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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-11-0062.
IMMUNOBIOLOGY
From the Lymphocyte Cell Biology Section, Laboratory of
Immunology, Gerontology Research Center, National Institute on Aging,
National Institutes of Health, Baltimore, MD.
The consequences of T-cell activation depend exclusively on
costimulation during antigen-T-cell receptor interaction. Interaction between the T-cell coreceptor CD28 and its ligand B7 during
antigen-antigen receptor engagement results in full activation of T
cells, the outcomes of which are proliferation and effector
functions. The ability of CD28 to costimulate the production of
interleukin-2 (IL-2) explains the importance of this costimulation. The
signaling event mediated by CD28 engagement has been proposed to have 2 components: one is sensitive to the immunosuppressive drug cyclosporin A (CsA), and the other one is CsA-resistant. In this report, we demonstrate that the CsA-resistant pathway is sensitive to the immunosuppressive drug rapamycin. Treatment with rapamycin blocked IL-2
production after activation of human peripheral blood T cells with
phorbol ester (PMA) and anti-CD28 (CsA-resistant pathway), whereas this
drug did not have any effect on PMA plus ionomycin stimulation
(CsA-sensitive pathway). The inhibitory effect of rapamycin was on
messenger RNA stability and translation, rather than on IL-2
transcription or protein turnover.
(Blood. 2002;99:4517-4524) Costimulation plays an important role in the
outcome of T-cell activation. When T-cell antigen receptors (TCRs)
interact with antigen-major histocompatibility complex in the absence
of coreceptor engagement, T cells become nonfunctional, a state usually
referred to as anergy. However, if T cells receive costimulation during antigen-TCR interaction, T cells become functionally activated, and the
outcomes are proliferation and the generation of effector populations.
CD28 is one important coreceptor, which regulates T-cell proliferation
and survival. The ability to costimulate the production of
interleukin-2 (IL-2) and induce Bcl-XL gene expression
comprise important roles that CD28 signaling plays in T-cell
activation.1-3 Besides IL-2, CD28 costimulation also induces other important cytokine genes, including interferon- Although significant work has been done to elucidate the signaling
pathways involved in CD28 costimulation, a complete understanding of
these pathways is yet to be achieved. It is not clear whether CD28
engagement generates a unique signaling pathway that complements TCR
signaling or whether CD28 signaling potentiates TCR-mediated signaling
to decrease the threshold of T-cell activation.8,9 It has
been proposed that CD28 signaling has 2 components: one is calcium
dependent and sensitive to cyclosporin A (CsA) similar to TCR
signaling, and the other one is calcium independent and resistant to
CsA.10 One of the first signaling molecules directly involved with activated CD28 was phosphatidylinositol 3-kinase (PI3-K),
although the role of PI3-K in CD28 signaling remains controversial.11-13 Recently, it has been shown that
transgenic mice or primary T cells carrying a CD28 mutant that lacks
ability to bind PI3-K were capable of distinguishing proliferative
signals from survival signals. These cells were unable to express
Bcl-XL, whereas their ability to proliferate and to produce
IL-2 were intact.14-16 Previously, it has been shown that
signals originating from TCR and CD28 converge on the activation of jun
N-terminal kinase (JNK),17 but Vav-1, a protooncogene
product, has been shown to be the point of integration for the 2 activation signals and lies upstream to the JNK signaling
pathway.18-21
Rapamycin is a macrolide antibiotic with potent immunosuppressive
properties. Rapamycin is structurally related to the well-known immunosuppressant FK506 and binds to the same intracellular receptor FKBP12 (FK506-binding protein).22 Interestingly, although
both FK506 and rapamycin bind to the same intracellular receptor, the principal target proteins are different for these 2 compounds. Whereas
FK506-FKBP12 inhibits the serine-threonine phosphatase calcineurin,
rapamycin-FKBP12 inhibits the serine-threonine kinase mTOR (mammalian
target of rapamycin)/FRAP (FKBP12-rapamycin-associated protein).23-28 Rapamycin treatment has been shown to cause
G1 arrest in a variety of cell types, including T
cells.29,30 The inhibition of T-cell proliferation is
primarily due to the blockage of IL-2 signaling,31,32
supporting the clinical use of rapamycin as an
immunosuppressant.33 It has been well established that
mTOR is a key regulator of translation,34 and 2 of the
most important components in the translational process that are
regulated by mTOR are 4EBP-1 and p70s6k.34
4EBP-1 is a repressor of eukaryotic initiation factor (eIF4E) and is
regulated by a phosphorylation/dephosphorylation
mechanism.35 The availability of the eIF4E is critical for
translation and is dependent on the phosphorylation status of 4EBP-1.
