Activation of the A2A adenosine receptor (A2AR) during reperfusion of various tissues has been found to markedly reduce ischemia-reperfusion injury. In this study, we used bone marrow transplantation (BMT) to create chimeric mice that either selectively lack or selectively express the A2AR on bone marrow-derived cells. Bolus i.p. injection of the selective A2A agonist, 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester (ATL313; 3 μg/kg), at the time of reperfusion protects wild-type (wt) mice from liver ischemia-reperfusion injury. ATL313 also protects wt/wt (donor/recipient BMT mouse chimera) and wt/knockout chimera but produces modest protection of knockout/wt chimera as assessed by alanine aminotransferase activity, induction of cytokine transcripts (RANTES, IFN-γ-inducible protein-10, IL-1α, IL-1-β, IL-1Rα, IL-18, IL-6, and IFN-γ), or histological criteria. ATL313, which is highly selective for the A2AR, produces more liver protection of chimeric BMT mice than 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester, which is rapidly metabolized in mice to produce 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid, which has similar affinity for the A2AR and the proinflammatory A3 adenosine receptor. GFP chimera mice were created to show that vascular endothelial cells in the injured liver do not account for liver protection because they are not derived by transdifferentiation of bone marrow precursors. The data suggest that activation of the A2AR on bone marrow-derived cells is primarily responsible for protecting the liver from reperfusion injury.

Adenosine is produced in response to ischemia or inflammation and protects tissues from injury. Exposure of tissues to adenosine before ischemia reduces subsequent injury in the CNS (1) and myocardium (2) due to preconditioning mediated by A1 or A3 adenosine receptors. In contrast, activation of the A2A adenosine receptor (A2AR)3 before ischemia does not reduce subsequent ischemic injury, but A2AR activation during reperfusion reduces tissue damage to kidney (3), heart (4), skin (5), lung (6), and spinal cord (7). Ohta and Sitkovsky (8) concluded that endogenous adenosine protects the liver from injury caused by toxins (Con A and endotoxin) by demonstrating enhanced hepatic injury in mice lacking acloraza, the A2AR gene. Recently, we found that endogenous adenosine also protects the liver from ischemia-reperfusion injury (IRI), particularly of short duration, but much greater protection is imparted to the liver by infusing the synthetic A2A agonist, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohex-anecarboxylic acid methyl ester (ATL146e), during reperfusion following ischemia (9). ATL146e is effective in wild-type (wt), but not A2AR knockout (ko), mice or in mice also treated with the A2AR antagonist, 4(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]-ethyl)phenol.

In the current study, we sought to investigate the contribution of the A2AR on bone marrow-derived cells to protection of liver from IRI. Our approach was to prepare chimeric mice by irradiation and bone marrow transplantation (BMT), resulting in animals that either selectively lack or selectively express the A2AR on bone marrow-derived cells. Chimeric animals designated ko/wt were created by transplanting A2AR ko marrow to wt-irradiated recipients. Chimeric animals designated wt/ko were created by transplanting wt marrow to A2AR ko recipients. In addition, we created GFP/wt BMT chimeric mice that were used to determine which cells are derived from donor marrow in chimeric mice. The results indicate that activation of the A2AR on bone marrow-derived cells is primarily responsible for protecting the liver from reperfusion injury.

The ko locus of B6;129P-adora2atm1chen mice that express an ablated A2AR gene on a mixed genetic background (10) was moved to a C57BL/6 background by monitoring 96 microsatellites for five generations of marker-assisted breeding. In the resulting mouse line, DNA derived from the 129 strain can be detected only in an 8-cm region between D10Mit31 and D10Mit42 surrounding the adora2a locus on chromosome 10.

C57BL/6 mice (Hilltop) or congenic A2AR ko mice were irradiated and used as recipients for BMT experiments. Donor marrow was derived from four different mouse strains: 1) typical C57BL/6 mice that are negative for CD45.1; 2) a substrain of C57BL/6 mice that are positive for CD45.1; 3) A2AR ko mice; or 4) green mice expressing GFP in all cells. Male donor mice (12 wk old; 25–28 g) were anesthetized with Nembutal (20 μg/g) and sacrificed by cervical dislocation. The marrow from the tibia and femur were harvested under sterile conditions. The marrow cavity was flushed with RPMI 1640 medium (Invitrogen Life Technologies) + 10% FCS and drawn through a 22-gauge needle and then through a 25-gauge needle to obtain a suspension of ∼50 million nucleated bone marrow cells. These were washed, resuspended, and counted. Recipient mice (male, 8–10 wk old, 22–25 g) were irradiated twice at 4-h intervals with 600 rad. Immediately following the second irradiation period, 3 × 106 bone marrow cells were injected i.v. Irradiated/transplanted mice were housed in microisolators for at least 8 wk before experimentation and given autoclaved food and water containing 5 mM sulfamethoxazole and 0.86 mM trimethoprim.

