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Published as doi: 10.1096/fj.07-100685.
 (The FASEB Journal. 2008;22:2263-2272.)
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Ecto-5'-nucleotidase (CD73) -mediated extracellular adenosine production plays a critical role in hepatic fibrosis

Zhongsheng Peng*, Patricia Fernandez*, Tuere Wilder*, Herman Yee{dagger}, Luis Chiriboga{dagger}, Edwin S. L. Chan* and Bruce N. Cronstein*,1

* Department of Medicine, Divisions of Clinical Pharmacology and Rheumatology; and

{dagger} Department of Pathology, New York University School of Medicine, New York, New York, USA

1Correspondence: Department of Medicine, Division of Clinical Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. E-mail: cronsb01{at}med.nyu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A2A receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl4, ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl4 or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl4, ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl4 or TAA. There were far fewer {alpha}-smooth muscle actin positive hepatic stellate cells in CCl4-treated KO mice than that in WT mice. After CCl4 treatment, the mRNA level of A1, A2A, A2B, and A3 adenosine receptors, tumor necrosis factor-{alpha}, interleukin (IL) -1β, IL-13r{alpha}1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1{alpha}1, Col3{alpha}1, and transforming growth factor-β1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13r{alpha}2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.—Peng, Z., Fernandez, P., Wilder, T., Yee, H., Chiriboga, L., Chan, E. S. L., Cronstein, B. N. Ecto-5'-nucleotidase (CD73) -mediated extracellular adenosine production plays a critical role in hepatic fibrosis.


Key Words: stellate cell • carbon tetrachloride • thioacetamide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADENOSINE, A NATURALLY OCCURRING nucleoside, is present in and released from nearly all mammalian tissues and organs where it can act in an autocrine and paracrine manner to regulate a variety of physiological processes (1 , 2) . Among its actions, adenosine mediates tissue protection and repair by increasing the ratio of oxygen supply to demand (vasodilatation), protecting against ischemic damage by cell conditioning, suppressing inflammation, and promoting angiogenesis (3) . The effects of adenosine are mediated by a family of four G protein-coupled receptors, A1, A2A, A2B, and A3, each of which has a unique pharmacological profile, tissue distribution, and effector coupling. We have recently reported strong evidence that adenosine and adenosine A2A receptors play a critical role in the development of fibrosis in the skin (4) and in the liver (5 , 6) .

Extracellular adenosine arises from either an increase in intracellular adenosine, which is released into the extracellular space, or by the release of adenine nucleotides, which are dephosphorylated extracellularly to adenosine (7) . Extracellular ATP and ADP can be converted into AMP by extracellular apyrases such as nucleoside triphosphate phosphohydrolase (CD39) and alkaline phosphatase (8 9 10) , and AMP can then be converted into adenosine by ecto-5'-nucleotidase (CD73) or alkaline phosphatase (9 10 11) . CD73 is up-regulated after hypoxia and plays a role in increasing local extracellular adenosine levels (12 13 14) . Thus, CD73 contributes to adenosine-mediated effects on coronary blood vessels and in the kidney (15 , 16) .

Ethanol, viruses, drugs, and metabolic derangements damage hepatocytes and stimulate fibrosis leading to cirrhosis. Ethanol is well known to stimulate increased extracellular adenosine concentration in vitro through its action on the nucleoside transporter, and ethanol ingestion increases purine release into the bloodstream and urine in normal volunteers (17 , 18) . In prior work, we have found that deletion or blockade of adenosine A2A receptors prevents the development of hepatic fibrosis induced by thioacetamide (TAA) or CCl4, indicating that extracellular adenosine plays a role in the pathophysiology of hepatic fibrosis (5) . However, the mechanisms governing adenosine formation in injured liver are not known and the role of CD73 and extracellular formation of adenosine from adenine nucleotides has not been established in the liver. Here we report evidence that extracellular formation of adenosine by the action of CD73 plays a critical role in the development of hepatic fibrosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57BL/6 [wild-type (WT)] mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Ecto-5'-nucleotidase knockout (CD73KO) mice were generated as described previously (19) and backcrossed more than 10 generations onto a C57BL/6 background. Animals were bred in the animal facilities of the School of Medicine of New York University. All experimental mice were 6- to 8-wk-old male mice. All experimental procedures were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of School of Medicine of New York University.

