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(Journal of Leukocyte Biology. 2002;72:1027-1036.)
© 2002 by Society for Leukocyte Biology

The A2A receptor mediates an endogenous regulatory pathway of cytokine expression in THP-1 cells

Khaled Bshesh*, Bin Zhao*, Donn Spight*, Italo Biaggioni{dagger}, Igor Feokistov{dagger}, Alvin Denenberg*, Hector R. Wong* and Thomas P. Shanley*

* Division of Critical Care Medicine, Children’s Hospital Medical Center and Children’s Hospital Research Foundation, Cincinnati, Ohio; and
{dagger} Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee

Correspondence: Thomas P. Shanley, M.D., Division of Critical Care Medicine, OSB-5, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039. E-mail: shant0{at}chmcc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine is an endogenous nucleoside that regulates numerous cellular functions including inflammation. Adenosine acts via cell-surface receptors subtyped as A1, A2A, A2B, and A3. The A2A receptor (A2AR) has been linked to anti-inflammatory effects of adenosine. Furthermore, microarray analysis revealed increased A2AR mRNA in lipopolysaccharide (LPS)-stimulated monocytes. We hypothesized that endogenous adenosine inhibited LPS-mediated tumor necrosis factor (TNF) production via A2AR stimulation. Using THP-1 cells, our results demonstrated that LPS increased expression of cellular A2AR and adenosine. A2AR agonism with 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine (CGS 21680) after LPS decreased TNF production in a dose- and time-dependent manner, whereas A2AR antagonism significantly increased TNF and blocked the inhibitory effect of CGS 21680. This inhibitory pathway involved A2AR stimulation of cyclic adenosine monophosphate (cAMP) to activate protein kinase A, resulting in phosphorylation of cAMP response element-binding protein (CREB). Phospho-CREB had been shown to inhibit nuclear factor-{kappa}B transcriptional activity, as was observed with CGS 21680 treatment. Thus, following immune activation with LPS, endogenous adenosine mediates a negative feedback pathway to modulate cytokine expression in THP-1 cells.

Key Words: adenosine • TNF • CREB • NF-{kappa}B


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of cells derived from the monocyte lineage, including macrophages, is felt to be a key initiating event in the development of acute inflammatory disease states. For example, endotoxin activation of circulating monocytes results in the expression of a number of proinflammatory genes [e.g., tumor necrosis factor (TNF)] that participate in the cascade of mediators causing the clinical signs associated with sepsis and septic shock. Our laboratory has been interested in identifying endogenous host factors that may serve to negatively modulate this response. Previous investigations had described the ability of some adenosine analogues to negatively modulate the cytokine response to a number of inflammatory stimuli including endotoxin [1 2 3 ]. Investigators have also demonstrated that the administration of adenosine [4 ] or the inhibition of adenosine deaminase [5 ] could reduce in vivo inflammatory responses to endotoxin or hemorrhagic shock. Together, these studies suggested that adenosine might mediate anti-inflammatory effects.

Adenosine is an endogenous nucleoside that is ubiquitously produced and regulates a number of physiological properties. Adenosine is produced during metabolism of adenosine phosphates. Normally, adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate are metabolized to adenosine 5'-monophosphate (AMP), and in the presence of adequate cellular energy, AMP is reconverted to phosphates. However, in the absence of sufficient energy supplies or in the context of cellular stress, the 5'-nucleotidase converts AMP to adenosine [6 ]. The release of adenosine can result in a variety of physiologic sequelae, depending on which adenosine receptor is engaged on the cell surface.

Following the cloning of adenosine receptors over the past decade, investigators have gained significant insight into their role in mediating various adenosine effects. To date, four adenosine receptors have been identified by pharmacological and molecular studies. These receptors are subtyped as the A1, A2A, A2B, and A3 receptors (A1R, A2AR, A2BR, and A3R, respectively), each of which has been ascribed a number of cellular physiologic properties (reviewed ref. [7 ]). These receptors associate with G-protein signaling pathways: A1 and A3, with the inhibitory G-protein GI and A2A and A2B, with the stimulatory G-protein GS. Thus, A1R and A3R are negatively coupled to adenylate cyclase with stimulation resulting in decreased cyclic adenosine monophosphate (cAMP). Conversely, A2AR and A2BR are positively coupled to adenylate cyclase so that stimulation increases cAMP levels [8 , 9 ]. Elevations in cAMP can in turn activate protein kinase A (PKA), resulting in the phosphorylation of the cAMP response element-binding protein (CREB) [10 ], with possible downstream effects on additional signal transduction pathways including nuclear factor (NF)-{kappa}B [11 ].

