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Originally published In Press as doi:10.1074/jbc.M510653200 on March 13, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12421-12427, May 5, 2006
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The cGMP/Protein Kinase G Pathway Contributes to Dihydropyridine-sensitive Calcium Response and Cytokine Production in TH2 Lymphocytes*

Bruno Gomes{ddagger}, Magali Savignac{ddagger}1, Marilena Djata Cabral{ddagger}1, Pierre Paulet{ddagger}, Marc Moreau§, Catherine Leclerc§, Robert Feil, Franz Hofmann, Jean-Charles Guéry{ddagger}, Gilles Dietrich{ddagger}, and Lucette Pelletier{ddagger}2

From the {ddagger}INSERM, 1U563, Centre de Physiopathologie de Toulouse Purpan, F-31024 Toulouse Cedex 3, France, §CNRS, Unite Mixte de Recherche 5547, Université Paul-Sabatier, F-31062 Toulouse Cedex 4, France, and Institut für Pharmakologie und Toxikologie der Technischen Universität München, G-80802 München, Germany

Received for publication, September 29, 2005 , and in revised form, January 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
T-helper cells of type 2 (Th2) constitute a distinct subset of CD4+ T lymphocytes (1) involved in the elimination of extracellular pathogens (2). However, exacerbated Th2 responses are also associated with allergic manifestations, including asthma. Specific signaling pathways are dedicated to cytokine production by Th2 cells (3–8). Calcium-dependent signaling pathway is necessary and sufficient for Il4 gene expression. Transfection of non-IL-4-producing T lymphocytes with constitutively active calcineurin induces Il4 gene promoter activity (9, 10). Calcium-independent pathways also contribute to Th2 cell differentiation and IL-4 production upon T cell receptor (TCR)3 stimulation. For example, the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) facilitates Gata-3-mediated chromatin remodeling at Th2 cytokine loci (11).

TCR engagement results in phosphorylation events and scaffolding of various adapters and enzymes. This TCR signaling complex, called signalosome, integrates signaling pathways into transcriptional events (12). One of these pathways is orchestrated by the second messenger calcium. Activated phospholipase C {gamma}1 cleaves phosphatidylinositol (4, 5)-biphosphate into inositol (1, 4, 5)-triphosphate (IP3) and diacylglycerol (DAG). DAG leads to protein kinase C and Ras-dependent mitogen-activated protein kinase activation (13), whereas IP3 mobilizes intracellular calcium stores. Calcium mobilization is followed by a calcium inflow through calcium release-activated calcium (CRAC) channels (reviewed in Ref. 14) and subsequent activation of the nuclear factor of activated T cells (NFAT). Once dephosphorylated by calcineurin, a calcium-activated phosphatase, NFAT translocates to the nucleus and binds together with other transcription factors to target genes, including cytokine genes (discussed in Ref. 12).

Intracellular calcium concentration was reported to be higher in resting Th2 lymphocytes than in other T cell subsets. Only low calcium concentration increase was measured in activated Th2 lymphocytes (3–5, 15–17), suggesting an original [Ca2+]i regulation mechanism in Th2 lymphocytes. We have previously shown that in Th2 cells TCR engagement involved dihydropyridine receptor (DHPR)-dependent calcium inflow (18). DHPR were also detected in other immune cells including natural killer (NK) cells (19), dendritic cells (20), B lymphocytes (21, 22), and naive T cells (23, 24). In B cells, DHPR-dependent [Ca2+]i increase subsequent to antigen receptor stimulation was shown to be mediated by cGMP (21).

In this study, we wondered whether cGMP was involved in the control of calcium response and cytokine production in Th2 lymphocytes. Our data show that TCR-dependent cGMP production results in PKG activation, which promotes DHP-sensitive calcium inflow and IL-4 synthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Reagents—7–12-week-old OVA (323–339)-specific DO11.10 TCR transgenic BALB/c mice (25), BALB/c mice (Janvier Ets, Le Genest St. Isle, France), and 4–6-week-old PKG1-deficient (PKG1–/–) mice (26) on a 129/Sv genetic background were cared for in the animal facility of the Regional Committee on animal experimentation. All aspects of animal care were approved by our institutional review board for animal experimentation. The calcium channel antagonist R(+) Bay K 8644, sodium nitroprusside (SNP), 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) were purchased from Sigma. The hamster anti-mouse TCR H57–597 mAb (27) was from BD Biosciences. Nicardipine was from Novartis (Basel, Switzerland). LY-83,583, an inhibitor of soluble guanylate cyclase, and spermine NONOate, a nitric oxide (NO) donor, were from Calbiochem (La Jolla, CA). The inhibitors of PKG(KT5823 and Rp-8-pCPT-cGMPS), of cAMP-dependent protein kinase (H-89), of PDE3 (trequinsin), of PDE4 (Ro-20-1724), and of PDE5 (zaprinast) were purchased from Calbiochem and cyclosporin A from Sandoz (Rueil Malmaison, France).