Toxin-induced hepatic release of adenosine in mice
WT and CD73KO mice were treated with single doses of CCl4 (0.1 ml in oil; 1:3 v:v, i.p.), TAA (100 mg/kg in PBS, i.p.), or ethanol (2 g/kg, 20% ethanol in PBS, i.p., followed 1 and 2 h later by injection of 20% ethanol, 1 g/kg), and control mice were given PBS alone. Animals were sacrificed after 20 h, and their livers were harvested. Liver slices were incubated in medium (Dulbecco modified Eagle medium /10% FBS) overnight, and adenosine was extracted from the culture supernatant and quantitated by HPLC as previously reported (5 , 20) . Results were normalized to the wet weight of the liver slices.

Toxin-induced acute hepatic damage
WT mice and CD73KO mice were treated with single doses of CCl4 (1.25 ml/kg CCl4 in corn oil; 1:3 25% v/v) or an equal amount of corn oil. Animals were killed after 12, 24, 48, and 72 h. Whole blood was collected, the serum was isolated, and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the clinical laboratory of Bellevue Hospital.

Murine model of hepatic fibrosis
The WT and CD73KO mice were treated with biweekly i.p. injections of CCl4 (1.25 ml/kg CCl4 mixed with corn oil; 1:3 25% v/v) or corn oil for 6 wk (n=15 per group) or with three i.p. injections of TAA (100 mg/kg TAA in PBS) or PBS per week for 8 wk (n=6). There were no deaths in any of the treatment groups. Mice were sacrificed by CO2 narcosis at the end of the treatment periods. Hepatic sections (5 cross-sections per liver) were harvested and stained with hematoxylin and eosin (H&E) or picrosirius red as described previously (21) . Digitized photomicrographs (entire cross-sections at x10; 5 sections per liver) were quantitated for total area of picrosirius red staining using SigmaScan Pro software v.5.0.0 (SPSS, Chicago, IL, USA), and fibrosis was calculated as a percentage of total hepatic area and expressed as the average of five randomly selected tissue sections from each liver. Hepatic inflammation was scored by a qualified pathologist in a blinded fashion using a modified Knodell scoring system (22 , 23) . Sections were scored for zonal necrosis, confluent necrosis, lobular inflammation, and portal inflammation (0–3 for each), and a composite score was calculated for each section (maximum score=12).

Quantification of hepatic hydroxyproline content
Hydroxyproline content in liver specimens was measured colorimetrically as described previously (24) .

Real-time polymerase chain reaction (PCR) quantitation of mRNA
Aliquots of liver tissue (~60–80 mg) were flash frozen and kept at –80°C until RNA isolation. The tissue was minced and homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA), and RNA was extracted according to the manufacturer’s instructions and then dissolved in sterile DEPC water and stored at –80°C. One microgram of sample RNA was transcribed to cDNA with the GeneAmp RNA Core Kit (Applied Biosystems, Branchburg, NJ, USA). Primer sequences for the genes studied are listed in Table 1 , and PCR was performed with the SYBR Green PCR Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions and carried out on the Mx3005P Q-PCR system (Stratagene, La Jolla, CA, USA) in 50 µl volume. The number of copies for each amplicon was calculated by Mx3005P software, normalized to GAPDH, and expressed as a dimensionless ratio.


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  		Table 1. Oligonucleotide primer sequences used for real-time PCR

Western blot or enzyme-linked immunosorbent assay
Liver was minced and homogenized in tissue protein extraction reagent (Pierce, Rockford, IL, USA) and extracted according to the manufacturer’s instructions. Western blot analyses were performed using anti-matrix metalloproteinase (MMP) -13 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) and anti-β-actin monoclonal antibody (Abcam, Cambridge, MA, USA), and after digital visualization, the relative protein concentration was quantitated using the Gel Logic 100 Image System (Kodak, New Haven, CT, USA) and normalized to β-actin. Enzyme-linked immunosorbent assay was performed with mouse IL-13 immunoassay kit (R&D Systems) following manufacturer’s instructions, and each sample was quantitated in duplicate.

