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Published as doi: 10.1096/fj.07-101097.
 (The FASEB Journal. 2008;22:3276-3286.)
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Differential expression of 5'-UTR splice variants of the adenosine A2A receptor gene in human granulocytes: identification, characterization, and functional impact on activation

Simone Kreth, Carola Ledderose, Ines Kaufmann, Gabriele Groeger and Manfred Thiel

Department of Anaesthesiology, Ludwig Maximilians University of Munich, Germany

1 Correspondence: Clinic of Anaesthesiology, Klinikum Grosshadern, Marchioninistr. 15, D-81375 Munich, Germany. E-mail: mthiel{at}med.uni-muenchen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A key step in the pathogenesis of sepsis is the excessive and uncontrolled activation of polymorphonuclear neutrophils (PMNs). An inflammation-controlling function of adenosine receptors has been presumed; however, their role in PMN of sepsis patients is poorly defined. We investigated the expression of adenosine receptors in resting and lipopolysaccharide (LPS) -activated human PMNs, and in PMNs of sepsis patients. Our studies revealed that native human PMNs express almost equal distributions of all four adenosine receptor subtype transcripts, whereas exclusively the A2A receptor (A2AR) was up-regulated in LPS-stimulated PMNs and PMNs of sepsis patients. As a possible mechanism, we identified and fully characterized eight 5'-untranslated region (UTR) splice variants of the A2AR gene resulting from alternative transcription and/or splicing of five noncoding exons. We report a differential, activation state-specific expression of 5'-UTR variants within the same cell type, indicating a new mechanism to modulate gene expression: In resting human PMNs, mainly A2AR transcripts with long 5'-UTRs are expressed, whereas in stimulated PMNs and PMNs of septic patients, short 5'-UTRs predominate. Transcripts with short 5'-UTRs are more efficiently translated into protein. The correlation between changes of transcript patterns and A2AR up-regulation offers interesting clues regarding the course of sepsis.—Kreth, S., Ledderose, C., Kaufmann, I., Groeger, G., Thiel, M. Differential expression of 5'-UTR splice variants of the adenosine A2AR gene in human granulocytes: identification, characterization, and functional impact on activation.


Key Words: sepsis • 5'-untranslated region • alternative transcripts


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POLYMORPHONUCLEAR NEUTROPHILS (PMNS) or leukocytes serve as the first line of innate defense against invading microorganisms. They are potent in their antimicrobial capacities but nonselective in their targets; therefore, PMN activation is a double-edged sword. PMNs fulfill the task of eradicating an infection or responding to local tissue injury at the cost of significant collateral damage (1) . This can cause life-threatening cell and tissue damage leading to severe inflammatory diseases such as sepsis, which comprises a complex clinical syndrome resulting from excessive and uncontrolled host responses to infection (2) . Therefore, the immune response must be tightly controlled by highly evolved and effective down-regulating mechanisms (3) .

The endogenous purine nucleoside adenosine has been shown to be physiologically involved in inhibiting activated immune cells and in protecting tissue from acute inflammatory damage (4) . Adenosine is released from metabolically active cells and accumulates in inflamed and damaged tissues (5) . It is a potent mediator that modulates numerous cell functions and exerts its effects via binding to four subtypes of G-protein coupled adenosine receptors: A1, A2A, A2B, and A3 (6) . Activation of high-affinity A1 and low-affinity A3 receptor inhibits adenylyl cyclase activity, whereas activation of high-affinity A2A and low-affinity A2B adenosine receptors causes accumulation of intracellular cAMP. Antiinflammatory effects of adenosine are predominantly mediated by A2A receptors (7 , 8) and, as a second-line mechanism, by A2B receptors (9 , 10) . Furthermore, some data indicate that the A3 subtype is also involved (11 12 13) . The extent of immunosuppression achieved by the activation of the adenosine signaling system depends not only on the extracellular adenosine levels but also on the expression of adenosine receptors, which has been shown to be cell type-specific (14) .

In this context it is noteworthy that—although an important topic with high clinical relevance—the expression of adenosine receptors by resting and stimulated leukocytes, and, in particular, by PMNs of sepsis patients, has not been analyzed conclusively before. Pharmacological and functional assays suggest that human PMNs express all four subtypes of adenosine receptors (15 16 17 18) . Concerning adenosine receptor mRNA expression, only semiquantitative data are available (19 , 20) , and uncertainties continue to exist: 1) whether all four subtypes are expressed on human PMNs; and 2) in which amounts the transcripts of adenosine receptor subtypes are expressed relative to each other.

Regulation of adenosine receptor expression in human PMNs has only been analyzed in in vitro stimulation assays so far. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and functional analyses, these data provided evidence that A2A receptor expression increases after stimulation with lipopolysaccharide (LPS) or with tumor necrosis factor {alpha} (TNF-{alpha}) (19) . The clinical relevance of these findings needs to be elucidated, particularly with regard to previously published studies that documented clinical importance of A2A receptor regulation in circulating blood cells of patients with congestive heart failure and in lung parenchyma of patients with chronic obstructive pulmonary disease (COPD) (21 , 22) .

Here, we provide evidence by real-time PCR that human PMNs express all four adenosine receptor subtypes in an almost equal relative distribution. After LPS stimulation, A2A receptor (A2AR) mRNA expression increases 5-fold, whereas the transcripts of A1, A2B, and A3 receptors do not change significantly. Furthermore, we investigated these issues in granulocytes of patients suffering from severe sepsis or septic shock. Expression patterns of adenosine receptor mRNA found in PMNs of sepsis patients are almost identical with those of in vitro-stimulated PMNs, which indicates a sepsis-related A2AR up-regulation. The molecular pathways underlying the observed changes in A2AR gene expression in human PMNs have never been investigated before. They may be due to control mechanisms at the transcriptional, post-transcriptional, or translational level. Prior studies have shown that in other cell types (e.g., mouse macrophages, primary human endothelial cells, human epithelial cells), LPS, interleukin-1 (IL-1), and TNF-{alpha} stimulate A2AR expression and that NF-{kappa}B and its regulatory sites may play an important role in this scenario (23 24 25) . However, direct evidence for NF-{kappa}B stimulating A2AR expression could only be given for IL-1 (26) . It could, therefore, not be excluded that further mechanisms in the regulation of A2AR expression exist. In the current study, we investigated the 5'-UTR of the A2AR gene as a possible gene expression regulating structure.

