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Originally published online as doi:10.1189/jlb.0505249 on December 30, 2005

Published online before print December 30, 2005
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(Journal of Leukocyte Biology. 2006;79:574-585.)
© 2006 by Society for Leukocyte Biology

Differential expression of adenosine receptors in human neutrophils: up-regulation by specific Th1 cytokines and lipopolysaccharide

Andrée Fortin, Danielle Harbour, Maria Fernandes, Pierre Borgeat and Sylvain Bourgoin1

Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Pavillon C.H.U.L. et Université Laval, Département d’Anatomie et de Physiologie, Canada

1 Correspondence: Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Pavillon C.H.U.L., 2705 Blvd. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: Sylvain.Bourgoin{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Four types of adenosine receptors have been identified in different tissues and cell types, namely, A1, A2A, A2B, and A3 receptors. We report that A2AR but not A2BR mRNA in freshly isolated polymorphonuclear neutrophils (PMN) is maximally up-regulated after 4 h stimulation with lipopolysaccharide (LPS) or tumor necrosis factor {alpha} (TNF-{alpha}) and to a lesser extent, with interleukin (IL)-1ß. These effects were maintained up to 21 h. Consistent with changes in A2AR mRNA expression, up-regulation of A2AR protein was also detected after 4 h of LPS or TNF-{alpha} exposure. Up-regulation of A2AR protein expression was transient and returned to near basal levels after 12 h or 16 h stimulation with TNF-{alpha} or LPS, respectively. Conversely, IL-1ß failed to promote A2AR protein expression. Suppression of thapsigargin-induced leukotriene synthesis by the selective A2AR agonist CGS-21680 was found to be more pronounced when PMN were cultured for 4 h with LPS or TNF-{alpha}. In contrast, the up-regulation of A2AR has no impact on CGS-21680-induced inhibition of phospholipase D activation and superoxide production in response to formyl-Met-Leu-Phe. These results demonstrate that the A2AR is up-regulated by specific T helper cell type 1 cytokines and LPS. Although this could represent a potential feedback mechanism to control inflammation, the effect of A2AR up-regulation varied depending on the stimulus used to stimulate PMN functional responses after their incubation with proinflammatory mediators.

Key Words: A2AR • TNF-{alpha} • leukotrienes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine is a potent, endogenous, anti-inflammatory agent that modulates numerous physiological functions including vasodilatation, suppression of cardiac rate and contractility, neurotransmitter release, and inhibition of platelet aggregation [1 ]. Adenosine is released into the extracellular space under normal conditions and at higher concentrations during hypoxia and ischemia [2 ]. Damaged cells and tissues also secrete adenosine [3 ].

Once in the extracellular milieu, adenosine exerts its biologic effects through four distinct receptor subtypes, namely, A1R, A2AR, A2BR, and A3R [1 , 4 , 5 ]. The expression of these receptors by leukocytes was determined using pharmacological and functional assays [6 ]. It is known that human polymorphonuclear neutrophils (PMN) express A1R protein [7 , 8 ], A2AR, and A2BR mRNA [9 ]. The activation of A1R enhances chemotactic responses [7 , 8 , 10 ] and adherence to endothelial cells, whereas activation of A2AR reduces the oxidative burst [11 ]. PMN also express A3R mRNA, and A3R has been, at least in part, implicated in the suppression of PMN degranulation [12 , 13 ].

Most of the anti-inflammatory actions of adenosine are generated through A2AR, although the A3R subtype might also be involved [13 14 15 ]. Extracellular adenosine acts via signaling through A2AR (coupled to a heterotrimeric Gs-protein), which stimulates adenylyl cyclase, leading to intracellular cyclic adenosine monophosphate (cAMP) accumulation. The inflammatory process is then suppressed by a negative feedback mechanism involving the inhibition of molecular intermediates of proinflammatory signaling pathways by cAMP [16 ]. In this way, adenosine decreases many PMN functions including phagocytosis [17 ], adherence to endothelial cells [11 ], leukotriene (LT) synthesis [18 ], and phospholipase D (PLD) activity in response to various stimuli [19 , 20 ].

Activation of A2AR by adenosine and its analogs also modulates the production of inflammatory cytokines such as interleukin (IL)-12, tumor necrosis factor {alpha} (TNF-{alpha}), IL-10, and interferon-{gamma} (IFN-{gamma}) [21 22 23 24 ]. Genetic evidence suggests that A2AR-mediated inhibition of the nuclear factor-{kappa}B transcription activity is a critical aspect of the suppression of lipopolysaccharide (LPS)-stimulated expression of proinflammatory cytokines in vivo [25 ].

It is now well establish that the expression and function of A2AR and A2BR are up-regulated by T helper cell type 1 (Th1) cytokines in cells of the immune system [26 ]. For example, TNF-{alpha} and IL-1ß up-regulate the expression and function of A2AR in human monocytic THP-1 cells [23 ], whereas IFN-{gamma} enhances expression of A2BR and promotes macrophage deactivation by adenosine [27 ]. Differentiation of monocytes to macrophages is also associated with a transient increase in the expression of A2AR [28 ]. In addition, exposure to LPS enhances expression of A2AR in THP-1 cells [23 , 29 ] and down-regulates A1R and A3R expression by mature dendritic cells [30 ].

