Up-regulation of IL-4 production by the activated cAMP/cAMP-dependent protein kinase (protein kinase A) pathway in CD3/CD28-stimulated naive T cells
Koji Tokoyoda1,
Kazutake Tsujikawa1,
Hiroaki Matsushita1,
Yuichi Ono1,
Tamon Hayashi1,
Yohsuke Harada2,
Ryo Abe2,
Masato Kubo2 and
Hiroshi Yamamoto1
1 Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, 16 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
Correspondence to: K. Tsujikawa; E-mail: tujikawa{at}phs.osaka-u.ac.jp
Transmitting editor: T. Saito
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Abstract
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The signal transduction of the cAMP/cAMP-dependent protein kinase [protein kinase A (PKA)] pathway through multiple receptors is critical for many processes in all cell types. In T cells, the engagement of both the TCRCD3 complex and the CD28 co-stimulatory molecule also induces cAMP, and subsequently activates PKA. It is believed that elevation of cAMP levels in T cells is inhibitory of IL-2 production and T cell proliferation. However, the function and detailed signal transduction mechanisms of the cAMP/PKA pathway in naive Th cells are less well understood. In this study, we show that calcitonin gene-related peptide (CGRP) down-regulates IL-2 and IFN-
production and up-regulates IL-4 production to promote Th2 differentiation by moderate activation of the cAMP/PKA pathway via the CGRP receptor in the presence of a CD3/CD28 co-stimulation signal. The IL-4 production and transcriptional activation of Th2 cytokine mRNAs were also reproduced by the addition of a cAMP analogue, dibutyryl-cAMP, in CD3/CD28-stimulated naive Th cells. More interestingly, cAMP/PKA activation in naive Th cells stimulated with anti-CD3 plus anti-CD28 mAb is essential for inducing IL-4 production and promoting Th2 differentiation; in addition, NF-AT is a downstream effector of the cAMP/PKA signaling pathway. These findings indicate that the cAMP/PKA pathway transduces the critical activation signal to Th2 polarization by a CD3/CD28 co-stimulation signal and a PKA activating reagent.
Keywords: calcitonin gene-related peptide, NF-AT, Th1, Th2
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Introduction
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Optimal activation of T cells requires both the engagement of the TCRCD3 complex, and interactions between co-stimulatory molecules such as CD28 (1) and ligands B7-1 (CD80) and B7-2 (CD86), which are expressed on antigen-presenting cells (APC) (24). Stimulation of the TCRCD3 complex in the absence of the CD28-mediated co-stimulatory signal drives T cells to enter a state of anergy or else to undergo apoptosis (57). Conversely, induction of T cell anergy can be prevented by stimulation of CD28 with B7 molecules or anti-CD28 antibody (8). Moreover, the CD28-mediated co-stimulatory signal in T cells augments the proliferation and secretion of multiple T cell cytokines such as IL-2, IFN-
and IL-4 (9,10). Murine CD4+ T cells have been divided into functionally distinct subsets based on their cytokine production profiles. Th1 cells produce IL-2 and IFN-
, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13. Th cell-derived cytokines participate in the development and progression of a number of disease states, direct normal immune responses, and regulate naive CD4+ T cell differentiation. It is, therefore, essential to understand the signal transduction mechanisms that regulate cytokine production in Th cells.
In recent years, understanding of TCRCD3 signal transduction has progressed significantly. On the other hand, CD28-mediated co-stimulatory signals are not well understood; however, some key molecules that transduce CD28 signaling have been identified. CD28 co-stimulation signal induces phosphorylation and activation of a proto-oncogene Vav (11). Vav acts as a guanine-nucleotide exchange factor for the small Rho family GTPases Rac and CDC42, allowing these molecules to switch from the inactive GDP-bound state to an active, GTP-bound state. Active forms of Rac and CDC42 then activate p21-activated kinase, leading to the activation of extracellular signal-regulated protein kinase (ERK) pathway. Other mitogen-activated protein kinase (MAPK) pathways, i.e. c-Jun N-terminal kinase (JNK) and p38 MAPK, appear to be involved in signal transduction of the CD28 co-stimulatory molecule as well. ERK and JNK phosphorylate c-fos and c-jun proteins respectively, which assemble to form the activated protein-1 (AP-1) transcription factor. AP-1 binds to the IL-2 promoter and then induces IL-2 production for T cell proliferation (12,13). Additionally, JNK has been shown to play an important role in IL-2 mRNA stabilization (14). Moreover, p38 MAPK has been reported to be preferentially required for Th2-type responses in human CD4+CD45RO+ T cells and Th2 effector cells (15).
There is growing evidence that the nervous system and the immune system cross-talk through common molecules and their receptors. In a previous study, we demonstrated that calcitonin gene-related peptide (CGRP), a 37-amino-acid neuropeptide, modulates the profile of IFN-
and IL-4 production from mouse splenocytes and Th cell clones stimulated with the appropriate antigen (16). CGRP plays important roles as a neurotransmitter/neuromodulator in the central nervous system and as a potent vasodilator when secreted from peripheral, perivascular nerves (17,18). These physiological functions are mediated through its binding to a specific receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) (19). CGRP and its receptor are found to be expressed in various organs such as brain, lung, thyroid, thymus and spleen (20,21). Recently, we cloned the mouse cDNA counterpart of the human CGRP receptor composed of CRLR and RAMP1, and clarified expression of the CGRP receptor on T cells (22). These results indicate that signaling through CGRP receptors modulates signal transduction by engagement of TCR and CD28 in T cells. Since CGRP receptors are coupled with Gs
protein (23), we hypothesized that the cAMP/PKA pathway is likely to be involved in the regulation of cytokine production by CGRP in activated T cells.