The origin of leukocytes in chimeric mice was determined 6 wk after BMT. Leukocytes were prepared from peripheral blood or spleen digested for 30 min with collagenase D, filtered through 40-μm nylon mesh, and washed with PBS. Harvested spleen leukocytes were placed in 1.5 mM NH4Cl to lyse RBC and then resuspended in 600 μl of PBS containing 1% BSA. After blocking nonspecific Fc binding with anti-mouse CD16/CD32 (553141; BD Pharmingen), spleen leukocytes were incubated on ice for 30 min with R-PE-conjugated mouse anti-mouse CD45.1 mAb (clone A20, 553776; BD Pharmingen) and either allophycocyanin-conjugated rat anti-mouse CD11b (Mac-1α-chain) mAb (553312; BD Pharmingen), FITC-conjugated rat anti-mouse CD8a mAb (553030; BD Pharmingen), or allophycocyanin-conjugated rat anti-mouse CD4 mAb (553051; BD Pharmingen). Subsequent flow cytometry data acquisition and analysis were performed using a FACScan and CellQuest software (BD Biosciences).

Mice expressing GFP in all cells were used as bone marrow donors to produce GFP/wt mouse chimera expressing GFP only in bone marrow-derived cells. GFP/wt chimeric mice were used to identify the source (donor or recipient) of liver vascular endothelial cells. Mouse livers were excised and fixed by immersion in 3.7% paraformaldehyde in PBS for 20–24 h and washed three times in PBS. Mouse livers were excised and fixed by immersion in 3.7% paraformaldehyde in PBS for 20–24 h, washed three times in PBS, and placed in cassettes in 70% ethanol for paraffin embedding. Five-micrometer sections were rehydrated and blocked for 20 min with 0.45% hydrogen peroxide in filtered deionized water. Sections were preincubated in TBS (pH 7.8) and blocked with 0.5% fish skin gelatin (TBS/FSG) + 10% normal serum (secondary host) + 2 drops/ml avidin block (Vector Laboratories) for 60 min. A rabbit polyclonal anti-GFP Ab (1:3500; Novus Biologicals) in TBS/FSG + 7.5% normal serum + 2 drops/ml biotin block was applied for 2–3 h at room temperature and then overnight at 4°C. After washing four times for 5 min in TBS/FSG, secondary Ab (6 μg/ml) was applied for 60 min at room temperature and washed again four times for 5 min in TBS before applying Vectastain Elite ABC reagent (Vector Laboratories) for 30 min. Chromogenic visualizations was achieved using 1 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (DAKO) for 5 min. Sections were counterstained with hematoxylin I (Richard-Allan Scientific).

Mice were anesthetized by i.p. injection of 100 mg/kg ketamine and 10 mg/kg xylazine and then injected s.c. with 50 μg/kg glycopyrrolate to prevent excess salivation and possible suffocation. Eye lubricant was applied to prevent ocular dehydration. Mice were placed under a heating lamp and on a 37°C heating pad. In some animals, rectal temperature was monitored with a TCAT-1A temperature control unit (Physitemp) and was found to be 35°C ± 1°C and did not differ between drug treatment groups. Careful monitoring of body temperature is particularly important in these experiments because A2AR agonists are vasodilators that can produce hypothermia, although this requires higher doses than were used here. We used a partial hepatic ischemia model that spares the right lobe. This model avoids intestinal congestion, sepsis, and peritonitis and is not lethal during liver ischemia times up to 60 min. Each mouse was placed in a supine position. A midline laparotomy and incision of the Linea Alba exposed the peritoneal cavity. The stomach and duodenum were displaced caudally to expose the hepatic triad and caudate lobes. The caudate lobe was separated gently from the left lobe and displaced from the right upper and lower lobes caudally to clearly view the hepatic triad above the bifurcation of right lobes, median lobe, and left lobe. A microaneurysm clip was applied to the hepatic triad above the bifurcation to clamp the flow of the hepatic artery, portal vein, and bile duct. The peritoneum was closed after superfusion with 200 μl of warm saline supplemented with 50 U/kg heparin. After 60 min of ischemia, the peritoneum was reopened, and the microaneurysm clip was removed. Immediately before reperfusion was initiated, each mouse received either a single bolus i.p. injection of 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester (ATL313; 3 μg/kg) or an i.p. loading dose of ATL146e (1 μg/kg) and a s.c. osmotic minipump (model 1003D; ALZET) to release 10 ng/kg/min ATL146e or vehicle during the entire 24-h reperfusion period. The surgical wound was closed with metal staples, and mice were maintained on the heating pad until the anesthetic wore off.

