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(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{ddagger}, Jana Bodorova{dagger}, Cathy Bare*, Marvin S. Reitz, Jr§ and Ronald E. Gress*

* Experimental Immunology Branch, Division of Basic Sciences, and
{ddagger} Transplantation Therapy Section, Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
{dagger} 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


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ABSTRACT
 
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)-{gamma} and failed to express the macrophage inflammatory protein (MIP)-1{alpha} 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
 
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 {kappa}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-{kappa}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)-{gamma}, 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){alpha} 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-{gamma} and failed to express MIP-1{alpha} 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
 
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 [{alpha}-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
 
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-{gamma}, ICERI{gamma} and ICERII{gamma} (denoted as ICER{gamma} 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-{gamma} (ICERI{gamma} and ICERII{gamma}; denoted as ICER{gamma}) [4 ]. ICER and ICER{gamma} 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).

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-{gamma}, 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{alpha} 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-{gamma}) or chemokine (MIP-1{alpha} 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-{gamma} promoter reflect differential expression of IFN-{gamma} in Th 1 versus Th 2 phenotypes of T cells [30 ]. However, in humans, a high level of expression of IFN-{gamma} occurs after restimulation in both Th cell subsets, a finding attributable to a uniform hypomethylation of the IFN-{gamma} 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-{gamma} expression (Fig. 3 , lane 12) than are Th 1-like cells (Fig. 3 , lane 6), which retain residual IFN-{gamma} 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-{gamma} 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-{gamma} when activated with P+I, ICER-transgenic thymocytes exhibited significantly decreased levels of IL-2 and IFN-{gamma} after activation (Fig. 4B) . Moreover, ICER-transgenic mice failed to express MIP-1{alpha} and MIP-1ß as well as lymphotactin and IFN-{gamma}-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) .



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Figure 4. ICER-mediated transcriptional attenuation of expression of cytokines and chemokines in ICER-transgenic mice. (A) Freshly isolated thymocytes from ICER-transgenic (lane pairs A and B) or control (lane pairs C and D) nontransgenic littermates were obtained, and total 35S-labeled cell lysates were assayed by immunoprecipitation, using a CREM-specific antiserum (single-lanes c) along with normal rabbit serum (single-lanes n). ICER-transgenic (+) or control nontransgenic (-) thymocytes were activated with PMA plus ionomycin for 3 h. RNAs were isolated and analyzed in the RNase protection assay for cytokine (B) and chemokine (C) production in parallel using the Riboquant mCK1 and mCK5 probe sets (PharMingen), respectively. Expression of both IL-2 and IFN-{gamma} was significantly reduced, along with expression of MIP-1{alpha}, MIP-1ß, lymphotactin, and IFN-{gamma}-inducible protein-10.

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 {alpha}/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 ).


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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.


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REFERENCES
 
    1
  1. 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]
  2. 2
  3. 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
  4. 3
  5. 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
  6. 4
  7. 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]
  8. 5
  9. Sassone-Corsi, P. (1994) Rhythmic transcription and autoregulatory loops: winding up the biological clock Cell 78,361-364[Medline]
  10. 6
  11. 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]
  12. 7
  13. 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.)
  14. 8
  15. 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]
  16. 9
  17. 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]
  18. 10
  19. 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]
  20. 11
  21. 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]
  22. 12
  23. 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]
  24. 13
  25. Paul, W. E., Seder, R. A. (1994) Lymphocyte responses and cytokines Cell 76,241-251[Medline]
  26. 14
  27. Romagnani, S. (1994) Lymphokine production by human T cells in disease states Annu. Rev. Immunol. 12,227-257[Medline]
  28. 15
  29. Baggiolini, M. (1998) Chemokines and leukocyte traffic Nature (London) 392,565-568[Medline]
  30. 16
  31. 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]
  32. 17
  33. 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]
  34. 18
  35. 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]
  36. 19
  37. 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]
  38. 20
  39. 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]
  40. 21
  41. 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]
  42. 22
  43. 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]
  44. 23
  45. 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]
  46. 24
  47. 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]
  48. 25
  49. 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]
  50. 26
  51. 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]
  52. 27
  53. 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]
  54. 28
  55. 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]
  56. 29
  57. 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]
  58. 30
  59. 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]
  60. 31
  61. 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]
  62. 32
  63. 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]
  64. 33
  65. 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]
  66. 34
  67. 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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