The regulation of the expression of the adenosine receptors has not been documented previously in PMN. As PMN play a key role in innate immunity and are exposed to Th1 cytokines in rheumatoid arthritis and other inflammatory diseases, we investigated the effect of Th1 cytokines and LPS on the function and expression of the adenosine receptor subtypes in PMN. We provide evidence that LPS, TNF-{alpha}, and IL-1ß differentially regulate the expression of A2AR at the mRNA and protein level. We also show that the up-regulation of the A2AR protein by LPS and TNF-{alpha} leads to a more pronounced suppression of thapsigargin-induced LT production by the A2AR-selective agonist CGS-21680.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
LPS, di-isopropylfluorophosphate (DFP), and cytochalasin B (CB) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada), and primers for polymerase chain reaction (PCR) were from Sigma Genosys (Oakville, Ontario, Canada). The penicillin-streptomycin-L-glutamine mix, Ficoll-Paque, Hanks’ balanced salt solution (HBSS), and RPMI-1640 medium were all obtained from Wisent Inc. (Montréal, QC). Triton X-100 was purchased from Fisher Scientific (Nepean, ON) and Dextran T-500, from Pharmacia Biotech (Baie d’Urfé, Québec, Canada). The FuGENE 6 transfection reagent and adenosine deaminase (ADA) were purchased from Roche Biochemicals (Laval, Québec, Canada). TRIZOL reagent and Moloney murine leukemia virus (MMLV)-reverse transcriptase (RT) kit were obtained from Gibco-BRL (Burlington, Ontario, Canada). Q-solution was purchased from Qiagen (Mississauga, Ontario, Canada). The human embryonic kidney (HEK)-293T cell line was purchased from GenHunter Corp. (Nashville, TN). CGS-21680 was from RBI (Oakville, Ontario, Canada) and SCH-58261, from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Thapsigargin was obtained from BioMol (Plymouth Meeting, PA).

Antibodies and cytokines
The anti-A2AR polyclonal antibody (antiserum, Cat. #AB1559) and A2AR peptide (Cat. #AG285) were obtained from Chemicon International (Temecula, CA). The peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) was purchased from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada) and the anti-hemagglutinin (HA).11 tag monoclonal antibody, from Babco (Cedarlane, Hornby, Ontario, Canada). Recombinant human (rhu)TNF-{alpha} was obtained from Knoll Pharmaceutical (Whippany, NJ). rhuIL-1ß, IFN-{gamma}, and granulocyte macrophage-colony stimulating factor (GM-CSF) were all purchased from Peprotech Inc. (Hornby, Ontario, Canada).

Human PMN purification, culture, and stimulation
PMN were isolated from heparinized venous whole blood of healthy adult volunteers as described previously with some modifications [19 , 31 ]. Volunteers participated in this study after obtaining informed consent to a protocol that was reviewed and approved by the Institutional Ethics Committee. In brief, whole blood was centrifuged and leukocytes obtained after erythrocyte sedimentation in 2% Dextran T-500. The leukocyte-rich supernatant was removed and spun down on Ficoll-Paque cushions (density 1.078 g/mL) at 600 g for 25 min. The mononuclear cell ring (monocytes and lymphocytes) was discarded except for the RT-PCR assay on A2BR mRNA expression (see Results). The PMN fraction, which pellets at the bottom of the tube, was harvested. Contaminating erythrocytes were lysed by a 20-s hypotonic shock in water, and normal osmolarity was reconstituted by the addition of HBSS. PMN, which were routinely of high viability (>95%), were then resuspended in HBSS (pH 7.4) containing 0.8 mmol/L Ca2+ but no Mg2+ to reduce nonspecific aggregation of the cells.

For total RNA extraction, the PMN concentration was adjusted to 107 cells/mL in HBSS (containing 1% autologous platelet-poor plasma (PPP) for LPS stimulation) and incubated at 37°C in sterile Ependorff tubes (107 cells/tube). PPP was prepared from platelet-rich plasma by centrifugation at 5585 g for 10 min.

For adenosine receptor solubilization, PMN were resuspended at a final concentration of 8 x 106 cells/mL in sterile RPMI-1640 culture medium supplemented with 10% PPP and 1% penicillin-streptomycin-L-glutamine mix. PMN (5x107) were then distributed in 25 cm2 cell culture flasks and incubated at 37°C. Stimulations were all carried out by addition of the proinflammatory substances directly in cell suspensions for the indicated times and concentrations.

Semiquantitative RT-PCR
After PMN stimulation, total cellular RNA was extracted using the TRIZOL reagent and precipitated with isopropanol. The RNA pellet was washed with 75% ethanol, resuspended in 10 µL diethylpyrocarbonate-treated, distilled water, and stored at –80°C. The yield and purity of the RNA preparations were estimated spectrophotometrically by measuring the absorbance at 260 nm and determining the A260/A280 ratio, respectively.

Total RNA (1 µg) from each sample was first reverse-transcribed into cDNAs, using the first-strand cDNA synthesis MMLV kit. Primers for the PCR reactions were designed from sequence data for the corresponding human cDNAs available from GeneBank: A1R (upper 5'-GGT GGC TGA TGT GG-3'; lower 5'-GCT TGC GGA TTA GGT AG-3'), A2AR (upper 5'-AGA TGG AGA GCC AGC CTC TGC-3'; lower 5'-GCT AAG GAG CTC CAC GTC TGG-3'), A2BR (upper 5'-GGC ACG GCG AAC ACT C-3'; lower 5'-TTG GTG GCA CTG TCT TTA CTG-3'), A3R (upper 5'-AAC GTG CTG GTC ATC TGC GTG GTC-3'; lower 5'-GTA GTC CAT TCT CAT GAC GGA AAC-3'), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; upper 5'-ATC TCT GCC CCC TCT GCT GA-3'; lower 5'-TCC ACC ACC CTG TTG CTG TA-3'). For the PCR reaction, 2.5 µL RT reaction product (10% of the total PCR reaction volume) was subject to amplification with 0.5 U Taq DNA polymerase in Q-solution. The MgCl2 content of the PCR buffer, the annealing temperatures, and number of cycles were individually optimized for each primer pair. The PCR reactions were carried out as follows: A2AR (28 cycles, 62.5°C annealing temperature), A2BR (38 cycles, 61.5°C annealing temperature), and A3R (32 cycles, 61.5°C annealing temperature). cDNA sequences were analyzed semiquantitatively using simultaneous amplification of the housekeeping gene GAPDH (30 cycles, 52°C annealing temperature).

For touch-down PCR (TD-PCR), following an initial melting period (5 min), a range of annealing temperatures, 65°C–53°C, was used (two cycles/°C) with nine final cycles at 53°C before the final extension period at 72°C (5 min). This protocol was performed for each primer pair (A1R, A2AR, A2BR, and A3R) on PMN and lymphocyte/monocyte (L/M) fractions.