Th2 Cell Differentiation—CD4+ T cells from DO11.10 mice (0.5 x 106 cells/ml) were suspended in RPMI 1640 supplemented with 10% fetal calf serum (ATGC, Noisy Le Grand, France), 1% pyruvate, 1% non-essential amino acids, 2 mM glutamine, 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 50 µM beta-mercaptoethanol. Cells were stimulated with irradiated BALB/c spleen cells and OVA 323–339 peptide (0.3 µM). For Th2 skewing, IL-4 (10 ng/ml) + anti-interferon {gamma} Ab (10 µg/ml) were added to the primary culture. CD4+ T cells from wild-type and PKG1–/– mice (0.5 x 106 cells/ml) were stimulated on plate-bound anti-TCR (3 µg/ml) + anti-CD28 mAbs (2 µg/ml) in Th2-polarizing conditions as above. On day 3–4 after stimulation, cells were split 1:3 with fresh medium. Viable T cells were recovered on Ficoll-Hypaque and restimulated in the same conditions every 7 days for 3 weeks.

To analyze cytokine production, T cells were distributed (2.5 x 104 cells/well) into 96-well flat-bottom plates containing either antigen-presenting cells (2.5 x 104 cells/well) + OVA peptide in the absence of exogenous cytokines and antibodies or plate-bound anti-TCR mAb (1 µg/ml) (28). Cytokine release was quantified by ELISA in supernatants collected 24 h later. cGMP intracellular concentration was determined by using ELISA according to the manufacturer's instructions (cGMP Enzymeimmunoassay Biotrak System; Amersham Biosciences).

Analysis of Intracellular Calcium Concentration—T cells were preincubated for 12 h with or without inhibitors, washed, loaded with 5 µM Fura-2 AM (28, 29), and stimulated with either soluble anti-TCR mAb (1 µg/ml) + anti-CD28 mAb (1 µg/ml) or 8-Br-cGMP (200 µM) or 8-Br-cAMP (200µM) or SNP (150µM) or spermine NONOate (300µM). [Ca2+]i increase was recorded at the single cell level by microspectrofluorometry as previously described (29). Images were recorded every 4 s for at least 400 s. Ionomycin was used as a positive control for each sample. Calcium concentration was calculated according to Ref. 30.

Imaging of NFAT Translocation—NFAT nuclear translocation was evaluated by confocal microscopy as previously described (31, 32). Three-round primed Th2 cells were cultured on plate-bound anti-TCR (5 µg/ml) + anti-CD28 (3 µg/ml) mAbs in the presence or in the absence of PKG inhibitors (KT5823 or Rp-8-pCPTcGMPS). Alternatively, cells were stimulated with either spermine NONOate (1 mM) or 8-BrcGMP (200 µM) for 4 h. Cells were then collected and plated onto Lab-Tek chambers (Calbiochem). They were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 10 min. Staining was performed with mouse anti-NFATc1 mAb (BD Biosciences) for 45 min. After extensive washing, NFATc1 was revealed using Alexa-488-labeled goat anti-mouse antibody (Molecular Probes, Eugene, OR). Cells were additionally treated with RNaseA (Sigma) for 10 min, and nuclei were stained with propidium iodide. Samples were mounted and examined with a Carl Zeiss LSM 510 confocal microscope using a x63 Plan-Apochromat objective (1.4 oil). We evaluated two parameters: the nuclear versus cytoplasmic NFAT ratio, analyzed by the ImageJ analysis software (NIH), and the percentage of cells that display NFAT nuclear translocation (defined as the frequency of cells in which nuclear/cytoplasmic NFAT ratio was superior to mean + 3 S.D. of the ratio in unstimulated cells).

IL-4 mRNA Quantification—Total RNA was isolated from 5 x 106 cells using the SV total RNA isolation system (Promega, Madison, WI) and reverse transcribed to cDNA using poly(dT) and Moloney murine leukemia virus reverse transcriptase (Invitrogen). IL-4 and hypoxanthine phosphoribosyl transferase (HPRT) mRNA were measured by real-time quantitative PCR using an ABI Prism 7000 sequence detection system. PCR was performed with the PCR SYBR Green sequence detection system (PerkinElmer). Primers for IL-4 were 5'-CGGTGAACTGAGGAAACTCTGTAG-3' and 5'-CACGGTGCAGCTTCTCAGTG-3' and for HPRT 5'-CTGGTGAAAAGGACCTCTCG-3' and 5'-TGAAGTACTCATTATAGTCAAGGGCA-3'. IL-4 mRNA was normalized to HPRT mRNA and quantified relative to mRNA expression in unstimulated Th2 cells.