The A2AR is ubiquitously distributed and activated at submicromolar concentrations of adenosine with major implications on immunologic functions. The development of high-affinity agonists and antagonists has assisted with the delineation of a number of functions mediated by the A2AR. Such agonists include: 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl carboxyaminoadenosine (CGS 21680), which was used in the current studies, and N6-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl)adenosine [12 ]. As several lines of research suggested that adenosine may mediate anti-inflammatory effects via the A2AR, we sought to define its role in modulating endotoxin-induced TNF expression in monocytes and to identify the signaling mechanism by which this occurred. The results presented support the concept that endogenously produced adenosine mediates a negative feedback loop modulating lipopolysaccharide (LPS)-induced TNF expression via the A2AR in THP-1 cells. Importantly, these inhibitory effects were observed even when A2AR agonism was achieved up to 8 h after LPS stimulation. This regulatory pathway appeared to involve cAMP activation of PKA with increased phosphorylation of CREB and subsequent modulation of NF-{kappa}B-driven transcription. These findings demonstrate the potential of using adenosine receptor signaling as a therapeutic target in some inflammatory disease states.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
To model human monocytes, the human monocytic leukemia cell line THP-1 was used (American Type Culture Collection, Manassas, VA). Cells were cultured in RPMI-1640 media containing 10% heat-inactivated fetal bovine serum and kanamycin. Cells were stimulated with 1 µg/ml media of LPS (Esherichia coli, serotype O55:B5; Sigma Chemical Co., St. Louis, MO) in all experiments, with the exception of transient transfection studies in which 10 µg/ml media was used.

Pharmacologic agents
CGS 21680, 8-(3-chlorostyryl)caffeine (CSC), Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS), and 8-(chlorophenylthio) cAMP were purchased from RBI Signaling Innovation (Natick, MA). 3-Isobutyl-8-pyrrolidinoxanthine (IPDX) was provided by I. Biaggioni (Vanderbilt University, Nashville, TN) and was used at a concentration of 10 µM, a dose previously shown to antagonize the A2BR but not the A2AR [13 , 14 ].

Flow cytometry for cellular expression of A2A
THP-1 cells were centrifuged and resuspended in media at 1 x 106 cells/ml and then differentiated using interferon-{gamma} (IFN-{gamma}; 10 µg/ml) at 37°C for 3 h to allow for optimal endotoxin responsiveness as previously reported [15 , 16 ]. Cells were then centrifuged (1200 rpm), resuspended in fresh media, and plated in 24-well plates. Control cells were left unstimulated, and the rest were stimulated with LPS (1 µg/ml). Cells were harvested at the time points indicated and fixed with 10% buffered formalin and stored at -70°C overnight. After thawing, the cells were treated with 10 mM EDTA in phosphate-buffered saline (PBS) and collected in Falcon 2050 tubes (Becton Dickinson, Franklin Lakes, NJ) with fluorescein-activated cell sorter (FACS) buffer solution [1x PBS, 1 gm bovine serum albumin (BSA), and 0.5% sodium azide]. Cells were centrifuged (1200 rpm, 5 min) at 4°C and then resuspended in 0.5 ml FACS buffer solution in new tubes. Cells were stained with 100 µl mouse monoclonal anti-A2AR antibody (1 µg/ml; R&D Systems, Minneapolis, MN) in FACS buffer or isotype-matched anti-immunoglobulin G (IgG)2a antibody (1 µg/ml; R&D Systems) and were incubated on ice for 1 h. Cells were washed with 2x with 1 ml FACS buffer and centrifuged (1200 rpm, 10 min, 4°C). Secondary fluorescein isothiocyanate (FITC)-conjugated goat antimouse antibody (Sigma Chemical Co.) at 1:100 dilution was added, and cells were incubated on ice for 1 h and were then centrifuged, washed twice, and resuspended in 0.2 ml FACS buffer. Cell staining, recorded as mean channel fluorescence (MCF) of A2A or isotype control, was measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). This methodology of fixation, storage, and staining likely identifies cell surface and intracellular A2AR protein.

Measurement of adenosine receptor mRNA
Total RNA was isolated using Trizol reagent followed by isopropanol precipitation as previously reported [17 ]. A human adenosine receptor gene expression array was custom-designed by Super Array, Inc. (Bethesda, MD) and performed according to the manufacturer’s instructions. In brief, gene-specific [32P]-labeled cDNA probes were generated from 5 to 10 µg total RNA using a gene-specific set of primers for reverse transcription. The cDNA probes were then hybridized with gene-specific cDNA fragments spotted on nylon membrane. The relative expression level of each gene was analyzed using a phosphoimager and ImageQuantTM software (Molecular Dynamics, Sunnyvale, CA) and normalized to ß-actin expression. Values are expressed as mean densitometry ± SEM relative to ß-actin.

