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Activation of T lymphocytes with mitogen results in a biphasic elevation of intracellular calcium
([Ca2+]i) due to Ca2+
release from internal stores followed by a sustained
Ca2+ influx across the plasma
membrane[1,2]. The latter phase is critical for
subsequent calcineurin activation and interleukin (IL)-2 gene expression in activated T
cells[3,4]. The voltage-independent calcium release activated
Ca2+ (CRAC) channels have been strongly suggested to be solely responsible for sustained
Ca2+ influx during mitogenic stimulation of T cells by lectins or
antibodies[2-4]. The evidence indicating that concanavalin (ConA)
induces Ca2+ influx through CRAC channels includes: (1) the
Ca2+ currents induced by mitogen stimulation and by passive
store depletion [using intracellular
ethyleneglycol-bis(b-aminoethyl
ether)-N,N,N¡¯,N¡¯-tetraacetic acid (EGTA) or an inhibitor
of endoplasmic reticulum Ca2+-ATPases that deplete internal
Ca2+ stores and irreversibly activate store-operated
Ca2+ (CRAC) channels in the plasma membrane] share the same properties, including ion selectivity, conductance,
Ca2+-dependent inactivation, and blockage by heavy metals
(Ni2+, Co2+, Mn2+,
Cd2+)[3,5]; (2) stimulation of T cells with lectins or antibodies
does not further increase Ca2+ influx or
Ca2+ current in T cells whose stores are already depleted by thapsigargin
(TG)[3,6]; (3) 1-{beta-[3-(4-methoxyphenyl)
propoxyl]-4-methoxy-phenethyl}-1H-imidazole (SK&F96365), a CRAC channel blocker, blocks
ICRAC in parallel with the
[Ca2+]i rise and induction of T cell activation markers by
TG[4]; and (4) a specific defect in
Ca2+ influx associated with the absence of
ICRAC was shown in nonresponsive T cells from patients with a severe
immunodeficiency[7], and, likewise, T cell activation with anti-CD3 evokes
Ca2+ release but not influx in Jurkat mutants selected specifically for the
absence of store-operated Ca2+
entry[8].
Cl- channels are involved in the proliferation of a variety of cell types, such as endothelial cells, glioma cells,
hepato-cytes, and vascular smooth muscle
cells[9-14]. Previous studies (including our own) have also shown that T cell activation
and proliferation were inhibited by Cl- channel blockers [4,4¡¯-diisothiocyanostilbene-2,2¡¯-disulfonic acid (DIDS),
5-nitro-2-(5-phenylpropylamino)-benzoate (NPPB) and tamoxifen] as well as by antisense oligonucleotides against
ClC-3[15-19]. We hypothesized that these
Cl- channel blockers affected T cell proliferation through
Ca2+-signaling and the interaction between
Cl- channels and CRAC channels that was involved in T lymphocyte activation and proliferation.
The present work was designed to further examine several key events in ConA-induced T cell activation and proliferation,
including Ca2+ signaling, IL-2 mRNA expression, and final T cell proliferation. The functional role of
Cl- and CRAC channels in T cell activation was confirmed by a
[3H]thymidine proliferation assay using several kinds of channel blockers. To investigate whether there is an interaction
between Cl- and CRAC channels in stimulated T cells, we further compared the effects of a combination of these channel
blockers and each alone on ConA-induced Ca2+
signaling, IL-2 mRNA expression, and final T cell proliferation by using
[3H]thymidine incorporation, Fura-2 analysis of
Ca2+ movement, RNase protection assay (RPA) of human IL-2 mRNA expression
and reverse transcription-polymerase chain reaction (RT-PCR) analysis of ClC-3 mRNA expression. Our data suggest an
interplay between DIDS-sensitive Cl- channels and CRAC-related
Ca2+ signaling during T cell activation and proliferation.