Plasmid DNA construct and HEK-293T transient transfection
A HA tag was added to the C terminus of the A2AR protein by amplifying a portion of the gene using the A2AR internal upper-primer (5'-CAG TGC CCG GGT CTT GGC-3') and HA lower-primer (5'-CCC TCA AGC GTA ATC TGG AAC ATC ATA TGG ATA GGA CAC TCC TGC-3'). The template used for PCR was the pSVL-A2AR plasmid containing the full-length A2AR coding sequence. The PCR product was then subcloned into the pCR2.1 cloning vector (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and sequenced. Finally, the pCR2.1-A2AR-HA construct was cut with BamHI and SmaI and subcloned into the pSVL-A2AR expression vector to replace the corresponding A2AR coding sequence. The final pSVL-A2AR-HA construct was sequenced to confirm the presence of the C-terminal HA tag.

HEK-293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-L-glutamine mix. The cells were collected, seeded on six-well culture plates (1.5x105 cells/well), and transfected at a confluence of 50–60% with a DNA complex containing 6 µL FuGENE 6 and 1 µg pSVL-A2AR-HA plasmid. Forty-eight hours after transfection, cells were collected and lysed for 10 min in 100 µL ice-cold 1% Triton lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, pH 8.0, 10% glycerol, 1% Triton X-100, and protease inhibitors). Samples were centrifuged at 4°C (8 min, 10,000 g), and supernatants were kept at –20°C until use.

Preparation of PMN membrane fractions and A2AR solubilization
After stimulation, PMN were pretreated with DFP at a final concentration of 1 mM for 10 min at room temperature. Cells were washed with ice-cold HBSS and centrifuged at 4°C (7 min, 700 g). Each pellet was resuspended with 2 mL ice-cold HEPES buffer [50 mM HEPES, 0.5 mM EGTA, 5 mM NaCl, 100 mM KCl, 3.5 mM MgCl2, 2.5 µg/mL aprotinin-leupeptin, 0.25 mM phenylmethylsulfonyl fluoride (PMSF)], sonicated on ice for 20 s at minimum power, and centrifuged for another 7 min at 700 g, 4°C. The supernatants were then centrifuged at 4°C for 45 min at 180,000 g to pellet the membranes. The membrane pellets were resuspended in 135 µL solubilization buffer [0.25 M phosphate buffer, pH 6.8, 300 mM NaCl, 2.5% sodium dodecyl sulfate (SDS), 2.5 µg/mL aprotinin-leupeptin, 0.25 mM PMSF]. After a quick spin, the SDS-soluble fractions were kept at 4°C until use.

SDS-polyacrylamide gel electrophoresis (PAGE) and A2AR immunoblotting
Samples were mixed with 3x Laemmli sample buffer for 30 min or less before loading the gel. The samples were not boiled to avoid aggregation/degradation of A2AR. Samples (30–60 µg/well) were loaded onto a 7.5–20% gradient SDS-PAGE gel, electrophoresed overnight, and transferred onto a polyvinylidene difluoride (PVDF) membrane.

Nonspecific sites were blocked by incubating PVDF membranes for 30 min at 37°C in a Tris-buffered saline (TBS)-Tween buffer (25 mM Tris-HCl, 190 mM NaCl, and 0.15% Tween-20, pH 8.0) containing 2% gelatin and 2% dry milk. After two washes with TBS-Tween buffer, blots were incubated overnight at 4°C in TBS-Tween buffer containing the anti-A2AR antibody, diluted 1:2500. The blots were then washed in TBS-Tween buffer containing 2% gelatin and incubated at 37°C in TBS-Tween-2% gelatin buffer containing horseradish peroxidase-linked secondary antibody (donkey anti-rabbit IgG) at a dilution of 1:20,000. Blots were then washed in TBS-Tween buffer, and proteins were visualized with the Renaissance detection system (Perkin Elmer Life Sciences, Wellesley, MA).

Measurement of 5-lipoxygenase (5-LO) products by reversed-phase (RP)-high-pressure liquid chromatography (HPLC)
After stimulation with various proinflammatory mediators (4 h), PMN were washed, and the concentration was adjusted to 5 x 106 cells/mL with 1x HBSS solution (with Ca2+, without phenol red). Cell suspensions were treated with 0.1 U/mL ADA and 10 µM CB for 5 min at 37°C, and incubated with the selective A2AR agonist CGS-21680 (10–8 M) for an additional 5 min. Following stimulation with 100 nM thapsigargin for 10 min, reactions were stopped, and samples were processed for RP-HPLC, as described previously [32 ].

Measurement of PLD activity
PMN were labeled by adding 1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 cells) directly in the cell suspension for 90 min before terminating the incubation with LPS or TNF-{alpha}. PMN were washed and resuspended at 107 cells/mL in HBSS. Cell suspensions (0.5 mL) were warmed at 37°C for 5 min and then pretreated for 5 min with 10 µM CB and 0.1 U/mL ADA to eliminate endogenous adenosine. PMN were stimulated with 100 nM formyl-Met-Leu-Phe (fMLP) for 10 min in the presence of 1% ethanol to monitor the formation of phosphatidylethanol (PEt) catalyzed by PLD. Incubations were stopped by adding 1.8 mL chloroform/methanol/HCl (50:100:1, v/v/v) and unlabeled PEt as a standard. Lipids were extracted, and the levels of [3H]PEt were quantified as described previously [31 ].

Superoxide measurements
Superoxide production was measured using the reduction of cytochrome c assay as described previously [33 ]. The cell suspensions (107 cells/mL) were preincubated for 4 h at 37°C, with or without the indicated concentration of LPS or TNF-{alpha}. The cells were diluted at 106 cells/mL and incubated in the presence of 130 µM cytochrome c, 10 µM CB, and 0.1 U/mL ADA for 5 min at 37°C before being stimulated for 5 min with 100 nM fMLP. The tubes were kept on ice for 15 min and centrifuged for 10 min at 1500 g. The amounts of superoxide anions in the supernatants were calculated from differences between the optical density readings at 550 and 540 nm using an extinction coefficient of 21.1.

