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Expression in Human Peripheral Blood T Cells: Implications for Cancer Immunotherapy1
Department of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, LA 70112
| Abstract |
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production, is impaired. To evaluate whether Bryo-1 plus IL-2 may
affect the activation pattern of T cells, we investigated the
expression of IFN-
mRNA and protein in human primary T cells.
Northern blot analysis and ELISAs demonstrated that Bryo-1 and IL-2
synergized to induce both IFN-
mRNA and protein expression. This
synergistic induction was seen within 3 h of treatment and with as
little as 10 U/ml IL-2 and 1.0 ng/ml Bryo-1. In vitro transcription
assays revealed that Bryo-1 plus IL-2 induced transcriptional
activation of the IFN-
gene. Furthermore, mRNA stability studies
indicated that this treatment also enhanced the IFN-
mRNA half-life.
Both CD4+ and CD8+ T cells responded to the
treatment with IFN-
expression. The induction of the IFN-
expression was decreased by a specific p38 mitogen-activated protein
kinase inhibitor, but not by a protein kinase C inhibitor. Our results
demonstrate for the first time that Bryo-1 in combination with IL-2
control IFN-
gene expression at both the transcriptional and
post-transcriptional levels through a p38 mitogen-activated protein
kinase-dependent process. Given the pivotal role that IFN-
plays in
the orchestration of an effective Th1 type of response, our results
suggest that Bryo-1 plus IL-2 may be a valuable combined therapy for
cancer treatment. | Introduction |
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, which are proinflammatory
cytokines endowed with antitumor and immunomodulatory properties
(17). Bryo-1 also induces the proliferation and activation
of B and T cells (18, 19, 20) and triggers tumor-specific T
cells that can traffic and mediate tumor regression (21, 22). Furthermore, Bryo-1 induces IL-2R
and IL-2R
chains
expression on human PBMC leading to an enhanced response to IL-2
(17, 19). Based on its direct antitumor activity and on
its broad immunomodulatory effects, Bryo-1 is being clinically
developed. Moreover, initial clinical studies have demonstrated that
Bryo-1 is a novel anticancer agent with biochemical and
immunomodulatory activities in cancer patients (19, 23).
IFN-
, a potent immunomodulatory cytokine produced by T cells and NK
cells, influences all aspects of the immune system, including the
development, activation, and maturation of monocytes/macrophages, T
cells, NK cells, and B cells (24). It is widely recognized
that IL-12 and IFN-
are key to the development of Th1 cells,
and current studies indicate that type 1 immune responses are strongly
correlated with antitumor immune activity (25, 26, 27, 28, 29, 30). These
reports clearly underscore the importance of IFN-
in the immune
response against tumors. The present study was designed to investigate:
1) whether treatment of human T lymphocytes Bryo-1 plus IL-2
induce IFN-
gene expression, and, if so, 2) to determine the
molecular mechanisms regulating the Bryo-1- and IL-2-induced IFN-
gene expression. Our results demonstrate for the first time that,
through mechanisms dependent on p38 mitogen-activated protein kinase
(MAPK) and independent of PKC, the antineoplastic agent Bryo-1
synergizes with IL-2 to induce IFN-
gene expression in freshly
isolated human peripheral blood T cells as well as in purified
CD4+ and CD8+ T cells.
Herein we also demonstrate that both transcriptional and
post-transcriptional levels of regulation control IFN-
gene
expression by this combined treatment. Lastly, we show that mRNA
expression induced by Bryo-1 and IL-2 leads to productive secretion of
IFN-
protein.