Immunohistochemistry
The slides from fixed, paraffin-embedded liver sections (4 µm) were prepared, and proteins were blocked by protein block (X0909; Dako, Copenhagen, Denmark) for 30 min. The primary antibody against {alpha}-smooth muscle actin ({alpha}-SMA; Abcam) was applied to tissue sections at a dilution of 1:500 and incubated overnight at 4°C. As a negative control, preimmune serum was used. Visualization was carried out after incubation with alkaline phosphatase-conjugated, affinity-isolated goat anti-rabbit immunoglobulin (Dako) and the Fast red substrate (Dako) followed by hematoxylin staining. {alpha}-SMA positive cells were counted in at least five random different high power fields (HPF) of each cross section (x400).

Statistical analysis
Data were expressed as means ± SD and were analyzed by Student’s t test or ANOVA analysis, as appropriate, with SPSS software 10.0. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Extracellular adenosine concentrations are higher in the supernatants of liver slices from WT than CD73KO mice
To determine whether CD73 plays any role in extracellular hepatic adenosine formation, the adenosine concentration in the supernatant of cultured liver slices was measured. Hepatic slices from WT mice treated with PBS accumulated more supernatant adenosine than liver slices from CD73KO mice (Fig. 1 ). As expected, after treatment with CCl4, ethanol, or TAA, adenosine concentrations rose markedly in supernatants of hepatic slices in both WT and CD73KO mice but much greater increases in adenosine concentration were observed in the supernatants of WT liver slices than CD73KO mice (Fig. 1) . Thus, CD73 plays an important, but not exclusive role, in extracellular adenosine formation in resting and toxin-challenged livers.


Figure 1
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  		Figure 1. The extracellular adenosine concentration is lower in supernatants of livers from CD73KO than those from WT mice. Mice were treated with PBS, CCl4, ethanol, or TAA; their livers were harvested and cultured; supernatant adenosine concentration was quantitated by HPLC; and results were normalized to the wet weight of the liver slice. The adenosine levels in the supernatant were much less in CD73KO mice than in WT mice whether the mice were treated with vehicle, CCl4, ethanol or TAA.

Liver function changes in CCl4-induced acute hepatic damage and CCl4- and TAA-induced hepatic fibrosis
Previous studies (5) have indicated that animals lacking adenosine A2A receptors suffer greater hepatic injury after CCl4 exposure than WT controls due to diminished adenosine A2A-receptor-mediated suppression of inflammation (25) . ALT was significantly more increased in WT than CD73KO mice 24 h after treatment (9770±323 vs. 7200±301 UI/l; n=4 in each group; P<0.01) but not when collected after chronic exposure to either CCl4 or TAA (data not shown).

CD73KO mice are resistant to CCl4- and TAA-induced hepatic fibrosis
After chronic CCl4 or TAA treatment, WT mice developed severe hepatic fibrosis or cirrhosis, with obvious fibrotic septa visible in picrosirius red-stained sections of the liver, but CD73KO mice suffered only mild fibrosis (Figs. 2 A, B and 3 A, B). Consistent with the histological appearance and histomorphometric measurements, the hydroxyproline content of hepatic tissue was much lower in CCl4- and TAA-treated CD73KO mice than in WT mice (Figs. 3C and 4 C). Modified Knodell scores were similar in WT and CD73KO mice (5.0±1.0 vs. 4.9±1.0) after CCl4 treatment. These results clearly demonstrate that CD73KO mice are protected against CCl4- and TAA-induced hepatic fibrosis without any discernible difference in the level of hepatic injury or inflammation between WT and CD73KO mice.


Figure 2
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  		Figure 2. CD73KO mice are protected from CCl4-induced hepatic fibrosis. WT and CD73KO mice were treated with CCl4 or vehicle, as described, and their livers were harvested. A) Hepatic sections were stained with H&E (top; vehicle treatment, x100; CCl4 treatment, x400:) or picrosirius red (bottom; x100). B) Quantification of picrosirius red staining was performed digitally using SigmaScan Pro v.5.0.0; data represent the percentage of total liver area stained by picrosirius red. The percentage of the hepatic area that was stained in vehicle-treated animals was 0.3 ± 0.1 and 0.3 ± 0.1% for both vehicle-treated WT and CD73KO mice (n=15), whereas after CCl4 treatment the livers of CD73KO mice were significantly less fibrotic (2.4±0.4 vs. 4.1±1.1%; n=15; P<0.01). C) Hydroxyproline content in the livers of animals treated with CCl4. The difference between the hydroxyproline content of livers from WT and CD73KO mice was highly significant (0.27±0.04 vs. 0.22±0.04 µg/mg, respectively; n=15; P<0.01).


