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Published as doi: 10.1096/fj.08-119701.
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(The FASEB Journal. 2009;23:473-482.)
© 2009 FASEB

Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs

Jörg Reutershan*, Irene Vollmer*, Stefanie Stark*, Rosalyn Wagner*, Kristian-Christos Ngamsri* and Holger K. Eltzschig*,{dagger},1

* Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany; and

{dagger} Mucosal Inflammation Program, Department of Anesthesiology and Perioperative Medicine, University of Colorado Health Science Center, Denver, Colorado, USA

1 Correspondence: Mucosal Inflammation Program, University of Colorado Denver, Department of Anesthesiology and Perioperative Medicine, 12700 E. 19th Ave., Mailstop B112, Research Complex 2, Room 7124, Aurora, CO 80045, USA. E-mail: holger.eltzschig{at}ucdenver.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular adenosine has been implicated as anti-inflammatory signaling molecule during acute lung injury (ALI). The main source of extracellular adenosine stems from a coordinated two-step enzymatic conversion of precursor nucleotides via the ecto-apyrase (CD39) and the ecto-5'-nucleotidase (CD73). In the present study, we hypothesized a critical role of CD39 and CD73 in mediating pulmonary neutrophil (PMN) transmigration during lipopolysaccharide (LPS) -induced lung injury. Initial studies revealed that pulmonary CD39 and CD73 transcript levels were elevated following LPS exposure in vivo. Moreover, LPS-induced accumulation of PMN into the lungs was enhanced in cd39–/– or cd73–/– mice, particularly into the interstitial and intra-alveolar compartment. Such increases in PMN trafficking were accompanied by corresponding changes in alveolar-capillary leakage. Similarly, inhibition of extracellular nucleotide phosphohydrolysis with the nonspecific ecto-nucleoside-triphosphate-diphosphohydrolases inhibitor POM-1 confirmed increased pulmonary PMN accumulation in wild-type, but not in gene-targeted mice for cd39 or cd73. Finally, treatment with apyrase or nucleotidase was associated with attenuated pulmonary neutrophil accumulation and pulmonary edema during LPS-induced lung injury. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in attenuating PMN trafficking into the lungs during LPS-induced lung injury and suggest treatment with their soluble compounds as a therapeutic strategy.—Reutershan, J., Vollmer, I., Stark, S., Wagner, R., Ngamsri, K.-C., Eltzschig, H. K. Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs.


Key Words: acute lung injury • ARDS • neutrophil • PMN • migration • chemotaxis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACUTE LUNG INJURY (ALI) is a syndrome characterized by acute hypoxemic respiratory failure in the setting of noncardiogenic pulmonary edema (1) . ALI significantly contributes to morbidity and mortality of critically ill patients (2) . Despite optimal management consisting of aggressive treatment of the initiating cause, vigilant supportive care, and the prevention of nosocomial infections, mortality ranges between 35 and 60% (1) . The pathogenesis of ALI is characterized by the influx of a protein-rich edema fluid into the interstitial and intra-alveolar spaces as a consequence of increased permeability of the capillary-alveolar barrier. In addition, experimental and clinical studies have demonstrated that pulmonary trafficking of polymorphonuclear leukocytes (PMNs) into the lungs resembles a key factor in the pathogenesis of ALI (3 4 5) . For example, recent studies of lipopolysaccharide (LPS) -induced acute lung injury demonstrated that attenuating PMN-trafficking via targeting chemokine receptors represents a therapeutic target in lung injury (5 , 6) . Similarly, neutrophil depletion has been shown to be protective in many animal models of ALI, and blocking the major PMN chemoattractant, interleukin 8 (IL-8), protects rabbits from lung injury following acid aspiration (7) . However, none of these principals have been introduced into clinical practice and at present, therapeutic approaches during ALI mainly focus on eliminating the originating cause in combination with supportive therapy.

