Trauma and Critical Care Research Labs, Departments of Surgery and Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
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ABSTRACT |
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We recently observed that prostaglandin E2 (PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fyn protein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59fyn kinase activity. The study allowed us to assess whether PGE2-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59fyn activation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30-50% suppression in the autophosphorylation and the kinase activity of p59fyn in T cells incubated with PGE2 or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE2-mediated inhibition of T cell p59fyn kinase activity. In contrast, no change in p59fyn autophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE2-mediated suppression of p59fyn autophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.
protein tyrosine kinase; src kinase; adenylate cyclase; phosphodiesterase; rat; prostaglandin E2
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INTRODUCTION |
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PROSTAGLANDIN E2 (PGE2) has been shown to suppress T cell interleukin-2 (IL-2) production and proliferation in a number of inflammatory conditions including burn, trauma, and sepsis (2, 9, 15, 20, 21, 33, 39, 40). In addition to inhibiting T cell proliferation, PGE2 has been shown to suppress macrophage antigen-presenting ability (4, 22). A decrease in T cell activation, either as a result of direct PGE2 effect or due to a defect in antigen presentation, could contribute to an overall decrease in host resistance and an increased susceptibility to both opportunistic and nonopportunistic pathogens.
The activation of T cells is primarily induced via stimulation of the T
cell receptor (TCR)-CD3 complex; it could be initiated in vitro via TCR
ligation with lectins or with the antibody against CD3 (1, 8, 12, 13).
The earliest biochemical events after TCR ligation are increases in the
activation of a number of protein tyrosine kinases including
p59fyn,
p56lck, and Zap-70 (19, 23, 28).
The activation of these kinases leads to phosphorylation of
phospholipase C- (PLC-
), which hydrolyzes phosphatidylinositol
4,5-bisphosphate (PIP2) into
inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol
(DAG) (8, 39). Whereas IP3
stimulates Ca2+ release from the
intracellular stores (7), DAG activates the protein tyrosine kinase C
activity (35). An increase in intracellular Ca2+ concentration
([Ca2+]i)
sustained for several hours precedes T cell activation and its
subsequent proliferation activity (42).
PGE2, acting on its receptors on T cells, stimulates adenylate cyclase to enhance the formation of cAMP (10, 11, 31, 38-40). The elevation of cAMP levels in T cells has been implicated in the inhibition of T cell IL-2 production, IL-2 receptor (IL-2R) expression, and proliferation (2, 5, 9, 20, 34, 38, 40). Our previous studies suggested that PGE2-mediated suppression of T cell proliferation could result from a disturbance in Ca2+ signaling (16, 17). Further, we have shown an inhibitory effect of PGE2 on p59fyn kinase activity, a component upstream to Ca2+ signaling (18). To investigate the mechanism of PGE2 modulation of p59fyn, the present study evaluated the effects of the incubation of T cells with cAMP analogues and with adenylate cyclase agonist forskolin on p59fyn activation. Also, this study ascertained the effects of adenylate cyclase inhibitor SQ-22536 on p59fyn kinase modulation by PGE2 to determine whether adenylate cyclase activation is vital to PGE2-mediated modulation of p59fyn in T cells.
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MATERIALS AND METHODS |
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Reagents.
PGE2, forskolin, and enolase were
purchased from Sigma (St. Louis, MO). Dibutyryl-cAMP (DBcAMP),
8-bromo-cAMP (8-BrcAMP), IBMX, and SQ-22536 were purchased from
Calbiochem-Novabiochem (La Jolla, CA). Monoclonal antibodies to
p59fyn (residues between amino
acids 85 and 206 of human Fyn protein) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat CD3 antibodies were
purchased from Pharmingen.
[-32P]ATP was
obtained from DuPont NEN (Boston, MA). The specific activity of
[
-32P]ATP is 3,000 Ci/mmol. Reagents for the SDS-PAGE were obtained from Bio-Rad
(Richmond, CA). An Immobilon-P membrane (polyvinylidene difluoride) for
the transfer of proteins was obtained from Millipore (Bedford, MA).