Figure 3
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  		Figure 3. CD73KO mice are protected from TAA-induced hepatic fibrosis. WT mice and CD73KO mice were treated with TAA or vehicle, as described, and their livers were harvested. A) Hepatic sections were stained with H&E (top; x200) or picrosirius red (bottom; x100). B) Quantification of picrosirius red staining was performed digitally using SigmaScan Pro v.5.0.0; data represent the percentage of total liver area stained by picrosirius red. The percentage of the hepatic area that was stained in vehicle-treated animals was 0.3 ± 0.1 and 0.3 ± 0.1% for WT and CD73KO mice, respectively (n=6), whereas after TAA treatment, the livers of CD73KO mice were significantly less fibrotic than WT mice (1.8±0.2 vs. 2.3±0.4%; n=6; P<0.05). C) Hydroxyproline content in the livers of animals treated with TAA; the difference between the hydroxyproline content of livers from TAA-treated WT and CD73KO mice was highly significant (0.21±0.02 vs. 0.18±0.02 µg/mg, respectively; n=6; P<0.05).


Figure 4
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  		Figure 4. {alpha}-SMA immunohistochemical staining of livers from vehicle- and CCl4-treated mice. A) Top panels: very few {alpha}-SMA-positive stellate cells are seen in livers of vehicle-treated mice. Bottom panels: many {alpha}-SMA-positive stellate cells are present in livers of CCl4-treated WT mice, whereas only a few are present in CD73KO mice (x400). B) Quantification of {alpha}-SMA-positive cells in CCl4-treated WT and CD73KO mice (31±3 vs. 18±2 {alpha}-SMA+ cells/HPF; n=10; P<0.01; x400).

Fewer activated hepatic stellate cells in CD73KO mice
To better understand how the loss of CD73 and with its resulting diminution in extracellular adenosine diminishes fibrosis, we studied the presence of activated hepatic stellate cells after toxin treatment. Hepatic stellate cells were distributed primarily in fibrotic septa, and there were significantly more {alpha}-SMA+ cells in the livers of CCl4-treated WT mice than CD73KO mice (Fig. 4 A, B).

Adenosine receptor mRNA expression increases after chronic CCl4 treatment
Because extracellular adenosine appeared to play such an important role in CCl4-mediated hepatic fibrosis, we determined the effect of CCl4 treatment on adenosine receptor expression and found that CCl4 increased mRNA expression from 2- to 4-fold for all four of the adenosine receptors in both WT and CD73KO mice (data not shown).

Col1{alpha}1 and Col3{alpha}1 mRNA expression is lower in CD73KO mice
Hepatic fibrosis is characterized by extensive deposition of extracellular matrix proteins, including collagen types I and III (26) . Col1{alpha}1 and Col3{alpha}1 mRNA levels were nearly identical in vehicle-treated CD73KO mice and WT mice but increased significantly more in the livers of the CCl4-treated WT mice than CD73KO mice (Fig. 5 ).


Figure 5
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  		Figure 5. Col1{alpha}1, Col3{alpha}1-procollagen, and TGF-β1 mRNA expression. Hepatic tissue Col1{alpha}1 (A) and Col3{alpha}1 (B) mRNA levels increased after CCl4 treatment, but the increase was significantly less in CD73KO than WT mice (Col1{alpha}1: 2.73±0.60 vs. 4.13±1.09; Col3{alpha}1: 2.91±0.55 vs. 5.07±0.81; n=10; P<0.01, respectively). Similarly, TGF-β1 (C) mRNA levels increased after CCl4 treatment, although the mRNA level for TGF-β1 was significantly less in CD73KO mice than the WT mice (4.94±1.25 vs. 7.28±2.14; n=10; P<0.01).

