The A2B

           adenosine receptor protects against inflammation and excessive vascular adhesion ([original](https://doi.org/10.1172/JCI27933)) ([raw](?raw))

Generation of A2B AR-KO/β-gal–knock-in mice

The mouse A2BAR gene (GenBank accession number AL596110) was cloned from a mouse ES-129/SvJ bacterial artificial chromosome library, and the A2BAR gene structure is shown in Figure 1A. The targeting vector, A2BAR-KO-β-gal was constructed as depicted in Figure 1A. A strategy was employed to delete exon 1, including the initiation codon, and to introduce instead a reporter gene with a stop codon and poly(A) at the 3′ end to terminate transcription. Exon 1 in the A2BAR gene encodes 3 transmembrane domains of A2BAR (53), and deleting it will remove the initiation methionine. The next in-frame methionine is not present until the fifth transmembrane domain (past the ligand-binding domain) (53). Hence, even if transcription is unexpectedly initiated past the transcription termination signal of the reporter gene, the truncated protein formed is not likely to fold or to be functional, as it will not bind a ligand. To confirm this contention, mRNA expression and functional studies were pursued as detailed below. Also of note, there are no annotated A2BAR pseudogenes in the mouse genome listed at the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) or potential unknown pseudogenes, based on using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10090) under permissive conditions. The human A2BAR pseudogene (GenBank accession number NG_000843) is typical of a retrotransposed pseudogene, meaning that it does not contain introns or the promoter (mRNA was reverse transcribed and then inserted in the genome). This type of pseudogene would not be targeted by our construct, because the flanking homologous regions are those of the promoter and the intron. To generate the targeting vector, a 3.6-kb fragment from intron 1 of the A2BAR gene generated by KpnI/XhoI endonuclease restriction digestion was first subcloned into a pPNT vector at KpnI/EcoRI sites between the neomycin (Neo) gene cassette and thymidine kinase (TK) cassette using a XhoI-EcoRI linker, yielding pPNT-A2BAR-intron. A 6-kb fragment of the A2BAR gene promoter was subcloned into the 5′ end of the gene encoding prokaryotic β-gal in the pBS-lacZ plasmid. The 9.4-kb NotI/XhoI A2BAR-β-gal fragment was then subcloned into pPNT-A2BAR-intron at the NotI/XhoI sites, 5′ of the Neo cassette. Recombinant A2BAR-KO-LacZ ES cells (mouse 129/SvJ derived) were obtained and cultured according to conventional methods (e.g., ref. 15). The positive recombinant ES colonies were first screened with Extend Long PCR (Roche Diagnostics), using the primer pair sense 5′-CAGCCTCTGTTCCACATACACTTCA-3′ and antisense 5′-AGCAGGGACTGGAAAGGTAGGTATT-3, yielding a 4-kb amplicon. The positively identified A2BAR-KO-β-gal ES cell clone 23 was injected into C57BL/6-derived blastocysts, which were subsequently implanted back into FVB foster mothers. Positive chimeric mouse lines were identified by coat color, and their offspring was screened by PCR and Southern blot analysis of tail DNA. PCR WT upstream primer 5′-CTCACACAGAGCTCCATCTT-3′ and downstream primer 5′-TCTGGCAGCAGCTTTGAT-3′ were used to produce a 485-bp fragment (both primers are located in the intron 1 area). Mutant upstream primer 5′-CAGCCTCTGTTCCACATACACT-3′ and downstream primer 5′-GGCACCTCTCCCTCCAAGACAC-3′ were used to generate a 900-bp fragment (both primers are located in the Neo cassette). The heterozygous mice were backcrossed to C57BL/6J strain mice for 4 generations to generate congenic C57BL/6J strain A2BAR gene mutant mice. In this study, the mice used were of 80% C57BL/6J background strain, confirmed by the PCR-based gene marker analysis MAX-BAX (Charles River Laboratories). Crossbreeding of the 80% C57BL/6J strain A2BAR mutant heterozygous mice generated the same background strain as WT or A2BAR gene–KO homozygous mice. In the experiments shown, control and knockout mice were strain-, sex-, and age-matched (8–12 weeks old unless otherwise indicated). All procedures were performed according to the Guidelines for Care and Use of Laboratory Animals published by the NIH. Throughout this study, all animals received humane care that was in agreement with the guidelines of and approved by the Institutional Animal Care and Use Committee of the Boston University School of Medicine. All analyses were repeated (n) to obtain averages ± SDs and subjected to 2-tailed Student’s t tests, as indicated in each figure. P values less than 0.05 were considered statistically significant.