Protein molecular weight markers and other reagents needed for the
preparation of lysis buffer, wash buffer, and kinase buffer were
obtained from Sigma. Nylon wool was obtained from Polysciences
(Warrington, PA). Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden).
T cell preparation. Adults rats (~250 g) were anesthetized (pentobarbital sodium; 65 mg/kg body wt) to remove the spleen and then were killed with a pentobarbital sodium overdose. Splenic T lymphocytes were isolated by previously described methods (16-18). Briefly, spleens were gently ground to prepare a single cell suspension. Red blood cells and the dead cells from the suspension were removed by density gradient centrifugation with Ficoll-Paque. Splenocytes appearing at the interface of Ficoll-Paque and the medium were collected and added to nylon wool-packed columns. These columns were preequilibrated with Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES, 5% FCS, and 50 mg/ml gentamicin. The columns containing cells were incubated at 37°C for 50-60 min. T cells were obtained by eluting the columns with 30-40 ml of HBSS at a flow rate of 1 drop/s. Flow cytometric analysis was carried out to assess the purity of the CD3-positive cells by using anti-CD3 antibodies. It was found that 90-95% of the cells were CD3 positive (data not shown).
Stimulation of T cells and lysate preparation. Rat splenic T cells were stimulated with soluble anti-CD3 antibodies (1 µg/ml) for 180 s at 37°C. The stimulation was stopped by cell solubilization in a phosphorylation lysis buffer (PLB). PLB was prepared by mixing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 1 mM MgCl2, 10 mM Na4P2O7, 200 µM Na3VO4, 0.5% Triton X-100, and 10% glycerol on ice for 45-50 min. Lysates were centrifuged at 10,000 rpm for 5 min at 4°C.
In some experiments, T cells were incubated with PGE2 (10 µM), forskolin (10 µM), or cAMP analogues (doses in the figure legends) for 2 h before stimulation with anti-CD3. These experiments allowed us to assess the effects of PGE2, forskolin, or cAMP analogues on anti-CD3-linked p59fyn autophosphorylation and kinase activity in T cells. For the measurements of SQ-22536 or IBMX effects, T cells were incubated with SQ-22536 or IBMX for 30 min and then with PGE2 or cAMP analogues, respectively, for an additional 2 h.Immunoprecipitation. Lysates were incubated with monoclonal antibodies to p59fyn protein for 1 h, and then the mixture was incubated with protein G-Sepharose beads for another 2 h (18). These incubations were carried out at 4°C. The precipitates were washed three times in PLB without added glycerol.
In vitro kinase assay.
This assay was performed by a previously described method (18). After
the final wash, immune complexes were collected and washed two times
with in vitro kinase buffer (50 mM Tris · HCl, pH
7.4, 10 mM MnCl2, 0.1% Triton
X-100). After these washes, kinase assays were performed by incubating
immune complexes first with 5 µg/ml acid-treated enolase and then for
30 min with 10 µCi
[-32P]ATP. These
incubations were carried out at room temperature (28°C). Samples
were analyzed by SDS-PAGE, and the proteins were transferred to the Immobilon-P membrane. Phosphoproteins were analyzed
by autoradiography, and the intensities of the bands were assessed by densitometry.
Reprobing the membranes. Membranes were reprobed for equal protein loading after stripping the antibodies. For stripping, membranes were incubated with stripping buffer (65 mM Tris · HCl, pH 6.8, 100 mM 2-mercaptoethanol, 2% SDS). The membranes were saturated with blocking buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20 supplemented with 10% BSA) for 1 h at room temperature or for 16-20 h at 4°C; this was followed by an incubation with anti-p59fyn antibody (1:200 dilution) at 4°C. The membranes were washed three times with wash buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20) and were incubated with a secondary antibody conjugated with horseradish peroxidase (1:3,000 dilution) and then washed. After the final wash, membranes were probed with enhanced chemiluminescence dye and proteins were autoradiographed.
Assessment of T cell cAMP levels. Splenic T cells were incubated with various reagents for 2 h, and the intracellular accumulation of cAMP was measured with an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI).