Statistics
The effect of cytokines and LPS treatments was assessed by ANOVA (one-way ANOVA) and the Dunnett’s post-test (P values<0.05 were considered to be significant).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A2AR and A3R mRNA are expressed on resting human PMN
In agreement with other reports, we detected mRNA for A2AR and A3R in PMN (Figs. 1 and 2 ). Although A1R primers amplified a PCR product of a predicted size [473 base pairs (bp)] in the human kidney, we did not detect A1R mRNA in PMN under our experimental conditions (Fig. 2A and 2B) . Similar data were obtained with another set of primers previously shown to amplify the message for A1R in human airway epithelial cells (data not shown) [34 ]. It is of note that although the A2AR and A3R mRNAs were expressed in PMN and L/M, the A2BR mRNA was only detected in L/M and not in PMN (Fig. 2A) .


Figure 1
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  		Figure 1. Kinetics of A2AR mRNA expression in stimulated PMN. mRNA was extracted from PMN treated with (A) LPS (1 µg/mL), (B) TNF-{alpha} (100 ng/mL), or (C) IL-1ß (100 ng/mL) in diluant medium [PPP, bovine serum albumin (BSA), or PBS] or medium alone [nonstimulated control (NS)] for the indicated times and subject to semiquantitative RT-PCR. Following electrophoresis on an agarose gel, the A2AR PCR product was quantified by densitometry and normalized to GAPDH. The data shown are the mean ± SEM from three separate experiments. *, P < 0.05; **, P < 0.01, versus control.

 

Figure 2
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  		Figure 2. Expression of adenosine receptor mRNA in PMN and L/M. Total mRNA was isolated from PMN or L/M stimulated with LPS (1 µg/mL), TNF-{alpha} (100 ng/mL), IL-1ß (17 ng/mL), IFN-{gamma} (1 ng/mL), GM-CSF (1.4 ng/mL), 1% PPP in medium, or medium alone (nonstimulated control) for 4 h. (A) Representative agarose gel analysis of TD-PCR (35 cycles) for A1R, A2AR, A2BR, and A3R is shown. (B) Representative agarose gel analysis of TD-PCR products to monitor A1R mRNA expression in human kidney and PMN obtained from four different volunteers. Semiquantitative RT-PCR analyses of A2AR (C) and A3R (E) in PMN and of A2BR (D) in L/M. Densitometric analyses were performed using the AlphaImager 3.3 program. The PCR product was quantitated and normalized using the internal standard GAPDH. The data shown are the means ± SEM A2AR, A2BR, or A3R/GAPDH ratios from four to seven separate experiments. *, P < 0.05; **, P < 0.01, versus control.

 
LPS and TNF-{alpha} have long-lasting effects on A2AR mRNA expression in PMN
We next examined the kinetics of mRNA expression after stimulation with LPS, TNF-{alpha}, or IL-1ß. LPS at 1 µg/mL induces a significant increase in the levels of A2AR mRNA expression (Fig. 1A) . The A2AR/GAPDH ratios increased from 0.43 ± 0.09 (no LPS) to 1.46 ± 0.11 after 6 h LPS exposure, a level that was maintained up to 21 h (data not shown). Stimulation with 100 ng/mL TNF-{alpha} also enhanced A2AR mRNA expression. As early as 2 h, a noticeable increase in the amounts of A2AR mRNA expression was detected and maintained after 6 h of cytokine exposure (Fig. 1B) . TNF-{alpha}-induced A2AR mRNA expression plateaued after 4 h, and the effect was maintained up to 21 h, the last time-point tested (data not shown). A significant enhancement of A2AR mRNA expression was only seen after 4 h of stimulation with 100 ng/mL IL-1ß, which was maintained until 6 h (Fig. 1C) . The A2AR/GAPDH ratio increased from 0.23 ± 0.03 (no IL-1ß) to 0.91 ± 0.05 after 6 h stimulation, respectively. As near-maximal accumulation of A2AR mRNA was detected after 4 h of incubation with LPS, TNF-{alpha}, or IL-1ß, this time-point was chosen for all other experiments.

LPS, TNF-{alpha}, and IL-1ß modulate A2AR mRNA expression
Having investigated the expression of the A2AR in PMN, we then assessed the effect of LPS and other inflammatory cytokines (at supramaximal concentrations generally reported in literature) on A2AR, A2BR, and A3R mRNA expression. After 4 h of stimulation with LPS (1 µg/mL) and TNF-{alpha} (100 ng/mL), a significant increase in the expression of A2AR mRNA was observed compared with the nonstimulated control (0.56±0.05; Fig. 2C ). The A2AR/GAPDH ratios reached 1.76 ± 0.18 and 2.52 ± 0.44 for LPS and TNF-{alpha}, respectively. Stimulation with IL-1ß (17 ng/mL) also increased the A2AR mRNA expression as compared with the nonstimulated control, although not significantly (A2AR/GAPDH ratio 1.10±0.22 vs. 0.56±0.05). IFN-{gamma} and GM-CSF had no impact on A2AR mRNA levels at the concentrations tested (1 ng/mL and 1.4 ng/mL, respectively).

The same stimuli were tested for their effect on the expression of A2BR and A3R mRNA on L/M and PMN, respectively (Fig. 2D and 2E) . None of the stimuli tested had an effect on expression of A2BR and A3R at the concentrations tested. Even after stimulation with Th1 cytokines and LPS, the A2BR message had not been detected in PMN, suggesting that it is not at all detectable under our experimental conditions (not shown).

We, as well as others, have shown that adenosine accumulates rapidly in neutrophil suspensions and suppresses several functional responses of PMN [19 , 32 ]. We, therefore, measured the expression of A2AR, A2BR, and A3R mRNA in the presence of ADA, which hydrolyzes adenosine to inosine. The addition of ADA (0.1 U/mL) to PMN suspensions did not affect the basal or stimulated levels of A2AR, A2BR, or A3R mRNA expression (data not shown). We can, therefore, exclude the possibility that endogenous adenosine and its degradation product inosine modulate the cytokine and LPS-induced expression of adenosine receptors, at least at the transcriptional level in human PMN. Hence, we concentrated our study on the regulation of the expression of A2AR in PMN by LPS, TNF-{alpha}, or IL-1ß in the absence of ADA.