Adenosine measurement
THP-1 cells were treated as described above. Adenosine measurements were performed as previously reported with minor modifications [18 , 19 ]. Briefly, all sample preparation steps were carried out at 4°C. Cells in suspension (80 million in 40 ml media) were transferred to a tube containing erythro-9(2-hydroxy non-3yl)-adenine (EHNA; A.G. Scientific, Inc., San Diego, CA), dipyridamole (Sigma Chemical Co.), and sodium orthovanadate (Sigma Chemical Co.), and the final concentrations were 5 µM, 0.25 µM, and 100 µM, respectively. The cell suspension with the above-added inhibitors was centrifuged for 3 min at 1500 rpm at 4°C. The media was discarded, and the pellet was deproteinized in 1 ml perchloric acid (0.6 N) with sonication for 10 s at speed 5 (Virsonic 475 membrane disruptor, Virtis Instruments Co., Gardiner, NY). After 60 min on ice, the deproteinized cell pellet was treated (to neutralize the supernate and precipitate all proteins) with 0.45 ml K2HPO4 (1.0 M at pH 12), centrifuged at 10,000 G for 10 min at 4°C, and filtered through a 0.2-µm syringe filter, and the supernatant was stored at -70°C for adenosine analysis. Adenosine levels were measured on a Beckman Model 126 high-pressure liquid chromatography (HPLC) system (Beckman-Coulter Instruments, Fullerton, CA). The supernatant (100 µl) was injected into a µ-Bondapak-C-18 column (Waters Assoc., Milford, MA) with a mobile phase of 0.1 M NH4H2PO4 at pH 4.5 and a linear gradient of 0–10% methanol over 45 min at a flow rate of 1 ml/min and detection at 254 nM. Sample peak areas were compared with standards, and results were expressed as pmol/mg cell protein. Protein content was measured using the Bradford assay (Bio-Rad Inc., Hercules, CA).

Measurement of TNF bioactivity
Cell-culture supernatants were removed, centrifuged (6000 rpm, 5 min), and stored at -70°C until evaluated for TNF activity using a standard WEHI cell cytotoxicity assay as previously reported [20 ].

Western blot analysis
Nuclear cell extracts were subjected to sodium dodecyl sulfate (10%)/polyacrylamide gel electrophoresis. The separated proteins were transblotted to nitrocellulose membrane (0.45 mM; Novex, San Diego, CA) for 2 h at 30 V. After transfer, the membranes were blocked with 20 mM Tris-HCL, pH 7.5, 500 mM NaCl, and 0.1% Tween 20 (T-TBS) containing 5% dry milk for 2 h at room temperature. Blots were incubated overnight with the primary antibodies: antiphospho-CREB-1 (sc-7978; Santa Cruz Biotechnology, Santa Cruz, CA) and antiphospho-p38 and antiphospho-extracellular regulated kinase (ERK)1/2 (both BioSource International, Camarillo, CA) in T-TBS with 5% BSA (1:2–500 dilution). After washing with T-TBS, secondary rabbit antigoat IgG (p-CREB) or goat antirabbit (p-p38 and p-ERK1/2) was added (1:5000 dilution) and incubated for 60 min. Blots were washed in T-TBS twice over 30 min, incubated in commercial enhanced chemiluminesence reagents (Amersham, Buckinghamshire, UK), and exposed to Kodak X-OMAT-AR photographic film (Kodak, Rochester, NY). Relative densitometry of the resultant bands was measured using ImageQuant 1.2 (Molecular Dynamics). All raw densitometric values were standardized to the value obtained from the band signal of control cells (assigned the value=1). Statistical analysis was performed on mean values ± SEM that were reported as relative to the change from control.

Determination of NF-{kappa}B transcriptional activity
To determine the transcriptional activity of NF-{kappa}B, transient transfections of THP-1 cells were performed using luciferase-linked, tandem NF-{kappa}B promoter construct 3X{kappa}B-Luc [21 ], kindly provided by Dr. Roland Schmid (University of Ulm, Germany) as previously reported [15 , 22 ]. Briefly, cells were centrifuged (1100 rpm, 10 min) and washed once in warmed saline-tris-borate solution (STBS) buffer. Cells were then resuspended in STBS buffer in a concentration of 2 x 106/ml followed by the addition of 10 µg/ml diethylaminoethyl dextran (Sigma Chemical Co.). Cells were separated into 6 ml aliquots, and 6 µg 3X{kappa}B-Luc or empty vector DNA constructs with a ß-gal-Luc reporter construct were added. Cell/dextran/DNA mixes were incubated at 37°C for 10–15 min until trypan blue inclusion reached approximately 20%. At this time, transfected cells were centrifuged (1100 rpm, 10 min), washed once with warmed STBS, and then resuspended in 10 ml fresh RPMI media and placed in T25 flasks (48 h, 37°C, 5% CO2) prior to being subjected to experimental conditions. For transient transfection assays, as no differentiation step occurred, cells were stimulated with 10 µg/ml LPS as previously reported [22 ]. Pretreatment with indicated agents was for 30 min prior to LPS stimulation. Cell lysates were harvested 4 h after LPS stimulation in 100 µl luciferase lysis buffer (Promega, Madison, WI), subjected to a freeze-thaw cycle, and centrifuged (4000 rpm, 5 min) prior to measuring luciferase activity according to the manufacturer’s instructions (Promega) using a Berthold AutoLumat LB953 luminometer. Luciferase activity is reported as light units corrected for total cellular protein. To determine nuclear translocation and DNA binding of NF-{kappa}B, electrophoretic mobility shift assays (EMSA) were performed as previously reported [17 ].