| Materials and Methods |
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Peripheral blood leukocytes were obtained from normal healthy
volunteers by leukapheresis using a Fenwall CS-3000 blood cell
separator (Fenwell Laboratories, Deerfield, IL). Lymphocytes were
separated by density gradient centrifugation on lymphocyte separation
medium (Organon Teknika, Durham, NC) and then purified in suspension
from the unfractionated mononuclear leukocyte preparation by
countercurrent centrifugal elutriation in a Beckman JE-6 elutriation
chamber and rotor system (Beckman Instruments, Palo Alto, CA) as
described previously (31). Total T,
CD4+, and CD8+ T cells were
purified with magnetic bits from the elutriated lymphocyte fraction
according to the manufacturers specifications (MACS; Miltenyi Biotec,
Auburn, CA). For any given experiment, only cell populations with a
purity of 9596% for total T cells or 9597% for
CD4+or CD8+ cells were used
in the experiments described below. Cell purity was assessed by flow
cytometry using mAbs against CD3, CD4, CD8, CD14, CD16, and CD19 (BD
Biosciences, San Jose, CA). Viability, as determined by the trypan blue
exclusion test, was >99%. T cells were cultured in RPMI 1640
(BioWhittaker, Walkersville, MD), supplemented with 100 U/ml
penicillin, 100 U/ml streptomycin, 2 mM glutamine, 20 mM HEPES (Life
Technologies, Gaithersburg, MD), and 10% heat-inactivated FBS
(HyClone, Logan, UT). T lymphocytes were cultured at the
indicated time points in a 100-mm2 tissue culture plates
(Corning Glass Works, Corning, NY) at 2 x 106 cell/ml in
medium alone or medium supplemented with either Bryo-1, highly purified
IL-2, or a combination of both biologicals. The specific activity of
IL-2 was 18 x 106 IU/mg; Chiron units are used throughout
the manuscript, 1 Chiron unit = 6 IU; LPS content, < 0.06 IU/ml. IL-2
was kindly provided by Cetus (Emeryville, CA) (32). Clinical grade
Bryo-1 was a gift from A. Fallavollita, Jr. (Cancer Therapy Evaluation
Program, Division of Cancer Treatment, Diagnosis, and Centers, National
Cancer Institute, National Institutes of Health, Bethesda, MD). Bryo-1
conversion factor is equal to 1.0 ng/ml = 0.904 nmol/l.
Dexamethasone (DEX) was purchased from Sigma (St. Louis, MO).
Bisindolylmaleimide (BI) and SB203580 were purchased from Calbiochem
(San Diego, CA). Cell-free supernatants were collected at the
designated time points, and cytokine secretion (IFN-
, IL-4, IL-12,
IL-13, IL-18) was evaluated using an ELISA according to the
manufacturers instructions (BD PharMingen, San Diego, CA).
Northern blot analysis
Human peripheral blood T cells were cultured in medium alone or
supplemented with the indicated reagents. Total RNA was extracted by
lysis with TRIzol (Life Technologies) and purified according to the
manufacturers specifications. Northern blot analysis was performed in
accordance with the previously described protocol (17).
Twenty micrograms of total RNA from each sample was electrophoresed
under denaturing conditions, blotted onto Nytran membranes (Schleicher
& Schuell, Keene, NH), and cross-linked by UV irradiation. Membranes
were prehybridized at 42°C in Hybrisol (Oncor, Gaithersburg, MD) and
hybridized overnight with 2 x 106 cpm/ml of
32P-labeled probe. Membranes were then washed
twice at 42°C for 15 min in 2x SSC (1x SSC = 0.15 M NaCl and
0.015 M sodium citrate (pH 7.0), 0.1% SDS and twice at 65°C for 20
min in 0.2x SSC, 0.1% SDS before being autoradiographed using Kodak
Biomax-MR (Eastman Kodak, Rochester, NY) films and intensifying screens
at -70°C. The human cDNA IFN-
probe (a gift from Dr. G. Ricca
(Rhone-Poulenc Rorer Biotechnology, King of Prussia, PA) and Dr. H. A.
Young (Laboratory of Experimental Immunology, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
MD) and the murine-18S-DECAtemplate probe (Ambion, Austin, TX) were
labeled by random priming using [
-32P]dCTP
(3000 Ci/mmol; Amersham, Arlington Heights, IL). For mRNA synthesis
inhibition, actinomycin D (Act-D; Sigma) was dissolved in ethanol at 1
mg/ml and used at a final concentration of 5 µg/ml as indicated in
the text. For protein synthesis inhibition experiments, cycloheximide
(CHX; Sigma) was used at a final concentration of 10 µg/ml. An
AlphaImager 2000 (alphaInnotech, San Leandro, CA) was used to analyze
the band intensities of the autoradiographs of the Northern blots. The
graphs were generated from the intensities of the distinct IFN-
mRNA
bands after being normalized to the relative abundance of 18S RNA
present in each sample.
Nuclear run-on
Nuclear run-on experiments were performed as previously
described (33). Briefly, nuclei were isolated from 5
x 107 cells/sample by lysing cells in 4 ml lysis
buffer (10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 10
mM NaCl, 150 mM sucrose, and 0.5% Nonidet P-40 (Sigma)) for 5 min on
ice. Nuclei were spun at 167 x g for 5 min at 4°C,
and pellets were resuspended in lysis buffer without Nonidet P-40.