We report here that CGRP inhibited IL-2 and IFN-
production, and augmented IL-4 production to promote Th2 differentiation by activation of the cAMP/PKA pathway in the presence of a CD3/CD28 co-stimulation signal. The IL-4 production and transcriptional activation of Th2 cytokine mRNAs were also reproduced by dibutyryl-cAMP (db-cAMP) in CD3/CD28-stimulated naive Th cells. Moreover, the CD3/CD28 co-stimulation signal also activates the cAMP/PKA pathway and then enhances NF-AT transcription activity to skew immune responses to the Th2 phenotype.
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Methods
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Mice
Specific pathogen-free male 5- to 7-week-old BALB/c mice were purchased from Charles River Japan (Kanagawa, Japan). Ovalbumin (OVA)-specific TCR
ß transgenic mice (DO 11.10 Tg) were kindly distributed by Dr Hiromichi Ishikawa (Keio University, Tokyo, Japan).
Antibodies and reagents
Anti-mouse IL-2 mAb (MM-101 and MM102-B) and anti-mouse IFN-
mAb (MM701) were purchased from Pierce (Rockland, IL). Biotinylated anti-mouse mouse IL-4 (BVD6-24G2), biotinylated anti-mouse mouse IFN-
(XMG1.2), anti-IFN-
(XMG1.2)FITC and anti-IL-4 (11B11)phycoerythrin were purchased from BD PharMingen (San Diego, CA). The mAb to CD8 (3155), B220 (RA3.3), MHC class II (345.3), Thy-1.2 (HO13.4.9) and CD3 (2C11, OKT3) were obtained from ATCC (Rockville, MA). The antigenic OVA synthetic peptide (residues 323339; ISQAVHAAHAEINEAGR) was synthesized by Fuso Pharmaceutical Industries (Osaka, Japan). CGRP was purchased from the Peptide Institute (Osaka, Japan), db-cAMP was purchased from Wako (Osaka, Japan), N-[2-((p-bromocinnamyl)aminoethyl)]-5-isoquinolinesulfonamide (H-89) was purchased from Sigma (St Louis, MO). PKI1422 was purchased from Calbiochem (La Jolla, CA). The pNF-AT72-luc was kindly provided by Dr Kenichi Arai (University of Tokyo, Tokyo, Japan); p(Ig
)3-luc was the generous gift of Dr Tatsuhiko Furukawa (Kagoshima University, Kagoshima, Japan). pCMVT-TK neo PKA-RG324D was kindly provided by Dr Tania H. Watts (University of Toronto, Toronto, Ontario, Canada) (24). PathDetect in vivo signal transduction pathway reporting systems [c-Jun trans-, Elk trans-, CHOP trans- and cAMP response element binding protein (CREB) trans-reporting systems] were purchased from Stratagene (La Jolla, CA). pSV-ß-galactosidase control vector was obtained from Promega (Madison, WI).
Preparation and stimulation of naive CD4+ T cells
To isolate naive CD4+ T cells, mouse splenocytes were incubated with anti-CD8 mAb (3155), anti-B220 mAb (RA3.3) and anti-MHC class II (345.3) mAb at 4°C for 30 min, and then were incubated on anti-mouse Ig-coated plates to eliminate B cells and CD8+ T cells. The CD4+ T cell-enriched population contained >80% CD4+ T cells. APC were prepared from splenic cells by cytotoxic killing treatment with anti-Thy-1.2 mAb (HO13.4.9) and a rabbit complement (Rockland, Gilbertsville, PA). The CD4+ T cells (106 cells/ml) were stimulated with antigenic peptide (OVA323339) in the presence of the splenic APC (5 x 106 cells/ml) or with plate-coated anti-CD3 mAb (2C11) and/or soluble anti-CD28 mAb (PV-1).
Measurement of cytokine concentrations
Purified CD4+ T cells were cultured on mAb-coated plates for 48 h and the cytokine concentrations in culture supernatants determined by ELISA. An in-house ELISA was employed to quantify IL-2, IFN-
and IL-4 as follows. Microtiter wells were coated overnight with anti-mouse IL-2, anti-mouse IFN-
and anti-mouse IL-4 mAb. Samples and the standards were diluted in RPMI 1640 supplemented with 2% FCS and incubated for 2 h at 37°C. After washing with PBS/0.05% Tween 20, each well was incubated for 1 h with biotinylated anti-mouse IL-2, anti-mouse IFN-
and anti-mouse IL-4 mAb, washed, incubated for 30 min with horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech, Little Chalfont, UK), and developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegaard & Perry, Gaithersburg, MD). Absorbance was determined at a wavelength of 405 nm. Quantities of IL-2, IFN-
and IL-4 were calculated according to the standard curves. The lower limit of detection was 10 U/ml for IL-2, 150 pg/ml for IFN-
and 6 pg/ml for IL-4.
Proliferation assays
CD4+ T cells (105 cells/well) in a 96-well plate were stimulated with plate-coated anti-CD3 mAb or plate-coated anti-CD3 plus soluble anti-CD28 mAb in the presence or absence of 1 nM CGRP for 18 h. Cells were pulsed with 1 µCi [3H]thymidine (Amersham Pharmacia Biotech) for 6 h, harvested and counted with a scintillation counter (Packard, Meriden, CT).