Flow Cytometric Analysis of IL-4 and IL-5 Intracellular Cytokine Synthesis—Th2 cells were stimulated on plate-bound anti-TCR mAb in the absence or in the presence of either nicardipine or LY-83,583 (10 µM). After 12 h of culture, cells were collected, resuspended at 106/ml, and stimulated with phorbol 12-myristate 13-acetate (50 ng/ml, Sigma) + ionomycin (0.5 µg/ml, Sigma) for 4 h. Two hours before cell harvest, 10 µg/ml of brefeldin A (Sigma) was added. Cells were then fixed with 4% paraformaldehyde (Fluka Chemie, Buchs, Switzerland). Intracytoplasmic staining was performed as previously described (33). Briefly, after washing and 10 min of incubation in saponin medium alone, cells were incubated with phycoerythrin-conjugated rat anti-mouse IL-4 (11B11) or anti-mouse IL-5 (TRFK5) antibodies (BD Biosciences) for 30 min at 4 °C. Cells were washed first in saponin buffer and then with phosphate-buffered saline to allow membrane closure. Data were collected on 20,000 CD4+ cells on an XL Coulter cytometer (Coultronics, Margency, France) and analyzed using the CellQuest software (BD Biosciences).

PCR Analysis—PCR conditions were 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min for 21 (beta actin) or 40 cycles (for PKG1). Primers for beta actin were 5'-TGGAATCCTGTGGCATCCATGAAAC and 5'-TAAAACGCAGCTCAGTAACAGTCCG and for PKG1 were 5'-ATTGTATGTACCCCGTGGAAT and 5'-TTGGTGAGTCTTCTCGGGTAA.

Western Blot Analysis—5 x 106 cells were lysed for 15 min in phosphate-buffered saline containing 4 mM EDTA, 1% Triton, 150 mM NaCl, 20 mM Tris-HCl, pH 8, protease inhibitor mixture (1 tablet/6 ml of buffer (Roche Diagnostics), aprotinin, leupeptin, and pepstatin (1 µg/ml, each), 1 mM phenylmethylsulfonyl fluoride, 5 mM tetrasodium pyrophosphate, 1 mM orthovanadate sodium, and 1 mM NaF. Lysates were spun at 13,000 rpm to remove insoluble material. Protein content was measured using a detergent-compatible protein assay (Bio-Rad). Samples (40 µg/lane) were subjected to 7.5% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were saturated and incubated overnight with anti-PKG1{alpha}/beta Ab (Santa-Cruz). Blots were developed with a rabbit anti-goat Ab and then a horseradish peroxidase-conjugated anti-rabbit Ab and the enhanced chemiluminescent system (Amersham Biosciences).

Statistical Analysis—Results are expressed as the mean ± S.D. Overall differences between variables were initially shown by using a Kruskal-Wallis test and subsequently confirmed by the Mann-Whitney U test.


Figure 1
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  		FIGURE 1.
cGMP is implicated in Th2 lymphocyte calcium response and Il4 gene expression. A, DO11.10 Th2 cells, collected 7 days after the third stimulation, were stimulated on plate-bound anti-TCR mAb (1 µg/ml) for 5 min. Cells were then lysed, and intracellular cGMP content was determined by ELISA (**, p < 0.005 when compared with unstimulated cells). B, Th2 lymphocytes were pretreated or not with DHPR antagonist (nicardipine, 10 µM) overnight. They were then washed and loaded with 5 µM Fura-2AM. Basal intracellular calcium concentration was recorded at the single cell level for ~60 s. The arrow indicates the addition of either 8-Br-cGMP (cGMP, 200µM) or 8-Br-cAMP (cAMP, 200 µM). Each curve represents the mean of 25–40 cells analyzed during one experiment and is representative of four independent experiments. C, Th2 cells were cultured with 8-Br-cGMP (cGMP, 200 µM) for 4 h. They were then stained with anti-NFATc1 antibody. Cells unstimulated or stimulated with plate-bound anti-TCR + anti-CD28 mAbs for 4 h were used as negative and positive controls, respectively. D, the histogram summarizes, for all cells within each field examined, the ratios between nuclear and cytoplasmic NFAT fluorescence (mean ± S.E., at least 100 cells for each condition; *, p < 0.01 when compared with unstimulated cells). E, Th2 cells were incubated for 6 h with 8-Br-cGMP (200 µM) in the presence or in the absence of cyclosporin A (CsA, 0.1 µg/ml). Th2 cells stimulated on plate-bound anti-TCR mAb (1 µg/ml) were used as positive control. IL-4 mRNA was quantified by real-time quantitative PCR and normalized to HPRT mRNA. Values were standardized relative to mRNA levels in unstimulated Th2 cells. Values are expressed as mean ± S.D. of two experiments performed in triplicate (*, p < 0.01; **, p < 0.005).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
cGMP Induces a Calcium Response, NFAT Translocation to the Nucleus, and IL4 Gene Expression in Th2 Lymphocytes—In B lymphocyte, cGMP was shown to be responsible for DHPR activation and subsequent calcium influx upon B cell receptor stimulation (21). Because we have recently shown that Th2 cells expressed DHPR (18), we investigated the role of cGMP in TCR-induced DHP-sensitive calcium response in these cells. DO11.10 cells were differentiated along the Th2 pathway by three weekly stimulations with antigen-presenting cells loaded with the OVA 323–339 peptide in the presence of IL-4 + anti-interferon {gamma} antibody (18). TCR stimulation for 2 (not shown) or 5 min (Fig. 1A) triggered a 2- to 4-fold increase in intracellular cGMP concentration, depending on the experiments. As assessed by microspectrofluorometry on Fura-2AM-loaded Th2 lymphocytes, cell-permeant 8-Br-cGMP promoted a sustained increase in [Ca2+]i (Fig. 1B). Abolition of the cGMP-induced [Ca2+]i increase in the absence of extracellular calcium (not shown) or in the presence of the DHPR antagonist (nicardipine) pointed out that cGMP promoted a DHP-sensitive calcium inflow (Fig. 1B). 8-Br-cAMP, another cyclic nucleotide known to promote Th2 cytokine production, had no effect on Th2 cell calcium response (Fig. 1B).