Upstream untranslated open reading frames [ORFs; 5'-untranslated region (UTR)] are well known to exert a major impact on gene expression and can serve as important regulatory elements (27) . 5'-UTR diversity frequently occurs in genes with critical biological functions, including G protein-linked receptors. Complex 5'-UTR exon structures have been described for the human melanocortin MC2, dopamine D3, and ADP-like GPR34 receptors (28 29 30) . For the human and the mouse A2AR, bioinformatic and molecular analyses suggested a complex organization of the 5'-UTR, with three variants of the noncoding exon 1 in the mouse and six exon 1 variants in the human gene and multiple transcription start sites (TSSs) (31) . While the murine exon 1 variants have been described in detail, the exact position, sequence, and boundaries of the human exons and the resulting transcripts have not yet been characterized. Moreover, the biological relevance needs to be proved.

In this study, the 5'-UTR of the A2AR was analyzed in human granulocytes. We revealed that human granulocytes express eight different A2AR transcripts, and we determined exactly the nucleotide sequence of the noncoding exons, including their 5' and 3' boundaries for the first time. Furthermore, we detected distinct changes in expression patterns taking place on PMN stimulation in vitro and in sepsis patients compared to healthy volunteers. On the basis of these findings, we assume that the 5'-UTR plays an important regulatory role in A2AR expression in human granulocytes and therefore may be an important regulatory mechanism influencing the development and the course of inflammatory diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient eligibility
The study was approved by the University of Munich Institutional Ethics Committee, and informed consent for blood withdrawal was obtained from healthy volunteers, from patients, or their relatives, respectively. Patients were considered eligible for this study if they met the criteria for severe sepsis or septic shock as defined by the members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee (32) . Healthy volunteers of comparable age and sex served as controls.

Preparation of blood peripheral neutrophils
Granulocytes were separated from whole blood of sepsis patients and from healthy volunteers as described previously (33) . Briefly, heparinized blood was mixed with an equal amount of dextran 60 (Deltaselect, Pfullingen, Germany), and erythrocytes were allowed to sedimentate for 45 min. The leukocyte-enriched supernatant was removed and centrifuged on a continuous Percoll density gradient. PMN leukocytes were harvested and washed with Hanks’ buffered salt solution (HBSS), and the number of cells was counted by a Coulter Counter®T540 (Coulter Electronics, Hialeah, FL, USA).

Cell stimulation
For LPS stimulation, cells were cultured in RPMI 1640 medium (Biofluids, Rockville, MD, USA) supplemented with 10% (v/v) fetal calf serum (FCS), 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1 mM HEPES, and 1 µg/ml LPS (Sigma-Aldrich GmbH, Taufkirchen, Germany) in 12-well plates at a density of 5 x 106 cells/well. After an incubation time of 6 h, cells were harvested by centrifugation and washed with HBSS.

Western blotting
Crude membrane preparations of native and LPS-treated granulocytes were obtained as described previously (34) . In brief, cells were washed, resuspended in ice-cold PBS, and disrupted by sonication (three 10-s periods) in the presence of complete protease inhibitors (Roche Diagnostics, Mannheim, Germany). Whole cells, nuclei, and other large debris were cleared by centrifugation at 300 g for 10 min at 4°C, and the crude membrane fraction of the cleared supernatant was separated by ultracentrifugation at 50,000 g for 30 min.

Protein concentrations were determined by Bradford protein assay (Bio-Rad Laboratories, München, Germany) with bovine serum albumin as standard. Samples (20 µg protein) were boiled for 5 min in 1x sample buffer containing 2-ME, electrophoresed on SDS-PAGE 4–10% acrylamide gels under reducing conditions using the Bio-Rad protein minigel system, and transferred to polyvinylidene difluoride membranes (Bio-Rad). Loading of protein samples was monitored by staining with Ponceau Red. After overnight blocking with 5% nonfat dry milk in 0.05% Tween 20 TBS, blots were incubated with monoclonal antibodies (Abs) against adenosine A2AR (clone 7F6-G5-A2; Upstate Biotechnology, Lake Placid, NY, USA); specificity was tested as described in ref. 35 . Ab-antigen (Ag) complexes were visualized using horseradish peroxidase (HRP) -conjugated Ab against mouse immunoglobulin G (IgG; Cell Signaling Technology, Danvers, MA, USA) and Femto chemiluminescent substrate (Pierce, Rockford, IL, USA). Prestained Protein Marker (Broad range 7720) was used and visualized with Anti-Biotin-Ab, HRP-linked (both from Cell Signaling Technology). To confirm equal loading, blots were reprobed with mouse mAb targeting β-actin (Cell Signaling Technology). The Gel-doc 2000 System (Bio-Rad Laboratories, München, Germany) was used for documentation and densitometric analysis of gel band intensities.

RNA isolation and cDNA reverse transcription
Total cellular RNA was isolated from native and LPS-treated granulocytes using the RNaequous Kit (Ambion, Austin, TX, USA) with subsequent DNase treatment (TURBO DNase, Ambion) following the manufacturer’s protocol. RNA was quantified using a spectrophotometer, and equal amounts from the different samples (1000 ng) were transcribed into cDNA. The RT reaction was performed using mixed random and oligo-dT primers and Superscript III RT (Invitrogen, Carlsbad, CA, USA), as per manufacturer’s instructions.

RT-PCR
RT-PCR was performed using the primer pairs listed in Table 1 (synthesized by MWG Biotech, Ebersberg, Germany). The following cycling conditions were applied: 94°C for 5 min denaturing; 35 cycles of 94°C for 30 s, 57°C for 20 s, 72°C for 30 s; and a final extension at 72°C for 10 min. The PCR products were separated on 2% agarose gels and stained with ethidium bromide.


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  		Table 1. Primer sequences

Real-time PCR
Real-time quantitative PCR (qPCR) was performed in duplicates with the Light Cycler480 instrument (Roche Diagnostics, Mannheim, Germany). For adenosine receptor quantification, Roche’s qPCR Mastermix and highly specific fluorogenic primer-probe sets synthesized by Primerdesign (Southampton, UK) were used. The thermal cycler conditions comprised 45 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 15 s.

Quantification of adenosine A2AR splice variants was performed using Roche’s SYBR green and primer sets listed in Table 1 (synthesized in HPLC grade quality by MWG Biotech). Cycling conditions were 45 cycles of 95°C for 10 s, 57°C for 8 s, and 72°C for 8 s.