Whole blood was harvested from an incision through an axillary artery and placed on ice before centrifugation at 14,000 × g for 20 min. Serum was collected and stored at −80°C. ALT was determined by using an ALT kit (Pointe Scientific) using a Spectra MAX 190 plate reader with SOFTmax PRO software, version 3.1.2 (Molecular Devices). A 200-μl aliquot of a prewarmed (37°C) mixture of l-alanine and α-ketoglutaric acid was added to 20 μl of undiluted and/or saline-diluted serum in a 96-well plate. After a 1-min incubation at 37°C, the plate was scanned at 340 nm at 9-s intervals for 60 s, and the rate of change in absorbance converted into Sigma-Frankel units (1 IU = 0.482 Sigma-Frankel U).

Total liver RNA was extracted from homogenized tissue with RNAzol B (Leedo Medical Laboratories) and subjected to 1.5% agarose gel electrophoresis to assess for the integrity of RNA before solution hybridization. Cytokine and chemokine mRNA expression were assessed with the RiboQuant Multiprobe RNAase protection system (BD Pharmingen), according to the manufacturer’s protocol. In brief, mRNA-specific RNA probes were labeled with [32P]UTP using multiprobe template sets, mCK2b for IL-12p35, IL-12p40, IL-10, IL-1α, IL-1β, IL-1Ra, IL-18, IL-6, and IFN-γ, and mCK5c for Ltn, RANTES, MIP-1β, MIP-1α, MIP-2, IFN-γ-inducible protein (IP)-10, MCP-1, TCA-3, and eotaxin. RNA was subjected to solution hybridization at 56°C and then digested by adding RNase. Protected fragments were separated by electrophoresis through 5% polyacrylamide gels at 100 mV. After ∼4 h of electrophoresis, gels were exposed to Kodak MS film at −80°C with intensifying screens.

Livers were harvested after 24 h reperfusion, fixed in 4% paraformaldehyde in PBS (pH 7.4), and embedded in paraffin. Four-micrometer sections were subjected to standard H&E staining.

Four-micrometer tissue sections were incubated with rat anti-mouse neutrophil Abs (1 μg/ml) followed by a biotinylated goat anti-rat secondary Ab. The peroxidase reaction was performed according to the manufacturer’s protocol (Vectastain ABC Elite kit), and reaction times for sections from sham control and experimental animals were identical.

Blood samples collected from animals at various time after the i.v. injection of ATL146e or ATL313 were mixed with 10 U/ml heparin. In some cases, ATL146e or ATL313 was added to blood in vitro. Blood aliquots (200 μl) were pipetted into 400-μl acetonitrile containing 0.3 μg/ml (2S,3R,4S,5R)-5-(6-amino-2-iodo-9H-purin-9-yl)-N-cyclopropyl-tetrahy-dro-3,4-dihydroxyfuran-2-carboxamide as an internal standard, centrifuged for 15 min at 14,000 × g, and the supernatants were collected and dried under vacuum at 4°C with centrifugation. The residue was resuspended in 200 μl of MeOH/dH2O (50:50), sonicated for 15 min, applied to a Millipore Microcon YM-100 filter cartridge, and centrifuged for 30 min at 14,000 × g. The filtrates were applied to a Waters Symmetry C8 2.1 × 150-mm column and eluted with methanol/aqueous 0.1% formic acid. The methanol concentration in the chromatography buffer as follows: 0–2 min, 40% isocratic; 2–10 min, 40–60% linear gradient; and 10–20 min, 60% isocratic. Compounds were detected by tandem mass spectrometry in positive ion mode using electrospray ionization with a Thermoelectron LCQ Advantage mass spectroscopy detector. Peak integration of internal standard peaks and ATL146e, ATL146a, and ATL313 was by the Excalibur program, allowing for the calculation of sample concentration.

