(Journal of Leukocyte Biology. 2001;69:1053-1059.)
© 2001
by Society for Leukocyte Biology
Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER)
Josef Bodor*,
Lionel Feigenbaum
,
Jana Bodorova
,
Cathy Bare*,
Marvin S. Reitz, Jr
and
Ronald E. Gress*
* Experimental Immunology Branch, Division of Basic Sciences, and
Transplantation Therapy Section, Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
Transgenic Mouse Model, Science Application International Corporation, National Cancer Institute Frederick Cancer Research and Development Center, Frederick, Maryland 21702; and
Institute of Human Virology, University of Maryland, Baltimore, Maryland 21201
Correspondence: Dr. Ronald E. Gress, National Cancer Institute, Experimental Immunology Branch, Bldg. 10, Rm. 4B14, 10 Center Dr., Bethesda, MD 20892-1360. E-mail:
gressr{at}dc10a.nci.nih.gov
 |
ABSTRACT
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Depending on the nature of the costimulation of T lymphocytes,
expression of regulatory cytokines and chemokines is either susceptible
or resistant to cyclic AMP (cAMP)-mediated inhibition. Our data show
that cAMP-mediated inhibition of endogenously expressed cytokines,
which is characteristic for T helper (Th) 1- and Th 2-like phenotypes,
correlates with the induction of a potent transcriptional repressor,
inducible cAMP early repressor (ICER), in both subsets of T cells
activated under conditions of suboptimal interleukin-2 (IL-2)
expression. Importantly, Th-specific expression of certain chemokines
is also susceptible to cAMP-mediated transcriptional attenuation. To
determine whether ICER per se, rather than forskolin-mediated elevation
of intracellular cAMP, is responsible for the observed inhibitory
effect, we generated transgenic mice expressing ICER under the control
of a lymphocyte-specific lck promoter. On stimulation,
transgenic thymocytes overexpressing ICER exhibited reduced levels of
IL-2 and interferon (IFN)-
and failed to express the macrophage
inflammatory protein (MIP)-1
and MIP-1ß genes. Splenic T cells
from ICER-transgenic mice showed a defect in proliferation and lacked a
mixed lymphocyte reaction response, implying that ICER-mediated
inhibition of cytokine and chemokine expression might play an important
role in T-cell inactivation.
Key Words: transcription factors • cytokines • chemokines
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INTRODUCTION
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The inducible cAMP early repressor (ICER) belongs to the cyclic
AMP (cAMP) response element (CRE)-binding protein (CREB)
[1
] and CRE modulator (CREM) [2
] family
of basic leucine zipper (bZIP) transcription factors [3
]
and acts as a dominant negative regulator of the cAMP-dependent protein
kinase A pathway of signal transduction [4
]. ICER, which
was initially found in the pineal gland, has been described as a
repressor of cAMP-induced transcription that is driven by rhythmic
adrenergic signals [5
]; it has been implicated in the
regulation of several physiological functions of the
hypothalamo-pituitary-gonadal axis (for a review, see ref. 6
). The
expression of ICER was once believed to occur exclusively in the
hypothalamo-pituitary-gonadal axis. Subsequently, ICER expression has
been shown to occur in the immune system, where it is proposed to act
as a repressor of T-cell proliferation and effector functions
[7
].
Here we demonstrate that on cAMP-mediated induction, ICER proteins
reached high levels in T cells and effectively competed with and
thereby repressed transcription mediated by bZIP proteins (e.g., by the
ubiquitously expressed activator CREB). Such competition for CREB is
presumably highly efficient in T cells because they do not express
activator forms of CREM [7
], which are constitutively
expressed in a stage-specific fashion in tissues of the
hypothalamo-pituitary-gonadal axis [8
]. ICER does not
possess a transactivation domain, an element required for the
recruitment of CREB-binding protein (CBP). Therefore, the binding of
ICER to the CRE might lead to the uncoupling of CBP or its homologue,
p300 (CBP/p300), abrogating early stages of transcriptional initiation
because of the lack of CBP/p300-associated histone acetyltransferase
activity. This results in a failure to maintain the transcriptionally
competent conformation of chromatin [9
]. Furthermore, in
the absence of a CBP-p300 complex, interactions of the nuclear factor
of activated T cells (NFAT) and nuclear factor 
B are likely to be
affected because their full transcriptional activity is dependent on
interaction with CBP-p300 [10
, 11
].
Therefore, it is possible that ICER’s competition with bZIP proteins
(e.g., CREB), bound to CRE-like motifs positioned adjacent to NFAT or
nuclear factor-B binding sites in the context of cytokine and
chemokine promoters, uncouples CBP-p300 and thereby aborts crosstalk
between Rel- and bZIP-mediated transcription [12
].