Measurement of cAMP amount
CD4+ T cells (106 cells/well) in a 96-well plate were stimulated with anti-CD3 mAb, anti-CD28 mAb or CGRP. After 15 min incubation, the cells were lysed with 20 µl lysis buffer. cAMP levels in the lysate were measured by the non-acetylation method using an enzyme immunoassay system (Amersham Pharmacia Biotech) according to the manufacturers instructions.
Cytokine mRNA analysis
The relative mRNA expression of 23 cytokines was analyzed with GEArray (SuperArray, Bethesda, MD) according to the manufacturers protocol. In brief, CD4+ T cells (106 cells/ml) were stimulated with 1 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb in the presence or absence of 5 µM db-cAMP. After 48 h incubation, total RNA was extracted with TRIzol reagent (Gibco/BRL, Grand Island, NY). Total RNA (5 µg) from each sample was reverse transcribed into cDNA with MMLV reverse transcriptase (Gibco/BRL) in the presence of [
-32P]dCTP (Amersham Pharmacia Biotech). The synthesized cDNA probes were hybridized to cytokine gene-specific cDNA fragments that were spotted on the GEArray membrane. The amount of radioactive signaling from the hybridized probes was analyzed with a BAS1500 phosphoimager (Fuji, Kanagawa, Japan). The signal from expression of each cytokine gene was normalized to the signal derived from GAPDH on the same membrane and expressed as cytokine mRNA arbitrary units. These units were calculated with the following formula. Cytokine mRNA arbitrary units = (cytokine signal background signal)/(GAPDH signal background signal) x 100.
In vitro PKA assay
CD4+ T cells (106 cells/well) in a 48-well plate were stimulated with 1 nM CGRP and/or 10 µg/ml plate-coated anti-CD3 plus 1 µg/ml soluble anti-CD28 mAb. After 1 h incubation, the cells were lysed in the lysis buffer containing 1 µM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1.8 µg/ml iodoacetamide, and PKA activity in lysates was determined by a PKA radioimmunoassay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturers instructions.
Induction of Th cells and intracellular cytokine staining
CD4+ T cells (106 cells/well) in a 48-well plate were stimulated with plate-coated anti-CD3 mAb and soluble anti-CD28 mAb in the presence or absence of CGRP or H-89 for 2 days. After 5 days, the T cells were re-stimulated with anti-CD3 mAb for 4 h in the presence of 2 µM monensin (Sigma). Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X. After blocking with PBS containing 3% BSA, cells were stained with anti-IFN-
FITC and anti-IL-4phycoerythrin, as described previously (25). Flow cytometric analysis was performed on FACSCalibur with CellQuest software (Becton Dickinson).
DNA transfection and luciferase assay
Jurkat cells expressing the mouse CD28 (Jurkat-CD28) were cultured in RPMI 1640 supplemented with 10% FCS (26). Jurkat-CD28 cells were transfected with 5 µg of pNF-AT72-luc or p(Ig
)3-luc for measuring NF-AT or NF-
B activity respectively, or 5 µg of pFR-luc concomitant with 0.5 µg of pFA-CREB, pFA-CHOP, pFA-c-jun or pFA-Elk for measuring CREB, p38, JNK or ERK activities respectively, using a Genepulser (Bio-Rad, Hercules, CA). After a 24-h incubation, the transfected cells were stimulated with plate-coated anti-human CD3 mAb (OKT3) or anti-CD3 plus soluble anti-CD28 (PV-1) mAb. In Fig. 6(B), Jurkat-CD28 cells were co-transfected with 5 µg of pNF-AT72-luc and 2 µg of pSV-ß-galactosidase control vector in combination of 15 µg of pCMVT-TK neo vector or pCMVT-TK neo PKA-RG324D vector. A pSV-ß-galactosidase control vector was used for normalizing the transfection efficiency. The transfectants were stimulated with anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb for 24 h. The cells were harvested and lysed in cell culture lysis reagent (Promega), followed by measurements of luciferase activity on a Lumat LB9501 luminometer (Berthold, Bundoora, Australia) or ß-galactosidase activity.
Gel mobility shift assay
Nuclear extracts were prepared from CD4+ T cells stimulated with 10 µg/ml plate-coated anti-CD3 mAb and 1 µg/ml soluble anti-CD28 mAb in the presence or absence of 1 nM CGRP or 20 µM H-89 for 6 h. Gel mobility shift assays were carried out as outlined by the manufacturer (EMSA kit, NF-ATc probe; Panomics, Redwood City, CA).
Statistical analysis
Results are presented as means ± SD. For the two-group comparison, the statistical significance of differences was determined by Students t-test for unpaired data.
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Results
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CGRP up-regulates IL-4 production from CD3/CD28-stimulated CD4+ T cells
CGRP is thought to be one of the neuropeptides involved in cross-talk between the neuronal system and the immune system via its specific receptor. To examine the effect of CGRP on cytokine production and proliferation, we stimulated naive CD4+ T cells with anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb in the presence or absence of 1 nM CGRP. The proliferative responses, and IL-2, IL-4 and IFN-
secretion were analyzed at 24 and 48 h respectively. When T cells were stimulated with plate-coated anti-CD3 mAb alone, CGRP significantly inhibited the production of IL-2 (20%), IFN-
(15%) and IL-4 (90%), and T cell proliferation (90%) (Fig. 1). The addition of soluble anti-CD28 mAb clearly augmented the production of three cytokines and T cell proliferation. Under these circumstances, addition of 1 nM CGRP in the presence of CD28 co-stimulatory signal reduced the production of IL-2 and IFN-
as well as T cell proliferation; however, IL-4 production was significantly increased (3.2-fold) by CGRP (Fig. 1C). CGRP at concentrations of 1100 nM revealed a dose-dependent effect on cytokine production from CD3/CD28-stimulated T cells (data not shown).