Nuclei were pelleted again as described above and resuspended in 150
µl freezing buffer (50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM
MgCl2, and 0.1 mM EDTA). Run-on assays were
performed by adding 150 µl 2x transcription buffer (20 mmol,
Tris-HCl (pH 8.0), 300 mmol KCl, 10 mM MgCl2, 200
mM sucrose 20% glycerol, 1 mmol dithiothreitol, and 0.5 mmol each of
ATP, GTP, and CTP) and 100 µCi 800 Ci/mmol
[
-32P]UTP (New England Nuclear, Boston, MA)
to 150 µl nuclei suspension. The samples were incubated at 29°C for
30 min. Thirty microliters of 200 mmol CaCl2 and
30 µl 1 U/ml RNase-free DNase 1 (Promega, Madison, WI) were added to
each reaction and further incubated for 10 min at 29°C. Labeled
transcripts were isolated using TRIzol (Life Technologies) and purified
according to the manufacturers specifications. Equal amounts of
radioactivity (
2 x 106 cpm labeled RNA)
were added in 2 ml Hybrizol (Oncor, Gaithersburg, MD) to Nytran
membranes on which 500 ng denatured full-length human IFN-
cDNA (1.0
kb, lacking most of the 3'-untranslated region) and chicken
-actin
cDNA (1.8 kb, HindIII fragment; Oncor) were immobilized
using a slot-blot apparatus (Life Technologies) and a UV cross-linker
(Fisher Scientific, Pittsburgh, PA). Hybridization was conducted at
42°C for 48 h. Filters were washed twice at 42°C for 15 min
with 2x SSC/0.1% SDS and twice at 65°C for 20 min with 0.2x
SSC/0.1% SDS. Filters were then autoradiographed at -70°C. Data
were normalized for the content of
-actin present in each sample
using an AlphaImager 2000 (alphaInnotech).
| Results |
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mRNA expression in
human T lymphocytes
To determine whether Bryo-1, IL-2, or the combination of these two
agents induced IFN-
mRNA expression in peripheral blood human T
cells, T lymphocytes were cultured for 12 h in medium alone or in
the presence of 1.0 ng/ml Bryo-1, 50 U/ml IL-2, or the combination of
the two agents. Total RNA was extracted, and Northern blot analysis was
performed. As shown in Fig. 1
, no basal
expression of IFN-
mRNA was detected in the medium control.
Stimulation with Bryo-1 or IL-2 failed to induce IFN-
mRNA
expression, whereas treatment of T cells with the combination of Bryo-1
and IL-2 led to a mayor induction of IFN-
mRNA. Dose-response
experiments were performed to determine the optimal concentration of
Bryo-1 and IL-2 needed to induce maximal IFN-
mRNA expression. T
lymphocytes were cultured in medium, Bryo-1 (1.0 ng/ml), or IL-2 (50
U/ml) alone or in the presence of various combined concentrations of
Bryo-1 and IL-2. After 12 h stimulation, total RNA was extracted
and analyzed by Northern blot for IFN-
mRNA expression. As shown in
Fig. 2
, Bryo-1 plus IL-2 induced IFN-
mRNA in a dose-dependent manner. As little as 10 U/ml IL-2 in
combination with 1.0 ng/ml Bryo-1 (Fig. 2
, lane 4) was
sufficient to induce a modest, but reproducible expression of IFN-
mRNA. Subnanomolar concentrations of Bryo-1 in combination with 50 U/ml
IL-2 induced a significant expression of the IFN-
transcript
(lane 6), while 1.0 ng/ml Bryo-1 plus 50 U/ml IL-2
were required for maximal expression of IFN-
mRNA (lane
5). Therefore, a combined dose of 50 U/ml IL-2 and 1.0 ng/ml
Bryo-1 was used in all subsequent experiments. Neither Bryo-1 alone
(lane 2) nor the combination of 50 U/ml IL-2 plus
polyethylene glycol, the diluent for Bryo-1 (lane 8),
induced the expression of IFN-
mRNA.
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mRNA induction by the combination
of Bryo-1 and IL-2, T cells were incubated in medium alone or in the
presence of 1.0 ng/ml Bryo-1 plus 50 U/ml IL-2 for the indicated
lengths of time. Total RNA was extracted, and Northern blot analysis
was performed to detect IFN-
mRNA expression. As shown in Fig. 3
mRNA was
detected within 3 h after Bryo-1 plus IL-2 stimulation. IFN-
message expression was further increased at 6 h and reached
maximal levels by 12 h. A small decrease in IFN-
mRNA amounts
was noted by 24 h, the last time point examined.
