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Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Furthermore, when expressed in
Th2-skewed cells, caNFATc1 appears to attenuate Th2 differentiation by
decreasing production of IL-4 and promoting the expression of IFN-.
Finally, we find that caNFATc1 enhances expression of functional
P-selectin glycoprotein ligand-1, up-regulates Fas ligand expression,
and increases susceptibility to activation-induced cell death, cellular
traits that are preferentially associated with Th1 effector cells.
Taken together, these results suggest that sustained NFAT signaling,
mediated by ectopic expression of caNFATc1, acts to promote a Th1-like
pattern of gene expression and thereby serves to highlight the
important relationship between the degree of NFAT signaling and the
qualitative pattern of gene expression induced during T cell
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and are involved in the regulation
of cell-mediated immune responses, whereas Th2 cells primarily produce
IL-4 and IL-5 and regulate humoral immunity (1). This
process of Ag-induced T cell activation and differentiation proceeds
via a complex genetic program of transcriptional events involving the
regulated expression of numerous genes, including those encoding
cytokines, cytokine receptors, and other immunoregulatory molecules
(2, 3). Analysis of the cis-acting regulatory
elements of many of these genes, including IL-2, IFN-, IL-3, IL-4,
IL-5, IL-8, IL-13, TNF-
, GM-CSF, CD25, CD40 ligand
(CD40L),4 and Fas
ligand (FasL), has revealed the presence of numerous functional binding
sites for the NFAT family of transcription factors (3, 4).
Because this family of transcription factors is known to be highly
inducible in response to TCR stimulation (3, 4, 5), NFAT
family members are believed to play a pivotal role in the T cell
activation-induced transcriptional response during Th cell
differentiation. NFAT proteins are regulated primarily at the level of their subcellular localization by a TCR-dependent signaling pathway involving the calcium/calmodulin-dependent phosphatase, calcineurin (3, 4, 5). NFAT family members are normally located in the cytoplasm of resting cells in a hyperphosphorylated latent form, but, following TCR stimulation and the ensuing increase in the intracellular calcium concentration ([Ca2+]i), they are dephosphorylated by activated calcineurin, triggering their rapid nuclear import and causing an increase in their intrinsic DNA binding activity (3, 4, 5). This calcineurin-mediated activation pathway is strongly opposed by a number of specific protein kinases, which act to directly rephosphorylate NFAT proteins, resulting in a decrease in their intrinsic DNA binding activity and their rapid nuclear export, thereby attenuating NFAT-dependent transcription (6, 7, 8, 9, 10, 11, 64). As a result of the opposing influences of calcineurin and the regulatory NFAT kinases, NFAT activity is readily reversible, highly dynamic, and extremely sensitive to changes in [Ca2+]i (6, 7, 12). This aspect of NFAT regulation is likely to be of particular significance, because the extent and duration of NFAT activation have recently been shown to be important parameters that can significantly affect the qualitative pattern of gene transcription induced during T cell activation (13).
The calcium-regulated NFAT family of transcription factors is comprised
of four known members: NFATc1 (NFATc/NFAT2), NFATc2 (NFATp/NFAT1),
NFATc3 (NFATx/NFAT4), and NFATc4 (NFAT3) (14, 15, 16, 17, 18). To gain
insights into the in vivo functional roles of the NFAT family, several
groups have generated mice deficient in one or more of these NFAT
genes. While these studies have revealed prominent functions for NFATc1
in cardiac valve morphogenesis (19, 20) and for NFATc2 in
the regulation of chondrogenesis (21), the precise
functions of NFAT family members in the immune system are less clear.
Due to embryonic lethality of the NFATc1 knockout, the role of this
protein in the immune system has been analyzed by the generation of
chimeric mice using the RAG-/- blastocyst
complementation system (22, 23).
NFATc1-/- chimeric mice exhibit a decrease in
thymocyte repopulation and impaired peripheral T cell proliferation. In
addition, both splenocytes and lymph node cells from these mice have
been shown to produce diminished levels of IL-4, but essentially normal
levels of IL-2 and IFN-, in response to anti-CD3 mAb
stimulation. These latter results suggest that NFATc1 is
specifically involved in the regulation of Th2 cytokines. Mice
deficient in NFATc2 develop age-related splenomegaly and accumulation
of T cells with an activated phenotype (24, 25).
NFATc2-deficient T cells exhibit a normal primary proliferative
response but a greatly enhanced secondary response (24, 25). The absence of NFATc2 does not appear to have a significant
effect on the production of either IL-2 or IFN-, although the
production of IL-4 is markedly enhanced in secondary immune
responses (25, 26). NFATc3 is primarily expressed in
thymocytes and, correspondingly, NFATc3-deficient mice exhibit impaired
thymocyte maturation (27). NFATc3-deficient peripheral T
cells exhibit an activated phenotype but produce normal levels of
activation-induced cytokines (27).