Statistics
All values were expressed as mean ± SEM. Individual group means were then compared with Student’s two-tailed t-test or ANOVA as indicated. Significance was defined by P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LPS increases cellular expression of A2AR
To determine if the previous observation of increased A2AR mRNA [23 ] was associated with increased expression of A2AR protein, FACS analysis was performed as described above. A representative FACS histogram is shown in Figure 1A and demonstrated the increase in FITC-based signal with the anti-A2AR antibody when compared with the IgG isotype control antibody. Figure 1B is a representative graph of three separate experiments showing the increase in MCF for A2AR expression versus time. In this experiment, a significant increase in A2AR expression was seen by 2 h after LPS stimulation and remained increased up to 24 h. The maximum increase was seen 8 h after LPS. Figure 1C displays the summation of mean data points from all three experiments expressed as a fold-change from the baseline expression of unstimulated cells. As an average, increases in A2AR expression were observed from 2 to 24 h after LPS stimulation with peak increase seen at 4 h; i.e., this time point was chosen for subsequent post-stimulation experiments. It should be noted that IFN-{gamma} treatment (3 h) is required to differentiate the THP-1 cells to allow for a heightened responsiveness to LPS stimulation [15 ]. As shown in Figure 1B , very modest increases in A2AR were seen during the differentiation stage in some experiments; however, on average, no increase above baseline expression was seen (Fig. 1C) , and no effect on A2AR expression from IFN-{gamma} treatment alone was ever observed beyond 4 h (data not shown).



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  		Figure 1. (A) Representative histogram of fluorescent emission from A2AR-labeled or isotype-matched IgG-labeled THP-1 cells. (B) Representative graph (n=3) of the effect of IFN-{gamma} differentiation and subsequent LPS stimulation on expression of A2AR over time as measured by MCF determined by flow cytometry. Data points are mean ± SEM MCF for n = 6 per time point.*, P < 0.05; **, P < 0.01 compared with 3-h time point (pre-LPS baseline). (C) Graphic depiction of the average fold-change from baseline expression of A2AR from three separate experiments. (D) Average fold-change expression of mRNA for A2A, A2B, and A3 as compared with baseline culture conditions (Culture). Cells were harvested for mRNA extraction following differentiation (IFN) and 4 h after stimulation with LPS (1 µg/ml media) or PBS. Values are expressed as mean relative densitometry as a percent of ß-actin mRNA expression.

 
Gene expression results indicated that the THP-1 cells preferentially express mRNA encoding A2AR. As a percentage of ß-actin expression, THP-1 cells under culture conditions expressed levels of A2AR, A2BR, and A3R of 1.5 ± 0.09, 0.38 ± 0.04, and 0.40 ± 0.03%, respectively (Fig. 1D) . Cells harvested immediately after differentiation did not demonstrate a significant increase in the level of expression for any of the receptors. However, 4 h after LPS stimulation (1 µg/ml), a significant increase in A2AR mRNA was observed (to 3.5±0.15%) as compared with PBS-stimulated cells (2.0±0.05; Fig. 1D ). In contrast, no increase in A2BR nor A3R mRNA was observed after LPS stimulation (Fig. 1D) . Therefore, the ratio of expression of A2A:A2B:A3 was approximately 10:1:1. These results confirmed that the increase in mRNA for the A2AR in LPS-stimulated monocytes similar to data previously reported [23 ] was associated with increased protein expression of the gene product.

LPS stimulation increases adenosine
In these studies, adenosine from LPS-stimulated THP-1 cells was measured by HPLC. As shown in Table 1 , significant increases in adenosine were found at 4 h following LPS stimulation. These determinations were performed in the presence of adenosine deaminase and adenosine reuptake inhibition to prevent the rapid degradation of adenosine in cell culture conditions [24 ]. Mean data from three separate experiments (n=3 samples per condition per experiment) are shown. The average increase in adenosine from LPS-stimulated cells was 5.5-fold when compared with unstimulated control cells (Table 1) . Together, the A2AR and adenosine data supported the concept that in monocyte-derived cells, stimulation with LPS increased endogenously produced adenosine as well as expression of the A2AR. We next designed studies to examine whether these mediated a regulatory or negative-feedback pathway-modulating cytokine (TNF) production.