Relative mRNA expression was calculated with the Relative Quantification software (Roche Diagnostics), using an efficiency-corrected algorithm based on the mathematical model of Pfaffl (36) , with standard curves and reference gene normalization against 18s rRNA. 18s rRNA was determined to be an appropriate gene for normalization, as described in ref. 37 .

5'-Rapid amplification of cDNA ends (RACE)
5'-RACE reactions were performed with 5 µg total RNA starting material using the FirstChoice RLM-RACE kit (Ambion). Gene-specific primers for 5'-RACE (outer primer) and nested PCR (inner primer) reactions are given in Table 2 . RNA processing and adaptor ligations were performed according to the manufacturer’s instructions. Outer and inner 5'-RLM-RACE-PCRs were performed using Ambion’s SuperTaq DNA polymerase and cycling conditions as follows: after an initial denaturation for 3 min at 94°C, 35 cycles at 94°C for 30 s, 62°C (or 63°C) for 30 s, and 72°C for 30 s were performed, followed by a final extension at 72°C for 7 min in a DNA MasterCycler (Eppendorf, Hamburg, Germany). PCR products were visualized on 2% agarose gels, and fragments of interest were cloned into the PCR-TOPO Vector (Invitrogen).


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  		Table 2. Characteristics of A2AR transcript isoforms

Coupled transcription/translation
A fragment covering the full-length A2AR mRNA sequence from exon 2 to the 3'-UTR was amplified using a cDNA prepared from human PMNs and primers A2AR-For and -Rev (listed in Table 1 ), introducing an XbaI restriction site in the 5' end and a NotI restriction site in the 3' end. The fragment was cloned into the pTNT vector (Promega, Madison, WI, USA) using the restriction sites KpnI and NotI in the vector’s multiple cloning site. The 5'-UTR sequences were amplified using a cDNA prepared from native and LPS-stimulated human PMNs and combinations of exon 1 specific primers (listed in Table 1 ), thereby introducing an XhoI site in the 5' end and an XbaI site in the 3' end of all PCR products. The obtained 5'-UTRs were ligated into the restriction sites XhoI and XbaI of the pTNT-A2AR vector construct. All vector constructs were verified by sequencing analysis. Coupled in vitro transcription and translation was performed on these plasmids using a TNT Quick Coupled Transcription/Translation System (Promega) and both the TranscendTM Non-Radioactive Translation Detection System (Promega) and 35[S]-methionine, according to the manufacturer’s instructions. The biotinylated products (2 µl of 50 µl translation reaction each) were separated by 10% SDS-PAGE and visualized by binding of Streptavidin-HRP (Promega), or, in case of radioactive labeling, products were analyzed by exposing the SDS-PAGE to a PhosphorImager screen (analysis with PhosphorImager CycloneTM, Perkin Elmer, Waltham, MA, USA). The in vitro translated products (12.5 µl of each translation reaction) were also subjected to Western blot analysis as described above. The amount of plasmids used (1 µg) was within a range of saturating conditions where the outcome of the transcription/translation reactions was independent of the differences in target DNA concentration introduced by the different lengths of the 5'-UTRs.

Sequencing analysis
Cloned fragments were analyzed by automatic sequencing using the ABI PRISM Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA, USA) and an automatic sequencer (ABI PRISM Cycle sequencing, Perkin Elmer, Boston, MA, USA) and sequencing files were analyzed with Sequencher 4.0.5 software (Gene Codes Corp., Ann Arbor, MI, USA). Nucleotide sequences were compared using the basic local alignment search tool (BLAST; National Center for Biotechnology Information, Bethesda, MD, USA), and nucleotide sequences were submitted to GenBank (accession numbers shown in Table 2 ).

Statistical analyses
All data were analyzed using SigmaPlot 9.0 and SigmaStat 3.5 software (Systat Software, Inc., San Jose, CA, USA). If otherwise not stated, values given represent means ± SE. Intergroup comparisons were performed by unpaired t test or Mann-Whitney test, if data were normally or not normally distributed, respectively. Two-tailed levels of statistical significance are indicated by values of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In human PMNs, A2AR mRNA and protein expression is significantly increased after stimulation with LPS
To evaluate mRNA expression of the A1, A2A, A2B, and A3 receptors in resting human PMNs, we performed real-time PCR experiments with highly specific fluorogenic primer-probe sets (sequences are given in Table 1 ). We detected transcripts of all four subtypes of adenosine receptors in an almost equal proportion, with the A3R mRNA being slightly more abundant than that of the other three (Fig. 1 A).


Figure 1
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  		Figure 1. LPS-stimulation increases A2AR mRNA and protein expression in human granulocytes. A) Adenosine receptor mRNA levels in native human granulocytes, as assessed by real-time quantitative RT-PCR. RNA was prepared from native PMNs and subsided to quantitative RT-PCR. Data were calculated relative to 18sRNA using an efficiency-corrected algorithm. Results are expressed as the mean ± SE percentage of total adenosine receptor mRNA analyzed in triplicate determinations in 7 independent experiments performed with different donors. B) RNA was prepared from both native and LPS-stimulated (1 µg/ml LPS for 6 h) human PMNs of the same donor and subsided to quantitative RT-PCR. Data were calculated relative to 18sRNA using an efficiency-corrected algorithm. Results are expressed as the mean ± SE change of subtype-specific adenosine receptor mRNA relative to native PMNs, analyzed in triplicate determinations in 6 independent experiments performed with 6 different donors. *P < 0.01 vs. native PMNs. C) Membrane fractions from both native and LPS-stimulated (1 µg/ml LPS for 6 h) human PMNs of the same donor were prepared, and protein content was determined. Equal amounts were analyzed by Western blotting using an A2AR protein- and a β-actin specific monoclonal Ab. A representative Western blot experiment (n=4) is shown. D) Densitometric quantification of A2AR protein expression. Results are expressed as the mean ± SE percentage of A2AR protein expression; 4 independent experiments. *P < 0.01 vs. native PMNs.

Treatment with LPS at 1 µg/ml for at least 5 h induced a more than 4-fold increase of A2AR mRNA (4.2±0.25, n=6; P>0.01), whereas transcripts of A1R, A2BR, and A3R did not change significantly (Fig. 1B ). Extending LPS exposure up to 20 h did not lead to any further increase of adenosine receptor transcript levels. With respect to granulocyte viability, stimulation times longer than 20 h proved to be not applicable under our experimental conditions (data not shown).