Figure 2
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  		FIGURE 2.
Guanylate cyclases and DHPR are involved in calcium signaling and Th2 cytokine production. A, Th2 lymphocytes were or were not treated with DHPR antagonist (nicardipine, 10 µM) or guanylate cyclase inhibitor (LY-83,583, 10 µM) overnight and were loaded with 5 µM Fura-2AM. Basal intracellular calcium concentration was recorded at the single cell level for ~60 s. The arrow indicates addition of anti-TCR (1 µg/ml) + anti-CD28 (1 µg/ml) mAbs. Experiments were performed in normal medium containing 0.5 mM calcium in the absence of inhibitor (medium w/o inhibitor) and in Ca2+-depleted extracellular medium ([Ca2+]ext = 0). Each curve represents the mean of 15–45 cells analyzed during one experiment and is representative of three independent experiments. B and C, Th2 cells were stimulated on plate-bound anti-TCR mAb (1 µg/ml) for 24 h with increasing concentrations of nicardipine or LY-83,583. IL-4 (B) and IL-5 (C) supernatant contents were determined by ELISA. Values are expressed as mean ± S.D., of two experiments performed in triplicate (*, p < 0.01 when compared with cells stimulated in the absence of drugs). D, Th2 lymphocytes were stimulated overnight on plate-bound anti-TCR mAb (1 µg/ml) in the absence or in the presence of either LY-83,583 (10 µM) or nicardipine (10 µM). Intracellular staining is shown of IL-4 (upper panels) and IL-5 (lower panels) in the absence (white areas) or in the presence of drugs (gray areas). One representative experiment of three is shown.

 
We then wondered whether the 8-BrcGMP-induced [Ca2+]i increase was sufficient to induce NFAT translocation to the nucleus. Th2 cells incubated for 4 h in the presence of 8-Br-cGMP exhibited a significant NFAT translocation as shown by confocal microscopy (Fig. 1, C and D). Depending on the experiments, NFAT was found to be translocated to the nucleus in 2–11% of resting Th2 lymphocytes, 30–50% of cGMP-treated cells, and 41–75% of anti-CD3 + anti-CD28-stimulated lymphocytes (not shown). Because Il4 gene expression is regulated by the calcium-dependent calcineurin pathway (9), we assessed whether cGMP triggered Il4 gene expression. 8-Br-cGMP induced a strong IL-4 mRNA increase. The inhibitory effect of cyclosporin A testified to the involvement of calcium-calcineurin pathway (Fig. 1E). Contrasting with TCR-stimulated Th2 cells, IL-4 was not detected at the protein level in supernatants collected 24 h after cGMP stimulation (not shown). The lower calcium response (Fig. 1, C and D) and Il4 gene expression (Fig. 1E) induced by cGMP could account for this difference.

An Inhibitor of Guanylate Cyclases, LY-83,583 Strongly Reduces Calcium Response and Type-2 Cytokine Production upon TCR Stimulation—[Ca2+]i increase induced by stimulation of Th2 lymphocytes with a combination of both anti-TCR and anti-CD28 mAbs was abolished by LY-83,583 (Fig. 2A). The calcium response was also inhibited either in the absence of extracellular calcium or in the presence of a DHPR antagonist (nicardipine) (Fig. 2A). Data were confirmed with another DHPR antagonist, R(+) BayK 8644 (not shown). Treatment of TCR-activated Th2 cells with LY-83,583 as well as nicardipine resulted in dose-dependent inhibition of IL-4 (Fig. 2B) and IL-5 (Fig. 2C) release. Intracellular IL-4 and IL-5 staining revealed that LY-83,583 and nicardipine dramatically reduced the number of IL-4- and IL-5-producing cells (Fig. 2D).