Differences in plasma ALT or liver myeloperoxidase in multiple groups of chimeric mice were determined by one-way ANOVA and posttesting using Bonferroni’s multiple comparison method.

In wt C57BL/6 mice subjected to 60 min of liver ischemia and 24 h of reperfusion, treatment with the selective A2A agonist ATL146e during the reperfusion period resulted in an 84% decrease in liver damage as assessed by serum levels of ALT (Fig. 1). As we have reported previously (9), this protection is completely lost in congenic C57BL/6 mice lacking the A2AR gene, indicating that it is mediated by the A2AR and not by other adenosine receptor subtypes. To determine which cells mediate protection by ATL146e, we created chimeric mice through the use of lethal irradiation and BMT. The repopulation efficiency of various hematopoietic lineages was determined by flow cytometry (n = 4) using CD45 as a mouse strain marker (11). CD45 found in C57BL/6 mice is replaced by the alloantigen CD45.1 in B6.SJL-Ptprca Pep3b/BoyJ mice (12). Cells arising from different mouse strains can be distinguished using selective Abs and FACS. Following lethal radiation and BMT, the repopulation of B lymphocytes, monocytes/macrophages, neutrophils, and dendritic spleen cells was >97% as shown in Fig. 2. Somewhat lower repopulation of CD4+ and CD8+ T cells (83–85%) can be attributed to radiation resistance of T lymphoblasts.

FIGURE 1.

Effects of ATL146e on liver IRI. Livers from WT or A2AR ko mice were subjected to 1 h of ischemia and 24 h of reperfusion. Serum ALT was collected at 24 h. ATL146e or vehicle was added at the time of reperfusion as described under Materials and Methods. Each bar is the mean ± SD of 10–12 mice.

FIGURE 1.

Effects of ATL146e on liver IRI. Livers from WT or A2AR ko mice were subjected to 1 h of ischemia and 24 h of reperfusion. Serum ALT was collected at 24 h. ATL146e or vehicle was added at the time of reperfusion as described under Materials and Methods. Each bar is the mean ± SD of 10–12 mice.

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FIGURE 2.

Repopulation of leukocytes in recipient mice after BMT. Irradiated mice lacking CD45.1 received bone marrow cells from CD45.1-positive animals. Six weeks after transplantation, spleen cells were harvested and examined by FACS for CD45.1 and leukocyte markers. The percentage of specific cell lineages (to the right of vertical lines) and positive for the donor epitope CD45.1 (above horizontal lines) is indicated on each panel.

FIGURE 2.

Repopulation of leukocytes in recipient mice after BMT. Irradiated mice lacking CD45.1 received bone marrow cells from CD45.1-positive animals. Six weeks after transplantation, spleen cells were harvested and examined by FACS for CD45.1 and leukocyte markers. The percentage of specific cell lineages (to the right of vertical lines) and positive for the donor epitope CD45.1 (above horizontal lines) is indicated on each panel.

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Because of the effects of radiation, BMT chimeric mice may be somewhat less immunocompetent that normal mice. Therefore, we used wt/wt (donor/recipient) BMT chimeric mice as controls for the possible immunosuppressive effects of BMT. Fig. 3 shows that ATL146e strongly inhibits liver IRI in wt/wt BMT chimera but fails to exert any protection in ko/wt, wt/ko, or ko/ko BMT chimera. By contrast, another A2AR-selective agonist, ATL313, attenuates liver IRI in all BMT chimeric mice, except ko/ko chimera.

FIGURE 3.

Assessment of liver injury in BMT chimeric mice. Irradiated wt or A2AR ko mice received bone marrow transplants from wt or ko mice to make wt/w, wt/ko, ko/wt, or ko/ko (donor/recipient) BMT chimeric mice. Mice were subjected to liver ischemia for 1 h followed by reperfusion for 24 h, at which time serum ALT levels were measured. Vehicle, ATL146e, or ATL313 was delivered immediately at reperfusion as described under Materials and Methods. Each bar represents the mean ± SD of 10–12 animals. ∗, p < 0.05 vs vehicle; #, p < 0.001 vs vehicle.

FIGURE 3.

