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The Journal of Immunology, 2004, 172: 5206-5212.
Copyright © 2004 by The American Association of Immunologists

Dihydropyridine Receptors Are Selective Markers of Th2 Cells and Can Be Targeted to Prevent Th2-Dependent Immunopathological Disorders1

Magali Savignac2,*, Bruno Gomes2,*, Alexandra Gallard*, Stéphane Narbonnet*, Marc Moreau{dagger}, Catherine Leclerc{dagger}, Pierre Paulet*, Bernard Mariamé*, Philippe Druet*, Abdelhadi Saoudi*, Gilbert J. Fournié*, Jean-Charles Guéry* and Lucette Pelletier3,*

* Institut National de la Santé et de la Recherche Médicale Unité 563, Institut Federatif de Recherche 30, Hôpital Purpan, Toulouse, France; and {dagger} Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5547, Université Paul Sabatier, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Th1 cells that produce IFN-{gamma} are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4+ T cells from BN rats into (LEW x BN)F1 hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In terms of cytokine productions and functions (reviewed in Ref. 1), CD4+ T cells are heterogeneous. Th1 cells produce IFN-{gamma}, IL-2, and TNF{beta} and contribute to the eradication of intracellular pathogens. Th2 cells produce IL-4, IL-5, IL-6, and IL-13 and are involved in the elimination of helminths.

Classically, TCR stimulation is known to induce tyrosine kinase activation, including that of p56lck and ZAP-70, leading to phospholipase C{gamma}1 activation and generation of inositol 3,4,5-triphosphate. Inositol 3,4,5-triphosphate mobilizes calcium from the endoplasmic reticulum, which generates a calcium influx from extracellular medium through calcium release-activated calcium channels for refilling the stores and amplifying the signal.

However, how the calcium response is controlled in Th2 cells remains unclear. Indeed, for example, ZAP-70 (2) and phospholipase C{gamma} (3) have been reported to be dispensable for IL-4 production. Furthermore, whereas calcium-dependent signaling was crucial for IL-4 synthesis (4, 5), the increase in intracellular calcium concentration ([Ca2+]i)4 was much lower in Th2 than in Th1 cells on TCR stimulation (6, 7, 8)

Heavy metals (mercury or gold) are T cell polyclonal activators resulting in preferential early expression of IL-4 in genetically predisposed animals (9, 10). In studying how heavy metals act, we discovered a new signaling pathway implicated in IL-4 synthesis and involving dihydropyridine (DHP)-sensitive channels (11). Indeed, in IL-4-producing T cell hybridomas, HgCl2 was shown to trigger an entry of calcium from the extracellular medium which was required for IL-4 gene expression and was abolished by DHPR antagonists (12). In addition, incubation with DHPR agonists induced an influx of calcium and IL-4 gene expression.

DHP-sensitive calcium channels are well known in excitable cells as the {alpha}1 subunit of voltage-operated calcium channels (13), which explains the use of DHPR antagonists in the treatment of hypertensive patients. The existence of DHPR is now reported in several types of nonexcitable cells including immune cells (dendritic, B lymphocytes, NK; Refs. 14, 15, 16). However, the presence of these channels in T cells and their role are matters of debate.

In this study, our aim was to look for the expression of DHP-sensitive channels during Th1 and Th2 cell differentiation, to assess their role in IL-4 and IFN-{gamma} secretion, and to test the effect of DHPR antagonists on the course of Th1- and Th2-mediated diseases. We show that: 1) DHPR are induced during Th2 but not Th1 cell differentiation; 2) DHPR agonists and antagonists modulate the TCR-dependent increase in [Ca2+]i and IL-4 production by Th2 cells, whereas they do not modify the Th1 cell responses; and 3) the administration of nicardipine, a DHPR antagonist, is beneficial in three models of Th2-mediated immunopathology but does not prevent experimental autoimmune encephalomyelitis (EAE), an experimental model of Th1-mediated autoimmune disease.

These results underline that TCR-dependent calcium signaling differs between Th2 and Th1 cells and suggest that DHPR are involved in the rise of [Ca2+]i on stimulation through the TCR in Th2 cells. Drugs targeting DHP-sensitive calcium channels may be beneficial in the treatment of pathologies associated with Th2 cell dysregulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male OVA323–339-specific DO11.10 TCR-transgenic BALB/c mice (17), BALB/c mice (Janvier Ets, Le Genest St. Isle, France), BN rats, Lewis (LEW) rats, and (LEWx BN)F1 hybrids (Janvier), 7 to 13 wk old, were cared for in our animal facility according to the Helsinki principles.

