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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2308-2318
Downregulation of JAK3 Protein Levels in T Lymphocytes by Prostaglandin
E2 and Other Cyclic Adenosine Monophosphate-Elevating
Agents: Impact on Interleukin-2 Receptor Signaling Pathway
By
Vladimir Kolenko,
Patricia Rayman,
Biswajit Roy,
Martha K. Cathcart,
John O'Shea,
Raymond Tubbs,
Lisa Rybicki,
Ronald Bukowski, and
James Finke
From the Departments of Immunology, Cell Biology, Clinical Pathology,
Biostatistics and Epidemiology, and Hematology-Oncology, Cleveland
Clinic Foundation, Cleveland, OH; and Lymphocyte Cell Biology Section,
Arthritis and Rheumatism Branch, National Institute of Arthritis and
Musculoskeletal and Skin Diseases, Bethesda MD.
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ABSTRACT |
The Janus kinase, JAK3 plays an important role in interleukin-2
(IL-2)-dependent signal transduction and proliferation of T
lymphocytes. Our findings show that prostaglandin E2
(PGE2) can inhibit upregulation of JAK3 protein in naive T
cells and can downregulate its expression in primed cells. Reduction in JAK3 was selective because expression of other tyrosine kinases (JAK1,
p56lck, and p59fyn) and signal transducer and
activator of transcription (STAT)5, which are linked to IL-2 receptor
(IL-2R) signaling pathway, were not affected. Inhibition of JAK3 may be
controlled by intracellular cyclic adenosine monophosphate (cAMP)
levels, as forskolin, a direct activator of adenylate cyclase and
dibutyryl cAMP (dbcAMP), a membrane permeable analogue of cAMP
suppressed JAK3 expression. Moreover, 3-isobutyl-1-methylxanthine
(IBMX), an inhibitor of cAMP phosphodiesterase, potentiated
PGE2-induced suppression of JAK3. In naive T cells, but not
primed T cells, PGE2 and other cAMP elevating agents also
caused a modest reduction in surface expression of the common gamma
chain ( c) that associates with JAK3. The absence of JAK3, but not
IL-2R in T cells correlated with impaired IL-2-dependent signal
transduction and proliferation. The alteration in IL-2 signaling
included decreased tyrosine phosphorylation and DNA binding activity of
STAT5 and poor induction of the c-Myc and c-Jun pathways. In contrast,
IL-2-dependent induction of Bcl-2 was unaffected. These findings
suggest that suppression of JAK3 levels may represent one mechanism by
which PGE2 and other cAMP elevating agents can inhibit
T-cell proliferation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
T LYMPHOCYTE ACTIVATION through antigen
presentation to the T-cell receptor-CD3 complex (TCR-CD3) results in
the passage from G0 to G1 of the cell cycle and in the development of
T-cell competency.1-3 This process is prerequisite for
further cell cycle progression1-3 and involves activation
of genes that encode cytokines and receptors such as interleukin-2
(IL-2) and the IL-2 receptor (IL-2R), respectively.3,4
T-cell activation results in expression of IL-2R (55 kD),
common chain (c) (64 kD), and an increase in the level of
IL-2R (75 kD).4-5 The high-affinity IL-2R contains all
three chains, while expression and heterodimerization of the and
chains alone is sufficient to mediate IL-2-dependent signaling.4-7 IL-2 binding to its receptor controls
movement from G1 into S phase of the cell cycle and therefore plays an important role in T-cell proliferation.3,4
Signal transduction through the IL-2R is initiated by activation of
tyrosine kinases associated with IL-2R and c.3,4,8-15 This includes Janus family protein tyrosine kinase (PTK) JAK3 that is
associated with the carboxy-terminal region of c and JAK1 that is
associated with the serine-rich region of the chain.3,4,12-15 The src family of kinases (Lck, Fyn, and
Lyn) are activated through their interaction with the acidic region of
the IL-2R chain, whereas the Syk-Zap-70 family of PTK interacts with
the serine-rich region of the chain.11,16 Very early
events in IL-2-dependent signaling involves tyrosine phosphorylation
and activation of the JAK kinases that may precede activation of the
Src and Syk-Zap-70 kinases and the phosphorylation of a number of
substrates.17 These early events ultimately result in the
induction of three distinct signaling pathways linked to the IL-2R that
are defined by the expression of c-myc, c-fos/jun, and Bcl-2,
respectively.3,4 Activation of these pathways leads to
movement of T cells into the S phase of the cell cycle.4
Prostaglandin E2 (PGE2) is known to inhibit
T-cell activation.18-20 The mechanism of suppression
appears to be through increasing levels of the intracellular second
messenger, cyclic adenosine monophosphate (cAMP), that binds to its
receptor, protein kinase A (PKA).21 IL-2 and IL-2R gene
expression are both targets of PGE2-induced suppression,
possibly through the inhibition of early events in T-cell signaling
that include calcium influx and phosphatidylinositol
breakdown.22,23 PGE2 is also known to inhibit
the DNA binding activity of the transcription factor, NF B to the
IL-2 transcriptional start site thereby blocking formation of
IL-2.24 Increasing cAMP levels and activation of PKA also alters PKC-induced transcriptional regulation of members of the jun and fos family of genes.25 More recent
studies indicate that PGE2-induced inhibition of T-cell
proliferation is mediated through blocking IL-2-dependent G1-S
transition.26,27 However, little is known about the effect
PGE2 has on signal transduction elements that are linked to
the IL-2R.
