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Originally published online as doi:10.1189/jlb.0705369 on December 30, 2005

Published online before print December 30, 2005
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(Journal of Leukocyte Biology. 2006;79:453-462.)
© 2006 by Society for Leukocyte Biology

A role of PP1/PP2A in mesenteric lymph node T cell suppression in a two-hit rodent model of alcohol intoxication and injury

Xiaoling Li, Martin G. Schwacha, Irshad H. Chaudry and Mashkoor A. Choudhry1

Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham

1 Correspondence: Center for Surgical Research, University of Alabama at Birmingham, Volker Hall G 094, 1670 University Boulevard, Birmingham, AL 35294. E-mail: choudhry{at}.uab.edu

ABSTRACT

This study examined the role of protein phosphatase type-1 (PP1), type-2A (PP2A), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in altered mesenteric lymph node (MLN) T cell function in a two-hit model of alcohol (EtOH) intoxication and burn injury. Male rats (250 g) were gavaged with EtOH to achieve a blood EtOH level of ~100 mg/dL prior to burn or sham injury (25% total body surface area). MLN T cells harvested 24 h after injury show a significant decrease in p38 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. Treatment of cells with inhibitors of PP1/PP2A [calyculin A (CA) and okadaic acid (OA)] prevented the suppression in T cells p38 and ERK-1/2 activation. In addition, the suppression in interleukin-2 and interferon-{gamma} production was attenuated in T cells cultured in the presence of CA and OA. MKP-1 inhibitor triptolide did not prevent the suppression in T cells p38/ERK-1/2 and cytokine production. Furthermore, there was a significant decrease in PP1{alpha} phosphorylation (Thr320) and an increase in PP2A (Tyr307) phosphorylation in T cells following a combined insult of EtOH intoxication and burn injury. As phosphorylation of PP1 at Thr320 and PP2A at Tyr307 led to an inhibition of their enzymatic activities, the decrease in the PP1{alpha} phosphorylation correlates with an increase in its enzyme activity. Thus, these results suggest that activation of PP1 is likely to play a predominant role in T cell suppression following a combined insult of EtOH intoxication and burn injury.

Key Words: T cell • mitogen-activated protein kinases • p38 • ERK-1/2 • IL-2 • IFN-{gamma} • thermal injury

INTRODUCTION

Alcohol (EtOH) intoxication increases the risk of infectious complications in trauma and burn patients [1 2 3 4 5 6 7 8 ]. Previous studies from our laboratory and others have shown that a combined insult of EtOH intoxication and burn injury impairs intestinal T cell function and increases gut bacterial translocation [1 , 9 10 11 ], which has been observed following burn injury in a number of studies [1 , 12 13 14 15 ]. Furthermore, previous studies suggest that intestine-derived bacteria and their products play significant roles in initiating/exacerbating multiple organ dysfunction in acutely injured patients as well as in patients with EtOH intoxication [1 , 14 , 16 ]. The mechanism by which a combined insult of EtOH intoxication and burn injury influences T cell function remains to be established.

Previous studies have shown that stimulation of T cell via T cell receptors (TCRs) results in a cascade of signaling events including the protein phosphorylation [1 , 17 18 19 ]. Although the majority of proteins is phosphorylated on tyrosine, threonine, or serine, some have been reported to phosphorylate on two residues. Among them are mitogen-activated protein kinases (MAPK) [18 ], which phosphorylate on tyrosine and threonine and thus, are referred to as dual phosphorylated proteins. There are three major MAPK-dependent pathways, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. ERK is 42 kD and 44 kD and is referred to as ERK-1 and-2, respectively. Similarly, JNK is a complex of 46 kD and 54 kD proteins. Previous studies from our laboratory have shown a correlation between a decrease in p38/ERK-1/2 activation (i.e., phosphorylation) and interleukin (IL)-2 production in mesenteric lymph node (MLN) T cells following EtOH intoxication and burn injury [20 , 21 ]. MAPK mediates a broad range of physiologic processes, including various aspects of immune responses [18 , 21 , 22 ]. The activation of MAPK is also regulated by protein phosphatases (PP). Similar to kinases, PP is also a large family consisting of protein tyrosine, serine, and threonine kinases [18 , 19 , 22 23 24 25 26 ]. T lymphocytes express several phosphatases [hemopoietic protein tyrosine phosphatase, VH1-related, VHR-related MKPX, and the serine/threonine-specific phosphatases type-1 (PP1) and type-2A (PP2A)], which dephosphorylate MAPK. Previous studies have suggested that PP1 and PP2A play a role in activation of p38 and ERK [18 , 19 , 22 , 25 26 27 28 ]. These studies suggest that PP2A inactivates MAPK by dephosphorylating threonine residues on MAPK. In addition, MAPK phosphatases (MKP), also referred as dual specificity phosphatases (DSP), are shown to regulate MAPK activation [22 , 28 29 30 31 ]. More than 12 members of the MKP family are known to date. Among them, MKP-1 is found to be critical in p38 and ERK-1/2 regulation [22 , 28 29 30 31 ]. Altogether, these studies suggest that activation of MAPK is regulated at multiple levels.

