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The Journal of Immunology, 2004, 172: 7804-7812.
Copyright © 2004 by The American Association of Immunologists

Down-Regulation of IL-2 Production in T Lymphocytes by Phosphorylated Protein Kinase A-RII{beta}1

Michael R. Elliott*, Ryan A. Shanks{dagger}, Islam U. Khan{dagger}, James W. Brooks{ddagger}, Pamela J. Burkett{ddagger}, Brandy J. Nelson{ddagger}, Vasileios Kyttaris§, Yuang-Taung Juang§, George C. Tsokos§ and Gary M. Kammer2,*,{dagger}

* Department of Microbiology and Immunology and {dagger} Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157; {ddagger} BD Biosciences, Lexington, KY 40511; and § Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The {beta} isoform of the type II regulatory subunit (RII{beta}) of protein kinase A suppresses CREB transcriptional activity and c-Fos production in T cells following activation via the TCR. Because CREB is an integral nuclear transcription factor for IL-2 production by T cells, we tested the hypothesis that RII{beta} down-regulates IL-2 expression and IL-2 production in T cells. Stable transfection of RII{beta} in Jurkat T cells led to an ~90% reduction in IL-2 mRNA and IL-2 protein following T cell activation. The inhibition of IL-2 production was associated with phosphorylation of the RII{beta} subunit at serine 114 (pRII{beta}) and localization of pRII{beta} in intranuclear clusters. A serine 114 phosphorylation-defective mutant, RII{beta}S114A, did not form these intranuclear clusters as well as wild-type RII{beta}, and did not inhibit IL-2 mRNA and protein synthesis, indicating that serine 114 phosphorylation is required for both nuclear localization and down-regulation of IL-2 production by RII{beta}. In contrast to its effect on IL-2, RII{beta} induced constitutive up-regulation of CD154 mRNA and cell surface expression. Thus, pRII{beta} differentially regulates gene expression following T cell activation. Unexpectedly, we also found that stable overexpression of another protein kinase A regulatory subunit, RI{alpha}, had the opposite effect on IL-2 expression, causing a 3- to 4-fold increase in IL-2 production following stimulation. In summary, our data demonstrate a novel mechanism by which serine 114 phosphorylation and nuclear localization of RII{beta} controls the regulation of gene expression in T cells.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For three decades it has been proposed that the second messenger cAMP diminishes the immune response by modifying T lymphocyte effector functions (1, 2). Heightened intracellular concentrations of cAMP have been temporally associated with inhibition of Ag- or mitogen-stimulated T cell proliferation, IL-2 production, and cytotoxicity (3, 4, 5). More recent evidence suggests that constitutive, low-level cAMP turnover is also required for T cell homeostasis (6, 7). However, the downstream targets of cAMP and the precise mechanisms by which elevated concentrations of the cyclic nucleotide influence T cell effector functions remain incompletely understood. Both immunogenic and tolerogenic responses depend upon mitogenesis and the production of cytokines such as IL-2 (8), underscoring the relevance of this second messenger in the competent immune response.

The cAMP/protein kinase A (PKA) 3 pathway is an integral signal transduction system in T cells (2). In its holoenzyme form, PKA is the principal intracellular receptor for cAMP, and exists as two isozymes, type I (PKA-I) and type II (PKA-II) (9, 10). The inactive, tetrameric form of PKA consists of two catalytic (C) subunits bound by a dimer of regulatory (R) subunits, R2C2. To date, four R subunit isoforms (RI{alpha}, RII{alpha}, RI{beta}, and RII{beta}) and three C subunit isoforms (C{alpha}, C{beta}, or C{gamma}) have been identified (9). Both PKA isozymes are activated by the binding of cAMP to R subunits, as depicted in this equation: R2C2 + 4cAMP {leftrightarrow} R2cAMP4 + 2C (9, 10, 11). Although the four R subunit isoforms are highly homologous, variations in the primary amino acid sequences lead to unique biochemical and functional characteristics, including differences in cAMP binding affinity and subcellular distribution (11).

In T cells, PKA-II localizes predominantly to the Golgi-centrosome region via direct interaction with A kinase anchoring proteins (AKAPs) (12, 13). However, both RII{alpha} and RII{beta} subunits have also been identified in the nuclei of multiple cell types, including human T cells (14, 15, 16, 17, 18). Although the mechanisms by which RII subunits localize to the nucleus remain incompletely understood, both RII{alpha} and RII{beta} but not RI subunits possess a putative nuclear localization sequence, KKRK, that has been linked to the nuclear localization of RII{beta} in transformed murine fibroblasts (19). Of potential pathophysiologic relevance was our recognition that the nuclei of T cells from some subjects with the autoimmune disease systemic lupus erythematosus (SLE) possess increased RII{beta} protein compared with healthy controls (18). To date, however, little is known about the regulation of PKA-II activity in T cells or the biologic significance of nuclear RII{beta} in modulating T cell effector functions.

A key feature that distinguishes RI and RII subunits is the presence of a consensus PKA phosphorylation site adjacent to the C subunit binding domain of both RII{alpha} (Ser99) and RII{beta} (Ser114). In vitro studies have shown that these sites are autophosphorylated by the PKA-C subunit (20, 21). There is also evidence that RII autophosphorylation decreases the affinity of RII for C subunit and increases its affinity for AKAPs (20, 21). Importantly, mutation of Ser114->Ala blocks RII{beta} nuclear localization and prevents its potent inhibitory effects on transformed fibroblast growth (19). However, little information is available regarding the in vivo regulation of RII{beta} phosphorylation at Ser114, or what biologic effects Ser114 phosphorylation has on the subunit’s function within the T cell nucleus.

Our interest in PKA-II has been focused on the role of the RII{beta} subunit in the nucleus of T cells. The finding that RII{beta} levels are abnormally high in the nuclei of T cells of some SLE patients (18) raised the possibility that this subunit might contribute to the immunopathogenesis of SLE by modifying gene expression and, therefore, effector functions such as cytokine production. Indeed, T cells overexpressing RII{beta} exhibit partitioning of the subunit between the cytosol and nucleus, down-regulation of CREB transcriptional activity, and diminished c-fos promoter activity and c-Fos protein production (22). These results provide evidence that RII{beta} acts as a repressor of CREB-dependent gene transcription in activated T cells.