Here we show that PGE2 inhibited induction of JAK3
expression in naive T cells and downregulated its expression in primed cells. The suppression of JAK3 was selective, as PGE2 had
minimal effect on expression of other tyrosine kinases linked to the
IL-2R. Moreover, JAK3 expression may be regulated by an increase in
cAMP, as forskolin and dibutyryl cAMP (dbcAMP), which increase cAMP levels, induced a similar defect. This reduction in JAK3 resulted in
impaired phosphorylation and DNA binding activity of signal transducer
and activator of transcription (STAT)5 and decreased induction of c-Myc
and c-Jun, but not Bcl-2. The block in IL-2-dependent proliferation
and signaling in both naive and primed T cells coincided with
suppression in JAK3 rather than with a reduction in IL-2R expression.
Because JAK3 is critical to IL-2-dependent signaling and
proliferation, its sensitivity to PGE2 and other agents
that elevate cAMP may make it a prime target for suppressing
IL-2-dependent cell cycle progression in lymphocytes.
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MATERIALS AND METHODS |
Antibodies and reagents.
PGE2, 3-isobutyl-1-methylxanthine (IBMX), dbcAMP,
isoquinolinesulfonamide dihydrochloride (H-89), and myristoylated
protein kinase inhibitor (PKI) amide were purchased
from Biomol (Plymouth Meeting, PA). Forskolin, phorbol 12,13-dibutirate
(PDB), and ionomycin were obtained from Sigma (St Louis, MO).
Recombinant human IL-2 was provided by Chiron Therapeutics (Emeryville,
CA). Anti-CD3 antibody (OKT3) was purchased from Ortho (Raritan, NJ).
Phytohemagglutinin (PHA) was purchased from Difco (Detroit, MI).
Antibody to Jak3 was prepared as previously described.28
Protein kinase assay kit was obtained from Calbiochem (San Diego, CA)
and the cAMP EIA kit was purchased from Cayman Chemical Co (Ann Arbor,
MI). Antibody to JAK1 and STAT5 was purchased from Transduction
Laboratories (Lexington, KY), whereas antibody to p59fyn
was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Antibody to p56lck was purchased from UBI (Lake Placid,
NY). Antibodies to (CD25) and (CD122) chains of the IL-2R were
obtained from Becton Dickinson (Mountain View, CA) and
the anti-c chain antibody (3B5) was prepared as previously
described.29 Antibodies to c-Myc (9E10), c-Fos, and Bcl-2
were purchased from Santa Cruz Biotechnology Inc. Mouse monoclonal
antiphosphotyrosine Ab, 4G10 was purchased from UBI. Horseradish
peroxidase-conjugated sheep antimouse Ig (Cat. No. NA9310) and donkey
antirabbit (NA934) were purchased from Amersham (Arlington Heights,
IL). The following antibody-coated beads were purchased from
Miltenyi Biotech (Sunnyvale, CA): anti-CD14, anti-CD16, and
anti-CD19. Fluorescein isothiocyanate (FITC)-conjugated anti-CD45RB and
phycoerythrin (PE)-conjugated anti-CD5
antibodies were purchased from Becton Dickinson (Mountain View, CA).
Medium used for the culture of T cells was RPMI 1640 (Bio-Whittaker,
Walkersville, MD) supplemented with 10% fetal calf serum (FCS)
(Hyclone, Logan, UT), 200 mmol/L L-glutamine, gentamycin (50 mg/L), 100 mmol/L sodium pyruvate, and 10 mmol/L nonessential amino acids. Cell lines used in these studies included the transformed T-cell lymphoma, Jurkat and a primary CD4+ human T-cell line (CD4K) that
recognizes autologous renal cell cancer.
Isolation of peripheral blood-derived T lymphocytes.
Peripheral blood T lymphocytes (T-PBL) from healthy volunteers were
isolated and purified as previously described.30,31 PBL
were isolated using Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation. Thereafter, T
cells were depleted of macrophages, B cells, and natural killer (NK)
cells by incubating with microbeads coated with antibodies to CD14, CD19, and CD16, respectively. Negative selection by magnetic separation was performed as previously reported30,31 (Miltenyi Biotech Inc). The T-cell isolation procedure yielded cells that were more than
92% positive for CD3.
To obtain primed T cells, highly enriched populations of
CD3+ cells purified from the peripheral blood of healthy
volunteers were cultured at a density of 1 × 106/mL
for 3 days in complete RPMI medium supplemented with PHA (10 µg/mL).
Immunocytometry.
Surface expression of IL-2R (CD122), IL-2R (CD25), and c on
peripheral blood-derived T cells was detected using immunofluorescence based flow cytometry before and at various times after stimulation with
anti-CD3 and IL-2 in the presence and absence of cAMP enhancing agents.32 The following monoclonal antibody (MoAb)
conjugates were used to phenotypically identify and quantitate
lymphocytic subsets: anti-CD3-PE and anti-CD25-PE from Becton
Dickinson; anti-IL-2R-FITC from Endogen (Cambridge,
MA) and anti-c (3B5). All analysis was performed for
3,000 or 10,000 event list mode files acquired through a forward versus
orthogonal scatter gate predefined for CD45+
CD14 cells. Matched isotypic controls were used for
each particular subclass of Ig and system used.
Analyses on the FACScan (Becton Dickinson) were performed using an
argon ion laser (Spectra Physics Laser, Mountain View, CA) with 15 mW of 488 nm excitation. Live gating of the
forward and orthogonal scatter channels was used to exclude debris and to selectively acquire lymphocytes events. All values presented are
based on percent lymphocytes as determined by light scatter. Individual
fluorescence populations were determined through the use of acquisition
and contouring/quadrant analysis software (Cell Quest; Becton
Dickinson). Results were reported as a percentage of CD3+
IL-2R, CD3+ CD25+, and
CD3+c+ cells in suspension corrected for
nonspecific binding of isotypic controls. The individual contour plots
were derived by joining dots of the same density.