Reagents

The DHPR agonist, S(–) Bay K 8644 (BayK–), the DHPR antagonist R(+) Bay K 8644 (BayK+), ionomycin, PMA, and thapsigargin were from Sigma-Aldrich (St. Louis, MO); hamster anti-mouse TCR mAb H57-597 mAb (18) was from BD PharMingen (San Diego, CA).

Th1-Th2 cell differentiation

CD4+ T cells from DO11.10 mice (0.5 x 106 cells/ml) were suspended in RPMI 1640 supplemented with 10% FCS (ATGC Biotechnologie, Noisy Le Grand, France), 1% pyruvate, 1% nonessential amino acids, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. Cells were stimulated at 37°C in 5% CO2 with irradiated BALB/c spleen cells and OVA323–339 peptide (0.3 µM). For Th1 skewing, IL-12 (5 ng/ml) plus anti-IL-4 (10 µg/ml) were added to the primary culture. For Th2 skewing, IL-4 (10 ng/ml) + anti-IFN-{gamma} (10 µg/ml) were included. On days 3–4 after stimulation, cells were split 1:3 with fresh medium. Viable T cells were recovered on Ficoll-Hypaque (Tebu, Le Perray-en-Yvelines, France) and restimulated in the same conditions every 7 days. In experiments in which we tested the effect of DHPR antagonists on cytokine production or proliferation, complete medium containing or not fresh BayK+ was used for replacing the culture medium every 2 days.

For analysis of cytokines by ELISA (BD PharMingen), T cells were plated (2.5 x 104 cells/well) onto 96-well flat-bottom plates that contained APC (25 x 104 cells/well) plus OVA peptide without exogenous cytokines or Abs. Alternatively, T cells were stimulated by plate-bound anti-TCR mAb (1 µg/ml) as described (12). Supernatants were collected 24 h later.

T-bet and GATA-3 expression

RNA (5 x 106 cells) was isolated using the SV total RNA isolation system (Promega, Madison, WI) and reverse transcribed to cDNA using polydeoxythymidylate and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD). The expression of the transcription factors GATA-3 (Th2 specific) and T-bet (Th1 specific) was analyzed by real time quantitative PCR (19) using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) to verify the efficiency of Th1 and Th2 differentiation. PCR was performed with the PCR SYBR Green sequence detection system (PerkinElmer, Norwalk, CT). Primers were: for GATA-3, 5'-gccatgggttagagaggcag-3' and 5'-ttggagactcctcacgcatgt-3'; for T-bet, 5'-cggtaccagagcggcaagt-3' and 5'-cagctgacccaagaggaatca-3'; and for hypoxanthine phosphoribosyltransferase, 5'-ctggtgaaaaggacctctcg-3' and 5'-tgaagtactcattatagtcaagggca-3'.Quantification of target gene expression was calculated by correcting the values relative to the expression of hypoxanthine phosphoribosyltransferase.

DHPR staining

BayK– (100 µM) was added or not to T cells before staining with 250 nM (4,4-difluoro-7-steryl-4-bora-3a,4a-diaza-s-indacene 3-propionic acid)-DHP: (–) ST-BODIPY-DHP (S7445; Molecular Probes, Eugene, OR) at 37°C in a dark chamber. Cells were then analyzed by confocal microscopy as described (12).

RT-PCR

RNA was isolated and reverse transcribed to cDNA as described above. The PCR conditions were 94°C for 45 s, 60°C (for {beta}-actin) or 55°C (for {alpha}1 subunits) for 45 s, and 72°C for 1 min for 21 ({beta}-actin) or 40 cycles (for {alpha}1 subunits). Primers for {beta}-actin were: 5'-tggaatcctgtggcatccatgaaac-3' and 5'-taaaacgcagctcagtaacagtccg-3'. We used primers specific for conserved regions (IIIS6 and IVS6) of the L-type calcium channel {alpha}1 subunits: 5'-ttcttcatgatgaacatctt-3' and 5'-catgtagaagctgatgaa-3' (20).

CFSE assays

T cells were suspended in warm RPMI containing 2 µM CFSE (Molecular Probes) for 10 min. Then, T cells were washed with cold RPMI, suspended in complete culture medium, and subjected to proliferation assays. T cells were stimulated on plate-bound anti-TCR mAb (1 µg/ml) plus anti-CD28 mAb (0.2 µg/ml) in the presence or absence of IL-2 (10 U/ml). Cells were analyzed after 48 h of culture by flow cytometry.