To further characterize PMN trafficking during ALI, we hypothesized a contribution of extracellular adenosine in attenuating PMN accumulation in LPS-induced lung injury. Extracellular adenosine can signal through any of 4 adenosine receptors (ARs), the A1AR, A2AAR, A2BAR, and A3AR (8) . Particularly, the A2AAR and A2BAR have been suggested in attenuating acute inflammation in different models. Among the first studies to demonstrate an anti-inflammatory role of the A2AAR in vivo utilized a murine air-pouch model of acute inflammation, where Cronstein et al. demonstrated that the anti-inflammatory effects of methotrexate can be completely blocked by antagonists of the A2AAR, and thereby demonstrating an anti-inflammatory role of A2AAR signaling in acute inflammation in vivo (9) . Other studies extended these findings of A2AAR-dependent attenuation of inflammation into other models (e.g., acute lung injury) using genetic approaches (10 11 12 13) . Moreover, other genetic in vivo studies found a contribution of A2BAR signaling in attenuating acute inflammation and PMN trafficking, for example, during vascular injury (14) or vascular inflammation (15) , ischemia (16 17 18) , vascular leakage (19 20 21) , or acute lung injury (22 , 23) .

As outlined above, previous studies demonstrated extracellular adenosine signaling in modulating neutrophil accumulation, particularly during conditions of acute injury (e.g., ambient hypoxia) (16 , 18 , 19 , 21 , 24 25 26 27 28) . During such conditions, extracellular adenosine mainly stems from the enzymatic phosphohydrolysis of precursor nucleotides to adenosine (8 , 29 30 31) . This is achieved by a two-step enzymatic process involving the ecto-apyrase (CD39, conversion of ATP/ADP to AMP) and the ecto-5'-nucleotdiase (CD73, conversion of AMP to adenosine). Both enzymes have been previously implicated in attenuating acute injury and inflammation in models of ambient hypoxia (20 , 21) , cyclic-mechanical stretch (23) , or bleomycin-induced lung injury (32) . Here, we pursued the contribution of CD39 or CD73 to PMN trafficking into the lungs during LPS-induced lung injury by combing genetic and pharmacological approaches.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Wild-type male C57BL/6 and C57BL/6/129SVJ mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice that were gene deficient for the ecto-apyrase cd39 (cd39–/– mice) or the ecto-5'-nucleotidase cd73 (cd73–/– mice) were previously characterized (33 , 34) . Experimental groups were matched in age, gender (male mice), and weight. All animal experiments were approved by the Animal Care and Use Committee of the University of Tübingen. Mice were 8 to 12 wk of age.

Differential blood cell counts
Increased blood cell counts in gene-deficient mice with targets that alter cell transmigration have been described (35) and will influence the analysis of migratory activity. To reveal possible differences between the different groups of mice, baseline differential blood counts were manually performed in cd39–/– and cd73–/– mice and their corresponding wild-type controls.

LPS-induced expression of CD39 and CD73
LPS-induced expression of CD39 and CD73 was determined by real-time RT-PCR. At indicated times after LPS exposure, lungs of wild-type mice were perfused free of blood, removed and stored at –80°C. Total RNA was isolated using the total RNA isolation NucleoSpin RNA II kit (Macherey-Nagel GmbH, Düren, Germany) as described previously (17) . RNA was washed, and the concentration was quantified. cDNA synthesis was performed using reverse transcription according to the manufacturer’s instructions (i-script kit; Bio-Rad, Hercules, CA, USA). The primer sets for the RT-PCR contained 1 µM sense and 1 µM antisense with SYBR Green I (Molecular Probes, Carlsbad, CA, USA). Primer sequences for murine CD39/CD73 were 5'-TACCACCCCATCTGGTCATT-3' and 5'-GGACGTTTTGTT TGGTTGGT-3' (sense/antisense) and 5'-CAAATCCCACACAACCACTG-3' and 5'-TGCTCACTTGGTCACAGGAC-3', respectively. The primer set was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, 72°C for 1 min. Murine β-actin (sense primer, 5'-ACATTGGCATGGCTTTGTTT-3' and antisense primer, 5'-GTTTGCTCCAACCAACTGCT-3) in identical reactions were used to control for the starting template. Levels and fold change in mRNA were determined as described previously (36) .

Murine model of acute lung injury
Up to 4 mice were exposed to aerosolized LPS in a custom-built cylindrical chamber (20x9 cm) connected to an air nebulizer (MicroAir; Omron Healthcare, Vernon Hills, IL, USA). LPS from Salmonella enteritidis (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline (500 µg/ml) and mice inhaled LPS for 30 min. As previously shown, this mimics several aspects of acute lung injury, including PMN recruitment into all compartments of the lung, increase in vascular permeability (37) , release of chemokines, and disruption of the pulmonary architecture (38) . Control mice were exposed to saline aerosol.