Several toxins are highly specific inhibitors of members of the PP family of serine/threonine phosphatases [32 33 34 ]. Previous studies have used pharmacological agents calyculin A (CA) and okadaic acid (OA) to determine the role of PP1/PP2A. CA and OA are serine/threonine phosphatase-specific inhibitors and are shown to inhibit PP1 and PP2A [32 33 34 35 36 37 ]. Triptolide (TP) is shown to inhibit MKP-1 [28 , 31 , 32 , 38 ]. These inhibitors are membrane-permeable and potently inhibit phosphatase activity in intact cells. We used these inhibitors to identify the roles of PP1, PP2A, and MKP-1 in altered MLN T cell p38/ERK-1/2 pathways and a related decrease in T cell cytokine production following a combined insult of EtOH intoxication and burn injury. Our findings from these experiments suggest that PP1 is likely to play a predominant role in suppressed T cell p38/ERK-1/2 and cytokine production following a combined insult of EtOH intoxication and burn injury.

MATERIALS AND METHODS

Animals and reagents
Male Sprague-Dawley rats weighing 225–250 g were obtained from Charles River Laboratories (Wilmington, MA). Nylon wool was obtained from Polysciences, Inc. (Warrington, PA). Reagents for cell culture were obtained from Fisher Scientific (Atlanta, GA). Anti-rat-CD3 antibodies were purchased from PharMingen (San Diego, CA). Concanavalin A (Con A) was obtained from Sigma Chemical Co. (St. Louis, MO). OA was obtained from EMD Bioscience, Inc. (La Jolla, CA). CA was obtained from Cell Signaling Technology Inc. (Beverly, MA). We have used antibodies to p38 protein (Cat. #9712, NEN Biolabs, Beverly, MA); phospho-p38 (Thr180/Tyr182, Cat. #9211, NEN Biolabs); ERK-1/2 protein (Cat. #9102, NEN Biolabs); phospho-ERK-1/2 (Thr202/Tyr204, Cat. #9101, NEN Biolabs); PP1{alpha} protein (Cat. #2582, NEN Biolabs); phospho-PP1{alpha} (Thr320, Cat. #2581, NEN Biolabs); PP2A (Cat. #sc-14020, Santa Cruz Biotechnology, Inc., CA); and phospho-PP2A (Tyr 307, Cat. #sc-14020, Santa Cruz Biotechnology, Inc.). Respective secondary antibodies were also obtained from Cell Signaling Technology Inc. Reagents for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad (Richmond, CA). Immobilon P membrane (polyvinylidine fluoride) was obtained from Millipore (Bedford, MA). Protein molecular weight markers were obtained from Invitrogen (Carlsbad, CA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2 measurement were obtained from Biosource International (Camarillo, CA) and for interferon-{gamma} (IFN-{gamma}) measurement, from R&D Systems Inc. (Minneapolis, MN).

Rat model of acute EtOH intoxication and burn injury
As described previously [9 , 20 , 21 ], rats were divided randomly into four groups: saline + sham, EtOH + sham, saline + burn, EtOH + burn. In EtOH-treated groups, rats were gavaged with 5 ml 20% EtOH in saline. In saline groups, animals were gavaged with 5 ml saline. Four hours after gavaging, a time at which blood EtOH levels in EtOH-treated groups are in the range of 90–100 mg/dL [9 ], animals were anesthetized and transferred into a template, which was fabricated to expose 25% of the total body surface. Animals were then immersed in a water bath (95–97°C) for ~10 s. Sham-injured rats were subjected to identical anesthesia and were immersed in lukewarm water. The animals were dried immediately and given fluid resuscitation intraperitoneally with 10 ml physiological saline. Animals were allowed to recover from anesthesia, returned to their cages, and were allowed food and water ad libitum.