Analysis of [Ca2+]i

T cells were loaded with 5 µM Indo-1 AM (Sigma-Aldrich) as described (12). When we tested the effect of BayK+, this compound was added to T cells 30 min before the assay and was present throughout the test. Then, T cells were set down on plates coated with anti-TCR mAb (1 µg/ml) or were spun together with APC loaded or not with the peptide (T cell:APC ratio, 1:10 in a final volume of 0.5 ml) at 1500 rpm for various times. APC were RBC lysed spleen cells previously incubated in the presence or in the absence of the Ova323–339 peptide (10 µM) at 37°C for 1 h. The bound-free calcium ratio was recorded with an Elite Coulter cell sorter (Coultronics, Margency, France) (excitation wavelength 355 nm and emission wavelengths 405/475 nm). Results were analyzed with CellQuest software (BD Biosciences, Mountain View, CA). Ionomycin (1 µM) was used for each sample to determine [Ca2+]i according to published procedures (21).

Treatment of BN rats with heavy metals

Aurothiopropanol sulfonate (Allochrysine; ATPS) was kindly provided by Solvay Pharma (Suresnes, France). Eight BN rats were injected with ATPS (20 mg/kg body weight (bw) s.c., 3 times a week for 3 wk) and nicardipine (Loxen; 5 mg/kg bw/day i.p., 5 days of 7). This dose of nicardipine is a therapeutic dose used to normalize blood pressure in spontaneously hypertensive rats (22). Injections of Loxen began at the same time as the first injection of ATPS and were pursued up to sacrifice. Eight other rats were injected with ATPS only and 4 rats were injected with Loxen only. Eleven other rats were injected with HgCl2 (250 µg/kg bw s.c. three times/wk for 4 wk) including five that were also injected with nicardipine as described above. Serum Ig isotypes and anti-laminin Ab titers were measured once a week as described (23). A 24-h urine protein excretion was determined once a week by colorimetric assay (Bio-Rad, Hercules, CA). At the time of sacrifice, kidneys were snap-frozen in liquid nitrogen, and 4-µm cryostat sections were incubated with fluoresceinated sheep anti-rat IgG Abs for 30 min at 4°C. Then, the sections were washed three times in cold PBS. The intensity of glomerular IgG deposits was scored on a 0–3 scale independently by two of us (23).

Induction of chronic graft vs host reaction (cGVH)

To induce cGVH reaction, 10 (LEW x BN)F1 hybrids were irradiated (450 rad) and injected iv 24 h later with CD4+ T cells (107 cells/rat) from normal BN rats. Five of these rats were also injected with nicardipine as described above. Serum Ig isotypes, anti-laminin Ab titers, and proteinuria were determined once a week as described above. At the time of sacrifice, kidneys were processed as above.

Induction of Th1-mediated autoimmune disease in LEW rats

Eight LEW rats were injected in the hind footpads with 10 µg of myelin basic protein from guinea pig emulsified in 100 µl of CFA containing 2 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). Myelin basic protein as prepared in our laboratory as previously described (24). Animals were scored daily for clinical signs of disease on a severity scale ranging from 0 to 6: 0, normal; 1, limp tail; 2, hind limb weakness; 3, unilateral hind limb paralysis; 4, bilateral hind limb paralysis; 5, bilateral hind limb paralysis and incontinence; 6, moribund or dead. Four of eight LEW rats were additionally injected with nicardipine as above.

Statistical analysis

Results are expressed as the mean ± SD, and differences between groups were initially shown with the Kruskal-Wallis test and subsequently confirmed by the Mann-Whitney test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DHPR expression characterizes murine Th2 but not Th1 cells

We have previously shown that IL-4-producing T cell hybridomas expressed DHPR (12). To investigate whether DHPR were a marker of Th2 cells, we used transgenic T cells specific for the Ova323–339 peptide which were differentiated along the Th1 and Th2 pathways by weekly stimulation in the presence of IL-12/anti-IL-4 Ab and IL-4/anti-IFN-{gamma} Ab, respectively. Th1 cells produced large amounts of IFN-{gamma} but no IL-4 (Fig. 1A) and expressed T-bet and not GATA-3 after two rounds of stimulation (Fig. 1B). Th2 cells produced IL-4 exclusively (Fig. 1A) and expressed GATA-3 but not T-bet (Fig. 1B). Then, we looked for the sequential expression of DHPR by confocal microscopy using a fluorescent DHP. The staining was faint in naive (not shown) and in Th1 cells (Fig. 2, A and B) and was not modified by preincubation of cells with an excess of DHPR agonist (Fig. 2B). In contrast, Th2 cells were unequivocally labeled with DHP after two rounds of stimulation (Fig. 2A). The staining remained stable thereafter (Fig. 2B and data not shown). Furthermore, it was reduced by preincubation with an excess of the BayK– DHPR agonist (Fig. 2B).



