Cell Biochem Biophys (2014) 68:497–509

DOI 10.1007/s12013-013-9728-z

ORIGINAL PAPER

Delphinidin Activates NFAT and Induces IL-2 Production

Through SOCE in T Cells
Evelyn Jara • Marı́a A. Hidalgo • Juan L. Hancke •
Alejandra I. Hidalgo • Sebastian Brauchi •
Luisa Nuñez • Carlos Villalobos • Rafael A. Burgos

Published online: 13 August 2013

Ó Springer Science+Business Media New York 2013

Abstract Delphinidin is an anthocyanidin that possesses
antioxidant and anti-inflammatory effects; however, some
reports suggest that delphinidin has pro-inflammatory
properties. For this reason, we assessed the effect of del-
phinidin on cytokine production in T cells. We demon-
strated that delphinidin increased the cytosolic-free Ca2?
concentration by releasing Ca2? from intracellular stores
and increasing Ca2? entry. The putative Ca2? release
activated Ca2? (CRAC) channel inhibitors BTP2 and
gadolinium reduced the calcium entry stimulated by the
anthocyanidin. Delphinidin induced nuclear factor of acti-
vated T cells (NFAT) translocation and NFAT-Luc activity
in Jurkat cells and was dependent on the CRAC channel
and calcineurin pathway. Delphinidin increased the mRNA
expression and production of IL-2 in Jurkat cells and was
inhibited by BTP2 and cyclosporine A. Using peripheral
blood lymphocytes, we demonstrated that delphinidin
increased the production of IL-2 and IFN-c and was
inhibited by BTP2. Taken together, our results suggest that
delphinidin exerts immunostimulatory effects on T cells by
Electronic supplementary material The online version of this
article (doi:10.1007/s12013-013-9728-z) contains supplementary
material, which is available to authorized users.
E. Jara  M. A. Hidalgo  J. L. Hancke  A. I. Hidalgo 
R. A. Burgos (&)
Institute of Pharmacology and Morphophysiology, Universidad
Austral de Chile, P.O. Box 567, Valdivia, Chile
e-mail: rburgos1@uach.cl
S. Brauchi
Institute of Physiology, Faculty of Medicine, Universidad
Austral de Chile, Valdivia, Chile
L. Nuñez  C. Villalobos
Spanish Research Council, Institute of Molecular Biology
and Genetics, University of Valladolid, Valladolid, Spain
increasing cytokine production through CRAC channel and
NFAT activation.
Keywords Delphinidin  Calcium  T cells  NFAT 
Cytokines
Abbreviations
SOCE Store-operated calcium entry
BTP2 N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-
carboxamide
CRAC Ca2? release-activated Ca2?
Introduction
Previous studies have reported that anthocyanidins, which
are commonly found in pigmented fruits and vegetables,
stimulate an immune response by augmenting pro-inflam-
matory cytokine expression [1–5]. Delphinidin is one of the
most important anthocyanidins found in berries and dark
grapes [6]. It has been suggested to be beneficial to human
health in many ways, such as providing antioxidant and
anticancer effects [7, 8]. In RAW 264.7 macrophages,
delphinidin increased the TNFa production induced by
LPS/IFN-c and was the most potent of all anthocyanidins
tested, which included malvidin, peonidin, and pelargoni-
din [9]. Despite these previous studies, other authors have
noted that delphinidin exerts anti-inflammatory effects on
Jurkat cells by suppressing NF-jB acetylation and inhib-
iting TNFa and IL-6 production when the Jurkat cells were
induced by LPS [10]. Thus, the effect of delphinidin on T
cell cytokine production is not clear.