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Fig. 1. Effect of CGRP on cytokine production and proliferation of naive CD4+ T cells stimulated with anti-CD3 mAb alone or with anti-CD3 plus anti-CD28 mAb. Naive CD4+ T cells (106 cells/ml) were stimulated with 10 µg/ml anti-CD3 mAb alone or 10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb in the presence or absence of 1 nM CGRP. After a 48-h incubation, concentrations of IL-2 (A), IFN- (B) and IL-4 (C) in the culture supernatants were determined by ELISA. For proliferation activity, T cells (2 x 105 cells/well) were plated in 96-well plates and stimulated with anti-CD3 mAb alone or with anti-CD3 plus CD28 mAb in the presence or absence of 1 nM CGRP for 24 h. Six hours after 1 µCi [3H]thymidine addition, T cells were harvested for scintillation counting (D). *P < 0.05, **P < 0.01, ***P < 0.001.
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Interaction of B7 and CD28 is essential for up-regulation of IL-4 production by CGRP
Next, we examined the effect of CGRP on initial IL-4 production in antigen-induced responses. For this purpose, CD4+ T cells purified from splenocytes of OVA-specific TCR transgenic mice, DO11.10 Tg, were stimulated with 1 µM OVA323339 peptide and APC in the presence or absence of 1 nM CGRP. As shown in Fig. 2, CGRP augmented IL-4 production as much as 2.5-fold compared to the control T cells, which is a similar result to that achieved by stimulation with anti-CD3 plus anti-CD28 mAb (Fig. 1C).

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Fig. 2. The CD28 co-stimulation signal is essential for CGRP-induced IL-4 production from antigenic peptide-stimulated T cells. CD4+ T cells (106 cells/ml) from DO.11.10 Tg mice were stimulated with 1 µM OVA323339 peptide and APC (5 x 106 cells/ml) in the presence or absence of 1 nM CGRP. CTLA-4Ig (10 µg/ml) was added to the culture at the same time as the CGRP stimulation was performed. After a 48-h incubation period, IL-4 levels in the culture supernatants were measured by ELISA. Values are means ± SD (n = 4). med.: medium. **P < 0.01, ***P < 0.001.
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The B7 family molecules, such as B7-1 and B7-2, expressed on APC bind to CD28 expressed on T cells and this process is critical to induce full activation of T cells in primary responses. To directly examine the relevance of the CD28-derived co-stimulatory signal in the up-regulation of IL-4 production by CGRP, we blocked the B7CD28 interactions using CTLA-4Ig, a soluble fusion protein made from the extracellular portion of CTLA-4 linked to the Fc portion of IgG (27). The addition of CTLA-4Ig completely inhibited up-regulation of IL-4 production by CGRP, indicating that signal transductions through both the CGRP receptor and the CD28 co-stimulatory molecule were coupled to IL-4 production.
Effect of db-cAMP on Th2 cytokine production from CD3/CD28-stimulated CD4+ T cells
CGRP receptors have been reported to couple with G
s and Gß
subunits of G proteins. After binding of CGRP to its receptors, the G
s subunit activates adenylyl cyclase, thereby increasing intracellular cAMP levels. Therefore, we determined cAMP levels in naive CD4+ T cells stimulated with anti-CD3 mAb and/or anti-CD28 mAb in the presence or absence of CGRP for 15 min (Fig. 3A). CGRP (1 nM) alone induced
2.5-fold cAMP production compared to that of the control level, but stimulation with anti-CD3 mAb or anti-CD28 mAb did not have this inductive effect. The combination of anti-CD3 plus anti-CD28 mAb markedly increased the cAMP level and this increase was further augmented by the addition of CGRP. The changes in cAMP levels by the stimulation of anti-CD3/CD28 mAb or anti-CD3/CD28 mAb plus CGRP were similar to the changes in IL-4 production (Fig. 1C), although the stimulation with either anti-CD3 mAb or anti-CD28 mAb failed to induce production of both the cAMP elevation and the initial IL-4 production. These results indicated that elevation of the cAMP level plays an important role on the initial IL-4 production induced by CD3/CD28 co-stimulation.

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Fig. 3. Effects of db-cAMP on cytokine production of naive CD4+ T cells stimulated with anti-CD3 plus anti-CD28 mAb. (A) Increase in intracellular cAMP levels by CGRP stimulation and/or CD3/CD28 co-stimulation. CD4+ T cells (106 cells/ml) were stimulated with the combination of 1 nM CGRP, 10 µg/ml anti-CD3 mAb and 1 µg/ml anti-CD28 mAb. T cells were lysed at 15 min after stimulation and cAMP levels in the lysates were measured by EIA. Values are means ± SD (n = 36). (B) CD4+ T cells (106 cells/ml) were stimulated with 10 µg/ml anti-CD3 mAb alone or 10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb in the presence of various concentrations of db-cAMP. IL-4 levels in culture supernatants were determined by ELISA. Values are means ± SD (n = 4). (C) CD4+ T cells (106 cells/ml) were stimulated with 10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb in the presence or absence of 5 µM db-cAMP. After a 48-h incubation period, total RNA was isolated and reverse transcribed to cDNA in the presence of [ -32P]dCTP. Labeled cDNAs were hybridized to GEArray membranes. Cytokine mRNA levels were calculated as arbitrary units, relevant to GAPDH.