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gene expression by Bryo-1 plus IL-2
treatment
To investigate whether the induced expression of IFN-
by Bryo-1
and IL-2 involved the activation of IFN-
gene transcription, nuclear
run-on experiments were performed. Human T cells were incubated in
medium alone or supplemented with 1.0 ng/ml Bryo-1 plus 50 U/ml IL-2.
The nuclei were isolated at 4 and 6 h after treatment, and nuclear
run-on assays were performed. As shown in Fig. 4
, the IFN-
gene was not
transcriptionally active in medium-treated control cells. On the other
hand, Bryo-1- plus IL-2-treated cells displayed a mayor induction of
IFN-
gene transcription, which was further augmented at 6 h
posttreatment. These results indicate that the induction of IFN-
mRNA in Bryo-1- plus IL-2-treated cells was associated at least in part
with the transcriptional activation of this gene. Next, experiments
were performed to determine whether the combined Bryo-1 and IL-2
treatment influenced the stability of IFN-
mRNA. Inasmuch as IFN-
mRNA is not constitutively present in human T lymphocytes, we compared
the half-life of Bryo-1- plus IL-2-induced IFN-
mRNA with that of
cells treated with Bryo-1 plus the potent T cell activator PMA. T cells
were incubated for 12 h in medium alone or supplemented with 1.0
ng/ml Bryo-1 plus 50 U/ml IL-2 or 1.0 ng/ml Bryo-1 plus 10 ng/ml PMA.
After the 12-h incubation period, Act-D was added to the cultures for
the indicated lengths of time to block further RNA transcription.
Northern blot analysis revealed that IFN-
mRNA decayed with
different kinetics in Bryo-1- plus PMA-treated samples compared with
Bryo-1- plus IL-2-treated cells (Fig. 5
).
The level of IFN-
mRNA in Bryo-1- plus PMA-treated cells decreased
by 50% (t1/2) after 55 min, and
IFN-
mRNA became almost undetectable after 4 h of Act-D
treatment. On the other hand, Bryo-1- plus IL-2-treated cells displayed
an enhanced IFN-
mRNA stability, resulting in a
t1/2 of 3 h and 20 min. Furthermore, almost
40% of the IFN-
mRNA was still present in the Bryo-1- plus
IL-2-treated cells after 6 h. Taken together, these results
demonstrate that treatment with Bryo-1 and IL-2 induced IFN-
gene
expression in human peripheral blood T cells through a dual mechanism
involving transcriptional and post-transcriptional levels of
regulation.
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secretion in human T
lymphocytes
To evaluate whether the induced expression of IFN-
mRNA led to
protein secretion, supernatants from stimulated human T lymphocytes
were assayed for the presence of IFN-
. T cells were cultured for
24 h in the absence or presence of 1.0 ng/ml Bryo-1 plus 50 U/ml
IL-2, and their supernatants were analyzed by ELISA. As shown in Table I
, medium-treated T lymphocytes have no basal production
of IFN-
. Treatment with Bryo-1 or IL-2 alone also failed to induce
IFN-
protein expression, while the combined treatment of Bryo-1 plus
IL-2 induced a significant secretion of IFN-
. These results indicate
that Bryo-1- plus IL-2-induced IFN-
mRNA expression in human T
lymphocytes is associated with the expression of IFN-
protein in
culture supernatants. Overall these results provide the first evidence
indicating that Bryo-1 and IL-2 act synergistically to induce IFN-
gene expression and secretion in human T cells
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mRNA induction by
Bryo-1 plus IL-2
To determine whether active protein synthesis was necessary for
the Bryo-1 plus IL-2 induction of IFN-
mRNA, T cells were incubated
for 4 h in the absence or the presence of 1.0 ng/ml Bryo-1 plus 50
U/ml IL-2 and in the absence or the presence of the protein synthesis
inhibitor CHX. As shown in Fig. 6
, the
addition of CHX to Bryo-1- and IL-2-treated T cells did not decrease
the induced IFN-
mRNA expression. Interestingly, addition of CHX to
Bryo-1- and IL-2-treated T cells caused a major increase in the
induction (>2-fold) of IFN-
mRNA expression over the level seen in
Bryo-1- plus IL-2-treated samples. These results suggest that the
Bryo-1 plus IL-2 induction of IFN-
mRNA is not dependent on de novo
protein synthesis.
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gene expression by Bryo-1 plus IL-2 is blocked
by DEX, but not by PKC, inhibition
Inhibitors are powerful tools that may help to dissect the
biochemical mechanisms responsible for gene regulation. DEX has been
shown to block both NF-
B nuclear translocation (34, 35)
and AP-1/CREB interaction with IFN-
promoter (36, 37).