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In T cells, activation through the T cell receptor (TCR)
complex leads to a rapid increase in the cytoplasmic Ca2?
concentration ([Ca2?]i), which triggers signals that are
essential for T cell activation, proliferation, gene expres-
sion, cytokine production and differentiation [11]. Calcium
signaling mechanisms include the inositol 1,4,5-trisphos-
phate (IP3)-induced release of Ca2? from the endoplasmic
reticulum (ER) and store-operated Ca2? entry (SOCE)
through CRAC channels [12].
SOCE-mediated Ca2? signals regulate the expression of
cytokines that are critical for immune responses. Deriva-
tives of 3, 5-bistrifluoromethyl pyrazole (BTP), such as
BTP2, inhibit T cell SOCE-dependent calcium signaling
and the subsequent cytokine production at the transcrip-
tional level [13]. The control of IL-2 gene expression in T
cells is predominately linked to the nuclear factor of acti-
vated T cells (NFAT), which is regulated in a dynamic
manner by Ca2? levels [14]. In unstimulated cells, NFAT is
located in the cytoplasm in a highly phosphorylated form.
An increase in intracellular Ca2? activates calcineurin
(Cn), which is a serine-threonine phosphatase that dephos-
phorylates NFAT and thus induces NFAT’s translocation
into the nucleus [15, 16]. Once in the nucleus, NFAT binds
to specific DNA sequences to activate the transcription of
NFAT-dependent genes [17–19].
Impaired Ca2? signaling in T cells is linked to several
inherited immunodeficiency diseases, such as severe
combined immunodeficiency (SCID) [20, 21]. While SCID
patients were originally identified by their susceptibility to
recurrent infections, it was later revealed that these patients
were completely deficient in CRAC channel function
[20–23]. The lack of SOCE in these patients is associated
with severely compromised T cell proliferation and a
decreased production of cytokines, such as IL-2, IL-4,
IL-10, IFN-c, and tumor necrosis factor (TNFa); patients
also exhibited markedly impaired antibody responses to T
cell-dependent antigens [22–25]. In addition, T cells from
SCID patients show a pronounced attenuation of NFAT
dephosphorylation and nuclear translocation in response to
TCR stimulation or treatment with pharmacological agents,
such as ionomycin or thapsigargin; however, these cells
maintain intact AP-1 and NF-jB activations [26]. In this
study, we demonstrate that delphinidin activates NFAT and
induces cytokine production through increased levels of
SOCE in T cells.
Materials and Methods
Reagents
Delphinidin was purchased from Extrasynthese (France)
and dissolved in DMSO at a stock concentration of
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Cell Biochem Biophys (2014) 68:497–509
125 mM. Each stock aliquot of delphinidin was frozen
at -80 °C, and a vial was dissolved for each experiment
using HEPES buffer. Fura-2/acetoxymethyl ester (AM) and
anti-mouse Alexa 488 (#A11017) were purchased from
Molecular Probes (InvitrogenTM, CA, USA). N-(4-[3,5-bis
(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-
thiadiazole-5-carboxamide (BTP2) was obtained from
Calbiochem (Darmstadt, Germany). Gadolinium (Gd3?),
cyclosporine A, and HistochoiceTM were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). RPMI 1640
medium, penicillin, and fetal bovine serum (FBS) were
purchased from Hyclone (Logan, UT, USA). IL-2 ELISA
kits were purchased from Biosource Europe S.A. (Niv-
elles, Belgium). A monoclonal antibody against NFATc1
was obtained from BD Transduction Laboratories (San
Diego, CA, USA). Anti-mouse IgG-HRP was obtained from
Cell Signaling (Beverly, MA, USA). Lymphocyte Separa-
tion Medium was obtained from Mediatech (Mediatech,
Inc., VA, USA). The RNeasyÒ Plus Mini Kit was obtained
from Qiagen, Inc. (CA, USA). The IL-2 and IFN-c BD
OptEIA TM Kits were obtained from BD Biosciences. All
other reagents and chemicals were purchased from Merck
(Darmstadt, Germany).
Jurkat E6-1 Cell Culture
The Jurkat E6-1 cell line (ATCCÒ CRL-1573TM) was
cultured in RPMI 1640 medium supplemented with 10 %
FBS, 1 mM sodium pyruvate, and 2 mM L-glutamine at
37 °C in a humidified incubator with 5 % CO2.
Isolation of Peripheral Blood Lymphocytes
Lymphocytes were separated from blood samples obtained
from healthy volunteer donors using protocols approved by
the Ethical Committee; specifically, lymphocytes were
separated by flotation in Lymphocyte Separation Medium
using the manufacturer’s protocol and then utilized in
ELISA assays.
Dye Loading
Jurkat E6-1 cells were suspended in HBSS at a concen-
tration of 20 9 106 cells/mL and incubated with 1 lM
Fura-2/AM for 30 min at 37 °C. The cells were then
washed twice, divided into 2 9 106 cells/mL aliquots and
maintained at 4 °C in the dark until use. Cells were pelleted
by centrifugation at 1,200 rpm for 6 min and resuspended
in 2 mL of HEPES buffer (20 mM HEPES, pH 7.2;
140 mM NaCl; 10 mM glucose; 1 mM KCl; 1 mM Ca2?
chloride (CaCl2) and 1 mM MgCl2). In some experiments,
the cells were suspended in Ca2?-free HEPES buffer with
Cell Biochem Biophys (2014) 68:497–509
0.3 mM Tris-EGTA that was added 20 s prior to the
experiment. Experiments involving cationic inhibitors (i.e.,
Gd3?) or an influx of strontium ions (Sr2?) were performed
in Ca2?-free HEPES medium without EGTA to avoid the
chelating effect of these compounds.
Fluorescence Imaging of Cytosolic Calcium
Experiments with Fura-2/AM-loaded Jurkat cells were per-
formed using an inverted fluorescence microscope (Zeiss
Axiovert S100 TV). Jurkat cells were grown on coverslips
coated with fibronectin (20 lg/mL) and incubated with
1 lM Fura-2/AM for 90 min at room temperature in the
dark. Images were captured using an OrcaER camera
(Hamamatsu Photonics, Hamamatsu, Japan). Finally, the
records and images were analyzed using Aquacosmos 2.0
image analysis software.
The Measurement of Calcium (Ca2?) and Strontium
(Sr2?) Influx
To assess SOCE, Fura-2/AM-loaded Jurkat E6-1 cells
(2 9 106 cells/mL) were suspended in Ca2?-free HEPES
buffer, and 0.3 mM EGTA was added to each cuvette 20 s
prior to the experiment. At 100 s, 50 lM delphinidin was
added, and after 200 s of recording, one pulse of 1 mM
Ca2? or Sr2? was administered and recorded for 400 s. To
investigate delphinidin-mediated SOCE activation, Fura-2/
AM-loaded Jurkat E6-1 cells were preincubated with Gd3?
or BTP2 for 15 min, and then, the influx of Ca2? and Sr2?
was recorded as the 340:380 excitation ratio measured at a
509 nm emission using a LS55 thermoregulated spectro-
fluorimeter (Perkin-Elmer, MA, USA).
The Assessment of Putative SOCE Inhibitors
on Ca2? Influx
To assess the effect of putative SOCE inhibitors, different
approaches were used. First, Fura-2/AM-loaded Jurkat E6-
1 cells (2 9 106 cells/mL) were suspended in HEPES
buffer without Ca2?, and 0.3 mM EGTA was added to
each cuvette 20 s prior to experiment initiation. After 100 s
of recording, 50 lM delphinidin was added. In the second
approach, Fura-2/AM-loaded Jurkat E6-1 cells (2 9 106
cells/mL) were suspended in HEPES with Ca2? buffer and
preincubated with the inhibitors for 15 min. After 100 s,
50 lM delphinidin was added; the dye fluorescence exci-
tation ratio was recorded for 200 s; and the area under the
200 s curve (AUC200) was estimated. For the final
approach, Fura-2/AM-loaded Jurkat E6-1 cells (2 9 106
cells/mL) were suspended in Ca2?-free HEPES buffer and
preincubated with the inhibitors for 15 min; then, 0.3 mM
EGTA was added to each cuvette 20 s prior to the
499
experiment. After 100 s of recording, 50 lM delphinidin
was added; at 300 s, a pulse of 1 mM CaCl2 was given and
registered for 150 s. The area under the 150 s curve
(AUC150) was determined. The Ca2? influx was recorded
as the excitation ratio (340/380) measured at a 509 nm
emission using an LS55 thermoregulated spectrofluorime-
ter (Perkin-Elmer, USA).
Immunofluorescence
The nuclear distribution of NFAT was measured using
immunofluorescence. Jurkat E6-1 cells were preincubated
with 10 lM BTP2, 1 lg/mL cyclosporine A or a vehicle
for 15 min and then stimulated with 50 lM delphinidin
for 30 min. Cells were centrifuged using a Cytospin for
10 min at 2009g onto 2 mg/mL poly-L-lysine-coated
slides. The cells were next fixed with HistochoiceTM for
10 min at room temperature and then permeabilized in
0.3 % Triton-X 100/PBS for 15 min. Nonspecific binding
was blocked by incubating the cells with PBS containing
1 % BSA and 5 % nonfat milk for 1 h at room temperature.
Cells were incubated with the NFATc1-specific monoclo-
nal antibody (1:200 dilution) overnight, followed by an
incubation with an anti-mouse Alexa 488 secondary anti-
body. Covered slides were analyzed using confocal fluo-
rescence microscopy (LSM5 PASCAL microscope, Zeiss,
Germany). The nuclear NFAT signal intensity was regis-
tered using Image Pro Plus v4.5 (Media Cybernetics, Inc.,
USA).
Analysis of NFAT Dephosphorylation by Immunoblot
For the detection of NFAT phosphorylation, Jurkat E6-1