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To investigate whether a cAMP analogue, db-cAMP, had the same effect as CGRP on IL-4 production, we stimulated naive CD4+ T cells with anti-CD3 mAb and anti-CD28 mAb in the presence of various concentrations of db-cAMP, and IL-4 production was assayed after 48 h. The addition of db-cAMP reduced IL-4 production from the T cells stimulated with anti-CD3 mAb alone; however, the same treatment increased IL-4 production from T cells stimulated with anti-CD3 plus anti-CD28 mAb in a dose-dependent manner (Fig. 3B). The addition of 5 µM db-cAMP with CD3 stimulation revealed
45% inhibition of the IL-4 level, whereas the same treatment, together with CD3/CD28 co-stimulation, produced
2.3-fold increase. On the other hand, db-cAMP inhibited IL-2 and IFN-
production both with CD3 stimulation alone and in conjunction with CD28 co-stimulation in a dose-dependent manner, although db-cAMP had no effect on the survival of T cells at concentrations <5 µM (data not shown).
Next, we examined the specificity of db-cAMP-dependent cytokines production in naive CD4+ T cells stimulated with anti-CD3/CD28 mAb using a cytokine array. Total RNA was isolated from CD4+ T cells that had been cultured for 48 h with anti-CD3 plus anti-CD28 mAb in the presence or absence of 5 µM db-cAMP. The RNA from each sample was reverse transcribed into cDNA in the presence of [32P]dCTP and the synthesized 32P-labeled cDNAs were hybridized to a GEArray cytokine membrane in which 23 cytokine gene-specific cDNA fragments, including IL-1 to IL-18, granulocyte colony stimulating factor, LT-ß, IFN-
, tumor necrosis factor (TNF)-
and TNF-ß, were spotted. The signals from each spot on the membrane were then scanned with a phosphoimager. To eliminate potential variation in RNA quantification between samples, the signal from expression of each cytokine gene was corrected by that of GAPDH (as 100 arbitrary units) on the same membrane and was expressed as an arbitrary unit. Since GEArray is a semiquantitative method of mRNA expression used for screening purposes, we considered a 3-fold deviation of the signal from the control signal as a significant difference. As shown in Fig. 3(C), the concomitant stimulation of db-cAMP with anti-CD3/CD28 mAb induced marked increases in IL-4, IL-5, IL-10 and IL-13 mRNA from CD4+ T cells; however, the changes in IFN-
or IL-2 mRNA levels were neither obvious nor suppressed respectively. We confirmed the transcriptional activity of IL-4, IL-5, IL-10 and IL-13 with semiquantitative RT-PCR in additional experiment (data not shown). Those results were clearly consistent with the results from the cytokine array. It is of note that the cytokine mRNAs increased by db-cAMP stimulation belonged to Th2 cytokines, indicating that cAMP-mediated signaling is involved in Th2, but not Th1, cytokine production.
CD28 co-stimulation signal activates PKA to produce IL-4
cAMP activates PKA to transduce PKA signaling downstream. Since it was observed that CGRP and CD3/CD28 co-stimulation increased cAMP production, we determined the PKA activity by the following method. Naive CD4+ T cells were stimulated with 1 nM CGRP and/or anti-CD3 plus anti-CD28 mAb for 1 h, and PKA activity in cell lysates was measured by a PKA-specific substrate, kemptide. As shown in Fig. 4(A), PKA activity was equally increased by stimulation with CGRP or anti-CD3 plus anti-CD28 mAb. Concomitant stimulation with CGRP and anti-CD3/CD28 mAb further enhanced PKA activity. Since CD3/CD28 co-stimulation induced the elevation of both cAMP levels and PKA activity, we hypothesized that the activation of the cAMP/PKA pathway by CD3/CD28 co-stimulation may play a role to regulate Th2 cytokine production in CD4+ T cells. To confirm this hypothesis, we first examined the effects of PKA inhibitors, H-89 and PKI1422, on IL-4 production from CD4+ T cells stimulated with anti-CD3 mAb or anti-CD3 plus anti-CD28 mAb for 48 h. The PKA inhibitors markedly inhibited IL-4 production from T cells stimulated with anti-CD3 plus anti-CD28 mAb, while they had no effect on IL-4 production from T cells stimulated with anti-CD3 mAb alone (Fig. 4B). These results led to the possibility that the cAMP/PKA pathway plays an important role in IL-4 production from CD4+ T cells via CD3/CD28 co-stimulation, but not via CD3 stimulation. On the other hand, the PKA inhibitors did not completely suppress IL-4 production by CD3/CD28 co-stimulation, suggesting that the cAMP/PKA pathway is a major signal transduction pathway for IL-4 production in naive CD4+ T cells; other pathways, such as the Ca2+/calcineurin pathway, are also likely to be involved in IL-4 production.