T cell preparation
Twenty-four hours after injury, rats were anesthetized, and via midline incision, intestine was exposed. MLN were removed aseptically. Isolated MLN were gently crushed to prepare single-cell suspension in Hanks’ balanced salt solution (HBSS; Fisher Scientific) supplemented with 10 mM HEPES, 50 µg/ml gentamicin, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cell suspension was incubated with nylon wool packed columns. These columns were pre-equilibrated with HBSS containing 10 mM HEPES, 50 µg/ml gentamicin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% fetal calf serum (FCS). The columns containing cells were incubated at 37°C for 50–60 min. T cells were obtained by eluting the columns with 15 ml HBSS at a flow rate of one drop/s [9 , 20 , 21 ]. T cells thus obtained were found to be ~95% positive for anti-CD3 [39 ].

T cell stimulation and lysate preparation
As described previously [9 , 20 , 21 ], nylon wool-purified T cells were stimulated with or without anti-CD3 (1 µg/ml) or Con A (5 µg/ml) for 5 min and lysed. In experiments determining the effect of phosphatase inhibitors, cells were incubated for 30 min in the presence or absence of various concentrations of calyculin A, okadaic acid and triptolide prior to their stimulation with anti-CD3 or ConA. Cells were lysed in a lysis buffer containing 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 min–1 h. Lysates were centrifuged; supernatants were harvested and stored at –70°C until analysis.

Measurement of p38 and ERK-1/2 phosphorylation levels
For the analysis of p38 and ERK-1/2 protein and phosphorylation, equal amounts of total protein from each sample preparation were analyzed on 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 5% dry milk) for 2 h at room temperature and incubated with the desired primary antibody (1:1000 dilution) at 4°C overnight. The membranes were washed five times with Tris-buffered saline supplemented with 0.05% Tween-20 (TBST). The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (1:2000 dilution) for 1 h at room temperature. The membranes were washed five times with TBST and probed using enhanced chemiluminescence dye, and phosphoproteins were autoradiographed [20 , 21 ].

Reprobing the membranes
Membranes were reprobed for the desired protein after stripping the antibodies. For stripping, membranes were incubated with Western blot stripping buffer (Pierce, Rockford, IL) for 30 min at room temperature. The membranes were washed five times with TBST. Membranes were saturated and immunoblotted with specific antibodies [20 , 21 ].

Measurement of T cell cytokines
Nylon wool-purified T cells were resuspended at a density of 5 x 106 cells/ml in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 50 µg/ml gentamicin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. As described previously [9 , 20 , 21 ], 100 µl T cell suspension was cultured in a 96-well plate at 37°C and 5% CO2 with anti-CD3 (wells precoated with 200 µl 2 µg/ml anti-CD3 suspension) or Con A (5 µg/ml) in the presence or absence of CA (1 nM), OA (1 nM), or TP (10 nM). After 48 h of culture, supernatants were harvested and tested for IL-2 and IFN-{gamma} production using an ELISA kit according to the manufacturer’s instructions.

Statistical analysis
The data, wherever applicable, are presented as means ± SE and were analyzed using ANOVA statistical program (statistical package for Social Sciences Software Program, Version 2.0, Sigma Stat). P < 0.05 between groups was considered statistically significant.

The experiments described here were carried out in adherence to the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and are approved by the Institutional Animal Care and Use Committee, University of Alabama at Birmingham.

RESULTS

p38 and ERK-1/2 phosphorylation and total protein contents in MLN T cell
The phosphorylation of p38 and ERK-1/2 in T cells with anti-CD3 stimulation is shown in Figure 1 . In unstimulated T cells, there was no demonstrable phosphorylation of p38 and ERK-1/2 in T cells obtained from rats receiving a combined insult of EtOH intoxication and burn injury compared with sham rat T cells (data not shown). The stimulation with anti-CD3 resulted in a several-fold increase in the phosphorylation of p38 and ERK-1/2 in T cells derived from all four experimental groups of animals. Anti-CD3-mediated p38 and ERK-1/2 phosphorylation in T cells obtained from rats receiving EtOH alone was not found to be significantly different from T cells derived from sham-injured rats receiving saline alone. Although there was a tendency of a decrease (~20%) in anti-CD3-mediated p38 and ERK-1/2 phosphorylation in burn-injured rat T cells compared with sham-injured rats, this decrease was not found to be significantly different. A more than 60% decrease in anti-CD3-mediated p38 (Fig. 1A) and ERK-1/2 (Fig. 1B) phosphorylation was observed in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with those derived from rats receiving sham injury regardless of their EtOH intoxication. Furthermore, p38 and ERK-1/2 phosphorylation in MLN T cells from EtOH-intoxicated, burn-injured rats was nearly 40% less compared with T cells derived from rats receiving burn injury alone in the absence of EtOH intoxication. There was no demonstrable change in the p38 and ERK-1/2 total protein content in any group.