Stimulation of A2AR mRNA expression occurs at low cytokine concentrations
We next examined the concentration-response curves of LPS, TNF-{alpha}, and IL-1ß on the levels of A2AR mRNA expression in cultured PMN. Stimulation with 1 ng/mL LPS was sufficient to induce a significant increase in the expression of A2AR mRNA (Fig. 3A ) as compared with control (A2AR/GAPDH ratio of 1.64±0.16). The A2AR/GAPDH ratio reached 2.49 ± 0.17 at 2.5 µg/mL LPS. Stimulation with TNF-{alpha} induces a similar increment in the amounts of A2AR mRNA. A significant increase in the levels of mRNA expression was observed at 1 ng/mL, corresponding to an A2AR/GAPDH ratio of 2.2 ± 0.11, which was maintained up to 2.5 µg/mL TNF-{alpha}, the highest concentration tested (Fig. 3B) . Although not significant, a slight increase in the amounts of A2AR mRNA was detected from 10 ng/mL to 2.5 µg/mL IL-1ß. The levels of A2AR mRNA were not further enhanced by higher concentrations of IL-1ß (Fig. 3C) . Finally, using the same range of concentrations (1 ng/mL–2.5 µg/mL), no increase in the levels of A2AR mRNA was observed in response to stimulation with IFN-{gamma} (Fig. 4 and data not shown). This cytokine was, therefore, used as our negative control for A2AR expression. For comparison, cell suspensions were stimulated with optimal concentrations of TNF-{alpha} (10 ng/mL), IL-1ß (10 ng/mL), and LPS (1 µg/mL). As summarized in Figure 4 , A and B, TNF-{alpha} and LPS were twofold more potent than IL-1ß in enhancing A2AR mRNA levels in human PMN.


Figure 3
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  		Figure 3. Dose response curves of the regulation of the expression of A2AR by LPS, TNF-{alpha}, and IL-1ß in PMN. mRNA was extracted from PMN treated with the indicated concentrations of (A) LPS, (B) TNF-{alpha}, and (C) IL-1ß for 4 h and subject to semiquantitative RT-PCR. Controls include PMN suspensions, incubated with or without (nonstimulated control) the diluant for LPS (PPP), TNF-{alpha} (BSA), or IL-1ß (PBS). Following electrophoresis on an agarose gel, the level of the A2AR PCR product was quantified by densitometry and normalized to GAPDH. The data shown are the mean ± SEM from three separate experiments. *, P < 0.05; **, P < 0.01, versus control.

 

Figure 4
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  		Figure 4. Comparison of optimal concentrations of stimuli on the A2AR mRNA expression. (A) Representative agarose gel analysis of semiquantitative RT-PCR amplified A2AR in PMN stimulated with LPS (1 µg/mL), TNF-{alpha}, IL-1ß, and IFN-{gamma} (a negative control) at 10 ng/mL for 4 h. (B) The amount of A2AR PCR product was quantified by densitometry and normalized to GAPDH. The data shown are the means ± SEM A2AR/GAPDH ratio from three separate experiments. **, P < 0.01, versus control.

 
A2AR protein is present in resting and stimulated PMN
To monitor the expression of A2AR at the protein level, an antibody to the C-terminal intracellular domain (30 amino acids) of human A2AR (from Chemicon International) was first characterized. HEK-293T cells, overexpressing A2AR fused to a HA tag, show an immunoreactive band at 45 kDa (Fig. 5A , lane 2) compared with nontransfected cells (Fig. 5A , lane 3). This immunoreactive band at 45 kDa was not detected in samples boiled in Laemmli’s sample buffer prior to SDS-PAGE electrophoresis (Fig. 5A , lane 1), indicating that boiling induces degradation and/or aggregation of A2AR, which is a glycosylated protein [35 ]. To further confirm the identity of the 45-kDa band in A2AR-transfected HEK cells, the samples were also probed with an antibody to the HA tag (anti-HA tag). As shown in Figure 5A , lane 4, the tagged A2AR was detected at ~45 kDa with the anti-HA antibody. As observed with the anti-A2AR antibody, the anti-HA antibody did not detect the HA-tagged receptor after boiling the samples (Fig. 5A , lane 5). The specificity of the A2AR antibody was also confirmed by the absence of an immunoreactive band at 45 kDa in immunoblots carried out in the presence of a competing peptide (data not shown).


Figure 5
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  		Figure 5. A2AR protein expression in stimulated neutrophils. (A) Characterization of the anti-A2AR antibody. HEK-293T cells were transfected with the pSVL-A2AR-HA plasmid as described in Materials and Methods. Whole cell lysates from transfected (lanes 1, 2, 4, 5) and nontransfected cells (lane 3) were boiled (lanes 1 and 5) or not (lanes 2–4) in Laemmli’s buffer. Samples (40 µg/lane) were loaded on a 10% SDS-PAGE gel, electrophoresed, and analyzed by immunoblotting with a specific anti-A2AR (lanes 1–3) or anti-HA (lanes 4 and 5) antibody. (B) Western blot analysis of A2AR expression in stimulated PMN. Total membranes were isolated from PMN stimulated with LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (10 ng/mL), IFN-{gamma} (10 ng/mL), or medium alone for 4 h. The samples were resolved on a 7.5–20% gradient SDS-PAGE (32 µg/lane) and analyzed by immunoblotting with an anti-A2AR antibody. A representative immunoblot is shown. (C) Quantification of A2AR protein expression (4 h stimulation) using the Image Gauge 3.0 program. The data are expressed as the mean ± SEM percentage of control (n=4). **, P < 0.01, versus control. (D) Extended time course of A2AR protein expression following stimulation with 1 µg/mL LPS (n=4) or 10 ng/mL TNF-{alpha} (n=3). The data are expressed as the percentage of nonstimulated control. *, P < 0.05, versus control.