Overall, the mild immunological phenotypes of the individual NFAT
knockout mice suggest a considerable degree of redundancy within the
NFAT family. This notion is supported by the much more severe
phenotypes of the NFAT double knockout mice. Thus,
NFATc2/NFATc3-/- mice exhibit a severe
lymphoproliferative disorder, and T cells from these mice are
hyperactive and produce markedly enhanced levels of Th2 cytokines
following primary stimulation (28). The enhanced cytokine
production and hyperactive state of T cells from
NFATc2-/-, NFATc3-/-,
and NFATc2/NFATc3-/- mice has led to the
surprising notion that NFATc2 and NFATc3 might play inhibitory roles in
the regulation of the immune response. Most interestingly,
NFATc1/NFATc2 doubly deficient T cells were found to be profoundly
impaired in their production of a large panel of cytokines including
IL-2, IL-4, IFN-, IL-10, GM-CSF, and TNF- (29),
indicating that the activities of both NFATc1 and NFATc2 are essential,
but largely redundant, with respect to activation-induced cytokine gene
expression. Based upon these observations, while it is clear that NFAT
proteins play a crucial role in the regulated transcription of T cell
effector cytokines, there does not appear to be a simple causal
relationship between an individual NFAT family member and the
expression of a specific cytokine gene(s). Accordingly, the precise
role of each individual NFAT family member and the contribution of
spatiotemporally distinct patterns of NFAT signaling to the
differential expression of cytokine genes during T cell differentiation
remain to be fully established.
In this study, we have used retroviral-mediated gene transfer to
introduce a constitutively active mutant form of NFATc1 (caNFATc1) into
primary murine CD4+ T cells to examine the
effects of sustained NFAT activity on the regulation of
CD4+ T cell gene expression. Our findings
indicate that sustained NFAT signaling caused by the ectopic expression
of NFATc1 enhances a number of cellular traits that are preferentially
associated with Th1 effector cells, including a selective increase in
the number of cells expressing the prototypical Th1 cytokine IFN-
(1), increased expression of functional P-selectin
glycoprotein ligand-1 (PSGL-1) (30), enhanced FasL
expression, and increased susceptibility to activation-induced cell
death (AICD) (31, 32, 33). We also find that ectopic
expression of caNFATc1 in developing Th2 cells decreases expression of
IL-4 and promotes the expression of IFN-. Collectively, these
results suggest that sustained NFAT signaling, mediated by ectopic
expression of caNFATc1, acts to promote a Th1-like pattern of gene
expression in primary murine CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-week-old female C57BL/6J and BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and DO11.10 TCR-transgenic x Rag-1-/- BALB/c mice were obtained from Dr. T. Barrett (Northwestern University Medical School, Chicago, IL). The mice were maintained in specific pathogen-free facilities at Northwestern University Medical School in accordance with Northwestern University animal care guidelines.
Cell culture
Phoenix-Eco packaging cells and primary T cells were
maintained at 37°C in 7.5% CO2 in DMEM (Life
Technologies, Rockville, MD) supplemented with 10% (v/v) FBS, 100 U/ml
penicillin G, 100 µg/ml streptomycin, 1% (v/v) nonessential amino
acids, 2 mM L-glutamine, 10 mM HEPES buffer, and 50 µM
2-ME. Recombinant murine IL-2, IL-4, and IFN- cytokines were
purchased from PeproTech (Rocky Hill, NJ). Anti-CD3 mAb (145-2C11),
anti-CD28 mAb (clone 37-51), anti-mouse IL-12 mAb
(C17.8), and anti-mouse IFN- mAb (XMG1.2) were all purchased
from BD PharMingen (San Diego, CA), and anti-mouse IL-4 mAb was
purified from hybridoma clone 11B11 tissue culture supernatants.
Retroviral plasmid constructs
The retroviral expression vector pMSCV-GFP (34) was
supplied by Dr. L. Van Parijs (California Institute of Technology,
Pasadena, CA). To generate pMSCV-caNFATc1-GFP, an N-terminal
hemagglutinin-tagged, C-terminal FLAG-tagged constitutively active
version of NFATc1 containing serine to alanine substitutions in the
conserved serine-rich domain and all three serine-proline repeats
(NFATc-mSRD-mSPx3; see Ref. 11) was inserted into
pMSCV-GFP downstream of the murine stem cell virus (MSCV) long terminal
repeat (LTR). The caNFATc1 cDNA used in this study is derived from the
716-aa human NFATc isoform originally isolated by Northrop et al.
(15). The pMSCV-H-2Kk retroviral
expression vector was created by replacing green fluorescent protein
(GFP) in the pMSCV-GFP retroviral expression vector with a
PCR-amplified truncated murine H-2Kk cDNA from
the pMACS Kk.II plasmid (Miltenyi Biotec, Auburn, CA).
pMSCV-caNFATc1-H-2Kk was created by introducing
the constitutively active form of NFATc1 into
pMSCV-H-2Kk.