To determine whether the observed LPS-induced increase in A2AR mRNA correlates with the A2AR protein expression, membrane fractions of native and LPS-stimulated cell were analyzed by Western blot using a monoclonal anti-A2AR Ab. As shown in Fig. 1C, an immunoreactive band at 45 kDa was detected in native and LPS-treated PMNs; a 2.3-fold increase in A2AR protein expression (2.3±0.5, n=5; P<0.01) was detected in PMNs incubated with LPS for 6 h (Fig. 1D ).

Granulocytes of sepsis patients express elevated levels of A2AR mRNA
To assess whether the changes in adenosine receptor mRNA expression on stimulation found in vitro have functional implications for inflammatory diseases in vivo, we analyzed PMNs of patients suffering from severe sepsis or septic shock (n=6). Expression patterns of adenosine receptor mRNA found in granulocytes of sepsis patients are highly similar to those of in vitro LPS-stimulated PMNs of healthy volunteers: A2AR mRNA is the predominantly expressed adenosine receptor transcript (5-fold more than A1R), followed by A2BR mRNA (2.5-fold more than A1R) and equal amounts of A1R and A3R mRNA (Fig. 2 ). These data indicated a sepsis-related A2AR up-regulation and justified analyses concerning possible mechanisms underlying these transcriptional changes.


Figure 2
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  		Figure 2. Granulocytes of sepsis patients express elevated levels of A2AR mRNA. RNA was prepared from PMNs of sepsis patients (n=6) and subsided to quantitative RT-PCR. Data were calculated relative to 18sRNA using an efficiency-corrected algorithm. Results are expressed as the mean percentage of total adenosine receptor mRNA analyzed in triplicate determinations. The relative distribution of adenosine receptor subtypes in PMNs of sepsis patients is shown (right), as compared to native and LPS-stimulated PMNs of healthy persons (left and middle).

In human PMNs, the 5'-UTR of the A2AR gene consists of 5 noncoding exons that lead to the expression of 8 different transcripts
For the human adenosine A2AR, a complex 5'-UTR with multiple upstream noncoding exons is described. Using 5'-RACE and RT-PCR experiments on human PMN RNA (RACE-primers are given in Table 1 ), we identified 5 variants of the noncoding exon 1 (designated E1A, E1B, E1C, E1D, and E1E) being expressed in human granulocytes (Fig. 3 B). We found 8 distinct A2AR transcripts in which the 5 different exon 1 variants alone or in combination are fused to a common splice acceptor region (position –273, relative to ATG at position 0), and to a common 2100 bp A2AR ORF (Fig. 3A ). Since leader exon variation is considered a manifestation of alternative promoter usage, mRNA transcripts consisting of a single exon 1 indicate the existence of different alternative promoters and TSSs. We also found mRNAs including more than one exon 1 sequence that are likely to be generated by alternative splicing. In transcripts E1Bshort and E1Cb, the leader exon is spliced to exon A, which then is spliced to the common acceptor site in exon 2 (Fig. 3A ). Furthermore, two unspliced A2AR transcripts exist (E1Along, found by 5'-RACE, and E1Blong, identified by RT-PCR and direct sequencing), both containing the complete genomic region spanning between exon E1A and E2 and E1B and E2, respectively.


Figure 3
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  		Figure 3. Genomic structure of the human A2AR and mRNA transcripts expressed in human granulocytes. A) The 5'-UTR of the A2AR gene consists of at least 5 different exon 1 variants: E1A, E1B, E1C, E1D, and E1E (size and genomic positions relative to ATG at position 0 are shown). These exon 1 variants are spliced (alone or in combination) to a common exon 2 splice acceptor region (position –273, relative to ATG at position 0), and to a common 2100 bp A2AR ORF that is located in exon 2. In human PMNs, 8 different A2AR transcripts could be detected: 1) 4 short transcripts in which the different exon 1 variant alone is fused to the common splice acceptor region; 2) 2 transcripts, including more than one exon 1 sequence: in transcripts E1Bshort and E1Cb, the leader exon is spliced to exon A, which then is spliced to the common acceptor site; and 3) 2 unspliced A2AR transcripts (E1Along and E1Blong) both containing the complete genomic region spanning between exon E1A and E2 and E1B and E2, respectively. B) 5'-RACE analysis of A2AR mRNA. mRNAs from native and LPS-stimulated human PMNs were subjected to 5'-RACE analysis. The PCR products were resolved on 2% agarose gel and stained with ethidium bromide. PCR products were subcloned into TOPO, and sequenced. MM, molecular marker; N, native PMNs; S, LPS-stimulated PMNs.

In human PMNs, the A2AR mRNA expression pattern distinctly changes after stimulation with LPS
To elucidate the physiological significance of the A2AR 5'-UTR isoforms, the expression of these mRNA variants was evaluated in human granulocytes by conventional RT-PCR. In each amplification reaction, a forward primer targeting a single exon 1 variant was combined with a common reverse primer targeted to exon 2. Design of primer pairs was based on the sequences obtained by the RACE experiments. Primer sequences are depicted in Table 1 , and all PCR products have been confirmed by DNA sequencing. As shown in Fig. 4 A, resting granulocytes of healthy persons mainly express the long versions of exon E1A and E1B (both containing the complete genomic region spanning between exon E1 and E2, including those regions that seem to be recognized as "intronic" in all other mRNA variants and therefore are spliced out) and exon E1D and E1E. No PCR bands, or only some that were extremely weak, were detectable for the short isoforms of exon E1A and E1B (E1Ashort, E1B short) and both E1C variants. Treatment with LPS for 6 h not only led to an increase in overall A2AR mRNA as described above, it also induced distinct changes in the A2AR mRNA expression pattern: transcription of E1Ashort, E1Bshort, and both E1C isoforms was induced, and expression of E1D appeared to be enhanced (Fig. 4A , right panel).