Assessment of liver injury in BMT chimeric mice. Irradiated wt or A2AR ko mice received bone marrow transplants from wt or ko mice to make wt/w, wt/ko, ko/wt, or ko/ko (donor/recipient) BMT chimeric mice. Mice were subjected to liver ischemia for 1 h followed by reperfusion for 24 h, at which time serum ALT levels were measured. Vehicle, ATL146e, or ATL313 was delivered immediately at reperfusion as described under Materials and Methods. Each bar represents the mean ± SD of 10–12 animals. ∗, p < 0.05 vs vehicle; #, p < 0.001 vs vehicle.

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These finding raise two questions: why is ATL313 more effective at protecting liver from IRI than ATL146e, and why does ATL146e fail to protect both ko/wt and wt/ko BMT chimeric mice? A possible explanation is suggested by our finding that ATL146e is rapidly (<30 s) converted to ATL146a when added to mouse or rat blood (Table I). Interestingly, this rapid metabolism occurs much more slowly (t1/2 > 30 min) in canine or human blood, suggesting that an esterase that converts ATL146e to ATL146a is much more active in rodent than in human or canine blood. No metabolism of ATL313 was observed in blood from any species, suggesting that replacement of the cyclohexyl moiety in ATL146e with a piperidine moiety in ATL313 confers resistance to esterase activity by converting the ester to the more stable nitrogen-containing carbamate. When injected into mice or rats, ATL146e was also rapidly and completely converted into ATL146a. When injected into dogs, ATL146e was converted to ATL146a with a t1/2 of ∼10 min, possibly due to liver esterase activity (Fig. 4). The potency and selectivity of ATL146e, ATL146a, and ATL313 to bind to recombinant adenosine receptor subtypes are summarized in Table I. Unlike ATL313 and ATL146e, which are potent and selective A2AR agonists, ATL146a has equal affinity for the A2AR and the A3 adenosine receptor. Hence, failure by ATL146e to inhibit liver injury may be due to latent A3 adenosine receptor agonist activity that exerts a proinflammatory effect, particularly in chimeric mice that have a reduced complement of cells that express the anti-inflammatory A2AR.

Table I.

Binding affinities of ATL146e, ATL146a, and ATL313 for recombinant human adenosine receptor subtypesa

Binding affinities of ATL146e, ATL146a, and ATL313 for recombinant human adenosine receptor subtypesa
Binding affinities of ATL146e, ATL146a, and ATL313 for recombinant human adenosine receptor subtypesa
a

Data are the mean ± SEM for binding to the high-affinity conformational state of recombinant human adenosine receptors expressed in HEK-239 cells as determined by competition for radioligand binding (34 ).

FIGURE 4.

Recovery of ATL146e and its metabolite, ATL146a, after i.v. injection into a dog. ATL146e (10 μg/kg) was injected 5 min before mixing a blood sample with acetonitrile. During HPLC, compounds were monitored in tandem mass spectrometry-positive ion mode using a Thermofinnigan LCQ mass spectrometer as described in Materials and Methods. Results are typical of three experiments.

FIGURE 4.

Recovery of ATL146e and its metabolite, ATL146a, after i.v. injection into a dog. ATL146e (10 μg/kg) was injected 5 min before mixing a blood sample with acetonitrile. During HPLC, compounds were monitored in tandem mass spectrometry-positive ion mode using a Thermofinnigan LCQ mass spectrometer as described in Materials and Methods. Results are typical of three experiments.

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It is notable that ATL313 produces greater protection of the liver from IRI in wt/ko than in ko/wt BMT mouse chimera. These data indicate that most, but not all, liver protection is mediated by A2ARs on bone marrow-derived cells. In contrast to the strong protective effect from liver IRI produced by ATL146e in wt/wt BMT chimeric mice, ATL146e appears to produce a small statistically insignificant increase in liver IRI in ko/wt BMT chimeric mice based on serum ALT. H&E staining reveals more uniform liver necrosis in ATL146e-treated than in vehicle-treated ko/wt chimeric animals (Fig. 5, C and D). Similarly, immunohistochemical staining for neutrophils suggests a small proinflammatory response in ko/wt animals in response ATL146e (Fig. 6, C and D). The clearest evidence that ATL146e causes a proinflammatory response in ko/wt mouse chimera is shown in Fig. 7. ATL146e increases IRI-stimulated induction of proinflammatory cytokines, particularly RANTES, IP-10, IL-1β, and IL-18. These data suggest that ATL146e produces a latent proinflammatory response that is unmasked when the A2AR is deleted partially in chimeric mice.