It is well established that T helper (Th) cells do not compose a
homogeneous population but rather are subdivided, on the basis of
cytokine expression, into at least two subsets, Th 1 and Th 2 (for a
review, see ref. 13
). Th 1 cells secrete predominantly interleukin
(IL)-2 and interferon (IFN)-, whereas Th 2 cells produce IL-4, IL-5,
IL-9, IL-10, and IL-13. There is good evidence that the ratio of the
abundance of Th 1 and Th 2 cells is relevant to the biology of a wide
variety of syndromes, including autoimmune diseases, allergic
conditions, and infectious diseases (for a review, see ref. 14
). Over
the past 10 years, the ability of numerous chemokines to attract
different types of blood leukocytes to sites of infection and
inflammation has been demonstrated [15
]. The
differential expression of chemokines and their receptors might
dictate, to a large extent, the migration and tissue homing of Th 1 and
Th 2 cells [16
, 17
]. The expression of
chemokine receptors could also result in different susceptibilities of
Th 1 and Th 2 cells to different strains of HIV using different fusion
coreceptors [18
]. Therefore, chemokines are part of an
effector and amplification mechanism relevant to polarized Th 1- and Th
2-mediated immune responses.
Here we demonstrate that cAMP-mediated inhibition of endogenously
expressed cytokines, a characteristic of Th 1 and Th 2 phenotypes,
correlated with the induction of ICER in both subsets of cells. It is
important that Th-specific expression of certain chemokines
[represented by macrophage inflammatory proteins (MIP-1) and
MIP-1ß] is also susceptible to ICER-mediated transcriptional
attenuation. To examine the direct role of ICER in cAMP-mediated
inhibition of cytokine and chemokine gene expression, we generated
transgenic mice expressing ICER under the control of the
lymphocyte-specific proximal lck promoter. On stimulation,
transgenic thymocytes overexpressing ICER exhibited reduced levels of
IL-2 and IFN- and failed to express MIP-1 and MIP-1ß. Moreover,
splenic T cells from ICER-transgenic mice showed a defect in
proliferation and lacked a mixed lymphocyte reaction response,
suggesting that ICER-mediated transcriptional attenuation of cytokine
and chemokine gene expression may compromise the T-cell response.
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MATERIALS AND METHODS
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Preparation of negatively selected T cells from human peripheral
blood lymphocytes by using superparamagnetic beads
Elutriated human peripheral blood lymphocytes (PBLs) were
prepared as detailed previously [19
]. PBL subpopulations
were fractionated by using superparamagnetic microbeads according to
the instructions provided by the manufacturer (Miltenyi Biotec, Auburn,
CA). Typically, the purity of negatively selected T-cell populations
was >95% CD3+ cells, with 
75% CD4+ cells
and 20% CD8+ cells (data not shown). After
paramagnetic-bead separation for T cells, <1% CD56+
CD16+ [natural killer (NK) cells], CD3+
CD56+ double-positive NK T cells expressing both T and NK
cell markers, CD19+ B lymphocytes, and CD14+
monocytes were detected (data not shown).
Stable polarization of human PBL T cells toward Th 1 or Th 2
phenotypes after polyclonal activation
Polarization was done by the method of Asselin et al.
[20
]. Polarized IL-12-derived Th 1-like or IL-4-derived
Th 2-like human PBL T cells were restimulated with either
phytohemagglutinin (PHA; Gibco-BRL, Rockville, MD) or phorbol myristate
acetate (PMA; 10 ng/mL) plus ionomycin (1 µg/mL) for 6 h in the
absence or presence of forskolin (0.1 mM final concentration). T
lymphocytes (containing >95% CD3+ cells) were typically
obtained after in vitro priming and polarization toward the Th 1- or Th
2-dominant phenotype. The non-CD3+ cells were
CD14+ (1% in Th 1 and <1% in Th 2), CD19+
(1% in Th 1 and 2% in Th 2), and CD16+ (1% in Th 1 and
5% in Th 2). In the experiments detailed here, the cells were cultured
for a total of 2 weeks prior to restimulation, and Th 1- and Th 2-like
populations shifted significantly toward the memory phenotype,
represented by the CD45RO marker (91% of CD45RO+ cells for
Th 1-like phenotype and 78% of CD45RO+ cells for Th 2-like
phenotype). Typically, the Th 1-like population contained 55%
CD4+ cells and 35% CD8+ cells. The Th 2-like
population usually contained 60% CD4+ and 15%
CD8+ cells (data not shown).