Both CREB and c-Fos are nuclear transcription factors that are integral for effective IL-2 transcription (23, 24). To examine the role of nuclear RII{beta} in regulating IL-2 expression in T cells, three questions were posed: 1) Is Ser114 phosphorylation necessary for nuclear localization of RII{beta} in T cells? 2) Does RII{beta} inhibit IL-2 expression? and 3) Is Ser114 phosphorylation important for RII{beta} to function as an inhibitor of IL-2? In this study, we use an Ab that specifically recognizes phosphorylated Ser114 RII{beta} (pRII{beta}) to demonstrate that human T cells possess constitutive pRII{beta}, and that pRII{beta} is primarily localized to the centrosome and nucleus. Also, we found that phosphorylation of Ser114 enhances intranuclear localization of RII{beta} and is required for suppression of IL-2 by RII{beta} in Jurkat T cells. Taken together, these results demonstrate that nuclear RII{beta} functions as a repressor of IL-2 in a Ser114 phosphorylation-dependent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Okadaic acid and calyculin A were purchased from Calbiochem (San Diego, CA). Anti-PKA Abs were purchased from BD Biosciences (Lexington, KY). Anti-phospho-Ser114-RII{beta} murine mAb (clone 24.1) was developed in collaboration with BD Biosciences. Anti-FLAG-HRP, anti-FLAG-Cy3, and anti-{beta}-actin Abs were purchased from Sigma-Aldrich. Goat anti-mouse IgG-Alexa Fluor-488 and TO-PRO-3 were purchased from Molecular Probes (Eugene, OR). FITC-conjugated Abs for flow cytometry were purchased from Caltag Laboratories (Burlingame, CA).

Plasmids

RII{beta}-FLAG and RI{alpha}-FLAG plasmids have been previously described (22). The cDNA encoding the phosphorylation mutant of RII{beta}, containing a serine to alanine substitution at amino acid 114 (RII{beta}S114A), was generously provided by Dr. Y. S. Cho-Chung (National Institutes of Health, Bethesda, MD). RII{beta}S114A was subcloned into the KpnI and XbaI restriction sites of the CMV-3x-FLAG-14 (Sigma-Aldrich) plasmid and the EcoRI site of the pGEX-3X plasmid as previously described (22). Bacterial expression constructs for untagged RII{alpha} and RII{beta} were generated by PCR subcloning of the full-length coding sequences into the pET-21a+ vector (Novagen, Madison, WI) as previously described (22), using the BamHI and NdeI sites for RII{alpha} and the EcoRI and NdeI sites for RII{beta}. IL-2-luciferase plasmid containing the luciferase reporter gene under control of the –600 to +51 region of the human IL-2 promoter was generously provided by Dr. G. Crabtree (Stanford University, Palo Alto, CA) (25). Thymidine kinase-Renilla luciferase plasmid was purchased from Promega (Madison, WI).

Recombinant proteins and in vitro phosphorylation

Purification of bacterially expressed GST-RII{beta} and GST-RII{beta}S114A was performed as previously described (22). Purification of untagged RII{alpha} and RII{beta} was performed similarly, except that purification of the untagged proteins from bacterial lysates was performed using cAMP-agarose affinity chromatography according to the method of Scott et al. (26). For in vitro phosphorylation of purified recombinant proteins, 2 ng/µl protein in PKA phosphorylation buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP, 2 mM EGTA, 5 mM DTT, 20 mM sodium fluoride) was incubated in the absence or presence of 15 U/µl PKA-C subunit (New England Biolabs, Beverly MA) for 10 min at 30°C. Reactions were stopped by adding SDS sample buffer and boiling for 5 min. Samples were analyzed by SDS-PAGE and immunoblotting.

Cell culture, transfection, and primary human T cell purification

Jurkat T cells (clone E6.1) were transfected by electroporation as previously described (22). For generation of stable transfectants, plasmid DNA was linearized with MfeI before electroporation. Twenty-four hours posttransfection, cells were cultured in medium containing 1 mg/ml Geneticin (Invitrogen, Carlsbad, CA), and passaged for a total of 4 wk. Individual subclones were obtained by limiting dilution and screened for stable expression by immunoblotting with anti-FLAG. Subclones were routinely maintained in medium containing 0.5 mg/ml Geneticin.

Primary human T lymphocytes were isolated and enriched from PBMCs of healthy donors using the human pan T cell isolation kit, Midi MACS (Miltenyi Biotec, Auburn, CA) as previously described (18).

Stimulation and IL-2 ELISA

Before stimulation, cells were cultured at 1 x 106 cells/ml in fresh medium without Geneticin for 18–24 h. Cells were then collected by centrifugation and resuspended at 2 x 106 cells/ml in fresh medium without Geneticin and stimulated with 10 ng/ml PMA + 1 µM ionomycin (IO), or an equivalent volume of DMSO for the times indicated in the figures. Following incubation, cells were harvested by centrifugation and supernatants and cell pellets were collected and stored at –70°C for later use. IL-2 levels were determined from cell-free culture supernatants using the IL-2 Quantikine kit (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions.

RNA extraction and PCR

RNA was harvested from cells using the RNeasy kit (Qiagen, Valencia, CA). RNA for real-time PCR was treated with DNase-I using the RNase-Free DNase kit (Qiagen). cDNA was synthesized from 1 µg of RNA in a final volume of 20 µl using the Superscript III First-Strand Synthesis System (Invitrogen). Semiquantitative PCR analysis was performed using 2 µl of cDNA in a 50-µl reaction of HotStarTaq PCR mastermix (Qiagen) according to the manufacturer’s instructions. PCR products were resolved on a 1.5% agarose-gel. Real-time PCR was performed with a Cepheid Smart Thermocycler (Sunnyvale, CA) by adding SYBR green to the reaction mixture. For each sample the fluorescence was measured in every PCR cycle and plotted as a curve. Primers used in this study: IL-2 (sense, 5'-ATGTACAGGATGCAACTCCTGTCTT; anti-sense, 5'-GTTAGTGTTGAGATGATGCTTTGAC; annealing temperature of 50°C); CD154 (sense, 5'-ATCCTCAAATTGCGGCACATGTCA; anti-sense, 5'-AAGCCAAAGGACGTGAAGCCAGTG; annealing temperature of 55°C); and GAPDH (sense, 5'-TCCGGGAAACTGTGGCGTGATGG; anti-sense, 5'-GC-CCTCCGACGCCTGCTTCACC; annealing temperature of 55°C).