Figure 4
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  		Figure 4. A2AR mRNA expression patterns of human PMNs distinctly change after stimulation with LPS. A2AR transcripts of human PMNs were analyzed by conventional RT-PCR and real-time PCR using transcript-specific primer pairs. Therefore, RNA was prepared from both native and LPS-stimulated (1 µg/ml LPS for 6 h) human PMNs of the same donor; 6 independent experiments with 6 different donors were performed. A) RT-PCR with exon-specific primer pairs. The PCR products were resolved on 2% agarose gel and stained with ethidium bromide. A representative agarose gel is shown. MM, molecular marker; C1, control PCR reaction performed with primer pair E1A and not reverse-transcribed RNA as a template to check for undigested DNA; C2 and C3, control RT-PCR reactions with primer pairs specific for β-actin with cDNA from native and stimulated PMNs, respectively, to check for equal template amounts. B, C) Adenosine A2AR transcripts in native (B) and LPS-stimulated (C) human granulocytes, as assessed by real-time quantitative RT-PCR. Data were calculated relative to 18sRNA using an efficiency-corrected algorithm. Results are expressed as the mean ± SE percentage of total A2AR mRNA analyzed in triplicate determinations in 6 independent experiments performed with different donors. n.d., not detectable.

Next, we performed real-time PCR experiments to determine exactly the proportion of the A2AR splice variants in resting human PMNs and to quantify the changes occurring on LPS-stimulation. To distinguish between the short and the long variant of E1A, E1B, and E1C transcript isoforms, the following methods were applied: 1) short PCR extension times were used that specifically amplified the short isoforms (verified by agarose gel electrophoresis), and 2) additional reverse primers were designed that specifically amplified the long transcript version. Primer sequences are diagrammed in Table 1 . Quantification of A2AR transcripts revealed exon E1Along to be predominantly expressed in resting human PMNs (45%), followed by E1Blong (21%), E1D (11%), and E1E (9%). Figure 4B shows the relative distribution of all detectible A2AR transcripts. After stimulation with LPS, transcription of all detectable mRNAs increased, due to the transcriptional A2AR up-regulation shown above. However, the extent of the increase differed considerably for each splice variant, leading to a completely changed A2AR mRNA expression pattern. Figure 4C shows the mean percentage of transcript isoforms of total A2AR mRNA: all exon 1 variants are expressed in an almost equal proportion, with exon E1Ashort being slightly more abundant (14%) than the other isoforms, and E1Bshort being the rarest transcript (9%).

A2AR mRNA expression patterns are highly similar in granulocytes of sepsis patients and in LPS-stimulated cells of healthy volunteers
The in vitro experiments led to the conclusion that in human PMNs, a shift from splice variants of the A2AR with long 5'-UTRs to those with relatively short 5'-UTRs takes place on LPS stimulation. We now investigated these issues in PMNs of patients suffering from severe sepsis or septic shock (n=6), which all exhibited A2AR mRNA expression patterns highly similar to those of in vitro LPS-stimulated PMNs of healthy persons (Fig. 5 A). Transcripts with short 5'-UTRs are the predominant mRNA species. Quantification of all transcripts proved an E1Along expression of 20% and almost equal proportions of E1Ashort, (15%), E1D (14%), and E1E (13%), followed by E1Cb (11%), E1Ca (9%), and E1Bshort (7%) (Fig. 5B ).


Figure 5
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  		Figure 5. In granulocytes of sepsis patients, A2AR mRNA expression patterns distinctly differ from those of healthy volunteers. RNA was prepared from PMNs of sepsis patients (n=6), and A2AR transcripts were analyzed by conventional RT-PCR and real-time PCR using transcript-specific primer pairs. A) RT-PCR with exon-specific primer pairs. The PCR products were resolved on 2% agarose gel and stained with ethidium bromide. A representative agarose gel is shown. MM, molecular marker. B) Adenosine A2AR transcripts in granulocytes of sepsis patients, as assessed by real-time quantitative RT-PCR. Data were calculated relative to 18sRNA using an efficiency-corrected algorithm. Results are expressed as the mean ± SE percentage of total A2AR mRNA analyzed in triplicate determinations in 6 independent experiments performed with different donors.

Translation efficiency of A2AR 5'-splice variants
The impact of different 5'-UTRs on the translation efficiency was analyzed using coupled transcription/translation in a reticulocyte lysate system. Plasmids harboring 7 different 5'-UTRs (denoted 5'-UTR: A-E in Fig. 3A ) placed in front of the A2AR coding region were analyzed. As both C transcripts were always found to be expressed in an equal proportion, Cb was regarded to be representative for both C-transcript isoforms. Translation products were resolved by SDS-PAGE followed by different analytic procedures. Biotinylated translation products were visualized by chemiluminescence, 35[S]-labeled products were analyzed with a PhosphorImager system, and translation reactions without labeling were subjected to Western blot analysis using an A2AR-specific Ab. In all cases, protein bands of compatible sizes (~45 kDa) were detectable. It clearly could be shown that transcripts with short 5'-UTRs (Ashort, Bshort, D, and E, with an emphasis on Ashort, D, and E) are more efficiently translated into protein than those with long 5'-UTRs (Fig. 6 ). Transcript isoform Cb exhibited almost no protein translation in all experiments, suggesting a minor role of the C-transcript isoforms in the regulation of A2AR expression. Low overall translation efficiencies required long exponation times, leading to a strong unspecific background. Therefore, reliable quantification of the translated protein was not possible.


Figure 6
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  		Figure 6. Transcripts with extended 5'-UTRs are less efficiently translated into protein. Plasmids harboring the different 5'-UTRs described in Fig. 3 A placed in front of the A2AR coding region were analyzed by in vitro coupled transcription/translation analysis. As both C transcripts were always found to be expressed in an equal proportion, Cb was regarded to be representative for both C-transcript isoforms. A representative Western blot analysis (n=3) of the products of the translation reactions probed with a monoclonal A2AR protein-specific Ab is shown; 12.5 µl samples of the translation reaction products were loaded on the gel.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A key step in the pathogenesis of sepsis is the excessive and uncontrolled activation of PMNs. An inflammation-controlling function of adenosine receptors has been presumed;

Immunomodulatory effects of adenosine via occupancy of adenosine receptors are numerous. Expression and regulation of adenosine receptors in human PMNs have not been analyzed conclusively so far; in particular, their role in PMNs of sepsis patients is poorly defined. In the present study, we show that native human PMNs express all four subtypes of adenosine receptor mRNA in an almost equal relative proportion. After stimulation with LPS for 6 h, A2AR mRNA expression increases 5-fold, whereas the transcripts of A1, A2B, and A3 receptors do not change significantly, and amounts of A2AR protein increase 2.6-fold. A2ARs have been conclusively implicated in the natural antiinflammatory mechanisms and are well known to inhibit overactive immune cells during acute inflammation, which limits inflammatory collateral tissue damage (8 , 38) . Insofar, it is not surprising that A2AR expression in human PMNs increases on stimulation. The need to develop a maximum inflammatory response to infectious agents on the one hand and the necessity to protect tissues from an overwhelming immune response on the other hand may in this way be kept in balance. The observed changes in A2AR mRNA expression may be due to transcriptional control mechanisms, which have not been investigated so far.

