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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2002-07-1992.
IMMUNOBIOLOGY
From the Signal Transduction Laboratory, Mogam
Biotechnology Research Institute, Gyunggido, Korea.
Rosmarinic acid (RosA) is a hydroxylated compound frequently found
in herbal plants and is mostly responsible for anti-inflammatory and
antioxidative activity. Previously, we observed that RosA inhibited
T-cell antigen receptor (TCR)- induced interleukin 2 (IL-2)
expression and subsequent T-cell proliferation in vitro. In this study,
we investigated in detail inhibitory mechanism of RosA on TCR
signaling, which ultimately activates IL-2 promoter by activating
transcription factors, such as nuclear factor of activated T cells
(NF-AT) and activating protein-1 (AP-1). Interestingly, RosA inhibited
NF-AT activation but not AP-1, suggesting that RosA inhibits
Ca2+- dependent signaling pathways only. Signaling events
upstream of NF-AT activation, such as the generation of inositol
1,4,5-triphosphate and Ca2+ mobilization, and tyrosine
phosphorylation of phospholipase C- Rosmarinic acid ( Recently, the inhibition of T-cell antigen receptor (TCR)-mediated
signaling was identified as one of the working mechanisms of many
anti-inflammatory drugs. Antirheumatic drugs, such as hydroxychloroquine,8 nonsteroidal anti-inflammatory
drugs,9 and a traditional Chinese antirheumatic medicine
TWHf10 were found to inhibit TCR-induced signaling events,
such as Ca2+ mobilization, the activation of p38
mitogen-activated protein kinase (MAPK), and the expression of CD40
ligand, despite the fact that their points of action are quite
different. Moreover, leflunomide represses the activity of 2 major Src
family protein tyrosine kinases, Fyn and lymphocyte-specific
cytoplasmic protein tyrosine kinase (Lck), which are
implicated in the signal transduction of T cells. Leflunomide also
blocks TCR-induced tyrosine phosphorylation of the phospholipase C- The T-cell response is initiated by the interaction between the antigen
presented in antigen-presenting cells and the TCR complex. Stimulation
of the TCR leads to the rapid activation of tyrosine kinases, which
phosphorylate a variety of signal-transducing proteins. A considerable
weight of evidence supports the view that the phosphorylation of
immunoreceptor tyrosine-based activation motifs within the TCR complex
is required to initiate TCR signaling.12-14 The
phosphorylation of immunoreceptor tyrosine-based activation motifs by
Lck recruits and activates Recent reports regarding the suppressive effects of various
anti-inflammatory agents on TCR signaling and our previous findings on
the antiproliferative effects of RosA on T cells led us to study the
role of RosA in TCR signaling. We found that RosA inhibited TCR-induced
NF-AT activation but not activating protein-1 (AP-1) activation, which
suggests that RosA specifically inhibits Ca2+-dependent
signaling pathways. This finding was further confirmed by the fact that
RosA effectively suppressed TCR-induced tyrosine phosphorylation of
PLC- Reagents
Antibodies
Cell cultures Jurkat T cells (ATCC) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), antibiotics (Gibco BRL, Grand Island, NY), and 50 µM -mercaptoethanol (Sigma).
Human peripheral blood mononuclear cells were isolated from adult
healthy donors by density-gradient centrifugation on Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden). Following isolation, cells were plated at a density of 2 × 106/mL and left in
tissue culture flasks at 37°C in 5% CO2 incubator for 2 hours to remove adherent cells. The resulting human peripheral blood
lymphocytes (hPBLs) were resuspended in complete RPMI medium and used
within 24 hours of isolation. The purified hPBLs consisted of 58.9% of
T cells and 11.3% of B cells according to the fluorescence-activated cell sorter (FACS) analysis using anti-CD3 and anti-CD19
antibodies, respectively.