Flow cytometry

Following treatment, 5 x 105 cells were washed once in PBS, followed by two washes in FACS buffer (1% BSA, 0.05% NaN3 in PBS). Cells were incubated with 5 µl of anti-CD69-FITC Ab, 10 µl of anti-CD154-FITC, or 10 µl of FITC-conjugated isotype control in 100 µl total volume of FACS buffer for 30 min at 4°C. Cells were washed twice in FACS buffer and fixed in 1% paraformaldehyde/PBS before analysis.

Luciferase assays

Jurkat T cells were transiently transfected with the amount of plasmid DNA indicated in each figure. Following treatment, luciferase activity was quantified from cell extracts using the Dual-Luciferase reporter assay system (Promega) as previously described (22). Firefly luciferase values were normalized to Renilla luciferase activity derived from inclusion of 2 µg of thymidine kinase-Renilla luciferase plasmid as an internal control in each transfection.

SDS-PAGE and immunoblotting

Whole cell extracts (WCE) were prepared and analyzed by 10% SDS-PAGE and immunoblotting as previously described (22). Briefly, cells were lysed in 1x SDS-sample buffer, sonicated for 10 1-s pulses, and boiled for 5 min before immunoblot analysis.

Confocal immunofluorescence microscopy

Approximately 1 x 105 cells were adhered to poly-L-lysine coated Cytoslides (Thermo-Shandon, Pittsburgh, PA) at 1000 rpm for 5 min in a Cytospin3 centrifuge (Thermo-Shandon). Cells were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized in 0.1% Triton X-100/PBS for 10 min, and blocked in 5% BSA/PBS for 10 min. Ab incubations were conducted for 1 h in 5% BSA/PBS at room temperature at the following concentrations: anti-pRII{beta}, 1:100; and anti-FLAG-Cy3, 1:1000. For peptide-blocking experiments, pRII{beta} Ab was preincubated with 100-fold molar excess of pRII{beta} immunizing peptide for 1 h with gentle agitation at room temperature. For DNA staining, cells were incubated with 10 mM TO-PRO-3 (Molecular Probes) in PBS for 10 min. Coverslips were mounted using Prolong reagent (Molecular Probes). Samples were examined using the Axiovert 100M laser-scanning confocal microscope (Zeiss, Thornwood NY).

Statistical Analysis

Statistical significance (p ≤ 0.05) was calculated by the paired Student’s t test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ser114 of RII{beta} is constitutively phosphorylated in T lymphocytes

To examine the phosphorylation status of RII{beta} Ser114 in T cells, we generated a mAb directed against pRII{beta}. The specificity of the Ab was tested by Western immunoblotting using rRII{alpha} and rRII{beta} incubated in the absence or presence of purified PKA-C subunit to promote phosphorylation. Although RII{alpha} and RII{beta} are highly conserved in this region (RII{alpha}: RRVSV, aa 96–100; RII{beta}: RRASV, aa 111–115), anti-pRII{beta} was highly specific for pRII{beta}, showing minimal cross-reactivity with phosphorylated RII{alpha} or unphosphorylated RII{beta} (Fig. 1A). Additionally, anti-pRII{beta} detected GST-RII{beta} but not GST-RII{beta}S114A containing a Ser114->Ala mutation at residue 114 (Fig. 1B). These data demonstrate that the pRII{beta} mAb specifically recognizes Ser114-phosphorylated RII{beta}.



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  		FIGURE 1. RII{beta} is constitutively phosphorylated in T lymphocytes. A, rRII{alpha} and rRII{beta} were incubated in the absence (–) or presence (+) of rPKA-C subunit to promote phosphorylation as described in Materials and Methods. Volumes corresponding to the indicated amounts of RII proteins (ng) were resolved on a 10% SDS-PAGE gel and probed with anti-phospho-Ser114-RII{beta} mAb (pRII{beta}). B, GST-RII{beta} or GST-RII{beta}S114A were incubated in the absence (–) or presence (+) of rPKA-C subunit, resolved on a 10% SDS-PAGE gel, and probed with anti-pRII{beta} or anti-RII{beta}. C, Equivalent amounts of WCE from primary human T cells (1° T cells), one female (F) and one male (M), Jurkat T cells, and Raji B cells were analyzed by 10% SDS-PAGE and immunoblotting with the Abs indicated to the left of the figure. Relative molecular weights in thousands are shown to the right of the panel.

 
To determine whether RII{beta} is constitutively phosphorylated in vivo on Ser114, we tested human primary T cells, a Jurkat T cell line, and a Raji B cell line for pRII{beta} content by immunoblotting. Constitutive pRII{beta} was detectable in all three cell types (Fig. 1C). It was remarkable that, although primary T cells contained much less total RII{beta} than Jurkat T cells, levels of pRII{beta} in primary T cells were comparable to Jurkat T cells (Fig. 1C). Thus, a portion of the total cellular RII{beta} in both cycling Jurkat and quiescent human T lymphocytes is constitutively phosphorylated at Ser114.

Protein phosphatase (PP)1-mediated dephosphorylation and nuclear localization of RII{beta} in Jurkat T cells

The finding that RII{beta} was constitutively phosphorylated at Ser114 in T cells led us to examine the role of PP in the dephosphorylation of RII{beta}. Treatment with the PP inhibitor calyculin A for 1 h enhanced Ser114 phosphorylation by >2-fold in Jurkat cells (Fig. 2A). By contrast, treatment with okadaic acid had no effect on pRII{beta} levels (Fig. 2A). Calyculin A inhibits PP1 and PP2A nearly equivalently (IC50 = 0.5–1.0 nM) and okadaic acid inhibits PP2A 100-fold more effectively than PP1 (IC50 = 0.2 nM) (27). Thus, the finding that calyculin A, but not okadaic acid, treatment enhances RII{beta} Ser114 phosphorylation demonstrates that PP1, but not PP2A, participates in RII{beta} Ser114 dephosphorylation in T cells. Because we were interested in studying the role of RII{beta} phosphorylation in the context of T cell activation, we also examined the effect of PMA plus IO on pRII{beta} levels. Activation of Jurkat T cells with PMA plus IO did not affect either the total amount or localization pattern of pRII{beta} (data not shown).