Measurement of A2B AR expression and activity in different tissues and cells, including smooth muscle cells and macrophages

Total RNA from different mouse tissues was prepared with Trizol (according to the manufacturer’s protocol; Invitrogen). RT-PCR was used (1 μg RNA, Moloney murine leukemia virus [M-MLV] reverse transcriptase, 1× first-strand buffer, 0.5 mM dNTP, 5 mM DTT, 0.5 U/μl RNase inhibitor, 5 μM random primers, all purchased from Invitrogen) to measure A2BAR mRNA expression. RNA was treated with RQ1 RNase-Free DNase (according to the manufacturer’s instructions; Promega, catalog no. M6101) and this was followed by RT-PCR performed in the presence or absence of reverse transcriptase (1 μg RNA, M-MLV reverse transcriptase, 1× first-strand buffer, 0.5 mM dNTP, 5 mM DTT, 0.5 U/μl RNase inhibitor, 5 μM random primers, all purchased from Invitrogen). The primer pairs were designed to produce a 746-bp fragment within exon 1 (PCR primers: sense, 5′-ATGCAGCTAGAGACGCAAGA-3′; antisense, 5′-GGAGCCAACACACAGAGCAA-3′). To optimally detect A2BAR mRNA, the number of PCR cycles in our system was set at 30. GAPDH mRNA expression, used as control, was set at 20 cycles (GAPDH primers generate 554-bp fragments and were: sense, 5′-TCACCATCTTCCAGGAG-3′ and antisense, 5′-GCTTCACCACCTTCTTG-3′). To amplify the second exon in A2BAR cDNA by RT-PCR, the following primers were employed: sense, 5′-CAAGTGGGTGATGAATGTGG-3′; antisense- 5′-TTTCCGGAATCAATTCAAGC-3′ (to generate a 448-bp fragment). The primers were selected using Primer3 primer design tool (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3\_www.cgi). The MacVector sequence alignment program (version 6.5.3; Accelrys) was used to ensure that the homology to A2AAR, A1AR, or A3AR was no greater than 75% (50% in most areas), with little homology at primer ends. Aberrant fragments potentially generated from other adenosine receptors will be of a different size than amplified A2BAR exon 2 (see Supplemental Figure 1A). Additionally, primers were analyzed using UCSC Genome Bioinformatics In-Silico PCR tool (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start) to ensure that there was no amplification of other gene sequences. Total RNA was isolated from mesenteric artery or kidney by immediate homogenization in Trizol reagent (Invitrogen) or using Versagene RNA Tissue Kit according to the manufacture’s protocol (Gentra). RNA was treated with DNase I (Promega) for 30 minutes at 37°C prior to reverse transcription consisting of 1 μg RNA, M-MLV reverse transcriptase, 1× first-strand buffer, 0.5 mM dNTP, 5 mM DTT, 0.5 U/μl RNase inhibitor, 5 μM random primers (all purchased from Invitrogen). A mock reaction lacing reverse transcriptase served as a control for DNA contamination. RT-PCR of exon 2 was performed with the above-mentioned primers with a program of 1 cycle at 95°C for 2 minutes, 30 cycles at 95°C for 55 seconds, 60°C for 55 seconds, 72°C for 55 seconds, and 1 cycle at 72°C for 5 minutes.