Flow cytometry
PBLs were analyzed before and after separations on
magnet-activated cell-sorting columns, using FACSort equipment (Becton
Dickinson, Paramus, NJ). Cells were stained at 4°C using
Ca2+- and Mg2+-free phosphate-buffered saline
with 0.5% bovine serum albumin and 0.025% sodium azide as a
diluent/wash fluorescence-activated cell sorting buffer. Nonspecific Fc
receptor binding was blocked by incubation with a 0.2-mg/mL solution of
human immunoglobulin G (Sigma Chemical Co., St. Louis, MO) for 10–15
min; then cells were triple stained with fluorescein isothiocyanate-,
phycoerythrin (PharMingen, San Diego, CA)-, and Tri-Color (Caltag,
Burlingame, CA)-conjugated antibody for 30 min. After being washed with
the cold fluorescence-activated cell sorting buffer, cells were fixed
in 1% paraformaldehyde in phosphate-buffered saline. Three-color
analyses were then performed.
Immunoprecipitation
Cells were metabolically labeled with 35S Translabel
(ICN Biomedicals, Costa Mesa, CA) according to established protocols
and lysed in radioimmunoprecipitation assay buffer [0.15 M NaCl, 50 mM
Tris-Cl (pH 7.2), 1% Triton X-100, 1% sodium deoxycholate, 0.1%
sodium dodecyl sulfate]. The lysate was supplemented with Complete
protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany),
clarified by centrifugation at 20,800 x g and 4°C
for 30 min, and precleared using protein A-Sepharose 4B beads
(Pharmacia, Uppsala, Sweden). Immune complexes were collected onto
protein A-Sepharose 4B beads that were prebound with CS4 CREM-specific
antiserum, rocked for 30 min at 4°C, and washed three times with
radioimmunoprecipitation assay buffer. Immune complexes were eluted
from beads with Laemmli sample buffer and resolved by sodium dodecyl
sulfate–15% polyacrylamide gel electrophoresis under reducing
conditions. The 35S signal was enhanced by treatment of the
gel with 2,5-diphenyloxazole (Sigma Chemical). 35S-labeled
proteins were detected by exposure of O-XAR film (Eastman Kodak,
Rochester, NY) for 1–10 days at -70°C.
RNase protection assay
RNA extraction was performed using an RNeasy kit (Qiagen,
Valencia, CA). The RNA probe for ICER, generated from pJL5 by either
XhoI or XbaI digestion, corresponds to the
full-length cDNA of human ICERII (described previously
[7
]). RNA probes hAPO3 and mAPO3 (PharMingen) were
labeled with [-32P]UTP using reagents from an RNA
probe kit (Ambion, Austin, TX). These probes were used for RNase
protection studies performed according to the protocol provided by
Ambion (RPAII ribonuclease protection assay kit).
Production and characterization of ICER-transgenic mice
A 0.36-kb fragment encompassing the ICER coding sequence, driven
by the proximal lck promoter, was introduced into a mouse
germ line by pronuclear microinjection [21
]. Of the
several independent founder lines generated, three were selected for
analysis on the basis of their levels of expression of the transgene.
These lines proved to be unstable over time. A different construct was
used to derive another set of founder lines which is not included in
this report. For measurements of lymphocyte proliferation, freshly
isolated lymphocytes (2 x 105) were cultured in
triplicate in 200 µL of Dulbecco’s modified Eagle medium
supplemented with 10% fetal calf serum (Gibco-BRL), using 96-well
tissue culture plates (Becton Dickenson Labware, Franklin Lakes, NJ).
Lymphocytes were activated for 48 h at 37°C by treatment with
PMA (10 ng/mL) and ionomycin (1 µg/mL), the anti-CD3 monoclonal
antibody 145.2C11 (10 µg/mL, immobilized on plastic tissue culture
plates), or concanavalin A (Con A; 2 µg/mL). Forty-eight hours after
activation, cells were labeled for 18 h by incubation in
[3H]thymidine-containing tissue culture medium (1
µCi/mL; specific activity, 2 Ci/mmol) (Amersham, Little Chalfont,
UK). Cells were collected onto glass fiber filter mats, and
[3H]thymidine incorporation was measured in a
scintillation counter. For measurement of allostimulation, splenocytes
from ICER-transgenic or nontransgenic mice were cocultivated with
either syngeneic splenocytes from CB57BL/6 mice or allogeneic
splenocytes from BALB/c mice for 48 h, after which
[3H]thymidine labeling was performed, and radioactivity
was determined as described above.