cells (5 9 106) were deprived of serum for 4 h, preincu-
bated with cyclosporine A (1 lg/ml) or vehicle for 30 min,
and then stimulated with 50 lM of delphinidin. Whole cell
lysates (60 lg of protein) were separated using SDS/PAGE
with 8 % gels and probed with an anti-NFATc1 mono-
clonal antibody (BD Transduction); labeled proteins were
detected with an enhanced chemiluminescence system
(Perkin-Elmer Life Science, Boston, MA, USA).
Bioluminescence Imaging of NFAT-Luciferase
Reporter Activity in Individual Living Cells
Jurkat F6 cells that stably expressed a luciferase reporter
gene under the control of triplet NFAT response elements
[27] were placed on fibronectin-coated glass coverslips and
subjected to time-lapse bioluminescence imaging to visu-
alize the dynamics of NFAT transcriptional activity in
living cells. Cells were introduced into a Zeiss incubator,
which was attached over an inverted Zeiss S100 TV
microscope, and subjected to bioluminescence imaging for
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16 h. Photonic emissions reflecting NFAT transcriptional
activity were taken every 15 min using a Hamamatsu
photon counting camera. Specific photonic emissions were
calculated as raw photonic emissions minus background
emissions (i.e., photonic emissions captured over 15 min in
a field devoid of cells). Ionomycin (500 nM) plus PMA
(100 nM), delphinidin (50 lM) or a vehicle treatment was
added at time 0.
Flow Cytometric Analysis of NFAT Translocation
in Jurkat Nuclei Preparations
For the detection of NFAT translocation by flow cytome-
try, we used the protocols described by Blaecke et al. In
brief, Jurkat E6-1 cells (1 9 106 cells/mL) were preincu-
bated with 10 lM BTP2 or 1 lg/mL cyclosporine A for
30 min and then stimulated with 50 lM delphinidin for
30 min. After the incubation period, the cells were washed
twice with PBS.
Nuclear preparations were achieved by incubating the cells
in 200 ll of Pipes-Triton solution, as described previously
[28]. After two washes in PBS, the nuclei were stained with
mouse anti-NFATc1 antibodies (5 lg/mL) for 30 min at
4 °C. The nuclei were washed twice with PBS, incubated for
30 min at 4 °C with anti-mouse Alexa 488 (1:200 dilution)
and finally washed twice with PBS. The nuclei were assessed
by flow cytometry on a FACSCanto II (Becton–Dickinson);
the results were analyzed using the FlowJo v7.6 software.
qRT-PCR
Total RNA was isolated from Jurkat E6-1 cells using the
RNeasyÒ Plus Mini Kit according to the manufacturer’s
instructions. Approximately 2 lg of total RNA, 0.5 lg of oligo
(dT)18 primer, 10 mM dNTPs, and 200 U of reverse trans-
criptase were used for each cDNA synthesis reaction. PCR
amplifications were performed using the BrilliantÒ II SYBRÒ
Green QPCR Master Mix (Stratagene) according to the man-
ufacturer’s instructions. All reactions were carried out using an
Mx300p thermal cycler (Stratagene, CA, USA). IL-2-specific
primers were used as follows: 50 -ACCAGGATGCTCACATT
TAAGTTTT-30 (sense) and 50 -GAGGTTTGAGTTCTTCTT
CTAGACACTG-30 (antisense).
IL-2 and IFN-c Measurements
Jurkat E6-1 cells (5 9 106 cells/mL) and peripheral blood
lymphocytes (PBLs) (5 9 105 cells/mL) were cultured in
24-well plates, preincubated with BTP2 (1, 5 or 10 lM),
cyclosporine A (1 lg/mL) or a vehicle for 15 min and then
stimulated with 50 lM delphinidin. Supernatants were
collected at 48 h post-stimulation and analyzed for IL-2
and IFN-c using the BD OptEIA TM Kit.
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Cell Biochem Biophys (2014) 68:497–509
MTT Assay
Jurkat cells (5 9 104 cells/mL) were incubated with a
vehicle or delphinidin (10, 50 or 100 lM) for 24 h. Cell
proliferation was assessed using the mitochondria-depen-
dent reduction of MTT to formazan. The reduction of MTT
to formazan was estimated by measuring OD570 values
with a microplate reader (Varioskan Flash Multimode
Reader, Thermo Scientific, USA).
Statistical Analysis
Results were analyzed using one-way analysis of variance
(ANOVA) and Dunnett’s multiple comparisons test. A
significance level of 5 % was used.
Results
Delphinidin Mobilizes Calcium in Jurkat Cells
Following TCR activation, increase in intracellular calcium
leads to the activation of PLCc1, increased levels of inositol-
1,4,5-trisphosphate (InsP3), and the binding of InsP3 to the
InsP3 receptor (InsP3R), thereby inducing the release of Ca2?
from the ER. The decreased Ca2? concentration in the ER
triggers the activation of the ‘‘capacitative,’’ or SOCE, path-
way, resulting in the opening of the store-operated Ca2?
channels in the plasma membrane, which are responsible for
the inward calcium current (Icrac). Because the Ca2? from the
ER and the extracellular calcium influx are two sources of this
cation, which are associated with T cell activation [11], we
assessed the effect of delphinidin on calcium mobilization by
measuring the calcium flux in Fura-2/AM-loaded Jurkat cells.
In situ calcium imaging in the presence of 1 mM CaCl2
revealed that during the perfusion of delphinidin a significant
increase of intracellular Ca2? was observed (Fig. 1a, b).
Treatment with varying concentrations of delphinidin (10, 50,
and 100 lM) induced a slow and steady increase in [Ca2?]i.
This effect was dose-dependent, as shown by representative
recordings and AUC150 measurements (Fig. 1c, d, respec-
tively), which increased following treatment with 50 or
100 lM delphinidin, compared to vehicle-treated cells (i.e.,
0.2 % DMSO). All concentrations of delphinidin used in this
experiment were not toxic to the Jurkat cells, as was demon-
strated in the MTT-based viability experiments (Fig. 1e).
The role of extracellular and intracellular calcium on the
calcium fluxes induced by 50 lM delphinidin was evalu-
ated (Fig. 2). Fura-2/AM-loaded cells were incubated in
Ca2?-free medium, and 0.3 mM EGTA was added 20 s
prior to delphinidin stimulation. Delphinidin treatment in
Ca2?-free medium induced a mild increase in intracellular
calcium compared to delphinidin in the presence of Ca2?,
Cell Biochem Biophys (2014) 68:497–509
A C
B D
Fig. 1 Delphinidin induces calcium fluxes in Jurkat cells. Fura-2/
AM-loaded Jurkat E6-1 cells were suspended in Ca2?-HEPES buffer
and then stimulated with delphinidin. The Ca2? influx was assessed
by in situ calcium imaging and spectrofluorimetric assays. a Repre-
sentative fluorescence imaging of cytosolic calcium increases induced
by treatment with 50 lM delphinidin (9400). The pseudocolor scale
reflects low (0) and high ratios [1]. b Representative measurements of
the effects induced by 50 lM delphinidin on the 340/380 ratio during
suggesting the contribution of an intracellular compartment
to this effect (Fig. 2a, b). In addition, we utilized BAPTA/
AM to chelate the intracellular calcium and to assess the
effect of delphinidin on ER Ca2? release. We observed an
almost complete inhibition of the calcium flux, which
suggests the inhibition of intracellular calcium release;
however, a mild increase in Ca2? was still recorded
(Fig. 2c). The calcium flux induced by 50 lM delphinidin
was completely blocked in the presence of 50 lM BAPTA/
AM and 0.3 mM EGTA in the absence of CaCl2 (Fig. 2d).
Putative SOCE Inhibitors BTP2 and Gd3? Reduce
the Delphinidin-Induced Ca2? Influx
In T cells, the main mechanism for the influx of extracel-
lular calcium across the plasma membrane is SOCE [11].
SOCE leads directly to a sustained increase in the
501
the fluorescence imaging of cytosolic calcium. c The effect of
delphinidin (10, 50, and 100 lM) on Fura-2/AM-loaded Jurkat cells
in the presence of Ca2?. d The dose response effect of delphinidin on
the AUC150 of the Fura-2/AM 340/380 ratio. e The effect of
delphinidin on Jurkat cell viability using MTT assay is shown. Each
bar represents the mean of at least three independent experiments.
*p \ 0.05 and **p \ 0.01 compared with vehicle
intracellular Ca2? concentrations and long-term functional
consequences [29].
To evaluate the contribution of SOCE on the increase of
intracellular calcium induced by delphinidin, BTP2, which
inhibits SOCE in Jurkat cells [13], was used. In the presence of
1 mM CaCl2, BTP2 significantly reduced the delphinidin-
induced calcium flux in Fura-2/AM-loaded Jurkat cells
(Fig. 3a; Supplementary Data 1A). In addition, we tested the
efficacy of Gd3?, a putative SOCE inhibitor, on delphinidin-
induced calcium flux in Fura-2/AM-loaded Jurkat cells
(Fig. 3d). It is widely known that lanthanides, such as Gd3?,
suppress CRAC channels in Drosophila S2 cells [30]. In the
presence of 1 mM CaCl2, the treatment of Fura-2/AM-loaded
Jurkat cells with 1, 5, or 10 lM Gd3? significantly reduced the
AUC200 induced by 50 lM delphinidin (Supplementary Data
1B), which suggests an interference with the Ca2? influx.
Next, we assessed the effect of BTP2 on the Ca2? influx
induced by delphinidin in the absence of external calcium
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Fig. 2 Ca2? chelation A B