VSMCs were isolated from aortas of 6-week-old WT or A2BAR-KO mice by enzymatic dispersion as previously described (54). Eighteen hours after seeding, VSMC culturing media was removed and replenished with 100 μl/well of fresh VSMC culturing media containing 5 μM MRS1754 [8-(4-[{(4-cyanophenyl)carbamoylmethyl}oxy]phenyl)-1,3-di(n-propyl)xanthine; Sigma-Aldrich, catalog no. M-6316]. After a 10-minute pretreatment, cells were stimulated by adding 5 μM NECA (Sigma-Aldrich) or 5 μM forskolin (7β-acetoxy-1α,6β,9α -trihydroxy-8,13-epoxy-labd-14-en-11-one; Sigma-Aldrich) for another 10 minutes. cAMP levels in VSMCs were measured using cAMP-Screen Chemiluminescent Immunoassay for Determination of cAMP Concentration Kit (Applied Biosystems, catalog no. T1500). Similar cAMP experiments were pursued using peritoneal macrophages. Mice were injected with 50 ml/kg Brewer thioglycollate medium (Sigma-Aldrich, catalog no. B 2551), and peritoneal macrophages were collected 3 days after injection. Cells were plated in 24-well plates (106 cells/well) and cultured overnight. After pretreatment with 5 μM MRS1754 for 10 minutes, cells were stimulated by adding 5 μM NECA or 1 μM CGS 21680 hydrochloride (Tocris Bioscience, catalog no. 1063) for another 10 minutes. cAMP levels were measured using Direct Cyclic AMP Kit (Assay Designs, catalog no. 900-066).

Primers used to amplify all adenosine receptor mRNAs by PCR

RNA preparation and PCR conditions were essentially as listed above for the A2BAR. For the A1AAR, the primers used were: sense, 5′-GCTTAGTCCCTCAGAATCACG-3′; antisense, 5′-CCCTTGTCCTTAGAGGTTCCA-3′; expected fragment of 436 bp. For the A2AAR, the primers were: sense, 5′-TGAAGGCGAAGGGCATCA-3′; antisense, 5′-CGCAGGTCTTTGTGGAGTTCT-3′; expected fragment of 117 bp. For the A3AAR, the primers were: sense, 5′-TAATGGGAAGGCAGGGATAAG-3′; antisense, 5′-GATGTAGGTGATGTTCAGCCA-3′; expected fragment of 284 bp. The PCR conditions included: 1 cycle at 95°C for 2 minutes, 95°C for 50 seconds, 60°C for 50 seconds, 72°C for 50 seconds, for 28 or 30 cycles, and 1 cycle of 5 minutes at 72°C.

Analysis of β-gal expression in tissue sections and in cells

β-gal assay in tissue sections at the light-microscopic level. Mice were anesthetized with isoflurane and perfused through the left heart ventricle with 20 ml PBS (pH 7.4) at a rate of 4 ml/min. Perfusion with fixative (30 ml freshly made 2% paraformaldehyde in PBS [pH 7.4]) continued for 15 minutes at 2 ml/min, followed by perfusion with PBS for 10 minutes. Various tissues were dissected from the perfused mouse and stored in ice-cold PBS, prior to staining for β-gal activity. Individual organs were cut into 1- to 2-mm-thick slices and stained with X-gal staining solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6η3H2O (Sigma-Aldrich, catalogue nos. P-8131 and P-9287, respectively), 2 mM MgCl2, in PBS] containing a final concentration of 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; American Bioanalytical, catalog no. AB02400-1000) or halogenated indolyl-β-d-galactoside, as indicated (Bluo-gal; Sigma-Aldrich, catalog no. B2904). Samples were incubated at 37°C for 6–12 hours on a rocking platform. After staining, samples were rinsed with PBS and stored in 4% paraformaldehyde at 4°C. Samples were embedded in paraffin and cut at a thickness of 5 μm. Sections were stained with H&E.