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  		FIGURE 2. PP1-mediated dephosphorylation and nuclear localization of RII{beta} in Jurkat T cells. A, Jurkat T cells were treated with 10 nM calyculin A, 0.5 µM okadaic acid, or left untreated for 1 h. Equivalent amounts of WCE were analyzed by immunoblotting with anti-pRII{beta} and anti-RII{beta}. B, Jurkat T cells were examined by confocal immunofluorescence microscopy following staining with anti-pRII{beta} alone (pRII{beta}), with the pRII{beta} immunizing peptide (pRII{beta} + peptide), or with the secondary Ab alone (2° alone). Differential interference contrast (DIC) image shows cells from the first panel of A. C, Jurkat cells stably transfected with RII{beta}-FLAG or RII{beta}S114A-FLAG were cultured for 1 h in the absence or presence of 10 nM calyculin A, and stained with anti-pRII{beta} (pRII{beta}, green), anti-FLAG-Cy3 (FLAG, red), and TO-PRO-3 (DNA, blue), and examined by confocal microscopy. pRII{beta} and FLAG images were overlayed (merged) to demonstrate areas of overlapping signal (yellow). Arrowheads point to centrosomes. Arrows point to clusters of intranuclear RII{beta}. All confocal images are of 1-µm-thick planar sections through the center of the cells.

 
Existing evidence indicates that nuclear translocation of RII{beta} in the NIH 3T3 fibroblast line requires Ser114 phosphorylation (19). Because RII{beta} is constitutively phosphorylated on Ser114 in T cells, we determined the subcellular localization of endogenous pRII{beta} in Jurkat T cells by confocal immunofluoresence microscopy. In untreated cells, pRII{beta} staining was identified predominantly in two regions: the Golgi-centrosome (Fig. 2B, arrowhead) and the nucleus (Fig. 2B, arrow). In the nucleus, pRII{beta} staining was visualized as both diffuse speckling and discrete clusters (Fig. 2B, arrow). The same pattern of staining was observed with three different Ab clones (data not shown). The specificity of the Ab was tested by preincubating anti-pRII{beta} with the pRII{beta} immunizing peptide. Fig. 2B (far right panel) shows that preincubation of the Ab with the pRII{beta} immunizing peptide reduced the signal to that of the secondary Ab alone. By contrast, incubation of the anti-pRII{beta} Ab with a nonphosphorylated form of the same peptide did not block the signal (data not shown). These results demonstrate that the pRII{beta} Ab specifically detects endogenous pRII{beta} in the nucleus of Jurkat T cells.

Based on the finding that endogenous pRII{beta} localized, in part, to discrete clusters in the nucleus, we determined the importance of Ser114 phosphorylation in the nuclear localization of RII{beta}. Jurkat T cells were stably transfected with constructs encoding FLAG-tagged wild-type RII{beta} (Jurkat-RII{beta}) or a mutant form of RII{beta} containing a Ser114->Ala mutation (Jurkat-RII{beta}S114A), treated in the absence or presence of calyculin A to enhance RII{beta} phosphorylation, and examined by confocal immunofluorescence microscopy. Calyculin A treatment enhanced pRII{beta} staining in Jurkat-RII{beta} cells and caused an increase in the formation of intranuclear clusters compared with untreated cells (Fig. 2C). A similar pattern of nuclear staining was observed in the Jurkat-RII{beta} cells using a mAb directed against the FLAG epitope to detect the ectopically expressed RII{beta} (Fig. 2C). The comparable staining patterns observed with anti-pRII{beta} and anti-FLAG Abs confirmed that the pRII{beta} mAb was indeed detecting RII{beta} (Fig. 2C). Moreover, the formation of intranuclear clusters by both endogenous pRII{beta} in nontransfected Jurkat cells (Fig. 2B) and the ectopically expressed RII{beta}-FLAG in stably transfected Jurkat-RII{beta} cells (Fig. 2C) confirms that the ectopically expressed RII{beta} localized similarly to endogenous RII{beta}. In contrast to RII{beta}, RII{beta}S114A displayed very weak intranuclear staining both before and following calyculin A treatment (Fig. 2C). Interestingly, the intranuclear clusters of endogenous pRII{beta} were less prominent in the RII{beta}S114A cells compared with the RII{beta} cells (Fig. 2C), suggesting that mutant RII{beta} protein may be interfering with the physiologic localization of endogenous nuclear pRII{beta}. Taken together, these data demonstrated that nuclear pRII{beta} localized to discrete regions within the nucleus, and that phosphorylation of Ser114 is required for this localization.

RII{beta} and RI{alpha} differentially regulate IL-2 promoter activity

Because RII{beta} significantly down-regulates CREB transcriptional activity and c-Fos production in Jurkat T cells (22), we postulated that RII{beta} may subsequently reduce IL-2 expression. As an initial approach, we assessed the effect of RII{beta} on the activation of the IL-2 promoter following stimulation of Jurkat T cells with PMA plus IO. Cells were transiently transfected with plasmids encoding RII{beta}-FLAG and the IL-2-luciferase plasmid containing the luciferase reporter gene under control of the –600 to +51 sequence of the human IL-2 promoter. Following stimulation with PMA plus IO for 6 h, luciferase activity was quantified in cell extracts as a measure of IL-2 promoter activity. RII{beta} inhibited IL-2 promoter activity by 35% (n = 3, p = 0.046) compared with empty vector control (Fig. 3A). As an additional control, we also determined the effect of another PKA-R subunit, RI{alpha}, on IL-2 promoter activity. In sharp contrast to the suppression by RII{beta}, RI{alpha}-FLAG cotransfection increased IL-2 promoter activity >3-fold following stimulation (n = 3, p = 0.018) (Fig. 3A). Neither RII{beta} nor RI{alpha} had any substantial effect on IL-2 promoter activity in the absence of stimulation (data not shown). The contrasting effects of RI{alpha} and RII{beta} on IL-2 promoter activity were even more striking considering that the transient overexpression of RI{alpha}-FLAG was consistently less than that of RII{beta}-FLAG (Fig. 3B).