The human A2AR gene consists of two coding exons separated by a single intron leading to the translation of a 45 kDa protein. In view of this genetic organization, the existence of multiple mRNA transcripts with varying regulatory susceptibility appears to be unlikely. In the current study, we investigated the 5'-UTR of the A2AR gene in human PMNs as a possible gene expression regulating structure, and we showed that the genetic structure of the human A2AR is more complex than previously reported. The 5'-UTR of eukaryotic mRNAs plays a crucial role in the post-transcriptional regulation of gene expression through the modulation of translation efficiency, message stability, and subcellular localization. Structured 5'-UTRs frequently occur in certain classes of genes, including many genes involved in the control of cellular growth, differentiation, embryonic development, and stress (39 40 41) . For the human and the murine A2AR, such a complex organization of the 5'-UTR was suggested by Yu et al. (31) in a single report using bioinformatic and RT-PCR analyses. While the murine 5'-untranslated exons were described in detail, including the complete sequence data, uncertainties continue to exist as to the exact position, the nucleotide sequence, and the 5' and 3' boundaries of the human exons. Furthermore, neither have the resulting transcripts been characterized nor the biological relevance of these findings proved.

In the present study, we have identified and fully characterized the A2AR 5'-UTR in human granulocytes. Using 5'-RACE and genomic sequencing, 5 distinct untranslated exons (E1A, E1B, E1C, E1D, and E1E) were found, spanning over 9.4 kb on chromosome 22. All of the exon 1 transcript variants have a unique splice donor site and share a common splice acceptor site in exon 2. Since leader exon variation is considered a manifestation of alternative promoter usage (42 , 43) , transcripts consisting of a single exon 1 are most likely be generated by the use of different alternative promoters and TSSs. We have also identified mRNAs including more than one exon 1 sequence; those may be generated by alternative promoter usage plus alternative splicing. Because the 5'-UTR part downstream from the splice acceptor site contains an inframe TGA stop codon located 21 bp upstream from the ATG translation start site, all the different transcripts do not alter the coding sequence of the A2AR protein, but they may serve as regulators of gene expression.

Initiation of translation is one of the most important steps that influences the level of gene expression, and—based on the scanning model for translation—an efficient translation needs 5'-UTRs that are moderately short, have a low GC content (<65%), are unstructured, and do not contain upstream AUGs (uAUGs) (38 , 44 , 45) . We analyzed the 8 A2AR 5'-UTR variants with respect to these features using the Mfold algorithm (46) to identify complex secondary structures and to calculate the Gibbs free energy values. As shown in Table 2 , the splice variants Along and Blong, which are predominantly expressed in resting human granulocytes, have features likely to adversely impact translation, most notably extensive secondary structures (as reflected in free energy values), and the presence of many uAUGs. Therefore, translation efficiency of these mRNA variants is expected to be rather poor. After stimulation with LPS for 6 h, transcripts with significantly shorter 5'-UTRs bearing features that may positively influence translation (such as lower GC contents and free energy values and fewer uAUGs) are the predominant mRNA species, whereas the expression of Along and Blong is considerably decreased. The short variants D and E are expressed in both native and stimulated PMNs with a proportion of 9–14%, which indicates that these short transcripts may fulfill "housekeeping" functions to ensure basal A2AR expression levels in native PMNs.

We demonstrated that all of these findings obviously can be transferred directly to sepsis, which is characterized by excessive activation of granulocytes: PMNs of sepsis patients exhibit elevated levels of A2AR mRNA, with transcript expression patterns predominantly containing the short 5'-UTR splice variants. This finding suggests that the presently reported distinct changes of A2AR mRNA expression taking place on granulocyte activation are regulated by activation state-specific alternative promoter usage and/or splicing. Because the A2AR is well known to "put the brake on inflammation" (47) , we suppose that these complex patterns of regulatory mechanisms to control the expression of this gene are a key point in the clinical course of sepsis.

Generally, splice variants of untranslated leader exons are associated with alternative promoter usage, which is well known to result in tissue- or cell type-specific gene expression. To the best of our knowledge, this is the first report demonstrating a differential, activation state-specific expression of 5'-UTR variants within the same cell type, which indicates a new, previously not considered possibility to achieve diversity of gene expression.

The exact determination of translation efficiencies of the 8-splice variants found in human PMNs remains elusive because primary human granulocytes are barely suitable for transfection experiments. Even the recently reported new method of nucleofection resulted in poor transfection efficiencies, which restricted subsequent analyses to experiments that use single cells (48) . Transferred to our experimental setting, these limitations would be further complicated by the necessity of stimulation with LPS for several hours, requiring fully viable cells as a prerequisite. Therefore, reporter gene assays to determine the exact translation efficiency of the A2AR transcripts in human PMNs are not a promising approach within the framework of this study. The same applies to experiments with the cell lines HEK293 and HL60, which either lack the TLR4 (49) or possess incomplete LPS-responsive signaling pathways (50) and are therefore not representative for the questions analyzed here. To obtain at least indirect experimental support for the analytical data, we performed in vitro transcription/translation studies with reticulocyte lysate. As expected, the translation efficiency of the A2AR protein (as a low abundant receptor protein) in its "physiological" genetical surrounding leads to low translation efficiencies, which did not allow a reliable densitometric quantification. However, Western blots of the translation reaction clearly showed that transcripts with short 5'-UTRs are more efficiently translated into protein. In particular, the transcript isoforms Ashort, D, and E appear to be most important in the up-regulation of A2AR expression.

Further extensive studies focusing on the analysis of sepsis patients are required to elucidate the exact relation between A2AR expression patterns and clinical course of sepsis. If a correlation exists among course, duration, and prognosis of the disease and differential expression of 5'-UTRs, the determination of A2AR transcripts may be a promising and easy-to-handle new prognostic marker, which might be a useful clinical tool to improve the treatment concepts of sepsis.

In summary, the present study suggests that in sepsis as an example for human inflammatory diseases, transcriptional and/or splicing mechanisms of the A2AR found in activated PMNs are altered from transcripts with long to those with short 5'-UTRs, which contributes to up-regulation of A2AR gene expression. This may be an important event determining the further course of the disease by limiting the inflammatory process and thereby preventing further collateral tissue damage.