Luciferase assay A total of 2 × 106 Jurkat T cells was transfected with 2 µg NF-AT- or AP-1-Luciferase reporter plasmids (gifts from Gerald Crabtree, Stanford University, Stanford, CA) using Lipofectamine according to the manufacturer's protocol (Gibco BRL). After incubating with DNA-Lipofectamine mixtures for 24 hours, cells were preincubated in the presence or in the absence of RosA (30 µM) for 2 hours before being stimulated. Cells were activated either with anti-CD3 mAb (5 µg/mL, UCHT1, IgG2a isotype) coated on a plate or with PMA (5 ng/mL) and ionomycin (0.5 µg/mL) for 16 hours. In the RosA-treated group, RosA was present throughout the 16-hour incubation process. After stimulation, the cells were washed, lysed, and assayed for luciferase activity, according to the manufacturer's instructions (Luciferase Assay System kit; Promega, Madison, WI) with a Microplate luminometer LB96V (Perkin-Elmer, Foster City, CA).Ca2+ mobilization assay Jurkat T cells were washed twice with Hanks balanced salt solution (HBSS) containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4), 1 mM MgCl2, 2 mM CaCl2, and 20 mM dextrose, and then incubated with 2 µM fura-2/AM (Calbiochem) for 45 minutes at 37°C. Cells were washed extensively and resuspended at 5 × 106/3 mL in HBSS.46 For calcium studies in primary human T cells, hPBLs were loaded in calcium buffer (25 mM HEPES, 125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.1% glucose, 500 µM MgCl2, 10 mM CaCl2, 0.1% bovine serum albumin [pH 7.4]) with fura-2/AM (10 µM) for 30 minutes at 37°C.8 Loaded cells were washed once, resuspended at 5 × 106/3 mL in calcium buffer, and immediately placed on ice. Fura-2-loaded cells were placed in a water-jacked cuvette and preincubated in the absence or presence of various concentrations of RosA for 15 minutes at 37°C. Following 100 to 150 seconds of data acquisition, cells were then incubated with anti-CD3 mAb (3 µg/mL, UCHT1, IgG1 isotype) or OKT3 ascites (1:1000), and fluorescence levels were measured using a Luminescence spectrometer LB50B (Perkin-Elmer) before and after stimulating with antimouse IgG antibody (10 µg/mL) to crosslink the anti-CD3 mAb. Data are presented in arbitrary units as a function of fluorescence (relative intracellular calcium) versus time.Measurement of IP3 Jurkat T cells (2 × 106/reaction) were incubated in the presence or absence of RosA for 4 hours at 37°C before harvesting. The cells were activated by incubating with OKT3 ascites (1:200), and cross-linking OKT3 with goat antimouse IgG antibody (40 µg/mL) for 3 minutes. The TCR-mediated stimulation was terminated by adding 10 µL ice-cold 100% trichloroacetic acid and then incubating on ice for 15 minutes. IP3 was quantified in duplicate with the use of an IP3 [3H] Radioreceptor Assay Kit (NEN Life Science Products, Boston, MA) and by liquid scintillation counting according to the manufacturer's instructions.Cell stimulation, lysis, immunoprecipitation, and Western blot analysis Jurkat T cells or hPBLs were stimulated by incubating with OKT3 or OKT4 ascites (1:200), and this was followed by cross-linking OKT3/4 with goat antimouse IgG antibody (40 µg/mL) for the indicated time at 37°C. For immunoprecipitation or Western blot, Jurkat T cells (5 × 107) or hPBLs (4 × 107) were lysed with ice-cold lysis buffer (50 mM Tris (tris(hydroxymethyl)aminomethane) [pH 8.0], 150 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 0.5% deoxycholate, 1% Nonidet P-40 or Brij97, 1 mM Na3VO4, 5 mM NaF, and one tablet proteinase inhibitor per 50 mL lysis buffer) for 1 hour on ice and then centrifuged at 12 000g at 4°C for 30 minutes. The supernatant was saved, precleared for 1 hour at 4°C by incubating with 25 µL mouse or rabbit serum-conjugated agarose (Sigma), and then incubated with the appropriate antibodies overnight at 4°C. This was followed by incubation with 25 µL protein A-conjugated agarose (Sigma) for 3 hours at 4°C. Immunoprecipitates were then either subjected to an in vitro kinase assay or immunoblotting. For immunoblot, the immunoprecipitated complexes were washed 3 times in ice-cold lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose membranes (Bio-rad, Hercules, CA). Blots were blocked overnight at 4°C and probed with the indicated antibodies in Tris-buffered saline (2 mM Tris [pH 7.5], 30 mM NaCl), which included 0.05% Tween 20 and 5% skim milk. Signals were detected using horseradish peroxidase-labeled secondary antibodies (antimouse and antirabbit, 1 µg/mL; Transduction Laboratories). Where indicated, blots were reprobed after stripping blots at 60°C for 30 minutes in 62.5 mM Tris (pH 6.7), 2% SDS, and 100 mM-mercaptoethanol. Blots were finally detected by the enhanced chemiluminescence (ECL; Amersham,
Arlington Heights, IL).
