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*
Departments of Physiology and Surgery, and Burn and Shock Trauma Institute, Loyola University Chicago Medical Center, Maywood, IL 60153; and
Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PLC-,3 9).
Activation of PLC- hydrolyzes phosphatidylinositol 4,5-bisphosphate
(PIP2) into inositol 1,4,5-triphosphate (IP3)
and 1,2-diacylglycerol (DAG) (5, 6, 10, 11). Whereas IP3
stimulates Ca2+ release from the intracellular stores, DAG
activates the protein kinase C activity (12, 13, 14, 15). An increase in
intracellular Ca2+ ([Ca2+]i)
sustained for several hours precedes T cell activation and its
subsequent proliferation activity (13, 14, 16, 17, 18, 19).
A suppression in the T cells’ proliferative response and their
production of IL-2 has been observed following sepsis, burn, and trauma
(20, 21, 22). Recent studies from our and other laboratories have
implicated a role of prostaglandin E2 (PGE2) in
the sepsis-associated suppression of T cell proliferation and IL-2
production (23, 24, 25, 26). Additionally, we have shown that PGE2
suppression of T cell proliferation and IL-2 production could result
from a suppression in Ca2+ signaling (25). The altered
Ca2+ signaling could result from either a direct effect of
PGE2 on [Ca2+]i homeostasis or
via an effect on upstream signaling events such as the phosphorylation
of PTKs. June et al. (27) have suggested that an inhibition in PTK
phosphorylation could prevent TCR-mediated signal transduction
including the [Ca2+]i elevation and
production of IL-2. Among the tyrosine proteins that are phosphorylated
after TCR simulation, p59fyn,
P56lck, and ZAP70 have been recognized as
playing important roles in T cell signal transduction pathways (3, 28).
P56lck is associated with CD4 and CD8 molecules
(29), and is believed to be important in the generation of signals
through these molecules. ZAP70 is associated with the 
-chain of TCR
(30). p59fyn has been proposed to be
instrumental in TCR-mediated signaling (31). Several studies have shown
that the overexpression of fyn protein results in enhanced TCR-mediated
Ca2+ mobilization and T cell IL-2 production (3, 28, 32, 33, 34). Rigley et al. (35) suggested that
p59fyn can regulate Ca2+ influx via
a mechanism that is independent of PIP2 hydrolysis. That
p59fyn is an integral component of the
TCR-mediated signaling pathways is indicated by the
co-immunoprecipitation of p59fyn with TCR (31),
and the modulation in the signaling cascade with manipulations in the
levels of p59fyn in transgenic mice (32, 36).
The present study evaluated the effects of PGE2 on the
phosphorylation and activation of p59fyn. For
the assessment of PGE2 effects on the phosphorylation and
activation of p59fyn in T cells during sepsis,
septic rats were treated with indomethacin to block the endogenous
PGE2 synthesis (23, 24, 25, 26), and to monitor its effect on
activation of p59fyn and its kinase activity
toward its target substrate. Further, we ascertained
PGE2-related changes in the p59fyn
autophosphorylation and its mediation of tyrosine phosphorylation of
other proteins in T cells incubated with and without PGE2,
in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli (American Type Culture Collection (ATCC), Rockville, MD; 25922) and Bacteroides fragilis (ATCC 25285) were obtained from Dr. Kenneth D. Thompson, University of Chicago, Chicago, IL. Con A, PGE2, enolase, and indomethacin were purchased from Sigma Chemical Co., St. Louis, MO. mAbs to p59fyn (residues between 85–206 of human Fyn protein) were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Anti-phosphotyrosine mAbs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-rat CD3 Abs were purchased from PharMingen, San Diego, CA. 32P-ATP was obtained from DuPont NEN, Boston, MA. Molecular weight markers were purchased from Sigma and other reagents for the SDS-PAGE were obtained from Bio-Rad, Richmond, CA. Immobilon P membrane (polyvinylidine fluoride) for the transfer of proteins was obtained from Millipore, Bedford, MA. Reagents needed for the preparation of lysis buffer, wash buffer, and kinase buffer were obtained from Sigma. Nylon wool was obtained from Polysciences, Inc., Warrington, PA. Ficoll-Paque was purchased from Pharmacia, Uppsala, Sweden.