PMN trafficking in the lung
PMN recruitment into the different compartments of the lung (pulmonary vasculature, interstitium, alveolar airspace) was assessed as described (37) . Briefly, 24 h after LPS exposure, intravascular PMNs were labeled by intravenous injection of Alexa 633-labeled GR-1 to murine PMN. After 5 min, mice were euthanized, and nonadherent PMN were removed from the pulmonary vasculature by flushing 10 ml of PBS at 25 cmH2O through the spontaneously beating right ventricle. Bronchoalveolar lavage (BAL) was withdrawn, and lungs were removed, minced, and digested in the presence of excess unlabeled anti-GR-1 to prevent possible binding of the injected antibody to extravascular PMN. A cell suspension was prepared by passing the digested lungs through a 70-µm cell strainer (BD Falcon, Bedford, MA, USA). Total cells in BAL and lung were counted and the percentage of PMNs determined by flow cytometry. In the BAL, PMNs were identified by their typical appearance in the forward/sideward scatter and their expression of CD45 (clone 30-F11), 7/4 (clone 7/4), and GR-1 (clone RB6–8C5). In the lung, the expression of GR-1 was used to distinguish intravascular (CD45+7/4+GR-1+) from interstitial (CD45+7/4+GR-1–) PMNs, which were not reached by the injected antibody (37) .

Pulmonary microvascular permeability
Pulmonary microvascular permeability in cd39–/– and cd73–/– mice and their appropriate wild-type control mice was determined by measuring extravasation of Evans blue dye (39) . Evans blue (20 mg/kg; Sigma-Aldrich) was injected intravenously 30 min prior to euthanasia. Lungs were perfused through the spontaneously beating right ventricle to remove intravascular dye. Lungs were removed, and Evans blue was extracted, as described previously (40) . The absorption of Evans blue was measured at 620 nm and corrected for the presence of heme pigments: A620 (corrected) = A620 – (1.426xA740+0.030) (41) . Extravasated Evans blue was determined in the different animal groups 6 h after LPS or saline inhalation and calculated against a standard curve (µg Evans blue dye/g lung).

Pharmacological inhibition of CD39
In subsets of experiments, mice were pretreated with a nonspecific inhibitor of E-NTPDases (POM-1, Na6[H2W12O40]) (42 43 44) , and LPS-induced PMN migration into the BAL was determined. This compound was synthesized at the Pharmaceutical Institute, Pharmaceutical Sciences (Bonn, Germany). Mice were treated with POM-1 (5 mg/kg i.p.) or sterile saline as vehicle control 30 min prior to LPS exposure, as described previously (42 , 43) .

Effect of soluble CD39 and CD73 on PMN migration and microvascular permeability
In some experiments, wild-type mice were treated with soluble apyrase (conversion of ATP/ADP to AMP, Sigma-Aldrich) or nucleotidase (conversion of AMP to adenosine, Sigma-Aldrich; purified from Crotalus atrox venom) (23 , 43) . Mice were pretreated with apyrase (10 U i.p.), nucleotidase (10 U i.p.) (12) , or a combination of both 30 min prior to LPS exposure. PMN accumulation into the BAL and microvascular permeability were determined as described above. Control animals received sterile saline as vehicle control.

Immunohistochemistry
Cd39–/–, cd73–/–, or corresponding wild-type control mice were euthanized 24 h after exposure to LPS. Control mice did not receive LPS. The pulmonary circulation was perfused free of blood, the trachea was cannulated, and the lung was inflated with 4% paraformaldehyde (PFA) for 10 min at 25 cmH2O. The lungs were subsequently removed and fixed in PFA for 24 h. Paraffin-embedded sections (5 µm) were stained for PMNs, using the avidin-biotin technique (Vector Laboratories, Burlingame, CA, USA), as described previously (45) . Briefly, deparaffinized and rehydrated sections were incubated with avidin, 10% rabbit serum, and 0.5% fish skin gelatin oil (FSGO) for 1 h to block nonspecific binding. After washing with PBS, a specific antibody against mouse neutrophils (clone 7/4; Caltag, Burlingame, CA, USA) was added (1 µg/ml) and incubated overnight. Sections were then washed and incubated with 5 µg/ml biotinylated rabbit anti-rat immunoglobulin G (IgG; Vector Laboratories) for 1 h, followed by avidin-biotin-peroxidase complexes (Vectastain Elite ABC kit, Vector Laboratories), washed with PBS, incubated with diaminobenzidine (DAB kit; Vector Laboratories), and counterstained with hematoxylin. Some sections were stained with the appropriate isotype and served as negative controls.