Materials and methods
Isolation of human T lymphocytes Human venous blood was obtained from the Guangzhou Blood Center and T
lymphocytes were isolated as described
previously[20]. Briefly, venous blood from healthy adult donors was collected in heparinized
tubes and diluted with RPMI-1640 containing 25 mmol/L
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid
(HEPES), 10% heated-activated fetal calf serum (FCS), 2 mmol/L
glutamine, 50 mg/mL streptomycin, and 50 U/mL penicillin. Peripheral blood
mononuclear cells were centrifuged on Ficoll-Paque (Shanghai Reagent Institute, Shanghai, China), and then transferred to
a sterile nylon wool fiber column (Polysciences Inc, Warrington, USA) pre-equilibrated with RPMI-1640/10% FCS at 37 ¡ãC for
45 min. The column was eluted with pre-warmed RPMI-1640/10% FCS, and the cells were washed 3 times with this medium.
Cell populations obtained in this way were ³95% T lymphocytes, which were measured by fluorescence-activated cell sorter
analysis with anti-CD3 antibody OKT3 (Ortho Pharmaceuticals, Raritan, NY, USA).
Proliferation assay Fresh isolated T lymphocytes were suspended in RPMI-1640/FCS and
2¡Á105 cells were placed in each well of a 96-well plate. The cells were preincubated with the compounds tested for 15 min and then exposed to mitogenic
lectin and ConA (5 µg/mL). At a concentration of 5 µg/mL, ConA induces significant proliferation of isolated human T
lymphocytes without any toxic effects, which has been confirmed in many previous
studies[1-3,15,16,18,21-23]. The cultures were
maintained in a humidified incubator at 37 ¡ãC (in 5%
CO2) for 72 h. Sixteen hours before the cells were harvested,
18.5¡Á103 Bq [3H]thymidine (Shanghai Atomic Energy Research Institute, Shanghai, China) was added to each well. The incorporated
radioactivity was measured in a Beckman scintillation counter (Beckman, USA), expressed as counts per minute (3
replicates/experiment). In order to exclude the direct cytotoxic effects of the tested compounds on T cells, the trypan blue exclusion
assay was used to determine cell viability at the end of incubation, and only samples with a viability of at least 95% were used
for further assay. At the concentration used, no cytotoxic effect on cell cultures was found.
[Ca2+]i measurements
[Ca2+]i was measured in a T lymphocyte suspension with a Fura-2 fluorescent probe using a
well-established method as described
previously[13]. Briefly, fresh isolated T lymphocytes
(1¡Á108 cells/L) were incubated in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM)/F12 medium containing Fura-2/AM (1 µmol/L) for 30 min at room temperature.
T cells were diluted with RPMI-1640/10% FCS (1:3) and kept at room temperature for 10 min to allow re-equilibration. Cells
were washed twice with a Ca2+-balanced salt solution (BSS; in mmol/L: NaCl 130, KCl 5,
MgCl2 1, CaCl2 1.5, HEPES 20, glucose
10, pH 7.4), and finally suspended in BSS
(2¡Á106-4¡Á106 cells/mL).
[Ca2+]i was monitored by using a fluorescence
spectrophotometer (RF-5000; Shimadzu, Kyoto, Japan) with dual excitation at 340 nm/380 nm and emission at 500 nm. In order to chelate
or remove the residual Ca2+ in
Ca2+-free medium, EGTA (50 mmol/L) was added into
Ca2+-free medium 1 min before determining
the release of intracellular Ca2+.
[Ca2+]i was determined from the relation:
[Ca2+]i
=Kd¡ÁSf380/b380
(R-Rmin)/(Rmax
-R), where Kd is 224 nm in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm;
R is the fluorescence ratio (340 nm /380 nm) of the intracellular Fura-2;
Rmax and Rmin are the maximal and minimal fluorescence
ratios obtained by addition of Triton X-100 (final concentration 0.09%) and EGTA (final concentration 3 mmol/L),
respectively[13]. Each tested compound was added to the cell suspension when the fluorescence intensity reached the stable state. The
measurement of [Ca2+]i using Fura-2 is well established in our
laboratory[13,24]. For every tested com-pound, we have
observed its effect alone on
[Ca2+]i for up to 15 min and can exclude non-specific effects due to the leakage of Fura-2 dye from
cells. All chemicals were purchased from Sigma (USA).