Figure 1
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  		Figure 1. p38 and ERK-1/2 phosphorylation (p-p-38 and p-ERK-1/2, respectively) and protein expression in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) for 5 min, and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation by Western blot, which was stripped and reprobed for p38 and ERK-1/2 total protein contents in various lanes. Blots were reprobed for ß-actin for equal protein loading in various lanes. Blots obtained from six animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with other groups.

 
PP1/PP2A phosphorylation, and MKP-1 protein expression in MLN T cell
There was no demonstrable phosphorylation of PP1 and PP2A in T cells obtained from rats receiving a combined insult of EtOH intoxication and burn injury compared with sham rat T cells (data not shown). The stimulation with anti-CD3 resulted in a substantial increase in phosphorylation of PP1 (as determined by antiphospho-PP1{alpha}) and PP2A in T cells derived from all four experimental groups of animals. The results, as shown in Figure 2 , suggest that anti-CD3-mediated PP1 (Fig. 2A) and PP2A (Fig. 2B) phosphorylation in T cells obtained from rats receiving EtOH was not found to be significantly different from T cells derived from sham-injured rats receiving saline. Although there was a tendency of a decrease in anti-CD3-mediated PP1 phosphorylation in burn-injured rat T cells compared with sham-injured rats, this decrease was not found to be significantly different. A significant decrease in anti-CD3-mediated PP1 phosphorylation (Fig. 2A) was observed in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving burn injury in the absence of EtOH intoxication or sham injury regardless of their EtOH intoxication. In contrast, the phosphorylation of PP2A (Fig. 2B) was increased significantly in T cells derived from rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving burn injury in the absence of EtOH intoxication or sham injury, regardless of their EtOH intoxication. There was no change in the PP1 and PP2A total protein contents in any group.


Figure 2
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  		Figure 2. PP1{alpha} and PP2A phosphorylation (p-PP1{alpha} and p-PP2A, respectively) and protein expression in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) for 5 min, and lysed. Lysates were analyzed for PP1{alpha} (A) and PP2A (B) phosphorylation by Western blot, which was stripped and reprobed for PP1{alpha} and PP2A total protein contents in various lanes. Blots were reprobed for ß-actin for equal protein loading in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with other groups.

 
In our studies, there was no difference in MKP-1 protein expression in any group (data not shown). In the present study, we were not able to determine MKP-1 phosphorylation, as phospho-specific MKP-1 antibody is not available.

The subsequent experiments were performed to determine the role of PP1/PP2A and MKP-1 in suppressed p38 and ERK-1/2 phosphorylation using their respective inhibitors. In these experiments, we did not include EtOH-alone and burn-alone groups, as p38, ERK-1/2, PP1, and PP2A activation was not significantly different in T cells derived from rats receiving EtOH plus sham and saline plus burn injury compared with sham animals.

Effect of PP1/PP2A and MKP-1 inhibitors on anti-CD-3-mediated p38 and ERK-1/2 phosphorylation in MLN T cell
To evaluate the role of PP1/PP2A in the phosphorylation of T cells p38 and ERK-1/2 after a combined insult of EtOH intoxication and burn injury, nylon wool-purified T cells were stimulated with anti-CD3 in the presence or absence of CA (1 nM) and OA (1 nM) for 5 min and lysed. Results as shown in Figure 3 suggest that although p38 (Fig. 3A) and ERK-1/2 (Fig. 3B) phosphorylation in response to anti-CD3 stimulation was significantly suppressed in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with sham-injured rats, the suppression in p38 and ERK-1/2 phosphorylation was prevented in T cells stimulated with anti-CD3 in the presence of PP1/PP2A inhibitors CA and OA. In contrast, stimulation of T cells in the presence of MKP-1 inhibitor TP (10 nM) did not prevent the suppression in T cells p38 and ERK-1/2 activation (Fig. 4 ).