Measurement of cAMP.
Freshly isolated T cells (1 × 106 cells/mL) were
incubated in RPMI 1640 at 37°C in the presence of 100 µmol/L of
the phosphodiesterase inhibitor IBMX for 30 minutes followed by
incubation for 5 minutes with PGE2 (10 µmol/L).
Intracelluler cAMP levels were measured according to a protocol
provided with cAMP EIA kit (Cayman Chemical Co).
Assessment of T-cell viability.
After T cells were cocultured in media, PGE2 (0.1, 1, and
10 µmol/L), dbcAMP (1 mmol/L) or forskolin (100 µmol/L), the number of viable cells was determined by trypan blue dye exclusion. We also
determined if PGE2, dbcAMP, and forskolin induced apoptosis in T cells using a DNA fragmentation detection kit from Oncogene Research Products (Cambridge, MA).
Assay for T-cell proliferation.
Proliferation of T cells was determined by the uptake of
[3H] thymidine.30 Lymphocytes were cultured
at a density of 5 × 104 cells/well in U bottom
96-well plates in triplicate for each culture condition. Cells were
stimulated with one of the following in the presence and absence of
different concentrations of PGE2, with and without IBMX;
(1) medium, (2) 1,000 IU/mL of recombinant IL-2 in the absence and (3)
presence of cross-linked anti-CD3 (OKT3). Two days after initiation of
culture, cells were pulsed with 1 µCi of [3H] thymidine
(6.7 Ci/mmol; NEN, Dupont, Wilmington, DE) and harvested 24 hours later
using a PHD harvester (Cambridge Technology Inc, Cambridge, MA). Filters were placed in scintillation fluid (Ecoscint, National Diagnostic, Manville, NJ) and counted in a -counter (Beckman, Fullerton, CA). Results are expressed as mean counts per
minute (cpm) (±standard error of mean [SEM] of triplicate samples).
Stimulation of T cells with cross-linked anti-CD3 antibody (OKT3) was
performed by preincubating flasks with 1 mol/L Tris buffer (pH 8.0)
containing 10 µg/mL of antibody for 30 minutes. Thereafter, the
flasks were washed twice with RPMI to remove unbound antibody and then
cells were added at a density of 1 × 106/mL for culturing.
Western blot analysis and immunoprecipitation.
Cells were lysed as previously described33 directly in
buffer (50 mmol/L Tris [pH 7.6], 150 mmol/L NaCl, 1% Triton X-100) containing protease and phosphatase inhibitors; aprotinin (5 µg/mL), leupeptin (2 µg/mL), sodium fluoride (50 mmol/L),
phenylmethylsulfonyl fluoride (PMSF) (1 mmol/L), and sodium
ortho-vanadate (1 mmol/L). Samples were placed on ice for 20 minutes
with occasional vortexing. Protein concentration was measured with a
commercial kit (Bio-Rad, Richmond, CA).
Equivalent amounts of proteins from whole cell lysates (10 µg) were
mixed with an equal volume of 2X Laemmli sample buffer, boiled, and
resolved by electrophoresis in 7.5%, 10%, and 12% sodium dodecyl
sulfate-polyacrylamide gels (SDS-PAGE). The proteins were transferred
from the gel to a nitrocellulose membrane using an electroblotting
apparatus (Bio-Rad) (15 V, 3 mA/cm2 for 24 minutes).
Membranes were incubated in blocking solution containing 5% nonfat dry
milk in TRIS-buffered saline overnight to inhibit nonspecific binding.
The membranes were then incubated with specific antibody (1 µg/mL)
for 2 hours. After washing in TRIS with 0.1% Tween 20 for 30 minutes,
membranes were incubated for another 30 minutes with horseradish
peroxidase-conjugated secondary antibody. The membranes were then
washed and developed with enhanced chemiluminescence (ECL Western
Blotting Kit; Amersham).
Immunoprecipitation of STAT5 was performed by adding 4 µg of the
anti-STAT5 Ab to 100 µg of cell lysate protein for 2 hours at
4°C. Thirty microliters of protein G-Sepharose-conjugated beads was
added for 1 hour. Immunoprecipitates were washed three times with lysis
buffer (50 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1% Triton X-100)
containing protease and phosphatase inhibitors; aprotinin (5 µg/mL),
leupeptin (2 µg/mL), pepstatin (5 µg/mL), sodium pyrophosphate (5 mmol/L), sodium fluoride (50 mmol/L), PMSF (1mmol/L), and sodium
ortho-vanadate (1 mmol/L). Proteins were eluted by boiling for 7 minutes in Laemmli sample buffer. Equivalent amounts of protein were
resolved on 7.5% SDS-PAGE and transferred to nitrocellulose membranes
for Western blot analysis.
In some experiments, densitometry scanning of immunoblots was performed
as follows. The developed X-omat AR film was placed on a white light
box by Fotodyn and its image captured by a High Resolution CCD camera
(Sierra Scientific, Sunnyvale, CA). NIH Image
1.57 (National Institutes of Health, Bethesda, MD) was
the program used to analyze the density of each band by graphically plotting the images and calculating the area under each peak. The
percent reduction in expression of a given protein by PGE2 and other agents that increase cAMP was calculated using T cells cultured in medium as the control.