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  		FIGURE 3. Effect of wild-type and mutant PKA-R subunits on IL-2 promoter activity. A, Jurkat T cells were transiently cotransfected with 10 µg of IL-2-luciferase, 10 µg of empty FLAG (vector), RII{beta}-FLAG (RII{beta}), RII{beta}S114A-FLAG (RII{beta}S114A), or RI{alpha}-FLAG (RI{alpha}). Twenty-four hours posttransfection, cells were treated with 50 ng/ml PMA + 1 µM IO for 6 h. Firefly luciferase activity was determined from cell extracts as described in Materials and Methods, and normalized to Renilla luciferase activity. The average of three independent experiments ± SD is shown. *, p = 0.046 for vector vs RII{beta}; **, p = 0.018 for vector vs RI{alpha}. B, WCE (25 µg) from the treated samples in A was analyzed by SDS-PAGE and immunoblotting with anti-FLAG and anti-{beta}-actin.

 
The different nuclear localization patterns of the wild-type and RII{beta}S114A mutant led us to consider the idea that Ser114-dependent nuclear localization of RII{beta} may be necessary for RII{beta} to inhibit IL-2 promoter activity. If this were so, then RII{beta}S114A would not be expected to inhibit this activity. Although wild-type RII{beta} inhibited promoter induction by 35%, the RII{beta}S114A mutant construct failed to inhibit, and actually modestly increased, IL-2 promoter activity (Fig. 3A). Relative expression of wild-type RII{beta}-FLAG and RII{beta}S114A-FLAG were observed to be approximately equivalent by immunoblotting with anti-FLAG (Fig. 3B). These results demonstrate that RI{alpha} and RII{beta} subunits of PKA differentially affect IL-2 promoter induction, and that the inhibition by RII{beta} is dependent on Ser114 phosphorylation. This suggests that nuclear RII{beta} can modify IL-2 promoter activity in T cells.

Regulation of IL-2 mRNA induction by PKA-R subunits

The inhibition of IL-2 promoter activity in Jurkat T cells by RII{beta} prompted us to investigate whether or not this inhibition was also seen at the level of endogenous IL-2 mRNA synthesis. To address this issue, Jurkat T cells were stably transfected with empty FLAG vector, RI{alpha}-FLAG, RII{beta}-FLAG, or RII{beta}S114A-FLAG expression constructs. Multiple individual subclones of each line were screened for FLAG protein expression by immunoblotting with anti-FLAG. Subcloned lines with similar levels of protein expression were tested for IL-2 mRNA production following treatment with PMA plus IO for 3 h. Semiquantitative PCR was performed using primers specific for IL-2 and the control gene GAPDH. IL-2 mRNA induction was almost completely inhibited in the Jurkat-RII{beta} cell line, whereas IL-2 mRNA content in the Jurkat-RII{beta}S114A line was similar to the vector control line (Fig. 4A). By contrast, the Jurkat-RI{alpha} cell line showed a marked increase in IL-2 transcript compared with the vector control line (Fig. 4A). To quantify the differences in IL-2 mRNA in these lines, real-time PCR was performed using RNA from the cell lines following stimulation. This technique revealed a >90% reduction of IL-2 mRNA in the Jurkat-RII{beta} line compared with the vector control and Jurkat-RII{beta}S114A cells, whereas the enhancement of IL-2 mRNA levels in the Jurkat-RI{alpha} line was >2-fold (Fig. 4B).



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  		FIGURE 4. RII{beta} selectively inhibits IL-2 mRNA induction. A, Jurkat cells stably transfected with empty FLAG vector, RII{beta}-FLAG, RII{beta}S114A-FLAG, or RI{alpha}-FLAG were treated in the absence (–) or presence (+) of 10 ng/ml PMA + 1 µM IO (PMA/IO) for 3 h. IL-2, CD154, and GAPDH mRNA levels were determined by RT-PCR, as described in Materials and Methods. Results shown are representative of at least two independent experiments. B, Jurkat stable transfectants were stimulated as in A, and IL-2 mRNA levels from the PMA/IO treated samples were quantified by real-time PCR as described in Materials and Methods. {diamondsuit}, Vector; {blacksquare}, RII{beta}; {blacktriangleup}, RII{beta}S114A; x, RI{alpha}. C, Jurkat stable transfectants were left untreated (empty) or treated with PMA/IO (filled) as in A, and CD69 cell surface expression was determined as described in Materials and Methods. The percentage of cells positive for CD69 following treatment is indicated in top right corner of each histogram. The fold increase in mean fluorescence intensities following treatment (MFI treated ÷ MFI untreated) are FLAG, 5.4; RII{beta}, 4.4; RII{beta}S114A, 7.3; and RI{alpha}, 7.8. D, Jurkat stable transfectants were cultured in fresh medium for 18 h and stained with either FITC-isotype control (light trace) or anti-CD154-FITC (dark trace) and analyzed by flow cytometry. The percentage of cells positive for CD154 is indicated in top right corner of each histogram.

 
To determine whether inhibition of IL-2 mRNA by RII{beta} was specific or reflected a general down-regulation of gene expression, we examined the induction of CD154 (CD40 ligand) mRNA in the stable transfectants under the same conditions used for IL-2 mRNA induction. In the absence of stimulation, CD154 mRNA was low or absent in the vector and Jurkat-RI{alpha} cells, whereas it was constitutively up-regulated in both the Jurkat-RII{beta} and Jurkat-RII{beta}S114A lines (Fig. 4A). Interestingly, constitutive CD154 transcript levels were much higher in the Jurkat-RII{beta} cell line compared with the Jurkat-RII{beta}S114A line, which suggests that RII{beta} Ser114 phosphorylation may be required for positive regulation of CD154 expression by RII{beta} (Fig. 4A). We also determined the proportion of cells constitutively expressing cell surface CD154 by flow cytometry, and found that 33% of Jurkat-RII{beta} cells expressed surface CD154 in the absence of stimulation compared with <5% for the other stably transfected cell lines (Fig. 4D). Following stimulation, CD154 transcript was up-regulated in all four cell lines (Fig. 4A). These results show that, in contrast to its effect on IL-2 mRNA induction, RII{beta} up-regulates CD154 transcript levels in a Ser114 phosphorylation-dependent fashion.