Probe generation Human IL-2 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA was ligated into
pBluescript II SK (+/-) vectors (Promega, USA), which contain the T3 and T7 RNA promoters. The plasmid DNA was isolated
and linearized with KpnI or XbaI. Then
[32P-CTP] (Dupont, USA)-labeled hIL-2 and human GAPDH riboprobes were
synthesized by using linearized DNA templates, T3 RNA polymerase and the Riboprobe
in vitro transcription system (Promega, USA). After extraction with phenol/chloroform and chloroform, and precipitation with ethanol, dried probes were dissolved
(3¡Á105 cpm/mL) in hybridization
buffer.
RPA and RT-PCR analysis Purified human T cells
(5¡Á106 cells/mL) were incubated with different blockers for 15 min, at 37
¡ãC, in 5% CO2, and then treated with 5 µg/mL ConA for 6 h. Total RNA was extracted using the acid guanidium
thiocyanate-phenol-chloroform
method[25].
The RPA was performed according to the manual supplied with the RNase protection kit (Ambion, USA). Briefly, a 15 µg
total RNA fraction of each sample was hybridized with 1 µL labeled hIL-2 and human GAPDH riboprobes. The hybridized
fragments protected from RNase A + T1 digestion were separated by electrophoresis on a standard 8% acrylamide/7M urea
sequencing gel. Dried gels were placed on X-ray film for 24 h at -70 ¡ãC. The bands on the X-ray film were quantified by
densitometry scanning (Koutron IBAS
2.0). The expression level of hIL-2 mRNA was expressed as the ratio of hIL-2/GAPDH.
For RT-PCR, 1 µg of the extracted RNA was used for synthesis of the first-strand cDNA using AMV reverse transcriptase
(Promega) according to the manufacturer¡¯s instruc-tions. PCR amplification was carried out with 100 ng DNA in a total
volume of 50 µL in a Gene Amp 9600 PCR system (Perkin-Elmer, Foster City, CA) for 25 cycles at 94 ¡ãC for
2 min, 55 ¡ãC (ClC-3) or 57 ¡ãC (b-actin) for 1 min, and extension at 72 ¡ãC for 1 min. The primers were
5¡¯-ATTAAATGGATATA-CCCTTTCTTG-3¡¯ (sense) and 5¡¯-TTGCAATTGTCAGGTC-TCTTCT-3¡¯ (antisense) for
ClC-3[26]; 5¡¯-GCTACAGCTTCA-CCACCAC-3¡¯ (sense) and 5¡¯-TACTCCTGCTTGCTGA-TCCAC-3¡¯ (antisense) for
b-actin[27]. The amplified products were
electrophoresed on 2% agarose gels alongside a
molecular weight marker. The PCR products were 288 and 498 bp in size for ClC3 and
b-actin, respectively. Because the primers for b-actin were designed to span two exons and an intron, RT-PCR with
b-actin primers controlled for genomic DNA contamination in the source RNA. The linear range of the PCR amplification was determined by carrying out the PCR for
varying number of cycles with a fixed quantity of RNA (1 µg), so that the expression levels of ClC-3 could be semi-quantified
by densitometry scanning and expressed as the ratio of
ClC-3/b-actin.
Statistical analysis Results are expressed as mean¡ÀSEM. The
IC50 values were estimated in each experiment on the basis
of a semi-logarithmic concentration-dependent curve. Data were analyzed with the
t-test using SPSS11. P<0.05 was
considered significant.