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Fig. 4. Effects of PKA on IL-4 production of naive CD4+ T cells stimulated with anti-CD3 plus anti-CD28 mAb. (A) Increase in PKA activity by CGRP stimulation and/or CD3/CD28 co-stimulation. CD4+ T cells were stimulated with 1 nM CGRP and/or 10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb. T cells were lysed at 1 h after stimulation and the PKA activity in the lysates was determined by radioimmunoassay. Values are means ± SD (n = 4). (B) Inhibitory effects of H-89 and PKI1422 on IL-4 production from CD3/CD28 activated-CD4+ T cells. CD4+ T cells (106 cells/ml) were stimulated with 10 µg/ml anti-CD3 mAb alone or 10 µg/ml anti-CD3 plus 1 µg/ml anti-CD28 mAb in the presence or absence of 20 µM H-89 or 30 µM PKI1422. After 48 h incubation, IL-4 levels in the culture supernatants were determined by ELISA. Values are means ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.
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Involvement of PKA on Th2 differentiation by CD3/CD28 co-stimulation
CD28 ligation on naive CD4+ T cells was shown to promote Th2 differentiation in vivo (28) as well as in vitro (29). Therefore, we studied the effect of H-89 on Th2 polarization by CD3/CD28 co-stimulation. CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 mAb in the presence or absence of H-89. Following a 7-day culture, the cells were re-stimulated with anti-CD3 mAb, and subjected to intracellular staining for IFN-
and IL-4. As shown in Fig. 5, CD3 stimulation alone polarized the Th0 cells toward the Th1 subset, whereas T cells stimulated with both anti-CD3 plus anti-CD28 mAb were polarized toward the Th2 subset. The addition of H-89 to Th0 cells stimulated with anti-CD3 plus anti-CD28 mAb almost completely reversed the effect of the CD28 co-stimulation signal, i.e. IFN-
production was augmented and IL-4 production was suppressed, resulting in marked block of Th2 polarization. On the other hand, concomitant addition of 1 nM CGRP with both anti-CD3 and anti-CD28 mAb to Th0 cells further increased IL-4 production compared to that of the absence of CGRP. These results suggested that the cAMP/PKA pathway mediates IL-4 production in initial T cell activation and is an essential process to generate Th2 responses via the CD28 co-stimulation signal.

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Fig. 5. Effects of H-89 or CGRP on Th1/Th2 polarization of CD4+ T cells. CD4+ T cells (106 cells/ml) were stimulated with 10 µg/ml anti-CD3 mAb and 1 µg/ml anti-CD28 mAb in the presence of either 20 µM H-89 or 1 nM CGRP. After 7 days, the primed CD4+ T cells were re-stimulated with anti-CD3 mAb for 4 h in the presence of 2 µM monensin, and stained for intracellular IFN- and IL-4. Values are means ± SD (n = 67). *P < 0.05, **P < 0.01.
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Regulation of NF-AT activation by the cAMP/PKA pathway in CD3/CD28 stimulation
Having observed the up-regulation of IL-4 gene expression by cAMP/PKA activation, we next studied the downstream signaling of the cAMP/PKA pathway and CD3/CD28. For this purpose, luciferase constructs that detect the activation of NF-AT, NF-
B, CREB, ERK, JNK and p38 signaling pathways were transfected in Jurkat-CD28 cells and then stimulated with anti-CD3 mAb alone or with anti-CD3/CD28 mAb in the presence of db-cAMP. As shown in Fig. 6(A), the stimulation with anti-CD3 plus anti-CD28 mAb increased the transcription activity of all signaling molecules examined at
2- to 5-fold the rate of that obtained by stimulation with anti-CD3 alone. When db-cAMP was added to Jurkat-CD28 stimulated with anti-CD3 mAb, a marked increase in CREB transcription activity was observed (Fig. 6B). This effect would be due to activation by direct phosphorylation of CREB by PKA. Interestingly, when Jurkat-CD28 was stimulated with anti-CD3 plus anti-CD28 mAb in the presence of db-cAMP, luciferase activities of CREB and NF-AT were markedly increased in comparison to those observed in the absence of db-cAMP (Fig. 6C).
To further examine whether the enhanced NF-AT activity was contributed to the activation of PKA pathway, this pathway was blocked with H-89 and expression of dominant-negative PKA (PKA-RG324D) in the NF-AT luciferase assay. As shown in Fig. 7(A), H-89 significantly inhibited NF-AT transcription activity induced by CD3/CD28 co-stimulation, but not with CD3 stimulation alone. Similarly, forced expression of PKA-RG324D also significantly inhibited NF-AT transcription activity activated by CD3/CD28 stimulation (Fig. 7B). Moreover, to address whether the cAMP/PKA pathway regulates NF-AT activation in not only Jurkat cell lines, but also naive CD4+ T cells, a gel shift assay was performed (Fig. 7C). NF-AT-binding activity on CD3/CD28 stimulation was enhanced by CGRP, a physiological activator of the cAMP/PKA pathway, and strongly inhibited by H-89, an inhibitor of the cAMP/PKA pathway. These results indicated that CD3/CD28 stimulation induces the activation of the cAMP/PKA pathway resulting in increased NF-AT transcription activity and that a moderate activator of the cAMP/PKA pathway in the presence of a CD3/CD28 co-stimulation signal is able to further enhance NF-AT activity to a much greater extent.