To determine the capacity of each cell type to express an activated phenotype, CD69 cell surface expression was quantified following stimulation with PMA plus IO. After 3 h of stimulation, CD69 expression was identified on ≥97% of cells of each line (Fig. 4C). In addition, cell surface expression of CD3-{epsilon} was found to be equivalent among the four cell lines both before and after stimulation, verifying that overexpression of the PKA-R subunits did not alter CD3-{epsilon} expression (data not shown). Taken together, these results indicate that pRII{beta} does not universally down-regulate gene expression; instead, pRII{beta} down-regulates IL-2 while up-regulating CD154 levels following activation.

RII{beta} inhibits IL-2 protein production in Jurkat T cells

To determine whether RII{beta} down-regulation of the IL-2 promoter and IL-2 mRNA inhibits endogenous IL-2 synthesis, the four stably transfected Jurkat T cell lines were tested for IL-2 secretion by ELISA following stimulation with PMA plus IO for 6 h. Unstimulated cells routinely produced very low or undetectable levels of IL-2 in all four cell lines (data not shown). Compared with the vector control line, cells stably overexpressing RII{beta} showed an 89% reduction in IL-2 secretion (1903 pg/ml ± 106 vs 218 pg/ml ± 21; n = 3, p = 0.0009), whereas cells overexpressing RII{beta}S114A inhibited IL-2 production by only 18% (1903 pg/ml ± 106 vs 1552 pg/ml ± 270; n = 3, p = 0.2) (Fig. 5A). By contrast, IL-2 production in the RI{alpha} stable transfectants was 3.6-fold higher than the vector control cells (1903 pg/ml ± 106 vs 6861 pg/ml ± 243; n = 3, p = 0.0009) (Fig. 5A). Similar results were obtained from multiple independently derived subclones of each transfected cell type (data not shown). Anti-FLAG immunoblot analysis of lysates from the four lines used in Fig. 5A revealed that the Jurkat-RII{beta} and Jurkat-RII{beta}S114A cells expressed similar levels of ectopic FLAG-tagged protein, whereas RI{alpha}-FLAG expression was ~50% lower than that of the RII{beta} lines (Fig. 5B, FLAG). These results demonstrated that RI{alpha} and RII{beta} have opposite effects on the production of endogenous IL-2 by Jurkat T cells following activation, and that Ser114 phosphorylation was a critical event for the effective inhibition of IL-2 by RII{beta}.



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  		FIGURE 5. Effect of PKA-R subunits on IL-2 secretion in Jurkat T cells. A, Jurkat T cells stably transfected with either empty FLAG vector, RII{beta}-FLAG, RII{beta}S114A-FLAG, or RI{alpha}-FLAG were cultured at 2 x 106 cells/ml in the presence of 10 ng/ml PMA + 1 µM IO for 6 h. Cell-free supernatants were tested for IL-2 production by ELISA. The average of three independent experiments ± SD is shown. *, p < 0.001 for vector vs RII{beta} and vector vs RI{alpha}. B, WCE (20 µg) from each stable Jurkat cell line was resolved on a 10% SDS-PAGE gel and immunoblotted with the Abs indicated on the left of the figure. The anti-RI mAb recognizes both RI{alpha} and RI{beta} isoforms, and the relative position of each is indicated with the appropriate symbol and arrow. Arrows with asterisks point to the ectopically expressed proteins that migrate slower than the endogenous proteins due to ~3-kDa FLAG epitope. One representative of at least two independent experiments for each blot is shown.

 
Using the anti-pRII{beta} mAb, we analyzed pRII{beta} levels in the stably transfected cell lines by immunoblotting. Endogenous pRII{beta} ran as a pair of bands in all four cell lines (Fig. 5B). The RII{beta}-FLAG appeared as a slower-migrating band compared with the endogenous protein due to the presence of the ~3-kDa FLAG epitope (Fig. 5B, pRII{beta}). The RII{beta}S114A mutant was not detected by the anti-pRII{beta} Ab, but the amount of endogenous pRII{beta} in these cells was comparable to the other lines (Fig. 5B, pRII{beta}). These results indicate that both endogenous and ectopically expressed RII{beta} were constitutively phosphorylated in Jurkat T cells, and that the RII{beta} Ser114->Ala mutation present in the RII{beta}S114A cell line prevented this phosphorylation in vivo.

Previous reports have shown that alterations in the relative expression of PKA-R and -C subunits can lead to compensatory changes in the levels of other PKA subunits (28, 29, 30). To address this possibility in the stably transfected Jurkat T cell lines, we examined the levels of PKA subunit proteins by immunoblotting. The amount of C subunit was constant between the four cell lines, indicating that the differential ability of these lines to produce IL-2 was not due to a difference in C subunit protein content (Fig. 5B, PKA-C). Immunoblotting with anti-RII{beta} showed that the Jurkat-RII{beta} and Jurkat-RII{beta}S114A stable lines expressed ~2-fold more RII{beta} protein compared with endogenous RII{beta} (Fig. 5B, RII{beta}). Immunoblotting with anti-RI Ab, which recognizes both RI{alpha} and RI{beta} isoforms, revealed no differences in the amounts of endogenous RI protein in the four cell lines (Fig. 5B, RI). However, it is notable that the amount of ectopically expressed RI{alpha} in the Jurkat-RI{alpha} line was very small compared with endogenous RI{alpha} (Fig. 5B, RI). These results demonstrate that small perturbations in the amounts of PKA-R subunits in Jurkat T cells significantly affected IL-2 production (Fig. 5B, RI).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There has been a long-standing question about the mechanism by which cAMP inhibits IL-2 production and T cell proliferation (1, 2, 3, 4). One mechanism may be via binding of cAMP to R subunits and activation of PKA. Because SLE T cells underproduce IL-2 in response to activation (31, 32, 33), and reduced PKA-II activity is associated with increased amounts of nuclear RII{beta} subunit (18), we recently undertook experiments to understand the biologic significance of nuclear RII{beta} in T cells. These results demonstrated that nuclear RII{beta} can interact with and inhibit CREB transcriptional activity, as well as inhibit c-fos promoter activity and c-Fos protein production in activated T cells (22). Both Fos and CREB are members of the basic leucine zipper family of transcription factors and are integral components of the transactivating complexes at the IL-2 promoter in T cells (24, 34). In CREBS119A transgenic mice, loss of T cell CREB activity leads to a substantial reduction in IL-2 production in activated T cells, highlighting the importance of CREB in regulating IL-2 expression (23). Therefore, we tested the hypothesis that nuclear RII{beta} regulates IL-2 transcription and IL-2 production.