In vitro kinase assay Jurkat cells (5 × 107) were immunoprecipitated with anti-Lck or Itk mAb (5 µg/mL), and kinase reactions were carried out with these immunoprecipitates (1/10 of the total immunoprecipitated complexes) at 30°C for 10 minutes in 15 µL kinase buffer (0.1 M Pipes-NaOH, pH 6.8, and 20 mM MnCl2) containing 0.37 MBq (10 µCi) 32P- -ATP
(Amersham, Buckinghamshire, United Kingdom) and 2 µL enolase (5 mg/mL; Sigma) as an exogenous substrate. Reactions were stopped by
adding 3 × reducing SDS sample loading buffer (New England Biolabs)
and analyzed by electrophoresis on a 12% SDS-PAGE. 32P
incorporation into enolase (41 kDa) or Itk (72 kDa) was measured by
exposure to Kodak (Rochester, NY) XAR film at  70°C, or to a BAS
2040 cassette and then quantified on a Bio-Imager analyzer (BAS 1000, FUJIX). The remainder of the Lck immunoprecipitates was used to detect
the autophosphorylating activity of Lck by running them on 10%
SDS-PAGE and performing Western blot analysis.
RosA inhibits NF-AT activation but not AP-1 in Jurkat T cells Our previous study indicated that RosA inhibits TCR-induced IL-2 promoter activation and subsequent T-cell proliferation.47 Moreover, this process seemed to occur at the membrane proximal point of TCR signaling, because RosA-mediated inhibition was limited to TCR-induced but not to PMA/ionomycin-induced IL-2 promoter activation.47 Because PMA and ionomycin can directly activate PKC and Ca2+ release, thus bypassing the requirement for the early activation of protein tyrosine kinases, our previous results strongly support the notion that RosA works upstream of PKC and Ca2+ flux. The IL-2 promoter contains several regulatory elements that can bind different transcription factors, such as NF-AT, AP-1, Oct-1, and nuclear factor B
(NF-B).35 Initially, we determined the effect of RosA
on 2 major transcription factors induced by TCR signaling, namely,
NF-AT and AP-1. Jurkat T cells were transiently transfected with either
NF-AT or AP-1 luciferase reporter plasmid, and luciferase activities
were determined after TCR or PMA/ionomycin stimulation in the presence
or in the absence of RosA. As shown in Figure
1, neither of these promoter activities was inhibited in response to PMA/ionomycin stimulation, which was in
agreement with our previous data. RosA treatment strongly repressed
TCR-induced NF-AT promoter activity but not AP-1. In several cases,
RosA-treated Jurkat cells showed a slight increase in AP-1 activity
(Figure 1). These results indicate that RosA inhibits TCR-induced IL-2
promoter activation by suppressing NF-AT activation, and RosA acts in
the membrane-proximal point(s) of the TCR signaling upstream of
PMA/ionomycin working sites.