Analysis of β-gal expression at the ultrastructural level. Mouse tissue was stained for β-gal activity as described above, except that Bluo-gal (halogenated indolyl-β-d-galactoside from Invitrogen; catalog no. 15519-010) was used as a substrate instead of X-gal. Tissues were processed for electron microscopy using a modification of a previously published protocol (36). Briefly, 25% glutaraldehyde (Polysciences Inc.) was diluted to 4.3% with 0.03 M sodium barbital–sodium acetate buffer (pH 7.4) in 0.07 M potassium chloride. The samples were kept in this glutaraldehyde solution overnight at 4°C. The samples were rinsed 3 times with sodium barbital–sodium acetate buffer containing potassium chloride for 15 minutes each. This was followed by dehydration in a graded series of ethanol starting with 50% ethanol, embedded in a 1:1 mixture of Araldite 502 (Ted Pella Inc.) and dodecenyl succinic anhydride at 60°C. After polymerization of the Araldite mixture, sections were cut on an LKB Ultratome V. Ribbons of sections showing gray, silver, or slightly gold interference colors were picked up on uncoated 200-mesh Athene Thin Bar copper grids.

β-gal assay in macrophages, polymorphonuclear cells, and bone marrow cells. Peritoneal macrophages were collected from 6-week-old WT or A2BAR-KO mice (4 mice per group). To this end, mice were anesthetized by inhalation of isoflurane and then injected with PBS (5 ml/mouse) in the peritoneal cavities. After 5 minutes of massaging, injected PBS was collected from the peritoneal cavities and spun down at 800 g, for 10 minutes, at 4°C. Red cells were lysed with 8 ml of prewarmed lysis buffer (17 mM Tris, 140 mM NH4Cl in H2O, pH 7.2) at 37°C, for exactly 10 minutes. Cell pellets were washed and centrifuged in 10 ml PBS twice. Cells were resuspended in 1 ml/well serum-free RPMI (RPMI-1640 medium supplemented with 4 mM l-glutamine, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM nonessential amino acids) and seeded on a 24-well plate. After 3 hours of incubation at 37°C and 5% CO2, the media with nonadherent cells (non-macrophages, polymorphonuclear cells) were removed. Attached macrophages were rinsed with PBS twice and fixed with 0.5% glutaraldehyde (Sigma-Aldrich, catalog no. G-7651) in PBS for 10 minutes at room temperature. Macrophages were then rinsed 2 times with PBS and stained in X-gal solution [4 mM K4Fe(CN)6η3H2O, 4 mM K3Fe(CN)6, 1 mM MgCl2, 1 mg/ml X-gal in PBS] for 7 hours at 37°C. Bone marrow cells were isolated from the femurs of 6-week-old WT or A2BAR-KO mice as described previously (55). Cell pellets were resuspended in fixative buffer for 20 minutes (0.5% glutaraldehyde, 0.02% Nonidet P-40 [NP-40] in 1× PBS without MgCl2), washed twice with PBS, and then incubated in 1 ml X-gal staining solution for 16–20 hours at 37°C. Cells were mounted onto slides, and blue precipitates (indicative of β-gal activities) were visualized via light microscopy.

Cytokine measurements in plasma and in liver

eBioscience ELISA Ready-SET-Go! kits testing for mouse TNF-α (catalog no. 88-7324-22), IL-6 (catalog no. 88-7064-22), IL-10 (catalog no. 88-7104-22), IL-2 (catalog no. 88-7024-22), IL-4 (catalog no. 88-7044-22), and IFN-γ (catalog no. 88-7314-22) were used to measure cytokine levels in plasma and in liver according to the manufacturer’s protocol. Liver protein was extracted as described in “Western blot analysis” below.

Blood cell count

Blood was harvested and blood cells were counted as we have described elsewhere (56, 57).