Statistical analysis
Statistical analysis was performed with JMP statistical software (ver. 7.0; SAS Institute, Cary, NC, USA). Differences between the groups were evaluated by one-way analysis of variance (ANOVA) followed by a post hoc Tukey test. Data are presented as means ± SD, and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD39 and CD73 transcript levels are increased following LPS exposure
Previous studies had shown that protective effects of CD39 and CD73 occur in conjunction with transcriptional responses (e.g., during hypoxia, cyclic mechanical stretch, or ischemia) (17 , 20 , 23 , 27 , 42 , 43 , 46) . Therefore, we pursued transcriptional responses of CD39 and CD73 following LPS inhalation. For this purpose, we exposed mice to aerosolized LPS from S. enteritidis via inhalation for 30 min and harvested the lungs at indicated time periods (30–180 min). As shown in Fig. 1 , transcript levels of CD39 and CD73 were significantly increased at 180 min following LPS exposure. Taken together, these studies demonstrate transcriptional induction of pulmonary CD39 and CD73 following in vivo exposure to aerosolized LPS.


Figure 1
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  		Figure 1. LPS induces expression of CD39 and CD73 in the lung. Lungs were homogenized at indicated time points after LPS exposure, and RNA was isolated. Data are presented as a fold increase over vehicle control; n = 4. C, control. *P < 0.05 vs. vehicle control without LPS.

Functional role of CD39 and CD73 in pulmonary PMN trafficking following LPS exposure
After having shown that CD39 and CD73 transcript levels are elevated during LPS-induced ALI, we next pursued the functional role of CD39 and CD73 in PMN trafficking into the lungs. Here, we first performed immunohistochemical studies of PMN accumulation 24 h following LPS exposure using previously characterized gene-targeted mice for cd39 (33) or cd73 (34) . As shown in Fig. 2 , LPS exposure of wild-type mice was associated with PMN accumulation in pulmonary tissues. However, these responses were clearly enhanced following gene-targeted deletion of cd39. Similarly, LPS-induced increases in pulmonary PMN accumulation were more pronounced in cd73–/– mice (Fig. 3 ).


Figure 2
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  		Figure 2. PMN infiltration into the lungs shown by immunohistochemistry. Cd39–/– mice or their corresponding wild-type controls were exposed to LPS. After 24 h, lungs were removed and fixed, and sections were stained with a specific neutrophil marker (7/4, brown cells). Control animals did not receive LPS. Images are representative of n = 4 experiments. Inset: isotype control.


Figure 3
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  		Figure 3. PMN infiltration into the lungs shown by immunohistochemistry. Cd73–/– mice or their corresponding wild-type controls were exposed to LPS. After 24 h, lungs were removed and fixed, and sections were stained with a specific neutrophil marker (7/4, brown cells). Control animals did not receive LPS. Images are representative of n = 4 experiments. Inset: isotype control.

To reveal alterations in the peripheral blood counts of cd39–/– and cd73–/– mice that may contribute to increased migratory activity, we next obtained leukocyte counts in the peripheral blood of cd39–/– or cd73–/– mice (Table 1 ). Total leukocyte, PMN, and lymphocyte counts were not different between gene-targeted mice and their corresponding controls (cd73–/– mice showed significantly increased monocyte counts).


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  		Table 1. Baseline cell counts

We then studied PMN trafficking into different pulmonary compartments following LPS exposure (vascular, interstitial or intra-alveolar compartment). As shown in Fig. 4 , PMN numbers in cd39–/– or cd73–/– mice were not elevated in the vascular compartment at 24 h after LPS exposure (Fig. 4A ). In contrast, LPS-associated increases in interstitial or intra-alveolar PMN numbers were significantly enhanced following genetic deletion of cd39 or cd73. Taken together, these data indicate that CD39 and CD73 are critical mediators to attenuate LPS-associated increases in interstitial and alveolar neutrophil accumulation.