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  		FIGURE 1. Characterization of Th1 and Th2 cells. OVA-specific TCR transgenic CD4+ T cells were stimulated every week with APC and the OVA323–339 peptide and differentiated along the Th1 and Th2 pathway. A, T cells were collected at different times after the beginning of differentiation (stim., stimulation) and cultured in the absence of exogenous cytokines/Abs for 24 h to assess IL-4 and IFN-{gamma} secretion. B, T-bet and GATA-3 expression was evaluated by sequential real time PCR during Th1 and Th2 cell differentiation. Results are expressed as mean + 1 SD of four experiments.

 


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  		FIGURE 2. Expression of functional DHPR on Th2 cell surface. A, Th2 and Th1 cells were stained 3 days after the second round of stimulation with ST-BODIPY-DHP. B, The histogram summarizes the results obtained with Th1 and Th2 cells after three rounds of stimulation. BayK– (100 µM) was or not added to T cells before staining with ST-BODIPY-DHP. Results are expressed as the mean + 1 SD of the fluorescence intensity/cell in arbitrary U (at least 35 cells analyzed in each group). *, p < 0.0001 compared with Th1 cells. The staining of Th2 cells was also markedly diminished when cells were preincubated with BayK– (p < 0.0001). C, T cells were loaded with indo-1AM and the bound-free calcium ratio was recorded before and after stimulation by the DHP agonist BayK– (BK–, 5 µM). Ionomycin (iono, 1 µM) was used as a positive control. One representative experiment of four. D, The histogram summarizes the effect of BayK– (BK–, 5 µM) in Th2 and Th1 cells tested after three rounds of stimulation. The DHP antagonist BayK+ (BK+, 10 µM) was or was not added to T cells 30 min before addition of BK–. *, p < 0.01 relative to the [Ca2+]i in unstimulated cells; **, p < 0.01 compared with the response of cells cultured in the absence of BK+.

 
In differentiated Th2 cells, the DHPR agonist BayK– induced an increase in [Ca2+]i which peaked at 30 s after stimulation and then decreased but remained higher than in unstimulated cells for 10–15 min (Fig. 2C). The calcium response induced by BayK– in Th2 cells was abolished when extracellular medium was deprived of calcium (not shown), demonstrating that DHPR were implicated in an extracellular calcium influx. The DHPR antagonist BayK+ reduced the peak of the increase in [Ca2+]i triggered by BayK– (Fig. 2D) in Th2 cells. No significant effect of BayK– or BayK+ was observed in naive (Fig. 2C and data not shown) or Th1 cells (Fig. 2, C and D).

The {alpha}1 subunits from L-type, voltage-dependent calcium channels form calcium pores and are defined as DHPR in excitable cells. PCR was done with consensual primers that amplify all {alpha}1 L-type calcium channel subunits. A band of the expected size (~900 bp) was detected in Th2 cells from day 5 after the first stimulation and thereafter. No PCR products were detected in Th1 cells (Fig. 3). This suggests that DHPR might be related to {alpha}1 L-type calcium channels, which have been described in other types of nonexcitable cells.



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  		FIGURE 3. Th2 cells express L-type calcium channel-related molecules. RNAs were sequentially extracted from 5 x 106 cells; cDNAs were prepared at various days (d) after one, two, or three rounds of stimulation (St) in Th1 and Th2 priming conditions. Serial 2-fold dilutions (1, 2, 4) of the cDNA were amplified by using consensual primers of {alpha}1 subunits from L-type calcium channels (LTCC) or {beta}-actin-specific primers. One representative experiment of four.

 
DHPR are implicated in IL-4 synthesis by Th2 cells

Stimulation with APC loaded with the peptide induced a sustained calcium response in Th2 and in Th1 cells (Fig. 4A). The DHPR antagonist, BayK+, diminished the calcium response in a dose-dependent manner in Th2 cells but did not affect the calcium response in Th1 cells (Fig. 4A). BayK+ also reduced TCR-induced IL-4 synthesis by Th2 cells as did nicardipine, another DHPR antagonist (Fig. 4B). As shown in Fig. 4B, BayK+ did not modify IL-4 secretion induced by thapsigargin (an inhibitor of the endoplasmic Ca2+ATPase), by ionomycin (a lipophilic calcium ionophore), or by PMA plus ionomycin (the combination of which bypasses TCR-induced early signaling events).



