
B-
mice failed to downregulate intracellular Bcl-xL
expression. In control experiments, SP CD4+ thymocytes
from these same wild-type and mI
B-
animals both failed
to demonstrate altered levels of intracellular Bcl-xL after
-CD3 administration (Fig. 9 D). Taken together, these
results demonstrated that the in vivo administration of
-CD3 mAb resulted in the specific downregulation of the
antiapoptotic gene bcl-xL in DP thymocytes and that this
reduced expression of Bcl-xL was, in turn, associated with
subsequent apoptotic cell death. In contrast, DP thymocytes from mI
B-
mice failed to downregulate Bcl-xL and were protected from
-CD3-mediated programmed cell
death.
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Discussion |
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In the studies described in this report, we have used
transgenic mice expressing a superinhibitory form of IB-
to study the function of NF-
B in T cells in vivo. The
dominant-negative approach used in these studies circumvents potential problems of functional redundancy and embryonic lethality that can complicate the analysis of gene
targeting experiments. In addition, because the transgene is
only expressed in thymocytes and T cells we can conclude
that the observed defects in T cell function are thymocyte or T cell autonomous. Our results have revealed several
important roles for NF-
B proteins in T cell development
and function. First in agreement with previous reports (33,
46) we showed that NF-
B is required for the development and/or survival of normal numbers of peripheral
CD8+ T cells, for the inducible expression of multiple cytokine genes, and for normal T cell proliferation in response to TCR-mediated T cell activation. However, our
studies have also revealed an unexpected and novel role for
NF-
B proteins in DP thymocyte apoptosis in response to
-CD3 mAb administration in vivo.
TCR engagement leads to the precisely
orchestrated transcriptional induction of >100 new genes
whose expression together determine the activated T
cell phenotype. Several inducible transcription factors
have been implicated as critical early regulators of activation-specific T cell gene expression. These include NF-AT,
CREB, AP1, and NF-B (60). Three of these, NF-AT,
CREB, and NF-
B, are present in an inactive form in resting T cells and are activated by posttranslational phosphorylation or dephosphorylation in response to TCR cross-linking. The precise role of each of these transcription
factors in regulating T cell activation remains unclear because many T cell genes contain binding sites for multiple
inducible transcription factors, and because it has been difficult to extrapolate from the results of transient transfection assays performed in immortalized T cell lines to in
vivo transcriptional regulatory pathways. Thus, for example, NF-AT, AP1, and NF-
B transcription factors can
each bind to functionally important sites in the IL-2 promoter in vitro (71). Moreover, transient transfection assays have suggested that each of these transcription factors
may play an important role in regulating the expression of
this T cell cytokine (75). However, gene targeting experiments have thus far failed to demonstrate a critical role
for NF-AT in positively regulating IL-2 expression and T
cell proliferation in normal T cells (77, 78). To circumvent these problems, we used a genetic approach involving overexpression of I
B-
A32/36 in transgenic mice to directly study
the role of NF-
B proteins in the development and activation of normal T cells in vivo. Our results demonstrated that
NF-
B is required for the inducible expression of the IL-2,
IL-3, and GM-CSF genes and for subsequent T cell proliferation in response to TCR stimulation. These findings are
consistent with similar defects in cytokine production and T
cell proliferation observed in mice lacking the c-Rel protein
(29) or expressing a constitutively active I
B-
transgenic
protein containing an NH2-terminal deletion (33).
Previous studies have suggested that the CD28 costimulatory pathway functions to activate cytokine gene transcription via an NF-B-mediated signaling pathway (59,
79). Interestingly, however, we and others (33) found
that the inhibitory effects of I
B-
A32/36 on
-CD3-mediated thymocyte proliferation could be partially reversed by
-CD28-mediated costimulation. This suggests that at least
some of the costimulatory effects of the CD28 pathway are
mediated via one or more NF-
B-independent pathways.