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RESULTS AND DISCUSSION
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ICER is readily inducible in human PBL T cells in response to
cAMP-elevating agonists
We reported previously that ICER mRNA is induced by cAMP-elevating
agonists [e.g., forskolin or prostaglandin E2
(PGE2)] in developing as well as mature human T
lymphocytes [7
]. To ascertain whether ICER mRNAs are
efficiently translated in mature T cells, we assayed for levels of ICER
proteins by immunoprecipitation of lysates prepared from human PBL T
cells at different times after treatment (Fig. 1
). Our data indicate that ICER protein accumulates in PBL T cells
for at least 18 h after the forskolin or PGE2
treatment. These findings correspond with those of earlier RNase
protection assays, which showed that the only detectable product of the
CREM gene in T lymphocytes is ICER, transcribed via the internal
cAMP-inducible P2 promoter [7
, 22
,
23
]. Immunoprecipitation of the whole-cell lysates with
antiserum raised against full-length CREM [24
] failed to
detect any constitutively expressed ICER or CREM protein prior to the
treatment in T lymphocytes isolated by negative selection (Fig. 1
;
compare CREM-specific antiserum denoted C in lane 1 with normal rabbit
control antiserum N in lane 2). However, after 3 h of forskolin
(F3) or PGE2 (P3) treatment (Fig. 1
, lanes 3 and 9,
respectively), distinct signals for both comigrating ICER isoforms,
ICERI and ICERII (denoted as ICER in Fig. 1
), and their comigrating
counterparts lacking exon-, ICERI and ICERII (denoted as
ICER in Fig. 1
), were detected [4
]. Accumulation of
ICER protein after forskolin or PGE2 treatment reached a
plateau 12 h (F12, P12) or 18 h (F18, P18) later (Fig. 1 ,
lanes 5 and 13). Our findings indicate that significant amounts of ICER
protein can be readily induced in mature human PBL T cells in response
to physiologically relevant ligands such as PGE2
[25
]. Furthermore, our data suggest that
PGE2 may activate a signal transduction pathway(s) to cause
the accumulation of stable ICER protein in human PBL T cells. These
observations differ substantially from earlier findings, obtained in a
study of developing human medullary thymocytes, in which the induction
of ICER after forskolin treatment was found to occur transiently, with
a peak seen at 3 h and a complete disappearance of detectable
ICER-specific protein evident by 12 h [22
]. In
contrast, in mature PBL T cells, ICER protein is stably induced for at
least 18 h after forskolin or PGE2 treatment.
Therefore, ICER may play an important role in cAMP-mediated
transcriptional attenuation in human PBL T cells.
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Figure 1. ICER is readily induced in human PBLs after forskolin (F) (0.1 mM final
concentration) or PGE2 (P) (500 ng/mL final concentration)
treatment. Immunoprecipitation of total 35S-labeled cell
lysates with CREM-specific antiserum (C) and normal rabbit serum (N)
demonstrates accumulation of ICER proteins in T cells (see Materials
and Methods) after 3, 12, and 18 h of forskolin (F3, F12, and F18,
respectively) or PGE2 (P3, P12, and P18, respectively)
treatment. After 3 h of forskolin or PGE2 treatment
(lanes 3 and 9, respectively), distinct signals were detected for both
the comigrating ICER isoforms (ICERI and ICERII, denoted as ICER) and
their comigrating counterparts lacking exon- (ICERI and
ICERII; denoted as ICER) [4
]. ICER and ICER
proteins were barely detectable in untreated (U) T cells (lanes 1 and
2) but were clearly detectable after 3 h of treatment (compare
lanes 3 and 4 and lanes 9 and 10, respectively), reaching robust levels
after 12 h of forskolin treatment (lane 5) or 18 h of
PGE2 treatment (lane 13).
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cAMP-mediated transcriptional attenuation of cytokine and chemokine
expression in Th 1- and Th 2-like cells correlates with induction of
ICER
To evaluate whether cAMP-mediated ICER induction correlates with
the transcriptional attenuation of cytokine and chemokine expression,
we first polarized human PBL T cells toward IL-12-derived Th 1-like and
IL-4-derived Th 2-like phenotypes [20
] and then
restimulated them with PHA in the absence or presence of forskolin
(Fig. 2
). Culture conditions during polarization gave rise only to partial
cytokine skewing after PHA restimulation. This could have been due to
the significant differences between Th 1- and Th 2-specific patterns of
cytokine expression in human as opposed to murine PBL T cells (for a
review, see ref. 26
). As noted above, RNase protection analysis
revealed that both IL-12 polarization (leading to a Th 1-like
population) and IL-4 polarization (leading to a Th 2-like population)
resulted in expression of IFN-, whereas the Th 2-like population
exhibited moderately increased levels of IL-4 and IL-5 (Fig. 2A
, lane
5), characteristic of the Th 2 phenotype. Furthermore, cAMP-mediated
inhibition of endogenously expressed cytokines, characteristic of both
the Th 1 and Th 2 phenotypes, correlated with the forskolin-mediated
induction of ICER in both subsets of T cells (Fig. 2B
and 2D)
. These
observations suggest that ICER may be responsible for the observed
inhibitory effect of the cAMP-mediated attenuation of cytokine
expression. It is interesting that, after PHA restimulation of in
vitro-polarized Th 1- and Th 2-like cells, only the cells with the Th
1-like phenotype expressed MIP-1ß, which was undetectable in the Th
2-like cells under these conditions (Fig. 2C)
. However, expression of
both MIP-1 and MIP-1ß was inhibited by forskolin (Fig. 2C
, lane
2), which correlated with forskolin-mediated induction of ICER (Fig. 2D
, lane 2). Collectively, these data suggested that ICER may play an
important role in inhibition of numerous cytokine and chemokine genes.