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  		Table 1. Adenosine Levels Measured from THP-1 Cells 4 h Following LPS Stimulation

 
A2AR stimulation decreases LPS-induced TNF production in a dose- and time-dependent manner
As on average, maximal A2AR expression was seen 4 h after LPS stimulation, we designed experiments to examine the effect of varying doses of the A2AR agonist CGS 21680 on subsequent TNF production after LPS stimulation. As shown in a representative experiment (Fig. 2 ), CGS 21680, added to the cells 4 h after LPS, significantly attenuated TNF production at 24 h in a dose-dependent manner, and near maximal inhibition was achieved at 10 µM when compared with vehicle [dimethyl sulfoxide (DMSO)]-pretreated cells. As shown in Figure 2A , DMSO at percent concentrations (vol:vol) corresponding to each dose of CGS 21680 tested, significantly decreased LPS-induced TNF production only at the highest dose tested (1%). However, a CGS 21680 effect beyond this DMSO effect was observed at all doses except 1 µM. Importantly, this effect of A2A agonism by CGS 21680 was inhibited by the addition of the A2AR antagonist CSC 1 h prior to the addition of CGS 21680 (Fig. 2) . In a separate study, DMSO (1% as vehicle control) or IPDX used at a final concentration of 10 µM [final (DMSO) equal to 1%], at which it had been demonstrated to be a selective A2B antagonist [14 ], was added to cells 3 h after LPS and 1 h prior to treatment with CGS 21680. As shown in Figure 2B , IPDX failed to significantly abrograte the inhibition of TNF mediated by CGS 21680. Together, these data supported the conclusion that TNF inhibition was most likely mediated by the A2AR; however, the lack of an adequately selective A2B agonist does not allow us to exclude a contribution of the A2BR.



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  		Figure 2. (A) Dose-dependent effect of CGS 21680 on TNF production. Four hours after LPS (1 µg/ml) stimulation, cells were treated with varying doses of CGS 21680 (0.1–100 µM) or the corresponding % DMSO as vehicle control (0.01–1%). TNF bioactivity in cell-culture supernatants at 24 h was measured as in Materials and Methods. Bars represent mean ± SEM for n = 8 samples per condition. There was an effect of DMSO on TNF production at the highest concentration used when compared with cells stimulated with LPS and no pretreatment (u, P<0.05 vs. none). However, CGS 21680 at 0.1, 10, and 100 µM significantly decreased TNF production as compared with vehicle (DMSO) control (*, P<0.01 vs. DMSO). Importantly, prior treatment with the A2AR antagonist CSC completely blocked this inhibitory effect of CGS 21680 ({dagger}, P<0.01 vs. 10 µM dose). Graph is representative of n = 3 experiments. (B) Differentiated THP-1 cells were stimulated with LPS (1 µg/ml) for 3 h prior to the addition of IPDX [10 µM with final (DMSO)=1%] or 1% DMSO prior to the addition of CGS 21680 or CGS 21680 alone. This selective A2B antagonist failed to abrogate the inhibition of TNF production mediated by CGS 21680 (*, P<0.05 vs. DMSO).

 
Because of the dynamics of the A2AR expression, we next examined the kinetics of A2AR-mediated inhibition of LPS-induced TNF production by varying the time of CGS 21680 treatment (using the 10-µM dose) after LPS stimulation from 1 to 8 h. A representative experiment (n=3 experiments) is shown in Figure 3 A . One hour after LPS stimulation, A2A receptor agonism with CGS 21680 did not significantly affect TNF expression versus vehicle (DMSO) control (Fig. 3A) . However, when CGS 21680 was added to THP-1, 2, 4, or 8 h after LPS, a significant inhibition of TNF production was observed in a manner similar to Figure 2A (4-h data: DMSO 2974±532 pg/ml vs. CGS 21680 1434.5±153 pg/ml, P=0.02). Figure 3B shows the mean percent inhibition of TNF expression mediated by CGS 21680 as compared with DMSO vehicle control as a function of the time from LPS stimulation of the THP-1 cells for all three experiments. As shown, A2AR stimulation resulted in progressively increased inhibition of TNF production as the post-treatment time was extended out to 8 h post-LPS.