Our results demonstrated four substantive findings. First, a portion of endogenous RII{beta} was constitutively phosphorylated at Ser114 in human primary and Jurkat T lymphocytes. Second, pRII{beta} localized within the nucleus. Third, RII{beta} strongly down-regulated IL-2 promoter activity, IL-2 mRNA, and IL-2 protein production, whereas, RI{alpha}, a subunit of the PKA-I isozyme, up-regulated IL-2 expression and IL-2 production. Fourth, the capacity of RII{beta} to repress IL-2 expression and IL-2 production was dependent on Ser114 phosphorylation.

In vitro experiments performed nearly three decades ago using partially purified RII proteins (i.e., both RII{alpha} and RII{beta}) found that the subunits were phosphorylatable by PKA-C subunit, and that this phosphorylation facilitated dissociation of the PKA holoenzyme into free R and C subunits (20). As a result, the residue in RII{alpha} (Ser99) and the homologous amino acid in RII{beta} (Ser114) were labeled autophosphorylation sites. Yet, to date, very little is known about how RII subunit phosphorylation is regulated in vivo or the significance of this event in cellular homeostasis in general and, in particular, in the T cell. To our knowledge, this report is the first identification of RII{beta} Ser114 phosphorylation in vivo. Using anti-pRII{beta} mAb, we demonstrated that RII{beta} is constitutively phosphorylated in primary human T lymphocytes as well as in lymphocyte cell lines. In addition, treatment with the PP inhibitor calyculin A markedly increased pRII{beta} in T cells. Thus, in both cycling and quiescent cells, RII{beta} may be continuously undergoing phosphorylation and dephosphorylation, and that dephosphorylation is mediated, in part, by PP1.

Confocal immunofluorescence microscopy revealed that pRII{beta} was localized predominantly to the nucleus and the Golgi-centrosome region in T cells. The attachment of RII subunits to the centrosome has been previously identified for both RII{alpha} and RII{beta} in nonhemopoietic cells, and is known to be mediated via AKAPs (35, 36). Although RII{alpha} and RII{beta} have been identified in the nucleus of a wide range of primary and transformed cell lines under a variety of conditions (14, 15, 16, 17), the mechanisms that potentiate RII nuclear localization are incompletely understood. Current data suggest that both the Ser114 and the putative nuclear localization sequence are integral sites for RII{beta} nuclear localization because mutation of either site blocks nuclear RII{beta} accumulation (19). Although our experiments did not address regulation of Ser114 phosphorylation in vivo, two key findings support the idea that RII{beta} Ser114 phosphorylation might mediate RII{beta} import to the nucleus in the T cell. First, constitutive RII{beta} Ser114 phosphorylation was identified in the nuclei of Jurkat and primary T cells. The extent of nuclear pRII{beta} was enhanced following treatment of cells with calyculin A, suggesting that Ser114 phosphorylation may promote nuclear translocation of RII{beta}. Second, the mutant, nonphosphorylatable RII{beta}S114A was nearly absent from the interior of the nucleus compared with wild-type RII{beta}, which could be seen as a ring around the periphery of the nucleus, as well as in clusters of variable size and intensity of staining within the nucleus. Both wild-type and RII{beta}S114A appeared to localize to the perinuclear region, although it is unclear at this time whether the proteins are associated with nuclear structures such as the nuclear envelope. So, while Ser114 is required for intranuclear localization of RII{beta}, it is currently not known how Ser114 phosphorylation affects localization to other regions of the nucleus or cell. Importantly, we also detected intranuclear clusters of RII{beta} with anti-FLAG Ab in the Jurkat-RII{beta} cell line, confirming that the pRII{beta} mAb is specifically recognizing RII{beta}, and that mutant RII{beta}S114A prevents this pattern of localization. Although the biologic significance of peripheral and clustered pRII{beta} in T cells remains to be established, these results suggest that RII{beta} is targeted to specific areas in the nucleus in a Ser114-dependent manner.

Established evidence suggests that the PKA-II isozyme may mediate the inhibition of T cell proliferation (37, 38). In this study, we present evidence that overexpression of the RII{beta} subunit of the PKA-II isozyme inhibits IL-2 production. Because IL-2 is a pivotal regulator of T cell proliferation, the inhibition of IL-2 by RII{beta} further supports the early concept that the PKA-II isozyme mediates inhibition of T cell proliferation (37, 38). Using a stably transfected wild-type Jurkat-RII{beta} cell line, we demonstrated profound suppression of IL-2 mRNA induction and IL-2 protein secretion following activation. Of central importance to this inhibitory effect on proliferation was RII{beta} phosphorylation at Ser114. Substitution of phosphorylation-defective Jurkat-RII{beta}S114A mutant cells, where the Ser114->Ala prevents phosphorylation, resulted in failure to inhibit IL-2 expression and IL-2 synthesis in the same experiments. Although Budillon et al. (19) first showed that phosphorylation of nuclear RII{beta} was required for differentiation of transformed fibroblasts, our experiments provide a novel role for RII{beta} Ser114 phosphorylation in the regulation of a cytokine that mediates T cell effector functions. Importantly, the presence of constitutive nuclear pRII{beta} in primary human T cells raises the possibility that this subunit may function in the regulation of gene expression.