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Figure 2. Forskolin-mediated transcriptional attenuation of cytokine and
chemokine expression in IL-12-derived (IL-12), Th 1-like and
IL-4-derived (IL-4), Th 2-like cells correlates with induction of ICER.
IL-12- and IL-4-derived Th 1- and Th 2-like populations of PBLs were
restimulated with PHA for 6 h (see Materials and Methods) either
in the absence (-) or the presence (+) of 0.1 mM forskolin (F), and
RNAs were scored for human cytokine (A) or chemokine (C) expression in
parallel with ICER (B and D), using an RNase protection assay (Ambion).
For evaluation of human cytokine expression (A), a Riboquant hCK1 probe
set was used, while for evaluation of human chemokine expression (C) we
used a Riboquant hCK5 probe set (PharMingen). Levels of cytokine (IL-4,
IL-5, IL-9, IL-13, and IFN-) or chemokine (MIP-1 and MIP-1ß)
expression in PHA-activated, IL-12-derived Th 1- and IL-4-derived Th
2-like populations after forskolin treatment are inversely related to
the levels of ICER mRNA (B and D). Templates for the analysis of hL32
and human glyceraldehyde-3-phosphate dehydrogenase housekeeping genes
were included to allow assessment of total RNA levels. Note that each
probe migrated more slowly than its protected band; this was due to
flanking sequences in the probe that were not protected by mRNA. Purity
of in vitro polarized T-cell populations with a Th 1- or Th 2-like
phenotype was evaluated by flow cytometry analysis prior to PHA
restimulation (see Materials and Methods).
|
|
cAMP-mediated down-regulation of cytokine gene expression in T
cells with Th 1- and Th 2-like phenotypes is critically
dependent on costimulation
The susceptibility or resistance of cytokine expression in Th 1-
and Th 2-like cells to cAMP-mediated inhibition is critically dependent
on restimulation. This effect is most notably reflected by endogenous
expression of IL-2. A suboptimal IL-2 expression level, induced by the
failure to deliver costimulatory signals, is believed to be crucial for
the induction and maintenance of T-cell anergy [27
].
Indeed, delivery of both signals, mimicked by treatment of the cells
with PMA and ionomycin, led to the expression of supraphysiological
levels of IL-2 in both Th 1- and Th 2-like cells, a situation likely to
promote resistance of cytokine expression to inhibition by forskolin
(Fig. 3
; compare lanes 3, 4, 9, and 10). In contrast, mitogenic
stimulation via PHA, which relays signals predominantly through the
T-cell receptor, induced suboptimal expression of IL-2, and cytokine
expression in these cells was more susceptible to inhibition by
forskolin (Fig. 3
; compare lanes 5, 6, 11, and 12). This apparent
disparity in the capacity of cAMP to inhibit cytokine expression in the
absence of IL-2 was previously noted in PGE2-mediated
inhibition of IL-4 and IL-5 expression, yet was not explained
[28
]. It has been proposed that physiological
differences that distinguish productive proliferation from anergy in T
lymphocytes are best characterized by the presence or absence of an
IL-2-mediated autocrine loop (for a review, see ref. 29
). Here we
demonstrate that signals that caused T cells to proliferate and produce
high levels of IL-2 were also likely to render them resistant to
cAMP-mediated inhibition. Our findings support the idea that, under
conditions of suboptimal costimulation, which results in low levels of
IL-2 expression, both Th 1 and Th 2 cells are more easily deprived of
cytokine expression than when costimulation leads to vigorous IL-2
expression. However, both treatments used for restimulation [phorbol
ester plus ionomycin (P+I) or PHA] were effective for expression of
other NFAT-driven cytokine genes, such as IL-4, IL-5, and IL-13,
required for the effector functions of the Th 2 phenotype.
Forskolin-mediated ICER induction in P+I-stimulated PBL T cells is a
rather complex issue that is being addressed elsewhere (J. Bodor, J.