Purification and activation of primary murine CD4+ T cells
Splenocytes harvested from 6- to 10-wk-old mice and
CD4+ T cells were purified to >97% purity using
MACSelect CD4+ MicroBeads (Miltenyi Biotec),
exactly according to the manufacturer’s instructions. Purified
CD4+ T cells were activated in vitro in 24-well
plates (2 x 106 cells/well) coated with
anti-CD3 mAb (5 µg/ml) and anti-CD28 mAb (5 µg/ml) in
medium supplemented with 10 ng/ml murine (m)IL-2. For
Th1-polarizing conditions 5 ng/ml mIL-12 and 10 µg/ml anti-IL-4
mAb were added to the cultures, whereas 50 ng/ml mIL-4, 5 µg/ml
anti-IL-12 mAb, and 5 µg/ml anti-IFN- mAb were added for
Th2-polarizing conditions. Cells were removed from stimulation on the
third day and expanded in medium containing IL-2 for a total of 7 days.
Where indicated, cells were maintained in Th1- or Th2-polarizing
conditions for the entire 7-day culture period.
Retroviral production and infections
The Phoenix-Eco ecotropic packaging cell line provided by Dr. G. Nolan (Stanford University, Palo Alto, CA) (35) was transfected with the appropriate retroviral construct using Lipofectamine PLUS (Life Technologies). Viral supernatants were collected at 48 h and stored at -80°C. Activated CD4+ T cells were spin-infected at 24 and 48 h postactivation by centrifugation at 2000 rpm for 1.5 h at room temperature with 1.5 ml viral supernatant containing 6 µg/ml polybrene (Sigma-Aldrich., St. Louis, MO) and 10 µg/ml mIL-2. Where indicated, cells were infected in the presence of the appropriate Th1/Th2-skewing conditions. The viral supernatant was removed after the spin infection and replaced with medium containing 10 µg/ml mIL-2 and the appropriate cytokines and Abs as required.
Flow cytometric analysis
On day 7 after infection, cells were incubated with either medium alone or medium containing 12.5 ng/ml PMA plus 1 µM ionomycin as indicated. After 8 h of incubation, the cells were stained with either fluorochrome-conjugated specific mAbs or appropriate isotype control Abs and analyzed by flow cytometry. Anti-CD25 mAb (7D4), anti-CD154 mAb (MR1), anti-CD62L mAb (Mel-14), anti-CD45RB mAb (16A), anti-FasL (MFL3), and anti-H-2Kk mAb (36-7-5), together with appropriate isotype control Abs, were all purchased from BD PharMingen, while anti-mouse H-2Kk Ab (H100-27.R55) was purchased from Miltenyi Biotec. P-selectin-Ig fusion protein and control CD45-Ig fusion protein were provided by Dr. G. Kansas (Northwestern University, Chicago, IL) and Cy5-conjugated anti-human IgM was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences, Palo Alto, CA).
Intracellular cytokine assays and ELISA
CD4+ T cells were plated at 3 x
106 cells/well in a 24-well plate and
restimulated with medium alone, 12.5 ng/ml PMA (Calbiochem, La Jolla,
CA), 12.5 ng/ml PMA plus 1 µM ionomycin (Calbiochem), or 12.5 ng/ml
PMA plus 1 µM ionomycin plus 10 µg/ml FK506 as indicated for 6
h at 37°C, with the addition of 15 µg/ml brefeldin A
(Sigma-Aldrich) for the last 4 h. After fixation in 2%
paraformaldehyde, cells were permeabilized by incubating twice in
permeabilization buffer (PBS plus 0.5% BSA, 1 mM sodium azide, and
0.5% saponin) for 15 min at room temperature. The relevant
anti-cytokine or isotype control Abs were added at 0.5 µg/1
x 106 cells in permeabilization buffer and
incubated for 30 min at room temperature. Cells were then washed twice
in permeabilization buffer and twice in wash buffer (PBS plus 0.5% BSA
and 1 mM sodium azide), and then analyzed by flow cytometry.
PE-conjugated anti-IL-4 (BVD4-1D11), allophycocyanin-conjugated
anti-IFN- (XMG1.2), PE-conjugated anti-IL-2 (JES6-5H4),
along with the isotype control PE-conjugated rat IgG2b (A95–1) and
allophycocyanin-conjugated rat IgG1 (R3-34), were all purchased from BD
PharMingen.
For ELISA, cells transduced with either the
MSCV-H-2Kk or
MSCV-caNFATc1-H-2Kk virus were purified using
MACSelect Kk MicroBeads (Miltenyi Biotec),
washed, and restimulated (1.5 x 106
cells/ml) with 12.5 ng/ml PMA plus 1 µM ionomycin in a 24-well plate.
After 24 h the presence of IFN- and IL-4 in the culture
supernatants was determined using IFN- (KM-IFNG) and IL-4 (KM-IL4)
ELISA reagents from Endogen (Cambridge, MA). Supernatants were serially
diluted and the concentration of cytokine was determined in relation to
a reference standard using protocols provided by the manufacturer.