Hypophosphorylated 4EBP-1 binds to eIF4E and prevents it from forming a
productive mRNA cap binding complex, whereas phosphorylation of 4EBP-1
abrogates this interaction.36 Another target of mTOR is
p70s6k, which phosphorylates S6 protein of the 40S
ribosome, and is important in regulating the translation of an
essential family of mRNAs that contain an oligopyrimidine tract at
their transcription start site.37,38
Depending on the primary stimulation, CD28 can initiate multiple
pathways. One of these pathways is CsA resistant and has been suggested
to be involved in graft-versus-host disease during allogenic bone
marrow transplantation.39 Surprisingly, very little is
known about this pathway. In this report we demonstrate the effect of
rapamycin on the CsA-resistant pathway of CD28 costimulation. Rapamycin
treatment inhibited the production of IL-2 by activated human
peripheral blood T cells. The inhibition of IL-2 production was mainly
due to the blockage of the CsA-resistant pathway, because rapamycin had
no effect on the CsA-sensitive pathway. The mechanism of the effect of
rapamycin on the IL-2 production was observed at 2 levels: decreased
translation and decreased mRNA stability.
Cells and tissue cultures
Antibodies
IL-2 assay A total of 2 × 106 T cells/mL was stimulated with various conditions overnight at 37°C. In the case of CsA, rapamycin, or okadaic acid, cells were pretreated for 1 hour. After incubation, supernatants were collected, and the IL-2 was measured by enzyme-linked immunosorbent assay by using Human IL-2 Flexia Kit from Biosource (Camarillo, CA) according to the manufacturer's protocol.Electrophoretic mobility shift assay The preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA) were performed as described previously.40 The oligonucleotide corresponding to the consensus Stat-binding site from FcgRI promoter was tcgaCGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAGGc. During supershift assay, anti-Stat5A and B were added to the binding reaction 15 minutes before the addition of the radiolabeled probe.Northern blot analysis Total RNA was isolated by solubilization in guanidine thiocyanate as described by Chomczynski and Sacchi.41 Total RNA (10 µg) was electrophoresed on formaldehyde agarose gels and transferred to GeneScreen Plus membranes. A 400-base pair polymerase chain reaction product corresponding to a portion of the IL-2 complementary DNA (cDNA) was used as a probe. To study mRNA stability, T cells were stimulated with PMA/ CD28 in the presence or
absence of rapamycin (20 ng/mL) for 18 hours. Actinomycin D (10 µg/mL) was then added, and the cultures were then incubated further
for various periods of times. After incubations, cells were harvested,
and total RNAs were isolated for Northern blot analysis.
Western blotting and kinase assay Whole cell lysates were prepared as described previously.42 For Western blot analysis, 25 µg cell lysates were analyzed on 12% polyacrylamide gels. The proteins were electrotransferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Detection of specific phosphoproteins was carried out with ECL-Plus Western blotting kit according to the manufacturer's protocol (Amersham Biosciences, Piscataway, NJ). For kinase assay, 35 µg cellular lysates were immunoprecipitated by anti-JNK-1 antibody (1.5 µg). To capture immunocomplexes, protein G-agarose (10 µL 50% slurry) was first incubated with the antibody in the extraction buffer for 1 hour at 4°C. The antibody-protein G-agarose was then washed with extraction buffer without Triton X-100 and was exposed to the lysates for 1 more hour at 4°C. After extensive wash, immunocomplexes were incubated in kinase buffer (25 mM HEPES, pH 7.7, 20 mM MgCl2, 20 mM -glycerophosphate, 20 mM p-nitrophenylphosphate, 0.1 mM sodium ortho vanadate, 2 µg/mL E64, 10 µg/mL phenylmethylsulfonyl fluoride, and 2 mM
dithiothreitol) containing 0.1 mM adenosine triphosphate (ATP). The
kinase reaction was started by adding 2 µg GST-c-Jun (1-169) and 2 µCi (0.074 MBq) [ -32P] ATP (10 Ci/mmol
[37 × 1010 Bq], NEN Life Science Products, Boston,
MA), and the reaction mixture was incubated at 30°C for 30 minutes
with frequent mixing. The reaction was stopped by adding NuPAGE LDS
sample buffer (Invitrogen, Carlsbad, CA). The samples were boiled for 6 minutes and were analyzed on 10% NuPAGE Novex Bis-Tris Gels using MES
SDS buffer (Invitrogen).
Effect of rapamycin on the production of IL-2 by activated T cells It has been reported that IL-2 signaling in activated T cells is rapamycin sensitive, whereas IL-2 production is rapamycin insensitive.31,32 Other reports show that IL-2 production by activated T cells is sensitive to rapamycin.43,44 To investigate the effect of rapamycin on the IL-2 production by activated T cells, we used freshly isolated human peripheral blood T cells. As shown in Figure 1A, T cells stimulated with cross-linkedCD3 and CD28 produced a significant amount of
IL-2, and this production was suppressed significantly by the
pretreatment of the cells with rapamycin.