RosA blocks TCR-induced tyrosine phosphorylation of PLC- 1-mediated hydrolysis of
PIP232; therefore, we tested the activation
status of PLC-1. To examine the effect of RosA on the TCR-induced
tyrosine phosphorylation of PLC-1, Jurkat cells were preincubated
with various concentrations of RosA for 4 hours and then stimulated
with OKT3. Because PLC-1 is tyrosine phosphorylated within 2 minutes
and rapidly dephosphorylated right after, we stimulated Jurkat cells
for 2 minutes.26 PLC-1 immunoprecipitates were resolved
on 12% SDS-PAGE, and tyrosine phosphorylated PLC-1 was detected by
antiphosphotyrosine mAb (Figure 3, top
panels). The identity of PLC-1 was
confirmed by reblotting with anti-PLC-1 mAb (Figure 3, bottom
panels). RosA completely abrogated TCR-induced tyrosine phosphorylation
of PLC-1 at the concentration between 15 and 30 µM, however, and
failed to inhibit at 3 µM (Figure 3B, top panel). In conclusion,
these results suggest that reduced Ca2+ mobilization and
IP3 induction in RosA-treated cells (Figure 2) are due to
improper PLC-1 activation. Notably, RosA exerted no appreciable
effect on viability of Jurkat cells during the course of these
experiments and up to 16 hours at 30 µM (data not shown).
RosA does not modulate Lck kinase activity There are 3 well-known PTKs that are important for the full activation of PLC-1, namely, Lck, ZAP-70, and Itk. Their cooperative actions are essentially required to assemble signaling molecules and to
finally phosphorylate PLC-1.48 A significant amount of
debate has taken place over the identity of the kinase(s) that directly
phosphorylates PLC-1, although it is generally accepted that Itk is
responsible. Itk needs to be phosphorylated for activation and, at the
same time, requires adaptors to approach PLC-1. Lck activates Itk by
phosphorylation and indirectly provides adaptors by phosphorylating
ZAP-70, which in turn phosphorylates LAT and SLP-76.18,20,48,49 Finally, Lck-activated Itk may
phosphorylate PLC-1 in association with SLP-76 and LAT.
First, we determined the effect of RosA on Lck kinase activity by
immunoprecipitation kinase assay. Jurkat T cells preincubated in the
presence or absence of RosA were either untreated or activated by OKT3.
The catalytic activity of Lck in immunoprecipitates was measured by
determining its ability to phosphorylate an exogenous substrate,
enolase. The results obtained indicate that RosA treatment did not
suppress Lck-mediated phosphorylation of enolase (Figure 4A, top
panel), thus indicating that RosA does
not inhibit Lck kinase activity. Autophosphorylation of Tyr394
in the activation loop of Lck is necessary for its kinase activity,
probably by inducing steric changes that allow the catalytic region to
fold into an active structure.50 Consistent with this data
(Figure 4A, top panel), the present study showed that RosA did not
inhibit the autophosphorylation of Lck (Figure 4A, middle
panel).
Anti-CD3 activation alone minimally activates Lck, and maximal Lck activation is best induced by CD3/4 co-cross-linking. To determine the RosA effect on fully activated Lck, Jurkat cells were stimulated with OKT3/4 co-cross-linking in the presence or absence of RosA (Figure 4B). In agreement with other reports,26,51 OKT3/4 co-cross-linking induced Lck shift from p56 to p59, indicating serine and threonine phosphorylation on SDS-PAGE. RosA did not inhibit this Lck shift, an indicator of Lck activation. The inability of RosA to inhibit Lck kinase activity was further
confirmed by determining the phosphorylation status of several in vivo
Lck substrates. In response to TCR stimulus, Lck phosphorylates TCR
RosA does not inhibit the ZAP-70-mediated tyrosine phosphorylation of SLP-76 and LAT, subsequent association of LAT with SLP-76 or Grb2, and Erk1/2 activation According to Figure 5B, the Lck-mediated tyrosine phosphorylation of ZAP-70 was intact in the presence of RosA; therefore, it is likely that ZAP-70 should be fully active. To confirm this, we investigated the phosphorylation status and association of 2 ZAP-70 substrates, eg, LAT and SLP-76.18,20 SLP-76 was precipitated from Jurkat T cells in the absence or presence of a TCR stimulus. SLP-76 was phosphorylated within 2 minutes and decreased gradually during 30 minutes (Figure 6A). Maximal phosphorylation of LAT was seen between 30 seconds and 2 minutes of stimulation, and LAT was quickly dephosphorylated. There was no noticeable effect of RosA on the kinetics of SLP-76 phosphorylation and its association with LAT (Figure 6A). Kinetics of SLP76-associated LAT phosphorylation was also not affected by RosA. The tyrosine phosphorylation of LAT was further confirmed by LAT immunoprecipitation and Western blot analysis (Figure 6B). Tyrosine phosphorylation of LAT was readily detectable within 2 minutes of stimulation in RosA-treated Jurkat T cells. As was the case for ZAP-70, the phosphorylation of SLP-76 and LAT and their association were not inhibited by RosA treatment, suggesting that abolished tyrosine phosphorylation of PLC-1 in RosA-treated Jurkat T cells does not arise from a blockade
of ZAP-70 activation and the subsequent failure of LAT, SLP-76, and
PLC-1 to assemble.