Analysis of AICD
Cells transduced with either the MSCV-H-2Kk or MSCV-caNFATc1-H-2Kk virus were collected on day 7 after infection and H-2Kk-positive cells were purified using MACSelect Kk MicroBeads (Miltenyi Biotec). Cells were plated at 3 x 106 cells/well in a 24-well plate and incubated with medium alone or medium containing 12.5 ng/ml PMA plus 1 µM ionomycin for 24 or 48 h, at which point TUNEL assays were performed using a Fluorescein In Situ Cell Death Detection kit (Roche, Indianapolis, IN) exactly according to the manufacturer’s protocol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the functional effects of sustained NFAT signaling
on T cell gene expression, we have taken advantage of a
calcineurin-independent, constitutively active mutant version of NFATc1
(caNFATc1; NFATc-mSRD-mSPx3 in Ref. 11) that is known to
be constitutively localized to the nucleus and capable of binding DNA
with high affinity (11, 36). A retroviral vector encoding
caNFATc1 was generated by introducing the caNFATc1 mutant downstream of
the MSCV LTR and upstream of an internal ribosomal entry sequence-GFP
cassette (Fig. 1
), thereby allowing
expression of both caNFATc1 and GFP from a single bicistronic mRNA. A
modified form of this retroviral vector was also constructed by
substituting a truncated form of the murine MHC class I
H-2Kk cDNA for GFP, allowing us to track viral
transduction by the surface expression of H-2Kk
and to rapidly purify transduced cells using
anti-H-2Kk paramagnetic beads. Similar
results were obtained using either viral construct. Throughout our
studies, to control for any effects of viral infection itself,
comparisons were always made between cells infected with the caNFATc1
virus and a control virus lacking caNFATc1 coding sequences.
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To assess the initial effects of caNFATc1 on
CD4+ T cells, we first analyzed the expression of
several activation markers whose expression levels are known to change
following T cell activation. As shown in Fig. 2A, CD25 is expressed at low
levels on cells infected with the control virus and is up-regulated
following restimulation with PMA and ionomycin. In contrast, cells
transduced with the caNFATc1 virus and left unstimulated exhibited a
high level of CD25 expression that was comparable to that observed in
stimulated control cells (Fig. 2A). This result indicates
that ectopic expression of caNFATc1 is sufficient to drive high-level
expression of CD25 in the absence of other overt signals and is likely
to be mediated via the direct action of caNFATc1 on the transcription
of the CD25 gene, as functional NFAT binding sites have been
identified in the CD25 promoter (37). In addition to CD25,
caNFATc1 also enhanced the expression of CD40L, although in this case
the level of CD40L expression was further increased following
stimulation with PMA and ionomycin, indicating that, while
caNFATc1 can partially induce the up-regulation of CD40L,
other additional signaling pathways are necessary for maximal
expression. The enhancing effect of caNFATc1 on the expression of CD40L
is consistent with the presence of multiple NFAT binding sites within
the CD40L promoter region (38).
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C, CD62L is expressed
on a substantial number of nonstimulated control virus-transduced cells
and is down-regulated following stimulation with PMA and ionomycin. By
comparison, in the absence of stimulation, a significant number of
cells transduced with the caNFATc1 virus appear to have lost expression
of CD62L (Fig. 2C). Hence it appears that caNFATc1 is able
to partially mimic T cell activation signals resulting in the
down-regulation of CD62L, although the underlying mechanism responsible
for this effect is not known. Taken together, these data indicate that
the caNFATc1 mutant construct is transcriptionally active and capable
of mimicking T cell activation signals to affect the expression levels
of endogenous T cell activation marker genes. Ectopic expression of caNFATc1 synergizes with PMA to induce expression of IL-2 in CD4+ T cells
Because NFAT was originally implicated in the transcriptional
regulation of the IL-2 gene (3, 4, 5), we next
examined the effects of caNFATc1 on IL-2 gene expression. In
control virus-transduced cells, expression of IL-2 was undetectable in
either nonstimulated or PMA-treated cells but was expressed at high
levels following stimulation with both PMA and ionomycin, an effect
that was blocked by the addition of FK506 (Fig. 3). In contrast, PMA treatment alone was
sufficient to induce the expression of IL-2 in
caNFATc1-expressing cells, albeit in only a subset of the
population (19%). As expected, the majority of these cells produced
IL-2 following stimulation with both ionomycin and PMA. Notably,
expression of caNFATc1 was able to render cells partially resistant to
the inhibitory effects of FK506. These results indicate that by
bypassing the requirement for calcineurin-mediated activation caNFATc1
can synergize with PMA to increase the expression of the endogenous
IL-2 gene, although it is apparent that optimal
cellular expression of IL-2 requires additional FK506-sensitive,
calcium-dependent signaling events.