Previous studies have shown that the immunosuppressive drug CsA
drastically inhibits, but does not completely block, IL-2 production
following stimulation with Because the effect of rapamycin has been reported to be due to blocking
IL-2 signaling, it was important to demonstrate that no IL-2 signaling
was involved in the suppressive effect of rapamycin on the
CsA-resistant CD28 costimulatory pathway. We decided to use a sensitive
assay to monitor the involvement of IL-2 signaling. We performed EMSA
by using the consensus Stat-binding site from the Fc
Rapamycin affects PMA/CD28-mediated IL-2 message stability To determine whether the effect of rapamycin on IL-2 production was transcriptional or posttranscriptional, we performed Northern blot analysis using total RNAs. T cells were stimulated with PMA/CD28 in
the presence or absence of rapamycin for various periods of times, and
total RNAs were isolated and were analyzed by Northern blot analysis.
As shown in Figure 3A, IL-2 message was
detected as early as 2 hours, peaked about 6 hours, and stayed at high levels for at least 24 hours. Interestingly, pretreatment with rapamycin did not alter the induction of IL-2 message at the earlier time points, although this treatment did suppress the mRNA level at 24 hours. Figure 3B shows the graphical representation of the IL-2 mRNA
levels quantitated and normalized with the 18S ribosomal RNA. These
data suggest that rapamycin did not have any effect on IL-2 gene
transcription. We tested the effect of rapamycin on the transient
transfection assay by using an IL-2 promoter-reporter construct.
Rapamycin did not affect PMA/CD28-mediated promoter activity (data
not shown), which is in agreement with the recent report by Kane et
al.48 Therefore, rapamycin does not act at the level of
the IL-2 promoter. To examine the differential effect of rapamycin on
PMA/CD28 versus PMA/ionomycin-induced IL-2 mRNA levels, peripheral
blood T cells were stimulated with either medium alone, PMA/CD28, or
PMA/ionomycin in the presence or absence of rapamycin for 18 hours.
Total RNAs extracted from activated T cells were analyzed by Northern
blot analysis. As shown in Figure 3C, PMA/CD28-induced IL-2 mRNA
level was reduced by rapamycin pretreatment (compare lane 3 with lane
2), whereas rapamycin did not have any effect on PMA/ionomycin-induced
IL-2 mRNA level (lane 5 versus lane 4). The difference in the IL-2 mRNA
levels between PMA/CD28 versus PMA/ionomycin stimulation reflects
the difference in the IL-2 production by these 2 sets of stimuli
(Figure 1B,C).
Next we wanted to test the effect of rapamycin on IL-2 mRNA stability.
Peripheral blood T cells were treated with PMA/ Effect of rapamycin on the phosphorylation status of 4EBP-1 It has been well established that mTOR, the target of rapamycin, plays an important role in translation.34 One of the important components in translation that is directly regulated by mTOR is 4EBP-1, a repressor of eukaryotic initiation factor 4E (eIF4E), whose phosphorylation status dictates the initiation of translation.35 To examine the effect of PMA/CD28 stimulation on the phosphorylation status of 4EBP-1, peripheral blood T cells were stimulated with PMA/CD28 for various periods of time, and
the whole cell lysates were analyzed by Western blot analysis by using
an antibody specific for phospho-4EBP-1 that is phosphorylated on
Ser-65. As shown in Figure 4, multiple
phosphorylated forms of 4EBP-1 were observed as early as 3 hours after
stimulation, and the amount of phosphorylated proteins increased with
time. Pretreatment of these cells with rapamycin caused the
dephosphorylation of 4EBP-1, and this effect of rapamycin was observed
as early as 3 hours. The effect of rapamycin was specific to PMA/CD28
stimulation, because PMA/ionomycin-induced multiple phosphorylated
forms of 4EBP-1 were unaffected by rapamycin (data not shown). These
and the data presented above demonstrate that rapamycin inhibits
PMA/CD28-mediated IL-2 production by 2 mechanisms: first, decreased
translation of IL-2, which is the earliest effect, and second,
decreased mRNA stability, which is the later effect.
Effect of rapamycin on PMA/CD28-mediated JNK-1 activity To determine the signaling pathways involved in the rapamycin-mediated IL-2 message instability, we examined PMA/CD28-induced JNK-1 activity. JNK-1 has been shown to play an
important role in IL-2 mRNA stability in activated T
cells.49 Human peripheral blood T cells were stimulated
with PMA/CD28 for various periods of time. Whole cell lysates were
prepared, and the immunocomplex kinase assay was performed according to
the protocol described in "Materials and methods." As shown in
Figure 5A, PMA/CD28 treatment induced
kinase activity as early as 15 minutes, peaked around 30 minutes, and
maintained a high level of activity for at least 24 hours.