Figure 3
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  		FIGURE 3.
NO donors induce cGMP production, calcium response, and IL-4 expression in Th2 lymphocytes. A, DO11.10 Th2 cells were stimulated for 5 min either on plate-bound anti-TCR mAb (1 µg/ml), sodium nitroprusside (SNP, 150 µM), or NONOate (300 µM) in the presence or in the absence of the PDE5 inhibitor zaprinast (10 µM). Cells were lysed, and intracellular cGMP content was determined by ELISA and normalized to total protein contents (*, p < 0.01 when compared with unstimulated cells). B, modulation was assessed of intracellular calcium concentration by NONOate or SNP (arrow, 300 and 150 µM, respectively) in Th2 cells pretreated or not with nicardipine or LY-83,583 (10 µM). 3 mM EGTA-containing extracellular medium was designated [Ca2+]ext = 0. Each curve represents the mean of 25–40 cells analyzed during one experiment and is representative of four independent experiments. C, Th2 cells were incubated with SNP (150 µM) for 6 h in the presence or in the absence of cyclosporin A (CsA, 0.1 µg/ml). Th2 cells stimulated on plate-bound anti-TCR mAb (1 µg/ml) were used as a positive control. IL-4 mRNA was quantified by real-time PCR and normalized to HPRT mRNA. Values were standardized relative to mRNA levels in unstimulated Th2 cells. Values are expressed as mean ± S.D. of two experiments performed in triplicate (*, p < 0.01; **, p < 0.005). D, Th2 lymphocytes were cultured with NONOate (1 mM) for 4 h and stained with anti-NFATc1 antibody. The histogram summarizes, for all cells within each field examined, the ratios between nuclear and cytoplasmic NFAT fluorescence (mean ± S.E., at least 100 cells for each condition, three experiments performed in duplicate; *, p < 0.01 when compared with unstimulated cells). E, Th2 cells from PKG1–/– and +/+ mice were stimulated with NONOate (0.3 or 1 mM) for 24 h, and IL-4 production was measured by ELISA. Values are expressed as mean ± S.D., of stimulation assays done in triplicate on Th2 cells individually differentiated from three mice/group (*, p < 0.01, PKG–/– versus PKG+/+).

 
NO Donors Generate cGMP, Increase [Ca2+]i, Induce NFAT Nuclear Translocation and IL-4 Production—The role of guanylate cyclases was further examined by using NO donors that activate soluble guanylate cyclases (34). SNP induced a 2-fold increase in intracellular cGMP concentration, in the same range as TCR stimulation did (Fig. 3A). NONOate, a more potent NO donor than SNP, induced a 10-fold increase in intracellular cGMP concentration when compared with unstimulated cells (Fig. 3A). Zaprinast, an inhibitor of phosphodiesterase 5 (known to specifically promote cGMP degradation), did not enhance cGMP production, indicating that cGMP degradation did not minimize cGMP production (Fig. 3A). Both NO donors, SNP and NONOate, triggered a sustained [Ca2+]i increase (Fig. 3B). However, the calcium response induced by NONOate was more abrupt and higher than the one induced by SNP. The guanylate cyclase inhibitor LY-83,583 delayed and reduced SNP-induced calcium response (Fig. 3B). The SNP-induced [Ca2+]i increase was inhibited either in the absence of extracellular calcium or in the presence of DHPR antagonist nicardipine (Fig. 3B). SNP induced Il4 gene expression, which was abolished in the presence of cyclosporin A (Fig. 3C). We failed to detect any SNP-induced IL-4 protein secretion (not shown). According to its high potency to enhance cGMP production, NONOate induced NFAT nuclear translocation (Fig. 3D) in 60–70% of cells (not shown) and detectable IL-4 production (Fig. 3E). By contrast, the dose-dependent increase in IL-4 production triggered by NONOate was not observed in Th2 cells from PKG1–/– mice, suggesting a prominent role of PKG in mediating NONOate effect (Fig. 3E). Altogether, these data showed that guanylate cyclase activation resulted in cGMP production and a subsequent DHP-sensitive extracellular calcium inflow, responsible for IL-4 expression.