Growth factor, cytokine, and IL-13r{alpha}2 mRNA expression and IL-13 protein level after CCl4 treatment
Because of their central role in hepatic fibrosis, we quantitated transforming growth factor (TGF) -β1, tumor necrosis factor (TNF) -{alpha}, interleukin (IL) -1β, IL-13r{alpha}1, and IL-13r{alpha}2 mRNA expression in the livers of WT and CD73KO mice and IL-13 protein levels in hepatic lysates. There was no difference in the mRNA levels for these cytokines or the IL-13 level between vehicle-treated WT and CD73KO animals. In contrast, as with the message for collagen, there was a much more marked rise in the mRNA for TGF-β1 in the livers of CCl4-treated WT mice than CD73KO mice. Interestingly, mRNA for IL-13r{alpha}2, the decoy receptor for IL-13, increased more markedly in the livers of CCl4-treated CD73KO than WT control mice. No differences were observed with respect to increases in mRNA for TNF-{alpha}, IL-1β, IL-13r{alpha}1, or IL-13 level (Figs. 5C and 6 ).


Figure 6
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  		Figure 6. TNF-{alpha}, IL-1β, IL-13r{alpha}1, IL-13r{alpha}2 mRNA expression, and IL-13 protein level. Hepatic TNF-{alpha} (A), IL-1β (B), and IL-13r{alpha}1 (C) mRNA levels and IL-13 (E) protein levels were similar in WT and KO mice and increased equally in both WT and KO mice after CCl4 treatment (n=10), whereas IL-13r{alpha}2 mRNA (D) increased much more in CD73KO mice than in WT mice (8.8±1.9 vs. 5.6±1.0; n=10; P<0.01)

MMP and tissue inhibitor of matrix metalloproteinase mRNA expression increase after CCl4 treatment
In addition to increased synthesis and secretion, matrix degradation by proteases like the MMPs also regulates the development of fibrosis. In the liver, MMP-2, MMP13, and MMP-14 play a role in extracellular matrix degradation (27 28 29) , and previous work from our laboratory has indicated that adenosine regulates MMP expression (5) . There was a marked increase in the expression of mRNA for all of these proteins and increased MMP-13 protein in the livers of animals treated with CCl4; however, only MMP-13 mRNA and protein levels were significantly increased in CD73KO mice compared with WT mice (3.15±0.68 vs. 1.95±0.53; protein: 2.23±0.13 vs. 1.61±0.17; n=10 in each group; P<0.01, respectively).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Under resting conditions adenosine is present at low concentrations in the extracellular space of most organs and adenosine levels rise substantially in response to hypoxia, tissue injury or metabolic stress (2 , 30) . In previous work, we have demonstrated that CD73 is required for methotrexate- and H2O2-mediated adenosine release at a peripheral site of inflammation and that cultured human hepatoma cells (HepG2) and liver slices release more adenosine after in vivo challenge with toxins, including ethanol, methotrexate, and thioacetamide (5 , 31) . The results reported here indicate that CD73 plays an important role in adenosine generation in the liver, and, consistent with prior studies, the extracellular adenosine generated by CD73 is a critical mediator of hepatic fibrosis in two different murine models.

Our results indicate that CD73 accounts for only about one-half of the increase in extracellular adenosine levels in the hepatic supernates. It is likely that the remainder of the increase in extracellular adenosine is mediated by either direct adenosine export from liver cells or by alkaline phosphatase-mediated dephosphorylation of adenine nucleotides in the liver. Interestingly, adenosine and its receptors may also prolong the half-life of adenosine in the extracellular space by a feed-forward process. Nagy et al. (17 , 32 , 33) have reported that adenosine uptake by S49 lymphoma cells in vitro, and liver in vivo is mediated by the equilibrative nucleoside transporter and that ethanol diminishes uptake via increasing extracellular adenosine concentrations leading to adenosine-receptor-mediated inhibition of the nucleoside transporter.