In Jurkat T cells, TCR-stimulated, ZAP-70-mediated tyrosine
phosphorylation of the adapter proteins SLP-76 and LAT has been correlated with the efficient activation of Ras and
Erk.19,52,53 Tyrosine phosphorylation of LAT results in
the recruitment and SH2-dependent binding of the adapter molecule Grb2,
and thus the binding of SOS, an upstream activator of
Ras.25,28 Ras activates the Raf-MEK-Erk protein kinase
cascade, which controls the level and the activity of the Jun/Fos
dimeric transcription factor known as AP-1. The blot of LAT
immunoprecipitates used in Figure 6B was reprobed with anti-Grb2 mAb to
confirm RosA's effect on the association of LAT with Grb2 in each
fraction (Figure 6B, middle panel). Amounts of Grb2 coprecipitated with
tyrosine phosphorylated LAT following OKT3 stimulation were not changed
significantly by RosA treatment. Furthermore, immunoblotting for the
dually phosphorylated, activated forms of Erk1/2, a downstream effector of the Ras pathway, revealed that Erk1/2 activation occurs normally in
the presence of RosA (Figure 7). Because
Erk activation ultimately controls the activity of AP-1, this result
agrees well with the data, which showed that RosA does not inhibit
TCR-induced AP-1 activation (Figure 1).
RosA suppresses the TCR-induced tyrosine phosphorylation of Itk and subsequent Itk activation Itk is involved in the generation of critical second messengers (Ca2+, PKC) by performing tyrosine phosphorylation of PLC-1. Given the ability of RosA to inhibit the TCR-induced tyrosine
phosphorylation of PLC-1 and Ca2+ mobilization in Jurkat
T cells, we also assessed the ability of RosA to inhibit the
TCR-stimulated tyrosine phosphorylation and subsequent activation of
Itk. The tyrosine phosphorylation status of Itk from RosA-treated or
-untreated Jurkat cells was monitored by immunoprecipitation
(IP)-Western. Previously, it was shown that Itk was rapidly
tyrosine phosphorylated within 2 minutes and returns to basal level by
15 minutes in response to TCR stimulus.54 In our
experiments, Itk was maximally phosphorylated at 3 minutes; therefore,
we chose a 3-minute stimulus for determining Itk activation status. As
shown in Figure 8 (top panel),
stimulation of Jurkat cells with OKT3 greatly induced the tyrosine
phosphorylation of Itk, whereas preincubation of Jurkat cells with 30 µM RosA for 4 hours prior to stimulation significantly inhibited the
TCR-stimulated tyrosine phosphorylation of Itk. The nitrocellulose
membrane was subsequently stripped and reprobed with anti-Itk antibody
to confirm that approximately equal amounts of Itk had been
immunoprecipitated from each sample (Figure 8A, bottom panel).
To evaluate the concomitant change in the specific activity of Itk, we
also determined Itk kinase activity in vitro (Figure 8B). Because there
is no known substrate available for evaluating Itk kinase
activity,49 we determined autophosphorylation activity of
Itk. As can be seen in Figure 8B, the Itk immunoprecipitates from the
stimulated Jurkat cells showed strong autophosphorylating activity.