Figure 3
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  		Figure 3. Effect of PP1/PP2A inhibitors CA and OA on anti-CD-3-mediated p38 and ERK-1/2 phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) in the presence or absence of CA (1 nM) or OA (1 nM) and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with other groups.

 

Figure 4
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  		Figure 4. Effect of MKP-1 inhibitor TP on anti-CD-3-mediated p38 and ERK-1/2 phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) in the presence and absence of TP (10 nM) and lysed. Lysates were then analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with sham gavaged with saline.

 
In addition to anti-CD3, p38 and ERK-1/2 phosphorylation was measured in response to another T cells mitogen, Con A, and the results are shown in Figure 5 . Similar to anti-CD3, the suppression in Con A-mediated p38 (Fig. 5A) and ERK-1/2 (Fig. 5B) phosphorylation was prevented in cells, which were stimulated in the presence of CA and OA. TP did not prevent the suppression in MLN T cells p38 and ERK-1/2 phosphorylation following EtOH and burn injury (data not shown).


Figure 5
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  		Figure 5. Effect of PP1/PP2A inhibitors CA and OA on Con A-mediated p38 and ERK-1/2phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with Con A (5 µg/ml) in the presence and absence of CA (1 nM) or OA (1 nM) for 5 min, and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graph as mean ± SEM. *, P < 0.05, compared with other groups.

 
Effect of direct stimulation of T cell with PP1/PP2A and MKP-1 inhibitors on MLN T cell p38 and ERK-1/2 phosphorylation
In subsequent experiments, T cells were treated directly with CA (10 nM and 50 nM) and OA (10 nM and 1 µM), and the results from those experiments are shown in Figures 6 and 7 . These results suggest that CA dose-dependently increased p38 and ERK-1/2 phosphorylation over basal values in sham and EtOH plus burn-injured rat T cells (Fig. 6) . The increase in phosphorylation was more prominent (an approximate fivefold increase) at higher does of CA (50 nM). Although a CA-mediated increase in p38 and ERK-1/2 phosphorylation at higher concentration (50 nM) was found to be of similar magnitude in sham and EtOH plus burn-injured rat T cells, at lower concentration (10 nM), p38 and ERK-1/2 phosphorylation was nearly 20% lower (but insignificant) in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with sham-injured rat T cells. OA at low concentration (10 nM) did not increase p38 and ERK-1/2 phosphorylation over basal values (Fig. 7) . At higher concentration (1 µM), OA resulted in an approximate threefold increase in p38 and an approximate fivefold increase in ERK-1/2 phosphorylation in sham rat T cells. Furthermore, at this concentration, no difference in OA-induced p38 phosphorylation was found in T cells derived from rats receiving a combined insult of EtOH intoxication and burn injury (Fig. 7A) compared with sham-injured rats. Although there was tendency of a decrease in OA-induced ERK-1/2 phosphorylation in EtOH-intoxicated, burn-injured rat T cells (Fig. 7B) , this decrease was not found to be significantly different from that observed in sham-injured rats. Treatment of T cells with MKP-1 inhibitor TP did not influence p38 ERK-1/2 phosphorylation in any group (Fig. 8 ).


Figure 6
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  		Figure 6. Effect of direct stimulation with CA on p38 and ERK-1/2 phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) or with CA (10 and 50 nM) for 5 min, and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with their respective sham groups.

 

Figure 7
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  		Figure 7. Effect of direct stimulation with OA on p38 and ERK-1/2 phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) or with OA (10 nM and 1 µM) for 5 min, and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with their respective sham groups.

 

Figure 8
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  		Figure 8. Effect of direct stimulation with TP on p38 and ERK-1/2 phosphorylation in MLN T cells following EtOH intoxication and burn injury. MLN T cells were isolated (1x107 cells/ml), stimulated with anti-CD3 (1 µg/ml) or with TP (100 nM and 1 µM) for 5 min, and lysed. Lysates were analyzed for p38 (A) and ERK-1/2 (B) phosphorylation. Blots were stripped and reprobed for p38 and ERK-1/2 total protein in various lanes. Blots obtained from four animals in each group were analyzed using densitometry. Densitometric values for phosphorylation were normalized to the total protein and are shown in bar graphs as mean ± SEM. *, P < 0.05, compared with respective sham groups.