Figure 4
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  		FIGURE 4.
PKG1–/– Th2 lymphocytes display impaired calcium response and IL-4 production. A, cDNA of 3-week stimulated Th2 lymphocytes from PKG1+/+ and –/– mice were amplified with PKG1- and beta actin-specific primers. B, lysates of PKG1+/+ and –/– Th2 cells were immunoblotted with anti-PKG1{alpha}/beta Ab. C, intracellular calcium concentration was measured in PKG1–/– and +/+ Th2 cells loaded with Fura-2-AM and stimulated with anti-TCR (1 µg/ml) + anti-CD28 (1 µg/ml) mAbs (arrow). Each curve represents the mean of at least 40 cells and is representative of cells differentiated from 3 mice/group and tested in duplicate. D, NFAT translocation was assessed in PKG1–/– and +/+ Th2 lymphocytes stimulated on plate-bound anti-TCR + anti-CD28 mAbs for 4 h. The histogram summarizes, for each mouse, the ratios between nuclear and cytoplasmic NFAT fluorescence (mean ± S.E., at least 100 cells for each condition; *, p < 0.01 when compared with unstimulated cells). E, Th2 lymphocytes from PKG1–/– and +/+ littermates were stimulated on plate-bound anti-TCR mAb (1 µg/ml) for 24 h, and IL-4 content was determined in supernatant by ELISA. Values are expressed as mean ± S.D. of three experiments (from three litters comprising 6 PKG–/– versus 8 PKG+/+ mice) performed in duplicate (*, p <0.01).

 
Calcium Signaling and IL-4 Secretion Are Defective in Th2 cells from PKG Null Mice—PKG or cyclic nucleotide phosphodiesterases (PDE) are putative targets for cGMP. In turn, PDE enzymes regulate cAMP and cGMP levels. As shown in Fig. 4A, PKG1 mRNA was found in Th2 lymphocytes. PCR specificity was demonstrated by the absence of amplification in Th2 cells from PKG1–/– mice. PKG1 protein was detected in PKG1+/+ but not in PKG–/– Th2 cells (Fig. 4B). Then we investigated the involvement of PKG in TCR-induced calcium response and subsequent IL-4 production. CD4+ T cells from PKG1–/– mice and PKG1+/+ littermates were differentiated along the Th2 pathway by three weekly polyclonal stimulations in the presence of IL-4 + anti-interferon {gamma} antibody. Calcium response in PKG1–/– Th2 lymphocytes was dramatically reduced as compared with PKG1+/+ Th2 cells (Fig. 4C). NFAT nuclear translocation was also diminished in Th2 lymphocytes from PKG1–/– mice (Fig. 4D). Only 8–25% of PKG1–/– versus 60–78% of PKG1+/+ Th2 cells displayed NFAT nuclear translocation upon TCR stimulation. IL-4 production was severely reduced in Th2 lymphocytes from PKG1–/– mice as compared with their PKG1+/+ counterparts (Fig. 4E).


Figure 5
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  		FIGURE 5.
PKG plays a key role in Th2 lymphocyte calcium response and cytokine production. A, DO11.10 Th2 lymphocytes were pretreated overnight with PDE3 (Trequinsin, 10 nM) or PDE4 (Ro-20-1724, 10 µM) inhibitors. Cells were loaded with 5 µM Fura-2AM. Basal intracellular calcium concentration was recorded at the single cell level for ~60 s. The arrow indicates addition of anti-TCR (1 µg/ml) + anti-CD28 (1 µg/ml) mAbs. Each curve represents the mean of 20–35 cells analyzed during one experiment and is representative of three independent experiments. B, DO11.10 Th2 lymphocytes were pretreated overnight with cAMP-dependent protein kinase (H-89, 5 µM) or with PKG (KT5823, 5 µM; Rp-8-pCPT-cGMPS, 200 µM) inhibitors. Intracellular calcium concentration was recorded as in panel A. C, Th2 lymphocytes were stimulated with plate-bound anti-TCR + anti-CD28 mAbs in the presence or in the absence of Rp-8-pCPT-cGMPS (200 µM). They were then stained with anti-NFATc1 antibody. The histogram summarizes, for all cells within each field examined, the ratios of nuclear to cytoplasmic NFAT fluorescence (mean ± S.E., at least 45 cells for each condition; *, p < 0.01). D, DO11.10 Th2 cells were stimulated on plate-bound anti-TCR mAb (1 µg/ml) for 24 h with increasing amounts of KT5823 (ranging from 1 to 10 µM), Rp-8-pCPT-cGMPS (1–300 µM), trequinsin (10 nM), or Ro-20-1724 (10 µM). IL-4 content was determined by ELISA. Values are expressed as mean ± S.D., of two experiments performed in triplicate (*, p < 0.01 when compared with cells stimulated in the absence of drugs).

 
PKG, but Not PDE, Inhibitors Suppress Ca2+ Signaling and Th2 Cytokine Production—To assess whether PKG or PDE were important for calcium signaling in differentiated Th2 cells, inhibitors of PKG and PDE were tested on fully differentiated DO11.10 Th2 lymphocytes. cAMP concentration, which favors Th2 cytokine production, is closely regulated by PDE3 and PDE4 (35, 36); therefore, we tested increasing concentrations of PDE4 and PDE3 inhibitors. PDE4 (Ro-20-1724, 10 µM) and PDE3 (trequinsin, 10 nM) inhibitors, used at the highest non-toxic doses, did not prevent the [Ca2+]i increase (Fig. 5A) or IL-4 production induced by TCR stimulation (Fig. 5D). By contrast, TCR-induced calcium response was robustly diminished by PKG inhibitors (Fig. 5B). In agreement with our previous results showing that cAMP did not mediate a calcium influx (Fig. 1B), the neutralization of cAMP-dependent protein kinase activity did not alter TCR-induced calcium increase (Fig. 5B), indicating that cAMP-dependent protein kinase was not involved in the control of calcium response.