The precise mechanism by which RII{beta} down-regulates IL-2 expression remains to be established. One potential means is by inhibiting trans-acting factors at the IL-2 promoter. Accumulating evidence suggests that this assembly of multifactor transcriptional complexes at the IL-2 promoter is a key step in the induction of IL-2 transcription (39). CREB and AP-1 have been identified as integral components of the transcriptional apparatus that assemble on the proximal IL-2 promoter to initiate transcription, particularly in the –180 and –150 (CD28RE) regions (34, 39). Our recent data demonstrating the suppression of CREB activity and c-Fos protein production in Jurkat T cells (22) supports a model where RII{beta} might inhibit IL-2 expression by affecting the capacity of these factors to mediate IL-2 transcription. However, we would point out that partial inhibition (35%) of IL-2 promoter activity by RII{beta} does not seem to explain the nearly complete inhibition of endogenous IL-2 mRNA and IL-2 protein by RII{beta}, and may indicate that RII{beta} is affecting IL-2 transcription via distal cis-elements not contained in the promoter construct used, or that RII{beta} may be regulating posttranscriptional events important for IL-2 production. Another potential mechanism is the substitution of a phosphorylated transcription factor, cAMP response modulator, for CREB at the –180 site of the IL-2 promoter. Phosphorylated cAMP response modulator partially replaces CREB at the –180 site in T cell nuclei of some SLE subjects, forms a multimolecular complex with CREB binding protein and p300, and down-regulates IL-2 transcription and IL-2 production (33). Whether or not both potential mechanisms might be operative simultaneously and pRII{beta} contributes to formation of this multimolecular complex has not been determined. Nevertheless, it also seems reasonable to consider that RII{beta} may affect other, as-yet-unidentified steps in the induction of IL-2 transcription in addition to its effect on the promoter.

Given that pRII{beta} caused such profound inhibition of IL-2 generation, the question arose whether or not overexpression of the subunit might be causing a generalized down-regulation of gene expression in activated T cells. This was not the case. In fact, CD154 transcript was constitutively up-regulated in Jurkat-RII{beta} cells compared with either vector controls or Jurkat-RII{beta}S114A cells, and could be further increased by activation. Moreover, constitutive up-regulation of CD154 transcript was associated with an increased proportion of cells bearing cell surface CD154 protein. Furthermore, activation of all four cell lines induced nearly 100% of cells to express the surface activation marker, CD69, indicating that overexpression of RII{beta} and its subsequent Ser114 phosphorylation did not universally inhibit T cell gene expression following activation.

Although PKA-I is activated in response to TCR ligation (40, 41) and has been implicated in the regulation of TCR-dependent signaling events (42, 43), the extent that RI{alpha} overexpression augmented IL-2 production in activated Jurkat T cells was surprising. It is possible that the increased RI{alpha} levels may inhibit free C subunit activity at the plasma membrane and thus suppress the previously described inhibitory effect of PKA-I-mediated phosphorylation on T cell signaling (42). However, the observation that RI{alpha} overexpression enhances IL-2 following stimulation with PMA plus IO suggests that RI{alpha} may be affecting signaling events downstream of the TCR. At present, however, the mechanism by which stimulated Jurkat T cells overexpressing RI{alpha} produce high levels of IL-2 remains to be determined. To our knowledge, this is the first report demonstrating the potent effect of RI{alpha} overexpression on IL-2, although recent work from our lab has shown that overexpression of the PKA-RI{beta} subunit in SLE T cells leads to a partial reconstitution of IL-2 production following activation in these cells (44).

The finding that RI{alpha} augments IL-2 production in Jurkat T cells demonstrates two salient points. First, the suppressive effect of RII{beta} on IL-2 expression is specific and does not reflect a general property of PKA-R subunits. Second, PKA can mediate variable, and in some cases, opposing signals in T cells. Thus, the diametric IL-2 responses underscore the divergent roles of the RI{alpha} and RII{beta} subunits, and raise the possibility that the expression of specific genes can be modified by modulating PKA-I or PKA-II isozymes.

The findings reported in this study may contribute to a better understanding of the role of PKA dysregulation in the immunopathogenesis of SLE. Because T cells from some SLE patients exhibit significantly elevated levels of the RII{beta} in the nucleus, and because we have found a correlation between nuclear RII{beta} and suppression of IL-2, it is possible that deficient IL-2 production seen in SLE (31, 32) may be caused, at least in part, by elevated levels of nuclear RII{beta}. Furthermore, the expression of CD154 and cell surface CD154, which mediates B cell proliferation and Ab production, was enhanced by nuclear RII{beta}. In SLE T cells, basal cell surface expression of CD154 is elevated compared with normal and disease controls, and may contribute to aberrant Ig production (45). The enhanced baseline level of CD154 by RII{beta} in Jurkat T cells raises a question of whether nuclear RII{beta} might be involved in this process. Finally, a deficiency of T cell PKA-I activity has been identified in 80% of patients with SLE and is associated with decreased expression of RI subunits (46, 47). The finding that overexpression of RI{alpha} causes a significant increase in IL-2 production in activated Jurkat T cells raises the possibility that RI is a positive regulator of IL-2 production and thus the deficiency of RI subunits in SLE T cells may contribute to an impairment in IL-2 synthesis. Although in this study we have demonstrated the role for RI and RII subunits in regulating IL-2 production in Jurkat T cells, it will be important to determine whether the altered levels and localization patterns of PKA-R subunits in SLE T cells affects these processes. Overall, these results represent a promising avenue of inquiry for understanding the importance of PKA in regulating normal and aberrant T cell responses.


   Acknowledgments
 
We thank Teresa Call for technical assistance with the immunocytochemistry experiments, Ken Grant of the Microscopy Core Laboratory for assistance with the confocal microscope, and Natalie Walker of the Hematology Laboratory for assistance with the flow cytometry analysis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI46526 (to G.M.K.), AI42782, and AI49954 (to G.C.T.), the Diane Pulliam Scales Lupus Research Memorial Fund, and the Lupus Foundation of America, Winston-Triad Lupus Chapter, North Carolina Lupus Foundation, Inc. M.R.E. was supported in part by the National Institutes of Health National Research Training Grant AI07401. Back

2 Address correspondence and reprint requests to Dr. Gary M. Kammer, Section on Rheumatology and Clinical Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: gmkammer{at}hotmail.com Back

3 Abbreviations used in this paper: PKA, protein kinase A; AKAP, A kinase anchoring protein; C, catalytic; IO, ionomycin; PKA-I, PKA type I; PKA-II, PKA type II; PP, protein phosphatase; pRII{beta}, phosphorylated Ser114 RII{beta}; R, regulatory; SLE, systemic lupus erythematosus; WCE, whole cell extract. Back

Received for publication January 12, 2004. Accepted for publication April 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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