Bodorova, C. Bare, D. L. Hodge, H. Young, and R. Gress, submitted
for publication). These data indicate that, after P+I stimulation, T
cells could express significant amounts of ICER mRNA even in the
absence of forskolin. Moreover, levels of ICER mRNA seemed to be
further elevated in the presence of forskolin. The additive effect of
forskolin treatment suggests that forskolin-mediated ICER induction was
likely to use signal transduction pathways distinct from those engaged
after P+I restimulation. Collectively, these data suggest that the
outcome of ICER-mediated inhibition and subsequent differential
susceptibility of cytokine expression was fundamentally dependent on
the nature of the restimulation. Since differences in relative levels
of IL-2 were the most striking under P+I versus PHA restimulation, we
propose that IL-2 is likely to be one of the factors underlying
differential susceptibility of cytokine expression to cAMP-mediated
inhibition. It is important that only PHA restimulation, which slightly
stimulated IL-2 expression in Th 2-like cells, rendered IL-4, IL-5, and
IL-13 expression susceptible to almost complete cAMP-mediated
inhibition (Fig. 3
, lane 12).
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Figure 3. Differential susceptibility of cytokine expression to cAMP-mediated
inhibition in IL-12-derived (IL-12), Th 1-like and IL-4-derived (IL-4),
Th 2-like cells after restimulation. Polarized T cells were
restimulated for 6 h either with P+I [PMA, 10 ng/mL; ionomycin, 1
µg/mL (final concentrations)] or with PHA in the absence (-) or
presence (+) of forskolin (0.1 mM). After restimulation, RNAs were
scored in the RNase protection assay for cytokine expression, using a
Riboquant hCK1 human cytokine probe set (PharMingen). Also shown are
the corresponding RNase-protected probes after hybridization with yeast
RNA in the presence (lane 13) or absence (lane 14) of RNase.
|
|
In rodents, differential levels of methylation of the IFN- promoter
reflect differential expression of IFN- in Th 1 versus Th 2
phenotypes of T cells [30
]. However, in humans, a high
level of expression of IFN- occurs after restimulation in both Th
cell subsets, a finding attributable to a uniform hypomethylation of
the IFN- promoter in both subsets (for a review, see ref.
26
). Nevertheless, after PHA-mediated restimulation, Th
2-like cells were more susceptible to cAMP-mediated inhibition of
IFN- expression (Fig. 3 , lane 12) than are Th 1-like cells (Fig. 3
,
lane 6), which retain residual IFN- expression even in the presence
of forskolin. It is possible that the observed differential
susceptibility to cAMP-mediated inhibition is related to signaling via
other Th 1-specific pathways, such as p38 mitogen-activated protein
kinase, which has been reported to be relevant for IFN- expression
in Th 1 but not Th 2 cells [31
].
Defective expression of cytokines and chemokines in ICER-transgenic
mice is accompanied by impaired T-cell proliferation
To determine whether ICER per se is directly responsible for the
observed transcriptional attenuation of cytokine and chemokine
expression, we generated ICER-transgenic mice expressing the human
ICERII isoform. High-level expression of ICER (Fig. 4A
), under the control of the heterologous lck promoter,
allowed us to test the inhibitory role of ICER in the early expression
of cytokines and chemokines in activated thymocytes in the absence of
forskolin. In contrast to thymocytes from nontransgenic littermates,
which expressed normal amounts of IL-2 and IFN- when activated with
P+I, ICER-transgenic thymocytes exhibited significantly decreased
levels of IL-2 and IFN- after activation (Fig. 4B)
. Moreover,
ICER-transgenic mice failed to express MIP-1 and MIP-1ß as well as
lymphotactin and IFN--inducible protein-10, whereas background
expression of the protein known as RANTES (for regulated on activation,
normal T expressed and secreted), although modest, was almost
unaffected, suggesting a high specificity of ICER-mediated inhibition
(Fig. 4C) .
To determine whether overexpression of ICER might alter the development
or effector functions of transgenic lymphocytes, we examined whether
the differentiation of cells with constitutive ICER expression in the
lymphoid compartment was affected. This analysis revealed that the
total numbers of thymocytes and splenocytes were similar in
ICER-transgenic and nontransgenic control littermates (Fig. 5A
). Both transgenic and nontransgenic thymocytes expressed normal
levels of CD3 and T-cell receptor /ß, and there were normal
numbers of double-negative (CD4- CD8-),
double-positive (CD4+ CD8+), and
single-positive (CD4+ or CD8+) thymocytes and
splenic T cells in transgenic animals (data not shown). Thus, ICER
overexpression did not noticeably disrupt T-cell development. In
contrast, after activation by either Con A, immobilized anti-CD3
monoclonal antibody 2C11, or P+I (Fig. 5B)
, ICER-transgenic splenocytes
displayed proliferative defects of various extents. The most pronounced
differences between ICER-transgenic and wild-type lymphocytes were seen
after mitogenic stimulation with Con A. Treatment with P+I or with
antibody 2C11 resulted in less-pronounced differences in the
proliferation of splenocytes from ICER-transgenic mice (Fig. 5B)
. These
data indicate that the outcome of ICER-mediated inhibition is dependent
on stimulation. Moreover, allogeneic stimulation using splenocytes from
ICER-transgenic or nontransgenic mice, cocultivated with either
syngeneic splenocytes from CB57BL/6 (B6) mice or allogeneic splenocytes
from BALB/c mice in a mixed lymphocyte reaction, yielded markedly
different thymidine uptake levels (Fig. 5C)
. Thus, ICER-transgenic
splenocytes are clearly functionally distinct from their nontransgenic
counterparts.