Interestingly, pretreatment of rapamycin suppressed JNK-1 activity at
the 24-hour time point (Figure 5A, upper panel). The inhibition of JNK
activity was not due to the difference in JNK protein level, because
rapamycin treatment did not alter the JNK level (Figure 5A, lower
panel). The effect of rapamycin was not observed at earlier time points
as shown in Figure 5B. Pretreatment with rapamycin did not alter JNK-1
activity at the 1-hour assay (lane 4 versus lane 2), whereas it did
affect the activity at the 20-hour time point (lane 6 versus lane 5).
The time course of rapamycin treatments demonstrated a requirement of
at least 18 hours for the inhibition of JNK-1 activity (data not
shown). These data suggest the involvement of mTOR/FRAP in the
PMA/CD28-mediated JNK-1 activation.
As it has been reported by others,50 pretreatment with
LY294002, an inhibitor of PI3-K, inhibited PMA/ Previously it has been shown that PMA/ Role of PP2A in the rapamycin-mediated down-regulation of JNK-1 activity It has been reported that rapamycin-induced dephosphorylation of p70S6K and 4E-BP1 was mediated by PP2A.51 To examine the role of PP2A in the inhibition of PMA/CD28-induced JNK
activity by rapamycin, peripheral blood T cells were stimulated with
PMA plus CD28 for 19 hours in the presence or absence of rapamycin
and/or okadaic acid. As shown in Figure
6A, pretreatment with rapamycin inhibited PMA/CD28-induced JNK-1 activity (lanes 3 versus 2), and this inhibition was blocked by addition of okadaic acid to the rapamycin treatment (lanes 5 versus 3), whereas pretreatment with okadaic acid
alone did not affect PMA/CD28-induced JNK-1 activity (lanes 4 versus
2). These data suggest that rapamycin-induced blockage of JNK-1
activity was mediated by PP2A/PP1. To check whether the reversal of
JNK-1 inhibition by okadaic acid was reflected in the IL-2 mRNA
stability, Northern blot analysis was performed by using RNAs isolated
from the same experiment as described in Figure 6A. Like the JNK assay,
the suppression of PMA/CD28-induced IL-2 message by rapamycin was
reversed by simultaneous treatment with okadaic acid (Figure 6B, lanes
5 versus 3), whereas okadaic acid treatment alone did not alter
PMA/CD28-induced IL-2 mRNA level (Figure 6B, lanes 4 versus 2).
Figure 6C shows the IL-2 mRNA levels quantitated and normalized with
18S ribosomal RNA. These data strongly suggest the possible involvement
of JNK-1 in rapamycin-mediated IL-2 mRNA instability.
Rapamycin treatment of T cells inhibits IL-2 signaling and blocks
T-cell proliferation.31,32 In this report we demonstrate that rapamycin also blocks the production of IL-2 by activated human
peripheral blood T cells. This effect of rapamycin was not accounted
for by autocrine IL-2 signaling for the following reasons. First, we
could not detect any Stat5, an IL-2-specific Stat, in PMA/ Previous studies have shown that the activation of T cells with One of the first signaling molecules involved in CD28 signaling was
shown to be PI3-K.11-13 It has been shown that the
serine-threonine kinase Akt, a target of PI3-K, can provide some of the
CD28 costimulatory signals, eg, production of IL-2 and IFN- It has been well established that mTOR is a key regulator of
translation,34 and 4EBP-1 is a downstream target molecule
whose phosphorylation status dictates the initiation of
translation.35 The availability of the initiation factor
eIF4E is critical for the initiation of translation and is dependent on
the phosphorylation status of 4EBP-1. Hypophosphorylated 4EBP1 binds to
eIF4E, whereas hyperphosphorylation abrogates this
interaction.36 We demonstrate here that the
phospho-4EBP-1 induced by PMA/ The role of JNK in IL-2 message stability has been well
documented.49 We demonstrate here that JNK also plays an
important role in CsA-resistant PMA/ PMA/
The inhibitory effect on IL-2 signaling and subsequent blockage in
T-cell proliferation by rapamycin has been well
documented,31,32 and, because of its immunosuppressive
property, rapamycin has been approved for clinical use along with other
immunosuppressants.33 The data presented here regarding
the inhibition of IL-2 production by rapamycin provide additional
information on the mechanism of action of rapamycin in activated T
cells. It will be of great interest to identify the physiologically
relevant pathways that mimic the PMA/
Submitted November 27, 2001; accepted February 11, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-11-0062.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Paritosh Ghosh, Lymphocyte Cell Biology Section, Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr, Baltimore, MD 21224; e-mail: ghoshp{at}grc.nia.nih.gov.
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Abstract
Introduction