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RESULTS |
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Effect of stimulation or blockade of adenylate cyclase on
p59fyn.
The effects of PGE2
on p59fyn autophosphorylation and
its phosphorylation of enolase are shown in Fig.
1A.
There was no detectable phosphorylation of
p59fyn or enolase by
p59fyn in T cells without their
stimulation with anti-CD3 antibodies. Stimulation of T cells with
anti-CD3 resulted in increased autophosphorylation of
p59fyn as well as increases in its
kinase activity. The autophosphorylation of
p59fyn induced by anti-CD3 was
significantly suppressed in T cells incubated with
PGE2 compared with T cells
incubated without PGE2. Likewise, the phosphorylation of enolase by
p59fyn in
PGE2-treated T cells was
significantly lower than that observed in untreated T cells.
Densitometric analyses of 10 or more similar experiments suggested a
30-50% inhibition of p59fyn
autophosphorylation and its kinase activity in T cells treated with
PGE2 compared with the untreated T
cells (Fig. 1, C and
D). Figure
1B shows the equal-protein-loading
controls for blots shown in Fig. 1A.
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Effect of cAMP analogues on p59fyn.
T cells were incubated with various concentrations of DBcAMP before
their stimulation with anti-CD3, and then
p59fyn autophosphorylation and
kinase activity were assessed. Surprisingly, there was no difference in
p59fyn autophosphorylation and its
ability to phosphorylate enolase whether or not T cells were treated
with DBcAMP (Fig.
4A). We also assessed p59fyn
autophosphorylation and kinase activity in T cells after their incubation with 8-BrcAMP. 8-BrcAMP also failed to affect
autophosphorylation and kinase activity (Fig.
5A). The
lack of effect of cAMP analogues on
p59fyn could be due to an
insufficient increase in intracellular cAMP levels resulting from the
treatment of cells with these analogues. Intracellular levels of cAMP
in T cells were measured after their incubation with
PGE2, forskolin, or a cAMP
analogue (DBcAMP or 8-BrcAMP). The data from these experiments (Table
1) show that all three agents,
PGE2, forskolin, and the cAMP
analogues, increased cellular cAMP. Prior treatments of T cells with
SQ-22536 significantly inhibited
PGE2-induced elevation of
intracellular levels of cAMP. The potential role of intracellular cAMP
on p59fyn was further examined by
inhibiting T cell phosphodiesterases with IBMX. In these experiments,
cAMP analogues (DBcAMP and 8-BrcAMP) were added to T cells in the
presence of phosphodiesterase inhibitor IBMX. The results from these
experiments are shown in Fig. 5, A and
C. In the cAMP analogue-treated T
cells, the presumed further increase in cAMP after phosphodiesterase
inhibition did not have any effect on the activation of
p59fyn. The above results
suggested that although there was an increase in intracellular levels
of cAMP after the treatment of T cells with
PGE2, forskolin, or cAMP analogues
(DBcAMP or 8-BrcAMP), such increases in cAMP levels did not contribute
to the PGE2-related inhibition of
p59fyn phosphorylation and its
kinase activity.
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DISCUSSION |
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Whereas some studies have suggested that PGE2-mediated alteration in the transcriptional regulation of IL-2 and IL-2R results in attenuated T cell proliferation (2, 14, 33, 36), others have shown an impairment in the TCR-related early signaling events contributing to disturbed T cell functions (11, 15, 25, 30-33, 39). In our previous studies (16-18), we found that PGE2-mediated inhibition of p59fyn kinase activity could be a component of the early signaling events that impaired the downstream signaling, including the elevation in [Ca2+]i and the transcriptional regulation of IL-2. The present study evaluated a cross talk between the anti-CD3- and PGE2-mediated signaling pathways. Our data demonstrated that PGE2-mediated suppression of p59fyn is dependent on the activation of adenylate cyclase. However, the elevation of the intracellular cAMP level appeared to have no effect on the anti-CD3-mediated activation of p59fyn, suggesting that the block in p59fyn activation was dependent on the activation of adenylate cyclase but independent of cAMP.