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Figure 5. A proliferative defect in lymphocytes from ICER-transgenic mice. (A)
Cell yields from the thymus and spleen of transgenic and nontransgenic
animals. (B) Proliferation of splenocytes activated for 48 h with
Con A, PMA plus ionomycin (PMA + Ionophore), or immobilized
anti-CD3 monoclonal antibody (2C11) was measured by determining
[3H]thymidine incorporation (see Materials and Methods).
(C) Allogeneic response in a mixed lymphocyte reaction of C57BL/6
splenocytes from ICER-transgenic or nontransgenic control mice to
allogeneic BALB/c splenocytes. Irradiated, unfractionated splenocytes
from syngeneic C57BL/6 or allogeneic BALB/c splenocytes were added to
cultures containing unfractionated ICER-transgenic or nontransgenic
C57BL/6 splenocytes. After 48 h of coincubation, thymidine uptake
was measured as described in Materials and Methods.
|
|
Our findings for ICER-transgenic mice correlate with previously
reported observations of mice made transgenic with a dominant-negative
mutant of CREB defective in phosphorylation [32
]. The
mutant-CREB-transgenic mice were unable to produce IL-2 and manifested
a severe defect in T-cell proliferation. This phenotype may be due to
an impaired ability to recruit the transcriptional integrator CBP-p300.
Indeed, CBP-deficient mice also showed a general proliferation defect
[33
], supporting the notion that ICER or the
dominant-negative mutant of CREB may compete with endogenously
expressed CREB and thus abrogate recruitment of CBP-p300 (for a review,
see ref. 34
).
|
ACKNOWLEDGEMENTS
|
|---|
We especially thank Drs. Joel F. Habener, Howard A. Young, and
Richard J. Hodes for their critical readings of the manuscript. We are
grateful to the cell processing staff of the Department of Transfusion
Medicine, National Cancer Institute, NIH, and to Dr. Herbert Hagenau of
the Transgenic Mouse Model facility in Frederick for their unique
expertise and support.
Received September 28, 2000;
revised January 13, 2001;
accepted January 17, 2001.
|
REFERENCES
|
|---|
-
Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L., Habener, J. F. (1988) Cyclic AMP-responsive DNA-binding protein: structure based on cloned placental cDNA Science 242,1430-1433[Abstract/Free Full Text]
-
Foulkes, N. S., Borelli, E., Sassone-Corsi, P. (1991) CREM gene: use of alternative DNA binding domains generate multiple antagonists of cAMP-induced transcription Cell 54,739-749
-
Walker, W. H., Habener, J. F. (1996) Role of transcription factors CREB and CREM in cAMP-regulated transcription during spermatogenesis Trends Endocrinol. Metab. 7,133-138
-
Molina, C. A., Foulkes, N. S., Lalli, E., Sassone-Corsi, P. (1993) Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor Cell 75,875-886[Medline]
-
Sassone-Corsi, P. (1994) Rhythmic transcription and autoregulatory loops: winding up the biological clock Cell 78,361-364[Medline]
-
Lamas, M., Lalli, E., Foulkes, N. S., Sassone-Corsi, P. (1996) Rhythmic transcription and autoregulatory loops: nuclear pacemaker CREM Cold Spring Harbor Symp. Quant. Biol. 61,285-294[Abstract/Free Full Text]
-
Bodor, J., Spetz, A. L., Strominger, J. L., Habener, J. F. (1996) cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes Proc. Natl. Acad. Sci. USA/TITLE> 93,3536-3541(Erratum: Proc. Natl. Acad. Sci. USA 93, 8154, 1996.)