Animal model of sepsis
Male Sprague-Dawley rats (225 to 250 g) obtained from Harlan (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used. Sepsis was induced by implanting 1-cm3 presterilized fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (104 CFU) into the rat abdomen (22). Rats implanted with sterilized pellets without the bacteria are referred to as sterile. A febrile response (>38°C), decreased sensibilities to touch, diarrhea, eye hemorrhage, and piloerection were characteristic findings in septic rats on days 1 to 3 postimplantation. Whereas no mortality occurred in sterile rats, approximately 45% of all septic rats died between days 1 and 2 after implantation. Rats were killed 48 h after implantation.
Treatment of animals with indomethacin
Indomethacin (2 mg/kg) was administered i.p. initially 2 h prior to implantation, and 24 and 36 h after implantation (25). There was no significant difference in the mortality in the septic rats treated with and without indomethacin.
T cell preparation
Rats were killed to remove their spleens. Splenic T lymphocytes were isolated using the method previously described (25). Briefly, spleens were gently ground to prepare a single cell suspension. The red and dead cells from the suspension were removed by density gradient centrifugation with Ficoll-Paque. Splenocytes appearing at the interface of Ficoll and the medium were collected. To obtain a pure T cell preparation, splenocytes were incubated with nylon wool-packed columns. These columns were prequiliberated with HBSS supplemented with 10 mM HEPES, 5% FCS, and 50 gm of gentamicin per ml. The column containing cells was incubated at 37°C for 50 to 60 min. T cells were obtained by eluting the columns with 30 to 40 ml of HBSS at a flow rate of 1 drop per second. Flow cytometric analysis was performed to assess the purity of the CD3-positive cells using anti-CD3 Abs. In most of the preparation, 90 to 95% of the cells were CD3 positive (data not shown).
Stimulation of T cells and lysate preparation
T cells obtained from control, sterile, and septic rats were stimulated with Con A (10 µg/ml) or anti-CD3 Abs (1 µg/ml) for 180 s at 37°C. The stimulation was stopped by cell solubilization in a phosphorylation lysis buffer (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 to 50 min. Lysates were centrifuged at 10000 rpm for 5 min at 4°C.
Immunoprecipitation
Lysates were incubated with mAbs to p59fyn protein for 1 h, and then the mixture was incubated with protein G-Sepharose beads for another 2 h (37, 38). These incubations were conducted at 4°C. The precipitates were washed three times in phosphorylation lysis buffer without added glycerol.
Immunoblot
As described elsewhere (37, 38), immunoprecipitated protein after the third wash was analyzed by SDS-PAGE and transferred to Immobilon membranes using a semi-dry Trans-Blot system (Bio-Rad). 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 to 20 h at 4°C followed by an incubation with anti-phosphotyrosine Ab (1:1000 dilution) at 4°C. The membranes were washed three times with wash buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween-20). The membranes were incubated with a secondary Ab conjugated with horseradish peroxidase (1:5000 dilution) followed by washing. After the final wash, membranes were probed using enhanced chemiluminescence dye, and phopshoproteins were autoradiographed.
In vitro kinase assay
This was performed using the method described previously (37, 38). 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 of acid-treated enolase, and then for 30 min with 10 µCi of 32P-ATP. These incubation were conducted at room temperature (28°C). Samples were analyzed on SDS-PAGE (9%) and the proteins were transferred to the Immobilon membrane. Phosphoproteins were analyzed by autoradiography and the intensity of the bands were assessed using densitometry.