Results
Effects of Ca2+ and Cl- channel blockers on ConA-
induced T lymphocyte proliferation We first investigated whether CRAC and
Cl- channel blockers affected T cell
proliferation using an [3H]thymidine incorporation assay. As shown in Figure 1A, SK&F 96365, a common CRAC blocker,
significantly inhibited cell proliferation at concentrations of 3 to 30 µmol/L in a concentration-dependent manner, with an
IC50 value of 9.4¡À1.0 µmol/L, whereas nifedipine (0.1-100 µmol/L), an L-type VDCC inhibitor, did not significantly
affect T lymphocyte proliferation (Figure 1A). These data indicate that
Ca2+ entry through CRAC channels, but not VDCC,
plays a functional role in T cell proliferation. Next we tested the effects of several
Cl- channel blockers on human T cell proliferation. As shown in Figure 1B, DIDS significantly inhibited T cell proliferation at concentrations of 32 to 512 µmol/L in
a concentration-dependent manner, with an
IC50 value of 168.0¡À3.3 µmol/L, whereas no inhibitory effects on T cell prolifera
tion were observed in the presence of 100 µmol/L 9-anthracene carboxylic acid (9-AC;
n=5), 100 µmol/L niflumic acid (NFA;
n=6) or 400 µmol/L furosemide (n=5;
P>0.05, data not shown). Based on our previous findings that antisense oligonucleotides against ClC-3 decreased
[3H]thymidine incorporation and prevented T cell
proliferation[15], these data suggest that
Cl- channels, which are sensitive to DIDS, may play a functional role during T lymphocyte proliferation.
Because CRAC-mediated Ca2+ signaling is the predominant pathway during T cell activation and
proliferation[2], the above results prompted us to further study whether
Cl- channels would affect T cell proliferation through the
CRAC-mediated Ca2+ signaling pathway. Although 2 µmol/L DIDS did not significantly affect cell proliferation, the inhibitory effect
of SK&F96365 (10 µmol/L) on T cell proliferation was enhanced from 45.4%¡À5.3% (without DIDS) to 67.3%¡À4.6%
(with 2 µmol/L DIDS; n=6, P<0.01; Table 1). These data suggest that
Cl- channels may play a functional role in T cell
proliferation through CRAC-mediated Ca2+
signaling.
Effects of Ca2+ and
Cl- channel blockers on ConA-induced
Ca2+ entry Measurement of
[Ca2+]i using the Fura-2 probe
revealed that the resting
[Ca2+]i in human T cells was 67¡À5 nmol/L
(n=54), which was not altered by depolarization of T cells
with 50 mmol/L KCl (66¡À10 nmol/L, n=4,
P>0.05), indicating a lack of VDCC in the T cell plasma membrane. We then examined
the effects of 10 µg/mL ConA and 2 µmol/L TG on
[Ca2+]i in human T cells, and representative traces are superimposed in
Figure 2A. ConA (10 µg/mL) caused a transient and then a prolonged increase in
[Ca2+]i, which was due to the corresponding
release of Ca2+ from intracellular stores and the subsequent transmembrane
Ca2+ entry, as shown in previous
reports[3,5,22,28]. The increase in
[Ca2+]i reached steady state within 150 s, and subsequent exposure of the cells to 2 µmol/L TG caused a further
significant increase in
[Ca2+]i (n=7). However, 10
µg/mL ConA failed to further increase
Ca2+ influx in T cells whose stores were
already depleted by 2 µmol/L TG (n=7). The pattern of the increase in
[Ca2+]i stimulated by ConA was similar to that produced
by 2 µmol/L TG except that the peak and the sustained plateau of
[Ca2+]i caused by 10
µg/mL ConA were lower than those caused by 2 µmol/L TG
(P<0.01, n=7; Figure 2B). These data indicate that ConA only evokes some
Ca2+ release from TG-sensitive intracellular
Ca2+ stores, which is enough to induce the
Ca2+ influx responsible for T cell activa-tion and proliferation.
Our data are consistent with the previous reports showing that lectin induces
Ca2+ influx through the depletion of
TG-sensitive intracellular Ca2+
stores[2,3,5,29]. The above proliferation assay demonstrated that 5
µg/mL ConA produced significant proliferative effects on T cells, but an even higher concentration of ConA (10 µg/mL) could not deplete the TG-sensitive
intracellular Ca2+ stores completely; therefore, we used 5
µg/mL ConA as the stimulant for T cell activation in the subsequent
experiments.