Western blot analysis

Tissues were homogenized and lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, and aprotinin [2 μg/ml] freshly supplemented with 1× protease inhibitor cocktail (Roche Diagnostics). Lysates were vigorously vortexed and incubated on ice for 30 minutes, then frozen in liquid nitrogen and thawed at 37°C. Finally, lysates were cleared by centrifugation at 800 g for 10 minutes at 4°C. Fifty micrograms/sample of total protein was electrophoresed on 6% or 10% SDS-PAGE gels and transferred overnight at 4°C to Immobilon-P PVDF membranes (Millipore), and Western blotting was performed as described in ref. 58. The following antibodies (purchased from Santa Cruz Biotechnology Inc.) were used: rabbit polyclonal anti–E-selectin antibody (1:200 dilution; catalog no. sc-14011), goat polyclonal anti-vWF antibody (1:200 dilution; catalog no. sc-8068), rabbit polyclonal anti–ICAM-1 antibody (1:200 dilution; catalog no. sc-1511), mouse monoclonal anti–VCAM-1 antibody (1:1,000 dilution; catalog no. sc-13506), goat polyclonal anti–P-selectin antibody (1:200 dilution; catalog no. sc-6943), rabbit polyclonal anti–IκB-α antibody (1:1,000 dilution; catalog no. sc-371), mouse monoclonal anti-actin antibody (1:1,000 dilution; catalog no. sc-1616). Secondary antibodies included: goat anti-rabbit IgG-HRP (catalog no. sc-2004), donkey anti-goat IgG-HRP (catalog no. sc-2056), and goat anti-mouse IgG-HRP (catalog no. sc-2005).

Immunohistochemistry of macrophages and leukocytes in aortic and mesenteric tissue sections

Tissues were collected after fixation as described for β-gal staining. They were embedded with paraffin and cut at a thickness of 5 μm. After deparaffinization, rehydration and high-temperature antigen retrieval were performed as follows. Sections were placed in 10 mM citric acid, pH 6, and heated by 700-W microwave for 2 minutes 3 times, with 2-minute intervals between each time, followed by cooling down for 20 minutes. Paraffin sections were blocked with 10% normal goat serum (Vector Laboratories, catalog no. S-1000) for 1 hour at 37°C and then incubated overnight at 4°C with rat anti-mouse CD43 monoclonal antibody diluted 1:25 (BD Biosciences, catalog no. 552366) to detect leukocytes or with rat anti-mouse F4/80 monoclonal antibody (Serotec, catalog no. MCA497R) to detect macrophages. The staining was revealed using goat anti-rat biotinylated secondary antibody (Vector Laboratories, catalog no. BA-9400) at a dilution of 1:200 with an incubation time of 1 hour at 37°C. After 1× PBS wash, sections were incubated with ABC reagent (Vector Laboratories, catalog no. PK-6100) for 30 minutes at 37°C, and Vector DAB substrate (Vector Laboratories, catalog no. SK-4100) was used to develop the brown positive signal by incubating the sections for 5 minutes. Sections were counterstained with Shandon Gill 3 Hematoxylin (Thermo Electron Corp., catalog no. 6775009).

LPS-induced acute inflammation

WT or A2BAR-KO mice were given a single i.p. injection of LPS (E. coli serotype 0127:B8; Sigma-Aldrich, catalog no. L-4516) at 5 μg/g of body weight or of saline in a total volume of up to 100 μl (control). Mice were sacrificed at 1 hour, 3 hours, 16 hours, and 24 hours after LPS or saline administration and subjected to blood and tissue collection. Plasma and liver protein were used for cytokine measurements.

Bone marrow transplantation experiments

Generation of chimeric mice by bone marrow transplantation. Ten-week-old WT or A2BAR-KO female mice were irradiated with a total dose of 12.5 Gy from a 137Cs source (34). On the day of irradiation, bone marrow cells were harvested from 10-week-old WT or A2BAR-KO male donor mice femurs. The bone marrow cells were subjected to red blood cell lysis, as described previously (55), and injected into irradiated mice at a dose of 2 × 106 cells/recipient in 0.3 ml through the tail vein. Five weeks after irradiation, peripheral blood was collected via the retro-orbital sinus and prepared for blood DNA isolation (Gentra VERSAGENE DNA Blood Kits, catalog no. VGD-0050B1).