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Having demonstrated an effect of caNFATc1 on both T cell
activation markers and IL-2 production, we next wanted to test the
effects of caNFATc1 on production of cytokines associated with Th cell
subsets, namely IL-4 and IFN-. For these experiments
CD4+ T cells purified from C57BL/6 mice were
transduced with either the control or caNFATc1 retrovirus and expanded
under neutral nonskewing cytokine conditions, so as not to favor
differentiation toward either the Th1 or Th2 phenotype. As shown in
Fig. 4A, following
restimulation with ionomycin and PMA, T cells transduced with the
control virus were found to predominantly produce IL-4 (39% of cells),
with relatively few cells producing IFN- (10% of cells). Similar
results were obtained when nontransduced cells were analyzed (data not
shown). However, a striking difference in cytokine profile was observed
when T cells expressing caNFATc1 were analyzed. In this case, a marked
increase in the percentage of cells producing IFN- was detected (60
vs 10%; Fig. 4A). In fact, over the course of multiple
experiments we observed that ectopic expression of caNFATc1 resulted in
an average 4.8-fold increase (n = 8) in the percentage
of cells expressing IFN-. Similar results to those described above
were obtained when CD4+ T cells from BALB/c mice
were used (Fig. 4B), indicating that the selective
effect of caNFATc1 on production of IFN- is not dependent
on genetic background. In contrast to its marked effect on
the production of IFN-, expression of caNFATc1 appeared to
have little effect on IL-4 production under these conditions, even
though the IL-4 promoter is known to contain several functional NFAT
binding sites (41, 42). However, it is interesting to note
that, although the total percentage of IL-4 producers in cells
expressing caNFATc1 changed very little, the percentage of cells
expressing both IFN- and IL-4 was greatly increased, with a
corresponding reduction in the number of cells producing only IL-4
(Fig. 4, A and B).
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(data not shown). Because so
many of our activated T cells produced IL-4 upon restimulation, we were
concerned that this might obscure a potential effect of caNFATc1 on
IL-4 production. To address this issue, we repeated the experiment with
CD4+ T cells purified from mice expressing the
DO11.10 TCR transgene on the Rag-1-deficient background, as these cells
were likely to represent a relatively pure population of naive T cells
devoid of memory T cells. When control virus-transduced DO11.10 x
Rag-1-/- CD4+ T cells
were restimulated, they produced predominantly IFN- with essentially
no evidence of IL-4 production (Fig. 4C). Similar to our
previous results, retroviral-mediated expression of caNFATc1 resulted
in a significant increase in the number of IFN--expressing cells but
again had no effect on the expression of IL-4. Hence, it appears
that ectopic expression of caNFATc1 in CD4+ T
cells maintained under neutral conditions selectively enhances
expression of IFN- and has little apparent effect on the production
of IL-4.
caNFATc1 enhances the expression of IFN- in
developing Th1 cells
The effects of caNFATc1 on IFN- expression in
CD4+ T cells maintained under neutral conditions
prompted us to test the effects of caNFATc1 on cytokine production in
cells specifically skewed toward either the Th1 or Th2 phenotype. To
accomplish this, CD4+ T cells activated in the
presence of either Th1- or Th2-polarizing conditions were transduced
with either the control or caNFATc1 retrovirus and then maintained
under the appropriate skewing conditions. As expected, when control
virus-transduced cells maintained under Th1-skewing conditions were
restimulated, a large percentage of the cells were found to produce
IFN- (47%), with <1% of cells exhibiting production of IL-4,
clearly indicating the efficiency of our Th1-skewing conditions (Fig. 5A). Consistent with our
previous results, analysis of cells transduced with the caNFATc1 virus
revealed a pronounced increase in the percentage of IFN--expressing
cells (73%) compared with control cells (47%). In fact, not only did
we observe an increase in the total percentage of IFN--expressing
cells, but we also observed a noticeable increase in the level of
IFN- expression produced per cell, as measured by an increase in the
mean fluorescence intensity (514 vs 324). Importantly, expression of
caNFATc1 had no effect on IL-4 production in these cells.
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in developing Th2 cells
Next, we examined the effects of caNFATc1 on cytokine expression
in cells maintained under Th2-skewing conditions. As shown in Fig. 5B, stimulation of control virus-transduced cells expanded
under Th2-inducing conditions resulted in a population of cells
exclusively producing IL-4, clearly demonstrating the efficiency of our
Th2-skewing conditions. However, in marked contrast,
CD4+ T cells that were transduced with the
caNFATc1 virus under Th2-polarizing conditions did not exhibit the
expected Th2-like phenotype. Rather, we observed that following
restimulation 25% of the cells expressed IFN-, with 15% of these
cells producing solely IFN- and 10% producing both IFN- and
IL-4. Remarkably, in addition to the enhancing effects of caNFATc1 on
IFN- expression, we also observed an inhibitory effect of caNFATc1
on IL-4 expression, as the percentage of IL-4 producers in cells
transduced with the caNFATc1 virus (34%) was decreased compared with
the percentage of IL-4 producers observed in control cells (59%).
These differential effects of caNFATc1 on the expression of IL-4 and
IFN- in developing Th2 cells were confirmed by ELISA (Fig. 5C). Importantly, the effects of NFATc1 appear to be cell
autonomous, because simultaneous analysis of the nontransduced cell
population from the same culture indicate that they exclusively produce
IL-4 at levels comparable to control cells (Fig. 5B). This
latter result is especially important, because it indicates that the
cultures were efficiently stimulated and that the diminished IL-4
production in the caNFATc1-transduced cells is a direct result of the
intrinsic activity of caNFATc1.