interferes with delphinidin-
induced Ca2? flux. Jurkat cells
was loaded with Fura-2/AM,
resuspended in the presence or
absence of Ca2? and stimulated
with delphinidin. The Ca2?
influx was then measured using
spectrofluorimetric assays.
a The effect of treatment with
50 lM delphinidin on Fura-2/
AM-loaded Jurkat cells in the
presence of Ca2? is shown. The
effects of a b Ca2?-free solution
plus 0.3 mM EGTA, c 1 mM C D
Ca2? plus 50 lM BAPTA/AM,
and d Ca2?-free/0.3 mM EGTA
plus 50 lM BAPTA/AM on the
delphinidin-induced calcium
mobilization are shown

Fig. 3 Delphinidin-induced A D
Ca2? and Sr2? influxes are
inhibited by BTP2 and Gd3?.
The spectrofluorometric register
of the effect of BTP2 (10 lM)
(a) or Gd3? (10 lM) (d) on
delphinidin-induced Ca2?
movements in Fura-2/AM-
loaded Jurkat has been depicted.
In a second set of experiments,
Jurkat cells were stimulated
with 50 lM delphinidin in
Ca2?-free HEPES/0.3 mM B E
EGTA, and, then, a 1 mM
CaCl2 pulse was added. A
representative
spectrofluorimeter recording of
the effect of BTP2 (b) or Gd3?
(e) on the Ca2? influx induced
by delphinidin is shown. In a
third set of experiments, Fura-2/
AM-loaded Jurkat cells were
stimulated with 50 lM
delphinidin in Ca2?-free
HEPES. A pulse of 1 mM SrCl2 C F
was added, and the 340/380
ratio of fluorescence was
recorded. The effects of 10 lM
BTP2 or Gd3? on Sr2? influx
(c, f) are depicted