FIGURE 5.

Effects of liver IRI and ATL146e on histological evidence of liver damage. BMT mouse chimera, wt/wt (A and B) or ko/wt (C and D) were subjected to liver IRI and treated with vehicle (A and C) or ATL146e (B and D). Liver sections were stained with H&E after 24 h of reperfusion to reveal living (purple granular) or necrotic (pink agranular) tissue. The sections shown are representative of five similar experiments.

FIGURE 5.

Effects of liver IRI and ATL146e on histological evidence of liver damage. BMT mouse chimera, wt/wt (A and B) or ko/wt (C and D) were subjected to liver IRI and treated with vehicle (A and C) or ATL146e (B and D). Liver sections were stained with H&E after 24 h of reperfusion to reveal living (purple granular) or necrotic (pink agranular) tissue. The sections shown are representative of five similar experiments.

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FIGURE 6.

Effects of liver IRI and ATL146e on immunohistological evidence of neutrophil accumulation in liver. BMT mouse chimera, wt/wt (A and B), or ko/wt (C and D) were subjected to liver IRI and treated with vehicle (A and C) or ATL146e (B and D). Liver sections were immunostained for neutrophils (brown) after 24 h of reperfusion. The sections shown are representative of five similar experiments.

FIGURE 6.

Effects of liver IRI and ATL146e on immunohistological evidence of neutrophil accumulation in liver. BMT mouse chimera, wt/wt (A and B), or ko/wt (C and D) were subjected to liver IRI and treated with vehicle (A and C) or ATL146e (B and D). Liver sections were immunostained for neutrophils (brown) after 24 h of reperfusion. The sections shown are representative of five similar experiments.

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FIGURE 7.

Effects of liver IRI and ATL146e on the induction of cytokine and chemokine transcripts in wt/wt and ko/wt BMT chimeric mice. Mice were subjected to liver IRI and treated with vehicle (V) or ATL146e for 24 h. Transcripts for various cytokines and chemokines were assessed by RNase protection assays for BD Pharmingen multiprobe template set mCK-2b and template set mCK-5c. The results shown are representative for two to three replicate experiments with similar results. Little or no transcripts for cytokines was visible in mRNA prepared from sham animals not subjected to liver IRI (data not shown).

FIGURE 7.

Effects of liver IRI and ATL146e on the induction of cytokine and chemokine transcripts in wt/wt and ko/wt BMT chimeric mice. Mice were subjected to liver IRI and treated with vehicle (V) or ATL146e for 24 h. Transcripts for various cytokines and chemokines were assessed by RNase protection assays for BD Pharmingen multiprobe template set mCK-2b and template set mCK-5c. The results shown are representative for two to three replicate experiments with similar results. Little or no transcripts for cytokines was visible in mRNA prepared from sham animals not subjected to liver IRI (data not shown).

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If the A2AR on endothelial cells mediate liver protection and if bone marrow-derived cells repopulate liver endothelium, then the deletion of the A2AR on endothelial cell precursors could account for the absence of protection by ATL146e and ATL313 in ko/wt BMT chimera. Hence, it was important to evaluate the degree to which transdifferentiation of donor mouse-derived endothelial progenitor cells repopulate the vascular endothelium in recipient mice (Fig. 8). GFP/wt BMT chimera were examined 8 wk after BMT. Some animals were subject to IRI, i.e., 1 h of ischemia followed by 24 h of reperfusion. Initial attempts to detect GFP by immunofluorescence in liver sections were unsuccessful because of high nonspecific background fluorescence in liver, especially in necrotic areas. For this reason, GFP was detected using anti-GFP immunohistochemistry. Livers from wt mice were devoid of GFP immunoreactivity. All liver cells in sections of transgenic GFP livers contained intense brown immunoperoxidase, including endothelial cells (Fig. 8, A and B). Scattered GFP-immunoreactive cells were seen in livers from GFP/wt BMT chimera, but no immunoreactivity was detected on endothelial cells lining the lumen of blood vessels, either in uninjured livers (Fig. 8,C) or following IRI with reperfusion for 24 h (Fig. 8 D). We conclude that the livers of mice collected 8 wk after BMT with or without IRI of short duration (24 h) have no detectable endothelial cells derived from bone marrow precursors. Therefore, the poor ability of ATL146e and ATL313 to reduce IRI in ko/wt BMT chimera cannot be attributed to repopulation of liver endothelial cells by bone marrow precursors lacking the A2AR.