These two nuclear transcriptional complexes are known to play a major
role in IFN-
transcription. To investigate whether DEX affects
Bryo-1- plus IL-2-mediated IFN-
gene expression, human peripheral
blood T cells were cultured with DEX in the presence or the absence of
Bryo-1 and IL-2 for 4 h. As shown in Fig. 7
A, DEX significantly
inhibited IFN-
gene expression induced by the combined treatment.
These results demonstrate that IFN-
mRNA expression induced by
Bryo-1 and IL-2 was highly sensitive to DEX treatment and suggest that
IFN-
induction by this combined treatment may be dependent on
NF-
B and/or AP-1/CREB transcriptional regulatory factors.
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mRNA by Bryo-1 and IL-2, T cells
were treated with either medium or Bryo-1 plus IL-2 in the presence of
increasing concentrations of the specific PKC inhibitor BI, and IFN-
expression was evaluated. As shown in Fig. 7
mRNA at either 0.01
µM, the half-maximal inhibitory concentration
(IC50), or 0.1 µM (38, 39). On the
contrary, BI at doses 100 times the IC50 for PKC
did inhibit IFN-
induction by Bryo-1 plus IL-2. These findings
indicate that the Bryo-1- and IL-2-induced IFN-
gene expression
occurs by PKC-independent mechanisms.
The p38 MAPK inhibitor decreases IFN-
expression induced by
Bryo-1 plus IL-2
p38 MAPK has recently been reported to be involved in the ability
of T cells to express IFN-
(40, 41). To ascertain the
role of p38 MAPK in the present system, we investigated the effects of
SB203580, a p38 MAPK-specific inhibitor (42), on the
expression of IFN-
mRNA in human lymphocytes treated with Bryo plus
IL-2. Purified human peripheral blood T cells were pretreated with
SB203580 at the indicated doses for 20 min, and then cells were
activated with Bryo-1 plus IL-2 for 6 h. Total RNA was extracted,
and Northern blot analysis was performed. As depicted in Fig. 8
, 0.1 µM SB203580 inhibited Bryo-1-
plus IL-2-induced IFN-
mRNA expression. Further reduction of Bryo-1-
plus IL-2-induced IFN-
expression was observed when cells were
treated with 1 µM SB203580. We next tested whether SB203580 also
decreased IFN-
production by Bryo-1- plus IL-2-activated T cells.
Lymphocytes were cultured with Bryo-1 plus IL-2 in the presence or the
absence of increasing concentrations of SB203580, and IFN-
levels
were measured by ELISA in the supernatants after 24 h of culture.
As shown in Fig. 9
, SB203580 inhibited
the production of IFN-
by activated T cells in a dose-dependent
manner. The inhibitory effects of SB203580 observed in these
experiments (0.1 µM SB203580 decreased IFN-
production by
50%)
occurred with a potency similar to that previously reported to block
p38 MAPK activity in several cellular systems (40, 41, 42, 43).
Overall, these results suggest that in human primary lymphocytes the
induction of IFN-
expression by Bryo-1 plus IL-2 is at least
partially controlled by activation of p38 MAPK.
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mRNA expression in
human CD4+ and CD8+ T lymphocytes
To determine the pattern of IFN-
expression in the different
subset of T cells, highly purified CD4+ or
CD8+ T cells were cultured for 12 h in
medium alone or in the presence of 1.0 ng/ml Bryo-1 plus 50 U/ml IL-2.
Total RNA was extracted, and Northern blot analysis was performed. As
shown in Fig. 10
, no basal expression
of IFN-
mRNA was detected in medium-treated cells, whereas Bryo-1-
plus IL-2-treated CD4+ or
CD8+ T lymphocytes displayed comparable levels of
IFN-
mRNA expression.
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The differential roles of Th1- and Th2-type responses in
tumor-bearing hosts have been described, and several reports suggest a
correlation between generation of a predominant Th1-type response and
antitumor activity (25, 26, 27, 28, 29, 30). The presence of IL-12 and
IFN-
promotes a Th1 response, whereas IL-4 and IL-13 are associated
with a Th2 response (44, 45, 46). To evaluate whether Bryo-1
plus IL-2 also affect the expression of type 2 cytokines, we
investigated the profile of IL-4 and IL-13 expression. Primary human
lymphocytes were cultured with medium alone or medium supplemented with
Bryo-1 plus IL-2, and IL-4 and IL-13 levels were measured by ELISA in
the supernatants after 24 h of culture. As shown in Table II
, medium-treated cells did not produce either IL-4 or
IL-13. Bryo-1- plus IL-2-treated lymphocytes produced either very low
levels of IL-4 or no IL-4 at all. On the other hand, IL-13 production
was observed in all cells treated with the combination of Bryo-1 plus
IL-2. Functional IL-13R-
has not been directly demonstrated in human
T cells, and IL-13, unlike IL-4, does not induce proliferation of
mitogen-activated T cells (47). Taken together these
results suggest that Bryo-1 plus IL-2 may induce a pattern of cytokine
that is skewed toward a Th1-type response, i.e., high IFN-
/low
IL-4.