We next examined the effects of different
[Ca2+]i channel blockers on 5
µg/mL ConA-induced Ca2+ entry. When
Ca2+ entry reached a stable state, nifedipine was added to the cell suspensions, which did not affect the ConA-induced
Ca2+ entry for concentrations of 0.1-10 µmol/L (Table 2), confirming that there are no VDCC in T cells, as described
previously[2]. As shown in Table 2, SK&F96365 concentration-dependently inhibited ConA (5 µg/mL)-induced
Ca2+ influx at concentrations of 1-20 µmol/L. At a concentration of 20 µmol/L, SK&F96365 inhibited
Ca2+ influx by 52.9%¡À5.4%. SK&F96365 is an inhibitor of
Ca2+ entry through CRAC channels, as well as through
VDCC[23]. Because T cells do not contain VDCC, any effects of SK&F96365
should reflect its effects on CRAC channels. At concentrations of 0.5-2.0 µmol/L, DIDS also decreased
Ca2+ influx in a concentration-dependent manner (Table 2). Because DIDS (>3 µmol/L) began to produce quenching effects on Fura-2
fluorescence, we did not test the effects of higher concentrations of DIDS. The results described so far suggest that DIDS
affects
[Ca2+]i. To investigate whether the inhibition of
Ca2+ influx by DIDS is mediated through CRAC channels, the following
pharmacological approaches were used. As shown in Figure 2C, 2 µmol/L DIDS caused a 32.3%¡À5.1% decrease in ConA (5
µg/mL)-induced Ca2+ influx (n=7,
P<0.01), and subsequent exposure of cells to 10 µmol/L SK&F96365 further decreased
Ca2+ influx by 19.6%¡À5.2% (n=7,
P<0.01). However, when 10 µmol/L SK&F96365 caused a decrease in
Ca2+ influx by
35.8%¡À6.2%(n=7, P<0.01), subsequent addition of 2 µmol/L DIDS brought about no change in
[Ca2+]i (n=7,
P>0.05; Figure 2D). Within the concentration range of 1-20 µmol/L,
SK&F96365 serves as a CRAC channel blocker in T
cells[2,4,23], so when the CRAC-mediated
Ca2+ influx was prevented by 10 µmol/L SK&F96365, 2 µmol/L DIDS did not induce any change in
[Ca2+]i, suggesting that DIDS inhibits the ConA-induced
Ca2+ influx through interfering with the CRAC-mediated pathway. We also
tested whether DIDS directly affects ConA (5 µg/mL)-induced
Ca2+ release. Figure 2E shows that in
Ca2+-free solution (following the removal of
CaCl2 and after addition of 200 µmol/L EGTA), ConA (5 µg/mL) evoked a transient increase in
[Ca2+]i, which was due to
Ca2+ release from intracellular
Ca2+ stores, but the subsequent application of 2 µmol/L DIDS failed to
induce any change in [Ca2+]i
(n=6, P>0.05; Figure 2E). In addition, pretreatment of cells with 2 µmol/L DIDS did not induce any
change in [Ca2+]i in
Ca2+-free solution and further failed to alter subsequent ConA-induced
Ca2+ release (n=5, P>0.05, Figure
2F). These data indicate that 2 µmol/L DIDS had no direct effect on ConA-induced
Ca2+ release or resting
[Ca2+]i.