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  		FIGURE 4. Selective inhibition of TCR-dependent calcium response and IL-4 synthesis by DHPR antagonists in Th2 cells. A, Th2 and Th1 cells were tested at the third round of stimulation. Cells were loaded with Indo 1-AM. The DHPR antagonist BayK+ (BK+, 0.1–10 µM) was or was not added to T cells 30 min before the assay and remained present throughout the test. T cells were recorded for the basal bound-free calcium ratio, and put together with APC loaded with the OVA323–339 peptide for times ranging from 30 s to 10 min. *, p < 0.01 relative to cells without BayK+, n = 5. The experiment was repeated four times with similar results. B, After three rounds of stimulation performed in Th1 or Th2 conditions, T cells were cultured with APC and the OVA peptide (Ova), without inhibitor (inh.) or in the presence of 10 µM BayK+ or 10 µg/ml nicardipine (nicar.). As controls, T cells were stimulated with 1 µM ionomycin (iono), 20 nM thapsigargin (thapsi) or PMA (10 ng/ml) plus ionomycin (1 µM) with or without BayK+. The production of IL-4 was measured after 24 h of culture. *, p < 0.01 relative to cells cultured without inhibitor. Differentiated Th1 cells were also stimulated with the peptide. BayK+ (10 µM) or nicardipine (10 µg/ml) were present or not during the stimulation assay. IFN-{gamma} secretion was measured after 24 h of culture. Cultures were done in quadriplicates, and the experiment was repeated three times with similar results.

 
Then, we tested the effect of DHPR antagonists on IL-5 and IL-10 productions which were also calcium dependent. Nicardipine inhibited not only IL-4 but also IL-5 and IL-10 secretion in a dose-dependent manner, but it did not modify PMA plus ionomycin-induced cytokine synthesis (Table I).


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  		Table I. Nicardipine, an antagonist of DHPR, inhibits IL-4, IL-5, and IL-10 in a dose-dependent mannera

 
On the contrary, neither BayK+ nor nicardipine reduced IFN-{gamma} secretion by Th1 cells (Fig. 4B). This underlines the theory that Th2 cells are specific targets for DHPR antagonists.

Assuming that our conditions of stimulation might be suboptimal because we did not add exogenous IL-2, we stimulated Th2 cells in the presence of IL-2. BayK+ was present or not during the culture. The addition of IL-2 to effector Th2 cells did not modify the pattern of CFSE staining on stimulation with anti-TCR mAb (Fig. 5A) but increased IL-4 production (Fig. 5B). BayK+ had no or a minor effect on the proliferation with cells dividing twice in 48 h in all groups (Fig. 5A). However, BayK+ and nicardipine still inhibited the production of IL-4 even when the stimulation was achieved in the presence of IL-2 (Fig. 5B).



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  		FIGURE 5. An antagonist of DHPR inhibits TCR-dependent IL-4 secretion but does not impair proliferation or differentiation of Th2 cells. A, Th2 cells collected after two rounds of stimulation were labeled with CFSE and stimulated on plate-bound anti-TCR mAb (1 µg/ml) plus soluble anti-CD28 mAb (0.2 µg/ml). IL-2 (10 U/ml) and/or BayK+ (BK+, 10 µM) were present or not (w/o) during the proliferation assay. Cells were analyzed before and after 48 h of stimulation by flow cytometry. B, Th2 cells collected after two rounds of stimulation were stimulated on plate-bound anti-TCR mAb (1 µg/ml). IL-2 (5 U/ml) and/or DHPR antagonists (either 10 µM BayK+ or 10 µg/ml nicardipine) were present during the stimulation. Supernatants were collected 24 h later for IL-4 determination. C and D, T cells were primed under Th2 conditions plus IL-2 (10 U/ml). BayK+ was present or not during the culture. C, After 7 days of culture, T cells were labeled with CFSE and stimulated on plate-bound anti-TCR mAb plus anti-CD28 mAb (0.2 µg/ml). BayK+ was present or not during the proliferation assay. Cells were analyzed before and after 48 h of stimulation by flow cytometry. D, T cells were stimulated on plate-bound anti-TCR mAb. IL-2 (10 U/ml) and/or BayK+ was or not present during the stimulation assay. IL-4 production was measured 24 h later. inh., Inhibitor.