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Fig. 7. PKA regulated NF-AT activity stimulated with anti-CD3 plus anti-CD28 mAb. (A) An aliquot of 5 µg pNF-AT72-luc (NF-AT) was transfected into Jurkat-CD28 cells. The cells were stimulated with 0.1 µg/ml anti-CD3 mAb (OKT3) and 1 µg/ml anti-CD28 mAb in the presence or absence of 20 µM H-89 for 16 h. (B) An aliquot of 5 µg pNF-AT72-luc was co-transfected with 15 µg pCMVT-TK neo vector (mock) or pCMVT-TK neo PKA-RG324D (PKA-RG324D) into Jurkat-CD28 cells. The cells were stimulated with 0.1 µg/ml anti-CD3 mAb and 1 µg/ml anti-CD28 mAb. Following the stimulation, the emitted light was expressed as relative light units. The data represents means ± SD (n = 4). **P < 0.01. (C) Gel shift assay with NF-AT oligonucleotides as a probe. Naive CD4+ T cells were stimulated with anti-CD3 mAb or anti-CD3/anti-CD28 mAb in the presence or absence of CGRP or H-89 for 6 h and nuclear extracts were purified. An aliquot of 5 µg of protein was analyzed in a gel shift assay. Arrowhead indicates specific bands that bound to the NF-AT oligonucleotide and competition with 66-fold excess of unlabeled NF-AT oligonucleotide shows the specificity of the NF-AT interaction.
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Discussion
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The present study demonstrated that the cAMP/PKA signal transduction pathway is critical for commitment to Th2 differentiation via engagement of CD3 and CD28. Moreover, the cAMP/PKA pathway is further activated by CGRP in the presence of a CD3/CD28 co-stimulation signal; this process thereby induces the up-regulation of IL-4 production, and the down-regulation of both IL-2 and IFN-
production from mouse naive CD4+ T cells, resulting in the promotion of Th2 differentiation in the immune system.
cAMP accumulation by CGRP has been reported in certain types of cells, including immune cells such as thymocytes (30), splenocytes (31) and Th clones (32). Recently, the CGRP receptor was shown to be composed of CRLR and RAMP1 (33). CRLR is a heptahelical receptor coupled with Gs
and binding of CGRP to its receptors activates adenylyl cyclase (19). Since we have detected the expression of both CRLR and RAMP1 mRNAs in mouse CD4+ T cells (unpublished data), CGRP would induce cAMP elevation and PKA activation by a direct effect via its receptors on CD4+ T cells. In contrast, CD3 and CD28 molecules do not directly couple to adenylyl cyclase, although stimulation of anti-CD3 plus anti-CD28 mAb increased cAMP levels and PKA activity in mouse naive Th cells. Therefore, CD3/CD28 would possess a signal transduction mechanism that induces indirect activation of adenylyl cyclase. One possible candidate of the activator is phospholipase C (PLC)
1. CD3/CD28 engagement provokes rapid increases in both tyrosine phosphorylation and the catalytic activity of PLC
1 (34,35). The activated PLC
1 hydrolyzes phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG stimulate the release of Ca2+ from intracellular stores and activate protein kinase C (PKC) respectively. The increased intracellular free Ca2+ binds to calmodulin and the Ca2+/calmodulin complex activates adenylyl cyclase (36). PKC also activates adenylyl cyclase catalysts by phosphorylation (37,38). Therefore, the cross-linking of CD3 and CD28 would indirectly activate adenylyl cyclase via the PLC
1-mediated signaling pathway in T cells.
Studies on the effects of cAMP/PKA pathway on cytokine production from T cells have reported conflicting results. The consensus is that cAMP and its inducers suppress both T cell proliferation and the expression of Th1 cytokine genes. We also confirmed the inhibition of IL-2 and IFN-
production and T cell proliferation of CD3- or CD3/CD28-stimulated naive Th cells by CGRP or db-cAMP. In contrast, the function of the cAMP/PKA pathway as regards production of Th2 cytokines appears to be affected in different ways, depending on cell type, the method of cell stimulation and the concentration of cAMP or its inducers. Cholera toxin, which ribosylates the
subunit of the G stimulatory protein, leads to an accumulation of cAMP, and cAMP-elevating agents have no effect on IL-4 production and proliferation of Th clones (39,40). On the other hand, it was reported that a high concentration of db-cAMP (1 mM) suppresses both mRNA accumulation and secretion of IL-4 from human T cells stimulated with anti-CD3 plus anti-CD28 antibodies (41). Another report demonstrated that cAMP up-regulates IL-4 and IL-5 production from CD4+ T cells (42). In that study, mouse CD4+ T cells were stimulated with concanavalin A, IL-2 and irradiated splenocytes for 3 days, and then re-stimulated with phorbol 12-myristate 13-acetate and ionomycin or with anti-CD3 mAb. The conclusion of that particular study was that, in activated CD4+ T cells, cAMP induces a switch of lymphokine production towards a Th2-like phenotype through regulation at the transcriptional level. However, the effect of cAMP in primary naive CD4+ T cells stimulated with anti-CD3/CD28 antibodies has not been studied to date. We clarified for the first time that CGRP (in a concentration that induced
2-fold increase in the level of cAMP, compared to that of the control) and db-cAMP (at a concentration <5 µM) reduced IL-2 and IFN-
production, but augmented IL-4 production in anti-CD3/CD28 mAb-stimulated naive CD4+ T cells.