 
Effect of PP1/PP2A and MKP-1 inhibitors on T cell IL-2 and IFN-{gamma} production
To determine whether inhibiting PP1/PP2A or MKP-1 prevents the suppression in T cell IL-2 and IFN-{gamma} production, T cells were cultured with anti-CD3 in the presence or absence of CA (1 nM), OA (1 nM), or TP (10 nM) for 48 h, and supernatants were harvested for the measurement of IL-2 and IFN-{gamma} production. Furthermore, to determine the effect of direct stimulation with these inhibitors, T cells were cultured in the presence of CA (10 nM and 50 nM), OA (10 nM and 1 µM), or TP (100 nM and 1 µM) for 48 h, and supernatants were harvested for the measurement of IL-2 and IFN-{gamma} production. It is to be noted that IL-2 and IFN-{gamma} were not detected in the supernatants harvested from T cells cultured directly in the presence of PP1/PP2A or MKP-1 inhibitors in any group. IL-2 and IFN-{gamma} levels, after anti-CD3 stimulation, are shown in Figure 9 . There was a significant decrease in anti-CD3-mediated IL-2 and IFN-{gamma} production in T cells derived from EtOH and burn and injured rats compared with rats receiving sham injury alone. However, EtOH and burn-mediated decrease in IL-2 and IFN-{gamma} were prevented in T cells cultured in the presence of CA (1 nM) or OA (1 nM; Fig. 9A ). In addition to anti-CD3, T cell cytokine production capacity was measured in response to another T cell mitogen, Con A, and the results are shown in Figure 9B . Similar to anti-CD3, the suppression in Con A-mediated production of T cells IL-2 and IFN-{gamma} was prevented in the group of cells, which were cultured in the presence of CA and OA. MKP-1 inhibitor TP did not influence the IL-2 and IFN-{gamma} production in any group (data not shown).


Figure 9
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  		Figure 9. Effect of PP1/PP2A inhibitors CA and OA on MLN T cell IL-2 and IFN-{gamma} production following EtOH intoxication and burn injury. MLN T cells (5x106cells/ml) were cultured with anti-CD3 (wells precoated with 2 µg/ml, A) or Con A (5 µg/ml, B) in the presence or absence of CA (1 nM) and OA (1 nM) in a 96-well plate at 37°C and 5% CO2 (A). After 48 h of culture, supernatants were harvested for the measurement of IL-2 and IFN-{gamma}. Values are means ± SEM from six animals in each group. *, P < 0.05, compared with other groups.

 
We recognize that in this study, we have not measured IL-2 at an early time-point, but in a previously unpublished study, we did measure IL-2 and IFN-{gamma} production at 24 h after Con A stimulation. We found similar responses of IL-2 and IFN-{gamma} levels at an early time-point; however, the levels were substantially lower (approximately half the levels) compared with supernatants harvested 48 h after T cell stimulation.

DISCUSSION

In this study, we observed that although EtOH intoxication and burn injury alone do not affect T cell p38 and ERK-1/2 activation, the combined insult of EtOH intoxication and burn injury significantly decreases p38 and ERK-1/2 phosphorylation in MLN T cells. This is accompanied with an increase in PP1 activity and a decrease in PP2A activity in T cells following a combined insult of EtOH intoxication and burn injury. The suppression of anti-CD3-linked p38 and ERK-1/2 phosphorylation was prevented when T cells were stimulated with anti-CD3 in the presence of PP1/PP2A inhibitors CA and OA. Our results showed that cells cultured in the presence of PP1/PP2A inhibitors CA and OA had restored IL-2 and IFN-{gamma} production following EtOH and burn injury. However, MKP-1 inhibitor TP did not prevent the decrease in T cells p38, ERK-1/2, IL-2, and IFN-{gamma}. We confirmed these findings using another T cell mitogen, Con A, which requires a cell with MHC for T cell activation. However, in this study, we did not use Con A to determine the role of antigen-presenting cell; rather, we used Con A as an additional T cell mitogen to confirm whether alterations in T cells p38 and ERK-1/2 are only specific to anti-CD3 stimulation. Altogether, these results suggest that that the suppression in T cell p38 and ERK-1/2 phosphorylation and the related decrease in IL-2 and IFN-{gamma} production could result from an increase in activation of PP1 in T cells following a combined insult of EtOH intoxication and burn injury.