As assessed by confocal microscopy, PKG inhibitors (Rp-8-pCPT-cGMPS, Fig. 5C; KT5823, not shown) suppressed NFAT translocation to the nucleus. Rp-8-pCPT-cGMPS reduced the percentage of cells with NFAT translocated to the nucleus from 68 ± 6% to 28 ± 2%. PKG inhibitors suppressed IL-4 (Fig. 5D) and IL-5 (not shown) synthesis. Interestingly, Rp-8-pCPT-cGMPS did not affect IL-4 production by PKG1–/– Th2 lymphocytes, whereas it strongly inhibited IL-4 synthesis by control Th2 cells (Fig. 6). These data indicated that Rp-8-pCPT-cGMPS effects on IL-4 production by wild-type Th2 cells resulted from a specific inhibition of PKG. Altogether, these data pointed out a key role for PKG in Th2 lymphocyte calcium signaling.


Figure 6
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  		FIGURE 6.
PKG inhibitor does not modify IL-4 synthesis in PKG-deficient Th2 lymphocytes. Th2 lymphocytes from PKG1–/– and +/+ littermates (from a litter different from those used in Fig. 4E) were stimulated on plate-bound anti-TCR mAb. Cultures were done in the presence or in the absence of Rp-8-pCPT-cGMPS (200 µM). 24 h later, IL-4 content was determined in supernatants. Stimulation assays were performed in triplicates. The experiment includes three mice per group. Values are expressed as mean ± S.D. (*, p < 0.01 when compared with the wild-type group in the absence of inhibitor).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results strongly support a pivotal role for cGMP and PKG in calcium signaling and IL-4 synthesis in Th2 lymphocytes. Contribution of cGMP in Th2 signaling was underlined by the following data: 1) cell-permeant cGMP, or nitric oxide donors known to activate soluble guanylate cyclase, triggered a calcium response and IL-4 expression; 2) addition of guanylate cyclase inhibitor LY-83,583 suppressed both [Ca2+]i increase and Th2 cytokine production induced by TCR activation. Moreover, calcium signaling and IL-4 production were defective in Th2 lymphocytes from PKG null mice, supporting a major role for PKG in Th2 cell activation.

We showed that cGMP concentration was increased in stimulated Th2 cells in a similar range as the rise reported in B-cell receptor-activated B lymphocytes (21). Incubation of Th2 lymphocytes with cGMP or SNP induced calcium response and IL-4 mRNA transcripts. This up-regulation of IL-4 expression was due to calcium signaling because it was inhibited by cyclosporin A. However, cGMP or SNP could not induce detectable IL-4 protein. This could be explained by the fact that IL-4 production requires a sustained calcium response in time and amplitude, which may not be induced by cGMP and SNP. Indeed, NFAT nuclear translocation, which reflects the global calcium response, was weaker in cells stimulated by cGMP as compared with TCR-activated Th2 cells. Stimulation of Th2 cells with NONOate, a potent NO donor, induced a stronger calcium response and NFAT translocation than SNP, resulting in detectable IL-4 production. High levels of IL-4 synthesis, as observed in TCR-stimulated Th2 cells, involve other calcium-independent pathways that may not be activated by cGMP or SNP. Therefore, the cGMP pathway is likely necessary but not sufficient to induce optimal Th2 cytokine production.

Some effects of NO result from guanylate cyclase and PKG activation, raising the question of the involvement of NO in Th2 cell activation. NO is produced by many cell types, including dendritic cells, whereas its production in T lymphocytes is unlikely (37). In this report, we showed that cGMP was produced in purified Th2 cells after TCR activation in the absence of antigen-presenting cells, excluding an effect of NO produced by antigen-presenting cells. Thus, guanylate cyclase activation may be NO independent in T lymphocytes. As suggested by others (38), protein kinase C might directly activate guanylate cyclase upon TCR stimulation. Indeed, we have previously shown the involvement of protein kinase C in DHP-sensitive calcium inflow and IL-4 production in Th2 lymphocytes (28).