Determination of repopulation efficiency of bone marrow–derived cells in chimeric mice. Genotyping of sex chromosome–linked genes (Jaridld and Jaridlc) was used to confirm cross-sex transplantation of bone marrow–derived cells (from male donor to female recipient) as reported before. Jaridld and Jaridlc PCR upstream primer 5′-CCGCTGCCAAATTCTTTGG-3′ and downstream primer 5′-TGAAGCTTTTGGCTTTGAG-3′ were used to produce a 300-bp fragment in female mice and both 300-bp and 330-bp fragments in male mice. Six weeks after transplantation, bone marrow cells were isolated from the femurs of all 4 groups of mice (WT→WT, KO→WT, KO→KO, WT→KO) and used for β-gal staining according to the method described above. For each group, β-gal–positive (blue) cells were counted under microscopy in randomly chosen fields (with native A2BAR-KO and WT mice as controls). To further test the efficiency of transplantation under our protocol, bone marrow cells from GFP mice (Jackson Laboratory, strain name C57BL/6-Tg [UBC-GFP] 30Scha/J, stock no. 004353) were transplanted into WT or A2BAR-KO mice, which we used as transplant recipients. Six weeks after transplantation, bone marrow cells were isolated from recipients and mounted on slides. GFP-positive cells were counted under a fluorescence microscope with GFP transgenic mice and WT mice as controls (data not shown).

Sample collection from transplant-recipient chimeric mice and related assays. Six weeks after transplantation, chimeric mice were injected i.p. with 1 μg/g or 5 μg/g body weight of LPS. Blood and liver samples were collected at 0 hours, 1 hour, and 16 hours after LPS injection. Plasma was used for cytokine measurements as we described above. Proteins were also subjected to Western blot analysis, and blood cell count was determined, all as described above.

Tail-cuff and direct BP measurements, adenosine infusion-catheterization, and determination of heart rates

Tail-cuff systolic BP was obtained at baseline using a computerized tail-cuff system (BP 2000, Visitech Systems) as described in ref. 59. Arterial and venous catheterization was performed under anesthesia induced by sodium pentobarbital (50 mg/kg, i.p.) (60). A modified polyethylene catheter (PE-50) was introduced into the right iliac artery for direct BP recording, and silastic tubing was placed into the right iliac vein for adenosine infusion. Both catheters were tunneled subcutaneously and exteriorized at the back of the animal’s neck. Subsequently, they were filled with heparin in 0.9% saline solution, sealed with heat, and attached to the animal’s nape. After the surgery, the animals were returned to their cages and allowed an overnight recovery period. On the following day, the arterial catheter was connected to a BP transducer attached to a recorder (model 220S; Gould) for direct BP monitoring. Control BP was recorded for no less than 30 minutes, when the BP became stable. The venous catheter was connected to a Harvard infusion pump (Harvard Apparatus), and adenosine was administered at a dose of 200–600 μg/kg/min for 60 minutes. Heart rates were measured as described previously (13).

_Analysis of leukocyte rolling by intravital microscop_y

Mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g), and a midline incision was made through the abdominal wall to expose the mesentery and mesenteric venules of 200- to 300-μm diameters. Exposed mesentery was kept moist by periodic superfusion using PBS (without Ca2+ or Mg2+) warmed to 37°C. The mesentery was transluminated with a 12-V, 100-W, DC stabilized source. The shear rate was calculated using an optical Doppler velocity meter as described previously (61). Venules were visualized using a Zeiss Axiovert 135 inverted microscope (objective ×32) connected to an SVHS video recorder (AG-6730; Panasonic) using a silicon-intensified tube camera (C2400; Hamamatsu). Leukocyte interaction with the endothelium vessel wall was recorded for 10 minutes each in 2–3 unbranched venules per mouse. Recorded images were analyzed as follows: the number of leukocytes passing a given plane perpendicular to the vessel axis during 1 minute was counted; leukocyte rolling/minute/venule for each mouse was determined by taking the average of four 1-minute counts during the entire 10-minute recording. The rolling velocity was determined as the number of leukocytes that traversed over a 250-μm-long and 200- to 300-μm-wide segment. A leukocyte was considered to be adherent if it remained stationary for more than 20 seconds.