Protein phosphorylation is the most important post-translational mechanism used by eukaryotic cells to regulate various functions. The phosphorylation of a protein can create novel recognition motifs for protein–protein interactions and thereby, play a critical role in regulation of various cellular activities including gene expression, cell differentiation, and cell proliferation. However, it is important to note that the level of protein phosphorylation inside the cell is controlled by a multitude of factors, which include intracellular counterparts such as PP as well as extracellular signaling stimuli. Consequently, protein kinases and PP are key components of the signaling cascade through which cells respond to extracellular signals [17 18 19 , 22 , 25 ]. Thus, the net activity of a phosphoprotein at any given time depends on the proportion of molecules in phosphorylated and dephosphorylated states, which in turn depends on the relative activities of protein kinases and PP. Although it has been clear for many years that PP play a role in signal transduction pathways, little information has been available about how individual phosphatase is involved in regulation of cell function. Furthermore, no study to date has investigated their role in altered T cell function in an injury condition such as the one that is described in this study.

Two major families of phosphatases, namely, protein tyrosine phosphatases and protein serine/threonine, are present in eukaryotic cells [17 18 19 , 22 , 25 ]. The serine/threonine-specific family of PP can be divided into four groups: PP1, PP2A, PP2B, and PP2C, based on their preference of substrate, dependence of divalent ions, as well as sensitivity to inhibitor proteins or naturally occurring toxins [18 , 22 , 25 , 26 ]. However, PP1 and PP2A, because of their abundance and broad specificity, are considered the two principal phosphatases, which play a predominant role in p38 and ERK-1/2 regulation [18 , 22 , 25 , 26 ]. In addition to PP1 and PP2A, some DSP have been recognized as key players for inactivating different MAPK isoforms; this class of phosphatases has been designated MKP [18 , 22 , 25 , 26 ]. Although the MKP form a structurally and functionally distinct subclass among the large number of PP present in eukaryotic genomes, there is a great deal of confusion regarding their classification. In our study, we used TP, an inhibitor of MKP-1, and found that TP did not prevent the suppression in MLN T cell p38 and ERK-1/2 activity, suggesting that MKP-1 is not likely involved in altered T cell function following EtOH and burn injury. In addition, we measured MKP-1 protein expression (data not shown) but did not find any alteration, which suggests that the suppression in p38 and ERK-1/2 is likely independent of MKP-1.

We have determined the activation of PP1 and PP2A using phosphoantibodies specific for PP1{alpha} (Thr320) and PP2A (Tyr307). Previous studies have shown that phosphorylation of PP1{alpha} at Thr320 and PP2A at Tyr307 leads to inhibition of enzyme activity [40 41 42 43 ]. We found that T cells derived from rats receiving a combined insult of EtOH intoxication and burn injury exhibit a significant decrease in the phosphorylation of PP1{alpha} along with an increase in PP2A phosphorylation. As a decrease in the PP1{alpha} phosphorylation correlates with an increase in its enzyme activity, it is possible that an increase in PP1 plays a predominant role in suppressed p38 and ERK-1/2 activation following EtOH and burn injury. However, as there is a dynamic balance between phosphatases and kinases, more studies evaluating the phosphorylation of PP1, PP2A, p38, and ERK-1/2 at multiple time-points are needed to ascertain such a role of PP1 and PP2A. Furthermore, studies are also needed to delineate the mechanism of differential regulation of PP1 and PP2A following EtOH intoxication and burn injury.

Several lines of evidence suggest that burn injury without EtOH intoxication results in hyperactivation of macrophage, leading to production of inflammatory mediators including IL-10, tumor necrosis factor {alpha} (TNF-{alpha}), transforming growth factor-ß (TGF-ß), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) derivatives [1 , 5 , 44 45 46 47 ]. The findings from some of these studies suggest that T cell suppression (i.e., 1–10 days) after burn injury is secondary to the factors released by macrophage [1 , 5 , 44 45 46 47 ]. In contrast, other studies have shown a suppression of T cell function in the absence or presence of macrophages or other adherent cells [1 , 12 ]. The difference between these studies is likely a result of different animal models and the T cell sources used. Although the role of these macrophage-derived factors in altered MLN T cell function following a combined insult of EtOH intoxication and burn injury remains to be established, recent findings from our laboratory suggest that EtOH intoxication combined with burn injury results in a significant increase in blood corticosterone levels on Days 1 and 2 after injury [21 ]. The findings reported in this manuscript indicate that on Day 1 after injury, the suppression in MLN T cells p38/ERK-1/2 and cytokine production was evident only in the group that had the combined insult of EtOH intoxication and burn injury, and no significant suppression in MLN T cell proliferation and cytokine production was observed in rats receiving EtOH intoxication or burn injury alone. In contrast, on Day 2 after injury, there was a decrease in MLN T cell function following burn injury alone in the absence of EtOH intoxication, but the suppression was greater in the group receiving a combined insult of EtOH intoxication and burn injury compared with the T cells from rats receiving burn injury alone [9 , 20 , 21 ]. These findings suggest that EtOH intoxication prior to burn injury potentiates an early and a greater T cell suppression compared with the group receiving burn injury alone in the absence of EtOH intoxication.