In this regard, it is of interest that
-CD28 costimulation
has also been reported to increase IL-2 expression by stabilizing IL-2 mRNA (82). It will be of interest to determine
if this mRNA-stabilizing effect of CD28 signaling is regulated via an NF-
B-independent pathway.
The mechanism responsible for the observed reduction
in peripheral CD8+ T cells in the mIB-
mice remains
unclear. It may reflect unique roles for NF-
B in the signaling pathways required for the maturation or export of
mature CD8+ thymocytes. Alternatively, it may reflect a
defect in the survival of CD8+ T cells in the periphery of
the mI
B-
mice. In this regard, it is of interest that the
survival of peripheral naive SP CD8+ T cells has been reported to require continuous stimulation by class I MHC.
Thus, it is possible that this class I MHC-mediated signal is
transmitted through an NF-
B-dependent pathway (83).
Pro grammed cell death plays an important role in shaping the
T cell repertoire during thymocyte ontogeny. More than
95% of immature DP thymocytes die before they exit the
thymus (84). Thymocytes that express TCRs with low
avidity for antigen plus self-MHC fail to be positively selected and die by "neglect", whereas self-reactive DP T
cells are deleted during the process of negative selection after high affinity engagement of their TCRs by self-antigenic peptides and MHC (85, 86). Our results demonstrate
that NF-B proteins are necessary positive mediators of DP
thymocyte apoptosis in response to
-CD3 administration in vivo. These findings were somewhat surprising given
several recent studies that have reported that NF-
B proteins are potent inhibitors of TNF-
- and IL-1-mediated
apoptosis in fibroblasts (36, 87). However, our findings
are consistent with previous reports demonstrating that
NF-
B proteins can positively regulate apoptosis in certain
cell types in vitro including thymocytes and T cell hybridomas (42, 44). The different findings of these studies may reflect the fact that NF-
B can regulate the expression of distinct proapoptotic and antiapoptotic programs in different
cell lineages, at different developmental stages of a single
lineage, and/or in response to different extracellular signals.
At least three different apoptotic pathways have been
identified in thymocytes: (a) a TCR-mediated pathway (63,
88); (b) a glucocorticoid-responsive pathway (89, 90); and
(c) a irradiation-sensitive pathway (64). Recent evidence
has suggested that these pathways can be distinguished at a
molecular level. Thus, for example,
irradiation-induced
thymocyte apoptosis requires p53 and does not occur in
p53-deficient thymocytes. In contrast,
-CD3-mediated
thymocyte apoptosis is p53 independent (64, 91). Our results
are consistent with such a model in that the mI
B-
DP
thymocytes were protected from
-CD3-induced apoptosis
but remained sensitive to programmed cell death induced by
irradiation. Thus, we would suggest that NF-
B is not required for p53-dependent thymocyte apoptosis, but is a critical positive regulator of at least one p53-independent pathway of programmed cell death in DP thymocytes. In this
regard, it will be of interest to study DP thymocyte apoptosis
in p53-deficient, mI
B-
transgenic mice.
Our results suggest that NF-B mediates
-CD3-mediated apoptosis in wild-type DP thymoctyes by downregulating the expression of the antiapoptotic gene bcl-xL. Because
bcl-xL is the predominant antiapoptotic gene expressed in DP
thymocytes (68, 69) it is not surprising that its downregulation would predispose these cells to apoptosis. Such a model
is also consistent with previous studies that have demonstrated that constitutive expression of a bcl-xL transgene in
DP thymocytes protects them from
-CD3-mediated apoptosis (69). Recently, an alternatively spliced form of bcl-xL called bcl-x
has been shown to be expressed in DP thymocytes and to be regulated in response to TCR stimulation
(92). Because the Bcl-xL and Bcl-x
proteins are virtually
identical in size and are both recognized by the anti-Bcl-xL
antibodies and probes used in our studies, we cannot distinguish them by RNAse protection, Western blotting, or intracellular flow cytometry. Thus, it is possible that one or
both isoforms of Bcl-x contribute to the antiapoptotic effects
of the I
B-
A32/36 transgene. The effect of NF-
B on Bcl-xL
expression in DP thymocytes may be direct or indirect. That
is, NF-
B proteins might bind directly to the bcl-xL promoter and downregulate its activity, or alternatively, might
control the expression of other (positive and/or negative) regulators of the bcl-xL gene in thymocytes. In this regard, it will be of interest to further characterize the bcl-xL promoter and to study its regulation by NF-
B proteins.