   ACKNOWLEDGMENTS
 
The authors declare no competing financial interests.

Received for publication November 1, 2007. Accepted for publication May 8, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hoesel, L. M., Neff, T. A., Neff, S. B., Younger, J. G., Olle, E. W., Gao, H., Pianko, M. J., Bernacki, K. D., Sarma, J. V., Ward, P. A. (2005) Harmful and protective roles of neutrophils in sepsis. Shock 24,40-47[Medline]
  2. Peters, K., Unger, R. E., Brunner, J., Kirkpatrick, C. J. (2003) Molecular basis of endothelial dysfunction in sepsis. Cardiovasc. Res. 60,49-57[Abstract/Free Full Text]
  3. Sitkovsky, M. V., Lukashev, D., Apasov, S. H., Koshiba, M., Caldwell, C., Ohta, A., Thiel, M. (2004) Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu. Rev. Immunol. 22,657-682[CrossRef][Medline]
  4. Hasko, G., Cronstein, B. N. (2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 25,33-39[CrossRef][Medline]
  5. Cronstein, B. N., Levin, R. I., Belanoff, J., Weissmann, G., Hirschhorn, R. (1986) Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells. J. Clin. Invest. 78,760-770[Medline]
  6. Fredholm, B. B., IJzerman, A. P., Jacobson, K. A., Klotz, K. N., Linden, J. (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53,527-552[Abstract/Free Full Text]
  7. Ohta, A., Sitkovsky, M. V. (2001) Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 414,916-920[CrossRef][Medline]
  8. Sitkovsky, M. V., Ohta, A. (2005) The ‘danger’ sensors that STOP the immune response: the A2 adenosine receptors?. Trends Immunol. 26,299-304[CrossRef][Medline]
  9. Linden, J. (2006) New insights into the regulation of inflammation by adenosine. J. Clin. Invest. 116,1835-1837[CrossRef][Medline]
  10. Yang, D., Zhang, Y., Nguyen, H. G., Koupenova, M., Chauhan, A. K., Makitalo, M., Jones, M. R., St. Hilaire, C., Seldin, D. C., Roselle, P., Lambert, E., Schreiber, B. M., Guavas, H., Wagner, D. D., Raved, K. (2006) The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J. Clin. Invest. 116,1913-1923[CrossRef][Medline]
  11. Gessi, S., Varani, K., Merighi, S., Cattabriga, E., Iannotta, V., Leung, E., Baraldi, P. G., Borea, P. A. (2002) A3 adenosine receptors in human neutrophils and promyelocytic HL60 cells: a pharmacological and biochemical study. Mol. Pharmacol. 61,415-424[Abstract/Free Full Text]
  12. Gomez, G., Sitkovsky, M. V. (2003) Differential requirement for A2a and A3 adenosine receptors for the protective effect of inosine in vivo. Blood 102,4472-4478[Abstract/Free Full Text]
  13. Salvatore, C. A., Tilley, S. L., Latour, A. M., Fletcher, D. S., Koller, B. H., Jacobson, M. A. (2000) Disruption of the A3 adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J. Biol. Chem. 275,4429-4434[Abstract/Free Full Text]
  14. Linden, J. (2001) Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annu. Rev. Pharmacol. Toxicol. 41,775-787[CrossRef][Medline]
  15. Bouma, M. G., Jeunhomme, T. M., Boyle, D. L., Dentener, M. A., Voitenok, N. N., van den Wildenberg, F. A., Buurman, W. A. (1997) Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors. J. Immunol. 158,5400-5408[Abstract]
  16. Cronstein, B. N., Daguma, L., Nichols, D., Hutchison, A. J., Williams, M. (1990) The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively. J. Clin. Invest. 85,1150-1157[Medline]
  17. Falleni, A., Trincavelli, M. L., Macchia, M., Salvetti, F., Hamdan, M., Calvani, F., Gremigni, V., Lucacchini, A., Martini, C. (1999) A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization. J. Cell. Biochem. 75,235-244[CrossRef][Medline]
  18. Fredholm, B. B., Zhang, Y., van der Ploeg, I. (1996) Adenosine A2A receptors mediate the inhibitory effect of adenosine on formyl-Met-Leu-Phe-stimulated respiratory burst in neutrophil leucocytes. Naunyn Schmiedebergs Arch. Pharmacol. 354,262-267[Medline]
  19. Fortin, A., Harbour, D., Fernandes, M., Borgeat, P., Bourgoin, S. (2006) Differential expression of adenosine receptors in human neutrophils: up-regulation by specific Th1 cytokines and lipopolysaccharide. J. Leukoc. Biol. 79,574-585[Abstract/Free Full Text]
  20. Zhang, N., Yang, D., Dong, H., Chen, Q., Dimitrova, D. I., Rogers, T. J., Sitkovsky, M., Oppenheim, J. J. (2006) Adenosine A2a receptors induce heterologous desensitization of chemokine receptors. Blood 108,38-44[Abstract/Free Full Text]
  21. Varani, K., Laghi-Pasini, F., Camurri, A., Capecchi, P. L., Maccherini, M., Diciolla, F., Ceccatelli, L., Lazzerini, P. E., Ulouglu, C., Cattabeni, F., Borea, P. A., Abbracchio, M. P. (2003) Changes of peripheral A2A adenosine receptors in chronic heart failure and cardiac transplantation. FASEB J. 17,280-282[Abstract/Free Full Text]
  22. Varani, K., Caramori, G., Vincenzi, F., Adcock, I., Casolari, P., Leung, E., Maclennan, S., Gessi, S., Morello, S., Barnes, P. J., Ito, K., Chung, K. F., Cavallesco, G., Azzena, G., Papi, A., Borea, P. A. (2006) Alteration of adenosine receptors in patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 173,398-406[Abstract/Free Full Text]
  23. Murphree, L. J., Sullivan, G. W., Marshall, M. A., Linden, J. (2005) Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-kappaB in A(2A) adenosine receptor induction. Biochem. J. 391,575-580[CrossRef][Medline]
  24. Khoa, N. D., Montesinos, M. C., Reiss, A. B., Delano, D., Awadallah, N., Cronstein, B. N. (2001) Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells. J. Immunol. 167,4026-4032[Abstract/Free Full Text]
  25. Khoa, N. D., Montesinos, M. C., Williams, A. J., Kelly, M., Cronstein, B. N. (2003) Th1 Cytokines regulate adenosine receptors and their downstream signaling elements in human microvascular endothelial cells. J. Immunol. 171,3991-3998[Abstract/Free Full Text]