However, the Itk immunoprecipitates from RosA-treated Jurkat cells
showed remarkably reduced kinase activity. A half of the Itk
immunoprecipitates was used for Western blotting to quantify the amount
of Itk, and according to Western blot fairly equal amounts of Itk were
immunoprecipitated (Figure 8B, bottom panel). Taken
together, RosA inhibits TCR-induced tyrosine phosphorylation of Itk and
subsequent Itk activation. We conclude that RosA inhibits PLC- RosA equally inhibits TCR-induced intracellular Ca2+ mobilization and the tyrosine phosphorylation of Itk but not ZAP-70 in human peripheral blood lymphocytes We demonstrated that RosA inhibits Ca2+-dependent TCR signaling by acting upstream of Itk and PLC-1 in Jurkat cells. To
determine whether this inhibitory mechanism of RosA is a universal
feature in T cells, we evaluated several key signaling events,
including Ca2+ mobilization, tyrosine phosphorylation of
Itk and ZAP-70 in normal hPBLs. Because anti-CD3 antibody was used for
stimulation, we assumed that our observation in hPBLs is mostly
ascribed to T cells. Consistent with the effect in Jurkat T cells
(Figures 2A,8A), RosA inhibited TCR-induced Ca2+ flux and
tyrosine phosphorylation of Itk in a dose-dependent manner in hPBLs
(Figure 9A-B). However, tyrosine
phosphorylation of ZAP-70 was not affected by up to 30 µM RosA
(Figure 9C). Therefore, we conclude that RosA inhibits TCR-signaling of
hPBLs and Jurkat cells in a similar mechanism.
In this study, we further clarified the inhibition mechanism of
RosA on TCR signaling. Because RosA specifically inhibited NF-AT
reporter activation but not AP-1 (Figure 1), it is likely that RosA
suppresses IL-2 promoter activation by blocking
Ca2+-dependent TCR-signaling events. This notion was
confirmed by the observation whereby signaling events, such as the
IP3 increase and Ca2+ mobilization, required
for NF-AT activation were efficiently inhibited by RosA treatment
(Figure 2). Furthermore, the failure of RosA to inhibit the TCR-induced
phosphorylation of Erk1/2 explains (Figure 7) why RosA does not inhibit
AP-1 activation. Our previous study suggested that RosA works in the
membrane proximal point of TCR signaling, based on the fact that the
RosA-mediated inhibition of IL-2 promoter activation is restricted only
to TCR stimulation and not to PMA/ionomycin-mediated stimulation. This
led us to determine the phosphorylation status of PLC- PLC- Exactly how RosA down-regulates Itk activity has not been clarified. In
the present study, we only know that RosA inhibits Itk activity in a
ZAP-70-independent manner. Heyeck et al49 reported that
Lck directly phosphorylates and activates Itk. However, RosA did not
inhibit Lck kinase activity, given that (1) Lck kinase activity was
intact in in vitro kinase assay (Figure 4A) and (2) Lck substrates, eg
the It is known that the Ca2+-dependent pathway and the Ras/Erk
pathway cooperate in T cells to activate the IL-2 promoter through the
activation of the transcription factors NF-AT and AP-1.35 Although inhibiting NF-AT activation, RosA displayed no inhibition of
AP-1-mediated transcription on TCR stimulation (Figure 1). In
conjunction with the failure of RosA to inhibit the TCR-induced phosphorylation of Erk1/2, this result indicates that RosA inhibits the
Ca2+ signaling pathway but not the Ras/Erk pathway.
PLC- In summary, we propose that RosA inhibits PLC-
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1
and Itk activity
Top
Abstract
Introduction
-o-caffeoyl-3,
4-dihydroxyphenyl lactic acid; RosA) is a naturally occurring
hydroxylated compound widely distributed in Labiatae herbs,
such as rosemary, sweet basil, and perilla. RosA appears to be a
substance of considerable interest because it has a broad range of
applications, from food preservatives to cosmetics, and has medicinal
use because of its antimicrobial and antioxidant
activity.
-chain and consequently inhibits Ca2+
mobilization, interleukin 2 (IL-2) secretion, and IL-2 receptor expression.
) or PMA (5 ng/mL) and ionomycin (0.5 µg/mL) (
) for
16 hours. In the RosA-treated group, RosA was present throughout the
whole 16 hours of incubation. Jurkat cells without any stimulation are
marked as open bars (
). Shown are the relative light units (RLU),
indicated as the average RLU and standard error of 2 independent
experiments.
/