We have previously demonstrated that adenosine A2A receptors directly stimulate collagen production and that they play an active role in the pathogenesis of hepatic fibrosis (5) . In our prior studies, we have found that adenosine A2A-receptor occupancy directly stimulates collagen production by rat and human hepatic stellate cell lines and by primary hepatic stellate cells (5 , 6) . Indeed, using both pharmacologic inhibitors of signaling and siRNA approaches, we have demonstrated that adenosine A2A receptors directly stimulate stellate cell production of collagen I by a pathway dependent on protein kinase A, src, and extracellular regulated kinase 1/2, whereas transcription and translation of collagen III are mediated by p38MAPK (6) . The magnitude of the increase in collagen production due to adenosine A2A receptors is similar to that induced by TGF-β (6) . Adenosine also induces hepatic stellate cell differentiation (34) . Further evidence for the central role of adenosine A2A receptors in hepatic fibrosis comes from studies in which adenosine A2A-receptor knockout mice, but not wild-type or A3 receptor knockout mice, are protected from development of CCl4- or TAA-induced hepatic fibrosis. Moreover, caffeine, a poorly A2A-receptor selective adenosine receptor antagonist, and ZM241385, a more selective antagonist of the adenosine A2A receptor, but not the selective A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, diminished hepatic fibrosis in wild-type mice exposed to either CCl4 or thioacetamide, the same models studied here (5) . In the studies reported here, we found both reduced numbers of activated stellate cells and reduced collagen in the tissue of CCl4-treated CD73KO mice, and it is likely that both diminished stellate cell activation and diminished collagen production contributed to the reduction in hepatic fibrosis. Either way these results are consistent with a role for adenosine in hepatic fibrosis, a role mediated primarily, as we and others have previously reported, by adenosine A2A-receptor-mediated effects on stellate cells.

CD73 is a glycosyl phosphatidylinositol-linked molecule that is expressed on the extracellular surface of many different cell types. Biochemical study reveals a strictly polarized expression of CD73 in hepatocytes (35) , as has also been observed in hepatocytes in tissue culture (36) . Immunohistochemical and enzyme histochemical study demonstrates that CD73 distributes in stellate cells, in the connective tissue of the central vein and periportal triads, and in the biliary canaliculi of hepatocytes (37 , 38) .

CD73 is also a lymphocyte differentiation antigen, which has been shown to mediate lymphocyte binding to endothelial cells (39) . When CD73 expression is diminished, lymphocyte binding to endothelial cells diminishes. There was no change in hepatic inflammation in the mice studied here, and there were also no differences in the hepatic expression of mRNA for inflammatory cytokines or IL-13, a profibrotic cytokine, so the effects of CD73 deletion and diminished adenosine levels in the animals studied here is unlikely to be mediated by alterations in the quantity or quality of the inflammatory response.

Prior studies (5 , 25) indicate that deletion or blockade of adenosine A2A receptors lead to greater elevations in liver function abnormalities after hepatic insult, and we expected that CD73KO mice would have a greater increase in hepatic injury due to reduction of the agonist. Surprisingly, CD73KO mice had lower ALT levels after acute but not chronic hepatic injury by CCl4. One explanation for this finding is that the background strain in this study (C57BL/6) differed from the strain studied in the prior study (S129) and sensitivity to inflammatory hepatic injury might differ between the strains. Alternatively, deletion of the adenosine A2A receptor is complete in the animals studied previously, whereas the reduction in adenosine levels in the mice studied here was only partial. Others have noted that, due to receptor reserve, leukocytes are much more sensitive to the effects of stimulating adenosine A2A receptors than other tissues (40) and thus even the modest levels of adenosine present in the CD73KO mice are sufficient to diminish inflammation but insufficient to stimulate stellate cell activation and collagen production.

Adenosine and adenosine receptors play a role in fibrosis of other organs. Adenosine deaminase (ADA) -deficient mice die from pulmonary inflammation and fibrosis, and treatment of these mice with ADA prevents fibrosis and prolongs their lives (41 , 42) . In the lungs of ADA-deficient mice, adenosine stimulates IL-13 production and IL-13 reciprocally increases extracellular adenosine levels synergistically stimulating the development of pulmonary inflammation and fibrosis (41 , 42) , a process reversed by exogenous ADA. In the murine models studied, reciprocal adenosine-IL-13 synergy does not likely play a role in the development of fibrosis, since we found no increase in IL-13 protein levels in the livers of WT as compared with CD73KO mice. In other studies, adenosine A2A-receptor-deficient mice are protected from the development of dermal fibrosis after treatment with subcutaneous bleomycin, further supporting a role for adenosine and its receptors in fibrosis (4) . In contrast, Volmer et al. (43) found that CD73-mediated adenosine production prevents fibrosis in a model of bleomycin-induced lung fibrosis. It is likely that these disparate findings arise from the diverse actions of the various adenosine receptors in different tissues; as shown here and previously, A2A receptors play a dominant role in hepatic and dermal fibrosis, whereas A2B adenosine receptors play a dominant role in pulmonary fibrosis (2 , 42) .