Effects of Ca2+ and
Cl- channel blockers on ConA-induced IL-2 mRNA
expression In T cells, CRAC-mediated sustained
increase in [Ca2+]i activates several transcription factors, including
NF-kB, Oct/OAP, and NF-AT, then initiates the
transcription of IL-2 mRNA, which results in IL-2 production and finally proliferation of T
cells[2]. RPA analysis demonstrated that
ConA (5 µg/mL) increased IL-2 mRNA expression in human T cells after 6 h of incubation (662%¡À45% increase;
n=7, P<0.01). To examine the effects of ion channel blockers on ConA-stimulated IL-2 mRNA expression, cells were incubated with the
blockers for 30 min prior to exposure to ConA. As shown in Figure 3A, 10 and 100 µmol/L DIDS caused a decrease in
ConA-stimulated hIL-2 mRNA expres-sion, with inhibitory rates of 37%¡À8% and 69%¡À7%, respectively
(n=4, P<0.01 vs ConA-stimulation control), whereas 1 µmol/L DIDS had no effect on IL-2 mRNA expression in stimulated T cells
(n=4, P>0.05 vs ConA-stimulation control). Consistent with previous
reports[4], SK&F96365 also decreased IL-2 mRNA expression. We
observed that 1 µmol/L SK&F96365 decreased IL-2 mRNA expression by 12%¡À5%
(n=5, P>0.05 vs ConA-treatment group;
Figure 3B). The inhibition of ConA-stimulated hIL-2 mRNA expression was significantly enhanced by the combination of 1
mmol/L SK&F96365 and 1 µmol/L DIDS (n=5,
P<0.01 vs the corresponding ConA plus blocker group). The inhibitory rates for
DIDS, SK&F96365, and SK&F96365 plus DIDS were 2%¡À1%, 12%¡À5%, and 66%¡À9%, respectively. It has been demonstrated
that naive T cell stimulation induces IL-2 mRNA, which is present for at least 5 h. This
short burst of IL-2 mRNA allows for the
secretion of functional levels of IL-2
protein that are sufficient to induce naive T
cells to enter the cell
cycle[30]. In the present study, we harvested total mRNA after the co-incubation of cells with the tested compounds for 6 h. Thus, comparing the
effects of both channel blockers on IL-2 mRNA expression could reflect the functional role of the
Cl- channels during Ca2+-dependent T cell proliferation signaling cascades.
Effects of Ca2+ and
Cl- channel blockers on ConA-
induced ClC-3 expression Our recent studies support the notion that ClC-3 may be the potential functional player during T
cell proliferation[15,16]. Therefore, we examined the effects of DIDS and its synergistic effect together with SK&F96365 on
ClC-3 mRNA expression. As shown in Figure 4A, using primers specific to the human cDNA sequence of ClC-3, bands
corresponding to the expected fragment size (248 bp) were obtained from the total RNA prepared from human T lymphocytes
exposed to different treatments. ClC-3 transcripts were found in all samples, and were increased by ConA-treatment, which
was consistent with the ClC-3 protein expression described in human T lymphocytes using Western blot
analysis[16]. As summarized in Figure 4B using
ClC-3/b-actin ratios, the exposure of T cells to 1
mmol/L DIDS and 1 µmol/L SK&F96365 decreased ConA-induced ClC-3 mRNA expression by 16%¡À6% and 9%¡À4%, respectively
(n=6, P>0.05 vs ConA-treatment
group). Moreover, the combination of 1 µmol/L DIDS and 1 µmol/L SK&F96365 acted synergistically to diminish ClC-3
mRNA expression by 64%¡À10% (n=6, P<0.05
vs the corresponding ConA plus blocker group). The
b-actin bands corresponding to 500 bp are the same in all lanes. These data suggest that the effect of DIDS on the ConA-induced ClC3 message may
be linked to CRAC-mediated signaling during T cell activation.
Discussion
The present study demonstrates the following events: (1) DIDS, a chloride channel blocker, inhibits ConA-induced
human T lymphocyte proliferation in a concentration-dependent manner, but 9-AC, NFA, and furosemide have no significant
effect on T cell proliferation induced by ConA; (2) when combined with SK&F96365, DIDS enhances the inhibitory effect of
SK&F96365 on ConA-induced IL-2 mRNA expression and T cell proliferation; (3) both DIDS and SK&F96365 decreased
ConA-induced Ca2+ influx, but the mechanisms used by the two inhibitors may be different; (4) a combination of DIDS and
SK&F96365 enhanced the inhibition of ConA-induced ClC-3 mRNA expression relative to the effect of each alone.