Reprobing the membranes
Membranes were reprobed after stripping the Abs. For stripping, membranes were incubated with stripping buffer (65 mM Tris-HCl, pH 6.8, 100 mM 2-ME, 2% SDS). Membranes were saturated and immunoblotting was performed essentially following the protocol mentioned in immunoblot section. For the analysis of fyn we have used anti-fyn mAbs instead of anti-phosphotyrosine Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells obtained from control, sterile, and septic rats were lysed
in the lysis buffer. The total protein content in each sample was
assessed and analyzed for the presence of p59fyn
using the immunoblot technique. A representative immunoblot is shown in
Figure 1
A. No significant
change in the p59fyn protein levels was observed
in T cells obtained from the control, sterile, and septic groups of
rats. The protein contents of p59fyn in T cells
from control and experimental animals were also analyzed after
immunoprecipitation of p59fyn from the cell
lysate with anti-fyn Abs, and were not found to be different
between the control and experimental groups of animals (Fig. 1B). Densitometric analyses of the blots from
immunoprecipitated fyn proteins are shown in Figure 1C.
There seemed to be a decreasing trend in the fyn protein content in T
cells from septic and sterile compared with control rats; however, it
was not found to be statistically significant.
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T cells obtained from control and experimental animals were
stimulated with Con A for 3 min at 37°C and lysed. Fyn protein was
immunoprecipitated from the cell lysates using 1 µg of anti-fyn
Abs and analyzed by SDS-PAGE. Proteins were transferred to the membrane
and the phosphorylation of fyn protein was assessed by immunoblotting
with anti-phosphotyrosine Abs. A representative immunoblot showing
the phosphorylation of p59fyn protein is shown
in Figure 2A. No detectable
phosphorylation of p59fyn was observed in
unstimulated T cells (data not shown). The stimulation of T cells with
Con A resulted in the tyrosine phosphorylation of
p59fyn protein. There was no change in the
tyrosine phosphorylation of p59fyn in sterile
rat T cells compared with control rat T cells. In contrast, a
significant decrease in the phosphorylation of
p59fyn PTK was observed in the T cells obtained
from septic rats compared with the sterile and control rats’ T cells.
To assure that the observed inhibition in p59fyn
phosphorylation was primarily due to the septic injury and not due to
different amounts of protein loaded in the lanes, we stripped the blots
and reprobed for the content of p59fyn protein
in each lane using anti-fyn Abs. The results are shown in Figure 2B. There was no change in the p59fyn
protein contents among the lanes. These data suggested that the
attenuation in phosphorylation of p59fyn protein
was the result of septic injury. Finally, p59fyn
phosphorylation data blots were analyzed using densitometric analyses,
and the data from four or more similar experimental groups were pooled.
The control response was normalized to 1. As shown in Figure 2C, while there was no significant change in the
p59fyn phosphorylation in T cells obtained from
sterile rats compared with the control rats, a 30 to 40% inhibition in
the phosphorylation of p59fyn was observed in T
cells obtained from septic rats compared with control and sterile
rats.
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These experiments evaluated whether or not the above observed
inhibition in p59fyn tyrosine phosphorylation
could affect its ability to phosphorylate enolase, a known substrate
for Src kinases. Phosphoproteins were analyzed using in vitro kinase
assay and are shown in Figure 3A. There was no detectable
p59fyn autophosphorylation or fyn-mediated
phosphorylation of enolase in unstimulated T cells. Con A stimulation
of T cells resulted in p59fyn
autophosphorylation as well as in enolase phosphorylation by fyn. Both
the p59fyn autophosphorylation and enolase
phosphorylation by fyn in control T cells were not significantly
different from sterile rat T cells. In septic rat T cells, a
significant inhibition was observed in both the autophosphorylation of
fyn as well as its ability to phosphorylate the exogenous substrate
compared with the T cells from control and sterile rats. The data from
densitometric analyses pooled from several blots are presented in
Figure 3, C and D. The fyn autophosphorylation
was inhibited by 40 to 50% in septic rat T cells (Fig. 3C). Similarly, a more than 40% inhibition in
enolase phosphorylation by fyn was found in septic rat T cells compared
with control and sterile rat T cells (Fig. 3D). For
equal loading of fyn in various lanes, blots were stripped and
reprobed. No significant change in the amount of fyn was detected in
various lanes (Fig. 3B).