Expression of caNFATc1 enhances the expression of functional PSGL-1 and induces high level expression of CD45RB
The selective effects of caNFATc1 on the expression of IFN- and
the down-regulation of IL-4 production in Th2 cells suggested that
expression of caNFATc1 might favor a Th1-like pattern of gene
expression. To investigate this issue further we next analyzed the
effects of caNFATc1 on the expression of cell surface markers that have
been associated with the Th1 phenotype. While it has been difficult to
identify cell surface molecules that absolutely define either Th1 or
Th2 cells, a functional form of PSGL-1 has been shown to be
preferentially expressed on activated Th1 cells (30).
Using a P-selectin recombinant Ig fusion protein as a probe, we were
able to assay cells for their expression of functional PSGL-1. As shown
in Fig. 6A, cells transduced
with the caNFATc1 virus exhibited significantly higher levels of
expression of functional PSGL-1 following restimulation when compared
with control virus-transduced cells.
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but little IL-4, have been implicated
as effectors in a proinflammatory autoimmune disease, and have been
shown to protect BALB/c mice from infection with
Leishmania major (43, 44, 45). As
shown in Fig. 6B, CD4+ T cells
transduced with the caNFATc1 virus expressed significantly higher
levels of the CD45RB marker compared with cells transduced with the
control virus (
4-fold). Hence, sustained NFAT signaling mediated by
ectopic expression of caNFATc1 acts to enhance the expression of cell
surface markers that appear to be preferentially associated with cells
exhibiting a Th1 phenotype. Expression of caNFATc1 leads to increased expression of FasL and enhanced AICD
Peripheral T cells are known to undergo AICD in response to
repeated antigenic stimulation by a signaling pathway involving Fas and
FasL (46). Moreover, increased expression of FasL and
enhanced susceptibility to AICD are cellular properties that have
previously been shown to be preferentially associated with Th1 effector
cells (31, 32, 33). Because the NFAT family of transcription
factors has been implicated in the T cell activation-inducible
expression of FasL (47), we decided to investigate the
effects of caNFATc1 on the expression of FasL and on AICD. As shown in
Fig. 7A, T cells transduced
with the caNFATc1 virus exhibited higher levels of FasL expression
under either resting or stimulated conditions when compared with cells
transduced with the control virus. This enhancing effect of caNFATc1 on
FasL expression is consistent with the presence of functional NFAT
binding sites within the FasL promoter (47), suggesting a
direct transcriptional effect of caNFATc1 on FasL expression.
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B). Similar results
were obtained when apoptosis was verified by analysis of subgenomic
cellular DNA content (data not shown). Thus, caNFATc1 acts to enhance
expression of FasL and increase susceptibility to AICD, cellular traits
that have previously been associated with Th1 effector cells
(31, 32, 33). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While we found that ectopic expression of caNFATc1 clearly affected the
expression of the T cell activation markers CD25, CD40L, and CD62L, and
was able to synergize with PMA to induce expression of IL-2, our most
striking finding was the selective enhancing effect of caNFATc1 on the
expression of the prototypical Th1 cytokine, IFN-. In this regard,
we found that when CD4+ T cells were grown under
neutral conditions that did not favor either Th1 or Th2 development,
ectopic expression of caNFATc1 resulted in a substantial selective
increase in the percentage of cells producing IFN-. Interestingly,
under these conditions, we observed little, if any, effect of caNFATc1
on IL-4 production. This was surprising because NFAT has been shown to
regulate the activity of the IL-4 promoter in transiently transfected
cells, and T cells from NFATc1-deficient mice have been reported to
produce diminished amounts of IL-4 in response to anti-CD3 mAb
stimulation (22, 23, 48). However, while we did not see an
effect of caNFATc1 on the percentage of IL-4-producing cells per se, we
did observe an increase in the percentage of cells producing both
IFN- and IL-4, together with a concomitant reduction in cells
producing only IL-4, suggesting that the expression of caNFATc1 is
sufficient to induce IFN- expression in cells producing IL-4. These
double cytokine-producing cells are suggestive of Th0 cells, which have
been reported to be preferentially generated by repetitive rounds of
antigenic stimulation (49). Hence, it is possible that
expression of caNFATc1 mimics repeated T cell stimulation, thereby
favoring the generation of Th0 cells under neutral nonpolarizing
cytokine conditions.
Because we had found that ectopic expression of caNFATc1 was able to
selectively increase the percentage of IFN--producing cells under
neutral cytokine conditions, we were naturally interested in examining
the effects of caNFATc1 on IFN- and IL-4 expression in cells induced
to differentiate toward either the Th1 or Th2 phenotype. As might have
been expected from our previous data, cells expressing caNFATc1 that
were maintained under strong Th1-inducing conditions exhibited
increased numbers of cells producing IFN- compared with controls
(Fig. 5A). This effect of caNFATc1 on the expression of
IFN- is consistent with the presence of NFAT binding sites within
the cis-acting regulatory elements of the IFN-
gene and the ability of NFATc1 to transactivate an IFN- promoter
reporter construct in a transient transfection assay (50, 51). However, despite the presence of multiple functional NFAT
binding sites in the IL-4 promoter (41, 42), ectopic
expression of caNFATc1 in developing Th2 cells did not enhance the
production of IL-4. Quite to the contrary, expression of caNFATc1 in
cells skewed toward the Th2 phenotype actually diminished the
percentage of cells expressing IL-4 and unexpectedly promoted the
expression of IFN- (Fig. 5B). These effects of caNFATc1
in developing Th2 cells are all the more remarkable because it is well
established that under normal Th2-skewing conditions IL-4 acts to
strongly up-regulate expression of the endogenous IL-4 gene
and potently suppress the expression of IFN- (1). In
addition to its effects on cytokine gene expression, we also found that
ectopic expression of caNFATc1 enhanced the expression of a number of
cell surface molecules and cellular traits that appear to be
preferentially associated with Th1 effector cells, including enhanced
expression of functional PSGL-1 (30), high-level
expression of CD45RB (43, 44, 45), increased FasL expression,
and enhanced susceptibility to AICD (31, 32, 33). Taken
together, these data suggest that ectopic expression of caNFATc1
appears to preferentially favor a Th1-like pattern of gene
expression.