The mechanisms by which adenosine elevations lead to hepatic fibrosis are not completely established. Adenosine A2A-receptor occupancy directly stimulates hepatic stellate cell activation and increases collagen production and TGF-β1 mRNA expression (5 , 34) . TGF-β1 is a primary mediator of liver fibrosis (44) and may be the proximal stimulus for collagen production after adenosine receptor activation. Alternatively, the increase in TGF-β1 production may amplify the direct effects of adenosine A2A-receptor stimulation on stellate cell activation and collagen production. Either way, adenosine A2A-receptor activation and increased TGF-β1 expression are strongly associated with hepatic fibrosis (5) and the greater increase in TGF-β1 mRNA in livers of CCl4-treated WT than CD73KO mice is consistent with this mechanism.

Inflammatory cytokines play a role in hepatic fibrosis, and adenosine modulates the expression of these cytokines. IL-13 is a key mediator of tissue fibrosis and a potent stimulator and activator of TGF-β1, as well as a direct stimulus for collagen production (45) . Sugimoto et al. (46) found that IL-13 directly stimulates human hepatic stellate cells to produce collagen. In in vivo studies, IL-13 plays a central role in hepatic fibrosis primarily in parasite-induce hepatic fibrosis (47) . IL-13r{alpha}2 is a decoy receptor and inhibits IL-13’s profibrotic role (48 , 49) . In the lung, adenosine stimulates IL-13 production and IL-13 reciprocally increases extracellular adenosine levels synergistically stimulating the development of pulmonary inflammation and fibrosis, and the adenosine A2B receptor appears to play a role in this phenomenon (41 , 42 , 50) . In our study, the IL-13 protein level and IL-13r{alpha}1 mRNA were similar in the CCl4-treated WT and CD73KO mice, whereas message for the decoy receptor IL-13r{alpha}2 was significantly increased in CD73KO mice likely diminishing the effect of any IL-13 increase on hepatic fibrosis.

Ethanol ingestion, a major cause of hepatic cirrhosis/fibrosis, has long been known to increase levels of extracellular purines, particularly adenosine, both by increasing adenine nucleotide turnover and inhibiting adenosine uptake (18 , 51 52 53 54 55 56 57 58 59) , and many of the effects of ethanol on the central nervous system have been attributed to adenosine and adenosine receptor activation (56 , 57 , 59 60 61 62 63 64 65) . Interestingly, the pathogenic mechanisms most commonly adduced to explain the toxic effects of ethanol on the liver, hepatic hypoxia and diminished recycling of S-adenosylhomocysteine to S-adenosylmethionine (66 67 68) , may also promote an increase in extracellular adenosine. Thus, the demonstration that adenosine plays a critical role in experimental hepatic fibrosis is consistent with the known role of adenosine in the pharmacologic effects of ethanol ingestion. Data from epidemiologic studies provide further indirect evidence that adenosine plays a role in hepatic fibrosis and its associated mortality. Large epidemiologic studies suggest that coffee drinking diminishes, in a dose-dependent fashion, the risk of death from liver disease (69 70 71 72 73 74 75 76) , and the most abundant pharmacologic agent in coffee is caffeine, which is a nonselective antagonist of adenosine receptors. This finding suggests that more potent and selective adenosine receptor antagonists might be useful in the prevention of hepatic fibrosis.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the U.S. National Institutes of Health (GM56268, AR41911, and AA13336), King Pharmaceuticals, the General Clinical Research Center (M01RR00096), the Kaplan Cancer Center, and the Vilcek Foundation. B.N.C. is a consultant to and owns stock in King Pharmaceutical, which has patents on the use of adenosine A2A-receptor agonists to promote wound healing and use of A2A-receptor antagonists to inhibit fibrosis and a patent application for use of adenosine A1-receptor antagonists to treat osteoporosis and other diseases of bone. He is also a consultant for CanFite Biopharmaceuticals, Bristol-Myers Squibb, Cellzome, Tap Pharmaceuticals, Prometheus Laboratories, Regeneron (Westat, DSMB), Sepracor, Amgen, Endocyte, and Protalex. He is on the Honoraria/Speakers’ Bureaus for Tap Pharmaceuticals and Amgen and received stock from CanFite Biopharmaceuticals for membership in the Scientific Advisory Board.

Received for publication October 16, 2006. Accepted for publication January 17, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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