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts isolated from T cells were incubated in 25 µL
reaction mixture containing 20 mmol/L HEPES, 80 mmol/L NaCl, 0.1 mmol/L
EDTA, 1 mmol/L dithiorthreitol (DTT), 8% glycerol, and 2 µL of poly (dI-dC) (Pharmacia, Piscataway, NJ) for 15 minutes at 4°C. For antibody-mediated supershift studies, 5 µL of the anti-STAT5 antibody was added during the preincubation for 30 minutes
at room temperature before the addition of labeled probe. The probe
used for the EMSA was the radiolabled IRF1-GAS (Interferon Regulatory
Factor 1-Gamma Activating Site) that has affinity for STAT 1-6. The
probe was prepared by annealing the coding strand template to a 10-base
complementary primer and a nick enzyme was used to complete the
overhang in the presence of [-32P] deoxycytidine
triphosphate (dCTP). After the probe was added to the
nuclear extracts, the samples were incubated at room temperature for 20 minutes and then run on a 6% nondenaturing polyacrylamide gel.
The gels were dried and analyzed by autoradiography.
Statistical analysis.
The paired t-test was used to determine if results (ie, JAK3
protein levels) obtained from T cells cultured in media (with and
without stimulus) are statistically different from those where T cells
were cultured with cAMP elevating agents. The results are presented as
percent reduction in mean pixel number [(1-mean pixel number of
treated T cells/mean pixel number of media control T cells) × 100]. All statistical tests were performed using a 5%
level of significance.
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RESULTS |
PGE2 inhibits the induction of JAK3 in resting human
peripheral blood T lymphocytes and downregulates its expression in
primed cells.
Recent studies have shown that JAK3 is required to activate IL-2R
pathway for T-cell proliferation.4,14,17,34-36 Loss of JAK3
expression or kinase activity results in impaired activation of the
IL-2R signaling pathway.17 JAK3 protein expression is known
to be regulated in T lymphocytes.8,28 In resting
lymphocytes, JAK3 is constitutively expressed at low levels and
stimulation with either PMA/ionomycin or signaling through the TCR/CD3
complex is known to upregulate its expression
(Fig 1). The findings reported here show
that increased JAK3 expression induced by stimulation of naive
peripheral blood T cells with anti-CD3 plus IL-2 was blocked by
PGE2 when added at the beginning of culture (Fig 1). PGE2 at 10 µmol/L consistently had the greatest effect on
JAK3 expression (n = 3 experiments). Western blotting experiments
showed that the inhibition of JAK3 expression was selective, as
PGE2 had no effect on the levels of other IL-2R-linked
kinases, JAK1, p56lck, and p59fyn. Densitometry
scanning of the Western blots confirmed that after 48 hours of
stimulation JAK3 protein levels increased and that the presence of
PGE2 reduced its expression in three separate experiments
(mean 56.6 ± 8.6 standard deviation [SD] % reduction, P < .001).
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| Fig 1.
PGE2 inhibits upregulation of JAK3 in naive T
cells. (A) Highly enriched naive T cells were cultured for 48 hours
under the following conditions; medium alone; cross-linked anti-CD3 and
IL-2 (1,000 U/mL) alone and anti-CD3/IL-2 plus PGE2 with
and without IBMX. Cell lysates were then subjected to immunoblotting
using antibodies to JAK1, JAK3, p56lck, and
p59fyn. In this and all experiments, equal amounts of
protein were added to each lane. Densitometry readings of the
immunoblot showed the following reduction of JAK3 by PGE2
alone and with IBMX: 0% 0.1 µmol/L PGE2, 4% 1 µmol/L
PGE2, 40% 10 µmol/L PGE2, 63% 0.1 µmol/L
PGE2/IBMX, 68% 1 µmol/L PGE2/IBMX, and 76%
10 µmol/L PGE2/IBMX. (B) Proliferation was assessed by
the uptake of [3H] thymidine as described in Materials
and Methods. (C) Effect of PGE2 on cAMP production in
resting T cells. Freshly isolated T cells were incubated in RPMI 1640 at 37°C in the presence of 100 µmol/L of the phosphodiesterase
inhibitor IBMX for 30 minutes followed by incubation for 5 minutes with
PGE2 (10 µmol/L) before measuring cAMP levels. A
significant reduction in JAK3 expression was observed in three
experiments (P < .001).
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It is known that exposure of naive and activated peripheral blood T
cells to PGE2 results in increased production of
intracellular second messenger cAMP.37-39 In our
experiments, the cAMP production in T lymphocytes induced by 10 µmol/L PGE2/IBMX was up sixfold to eightfold higher than
in cells not exposed to PGE2/IBMX (Fig 1). Because the
suppressive activity of PGE2 appears to be mediated by
cAMP, we determined if other agents that increase cAMP would suppress
JAK3 expression. Here we determined if IBMX would potentiate the
suppression of JAK3 by stabilizing the levels of cAMP increased by
PGE2. Cyclic nucleotides are regulated by cAMP
phosphodiesterase and IBMX is known to block the action of this enzyme
resulting in an increase in intracellular levels of cAMP.21
At the concentration used in these experiments, IBMX (10 µmol/L) had
little effect on JAK3 levels however, it did increase the suppression
of JAK3 mediated by a suboptimal concentration of PGE2 (0.1 µmol/L PGE2) (Fig 1). The difference in JAK3 expression
in naive T cells cultured with or without PGE2/IBMX was
statistically significant (P = .004 for naive T cells [n = 3]). In the same experiments, it was noted that the suppression of
T-cell proliferation by PGE2 alone and when combined with
IBMX paralleled the inhibition in JAK3 expression (Fig 1).