As we have shown here, when compared to CD3 stimulation alone, CD3/CD28 co-stimulation up-regulated the transcription activities of NF-AT, NF-
B and CREB, as well as activated the ERK and p38 MAPK pathways in Jurkat-CD28 cells. The most interesting finding was that NF-AT transcription activity was markedly increased by db-cAMP in the presence of a CD3/CD28 co-stimulation signal. NF-AT is localized in the cytoplasm of resting T cells. TCR engagement activates the Ca2+/calmodulin-dependent protein phosphatase calcineurin and calcineurin dephosphorylates NF-AT, allowing NF-AT to translocate from the cytoplasm to the nucleus (4345). It has been reported that PKA suppress NF-AT translocation into the nucleus by phosphorylation (46). However, our study indicated that NF-AT transcription activity was activated by db-cAMP in the presence of CD3/CD28 co-stimulation; furthermore, the inhibition of PKA activity by the specific inhibitors suppressed NF-AT activity. From these results, we suggest that the cAMP/PKA pathway, activated by physiological stimulation and moderate concentrations of PKA activators, transduced a positive regulation signal enhancing NF-AT transcription activity. Recently, glycogen synthase kinase 3 (GSK3) was reported to regulate the nuclear export of NF-AT in T cells (47,48). Moreover, Fang et al. demonstrated that PKA could inhibit GSK3 activity by phosphorylation in some cell lines (49). Therefore, it is possible to infer that activated PKA might inhibit the nuclear export of NF-AT by inactivating GSK3 in CD4+ T cells and that a longer presence of NF-AT in the nucleus might augment the transcription of Th2 cytokine genes.
In our study, PKA inhibitor H-89 and dominant-negative PKA failed to inhibit NF-AT activation completely in CD3/CD28-stimulated T cells, whereas H-89 and PKI1422 markedly inhibited IL-4 production and promoted Th1 differentiation. These results suggest the existence of a transcription factor(s) other than NF-AT that is regulated by PKA for IL-4 production. We showed that CD3/CD28 co-stimulation induced a more significant activation of the p38 MAPK pathway and the CREB pathway than did CD3 stimulation alone. p38 MAPK is known to be an important integrator of TCRCD28 stimulation of cell proliferation and cytokine production in primary naive Th cells (50). Recently, it was reported that H-89 inhibited the phosphorylation of p38 MAPK by a cAMP-elevating agent, forskolin, whereas inhibitors of PKC, p70 (S6K) and phosphatidylinositol 3-kinase were ineffective in this regard (51). Moreover, Chen et al. showed that p38 MAPK is phosphorylated by the cAMP-dependent mechanism and that activated p38 MAPK phosphorylates GATA-3, resulting in promoting the production of both IL-5 and IL-13 in Th2 cells (52). GATA-3 is a critical transcription factor of Th2 cytokine gene expression, including the genes for IL-5, IL-13 as well as IL-4, via distal enhancers (53). It remains unclear whether or not PKA phosphorylates p38 MAPK in Th0 cells; however, it is possible that the cAMP/PKA pathway activates p38 MAPK from our luciferase assays and cytokine array experiments. Moreover, our preliminary experiment showed an increase in GATA-3 mRNA by neuropeptide stimulation (unpublished data).
The signal transduction pathway responsible for CREB phosphorylation in T cells also remains unidentified. The candidates are Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which is highly expressed in T cells, and PKA. In studies using CaMKIV knockout mice, memory T cells showed reduced CREB phosphorylation and secreted less amounts of IL-2, IFN-
and IL-4, compared to cells from wild-type mice, by stimulation of CD3/CD28 (54). These results suggest that CaMKIV-dependent CREB phosphorylation is important for the regulation of cytokine production in memory T cells. In contrast, naive T cells from CaMKIV-deficient mice are likely to normally produce cytokines and induce phosphorylation of CREB by CD3/CD28 co-stimulation, suggesting that PKA might regulate the phosphorylation of CREB in naive T cells.
In conclusion, the cAMP/PKA pathway transduces a critical signal for the regulation of IL-4 production of naive Th cells stimulated with CD3/CD28. The signaling pathway modulates transcription factors such as NF-AT and CREB, and the activation of the p38 MAPK pathway. The results of the present study emphasize the importance of the cAMP/PKA pathway for signal transduction in naive Th cells stimulated with CD3/CD28.
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Acknowledgements
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We thank Dr Hiromichi Ishikawa (Keio University, Tokyo, Japan) for DO 11.10 transgenic mice, Dr Kenichi Arai (University of Tokyo, Tokyo, Japan) for pNF-AT72-luc, Dr Tatsuhiko Furukawa (Kagoshima University, Kagoshima, Japan) for p(Ig
)3-luc and Dr Tania H. Watts (University of Toronto, Toronto, Ontario, Canada) for pCMVT-TK neo PKA-RG324D. This work is supported by Grants-in-Aid from the Ministry of Education, Culture, Sports Science and Technology of Japan, the Ministry of Health and Welfare of Japan, and the Long-range Research Initiative by the Japan Chemical Industry Association.
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Abbreviations
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AP-1activated protein-1
APCantigen-presenting cell
CaMKIVcalmodulin-dependent protein kinase IV
CGRPcalcitonin gene-related peptide
CREBcAMP response element binding protein
CRLRcalcitonin receptor-like receptor
DAGdiacylglycerol
db-cAMPdibutyryl-cAMP
ERKextracellular signal-regulated kinase
GSK3glycogen synthase kinase 3
H-89N-[2-((p-bromocinnamyl)aminoethyl)]-5-isoquinolinesulfonamide
JNKc-Jun N-terminal kinase
IP3inositol-1,4,5-trisphosphate
MAPKmitogen-activated protein kinase
OVAovalbumin
PKAprotein kinase A
PKCprotein kinase C
PLCphospholipase C
RAMP1receptor activity modifying protein 1
TNFtumor necrosis factor
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