Although the mechanism by which EtOH intoxication and burn injury influences the balance between PP1/PP2A and p38/ERK pathways remains to be established, previous studies have suggested a role of corticosterone in altered balance between protein kinases and PP. These studies showed that a short exposure of mast cells to glucocorticoids or dexamethasone results in the increased expression of the MKP-1 gene at the promoter level and attenuate proteosomal degradation of MKP-1 [29 , 30 ]. Moreover, studies have shown that OA prevented glucocorticoid-induced apoptosis in T cells [36 ]. Furthermore, corticotrophin was shown to up-regulate protein tyrosine phosphatases [48 ]. In addition, recent studies from our laboratory have shown a role for corticosterone in an altered p38 and ERK-1/2 pathway, and related decrease suppressed IL-2 production [21 ]. Although we did not find a role of MKP-1 in our present study, it is likely that increased corticosterone levels following a combined insult of EtOH intoxication and burn injury may up-regulate other phosphatases such as PP1/PP2A. Alternatively, a role of macrophage-derived factors (e.g., IL-10, TNF-{alpha}, TGF-ß, PGE2, and iNOS derivatives) in altered T cell signaling is also not ruled out. Thus, more studies are needed to identify such a role of these factors in T cell functional deficits following EtOH intoxication and burn injury.

MLN is the central lymph node that connects various parts of the intestine and plays a critical role in clearing bacteria originating from the intestine. Although multiple factors, including increased gut permeability and bacterial overgrowth in the intestine following a combined insult of EtOH intoxication and burn injury, may contribute to increased bacterial translocation, the process of infection involves not only the passage of bacteria from the intestinal lumen to extra-intestinal sites but also survivability of translocated bacteria in the extra-intestinal sites. Thus, the host immune defense becomes a much more critical component in the bacterial translocation process. As T cell-mediated immunity plays a role in defense against bacteria originating from intestine, suppression in T cell function as a result of an imbalance between PP1/PP2A and p38/ERK pathways may impair bacterial clearance, leading to increased bacterial multiplication and accumulation in MLN.

In summary, we found that PP1/PP2A inhibitors CA and OA prevented the decrease in p38 and ERK-1/2 activation in T cells following a combined insult of EtOH intoxication and burn injury. Several previous studies have used a high concentration of CA and OA to directly activate T cell p38 and ERK-1/2 via a mechanism, which is independent of TCR stimulation. Findings from those studies have suggested that CA and OA have differential affinities for PP1 and PP2A and that these inhibitors can inhibit PP1 and PP2A in a highly selective manner [33 34 35 36 ]. These studies suggest that although CA has nearly equivalent inhibitory activity against PP1 and PP2A, OA has 100-fold greater selectivity for PP2A over PP1 [33 34 35 36 ]. In our study, although we are unable to discern such effects of CA and OA, as there were no demonstrable differences between the effects of CA and OA on p38 and ERK-1/2, the findings of a significant decrease in PP1 phosphorylation (i.e., increase in activity) suggest that PP1 may play a predominant role in altered T cell p38/ERK-1/2 and a related decrease in IL-2/IFN-{gamma} production following EtOH and burn injury. Although a role of JNK along with p38 and ERK-1/2 has been suggested in T cell proliferation and IL-2 production [18 ], previous findings from our laboratory have shown that JNK may not be critical to T cell suppression in EtOH and burn-injured rats, as there was no change in JNK activity following EtOH intoxication and burn injury [21 ]. Thus, the suppression in p38 and ERK-1/2 is likely to play a major role in decreased T cell cytokine production following a combined insult of EtOH intoxication and burn injury.

ACKNOWLEDGEMENTS

This study was supported by NIH through AA12901.

Received July 6, 2005; revised October 7, 2005; accepted November 14, 2005.

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