We also determined whether PGE2 with and without IBMX would
downregulate JAK3 that is expressed in activated T cells. Western blotting was performed on T cells that had been stimulated with PHA for
72 hours and then cultured in the presence and absence of
PGE2 for 24 hours. Representative data are presented in
Fig 2 and shows that primed T cells express
significant levels of JAK3; however, coculture with 10 µmol/L
PGE2 reduced expression of JAK3 by 58% without having any
affect on the other kinases. In addition, IMBX (10 µmol/L) and
PGE2 (0.1 µmol/L) at concentrations not effective alone
did suppress JAK3 expression when combined (P < .001 for
primed T cells [n = 8]).
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| Fig 2.
PGE2 downregulated JAK3 expression in primed
T cells. CD3+ peripheral blood lymphocytes were
cocultured with PHA for 3 days. Primed cells were then cultured for 24 hours with medium IBMX, PGE2, or PGE2 plus IBMX
before performing a Western blot to detect JAK1, JAK3,
p59fyn, or p56lck. Representative data are
presented.
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The inhibition of JAK3 expression by PGE2 was induced by
dbcAMP and forskolin in T cells.
Additional experiments examined whether dbcAMP, an analog of cAMP, and
forskolin, a diterpin that activates adenylate cyclase, could suppress
JAK3 levels (Fig 3).21 Western
blotting was performed after naive T cells were stimulated with
anti-CD3/IL-2 for 24 and 48 hours in the presence and absence of cAMP
elevating agents. Both forskolin and dbcAMP inhibited the
stimulus-dependent expression of JAK3, whereas these agents had little
effect on the expression of p56lck, p59fyn, or
JAK1. The expression of STAT5 was also examined, as it is involved in
the IL-2-dependent signaling pathway.4,17,40,41 In
contrast to JAK3, the levels of STAT 5 were not affected by either
dbcAMP or forskolin.
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| Fig 3.
Forskolin and dbcAMP can mimic the effect of
PGE2 and selectively suppress JAK3 expression in naive and
primed T cells. (A) CD3+ lymphocytes were stimulated with
anti-CD3/IL-2 for 24 and 48 hours in the presence of PGE2
(10 µmol/L), Forskolin (100 µmol/L), or dbcAMP (1 mmol/L). Western
blotting was performed for JAK1, JAK3, p59fyn,
p56lck, and STAT5. (B) Primed T cells were cultured with
medium, Forskolin (100 µmol/L) or dbcAMP (1 mmol/L) for 48 hours
before Western blotting. Representative data from 1 of 4 experiments.
(C) Primary CD4+ T-cell line (CD4K) and Jurkat T cells
were cultured with dbcAMP (1 mmol/L) for 48 hours before Western
blotting. Representative data from 1 of 4 experiments are presented.
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Similar findings were observed when primed T cells were cocultured with
forskolin or dbcAMP (Figs 3 and 4).
Concentration-dependent suppression of JAK3 by dbcAMP is presented in
Fig 4 and illustrates that inhibition is seen with 0.1 mmol/L dbcAMP
(54% reduction), although 1 mmol/L is most active (84% reduction).
The time frame required for suppression of JAK 3 in primed T cells by
dbcAMP was also examined. Cells were stimulated with PHA for 3 days and then washed and cultured with dbcAMP for various lengths of time. As
seen in Fig 4, the reduction of JAK3 protein by dbcAMP was noticeable
after 6 hours of incubation (31% reduction) and was near completion by
24 hours (92% reduction). An analysis of all experiments showed a
significant reduction in JAK3 levels for both naive T cells (n = 5, P < .001) and for primed T cells (n = 8, P < .001)
cocultured with dbcAMP. The fact that both forskolin and dbcAMP could
mimic the suppression induced by PGE2 is consistent with
the idea that Jak3 expression is regulated by elevated cAMP levels. The
decreased expression of JAK3 induced by dbcAMP is not unique to normal
human T cells because this suppression was also noted with an
untransformed T-cell line (CD4K) and the transformed line, Jurkat
(Fig 3).
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| Fig 4.
Suppression of JAK3; concentration and time dependent
studies with dbcAMP. (A) Primed T cells were cultured for 24 hours with
various concentrations of dbcAMP before Western blotting. (B) Primed T
cells were cultured with 1 mmol/L dbcAMP for various lengths of time
before Western blotting. An analysis of all experiments showed a
significant reduction in JAK3 levels for both naive T cells (n = 5, P < .001) and for primed T cells (n = 8, P < .001) cocultured with dbcAMP.
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It is also important to note that the reduction of JAK3 expression in
naive and primed T cells was not due to reduced viability after
coculture with agents that increase cAMP. After incubation with
PGE2 (0.1, 1, and 10 mmol/L), dbcAMP (1 mmol/L),
or forskolin (100 µmol/L), the number of viable cells as determined
by trypan blue staining was comparable to that of T cells cultured in
medium alone (>90% trypan blue negative). We also showed that
PGE2 (10 mmol/L), dbcAMP (1 mmol/L), and
forskolin (100 µmol/L) did not induce any significant apoptosis in T
cells as determined by the terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) assay. The level of
apoptotic T cells cultured for 48 hours in PGE2 (6.9%),
dbcAMP (10.1%), and forskolin (7.4%) was low and not different from
that of T cells cultured in medium (7%).
In naive, but not primed T cells, cAMP elevating agents selectively
suppressed surface expression of the JAK3-associated c
chain without affecting IL-2R.