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| Discussion |
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gene expression
in freshly isolated human peripheral blood T cells. This work also
provides the first look at the molecular mechanisms involved in the
Bryo-1- and IL-2-induced expression of IFN-
mRNA. In agreement with
previous reports (49) we have shown that Bryo-1 alone does
not induce IFN-
in human lymphocytes. On the other hand, we have
demonstrated that as little as 10 U/ml IL-2 and 1.0 ng/ml Bryo-1 are
sufficient to induce IFN-
mRNA expression. Bryo-1 plus IL-2
synergized not only to induce IFN-
mRNA expression, but also to
induce high levels of IFN-
protein production similar to those
obtained with the potent IFN-
inducer IL-12 (50).
Induction of IFN-
gene expression occurred as early as 3 h
posttreatment and was further augmented up to 12 h. The rapid
induction of IFN-
mRNA in primary cultured T lymphocytes suggests a
direct effect of Bryo-1 and IL-2 on the expression of this gene rather
than a secondary effect mediated by another Bryo-1- and IL-2-inducible
gene. This conclusion is further supported by the absence of IL-12 and
IL-18 in the supernatant of lymphocytes treated with Bryo-1 plus IL-2
(data not shown). Our group has reported that in human peripheral blood
monocytes Bryo-1 is capable of up-regulating the expression of the
IL-2R
-chain while not affecting the constitutive expression of
IL-2R
-chain or inducing the expression of IL-2R
-chain
(17). In an attempt to determine whether Bryo-1 was
synergizing with IL-2 through the induction of the IL-2R, we examined
the surface expression of the three chains of the IL-2R complex on
Bryo-1-treated T lymphocytes. No significant increase in the expression
of any of the IL-2R chains at 3 and 6 h post-treatment was
observed (data not shown). In agreement with a previous report
(20), we noticed that Bryo-1 induces IL-2R
-chain only
after 24 h of treatment. These data demonstrate that the
synergistic mechanism(s) operating in the early induction of IFN-
mRNA by Bryo-1 and IL-2 does not involve up-regulation of the IL-2R
complex. To better understand the nature of Bryo-1 and IL-2 synergism,
we are currently undertaking efforts to further characterize the Bryo-1
signal transduction cascade leading to IFN-
gene regulation.
To further dissect the molecular mechanisms responsible for the Bryo-1-
plus IL-2-induced IFN-
mRNA expression, nuclear run-on experiments
were performed. These assays showed that the IFN-
gene was not
transcriptionally active in medium-treated T cells and that Bryo-1 plus
IL-2 treatment induced transcriptional activation of the gene. The
above-mentioned results from both the kinetics and run-on experiments
provide the first evidence that this combined treatment can exert
transcriptional control of the IFN-
gene and that this effect
contributes at least in part to the expression of IFN-
mRNA in
treated human T lymphocytes. It has been reported that PKC activation
or increased cAMP levels have a stabilizing effect on IFN-
mRNA
expression (51). In an attempt to determine whether
message stabilization was one of the mechanisms involved in the Bryo-1-
and IL-2-induced IFN-
gene expression, experiments with the
transcription inhibitor Act-D were performed. This study was
complicated due to the fact that IFN-
is not constitutively
expressed in T lymphocytes; therefore, it was not possible to measure
the IFN-
mRNA half-life in the absence of stimulation. We observed
that IFN-
mRNA decayed with different kinetics in Bryo-1- plus
IL-2-treated cells (t1/2 = 3 h
and 20 min) compared with that in Bryo- plus PMA-treated cells
(t1/2 = 55 min). These results
suggest a potentially important role for post-transcriptional
regulation of the IFN-
mRNA. Importantly, the
t1/2 of IFN-
induced by Bryo-1 plus IL-2
was much greater than the previously reported
t1/2 induced by IL-2 and PHA
(t1/2 = 1 h), IL-12 plus PHA
(t1/2 = 1 h and 15 min), or
IL-2, IL-12, plus PHA (t1/2 = 1
h and 55 min) (52). Rapid degradation of mRNAs encoding
many oncogenes and cytokines is regulated in part by A+U-rich elements
in their 3'-untranslated regions (53, 54). Proteins that
bind to A+U-rich elements in the 3'-untranslated regions of these
messages control their stability, and by doing so they also control the
levels and timing of expression (53, 54). Furthermore,
AU-rich sequences have been documented in the 3'-untranslated region of
IFN-
mRNA (55), strongly suggesting that IFN-
gene
expression is at least partly regulated through its mRNA stability.