Interestingly, our findings regarding the effects of caNFATc1 on the
expression of IFN- and IL-4 appear to conflict with the previous
analysis of mice deficient in NFATc1. In this regard, NFATc1-deficient
splenocytes or lymph node cells have been reported to produce
diminished levels of IL-4 in response to mitogenic stimulation, which
has been taken as evidence that NFATc1 is specifically involved
in the regulation of Th2 cytokines (22, 23). However, our
finding that ectopic expression of caNFATc1 acts to enhance expression
of the prototypical Th1 cytokine IFN-, yet inhibit expression of the
Th2 cytokine IL-4, suggests that NFATc1 per se is not likely to be
uniquely associated with the regulation of Th2 cytokines. Indeed,
recent analysis of NFATc1/NFATc2 doubly deficient T cells
(29) demonstrating a profound defect in cytokine
production relative to T cells deficient in either NFATc1 or NFATc2
alone (22, 23, 24, 25) suggests that there is in fact likely to be
a considerable degree of overlap between the specific cytokine target
genes of both NFATc1 and NFATc2. This notion is further supported by
experiments indicating that both NFATc1 and NFATc2 exhibit very similar
in vitro DNA binding specificities (4, 16, 17).
Accordingly, it is important to note that the effects on gene
expression that we observe with caNFATc1 may not necessarily be
specific to this NFAT family member per se, but rather may merely
reflect a general property of the sustained activity of the NFAT
signaling pathway caused by the ectopic expression of a constitutively
active NFAT mutant.
The Th1/Th2 cell fate decision is regulated by a number of different factors, including the cytokine environment, level of costimulation, and the degree of TCR stimulation (1, 52). In this latter case, high doses of Ag or antigenic peptides that exhibit high affinity for the TCR appear to favor the development of Th1 cells, whereas low doses of Ag or Ag with low affinity for the TCR have been shown to preferentially induce Th2 differentiation (52, 53, 54). These different types of antigenic stimulation are known to induce different degrees of TCR signaling that translate into the generation of distinct patterns of intracellular biochemical second messengers. Specifically, Ags with different affinities for the TCR are known to elicit distinct changes in the [Ca2+]i (54, 55): high-affinity Ags induce sustained/high-frequency calcium oscillations, whereas low-affinity Ags induce transient/low-frequency calcium oscillations. In fact, recent studies have demonstrated that the quantitative nature of the calcium signal can indeed influence the Th1/Th2 cell fate decision, with a strong increase in [Ca2+]i appearing to preferentially favor the development of Th1 cells (54, 56).
Given the exquisite calcium sensitivity of the NFAT family of transcription factors and their ability to respond to quantitatively distinct changes in [Ca2+]i in a highly stimulus-specific fashion (12), NFAT proteins are perfectly poised to translate quantitatively distinct Ag-induced changes in [Ca2+]i into specific transcriptional responses capable of influencing the outcome of Th cell differentiation. In fact, evidence to support a role for quantitative differences in the calcineurin/NFAT signaling pathway regulating the Th1/Th2 cell fate decision has been provided by the observation that partial inhibition of calcineurin, the direct upstream activator of NFAT proteins, can redirect Th1-skewed cells toward the Th2 phenotype (56). In this case, reduced signaling through the calcineurin/NFAT signaling pathway appears to favor a Th2 pattern of differentiation. Conversely, our current data demonstrate that increased and sustained signaling through the NFAT signaling pathway caused by ectopic expression of caNFATc1 appears to favor a Th1-like pattern of gene expression. In this instance we believe that, as a result of its constitutively active state, ectopic expression of caNFATc1 is able to phenocopy the effects of sustained signaling through the calcium/calcineurin signaling pathway, and just like a strong calcium signal (54, 56), is able to induce a transcriptional response that preferentially favors a Th1-like pattern of gene expression. Taken together with recent results demonstrating that the extent and duration of NFAT activation can significantly affect the qualitative pattern of gene transcription induced during T cell activation (13), these two complementary sets of data suggest that the degree of signaling through the calcineurin/NFAT signaling pathway is likely to play an important role in determining the differential pattern of cytokine gene expression induced during Th cell differentiation.