Because c and IL-2R interact with JAK3 and JAK1, respectively,
and both chains are required for IL-2R signaling and proliferation, the
impact that PGE2/IBMX had on their surface expression was examined along with the IL-2R chain. As previously reported, the
percentage of resting T cells that express IL-2R chains is low, but is
highly inducible after 48 hours of stimulation with anti-CD3/IL-2. In
agreement with previous studies, PGE2/IBMX caused a partial
reduction in surface expression of the chain (21% reduction,
Fig 5).22,39
PGE2/IBMX also modestly suppressed induction of c. After
48 hours of stimulation, there is a significant increase in the
percentage of T cells that express c and the presence of
PGE2/IBMX reduced the number of cells expressing c by
28% percent (Fig 5).4,5 In contrast, PGE2/IBMX
has no effect on the expression of IL-2R.
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| Fig 5.
In naive T cells PGE2/IBMX caused a partial
reduction in IL-2R and c surface expression, but had no effect on
IL-2R. Surface expression of IL-2R, IL-2R, and the common chain were examined on CD3+ peripheral blood T cells
before and 48 hours after stimulation with anti-CD3 plus IL-2 in the
presence and absence of PGE2/IBMX. chain expression was
determined by staining cells with 3B5. IL-2R and IL-2R were
detected by staining the anti-p55-PE and anti-p75-PE in combination
with CD3-FITC. The percent reduction in the number of T cells
expressing IL-2R chains was: 21% IL-2R, 0% IL-2R, and 28%
c. The percentage of cells in each quadrant is presented adjacent to
the histogram. Similar results were obtained in two additional
experiments.
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The impact PGE2/IBMX and other agents that increase cAMP
would have on downregulating IL-2R expression on primed T cells was also examined. After 3 days of stimulation, chain expression is
detectable, but at low levels, whereas the levels of the and c
remain elevated (Fig 6). In contrast to
results with naive cells, none of the agents that increase cAMP
downregulated IL-2R expression on primed T cells after 48 hours of coculture. At this time, JAK3 was reduced and representative
data with dbcAMP is presented (Fig 6).
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| Fig 6.
In primed T cells, dbcAMP did not downregulate IL-2R
expression, but did suppress JAK3 expression. (A) Highly purified T
cells were activated for 3 days with PHA and then cultured for an
additional 48 hours in medium alone or medium supplemented with 1 mmol/L dbcAMP. Surface levels of IL-2R, IL-2R, and the chain
were then determined by staining cells as described in the legend to
Fig 5. The percentage of cells in each quadrant is presented adjacent
to the histogram. (B) Aliquots of the same cells were subjected to
Western blotting to determine if dbcAMP suppressed JAK3. Representative
data from one of four experiments are presented.
|
|
Suppression of JAK3 expression by PGE2 correlates with
the inhibition of IL-2-dependent signaling pathways for induction of
c-Myc and c-Jun.
IL-2R is linked to at least three distinct signaling pathways to
activate the protooncogenes critical for IL-2-mediated cell proliferation.4,35 JAK3 appears to play a role in
activation of c-Myc and c-Jun, but not the Bcl-2 pathway, as shown by
overexpression of a dominant negative mutant of
JAK3.4,34,35 Suppression of JAK3 by PGE2/IBMX
and other agents that increase cAMP levels raised the issue of whether
IL-2-dependent signaling was impaired. To address this issue, we used
primed T cells because they provide a way to define the contribution
that a loss of JAK3 has on IL-2 signaling in cells that are insensitive
to suppression of IL-2R by cAMP elevating agents. After 3 days of
priming, T cells were cultured with PGE2/IBMX for 24 hours
and then stimulated with IL-2 for various times before performing
Western blot. Results of this study are shown in
Fig 7. In control cells, the increase in
expression of all the protooncogenes was detectable within 6 hours
after stimulation with IL-2 and remained elevated for at least 24 hours. As expected, JAK3 expression was significantly reduced by
PGE2/IBMX. In the absence of JAK3, the induction of c-Jun
by IL-2 was completely inhibited (96% reduction, 24 hours), whereas
the induction of c-Myc was only partially suppressed (54%, 24 hours).
On the other hand, the IL-2-dependent induction of Bcl-2 was minimally
affected (6% reduction) by the loss of JAK3. As seen in Fig 7, the
deficiency in JAK3 expression and in IL-2-dependent activation of
c-Jun/c-Myc correlated with impaired proliferation to exogenous IL-2.
Similar findings were observed when T cells were cocultured with
forskolin or dbcAMP (Fig 8).
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| Fig 7.
Suppression of JAK3 expression by
PGE2/IBMX correlates with the inhibition of IL-2-dependent
signaling pathways for induction of c-Myc and c-Jun. (A) After 3 days
of priming, T cells were cultured with PGE2/IBMX for 24 hours and then stimulated with IL-2 for various times before performing
Western blot using antibodies to JAK1, JAK3, c-Myc, c-Jun, and Bcl-2.
(B) Densitometry scan of immunoblot in (A) showed that after 24 hours
of coculture with 10 µmol/L PGE2/10 µmol/L IBMX JAK3
was reduced by 57%, which remained suppressed during IL-2 stimulation
(81% reduction). In PGE2/IBMX-treated cells, the
IL-2-dependent induction of c-Myc was partially suppressed (54%
reduction), whereas the induction of c-Jun was suppressed by 96%. In
contrast, PGE2/IBMX had little effect on the increase in
Bcl-2 induced by IL-2 stimulation (6% reduction, 24 hours). (C) Primed
cells were stimulated with IL-2 (1,000 U/mL) in medium alone or medium
supplemented with PGE2/IBMX. Proliferation was assessed
after 3 days of culture by measuring the uptake of [3H]
thymidine. Representative data are presented (n = 3).
|
|
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| Fig 8.