-
Lalli, E., Sassone-Corsi, P. (1995) Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor Proc. Natl. Acad. Sci. USA 92,9633-9637[Abstract/Free Full Text]
-
Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., Nakatani, Y. (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases Cell 87,953-959[Medline]
-
Garcia-Rodriguez, C., Rao, A. (1998) Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300 CREB-binding protein (CBP) J. Exp. Med. 187,2031-2036[Abstract/Free Full Text]
-
Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore, S., Shi, Y., Collins, T. (1997) CREB-binding protein/p300 are transcriptional coactivators of p65 Proc. Natl. Acad. Sci. USA 94,2927-2932[Abstract/Free Full Text]
-
Butscher, W. G., Powers, C., Olive, M., Vinson, C., Gardner, K. (1998) Coordinate transactivation of the interleukin-2 CD28 response element by c-Rel and ATF-1/CREB2 J. Biol. Chem. 273,552-560[Abstract/Free Full Text]
-
Paul, W. E., Seder, R. A. (1994) Lymphocyte responses and cytokines Cell 76,241-251[Medline]
-
Romagnani, S. (1994) Lymphokine production by human T cells in disease states Annu. Rev. Immunol. 12,227-257[Medline]
-
Baggiolini, M. (1998) Chemokines and leukocyte traffic Nature (London) 392,565-568[Medline]
-
Sallusto, F., Lenig, D., Mackay, C. R., Lanzavecchia, A. (1998) Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes J. Exp. Med. 187,875-883[Abstract/Free Full Text]
-
Bonecchi, R., Bianchi, G., Bordignon, P. P., D’Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A., Sinigaglia, F. (1998) Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s J. Exp. Med. 187,129-134[Abstract/Free Full Text]
-
Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., Lusso, P. (1995) Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells Science 270,1811-1815[Abstract/Free Full Text]
-
Czerniecki, B. J., Carter, C., Rivoltini, L., Koski, G. K., Kim, H. I., Weng, D. E., Roros, J. G., Hijazi, Y. M., Xu, S., Rosenberg, S. A., Cohen, P. A. (1997) Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells J. Immunol. 159,3823-3837[Abstract]
-
Asselin, S., Conjeaud, H., Minty, A., Fradelizi, D., Breban, M. (1998) Stable polarization of peripheral blood T cells towards type 1 or type 2 phenotype after polyclonal activation Eur. J. Immunol. 28,532-539[Medline]
-
Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs Proc. Natl. Acad. Sci. USA 82,4438-4442[Abstract/Free Full Text]
-
Bodor, J., Habener, J. F. (1998) Role of transcriptional repressor ICER in cyclic AMP-mediated attenuation of cytokine gene expression in human thymocytes J. Biol. Chem. 273,9544-9551[Abstract/Free Full Text]
-
Mao, D. L., Warner, E. A., Gurwitch, S. A., Dowd, D. R. (1998) Differential regulation and transcriptional control of immediate early gene expression in forskolin-treated WEHI7.2 thymoma cells. Mol. Endocrinol. 12,492-503[Abstract/Free Full Text]
-
Walker, W. H., Sanborn, B. M., Habener, J. F. (1994) An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription Proc. Natl. Acad. Sci. USA 91,12423-12427[Abstract/Free Full Text]
-
Goetzl, E. J., An, S., Smith, W. L. (1995) Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases FASEB J 9,1051-1058[Abstract]
-
Young, H. A., Ghosh, P. (1997) Molecular regulation of cytokine gene expression: interferon-gamma as a model system Prog. Nucleic Acid Res. Mol. Biol. 56,109-127[Medline]
-
Madrenas, J., Schwartz, R. H., Germain, R. N. (1996) Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists Proc. Natl. Acad. Sci. USA 93,9736-9741[Abstract/Free Full Text]
-
Hilkens, C. M., Vermeulen, H., van Neerven, R. J., Snijdewint, F. G., Wierenga, E. A., Kapsenberg, M. L. (1995) Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2 Eur. J. Immunol. 25,59-63[Medline]
-
Powell, J. D., Ragheb, J. A., Kitagawa-Sakakida, S., Schwartz, R. H. (1998) Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy Immunol. Rev. 165,287-300[Medline]
-
Young, H. A., Ghosh, P., Ye, J., Lederer, J., Lichtman, A., Gerard, J. R., Penix, L., Wilson, C. B., Melvin, A. J., McGurn, M. E., et al (1994) Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-gamma gene J. Immunol. 153,3603-3610[Abstract]
-
Rincon, M., Enslen, H., Raingeaud, J., Recht, M., Zapton, T., Su, M. S., Penix, L. A., Davis, R. J., Flavell, R. A. (1998) Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway EMBO J 17,2817-2829[Medline]
-
Barton, K., Muthusamy, N., Chanyangam, M., Fischer, C., Clendenin, C., Leiden, J. M. (1996) Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB Nature (London) 379,81-85[Medline]
-
Yao, T. P., Oh, S. P., Fuchs, M., Zhou, N. D., Ch’ng, L. E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., Eckner, R. (1998) Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300 Cell 93,361-372[Medline]
-
Bodor, J., Bodorova, J., Gress, R. E. (2000) Suppression of T cell function: a potential role for transcriptional
repressor ICER J. Leukoc. Biol. 67,774-779[Abstract]
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ABSTRACT
INTRODUCTION