The accumulation of intracellular cAMP after T cell stimulation with PGE2 is a common finding in a number of previous studies (10, 24, 25, 29, 38, 40). The correlation between elevated levels of cAMP and suppression of Ca2+ signaling and IL-2 transcription in T cells has been generally taken to indicate cAMP mediation of T cell dysfunction (2, 6, 15, 32, 34, 39, 41, 44). Our study has shown that although cAMP modulation did occur because of PGE2 action, cAMP itself was not required for the PGE2-mediated suppression of p59fyn. The inability of cAMP to modify p59fyn signaling in T cells is also supported by experiments in which intracellular cAMP hydrolysis was prevented by means of phosphodiesterase inhibition.
That cAMP might be involved in the suppression of TCR-mediated
signaling events was demonstrated primarily by studies using cAMP-elevating agents, cholera toxin, and forskolin (3, 5, 10, 31, 39,
40). Both cholera toxin and forskolin act on
Gs protein to stimulate adenylate
cyclase, leading to the accumulation of cAMP (3, 10, 25, 27). cAMP is
known to stimulate protein kinase A (PKA) in a number of cell types
including T cells (2, 5, 6, 26, 32, 43). Furthermore, PKA has been
shown to inhibit the PLC--mediated hydrolysis of
PIP2 to
IP3 and DAG (6, 24, 31, 32, 37,
38). An inhibition of PLC-
, PIP2 hydrolysis, and the
IP3-related elevation in
[Ca2+]i
has been observed after adenylate cyclase activation of T cells with
forskolin (24, 30-32, 44). Thus a direct activation of adenylate
cyclase via forskolin and cholera toxin leading to cAMP accumulation
could inhibit Ca2+ signaling in T
cells and thereby prevent their activation and the expression of T cell
functions. However, other studies have shown that although cholera
toxin-mediated activation of adenylate cyclase, subsequent to
Gs
activation, could effect
both an increase in cAMP and a decrease in the
Ca2+ signal in T cells, the
decrease in Ca2+ signaling with
increased accumulation of cellular cAMP could not be shown (3, 24, 25,
27, 29, 31). The intracellular Ca2+ signaling
decrease could result not only from
Gs
activation but also from an
interference with PLC-
activation caused by p59fyn inhibition. The latter
possibility is indicated by the dependence of PLC-
activation on
p59fyn activity (8, 13, 28). Our
previous studies had shown that PGE2-mediated inhibition of
p59fyn is correlated with
downregulation of the Ca2+ signal
(18). However, we do not know whether the downregulation of the
Ca2+ signal was due exclusively to
p59fyn inhibition or due
additionally to an inhibitory effect of
PGE2-mediated Gs
activation and cAMP
accumulation on the Ca2+ signal.
Because we have found that PGE2
downregulates p59fyn in a
cAMP-independent manner, we postulate that the
Ca2+ signal downregulation by
PGE2 is partly through
p59fyn inhibition and partly due
to accumulation of cAMP.
The precise mechanism for p59fyn
suppression has remained unclear. A speculative mechanism for
p59fyn inhibition could be that
PGE2-mediated activation of the G
protein and adenylate cyclase couples to some adapter protein, which
could lead to a downregulation of
p59fyn tyrosine kinase and/or an
upregulation of protein tyrosine phosphatases. An alternative
possibility could be that, as for cholera toxin, PGE2 activation of the G protein
and adenylate cyclase modifies the TCR- homodimer (
) chain (1,
8) located upstream of p59fyn, as
has been shown by Haack et al. (25). A modulation in the
-chain
could alter p59fyn
autophosphorylation and kinase activity.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of L. Amato.
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FOOTNOTES |
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This study was supported by National Institute of General Medical Sciences Grants RO1-GM-53235 and RO1-GM-56865.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. M. Sayeed, Trauma and Critical Care Research Labs, Dept. of Surgery, Stritch School of Medicine, Loyola Univ. Chicago, Maywood, IL 60153 (E-mail: msayeed{at}luc.edu).
Received 31 December 1998; accepted in final form 22 April 1999.
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