Figure 7
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  		FIGURE 7.
Schematic representation of the TCR-induced cGMP pathway involved in cytokine production in Th2 lymphocytes. T cell stimulation through the TCR would activate guanylate cyclases, resulting in cGMP level increase. Subsequent PKG activation would be responsible for DHP-sensitive calcium influx, resulting in NFAT translocation to the nucleus and expression of Il4 and Il5 genes. Activators or inhibitors used in this study are indicated.

 
cGMP may activate cGMP-dependent protein kinase and modulate non-selective cation cGMP-gated channels or cyclic nucleotide phosphodiesterases (discussed in Ref. 39). PDE family members encompass 11 isoenzymes with distinct patterns of substrate selectivity (40, 41). PDE3 is defined as cGMP inhibited and cAMP selective, PDE4 as cAMP specific, and PDE5 as cGMP specific (reviewed in Ref. 41). These enzymes are co-expressed in T lymphocytes and especially in Th2 lymphocytes (42). Thus, cGMP might have decreased PDE3 activity, resulting in an indirect increase in cAMP concentration that could regulate DHP-sensitive calcium influx through a cAMP-dependent protein kinase-dependent mechanism (discussed in Ref. 43). This hypothesis is unlikely because we showed that neither PDE3 nor PDE4 inhibitors, expected to increase cAMP levels, modified TCR-dependent calcium increase and IL-4 synthesis. These data are in agreement with previous work showing that PDE inhibitors had little effect on IL-4 production by a Th2 clone (42). Furthermore, addition of cAMP, as well as inhibition of cAMP-dependent protein kinase, did not affect calcium response in Th2 lymphocytes.

Contrary to PDE, our data emphasize a major role for PKG in cGMP-dependent signaling in Th2 lymphocytes. Indeed, TCR-induced [Ca2+]i increase, nuclear NFAT translocation, and IL-4 synthesis were strongly reduced in Th2 cells from PKG1–/– mice. Likewise, PKG inhibitors inhibited both [Ca2+]i increase and cytokine production by wild-type Th2 lymphocytes. PKG could also activate a calcium-independent signaling pathway controlling IL-4 production. By analogy to cardiac myocytes (44), it could be speculated that PKG might decrease nuclear exportation of NFAT through c-Jun NH2-terminal kinase 2 (JNK2) inhibition, resulting in an enhanced IL-4 transcription in Th2 cells.

Several studies support that DHPR are involved in calcium responses of non-excitable cells, including B lymphocytes (21), NK cells (19), dendritic cells (20), naive T cells (24), Th2 cells (18, 28), and epithelial cells (45, 46). It has been shown that signaling through the B cell receptor was coupled to a guanylate cyclase/cGMP-dependent pathway that controlled DHPR-dependent calcium entry (21). Here we have shown that in Th2 lymphocytes DHPR antagonization by nicardipine reduced cGMP-mediated calcium response, suggesting a role for DHPR in cGMP signaling. Although the structure of DHPR has not been completely elucidated in lymphocytes, DHPR had been related to the {alpha}1 calcium channel subunit in B cells (21) as well as in Th2 cells (18) and human T cells (24). In excitable cells, the NO/cGMP/PKG pathway is known to activate or repress DHPR-dependent calcium currents depending upon the cell type (47, 48). How this pathway operates in Th2 lymphocytes will require further investigation.

Our study, therefore, establishes the following sequence of events that lead to cytokine production in Th2 lymphocytes (Fig. 7). TCR stimulation results in guanylate cyclase activation, responsible for cGMP production. cGMP then activates PKG, which may, directly or not, control DHP-sensitive calcium influx. Intracellular calcium increase induces NFAT translocation to the nucleus and subsequent gene expression. Altogether, our data lend support for a new signaling pathway in Th2 lymphocytes in which cGMP acts as a major second messenger in Th2 calcium response and cytokine production.


   FOOTNOTES
 
* This work was supported by grants from the Ligue contre le Cancer, the Association pour la Recherche sur la Polyarthrite Rhumatoide, INSERM (progamm Progres), Agence Nationale pour la Recherche, the Association de Recherche contre le Cancer (to B. G. and M. S.), and la Fundaçao Calouste Gulbenkian (to M. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: INSERM U563, CHU Purpan, Place du Dr Baylac, Dept. of Genetics, Pavillon Lefebvre, BP 3028, 31024 Toulouse Cedex 3, France. Tel.: 33-5-62-74-45-01; Fax: 33-5-62-74-45-58; E-mail: Lucette.Pelletier{at}toulouse.inserm.fr.

3 The abbreviations used are: TCR, T cell receptor; Ab, antibody; 8-Br-cAMP, 8-bromoadenosine 3':5'-cyclic monophosphate; 8-Br-cGMP, 8-bromoguanosine 3':5'-cyclic monophosphate; DHP, dihydropyridine; DHPR, DHP receptor; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; mAb, monoclonal antibody; NFAT, nuclear factor of activated T cell; NO, nitric oxide; NONOate, spermine NONOate; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; SNP, sodium nitroprusside; NK, natural killer; HPRT, hypoxanthine phosphoribosyl transferase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Lory for helpful discussion and Fatima L'Faqihi and Mustafa Faroudi for their assistance.



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