 
To determine whether a correlation exists between the observed increase in A2AR at the mRNA and protein level, membrane preparations obtained from resting and stimulated PMN were analyzed by Western blot. As shown in Figure 5B , an immunoreactive band at 43–45 kDa was detected in membrane preparations from PMN treated with medium alone (control PMN, nonstimulated) or cytokines for 4 h. When compared with control, there was a 172.2 ± 22.2% (n=4, P<0.01) and 212.6 ± 12.1% (n=4, P<0.01) increase in the amounts of A2AR protein in PMN treated with 1 µg/mL LPS and 10 ng/mL TNF-{alpha}, respectively (Fig. 5C) . In contrast, the amount of receptor expressed by PMN was not modified after a 4-h stimulation with IL-1ß (91.5±5.7% of control PMN). Consistent with our data at the mRNA level, IFN-{gamma} failed to enhance A2AR protein expression (106.5±16.4% of control PMN). Finally, we also investigated the kinetics of receptor expression after incubation with LPS and TNF-{alpha} for up to 21 h (Fig. 5D) . A significant increase in A2AR expression was observed up to 12 h with LPS, after which protein levels returned to the baseline at 16 and 21 h stimulation. The expression A2AR induced by TNF-{alpha} was also transient, as assessed by densitometry analysis of immunoblots. Changes in A2AR protein expression were only significant after 4 h incubation (Fig. 5C) but not significantly different from control values after 12, 16, and 21 h of TNF-{alpha} exposure (Fig. 5D) .

TNF-{alpha} and LPS treatments promote the inhibitory effect of CGS-21680 on thapsigargin-induced LT biosynthesis
It is known that several signaling pathways, including PLD activation initiated by receptor-ligand interactions, are involved in the regulation of the oxidative burst. Although the intracellular messenger(s) mediating the inhibition of PMN functions by A2AR remain controversial, cAMP analogs and agents, which stimulate cAMP accumulation, have been shown to inhibit fMLP-induced, small GTPase activation or arachidonic acid (AA)-mediated stimulation of PLD and synthesis of 5-LO products [20 , 36 ]. Hence, we investigated the effect of proinflammatory mediators on LT synthesis following stimulation of PMN with thapsigargin, a potent inducer of LT synthesis [36 ]. Figure 6 shows that a preincubation of PMN suspensions with 10 nM CGS-21680, a selective A2AR agonist, inhibits thapsigargin-mediated synthesis of LTs by 30% in comparison with control cells (nonstimulated, no CGS-21680). When cell suspensions were incubated with TNF-{alpha} or LPS for 4 h, a more pronounced, inhibitory effect of CGS-21680 on thapsigargin-induced LT synthesis was observed (78% and 75% inhibition, respectively). Furthermore, the biosynthesis of 5-LO products was reduced by low concentrations of CGS-21680 (<10 nM) only after TNF-{alpha} and LPS exposure but not after incubation of cell suspensions with the two cytokines (IL-1ß or IFN-{gamma}), which fail to promote A2AR protein expression (Table 1 ).


Figure 6
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  		Figure 6. Effect of the A2AR agonist CGS-21680 on thapsigargin-induced LT synthesis in cytokine- and LPS-treated PMN. Following pretreatment with LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (10 ng/mL), or IFN-{gamma} (10 ng/mL) for 4 h at 37°C, PMN were treated with ADA (0.1 U/mL) and CB (10 µM) for 5 min at 37°C. After an additional 5 min incubation with or without CGS-21680 (10–8 M), cells were stimulated with thapsigargin (100 nM) for 10 min. Samples were processed for determination of LT synthesis as described in Materials and Methods. The quantity of LTs represents the sum of LTB4, 20-hydroxy-LTB4, and 20-COOH-LTB4. Control level of LT synthesis without CGS-21680 was 114 ± 9.3 pmol/106 cells (n=6). The amounts of LTs generated were 59.4 ± 16.1, 107.8 ± 11.7, 113 ± 8.9, and 54.6 ± 16.2 pmol/106 cells after PMN treatment with TNF-{alpha} (n=6), IL-1ß (n=3), IFN-{gamma} (n=3), and LPS (n=6), respectively. The data are expressed as the mean ± SEM percentage of the respective control without CGS-21680. Each experiment was performed in triplicate.

 

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  		Table 1. Inhibitory Effect of CGS-21680 on Thapsigargin-Mediated Synthesis of 5-LO Products

 
Up-regulation A2AR by TNF-{alpha} and LPS does not promote CGS-21680-mediated inhibition of fMLP-induced PLD activation and superoxide production
We have previously reported that adenosine released by PMN into the extracellular milieu exerts, through A2AR, an inhibitory constraint on fMLP-induced PLD activation [19 ]. Therefore, we next determined if TNF-{alpha}- and LPS-induced up-regulation of A2AR protein was associated with a more pronounced inhibition of fMLP-induced PLD activation. As shown in Figure 7A , preincubation of cell suspensions with 10 ng/mL TNF-{alpha} or 1 µg/mL LPS for 4 h enhanced fMLP-induced PLD activation by ~2.5-fold as compared with control PMN. It is surprising that TNF-{alpha} or LPS exposure did not promote the inhibitory effect of the A2AR agonist CGS-21680 on fMLP-stimulated PLD activation.


Figure 7
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  		Figure 7. Effect of the A2AR agonist CGS-21680 on fMLP-stimulated PLD activity and respiratory burst in TNF-{alpha}- and LPS-treated PMN. Following a pretreatment with LPS (1 µg/mL) and TNF-{alpha} (10 ng/mL) for 4 h at 37°C, PMN were incubated with ADA (0.1 U/mL) and CB (10 µM) for 5 min at 37°C. After an additional 5-min incubation with the indicated concentration of CGS-21680, cells were stimulated with 100 nM fMLP. (A) PMN were labeled with 1-O-[3H] alkyl-2-lyso-phosphatidylcholine and stimulated for 10 min in the presence of 1% ethanol, and samples were processed for determination of PLD activity as described in Materials and Methods. The results are from three independent experiments conducted in duplicate. (B) PMN were stimulated for 5 min as described in A, and superoxide anion production was evaluated as described in Materials and Methods. The results are from two independent experiments conducted in triplicate. (C) SCH-58261 (0.1 µM) antagonizes the inhibitory effect of 0.1 µM CGS-21680 on fMLP-induced PLD activation. SCH-58261 was added to the cell suspension 5 min before CGS-21680. The results are from four independent experiments. (D) SCH-58261 reverses the suppressive effect of CGS-21680 (0.1 µM) on fMLP-induced superoxide anion production in a dose-dependent manner. The results are from four independent experiments conducted in triplicate.