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Cell Biochem Biophys (2014) 68:497–509 503

(Fig. 3b). For these experiments, we used Fura-2/AM-loa-
ded cells that were preincubated with varying concentrations
of BTP2 in the absence of external calcium and in the pres-
ence of 0.3 mM EGTA. After 100 s of recording, 50 lM
delphinidin was added. We did not observe an inhibition of
the intracellular calcium movements. However, after 200 s,
a pulse of 1 mM CaCl2 was added. Under these experimental
conditions, pretreatment with BTP2 (5 and 10 lM) signifi-
cantly reduced the delphinidin-induced Ca2? influx, as
measured by the AUC150 (Supplementary Data 2).
Because delphinidin treatment induced intracellular
Ca2? release, we assessed whether Gd3? interfered with
this mechanism. To achieve this, we developed an exper-
imental protocol in the absence of external Ca2? and in the
presence of 0.3 mM EGTA. We added 50 lM delphinidin
to Fura-2/AM-loaded Jurkat cells either in the presence or
absence of Gd3? and then measured the level of fluores-
cence emitted. After a period of 200 s, a pulse of 1 mM
CaCl2 was incorporated. In the absence of external cal-
cium, delphinidin induced a strong Ca2? influx (Fig. 3e).
Gd3? did modify the intracellular Ca2? release, as mea-
sured by the AUC200 (Supplementary Data 3A). The
addition of 1 mM CaCl2 increased the fluorescence, sug-
gesting the existence of a Ca2? influx that was significantly
inhibited by Gd3? (Fig. 3e; Supplementary Data 3B).
phosphatase. Calcineurin activation is initiated by the
binding of calcium to calmodulin; once activated, calci-
neurin causes NFAT dephosphorylation, which is a key
step in NFAT nuclear translocation [35]. Because the
increase in calcium induced by delphinidin is able to
activate the NFAT pathway, we analyzed the effect of
delphinidin treatment on NFAT phosphorylation using
western blot analysis. While NFAT migrated as a single
protein band with an apparent molecular mass of 140 kDa
in unstimulated cells, NFAT migrated as multiple bands in
cells stimulated with 50 lM of delphinidin (Fig. 4a).
Delphinidin induced a marked NFAT dephosphorylation at
15 min post-stimulation that was sustained for 4 h. In
A
B

BTP2 and Gd3? Reduce the Delphinidin-Induced

Sr2? Influx in Jurkat Cells

CRAC channels are known to be permeable to other cat-

ions, such as Sr2? [31]; therefore, in a manner similar to
Ca2?, delphinidin could induce the Sr2? influx in Jurkat
cells. To test this hypothesis, we investigated the impact of
delphinidin on Sr2? entry into cells. Although Sr2? has a
lower affinity for Fura-2/AM compared to Ca2? and its
binding causes less fluorescence, their isosbestic points and
340:380 ratio profiles are similar [32]. Furthermore, Sr2?
has been used as an indicator of SOCE in previous studies
[33, 34]. In the absence of external calcium, delphinidin
treatment increased the Sr2? influx when 1 mM Sr2? was
added into the medium at 300 s (Fig. 3c). The influx of Fig. 4 Delphinidin induces NFAT activation in Jurkat cells.
Sr2?, but not an intracellular Ca2? release, was inhibited by a Western blot analysis of cell extracts after various periods of
treatment with delphinidin. Jurkat cells were treated with vehicle or
10 lM BTP2 (Supplementary Data 4A, C). The inhibitor
delphinidin for 0, 5, 15, 30, 45, 60, 120, and 240 min. The
Gd3? (10 lM) also blocked the Sr2? influx (Fig. 3f). electrophoretic mobilities of the upper and lower bands, which
However, in contrast to BTP2, Gd3? affected both the correspond to the phosphorylated and dephosphorylated forms of
release of intracellular calcium and the influx of Sr2? NFAT1, respectively, are indicated by arrows. The data shown are
representative of at least three separate experiments. Jurkat F6 cells
(Fig. 3f; Supplementary Data 4B, D).
were placed on fibronectin-coated glass coverslips and subjected to
time-lapse bioluminescence imaging to visualize the dynamics of
Delphinidin Activates the NFAT Pathway NFAT transcriptional activity in living cells. Bioluminescent images
in Jurkat Cells of accumulated photonic emissions during a 15 min period in a
control medium or in medium containing 50 lM delphinidin (the
pseudocolor scale ranges from 0 to 50 photons per pixel). Data are
In T cells, sustained calcium influx is associated with the representative of at least three independent experiments for each
activation of calcineurin, which is a calmodulin-dependent condition