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In these studies, T cells were stimulated with anti-rat CD3 or
Con A. The blots showing the fyn autophosphorylation and enolase
phosphorylation by fyn in T cells after their stimulation with Con A or
anti-CD3 Abs are given in Figure 4,
A and C. There was no apparent change in the
pattern of autophosphorylation of fyn as well as fyn-mediated
phosphorylation of enolase in T cells after their stimulation with Con
A or anti-CD3. Moreover, the kinase activity of
p59fyn in T cells obtained from sterile rats
treated with and without indomethacin was not significantly different.
As shown above, a significant inhibition in both fyn
autophosphorylation as well as the enolase phosphorylation by fyn was
observed in septic rat T cells after their stimulation with Con A or
anti-CD3. Indomethacin treatment of septic animals significantly
prevented the inhibition in fyn autophosphorylation as well as the
enolase phosphorylation. The data presented in Figure 4, E
and F represent the groups of T cells stimulated with Con A.
As shown in the figures, sepsis resulted in a 40 to 50% inhibition of
fyn autophosphorylation (Fig. 4E) and fyn-mediated
enolase phosphorylation (Fig. 4F) compared with
sterile rats. The inhibition in fyn autophosphorylation and its kinase
activity was evidently prevented when the rats were treated with
indomethacin. Protein-loading control of the blots is shown in Figure 4, B and D. Because we did not find any
difference in the stimulatory effects of Con A or anti-CD3 on T
cells’ p59fyn phosphorylation, we presumed the
effects of PGE2 blockade on p59fyn
phosphorylation would be similar whether the cells were stimulated with
Con A or anti-CD3.
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T cells from control rats were incubated for 2 h with or
without PGE2 (1 µM) at 37°C before their
stimulation with Con A or anti-CD3. The effects of PGE2
on fyn autophosphorylation and its phosphorylation of enolase are shown
in Figure 5, A and
C. Stimulation of control rat T cells with Con A or
anti-CD3 resulted in increased autophosphorylation of fyn as well
as its kinase activity. As shown in Figure 5, A and
C, autophosphorylation of p59fyn
induced by Con A or anti-CD3 was significantly suppressed in T
cells incubated with PGE2 compared with T cells incubated
in its absence. Similarly, phosphorylation of enolase by fyn in
PGE2-treated T cells was significantly lower than that
observed in untreated T cells. Densitometric analyses of six or more
experiments performed with anti-CD3 are shown in Figure 5, E and F. A nearly 40 to 50% inhibition of fyn
autophosphorylation was observed in T cells treated with
PGE2 compared with the untreated T cells (Fig. 5E). Similarly a 50 to 60% decrease in enolase
phosphorylation was observed by fyn from PGE2-treated T
cells compared with control T cells (Fig. 5F). Figure 5, B and D shows the equal protein-loading
controls in blots shown in Figure 5, A or C,
respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although various studies have shown the suppression in T cell proliferation during burn, sepsis, and trauma, the mechanism of the proliferative suppression during these injury states has remained largely unknown (26). Several lines of evidence suggested that the burn-, sepsis-, or trauma-associated inflammatory response is accompanied by increased production of PGE2 from phagocytic cells and monocytes (42, 43). On the basis of our present findings, an increase in the PGE2 levels in the various inflammatory conditions would seem to play a role in the T cell suppression through an attenuation in p59fyn activation followed by disturbances in Ca2+ signaling.
The tyrosine phosphorylation of fyn triggers its enzymatic activity and
leads to the phosphorylation of the substrate proteins including
PLC-1 (28, 34). PLC-1 phosphorylation in turn hydrolyzes
PIP2 into IP3 and DAG (44). The
IP3-mediated release of Ca2+ from intracellular
stores and the subsequent Ca2+ influx from the
extracellular space are well-established prerequisites for T cell
activation, proliferation, and lymphokine production (3, 11, 12, 13, 28).