What are likely to be the underlying molecular mechanisms that account
for the effects of caNFATc1 on gene expression? While the differential
expression of IFN- and IL-4 is known to be influenced by a variety
of cytokines (1), it is clear from our analysis of
caNFATc1-transduced and nontransduced cell populations that the effects
of caNFATc1 are cell autonomous and are therefore unlikely to involve
the production of a secreted paracrine factor. Hence, caNFATc1 is
likely to act in a cell intrinsic fashion to directly influence
cytokine gene expression. Because the cis-acting regulatory
elements of both IFN- and IL-4 are known to
contain functional NFAT binding sites (41, 42, 50, 51),
the most straightforward model to explain the differential effects of
caNFATc1 on the expression of IFN- and IL-4 involves the direct
action of caNFATc1 on the regulatory elements of these genes. While a
direct transcriptional effect of caNFATc1 could easily account for the
greatly enhanced expression of IFN-, it does not readily
explain the inhibitory effect of caNFATc1 on IL-4 expression
observed in developing Th2 cells. One possibility is that while
caNFATc1 might act as a positively acting transcription
factor at the IFN- locus by potentially promoting
chromatin remodeling of the locus and directly increasing
IFN- transcription, it may instead act as a
context-specific transcriptional repressor on the IL-4 promoter in
developing Th2 cells. Although no such repressive role for NFATc1 has
been described, it is interesting to note that another NFAT family
member, NFATc2, is believed to exhibit inhibitory activity and can
apparently inhibit the expression of IL-4 in vivo (26).
Whether, like NFATc2, NFATc1 can in some cases either down-regulate or
directly repress transcription of certain genes remains to be
determined. An alternative explanation for the inhibitory effect of
caNFATc1 on IL-4 production in developing Th2 cells is that ectopic
expression of caNFATc1 might interfere with the IL-4-dependent
signaling pathway responsible for inducing IL-4 gene
expression during Th2 differentiation. In this regard, IL-4 expression
is known to require the actions of both the GATA-3 and c-maf
transcription factors (57, 58), while in addition GATA-3
has been shown to play a role in repressing the expression of IFN-
in developing Th2 cells (59). Because NFAT proteins have
been reported to interact with c-maf (60) and NFATc1 is
known to specifically interact with another GATA factor, GATA-2 in
skeletal muscle (61), it is possible that caNFATc1
acts by directly antagonizing the activities of c-maf and GATA-3,
thereby blocking the induction of IL-4 expression and preventing the
repression of IFN- expression in developing Th2 cells. Finally, it
is possible that ectopic expression of caNFATc1 mediates its effects
via an indirect mechanism by inducing the expression of a target
gene(s) that in turn is either capable of differentially affecting the
expression of IFN- and IL-4, or is able to directly influence the
Th1/Th2 cell fate decision. In this case, a potential candidate gene is
T-bet, a recently described TCR-induced master regulatory
transcription factor gene implicated in the induction of Th1 cell
differentiation (62). Ectopic expression of T-bet has been
shown to selectively increase the number of cells expressing IFN- by
inducing chromatin remodeling of the IFN- locus
(62, 63). Moreover, expression of T-bet in Th2 cells has
been reported to redirect them toward the Th1 phenotype by inhibiting
production of IL-4, inducing IFN- gene expression, and
enhancing expression of the Th1 cell surface marker, PSGL-1
(62). While the phenotype induced by ectopic expression of
T-bet is strikingly similar to that induced by caNFATc1, it remains to
be determined whether caNFATc1 acts by up-regulating expression of
T-bet, or for that matter some other Th1-inducing gene product. Because
at present we are unable to exclude any of these non-mutually exclusive
possibilities, future experiments will be specifically designed to
determine the molecular mechanism(s) by which caNFATc1 differentially
affects cytokine gene expression in murine CD4+ T
cells.
In summary, we have shown that sustained activation of the NFAT signaling pathway caused by ectopic expression of caNFATc1 has a pronounced effect on gene expression in primary murine CD4+ T cells, where it appears to favor the induction of a Th1-like pattern of gene expression. These findings are consistent with recent results demonstrating the profound effect of the degree of NFAT signaling on the pattern of transcriptional events induced during T cell activation (13), and serve to underscore the potentially important relationship between the quantitative extent of NFAT signaling and the qualitative pattern of gene expression induced during Th cell differentiation.
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Acknowledgments |
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Footnotes |
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2 Current address: Laboratory of Immunology and Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852.
3 Address correspondence and reprint requests to Dr. Neil A. Clipstone, Department of Microbiology-Immunology, Room Tarry 6-701, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: n-clipstone{at}northwestern.edu
4 Abbreviations used in this paper: CD40L, CD40 ligand; FasL, Fas ligand; [Ca2+]i, intracellular calcium concentration; caNFATc1, constitutively active NFATc1; m, murine; AICD, activation-induced cell death; PSGL-1, P-selectin glycoprotein ligand-1; LTR, long terminal repeat; MSCV, murine stem cell virus; GFP, green fluorescent protein.
Received for publication September 20, 2001. Accepted for publication March 12, 2002.
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