 
DHPR are not involved in Th2 cell differentiation

To test the effect of DHPR antagonist on Th2 cell differentiation, BayK+ was added or not to the culture in Th2 priming conditions. Culture medium was replaced by fresh medium containing BayK+ or not every 2 days. After 7 days, we recovered the same number of viable T cells, whether or not BayK+ was present in the culture. Cells were then stimulated in the absence of BayK+, without IL-4 or anti-IFN-{gamma} mAb for 24 h. The presence of BayK+ in primary culture did not modify IL-4 synthesis (14 ± 2 ng/ml) relative to the production of IL-4 by cells that have been cultured in Th2 priming conditions without BayK+ (12 ± 3 ng/ml; data not shown). IL-2-dependent signaling is important for optimizing Th2 priming (25). Thus, we differentiated Th2 cells in the presence of IL-4, IL-2, and anti-IFN-{gamma} mAb, and we replaced the culture medium every 2 days with fresh medium containing IL-4, IL-2, and anti-IFN-{gamma} mAb. BayK+ was present or not during the culture. The proliferative response was not affected by BayK+ with cells dividing about four times during 48 h in both groups (Fig. 5C). After 7 days of culture, we recovered the cells and tested them for IL-4 secretion after 24 h of stimulation with the Ag. IL-2 but not IL-4 or anti-IFN-{gamma} mAb was present during the stimulation assay. The production of IL-4 was not modified when BayK+ was present during the culture but absent during the stimulation assay (Fig. 5D). However, BayK+ inhibited IL-4 synthesis (n = 4, p < 0.01 in all the combinations) when present during the stimulation assay (Fig. 5D).

The DHPR antagonist nicardipine prevents the Th2 cell-mediated autoimmune disease induced by heavy metals in BN rats

The gold salt ATPS, which induces an increase in serum IgE concentration and an immune-mediated glomerulopathy in predisposed patients, constantly triggers Th2-dependent immune disorders in BN rats. ATPS induced an increase in serum IgG1 (Fig. 6A) and IgE (Fig. 6B) concentration. Nicardipine strongly diminished the increase in serum IgG1 (Fig. 6A) and IgE (Fig. 6B) induced by ATPS. Nicardipine also suppressed antilaminin Ab production (Fig. 6C) and glomerular Ig deposits (data not shown). Indeed, the mean fluorescence intensity of Ig deposits was scored 1.5 ± 0.5 in ATPS-treated BN rats vs 0.2 ± 0.2 in rats that were additionally injected with nicardipine (p < 0.01). None of the rats, whether they were injected with nicardipine or not, displayed abnormal proteinuria (not shown).



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  		FIGURE 6. Inhibition of gold salt-induced Th2-dependent immune disorders by treatment with nicardipine, a DHPR antagonist. BN rats were injected three times a week with 20 mg/kg ATPS and received i.p. injections of nicardipine (1 mg/injection, 5 days a week) or not. Control rats were injected with nicardipine only. Serum IgG1 (A) IgE (B) concentrations and anti-laminin autoantibody (ab) production (C) were quantified every week. Anti-laminin Ab titer was expressed in arbitrary U/ml. *, p < 0.01 and **, p < 0.05, relative to BN rats injected with ATPS only.

 
Chronic injections of HgCl2 in BN rats induced a much more severe disease than the immune disorders induced by gold salts. Indeed, HgCl2 triggered a huge increase in serum IgE concentration superior to 20 mg/ml (Fig. 7A). The production of antilaminin Abs (Fig. 7B) correlated with linear glomerular IgG deposits (Fig. 7C). In addition, rats developed an heavy proteinuria (Fig. 7D) and displayed an important loss of bw at day 21 after the first injection of HgCl2 (–35 ± 5% compared with day 0 values). Nicardipine strongly reduced the increase in serum IgE concentration (Fig. 7A), the titer of antilaminin autoantibodies (Fig. 7B) and the intensity of glomerular IgG deposits (mean score 2 ± 0.45 in HgCl2-treated BN rats vs 0.5 ± 0.35 in rats that were additionally injected with nicardipine; p < 0.01; Fig. 7C). In addition, nicardipine suppressed the development of proteinuria induced by HgCl2 (Fig. 7D) and prevented any loss of bw.



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  		FIGURE 7. Inhibition of HgCl2-induced autoimmune glomerulopathy by nicardipine. BN rats were injected three times a week with 250 µg/100 g bw. HgCl2 and received i.p. injections of nicardipine (5 to 6 rats per group) or not. Serum IgE concentration (A) and anti-laminin autoantibody (ab) production (B) were quantified every week. C, Intensity of glomerular IgG deposits of representative rats that were injected with HgCl2 and received injections of nicardipine or not. D, 24-h proteinuria was determined once a week. *, p < 0.01 relative to BN rats injected with HgCl2 only.