An inhibition in the upstream event such as fyn activation could
potentially attenuate the [Ca2+]i signal via
altered PLC- and hence lead to T cell proliferative dysfunction.
There is also a possibility that fyn may not interfere with
PIP2 hydrolysis but may directly affect Ca2+
signaling using a hitherto unknown pathway suggested by some
studies (35, 45).
It remains unknown whether the fyn inhibition is caused by a direct
effect of PGE2 or by PGE2 interference with the
mitogen-induced signal transduction. PGE2 interaction with
its receptor results in an elevation of intracellular cAMP via an
up-regulation of adenylate cyclase (40). Previous studies have shown
that cholera toxin and forskolin, which up-regulate adenylate cyclase
and subsequently increase cAMP levels, inhibit the TCR-mediated
signaling via an attenuation in most proximal events of the cascade
such as CD3- (46) and PLC- phosphorylation (10). Whether or not
fyn attenuation in septic rat T cells or T cells pretreated with
PGE2 is affected by these PGE2-mediated second
messengers is not known. However, as observed in cholera toxin-treated
T cells, a PGE2-mediated modulation of CD3- could
attenuate fyn activation and hence the subsequent signaling events.
PGE2 could also affect the distal events of T cell
activation in addition to its effect on
[Ca2+]i and upstream signaling. There are a
number of studies suggesting that an inhibition in T cell proliferation
and IL-2 production could result from a disturbed transcriptional
regulation of IL-2 by PGE2 and other cAMP-elevating agents
(47, 48, 49). These studies suggest that PGE2 can inhibit T
cell proliferation and IL-2 production by modulating both the proximal
and distal events of T cell activation. The stimulation of TCR serves
as the primary signal needed for T cell activation but the cell
proliferation and IL-2 production could be influenced by the activation
of several other accessory signals generated through other surface
molecules (3, 5, 6). Among these, the activation of
P56lck by CD4, and of phosphotyrosine
phosphatases are important (3, 37). Whether or not PGE2
released during sepsis attenuates the accessory signals is yet to be
determined.
In summary, sepsis induced a 40 to 50% inhibition of T cell fyn phosphorylation and its kinase activity. This was similar to the inhibition of fyn kinase activity in PGE2-exposed T cells. Indomethacin treatment of rats prevented the septic-related suppression of fyn phosphorylation and kinase activity in T cells. These observations indicate that PGE2 released during sepsis could contribute to suppressed T cell proliferation and IL-2 production by inhibiting p59fyn activation. Because p59fyn activation precedes [Ca2+]i signaling in T cells, and our earlier studies showed indomethacin prevented the sepsis-related attenuation in T cell [Ca2+]i responses, our present findings suggest that the septic injury initially induces a p59fyn down-regulation to lead to the disturbances in the [Ca2+]i responses, lymphokine production, and proliferation in T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mohammed M. Sayeed, Burn and Shock Trauma Institute, 2160 South First Avenue, Maywood, IL 60153. E-mail address:
3 Abbreviations used in this paper: PLC-, phospholipase C-; PGE2, prostaglandin E2; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5- triphosphate; DAG, 1,2-diacylglycerol; PTK, protein tyrosine kinase; [Ca2+]i, intracellular [Ca2+].
Received for publication April 16, 1997. Accepted for publication October 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 by tyrosine phosphorylation. Science 250:1253., a possible mediators of T cell receptor function. J. Biol. Chem. 266:16277. chain. Cell 71:649.[Medline]
. J. Immunol. 158:2390.[Abstract]
(IL-2) production in patients with major trauma. J. Trauma 27:837.[Medline]
subunit. J. Immunol. 150:2599.[Abstract]
gene expression at multiple levels. J. Immunol. 148:2845.[Abstract]
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