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  		Figure 3. Time-dependent effect of CGS 21680 on TNF production. Cells were treated with 10 µM CGS 21680 or DMSO vehicle (0.1%) at the times indicated after LPS stimulation (1 µg/ml). TNF bioactivity in cell culture supernatants at 24 h was measured as in Materials and Methods. Bars represent mean ± SEM for n = 8 samples per condition. CGS 21680 at 2, 4, and 8 h after LPS stimulation significantly attenuated TNF production (*, P<0.01 vs. DMSO control for each time point). Graph is representative of n = 3 experiments. **, P < 0.05

 
A2AR antagonism in the context of LPS stimulation augments TNF production
We next tested the hypothesis that the A2AR up-regulation was a host response aimed at endogenously regulating inflammatory gene expression as reflected by TNF production. To test this hypothesis, cells were treated with the A2AR antagonist CSC at doses previously demonstrated to block A2AR signaling [25 , 26 ] simultaneously with LPS stimulation. As shown in Figure 4 , when the A2AR was blocked in the presence of LPS, a significant augmentation in TNF expression was observed. The lack of effect at the highest dose examined (5 mM) was a reflection of significant toxicity as determined by increased lactate dehydrogenase release under these conditions (data not shown). In a related experiment, LPS-stimulated THP-1 cells were cotreated with the selective A2B antagonist, IPDX, or DMSO as vehicle control. Under these conditions, no difference in the LPS-induced TNF response was observed: LPS + DMSO [(final)=1%] = 8.42 ± 0.75 ng/ml versus LPS + IPDX = 7.66 ± 0.63, P = 0.52, suggesting the observed effect was not being mediated via the A2BR. Taken together with the data presented in Figure 1 and Table 1 , these results supported the hypothesis that in response to LPS stimulation, endogenous adenosine down-regulates TNF production via the A2AR, which also undergoes increased expression.



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  		Figure 4. Effect of A2AR antagonism on TNF production. Cells were stimulated with LPS (1 µg/ml) in the presence of varying doses of the A2AR antagonist CSC. Treatment with 50 and 500 µM significantly augmented TNF production versus vehicle control-treated cells (0). Bars represent mean ± SEM for n = 8 samples per condition; **, P < 0.01 versus vehicle control-treated cells. The lack of effect at the 5 mM dose likely represents toxicity as noted in Results.

 
Proposed mechanism of action of A2AR-mediated cytokine inhibition
Having demonstrated the role that the A2AR played in modulating LPS-mediated TNF production, we next attempted to determine the signaling mechanism by which this occurred. It is known that the A2AR is positively coupled to cAMP such that receptor stimulation increases cAMP [6 ] to activate PKA, which subsequently phosphorylates CREB [27 ]. More recently, it has been shown that phospho-CREB, by binding to phosphorylated p65, a NF-{kappa}B subunit required for transcriptional activation, was able to inhibit NF-{kappa}B-driven gene expression [28 ]. To determine if this pathway played a role in the above observations, we first determined the effect of CGS 21680 on the accumulation of phospo-CREB in the nucleus of cells treated with CGS 21680. As shown in Figure 5 , CGS 21680 treatment caused a substantial increase in the phosphorylated form of CREB in nuclear extracts.



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  		Figure 5. Western blot analysis of the phosphorylated isoform of CREB in nuclear extracts of CGS 21680 (CGS; lanes 2 and 4)- or vehicle (Veh; lanes 3 and 5)-treated cells following LPS stimulation at the time points indicated. Increased signal for phospho-CREB was observed in those cells treated with CGS 21680. In contrast, nuclear proteins from unstimulated control cells (Unstim; lane 1) and cytosolic protein fractions from stimulated cells (Cyto; lane 6) showed little phospho-CREB. Blot is representative of four independent determinations.

 
We next determined the role of cAMP-dependent PKA activation in the above observations. We postulated that inhibition of cAMP-mediated PKA activation would block the inhibitory effect of CGS 21680 on LPS-mediated TNF production. Rp-cAMPS is an analogue of cAMP that can inhibit PKA activation [29 ]. Pretreatment of cells with a higher dose of Rp-cAMPS (1 mM), 1 h prior to addition of CGS 21680, significantly abrogated its inhibitory effect on LPS-stimulated TNF production (Fig. 6 ). Together, these data suggested that the A2AR pathway involved cAMP activation of PKA with increased phosphorylation of CREB.



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  		Figure 6. Effect of PKA inhibition on reversing the CGS 21680 inhibitory effect. As was depicted in Figures 2 and 3 , cells treated with CGS 21680 (10 µM) 4 h after LPS (1 µg/ml) stimulation significantly attenuated TNF production as compared with vehicle-treated cells (LPS bar; 57% decrease, P=0.02). Pretreatment with an inhibitor of cAMP activation of PKA (Rp-cAMPS, RP) at a dose of 1 mM significantly attenuated this CGS 21680-mediated inhibition (by 44%, P=0.014). Bars represent mean ± SEM for n = 8 samples per condition except NEG (negative control cells) and RPcAMP alone (n=6). No effect of RPcAMP alone was observed. Graph is representative of n = 2 experiments.