Fura-2 fluorescence analysis showed that ConA-treatment did not further increase
Ca2+-influx in T cells whose stores had
been already depleted by TG. This observation agrees with the recognized idea that CRAC channel is solely responsible for
Ca2+-influx during lectin-induced T cell
activation[2]. Thus, the effects of SK&F96365 on ConA-induced T cell activation and
proliferation can be attributed to the inhibition of
Ca2+-influx through CRAC channel.
A growing number of reports suggest that
Cl- channels play a functional role in the cellular proliferation process in T cells
and other cell types[12,13,17,19], which was further supported by our present work examining the effects of DIDS on several
pivotal steps during T cell proliferation, including early
Ca2+ signaling and IL-2 mRNA expression as well as later cell
proliferation. In our present and previous studies, DIDS has been shown to prevent cell proliferation in T lymphocytes
stimulated by lectins (ConA) and in rat aortic smooth muscle cells stimulated by
ET-1[13,18]. However, it should be noted that
DIDS is also an effective blocker of
Cl-/HCO3-
exchangers, ATP-sensitive K+ channels, and can bind to other
proteins[31,32]. Due to the lack of specific chloride
channel blockers, it is difficult to confirm whether
Cl- channels play a functional role in cell proliferation just using
Cl- channel blockers. To exclude several ion transports from the potential candidate list for cell proliferation, we first compared the effects
of chemically different Cl- channel blockers on ConA-induced T lymphocyte proliferation. As shown in our previous
studies[13,18], it appears that the effect of DIDS and NPPB on proliferation is not related to
Ca2+-activated or maxi Ca2+ channels,
because the Cl- channel inhibitors NFA
(Ca2+-activated Cl- channel blocker), SITS
(Ca2+-activated and maxi Cl- channel
blocker), and IAA-94 (Ca2+-activated
Cl- channel blocker) did not change the effect of ConA on T cell proliferation. Among
the ClC family members identified in immune cells (ClC-2, 3, 4, 5), ClC-2 has been excluded from the candidate lists for cellular
proliferation[17]. Another potential candidate may be the ClC-3
Cl- channel. Abundant expression of ClC-3 mRNA and protein
has been detected in T
lymphocytes[16,26]. Our previous work provided compelling evidence of the functional role of ClC-3
protein in ConA-induced T cell proliferation using antisense oligonucleotides against ClC-3. We found that the downregulation
of ClC-3 by antisense, but neither sense or missense, oligonucleotides against ClC-3 not only inhibited ConA-induced
ClC-3 expression but also decreased
[3H]thymidine incorporation and prevented T cell
proliferation[15,16]. The Cl-
current recorded in T cells shares similar pharmacological properties with the expressed ClC-3 currents in mammalian cell lines, such as
sensitivity to Cl- channel blockers including DIDS, NPPB and
tamoxifen[33,34]. These studies, along with the present work
examining the effects of Cl- channel blockers on several key signaling steps during T cell proliferation, strongly suggest that
ClC-3 channels may play functional roles during T cell proliferation signaling cascades. Although it is still unclear whether
the ClC-3 protein is a volume-regulated channel or a component thereof in some cell
types[35,36], the involvement of ClC-3 protein in cell proliferation has received strong support from studies carried out in our laboratory and other independent
studies[12]. On the other hand, the present study could not indicate whether ClC-3 is solely responsible for
Cl- channel activation by ConA in T cells. We have not accepted
Ca2+-activated Cl- channels
(ICl(Ca)) as potential candidates, because
NFA, a very common blocker of ICl(Ca)
did not alter T cell activation. Elucidating the exact nature of
Cl- channel activation in T cell proliferation will require further study.
In addition to their direct effect on T cell activation,
Cl- channels may participate in T cell activation through the regulation
of Ca2+ signaling. Although CRAC channels are not opened directly by depolarization,
Ca2+ ions entering the cell through CRAC channels feed back to inhibit the channel¡¯s activity by reducing the membrane potential.