While at present we are unable to determine the natural half-life of
the IFN-
mRNA, our data suggest that post-transcriptional mechanisms
are probably involved in the Bryo-1- plus IL-2-induced IFN-
expression.
The rapid induction of IFN-
mRNA expression suggested a direct
response independent of de novo protein synthesis. Indeed, we
demonstrated that CHX treatment did not block the early induction of
IFN-
mRNA expression by Bryo-1 and IL-2. On the contrary, we
observed a significant increase in IFN-
mRNA expression in cells
treated with CHX plus Bryo-1 and IL-2 compared with Bryo-1- and
IL-2-treated lymphocytes. These results indicate that IFN-
mRNA
expression induced by Bryo-1 plus IL-2 might be partially controlled by
a de novo synthesized repressor protein(s). This finding further
confirms and extends a previous report showing that CHX superinduced
IFN-
expression in PHA-blasted peripheral mononuclear cells
(52). It has been recently reported that the zinc finger
transcription factor yin-yang-1 may act as a repressor of IFN-
basal
transcription (36). Future efforts in our laboratory will
try to delineate the role of yin-yang-1 in IFN-
gene expression
induced by Bryo-1 and IL-2. Taken together our data from the kinetics
and CHX experiments indicate that early induction of IFN-
gene
expression by Bryo-1 plus IL-2 does not require de novo protein
synthesis, but it is probably down-modulated by a de novo synthesized
repressor protein(s).
In an attempt to further explore the molecular mechanisms that led to
the induction of IFN-
gene expression by Bryo-1 and IL-2, the
synthetic glucocorticoid DEX was used. We demonstrated that DEX
produces a major inhibition of Bryo-1- plus IL-2-induced IFN-
expression. DEX has been well characterized for its ability to block
nuclear translocation of NF-
B factors (34, 35) and for
interfering with binding of the transcriptional complex AP1/CREB-ATF to
the IFN-
promoter (37). NF-
B is a critical
regulatory element present in the 5'-flanking region of the IFN-
gene (56, 57), and we have recently demonstrated that
Bryo-1 enhances the expression of NF-
B in human monocytic cells
(C. S. Garcia et al., manuscript in preparation). Further work
will be needed to investigate the roles of these transcriptional
factors in the induction of IFN-
expression by Bryo-1 plus
IL-2.
Bryo-1 is known to be a potent ligand for PKC, and a large body of
evidence suggests that many of its biological effects are mediated
through the activation of PKC (3, 4, 5). However, Bryo-1 can
antagonize several PKC-mediated effects (6, 7, 58), and
recent work has indicated that Bryo-1 antitumor activity in a mouse
melanoma model was exerted through a PKC-independent mechanism
(59). In the present experimental model, the specific PKC
inhibitor BI did not affect the Bryo-1 plus IL-2 induction of IFN-
mRNA at either the IC50 of 0.01 µM or at 0.1
µM, a dose 10 times the IC50 (38, 39). In contrast, at concentrations 100 times the
IC50 of BI for PKC, BI was able to block Bryo-1
and IL-2 induction of IFN-
mRNA expression (Fig. 7
B). It
is noteworthy that this later concentration of BI not only inhibits
PKC, but also inhibits other kinases such as cAMP-dependent protein
kinase and phosphorylase kinase (38, 39). Overall, these
findings indicate that the Bryo-1- and IL-2-induced IFN-
gene
expression occurs via PKC-independent mechanisms. Our data also suggest
that other kinases, different from PKC, are key to the signal
transduction cascade induced by Bryo-1 plus IL-2 leading to IFN-
production. Since p38 MAPK has recently been involved in the ability of
T cells to express IFN-
(40, 41), we investigated the
role of p38 MAPK in Bryo-1 plus IL-2 signaling. We clearly demonstrated
that SB203580 inhibited the induction of IFN-
expression in a
dose-dependent manner. Although we did not measure p38 MAPK activity in
our model, the efficacy and specificity of SB203580 have been
extensively demonstrated in several cellular models
(40, 41, 42, 43). Taken together these results suggest that p38
MAPK activity is a fundamental component of the pathway leading to
IFN-
production by primary human lymphocytes activated with Bryo-1
plus IL-2. Future work in our laboratory will attempt to further define
the signal transduction events induced by Bryo-1 in T lymphocytes.