Suppression of JAK3 expression by forskolin and dbcAMP
correlates with the inhibition of IL-2-dependent signaling pathways
for induction of c-Myc and c-Jun. (A) After 3 days of priming, T cells
were cultured with forskolin (100 µmol/L) or dbcAMP (1 mmol/L) for 24 hours and then stimulated with IL-2 for various times before performing
Western blot using antibodies to JAK1, JAK3, c-Myc, and c-Jun. (B)
Densitometry scan of immunoblot in (A). After 24 hours of coculture
with forskolin (100 µmol/L) or dbcAMP (1 mmol/L), JAK3 was reduced
(43% and 49% correspondingly) and remained suppressed during IL-2
stimulation (79% and 94% reduction correspondingly). IL-2-dependent
induction of c-Myc was suppressed by 67% (forskolin) and 61%
(dbcAMP). The induction of c-Jun was suppressed by 77% (forskolin) and
69% (dbcAMP).
|
|
Because IL-2-mediated JAK3 activation results in subsequent tyrosine
phosphorylation of STAT5 protein, we examined the impact that JAK3
suppression induced by cAMP elevating agents had on STAT5 activation.
Primed T lymphocytes were cultured in the presence of 1 mmol/L dbcAMP
for 24 hours followed by IL-2 stimulation for 15 minutes
(Fig 9). The suppression of tyrosine
phosphorylation of STAT5 protein in dbcAMP-treated cells (68%
reduction) paralleled the downregulation of JAK3 expression (71%
reduction). These findings show that an early signaling event, STAT5
phosphorylation, linked to JAK3 activation is impaired by dbcAMP. The
loss of STAT5 phosphorylation resulted in impaired DNA binding
activity. As seen in Fig 9, IL-2 stimulation induced the binding of
STAT5 to IRF1-GAS probe in control T cells. The inducible band
represents STAT5 binding, as antibody to STAT5 eliminated the
expression of this complex in the EMSA. Pretreatment with dbcAMP
eliminated the IL-2 inducible STAT5 DNA binding complex. It also
reduced the constitutive complexes that bind IRF1-GAS.
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| Fig 9.
dbcAMP inhibits tyrosine phosphorylation of STAT5. (A).
Primed T cells were cultured for 24 hours in either medium alone or in
the presence of 1 mmol/L dbcAMP and then stimulated with IL-2 (1,000 IU/mL) for 15 minutes. Cell lysates were then prepared and
immunoprecipitated with anti-STAT5 antibody. Western blotting was
performed on aliquots of the immunoprecipitate to determine whether
STAT5 was tyrosine phosphorylated (4G10 Ab) and to assess the level of
STAT5 in the immunoprecipitates (anti-STAT5 Ab). (B) Aliquots of the
same cells were subjected to Western blotting to determine if dbcAMP
suppressed JAK3. (C) EMSA and supershift analyses were performed as
indicated in Materials and Methods using nuclear extracts from samples
shown in (A). The arrow indicates the migration of the IL-2 inducible
complex binding to IRF1-GAS sequence. The loss of this inducible band
in the presence of anti-STAT5 antibody shows that this band contains
STAT5.
|
|
It is well documented that elevation of cAMP results in activation of
cAMP-dependent protein kinase (PKA). To explore the involvement of PKA
in the inhibition of JAK3 expression, we preincubated lymphocytes with
PKA inhibitors H-89 (100 to 600 nmol/L) or myristoylated PKI amide
(75 µmol/L) followed by PGE2 or dbcAMP
treatment (n = 4). Surprisingly, both PKA inhibitors failed to restore
JAK3 expression in T cells exposed to PGE2, although PKA
activity was substantially inhibited (80% reduction by H-89 and 65%
reduction by myristoylated PKI amide). These results show that
cAMP-mediated inhibition of JAK3 expression is not perturbed when PKA
is substantially inhibited, thus suggesting that this cAMP-dependent
event is either PKA-independent or requires extremely low levels of PKA activity.
| |
DISCUSSION |
The Janus kinases represent a family of nonreceptor tyrosine kinases
involved in cytokine receptor induced signal
transduction.42 JAK3 is a unique family member because it
is present only in lymphoid and myeloid cells and is involved in signal
transduction linked to c, a component of receptors for IL-2, IL-4,
IL-7, IL-9, and IL-15.12,28,42-45 The importance of JAK3
and its interaction with c in lymphoid maturation and activation has
been recently documented in murine and human studies.46,47
B-cell development and T-cell maturation are impaired in homozygous
mutant mice where the JAK3 gene was disrupted by gene
targeting.46,47 Moreover, T cells present in these
JAK3-deficient mice are defective in proliferation and IL-2
production.46,47 In humans, mutation in c is known to
result in X-linked severe combined immunodeficiency (SCID), a defect in
immunity characterized by T-cell lymphopenia and the presence of
nonfunctional B cells.48 Genetic defects of JAK3 in
patients have also been identified that display immunodeficiencies similar to those seen in X-linked SCID.49 Furthermore,
B-cell lines established from JAK3-deficient patients display impaired signaling through the IL-2R and to a lesser degree through
IL-4R.17 These studies point to a critical role for JAK3
and c in the maturation and activation of lymphoid cells. The
findings presented here suggest that PGE2 and other agents
that increase cAMP may suppress T-cell function by inhibiting JAK3 and
to a lesser extent c expression. This suppression had negative
consequences for IL-2R signal transduction and cellular proliferation.