K+ and Cl- channels have been found to assist with
Ca2+ entry by maintaining a sufficiently negative membrane
potential[37]. Therefore, it is reasonable
to suggest that Cl- channels may be involved in T cell activation by regulation of the CRAC channels. Our data also indicated
that ConA-induced Ca2+ release was not significantly changed by DIDS, which further supports the finding that DIDS at low
concentrations has no effect on Ca2+ release in some cell
types[38,39]. Thus, it seems that the effect of DIDS on T cell activation
is due to the inhibition of CRAC-induced Ca2+
entry. This appears to be the first description at different molecular levels of
the interaction between Cl- channels and CRAC-mediated
Ca2+ signaling during T cell activation. Although the link between
Cl- channels and CRAC-mediated
Ca2+ influx cannot be fully explained by the quenching effects of DIDS (>3
µmol/L) on Fura-2
fluorescence, the concentration range of DIDS that produced effects on ClC-3, IL-2 mRNA expression and cell proliferation
agree with the characterization of Cl- channels determined in other
studies[19,28,34,40].
Although a sustained Ca2+ plateau is the critical trigger inducing downstream IL-2 gene expression and cell proliferation
in ConA-activated T cells, how the specific information embedded in the amplitude, duration and kinetics of
Ca2+ signals is transmitted to downstream selective gene expression in T cells remains an unanswered key question in cell
signaling[8]. In addition, the various sizes of commonly distributing
Ca2+ oscillations also adds more complexity to
Ca2+ signaling in T cells[8]. Therefore, T lymphocyte
Ca2+ signals are more than a binary switch in T cell activation, and different patterns of
Ca2+ signals may transmit according to the specific patterns of gene expression, which will elicit final selective responses in
activated T cells. The emerging complex Ca2+
signaling in T cell activation may also help to explain the inconsistencies in
effective DIDS concentrations in the present study. A concentration of 2 µmol/L DIDS significantly inhibited CRAC-induced
Ca2+ entry, but had no effect on IL-2 mRNA expression and final T cell proliferation. It is reasonable to assume that
DIDS-sensitive Cl- channels play a time-dependent functional role during the whole process of cellular proliferation. Acute
exposure to DIDS (less than a few seconds) at a low concentration may only transiently reduce
Ca2+ signaling and fail to affect later gene
expression and final cell proliferation; whereas long-term
exposure (up to a number of hours) to higher concentrations of DIDS may cause complete inhibition of specific
Ca2+ signaling, leading to the inhibition of specific gene expression and final T cell proliferation.
In summary, the results of our present study suggest that DIDS-sensitive
Cl- channels may play a functional role in
ConA-stimulated, Ca2+-dependent IL-2 mRNA expression and cell proliferation in T cells through CRAC-mediated
Ca2+ entry. Definitive molecular determination of the DIDS-sensitive
Cl- channels will require gene targeting experiments combined with
more specific pharmacological tools. ClC-3 may be responsible for the DIDS-sensitive
Cl- channel, which may serve as a novel interfering target during the signaling transduction of T cell activation and proliferation.
Footnotes
Abbreviations used in this paper: ConA, concanavalin A;
[Ca2+]i, intracellular free calcium concentration; CRAC,
Ca2+-release-activated Ca2+;
DIDS, 4,4¡¯-diisthiocyanostilbene-2,2¡¯-disulphonic acid; SK&F96365, 1-{beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxy-phenethyl}-1
H-imidazole; TG, thapsigargin; NPPB, 5-nitro-2-(5-phenylpropylamino)-benzoate; 9-AC, 9-anthracene carboxylic acid; NFA, niflumic acid;
STIS, 4-acetamido-4-isocyana-tostilbene-2,2-disulphonic acid;IAA-94, Indanyloxyacetic acid; RPA, RNase protec-tion assay; VDCC,
voltage-dependent Ca2+ channel.
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