FIGURE 8.

Source of endothelial cells in BMT chimeric mice. Liver sections were stained with hematoxylin and immunostained with anti-GFP Abs. Livers were derived from the following: (A) wt mouse following 1 h of ischemia and 24 h of reperfusion; (B) transgenic GFP mice; (C) GFP→WT bone marrow chimera; (D) GFP→WT bone marrow chimera following 24 h of reperfusion after 60 min of ischemia. Black arrows denote examples of endothelial cells, and white arrows denote GFP-immunoreactive cells derived from donor GFP animals. The results are representative of three experiments.

FIGURE 8.

Source of endothelial cells in BMT chimeric mice. Liver sections were stained with hematoxylin and immunostained with anti-GFP Abs. Livers were derived from the following: (A) wt mouse following 1 h of ischemia and 24 h of reperfusion; (B) transgenic GFP mice; (C) GFP→WT bone marrow chimera; (D) GFP→WT bone marrow chimera following 24 h of reperfusion after 60 min of ischemia. Black arrows denote examples of endothelial cells, and white arrows denote GFP-immunoreactive cells derived from donor GFP animals. The results are representative of three experiments.

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The purpose of this study was to determine whether bone marrow-derived cells are responsible for the substantial liver protection from IRI that is seen in response to A2AR activation during reperfusion. On the basis of the use of BMT to create chimeric mice, we conclude that most of the effect of the selective A2AR agonist, ATL313, to protect the liver from IRI are mediated by bone marrow-derived cells because little protection is noted in ko/wt chimeric mice that do not express the A2AR on bone marrow-derived cells, and substantial protection is noted in wt/ko chimera that express the A2AR only on bone marrow-derived cells. Nonbone marrow-derived cells or bone marrow-derived cells that are not efficiently repopulated within 8 wk of BMT also contribute somewhat to the effects of A2AR activation because ko/wt BMT chimeric mice are protected to a small degree by ATL313. Such cells might consist of hepatocytes, endothelial cells, vascular smooth muscle, or other parenchymal cells. They also could consist in part of cells of bone marrow origin that are not efficiently repopulated, such as a subset of ∼15% of T lymphocytes (Fig. 2) or Kupffer cells, which appeared not to be repopulated in GFP/wt BMT chimera (Fig. 8).

ATL146e failed to protect either ko/wt or wt/ko BMT chimeric mice from liver IRI. A possible explanation for this apparent paradox may be that ATL146e activates the A3 adenosine receptor, which has been shown to be proinflammatory in rodents by enhancing hypoxia-induced mast cell degranulation (13) and to enhance endotoxin-induced killing of mice (14). Tissue resident mast cell deregulation may contribute to inflammatory responses during reperfusion after ischemia. Increased mast cell-specific proteases, such as rat mast cell protease II, has been demonstrated in rat intestine and mesentery subjected to IRI, suggesting that mast cell degranulation occurs upon IRI and contributes to neutrophil recruitment.

Although ATL146e is a highly selective agonist of the A2AR based on binding to recombinant adenosine receptors (Table I), we found in this study that it is rapidly (t1/2 < 30 s) converted to the nonselective A2A/A3 agonist, ATL146a, when injected into rodents or mixed with rodent blood. By contrast, ATL146e has a t1/2 > 30 min when mixed with human or canine blood and a t1/2 of ∼10 min when injected into dogs. The anti-inflammatory effect of the A2AR predominates over the proinflammatory effect of A3 adenosine receptor activation in mice expressing a normal complement of A2A and A3, i.e., in wt mice and in wt/wt BMT chimeric mice. In BMT chimeric mice, a latent proinflammatory component of ATL146a action appears to largely neutralize residual anti-inflammatory activity. However, it is notable that there are no data to indicate that activation of the A3 adenosine receptor has a proinflammatory effect in nonrodent species. Unlike in rodent species, the adenosine receptor subtype that stimulates mast cell degranulation in dogs and human cells appears to be the A2B adenosine receptor (15, 16).