In summary, we have demonstrated that at doses pharmacologically
achievable, Bryo-1 plus IL-2 induce high levels of IFN-
expression
in primary human T cells without the need for pretreatment with PHA or
other mitogenic activator(s). The present study also provides the first
report dissecting the molecular mechanisms involved in the synergistic
induction of IFN-
by Bryo-1 plus IL-2. We demonstrated that in
primary T lymphocytes, through a process dependent on p38 MAPK and
independent of PKC activation, a dual mechanism involving
transcriptional and post-transcriptional levels of regulation is
responsible for the Bryo-1 plus IL-2 induction of IFN-
gene
expression and protein secretion. We found that this combined treatment
induces IFN-
gene expression in both CD4+ and
CD8+ T cells.
Studies in cancer patients and preclinical models have indicated that T
cell responses to tumor cells are impaired. Of particular relevance,
some reports have shown that in T cells from tumor-bearing animals
IFN-
production is deficient (60, 61). Several
investigators have also suggested that T cells from tumor-bearing hosts
have changed their cytokine production patterns from a Th1 to a Th2
pattern (25, 26, 27, 28, 29, 30, 44, 45). These studies have indicated
that IFN-
and IL-12 tend to promote the development of T cells with
a Th1-type pattern of cytokine expression while inhibiting the
development of Th2 cells (25, 26, 27, 28, 29, 30) Thus, it has been
hypothesized that the failure to protect against tumor is not due to
the lack of an immune response, but it is the result of the cytokine
pattern deviation that impairs the proper development of an antitumor
response. The present results suggest that Bryo-1 plus IL-2, by
inducing a pattern of cytokine expression that is strongly skewed
toward a Th1-type response (high IFN-
/low IL-4), may play a crucial
role in controlling the polarization of the immune response in a
clinical therapeutic setting. Future efforts in our laboratory will try
to determine whether Bryo-1 and IL-2 treatment can direct T cell
differentiation toward Th1 and, if so, whether this combined treatment
induces changes in the methylation status of the IFN-
gene and/or
induces the expression of the recently identified Th1 developmental
factor T-bet (62).
The differential regulation of IL-4 and IL-13 by Bryo-1 plus IL-2 clearly emphasizes the complex nature of the cytokine network. IL-13 production is more ubiquitous than IL-4 production, and in agreement with our observation others have reported that human T cells that otherwise have a Th1 phenotype produce IL-13 (63, 64, 65). IL-13 shares approximately 30% homology with IL-4 and seems to have several overlapping biological activities with IL-4 (66). However, there are several functions of IL-4 that cannot be mimicked by IL-13. For instance, IL-13 transgene expression does not reverse the IgG1 deficiency observed in IL-4-deficient (IL-4-/-) mice (46). Another major difference between IL-13 and IL-4 is the inability of the former to induce T cell proliferation, which is probably due to the lack of functional IL-13R on human T cells (47). Although it has been recently reported that IL-13 reverses the inhibitory effects of renal cell carcinoma on the functional differentiation of dendritic cells (67), at present the significance of Bryo-1- plus IL-2-induced IL-13 is not clear. Further studies are warranted to better understand the role of IL-13 in modulating the immune response against tumor.
Taking into account the well-characterized antineoplastic and immunomodulatory activity of both Bryo-1 and IL-2 and having shown in a murine model with B16-F10 melanoma cells that a combination of Bryo-1 and IL-2 had antitumor activity without significant toxicity (68), our group is currently conducting a National Cancer Institute-funded phase I clinical trial to evaluate the immune effects and toxicity of this combination in patients with cancer.
| Acknowledgments |
|---|
| Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Igor Espinoza-Delgado, Section of Hematology-Oncology, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Room 4C10, Baltimore, MD 21224. E-mail address: espinozaig{at}grc.nia.nih.gov ![]()
3 Abbreviations used in this paper: Bryo-1, bryostatin-1; Act-D, actinomycin D; BI, bisindolylmaleimide; CHX, cycloheximide; DEX, dexamethasone; IC50, half-maximal inhibitory concentration; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. ![]()
Received for publication September 6, 2000. Accepted for publication August 20, 2001.
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