 
It has been demonstrated that adenosine, through the occupancy of A2AR, suppresses fMLP-induced superoxide anion production [7 ]. As the products of PLD, phosphatidic acid (PA), and its metabolic product diacylglycerol, have been shown to activate a component of the NADPH oxidase in human PMN [37 , 38 ], a series of experiments was subsequently undertaken to define the impact of enhanced A2AR expression on fMLP-mediated superoxide anion production. Whereas preincubation with 10 ng/mL TNF-{alpha} or 1 µg/mL LPS markedly potentiated fMLP-induced PLD activation, these cell suspensions produced slightly lower amounts of superoxide anions (Fig. 7B) . These differences were not, however, statistically significant. As expected, the selective A2AR agonist CGS-21680 inhibited fMLP-mediated superoxide anion production in a dose-dependent manner (Fig. 7B) . However, the concentration-response curves of CGS-21680 were not altered by preincubations of PMN with TNF-{alpha} or LPS.

The involvement of A2AR in the regulation of PMN functional responses was further investigated using SCH-58261, the most selective antagonist for human A2AR [39 ]. When cell suspensions were preincubated with SCH-58261, the inhibitory effect of CGS-21680 on fMLP-mediated PLD activation (Fig. 7C) and fMLP-induced superoxide anion production (Fig. 7D) was antagonized in the control (nonstimulated) and TNF-{alpha}- and LPS-treated cells. As indicated in Figure 7C and 7D , a preincubation with 0.1 µM SCH-58261 was sufficient to totally block the suppressive effect of CGS-21660 on fMLP-induced PMN functional responses. Taken together, the above results suggest that an increment in the number of A2AR stimulated by TNF-{alpha} or LPS is not associated with a more pronounced inhibition of fMLP-induced functional responses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The incubation of human PMN without stimulus in vitro results in the rapid accumulation of endogenous adenosine, which has profound inhibitory effects on many cell functions, including adherence to endothelial cell layers by down-regulating the expression of ß2-integrins [40 ], generation of superoxide anions and phagocytosis [10 ], production of TNF-{alpha} [21 ], LT biosynthesis [18 , 41 ], and activation of PLD [19 ]. Several lines of evidence identify the A2AR as the adenosine receptor that mediates these inhibitory effects of adenosine on PMN. In the present study, we show that PMN express significant amounts of A2AR and A3R but not A1R and A2BR mRNA. In addition to A2AR and A3R, L/M were found to express A2BR but not A1R mRNA, suggesting differences in adenosine receptor subtype expression between cell types. Our results do not exclude the expression of A2BR and A1R by PMN but rather suggest that the amounts of these adenosine receptor transcripts are low in comparison with A2AR and A3R mRNA and not detectable using our TD/RT-PCR experimental conditions. The expression of A2BR was recently investigated in peripheral blood leukocytes with the A2BR mRNA being more abundant in neutrophils and lymphocytes than in monocytes, as estimated by real-time PCR [42 ]. Other studies have reported the expression of A2AR, A2BR, A3R, and A1R mRNA [9 , 12 , 43 ] and of functional A1R protein [7 , 8 , 10 ] by human PMN.

In various pathophysiological conditions, emigrated PMN are exposed to Th1 cytokines such as TNF-{alpha}, IL-1ß, or IFN-{gamma}, which are present in inflammatory exudates. In this current study, we show that IL-1ß, TNF-{alpha}, and LPS stimulation of human PMN increases the expression of A2AR mRNA but not of A3R, A2BR, and A1R mRNA. Moreover, TNF-{alpha} and LPS but not IL-1ß increased A2AR protein expression. It should also be noted that the kinetics of A2AR mRNA induction, which we report in response to TNF-{alpha} and LPS, is different from that of A2AR protein up-regulation. Although the amounts of A2AR protein had returned to baseline after 12 h and 16 h of TNF-{alpha} and LPS exposure, respectively, sustained induction at the transcript level was observed up to 21 h of PMN treatment, the last time-point sampled. Whereas IL-1ß slightly enhanced A2AR mRNA expression, the amount of transcript was less abundant compared with PMN treated with TNF-{alpha} or LPS. This may explain why up-regulation of A2AR protein was not detected in IL-1ß-stimulated cells. Although it is now well established that the modulation of adenosine receptor expression varies with cell types and stimuli, up-regulation of A2AR by Th1 cytokines and LPS in PMN corroborates the changes previously reported in A2AR expression by human monocytic THP1 cells stimulated with Th1 cytokines [23 ] and LPS [29 ]. It is interesting that an increase in the levels of A2AR expression has recently been reported in circulating lymphocytes and PMN from patients with chronic heart failure [44 ], a disease characterized by a strong inflammatory component. Moreover, the induction of A2AR expression by IL-1ß and TNF-{alpha} in human microvascular endothelial cells [23 ] and rat PC12 cells [45] indicates that the effect of Th1 cytokines is not restricted to hemopoietic cells. Similarly, neuronal A2AR are up-regulated after ischemia [46 ]. Other studies have shown that A2AR plays an important, protective role in ischemia-induced injury in various organs including neonatal brain [47 ].

IFN-{gamma} has recently been reported to up-regulate the gene and surface protein expression of the A2BR in murine bone marrow-derived macrophages [27 ] and to decrease A2AR in THP1 cells [23 ]. Similarly, hypoxic conditions increased A2BR and down-regulated A2AR expression in human endothelial cells [48 ]. Although A2BR mRNA was not detected in PMN using our PCR conditions, others have shown that the A2BR protein is expressed by PMN and down-regulated in pathophysiologic conditions such as systemic sclerosis [49 ]. Nevertheless, there was no change in the levels of expression of this adenosine receptor subtype by L/M (nor induction of A2BR mRNA in PMN) after 4 h of cell exposure to IFN-{gamma}. As previously reported in bone marrow-derived macrophages [27 ], it is also possible that IFN-{gamma}-induced expression of A2BR is a late event.