Neutrophil accumulation in the ischemic liver is reduced significantly by ATL146e administered at the time of reperfusion. Previous studies of liver damage using IRI models in rats and mice have implicated neutrophils that infiltrate the injured tissue as a source of reperfusion injury (17, 18). Activation of A2ARs suppresses the oxidative burst of neutrophils and inhibits leukocyte extravasation into inflamed tissues. However, a delay in the administration of ATL146e for >4 h after reperfusion results in loss of liver protection by ATL146e, although most neutrophils extravasate into the liver > 4 h after the beginning of reperfusion injury (9). Hence, the reduction in neutrophil accumulation with A2AR activation may be primarily due to acute effects on a small number of neutrophils or on other cells that reside in the liver or that accumulate within a few hours of the beginning of reperfusion.

Some candidate targets of A2A agonists are mast cells (19), platelets (20), macrophages (21), dendritic cells (22), and T lymphocytes (23). Macrophages and dendritic cells are APCs and serve to initiate inflammation in response to invading microorganisms. In addition, they have been postulated to play a role in IRI-induced inflammation. Activation of the A2AR has been shown to inhibit TNF-α and IL-12 release from macrophages (24). Immature dendritic cells do not make cAMP in response to adenosine, but following treatment with LPS, mature dendritic cells respond to adenosine, and this results in reduced IL-12 production in response to inflammation (25). Large numbers of T lymphocytes accumulate late (48–72 h) after reperfusion injury in tissues subjected to IRI. IFN-γ production follows a similar temporal pattern. The time course of T cell accumulation suggests that most of these cells do not participate in liver protection by A2A agonists. However, a role for a small number of T cells in provoking injury was suggested in a model of ex vivo cold IRI in which isolated livers from nude mice were less injured and released less IFN-γ (26) than did livers from wt mice. Kupffer cells can recruit CD4+ T lymphocytes and neutrophils by releasing MIP-2, KC, and IP-10 (27). We have recently shown that IFN-γ release from isolated CD4+ T cells is inhibited strongly by ATL146e and other A2A agonists (23). These considerations suggest that activation of the A2AR on liver macrophages and small numbers of T cells that are resident in the liver or that accumulate shortly after reperfusion following ischemia may contribute to the protection of the liver from reperfusion injury.

Early studies suggested that bone marrow-derived progenitor cells can only differentiate into hemopoietic lineages. Subsequent studies have shown that after transplantation, bone marrow cells can differentiate into various differentiated cell types, including vascular endothelial cells (28). Nevertheless, although hemopoietic stem cells clearly have the potential to transdifferentiate, this has been noted only during the repair of damaged tissues and has not be shown to occur in normal healthy tissues. For example, transdifferentiation was observed after chemical injury to muscle (29), in Duchenne’s muscular dystrophy (30), myocardial infarction (28), and tumor angiogenesis (31). Following BMT, endothelial cells derived from donor mice were found in blood vessels of chimeric mice that had been injured by wire, graft vasculopathy, or atherosclerosis (32, 33), but no donor cells were found in uninjured vessels, even after radiation treatment. These results are consistent with the conclusions of the current study in which GFP cells in GFP/wt BMT mouse chimera cannot be found in the endothelium or uninjured or even acutely injured (24 h of IRI) liver. Hence, weak activity of ATL313 to protect liver from IRI in A2AR ko/wt BMT chimera cannot be attributed to repopulation of liver blood vessels with A2A ko endothelial cells derived from donor bone marrow progenitors.

In conclusion, we have used BMT and A2AR ko mice to create chimeric mice that selectively lack or selectively express the A2AR on bone marrow-derived cells. The results suggest that most protection from liver reperfusion injury is due to activation of A2ARs on bone marrow-derived cells because protection by ATL313 is minimal in ko/wt BMT chimera and more substantial in wt/ko BMT chimeric animals.

J. Linden and M. Okusa own equity in Adenosine Therapeutics, LLC.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work is supported by National Institutes of Health Grant RO1-HL37942 and by the Dr. Ralph and Marian Falk Medical Research Trust.

3

Abbreviations used in this paper: A2AR, A2A adenosine receptor; IRI, ischemia-reperfusion injury; ATL146e, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; wt, wild type; ko, knockout; ATL146a, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclo-hexanecarboxylic acid; BMT, bone marrow transplantation; FSG, fish skin gelatin; ATL313, 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester; ALT, alanine aminotransferase; IP, IFN-γ-inducible protein.

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