It is important to emphasize that at present, we cannot
conclude that the proapoptotic effects of NF-B in DP
thymocytes are thymocyte autonomous. For example, it is
possible that
-CD3 administration causes peripheral T cell
activation resulting in cytokine expression that in a paracrine fashion causes DP thymocyte toxicity (93). In such a
model, decreased cytokine production by peripheral mI
B-
T cells in response to
-CD3 treatment could account for
the decreased DP thymocyte apoptosis seen in these animals. Indeed our results demonstrate decreased cytokine
production by thymocytes after TCR cross-linking (Fig.
5). However, this non-thymocyte-autonomous model is
less likely to account for the resistance to
-CD3-induced thymocyte death seen in the mI
B-
mice because DP
thymocytes from the mI
B-
mice are also resistant to
-CD3-mediated killing in FTOC. Thus, it would appear
more likely that NF-
B expression in DP thymocytes is directly required for
-CD3-induced apoptosis.
During the last several years, there has been considerable
interest in developing inhibitors of NF-B for the treatment of both autoimmune diseases and cancer. In the case
of cancer, such inhibitors might function to increase the
sensitivity of tumor cells to apoptotic cell death induced
both by cytokines such as TNF-
and by CTLs. In patients
with autoimmune disease, such inhibitors might function
by suppressing T cell activation in response to self-antigens.
Our finding that NF-
B can function to potentiate apoptosis in certain cell types and specifically in DP thymocytes raises several potential concerns about the systemic administration of NF-
B inhibitors. First, it is possible that some
tumor cells and/or virus-infected cells, like DP thymocytes, may actually demonstrate decreased apoptosis in response to NF-
B inhibitors. Thus, it is possible that potent
NF-
B inhibitors might potentiate tumorgenesis in some
tissues and might also reduce host responses against viral
pathogens by decreasing susceptibility to apoptosis. Similarly, our finding that NF-
B inhibitors protect DP thymocytes from apoptosis suggests the possibility that the
administration of such inhibitors to patients with autoimmune diseases might result in a failure in the negative selection of some DP thymocytes, thereby potentially increasing
the generation of autoreactive T cell clones. Given these
possibilities, it will be important to carefully assess the effects of NF-
B inhibitors on the apoptotic potential of different human tumor and virus-infected cells and on the
negative selection of DP thymocytes before their wide-spread use in human therapy.
![]() |
Footnotes |
---|
Address correspondence to Jeffrey M. Leiden, University of Chicago, Rm. B608 MC 6080, 5841 South Maryland Ave., Chicago, IL 60637. Phone: 773-702-1919; Fax: 773-702-1385; E-mail: jleiden{at}medicine.bsd.uchicago.edu
Received for publication 6 July 1998 and in revised form 5 October 1998.
Thore Hettmann is a Terry Fox Research Fellow. This work was supported in part by a grant from the NIAID to J.M. Leiden (2 R37 A129637-07) and by the Cancer Center of the University of Chicago, and by a grant from the NIAID to M. Karin (R01 AI43477-01).We thank C. Clendenin for help with the production of transgenic mice, J. Auger for technical assistance with FACS® analyses, and P. Lawrey and L. Gottschalk for help with the preparation of the manuscript and illustrations.
Abbreviations used in this paper
EMSA, electrophoretic mobility shift assay;
FTOC, fetal thymic organ culture;
HA, hemagglutinin;
IB-
, inhibitor
B-
;
mI
B-
, mutant I
B-
;
NF-
B, nuclear factor
B;
TUNEL, Tdt-mediated dUTP nick end labeling.
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