In the present study, we show for the first time in human PMN a heterologous regulation of adenosine receptor expression by Th1 cytokines and LPS. The predominant up-regulation of A2AR protein by LPS and TNF-{alpha}, along with adenosine release at inflammatory sites, suggests a predominant role for A2AR in suppressing the functions of inflammatory PMN. In various cell types, up-regulation of A2AR expression by IL-1ß, TNF-{alpha}, and LPS has resulted in enhanced production of cAMP in response to selective A2AR agonists [23 , 50 ]. We have previously shown that the occupancy of A2AR by adenosine or selective A2AR agonists, such as CGS-21680, promotes fMLP-induced cAMP accumulation in human PMN [51 ]. Other studies have shown that the ability of CGS-21680 to inhibit LTB4 synthesis varied depending on the stimulus used to stimulate LT biosynthesis [41 ]. These studies have established that CGS-21680 is a more potent suppressor of thaspsigargin-induced LTB4 synthesis compared with ionophore or other natural PMN agonists. Furthermore, occupancy of A2AR by adenosine or adenosine analogs led to a decrease in LT biosynthesis through an increase of cAMP levels and protein kinase A (PKA) activation [36 , 41 ]. Here, we show an increase in A2AR protein expression induced by TNF-{alpha} and LPS, which parallels the enhanced efficacy of CGS-21680 to inhibit thapsigargin-induced LT synthesis. These data suggest that the up-regulation of functional A2AR could in turn promote cAMP levels and PKA functions in activated PMN.

It was not possible to monitor the effect of A2AR up-regulation by cytokines and LPS on fMLP-induced LTB4 biosynthesis. It should be emphasized that although circulating PMN show minimal LT synthesis in response to fMLP or other natural agonists, exposure to proinflammatory agents such as TNF-{alpha} or LPS results in a significant enhancement of the capacity of PMN to produce 5-LO products [41 ]. Furthermore, these inflammatory cytokines have been shown to up-regulate fMLP receptor expression and paradoxically, to down-regulate LTB4 receptors (BLT1) in PMN [52 ] and monocytes [53 ]. It is notable that TNF-{alpha} and LPS treatments reduced by ~50% the biosynthesis of LTs in thaspigargin-activated PMN. Although the reason for this decrease in the capacity of PMN to produce 5-LO products is not clear, it may be explained by a down-regulation of LTB4receptors, as we previously demonstrated that thapsigargin (and AA as well) stimulates LT biosynthesis through a mechanism that requires an autocrine-stimulatory loop by LTB4 and BLT1 [20 , 54 ].

PMN priming with TNF and LPS has been shown previously to result in the up-regulation of agonist-stimulated PLD activity [55 , 56 ], which parallels the increase in superoxide generation and degranulation [37 , 38 ]. Although we report here a similar enhancement of fMLP-induced PLD activation by TNF-{alpha} and LPS, there was, to our surprise, no increase but rather a small decrease in superoxide anion generation. However, it should be highlighted that in other studies, the effect of TNF-{alpha} (and LPS as well) in priming the activation of NADPH oxidase has been studied after an incubation of PMN for 30 min or less [37 , 55 ], instead of 4 h in the present study. We also show that up-regulation of A2AR by TNF-{alpha} and LPS was not associated with an increase in the efficacy of CGS-21680 to inhibit fMLP-induced PLD activation and superoxide production, whereas the inhibitory effect of CGS-21680 on thapsigargin-induced LT synthesis was markedly potentiated. Although the exact mechanism of regulation must be clarified further, it may be explained by differences in the levels of fMLP receptor coupling to the signal transduction machinery. It has been reported that the half-life of circulating PMN, which is approximately 5 h, is prolonged up to 24 h in the presence of LPS and other agonists [57 ] and that LPS and TNF-{alpha} can delay PMN apoptosis, at least in part by preventing the loss of PLD1 protein [56 ]. The observation that CGS-21680 decreased the PLD response in cells primed with TNF-{alpha} or LPS to levels normally reached in unprimed PMN stimulated with fMLP without CGS-21680 may be of particular interest in the context of inflammation. It may have important consequences on the inflammatory responses in vivo in that activation of A2AR would suppress with greater efficacy the PMN functional responses that are not regulated by PLD-derived PA such as AA release and LT synthesis. Inflammatory cytokines and LPS do not only modulate the expression of G-protein-coupled receptors in leukocytes but also of other proteins [52 , 53 ]. For example, genomic and proteomic analysis during PMN activation and priming by LPS has identified early and late changes in gene expression [58 , 59 ]. More than 100 genes, including cytokines and chemokines, metabolic enzymes, signaling molecules, and transcription factors, were up-regulated or repressed after several hours of exposure to LPS.

In conclusion, this study demonstrates that PMN exposure to LPS and TNF-{alpha} up-regulates A2AR protein expression and promotes the inhibition by CGS-21680, a selective A2AR agonist, on thapsigargin-induced LT synthesis. It is generally appreciated that the differential up-regulation of A2AR expression by cytokines could represent one potential negative feedback through a mechanism involving the production of cAMP. The selective up-regulation of A2AR may promote leukocyte deactivation, thus protecting surrounding tissues from the deleterious effect of activated inflammatory cells. Although occupancy of A2AR clearly suppresses fMLP-induced functional responses, up-regulation of A2AR expression by TNF-{alpha} and LPS does not promote the inhibitory effect of CGS-21680 on fMLP-stimulated PLD activation and superoxide anion generation. We speculate that TNF-{alpha} and LPS also change some elements in fMLP-receptor coupling to the signal transduction machinery, thereby modulating the suppressive effect of adenosine on fMLP-induced functional responses. Thus, the physiological mechanisms that relate to the inhibition of PMN functional responses by adenosine in the context of acute inflammation remain to be clarified further.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and a senior scholarship from the Arthritis Society of Canada to S. B.

Received May 9, 2005; revised November 2, 2005; accepted November 15, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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