  26. Morello, S., Ito, K., Yamamura, S., Lee, K. Y., Jazrawi, E., Desouza, P., Barnes, P., Cicala, C., Adcock, I. M. (2006) IL-1 beta and TNF-alpha regulation of the adenosine receptor (A2A) expression: differential requirement for NF-kappa B binding to the proximal promoter. J. Immunol. 177,7173-7183[Abstract/Free Full Text]
  27. Ayoubi, T. A., Van De Ven, W. J. (1996) Regulation of gene expression by alternative promoters. FASEB J. 10,453-460[Abstract]
  28. Anney, R. J., Rees, M. I., Bryan, E., Spurlock, G., Williams, N., Norton, N., Williams, H., Cardno, A., Zammit, S., Jones, S., Jones, G., Hoogendoorn, B., Smith, K., Hamshere, M. L., Coleman, S., Guy, C., O'Donovan, M. C., Owen, M. J., Buckland, P. R. (2002) Characterisation, mutation detection, and association analysis of alternative promoters and 5' UTRs of the human dopamine D3 receptor gene in schizophrenia. Mol. Psychiatry 7,493-502[CrossRef][Medline]
  29. Engemaier, E., Rompler, H., Schoneberg, T., Schulz, A. (2006) Genomic and supragenomic structure of the nucleotide-like G-protein-coupled receptor GPR34. Genomics 87,254-264[CrossRef][Medline]
  30. Noon, L. A., Bakmanidis, A., Clark, A. J., O'Shaughnessy, P. J., King, P. J. (2006) Identification of a novel melanocortin 2 receptor splice variant in murine adipocytes: implications for post-transcriptional control of expression during adipogenesis. J. Mol. Endocrinol. 37,415-420[Abstract/Free Full Text]
  31. Yu, L., Frith, M. C., Suzuki, Y., Peterfreund, R. A., Gearan, T., Sugano, S., Schwarzschild, M. A., Weng, Z., Fink, J. S., Chen, J. F. (2004) Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses. Brain Res. 1000,156-173[CrossRef][Medline]
  32. Bone, R. C., Balk, R. A., Cerra, F. B., Dellinger, R. P., Fein, A. M., Knaus, W. A., Schein, R. M., Sibbald, W. J. (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 101,1644-1655[CrossRef][Medline]
  33. Thiel, M., Imendorffer, S., Chouker, A., Groh, J., Briegel, J., Anthuber, M., Kramling, H. J., Arfors, K. E., Peter, K., Messmer, K. (1998) Expression of adhesion molecules on circulating polymorphonuclear leukocytes during orthotopic liver transplantation. Hepatology 28,1538-1550[CrossRef][Medline]
  34. Khoa, N. D., Postow, M., Danielsson, J., Cronstein, B. N. (2006) Tumor necrosis factor-alpha prevents desensitization of Galphas-coupled receptors by regulating GRK2 association with the plasma membrane. Mol. Pharmacol. 69,1311-1319[Abstract/Free Full Text]
  35. Haschemi, A., Wagner, O., Marculescu, R., Wegiel, B., Robson, S. C., Gagliani, N., Gallo, D., Chen, J. F., Bach, F. H., Otterbein, L. E. (2007) Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages. J. Immunol. 178,5921-5929[Abstract/Free Full Text]
  36. Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29,e45[Abstract/Free Full Text]
  37. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 18,research0034.1-0034.11
  38. Pickering, B. M., Willis, A. E. (2005) The implications of structured 5' untranslated regions on translation and disease. Semin. Cell. Dev. Biol. 16,39-47[CrossRef][Medline]
  39. Kos, M., Denger, S., Reid, G., Gannon, F. (2002) Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha. J. Biol. Chem. 277,37131-37138[Abstract/Free Full Text]
  40. Payton, S. G., Haska, C. L., Flatley, R. M., Ge, Y., Matherly, L. H. (2007) Effects of 5' untranslated region diversity on the posttranscriptional regulation of the human reduced folate carrier. Biochim. Biophys. Acta 1769,131-138[Medline]
  41. Walsh, N. C., Cahill, M., Carninci, P. J., Okazaki, Y., Hayashizaki, Y., Hume, D. A., Cassady, A. I. (2003) Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene. Gene 307,111-123[CrossRef][Medline]
  42. Van der Velden, A. W., Thomas, A. A. (1999) The role of the 5' untranslated region of an mRNA in translation regulation during development. Int. J. Biochem. Cell Biol. 31,87-106[CrossRef][Medline]
  43. Nakanishi, T., Bailey-Dell, K. J., Hassel, B. A., Shiozawa, K., Sullivan, D. M., Turner, J., Ross, D. D. (2006) Novel 5' untranslated region variants of BCRP mRNA are differentially expressed in drug-selected cancer cells and in normal human tissues: implications for drug resistance, tissue-specific expression, and alternative promoter usage. Cancer Res. 66,5007-5011[Abstract/Free Full Text]
  44. Mignone, F., Gissi, C., Liuni, S., Pesole, G. (2002) Untranslated regions of mRNAs. Genome Biol. 3,reviews0004.1-0004.10
  45. Morris, D. R., Geball, E A. P. (2000) Upstream open reading frames as regulators of mRNA translation. Mol. Cell. Biol. 20,8635-8642[Free Full Text]
  46. Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31,3406-3415[Abstract/Free Full Text]
  47. Kirkpatrick, P. (2002) G-protein coupled receptors: putting the brake on inflammation. Nat. Rev. Drug Discov. 1,99-99
  48. Johnson, J. L., Ellis, B. A., Munafo, D. B., Brzezinska, A. A., Catz, S. D. (2006) Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes. BMC Immunol. 7,28[CrossRef][Medline]
  49. Sauter, K. S., Brcic, M., Franchini, M., Jungi, T. W. (2007) Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS. Vet. Immunol. Immunopathol. 118,92-104[CrossRef][Medline]
  50. Tamassia, N., Le Moigne, V., Calzetti, F., Donini, M., Gasperini, S., Ear, T., Cloutier, A., Martinez, F. O., Fabbri, M., Locati, M., Mantovani, A., McDonald, P. P., Cassatella, M. A. (2007) The MyD88-independent pathway is not mobilized in human neutrophils stimulated via TLR4. J. Immunol. 178,7344-7356[Abstract/Free Full Text]




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