 
Nicardipine prevents chronic GVH

cGVH disease that is thought to be Th2 mediated in humans occurs frequently in long term survivors of allogeneic hemopoietic stem cell transplantation (26). Clinical manifestations have been likened to those of systemic immune diseases such as progressive systemic sclerosis, systemic lupus erythematosus-like disease, or Sjögren’s syndrome. Some patients display membranous glomerulopathy. Thus, we tested the effect of nicardipine on the development of experimental cGVH reaction. We transferred 107 CD4+ T cells from BN rats into (LEW x BN) F1 hybrids that were injected or not with nicardipine. Donor alloreactive Th2 cells were activated and were responsible for the polyclonal activation of the recipient B cells, resulting in the increase of serum IgG1 (not shown) and IgE (Fig. 8A) concentrations, and the production of antilaminin autoantibodies (Fig. 8B). IgG were found deposited along the glomerular capillary walls (Fig. 8C) as previously described (27). Nicardipine substantially reduced all signs of cGVH (Fig. 8) including glomerular Ig deposits (mean score 1.2 ± 0.5 in rats that developed the cGVH reaction vs 0.3 ± 0.2 in nicardipine-injected rats; p < 0.05; Fig. 8C). Furthermore, treatment by nicardipine did not induce an acute form of GVH disease: all the rats survived; they did not lose bw and were free of skin lesions.



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  		FIGURE 8. Inhibition of cGVH reaction by nicardipine. (LEW x BN)F1 hybrids were injected with CD4+ T cells from BN rats and received i.p. injections of nicardipine or not. Serum IgE (A) concentrations and anti-laminin autoantibody (ab) production (B) were quantified each week. *, p < 0.01 relative to F1 rats that developed the cGVH reaction. C, Intensity of glomerular IgG deposits of representative rats that were injected with CD4+ T cells from BN rats and received injections of nicardipine or not. AU, Arbitrary U/ml.

 
Nicardipine does not protect from the development of EAE

LEW rats are susceptible to the development of EAE, a model of multiple sclerosis that is due to the emergence of Th1 cells specific of self Ags from the CNS. LEW rats immunized with myelin basic protein developed typical EAE as previously described. Nicardipine did not modify either the day of onset or the disease severity (Table II).


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  		Table II. Nicardipine does not improve the course of EAE in LEW ratsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that DHPR are markers of Th2 cells and are implicated in the calcium influx induced by T cell activation in these cells. Indeed, Th2 cells are specifically stained with DHP. DHPR agonist induces a calcium response that is inhibited by DHPR antagonist. DHPR antagonist also reduces TCR-dependent calcium response and IL-4 synthesis by Th2 cells but has no effect on the TCR-induced calcium signal or on the IFN-{gamma} secretion by Th1 cells.

In excitable cells, the {alpha}1 subunit of long-lasting (L-type) voltage-dependent channels is defined as DHPR. L-type calcium channels located at the plasma membrane are coupled, directly or not, to ryanodine receptors expressed at the surface of the endoplasmic reticulum and are implicated in the increase in [Ca2+]i upon membrane depolarization (discussed in Ref. 28). DHPR have also been described in non excitable cells including pancreatic {beta} cells (29), renal epithelial cells (30), osteoblasts (31), erythroleukemia cells (32), B lymphocytes (15), NK cells (16), and dendritic cells (14). The molecular identity of DHPR and how they are implicated in the calcium response in nonexcitable cells is unclear even if the presence of classical (29) or truncated forms of L-type calcium {alpha}1 subunits (32) as well as molecules related to L-type calcium channels (30, 31, 33, 34) has been reported in these cells.

The presence of DHPR, their molecular identity and their role in T cells is a matter of debate. DHPR agonists and antagonists have been tested by some groups on peripheral blood lymphocytes or on human Jurkat T cells. It was reported that human blood lymphocytes were stained with radioactive DHP, but the functional consequences were not studied (35). Young et al. (36) showed that BayK–, a DHPR agonist, induced an increase in [Ca2+]i in Jurkat T cells and concluded that this agonist may interact not only with voltage-gated calcium channels (L-type) but also with the store-operated calcium channels (calcium release-activated calcium) which are known to be expressed on T cells. A possible capacity of DHPR antagonists to inhibit store-operated calcium channels has also been described in some cells (37, 38). Our study did not detect DHPR in murine naive T and Th1 cells; indeed, the staining of these cells with ST-BODIPY-DHP is barely detectable, and we failed to detect a calcium signal in naive T or Th1 cells on exposure to BayK–. This discrepancy could be explained by differences in the origin of the T cells tested or by differences in the sensitivity of the techniques used for the detection of DHPR. In any case, our study shows that DHPR expression is induced or up-regulated in Th2 cells.