 
Finally, the effect of increased phosho-CREB on NF-{kappa}B nuclear translocation and DNA binding (EMSA) as well as transcriptional activity (using {kappa}B-dependent luciferase construct) was determined. The signal for NF-{kappa}B translocation and binding between cells treated with CGS 21680 versus vehicle was similar as determined by EMSA (representative blot shown in Fig. 7 , Upper). Analysis of the relative densitometry as compared with the signal from control cells from three blots failed to show a statistically significant difference among treatment groups (Fig. 7 , Lower). However, NF-{kappa}B-dependent luciferase expression, which directly reflects NF-{kappa}B-dependent transcription, was significantly attenuated by treatment with CGS 21680 (Fig. 8 ).



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  		Figure 7. Effect of CGS 21680 on LPS-induced NF-{kappa}B activation in THP-1 as determined by EMSA. Unstimulated cells (lane 1) and CGS 21680 treatment alone (lane 2) showed little signal for NF-{kappa}B nuclear translocation/binding. At the times indicated, LPS (1 µg/ml) induced a strong signal (lanes 3, 5, 7) that on average was not substantially affected by CGS 21680 treatment (lanes 4, 6, 8) The specificity of this signal for NF-{kappa}B was confirmed by the ability of excess, nonradiolabeled NF-{kappa}B oligoprobe to compete off the signal from LPS-stimulated cells that were not observed using excess, "cold" irrelevant competitor (Oct-1; data not shown). Graphic depiction of the mean ± SEM relative densitometry from three separate EMSA determinations is shown below.

 


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  		Figure 8. Effect of CGS 21680 on NF-{kappa}B-driven luciferase expression. In transient transfection assays using the 3X{kappa}B-Luc construct, LPS stimulation (10 µg/ml media) caused the typical 25- to 100-fold increase in luciferase expression as compared with unstimulated (NEG) cells (P<0.001). Treatment with CGS 21680 (10 µM) significantly attenuated LPS-mediated, {kappa}B-driven luciferase expression (29%, P<0.01). Values were determined in duplicate and are expressed as mean ± SEM (n=8 wells per condition) standardized to protein content of samples. Results are representative of three separate experiments in which the percent inhibition ranged from 24 to 41%.

 
Because of the dependence of TNF gene expression on the p38 and ERK mitogen-activated protein kinase (MAPK) pathways [30 ], we examined the effect of CGS 21680 on the LPS-induced phosphorylation and presumed activation of these kinases by Western blot analysis for the phospho-specific isoforms. As shown in Figure 9 , no inhibition of phosphorylation of p38 or ERK1/2 was observed following treatment with CGS 21680. Instead, in comparing the relative densitometry of the signal among treatment groups for three blots, a significant increase in the phosphorylated form of ERK1/2 was found at the 30- and 60-min time points.



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  		Figure 9. (Top) Western blot analysis of the phosphorylated isoforms of p38 (upper) and ERK1/2 (lower) in nuclear extracts of vehicle (lanes 2 and 4)- or CGS 21680 (lanes 3 and 5)-treated cells following LPS stimulation at the time points indicated. Modest increases in the signal for phospho-p38 and ERK1/2 were observed in those cells treated with CGS 21680. Nuclear proteins from unstimulated control cells (Unstim; lane 1) showed little phospho-p38 or ERK as compared with LPS (1 µg/ml) stimulation. Graphic depiction of the mean ± SEM relative densitometry from three separate Western blot determinations was shown below. *, P < 0.05 versus vehicle-treated cells in lanes 2 and 4.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sepsis and its resultant sequelae of multiple organ dysfunction syndrome remain a frequent cause of mortality in adult and pediatric intensive care units. In the past two decades, great insight has been gained toward understanding the molecular pathophysiology of the host immune response triggered by sepsis. In many cases, Gram negative enteric organisms release LPS or endotoxin from their cell walls, which causes a systemic activation of peripheral blood monocytes that release early cytokines such as TNF (reviewed in ref. [31 ]). Despite this improved understanding, it remains unclear as to why this dysregulated inflammatory response develops in some patients, and others appear capable of regulating the host immune response to preserve biologic homeostasis. We have continued to try to identify host factors produced in response to inflammatory challenges (e.g., endotoxin), which assist the host in this compensatory function.