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addition, we assessed the transcriptional activation of
NFAT following delphinidin treatment using Jurkat cells
that express luciferase under the control of triplet NFAT
response elements. These cells were subjected to time-
lapse bioluminescence imaging to visualize the dynamics
of NFAT transcriptional activity in live Jurkat F6 cells.
Treatment with 50 lM delphinidin increased NFAT tran-
scriptional activity until 4 h post-stimulation, after which
NFAT transcriptional activity achieved a steady state that
was sustained throughout the experimental time period
(16 h) (Fig. 4b). As a positive control, treatment with
ionomycin (500 nM) plus PMA (100 nM) also increased
the NFAT transcriptional activity, and a maximal level of
transcription was achieved at 6 h post-stimulation (data not
shown).
Delphinidin-Induced NFAT Activation and IL-2
Production are Dependent on SOCE and Calcineurin
As SOCE and calcineurin are critical for NFAT activation,
we analyzed the effect of delphinidin on NFAT translo-
cation via flow cytometric and immunofluorescence anal-
yses. The flow cytometric analysis of NFAT translocation
in nuclei purified from Jurkat E6-1 cells showed that del-
phinidin induced an increase in the amount of nuclear
NFAT compared to control cells. This increase was
blocked in Jurkat E6-1 cells that were preincubated with
BTP2, and the translocation levels observed under these
experimental conditions were similar to the levels observed
in controls (Fig. 5a). In addition, we corroborated the
nuclear translocation of NFAT using immunofluorescence
experiments. We observed that delphinidin increased the
presence of NFAT in the nuclei of Jurkat E6-1 cells
compared to control cells. Furthermore, the preincubation
of Jurkat E6-1 cells with BTP2 blocked the nuclear trans-
location of NFAT (Fig. 5b).
In T cells, calcium signals are a critical signal for the
activation of effector functions, such as IL-2 production
[11]. To assess whether delphinidin could exert an immu-
nostimulatory effect, we treated Jurkat cells with 50 lM
delphinidin and measured the resulting IL-2 mRNA and
protein levels. Delphinidin increased the expression of IL-2
mRNA after 24 h of incubation (Fig. 5c) and the produc-
tion of IL-2 protein after 48 h of incubation (Fig. 5f).
Higher concentrations of delphindin did not increase the
expression of IL-2 mRNA or proteins more than observed
with 50 lM.
To assess whether CRAC channels and NFAT are involved
in delphinidin-induced IL-2 production, Jurkat cells were
preincubated with BTP2 prior to delphinidin stimulation.
BTP2 pretreatment significantly inhibited IL-2 mRNA
expression (Fig. 5c) and IL-2 protein production (Fig. 5f).
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Cell Biochem Biophys (2014) 68:497–509
Next, cyclosporine A was used to study the role of
calcineurin on NFAT nuclear translocation induced by
delphinidin. Flow cytometric and immunofluorescence
analyses showed that the delphinidin-induced NFAT
nuclear translocation was calcineurin-dependent, as the
nuclear translocation was inhibited by cyclosporine A
treatment (Fig. 5d, e).
To assess whether NFAT participated in delphinidin-
induced IL-2 production, Jurkat cells were incubated with
cyclosporine A (1 lg/mL) for 30 min and then stimulated
with delphinidin. Treatment with 1 lg/mL cyclosporine A
significantly reduced IL-2 mRNA expression (Fig. 5c) and
IL-2 production (Fig. 5f).
In addition, we demonstrated that delphinidin induced
the production of IL-2 and IFN-c in PBLs (Fig. 6a, b,
respectively). In both cases, this effect was significantly
blocked by BTP2, which suggests that cytokine production
induced by delphinidin is also SOCE dependent (Fig. 6c,
d). We demonstrated that the IFN-c production induced by
delphinidin in PBLs was reduced with 1 lg/mL of cyclo-
sporine A (Supplementary Data 5).
Discussion
Polyphenols have been reported to exert a large number of
beneficial physiological effects on humans [36]. Within the
polyphenol group, anthocyanidins and their glycosylated
forms, anthocyanins, have gained significant attention due
to the numerous studies that indicate their potential human
health benefits [37, 38]. Delphinidin has been reported to
possess antioxidant properties [39], as well as to exert
antimutagenic [40] and neuroprotective effects [41, 42]. In
addition, delphinidin displays a variety of effects on blood
vessels [43] and platelets [44] and has been shown to
reduce the risk of coronary artery disease [45].
In the present work, we demonstrated that delphinidin
increases the intracellular calcium fluxes that induce
cytokine production in T cells.
We demonstrated through spectrofluorimetric experi-
ments that delphinidin induces a sustained calcium mobi-
lization in Jurkat E6-1 cells. Calcium imaging experiments
revealed that delphinidin treatment increased calcium
movement, which decreased when it was withdrawn at
6 min from the perfusion medium. In addition, in the
absence of external calcium, delphinidin-induced calcium
fluxes were reduced, but not abolished. Calcium mobili-
zation was abolished completely in the presence of EGTA
plus 50 lM BAPTA/AM, indicating that intracellular Ca2?
stores play an important role in delphinidin-induced cal-
cium mobilization.
Previously, treatment with 30 lM delphinidin was shown
to increase calcium fluxes in bovine aortic endothelial cells.
Cell Biochem Biophys (2014) 68:497–509 505