DHPR antagonists have also been described as inhibitors of calcium-activated potassium (KCa) channels in T cells which impairs hyperpolarization that is required for a sustained calcium response (39). Indeed, KCa channels are involved in providing the electrochemical driving force for Ca2+ entry into T cells. Such a property of DHPR antagonists could explain the immunosuppressive properties attributed to these drugs (40). However, such an effect does not explain why the TCR-dependent increase in [Ca2+]i and IFN-{gamma} production is not modified by DHPR antagonist in Th1 cells.

We detected PCR products by using consensual primers for L-type calcium channels in Th2 cells from day 5 after the first stimulation but not in Th1 cells after 35–40 rounds of amplification. The kinetics fits with the fact that functional DHPR are detected rather late, at the second round of stimulation. Thus, Th2 cells up-regulate L-type calcium channel-related molecule expression which may contribute to form functional DHPR. Preliminary data indicate that the PCR products were related to {alpha}1C (cardiac-neuronal) and {alpha}1D (neuroendocrine) subunits of L-type calcium channels in Th2 cells (M. Savignac, B. Gomes, D. Baup, C. Larroze, S. Narbonnet, M. Moreau, C. Leclerc, P. Paulet, D. Lagrange, B. Mariamé, et al., manuscript in preparation). Very recently, Kotturi et al. (41) used nested RT-PCR to identify a transcript encoding the retinal {alpha}1F subunit of L-type calcium channels in human and murine T cells. In addition, this study reported that DHPR agonist triggers a rise in [Ca2+]i at concentrations equal or superior to 50 µM and that nifedipine, a DHPR antagonist, reduced calcium signal, activation of the calcium-sensitive transcription factor NFAT, and IL-2 production by T cells. We were unable to detect a calcium signal on stimulation with DHPR agonist in naive or Th1 cells, but we used concentrations of DHPR agonist and antagonist that did not exceed 10 µM. These results suggest that T cells express DHPR, the number and the molecular identity of which depend on the state of differentiation.

Rats treated with nicardipine were protected against several experimental diseases resulting from Th2 cell activation such as cGVH disease and autoimmunity induced by heavy metals. By contrast, they did not prevent paralysis in the experimental model of Th1-cell mediated EAE. These results argued against a global immunosuppressive effect of calcium channel blockers at used doses. A direct effect of DHPR antagonists in inhibiting IL-4 synthesis by T cells was likely to explain the beneficial effect of DHPR antagonist in the models of Th2 cell-dependent diseases that we tested. It has been reported that diltiazem, another calcium channel blocker, impairs IL-12 production by dendritic cells (42), which could favor the development of Th2 cells. However, our data did not support the idea that DHPR antagonist impaired Th1 cell development because it did not improve the course of EAE, a Th1-mediated autoimmune disease.

This study indicates that Th1 and Th2 cells differ in TCR-dependent signaling pathways and that DHPR are markers of Th2 cells, opening the opportunity to selectively downgrade Th2-mediated immune disorders.


   Acknowledgments
 
We thank Fatima L’Faqihi for her assistance and Florence Capilla and Dr. Talal Al Saati for their expertise in histology. We are thankful to Professor S. Valitutti for critical reading of the manuscript.


   Footnotes
 
1 This study is supported by grants from Ligue Nationale Contre le Cancer, Association pour la Recherche sur la Polyarthrite Rhumatoide, Institut National de la Santé et de la Recherche Médicale (Programme Progress), and Association de Recherche Contre le Cancer (to M.S.). Back

2 M.S. and B.G. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Lucette Pelletier, Institut National de la Santé et de la Recherche Médicale Unité 563, Toulouse, France. E-mail address: Lucette.Pelletier{at}toulouse.inserm.fr Back

4 Abbreviations used in this paper: [Ca2+]i, intracellular calcium concentration; BayK–, S(–) Bay K 8644; BayK+, R(+) Bay K 8644; bw, body weight; DHP, dihydropyridine; ATPS, aurothiopropanol sulfonate sodium salt; EAE, experimental autoimmune encephalomyelitis; GVH reaction, graft-vs-host reaction; cGVH, chronic GVH reaction. Back

Received for publication October 6, 2003. Accepted for publication February 18, 2004.


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