In the current studies, we used a model of LPS-stimulated monocytes to identify a negative-feedback pathway mediated by adenosine stimulation of the A2AR. It was previously shown and confirmed in the present studies that LPS stimulation of human monocytes increases the mRNA for the A2AR [23 ]. By flow cytometric analysis, it was demonstrated that this increased gene expression was associated with increased expression of the A2AR protein. Endogenous signaling through the A2AR is likely to only be mediated by adenosine, although it remains incompletely disproven that inosine could also serve as a ligand. However, although we were able to demonstrate significant increases in adenosine by HPLC, little inosine was detected in these same samples (data not shown). It should be noted that adenosine is a purine, which undergoes rapid turnover. Particularly in the extracellular environment, it is rapidly degraded by adenosine deaminase or taken up by an adenosine reuptake pathway that can be inhibited by dipyridamole [24 ]. Thus, our measurement of adenosine was substantially facilitated by the use of the adenosine deaminase inhibitor, EHNA, and the reuptake inhibitor, dipyridamole, as had been shown previously. Using this strategy, it was clear that a significant amount of adenosine is generated by the monocyte in response to the stress of LPS stimulation. Whether this is the principal source in vivo remains unknown based on these current data, but it is possible that extracellular ATP may be an additional source of adenosine. Importantly, the cotreatment of cells with LPS and the A2AR antagonist but not an A2B antagonist resulted in a significantly augmented TNF expression supporting the concept that adenosine mediates an endogenous, negative-feedback pathway via A2AR stimulation. It should be emphasized that the lack of a sufficiently specific A2B agonist precludes us from completely excluding a role for the A2BR in these observations. However, with the data presented in the context of the predominant expression of A2AR in these cells in contrast to the A2BR and A3R (see Fig. 2B ), it remains likely that the A2AR plays the dominant role in this regulatory pathway.

The findings of cytokine inhibition were consistent with other observations in which exogenous administration of adenosine [4 , 32 ] or inhibition of adenosine deaminase [5 ] resulted in decreased inflammatory cytokine production under various experimental conditions. In an air-pouch model of carrageenan-induced neutrophil inflammation, the inhibition of adenosine kinase resulted in increased local concentration of adenosine and substantially reduced neutrophil influx [33 ]. When an A2AR antagonist was coadministered, this effect was reversed, suggesting the anti-inflammatory effect was mediated via this receptor. Similarly, when the mouse macrophage line, RAW 264.7, or human monocytes were pretreated with adenosine, an abrogated cytokine response to endotoxin was observed [1 , 34 ]. In vivo studies using endotoxemic mice have corroborated these findings in that adenosine pretreatment inhibited TNF production [35 ]; however, the receptor responsible for this inhibitory effect had not been elucidated. Subsequent studies suggested that this in vivo inhibitory effect of adenosine might be mediated via the A3R, as the A3R agonist 1-deoxy-1-(6[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-ß-D-ribofuron-amide (IB-MECA) prevented endotoxin-mediated mortality in mice [2 ]. However, the results presented clearly identify the A2AR as mediating the inhibition of TNF production from LPS-stimulated monocytes. Whether this is also the case in primary human monocytes remains to be confirmed.

Although the current studies are limited in scope by focusing on the single proinflammatory cytokine TNF, we prioritized TNF, as it has been identified as a key initiator of the complex cascade mediating the pathophysiologic consequences of endotoxic shock [36 ]. The data presented support the conclusion that A2AR stimulation can inhibit LPS-mediated TNF production in the cell line examined. Expression of TNF is a complex process involving transcriptional and post-transcriptional mechanisms [37 ]. Analysis of the TNF promoter sequence has identified several key transcriptional factors including NF-{kappa}B, Erg-1, ERK1/2, and activated protein-1 contributing to the production of TNF mRNA [22 , 38 ]. Post-transcriptional stabilization of TNF mRNA appears to be a p38-dependent process [30 ]. We have attempted to elucidate the signal transduction pathway involved in the cell model used. It appeared that A2AR stimulation activated PKA via increased cAMP, resulting in increased phosphorylation of CREB, which attenuated NF-{kappa}B-dependent transcriptional activity. These observations are consistent with previous investigations in which agents that increased intracellular cAMP or overexpression of PKA resulted in decreased, NF-{kappa}B-mediated transcription using a similar {kappa}B-dependent construct [11 , 28 ]. It is interesting that no changes in nuclear translocation and DNA binding (by EMSA) nor decreased p65 phosphorylation were observed in these studies also. This is in contrast to another investigation in which elevated cAMP decreased inducible nitric oxide synthase expression in association with a decreased NK-{kappa}B signal as measured by EMSA [39 ]. These studies, which were performed on hepatocytes, suggest this inhibitory signal transduction pathway may be cell-specific. The finding that A2AR stimulation increased ERK1/2 activation is consistent with other observations linking an increase intracellular cAMP with downstream activation of ERK1/2 [40 , 41 ], albeit in different cell types. Thus, it is possible that A2AR stimulation might also alter MAPK-regulated signaling in THP-1 cells.

In summary, the present data show that LPS stimulation of monocytes increases expression of the A2AR and its endogenous ligand adenosine. Together, this receptor-ligand pair mediates activation of PKA with increased phosphorylation of CREB, resulting in downstream effects on TNF production, most likely via inhibition of NF-{kappa}B-dependent transcription. These findings not only identify a key endogenous regulatory pathway but also suggest that the A2AR may be a valid therapeutic target even after activation of immune cells has occurred.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH Grants HD-28827-08 and K08 HL04291-02 to T. P. S.

Received October 23, 2001; revised May 21, 2002; accepted May 23, 2002.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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