A D

B E

C F

Fig. 5 Delphinidin-induced NFAT translocation and IL-2 mRNA
expression and cytokine production are blocked by BTP2 and
cyclosporine A. Jurkat cells were incubated with 10 lM BTP2 (a,
b) or 1 lg/mL cyclosporine A (e, f) for 15 min and then stimulated
with 50 lM delphinidin for 15 min. Jurkat cell nuclei were then either
subjected to flow cytometric analysis to determine the amount of
NFAT present (a, d) or to a cytospin of cells before being fixed onto
glass coverslips for immunofluorescence analysis using propidium
iodide and the NFATc1 antibody-Alexa Fluor488 (b, e). The bar
graph shows the ratio of the fluorescence between nuclear NFAT and
total cellular fluorescence. Jurkat cells were incubated with 10 lM
BTP2 (c) or 1 lg/mL cyclosporine A (e, f) for 15 min and then
stimulated with 50 lM delphinidin. RNA and supernatants were
extracted and subjected to analysis by qRT-PCR (c) or ELISA (f),
respectively. Each bar represents the mean ± SEM of at least three
independent experiments. **p \ 0.01 compared to treatment with
50 lM delphinidin alone

In the absence of external calcium, the mean peak of [Ca2?]I,
as induced by delphinidin, was significantly reduced by
70 ± 2 %, which suggests the involvement of both calcium
influx and internal calcium release [46].
The main mechanism for the entry of extracellular Ca2?
across the plasma membrane into T cells is via SOCE,
which occurs when InsP3-induced depletion of the ER
Ca2? stores triggers plasma membrane Ca2? entry through
Ca2? release-activated calcium (CRAC) channels [47].
We assessed the effects of two CRAC channel inhibi-
tors, Gd3? and BTP2. Both inhibitors reduced delphinidin-
induced calcium mobilization. In the absence of external
calcium, Gd3? and BTP2 inhibited calcium uptake when
1 mM CaCl2 was incorporated into the medium. However,
while Gd3? reduced the delphinidin-induced calcium
release from intracellular stores, BTP2 did not. This sug-
gests a more specific effect of BTP2 on the opening of
CRAC channels in Jurkat cells [13].
The inhibition of the calcium influx induced by BTP2
and Gd3? in Jurkat cells suggests that delphinidin activates
the uptake of extracellular calcium through CRAC chan-
nels. Consistent with these findings, Zitt et al. [13] dem-
onstrated that BTP2 specifically inhibited CRAC channels
in human peripheral blood CD4? and CD8? T cells and in