It is also possible that the reduction in JAK3 expression may affect
signaling via other receptors that are known to share
c.42-45
PGE2 and other cAMP elevating agents can regulate immune
responses by inhibiting T-cell activation events including IL-2 and IL-2R expression.18-27 This suppression may involve
PKA-mediated inhibition of phospholipase C-
(PLC) enzymatic activity, which in turn, blocks
phosphatidyinositol-turnover.22,23 Additional studies have
shown that cAMP elevation can alter expression and function of
transcription factors involved in IL-2 and IL-2R gene expression.
dbcAMP is known to inhibit the PKC-mediated induction of the jun and
fos family of genes.25 Moreover, c-Jun transcriptional activity is altered by dbcAMP through PKA antagonizing c-Jun N-terminal kinase activity.50 There are also reports that activation
of PKA can block DNA binding activity of NFB to B sequences in the IL-2 promoter.24 These studies illustrate that cAMP
elevating agents may act at multiple levels to alter gene expression
after TCR/CD3 signaling. It also appears that the induction of JAK3 in
naive cells after ligation of TCR/CD3 is sensitive to PGE2. Whether the defect in JAK3 expression is the result of transcriptional or posttranscriptional modification is currently under investigation.
In agreement with previous reports, our studies show that
PGE2 and other cAMP elevating agents can cause some
reduction in surface expression of IL-2R after stimulation via
TCR/CD3.22,39 It was also noted that PGE2 can
cause modest suppression of c expression after stimulation. In
contrast, we did not detect any significant reduction in IL-2R by
cAMP elevating agents, yet in another study, IL-2 binding to IL-2R
was decreased by forskolin.51 It is not clear what accounts
for the differences in results, however, measurement of IL-2 binding
versus surface expression and our use of peripheral T lymphocytes
versus cultured T-cell lines may be contributing factors. In our
studies, however, PGE2/IBMX treatment induced only modest
reduction of IL-2R and c in naive T cells and none in primed T
cells even though suppression of proliferation was complete. Therefore,
our findings are more consistent with the IL-2 unresponsiveness induced
by PGE2/IBMX treatment resulting from suppression of JAK3
function rather than from reduced IL-2R expression.
In our experiments, PKA inhibitors had no effect on JAK3 expression in
peripheral T lymphocytes exposed to PGE2. These results suggest that cAMP-modulated JAK3 suppression either requires very low
levels of PKA activity or is PKA-independent. It is well-documented that PKA-independent pathways are involved in cAMP signaling mechanism in addition to PKA-dependent pathways.52-54
Signaling through the IL-2R is dependent on the activation of several
PTKs; JAK3, JAK1, p56lck, p59fyn, and
Syk.8-11,13,15,16 Tyrosine phosphorylation of multiple substrates occurs after IL-2 binding to its receptor, and these include
the IL-2R chains ( and ) themselves, various kinases (JAK3, JAK1,
p56lck, and p59fyn), the adapter molecule
Shc3,4,8-11,13,16,55 along with STAT3 and
STAT5.40,41 Recent studies suggest that JAK3 plays an
important role in the phosphorylation of JAK1, IL-2R, and STAT5.17,35 IL-2-dependent signaling also results in
activation of phosphatidylinositol 3-kinase that regulates the kinase
activity of the 70-kD S6 protein kinase
(pp70s6k).56 Although the role each kinase
plays in IL-2-dependent signaling is not clearly defined, it appears
that the activation of these early events ultimately results in the
induction of three distinct signaling pathways that are defined by the
expression of c-Myc, c-Fos/Jun, and Bcl-2.3,4,34 Recent
studies suggest that IL-2-dependent G1-S transition is affected by
PGE2, and this may involve altering IL-2R linked signal
transduction pathways.27,51 Another study has shown that
forskolin can activate PKA and antagonize IL-2-dependent activation of
pp70s6k, as well as phosphatidylinositol
3-kinase.56 In contrast, PKA had no effect on
IL-2-mediated activation of MAP kinase showing that the inhibitory
effect was not global.56
The results presented here suggest that cAMP elevating agents can also
alter IL-2-dependent signaling by selectively downregulating the
expression of JAK3. In dbcAMP-treated lymphocytes, decrease in JAK3
protein expression was accompanied by proportional reduction of STAT5
tyrosine phosphorylation and DNA binding activity. T cells deficient in
JAK3 after treatment with cAMP elevating agents also display the same
alterations in IL-2-dependent induction of protooncogenes as lymphoid
cells expressing a dominant negative mutant of JAK3. In both cases,
there is no induction of c-Jun, reduced expression of c-Myc, and normal
upregulation of Bcl-2.34,35 Additional studies are clearly
needed to fully define the effect that cAMP elevating agents have on
IL-2R signaling and to delineate the mechanism responsible for the
selective suppression of JAK3 protein. The downregulation of JAK3
expression in T cells may represent a unique mechanism by which
PGE2 and other agents that elevate cAMP can suppress IL-2 responses.
| |
ACKNOWLEDGMENT |
We thank Jan Kodish for assisting in the preparation of this
manuscript. We also thank Dr Jerome Ritz for providing us with antibody
to the common chain.
| |
FOOTNOTES |
Submitted June 24, 1998; accepted November 30, 1998.
Supported by United States Public Health Service Grant No. CA56937.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Vladimir Kolenko, MD, PhD, The Cleveland
Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.
| |
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