123
506
A B
C D
Fig. 6 BTP2 blocks IL-2 and IFN-c production induced by delphin-
idin in PBLs. Lymphocytes were separated from blood samples and
then incubated with delphinidin (10, 30, 50, or 100 lM) for 48 h.
Next, the supernatants were extracted and subjected to ELISA
analysis for the presence of IL-2 (a) or IFN-c (b) production. PBL
cells were incubated with 1, 5, and 10 lM BTP2 for 15 min and then
Jurkat cells without affecting the other channels or trans-
porters [13]. BTP2 has been shown to block SOCE in T
lymphocytes by inhibiting some of the proteins putatively
involved in this phenomenon, which include members of
the transient receptor potential (TRP) superfamily, such as
TRPM4 [48].
In addition, we observed that delphinidin induced a
sustained Sr2? influx over time. This result corroborates
delphinidin-induced SOCE activation because the perme-
ability to both Ca2? and Sr2? was increased. Furthermore,
both BTP2 and Gd3? reduced delphinidin-induced Sr2?
influx, suggesting that their entry was CRAC-dependent.
However, the increase in fluorescence with Sr2? was
reduced compared to Ca2?. This result could be explained
by the lower affinity of Sr2? for Fura-2/AM or a result of
its binding, which might cause decreased fluorescence
compared to Ca2? [32]. In addition, the relative perme-
ability for Sr2? was lower compared to Ca2? in the Jurkat
cells, which exhibited a permeability ratio of 0.06 for Sr2?
versus 1 for Ca2? [49]. Furthermore, we determined that
delphinidin was not able to induce Ba2? influx in Jurkat
cells (data not shown). This result is consistent with the low
permeability reported for this cation in Jurkat cells that
were stimulated with 1 lM thapsigargin in Ca2?-free
K?-Ringer’s solution [49]. Moreover, in cytotoxic T cells,
the store-operated calcium influx is seven times more
selective for Ca2? over Sr2?, and 17 times more selective
for Ca2? over Ba2? [49]. The mechanism of the delphini-
din-induced activation of the CRAC channels remains
unknown. The use of delphinidin has been described in
123
Cell Biochem Biophys (2014) 68:497–509
stimulated with 50 lM delphinidin for 48 h; at that point, the
supernatants were extracted and subjected to ELISA analysis for the
presence of IL-2 (c) or IFN-c (d). Each bar represents the
mean ± SEM of at least three independent experiments. *p \ 0.05;
**p \ 0.01 compared to treatment with 50 lM delphinidin alone
LoVo/ADR cells, a doxorubicin-resistant metastatic human
colorectal adenocarcinoma cell line, and said to induce
cytotoxicity and increase the production of radical oxygen
species [50]. Because H2O2 can be involved in the acti-
vation of CRAC channels in Jurkat cells [51], and antho-
cyanins have shown anti-proliferative effect in leukemia
cells [52], this could represent a potential mechanism
implicated in the effect of delphinidin on T cells. However,
this theory was discarded because the concentration of
delphinidin used in T cells did not induce any toxic effects.
Here, using luminol, we were not able to not observe any
increase in the production of radical oxygen species (data
not shown).
In T cells, the sustained influx of Ca2? is associated with
the activation of calcineurin, which is a Ca2?/calmodulin-
dependent Ser/Thr phosphatase that dephosphorylates
NFAT; the dephosphorylation of NFAT enables NFAT
nuclear translocation and transcriptional activity [53, 54].
We assessed the NFAT phosphorylation status following
delphinidin treatment. Treatment with 50 lM of delphini-
din induced NFAT dephosphorylation. Moreover, the
strong NFAT dephosphorylation observed after 15 min of
delphinidin treatment was maintained through 240 min of
stimulation.
NFAT-h is a conserved module of *300 residues,
which regulates NFAT nuclear/cytoplasmic trafficking in
response to changes in intracellular Ca2? concentrations.
The engagement of the TCR or treatment of cells with a
Ca2? ionophore activates calcineurin. Calcineurin then
dephosphorylates the NFAT-h domain, which leads to the
Cell Biochem Biophys (2014) 68:497–509
unmasking of NFAT’s nuclear localization sequences and
subsequent NFAT nuclear translocation [55, 56]. In cells,
NFAT remains in the nucleus while Ca2? is elevated, but it
is rapidly phosphorylated and exported to the cytoplasm
following the termination of calcium signaling [17, 18].
Because delphinidin induced a strong and sustained Ca2?
flux in Jurkat cells, we assessed the extent of NFAT
translocation in response to delphinidin treatment. We
demonstrated that treatment with 50 lM delphinidin
resulted in increased NFAT nuclear localization in Jurkat
cells. In addition, BTP2 significantly inhibited this
response, suggesting that NFAT nuclear translocation is
CRAC channel-dependent. Previously, BTP2 was demon-
strated to inhibit NFAT activation in Jurkat cells and pri-
mary human T cells [13, 56, 57], which supports the
critical importance of CRAC channels in NFAT activation.
We also observed that cyclosporine A partially blocked the
delphinidin-induced NFAT nuclear translocation. Consis-
tent with these results, BTP2 has been shown to suppress
calcineurin activation induced by PMA/ionomycin in Jur-
kat cells, which further supports the role of CRAC channels
in calcineurin activation [58].
Finally, we demonstrated that delphinidin increased
NFAT transcriptional activity. Treatment with delphinidin
induced a sustained increase in NFAT-luciferase reporter
activity during the entire recording period.
These results suggest that delphinidin can induce the
transcriptional activation of NFAT-dependent genes in T
cells, including genes that code for cytokines, such as IL-2
and IFN-c [11]. Consistent with this hypothesis, we
observed that delphinidin increased the expression and
production of IL-2 mRNA and protein. Delphinidin treat-
ment enhanced IL-2 mRNA expression five times above
the levels observed in control cells while increasing IL-2
production by *250 pg/mL compared to control-treated
cells. BTP2 pretreatment significantly decreased delphini-
din-induced IL-2 mRNA expression and protein produc-
tion. In this context, the CRAC channels are fundamental
for the activation of T cell effector functions, particularly
because BTP2 is able to specifically inhibit calcium-
dependent gene expression, including IL-2, IL-5, and IFN-
c, in peripheral blood regulatory T lymphocytes and Jurkat
cells [56, 57]. Moreover, we observed that BTP2 can
reduce IL-2 and IFN-c production induced by delphinidin
in peripheral blood T lymphocytes. In addition, cyclo-
sporine A pretreatment resulted in a partial decrease of
delphinidin-induced IL-2 production. Because cyclospor-
ine A completely inhibits IL-2 production induced by
PMA/ionomycin in Jurkat cells [59], our results suggest
that delphinidin-induced IL-2 production involves other
calcium-dependent signaling pathways. The anti-inflam-
matory properties of delphinidin are well-known and have
been demonstrated previously [60]. However, in RAW
507
264.7 macrophages, delphinidin increased the TNFa pro-
duction induced by LPS/IFN-c [9]. The present finding on
T cells supports a more complex immunomodulatory effect
of delphinidin on leukocytes. Due to the low bioavailability
of anthocyanins, a potential therapeutical effect, should be
carefully considered to explain systemic effects. However,
because anthocyanins are present in foods and several
antecedents have suggested potentially positive effects in
gastrointestinal diseases, we did not discard the potential
local immunological modulations induced by these com-
pounds. In support of this assumption, anthocyanins have
been described as effective in animals models and humans
of colon cancer [61]. The IFN-c expressed by Th1 cells has
been described as an effective antitumoral cytokine in
colon adenocarcinoma and a possible autocrine enhancer of
cytolytic T cell capacity [62, 63].
In conclusion, delphinidin treatment induced Ca2? and
2?
Sr influx through CRAC channels, as indicated by the
ability of BTP2 and Gd3? to inhibit both events. Further-
more, BTP2 blocked delphinidin-induced NFAT activation,
which corroborates the involvement of SOCE in delphinidin-
mediated calcium signaling. Finally, delphinidin treatment
induced the expression and production of IL-2 mRNA and
protein, which is an effect that is mediated by SOCE and
NFAT activation, as both processes were inhibited by BTP2
and cyclosporine A. However, as IL-2 production was not
completely blocked by BTP2 and cyclosporine A, it is pos-
sible that other signaling pathways are involved in delphin-
idin-mediated T cell activation. Taken together, these results
suggest that delphinidin can increase the immune response
by stimulating cytokine production in T cells.
Acknowledgments This work was supported by Grants from Con-
sorcio de Tecnologı́a e Innovación para la Salud CTI-Salud (CTE-06),
Chile (CONICYT 21090900 and CONICYT AT-24100037).
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