Figure 4
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  		Figure 4. LPS-induced accumulation of PMNs in the different compartments of wild-type (solid bars), cd39–/–, and cd73–/– mice (open bars). Accumulation of PMNs in the vasculature (IV) (A), the lung interstitium (IS) (B), and the bronchoalveolar space (BAL) (C) were analyzed 24 h following LPS exposure. Values are means ± SD of n = 8 experiments. *P < 0.05 vs. vehicle control without LPS; #P < 0.05 vs. wild-type control within the same treatment group (±LPS).

LPS-induced pulmonary barrier dysfunction is enhanced in cd39–/– or cd73–/– mice
One of the pathophysiological hallmarks of ALI is attenuated capillary-alveolar barrier function, leading to noncardiogenic pulmonary edema (1) . To extend the evidence for CD39- and CD73-dependent attenuation of PMN trafficking during LPS-induced ALI, we next measured capillary-alveolar barrier function by using the albumin marker Evans blue in this model. As shown in Fig. 5 , increases in LPS-induced Evans blue accumulation were further enhanced in mice with genetic deletion of cd39 (Fig. 5A ) or cd73 (Fig. 5B ). In conjunction with the findings from above, these studies reveal a critical role of CD39 and CD73 in attenuating pulmonary PMN accumulation and LPS-induced barrier dysfunction during ALI.


Figure 5
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  		Figure 5. LPS inhalation induced increase in microvascular permeability in wild-type (solid bars), cd39–/–, or cd73–/– mice (open bars), as assessed by the extravasation of Evans blue. Data are means ± SD from 8 experiments. Note that LPS-induced increases in lung permeability were significantly higher in cd39–/– and cd73–/– mice vs. wild-type controls. *P < 0.05 vs. negative control without LPS; #P < 0.05 vs. wild-type control within the same treatment group (±LPS).

Pharmacological inhibition of nucleotide phosphohydrolysis enhances LPS-elicited PMN trafficking into the lungs
On the basis of these findings showing a critical role of extracellular nucleotide phosphohydrolysis in LPS-induced lung injury, we next pursued the hypothesis that attenuated nucleotide phosphohydrolysis in cd39–/– or cd73–/– mice is responsible for these observations. For this purpose, we used a recently described inhibitor of ecto-nucleoside-triphosphate-diphosphohydrolases, POM-1, a polyoxometalate (42 43 44) . If attenuated, extracellular phosphohydrolysis in cd39–/– or cd73–/– mice is responsible for enhanced pulmonary PMN accumulation and barrier dysfunction, POM-1 treatment would produce a similar phenotype in wild-type mice but would not affect PMN trafficking in cd39–/– or cd73–/– mice. For this purpose, we treated mice with POM-1 (5 mg/kg i.p.) or sterile saline as vehicle control 30 min prior to LPS exposure. As shown in Fig. 6 , increases in pulmonary PMN accumulation were enhanced in POM-1-treated mice. In contrast, POM-1 treatment was not associated with additional increases in pulmonary neutrophil accumulation in gene-targeted mice for cd39 or cd73. Taken together, these findings suggest that modulation of PMN trafficking by CD39 or CD73 involves their enzymatic function.


Figure 6
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  		Figure 6. The effect of the nonspecific CD39 inhibitor POM-1 on LPS-induced PMN transmigration into the alveolar space was tested. In wild-type mice, pretreatment with POM-1 significantly increased PMN accumulation in the BAL, consistent with increased PMN counts in cd39–/– mice. POM-1 did not affect PMN trafficking in cd39–/– or cd73–/– mice. Data are means ± SD from 4 experiments. *P < 0.05 vs. negative control without LPS; #P < 0.05 vs. wild-type control within the same treatment group (±POM-1).

Treatment with soluble apyrase or nucleotidase attenuates PMN accumulation in the BAL during LPS-induced ALI
On the basis of the above findings of increased lung injury with genetic or pharmacological inhibition of extracellular nucleotide phosphohydrolysis, we next pursued these findings in a therapeutic setting. Here, we treated mice with soluble apyrase (conversion of ATP/ADP to AMP) or nucleotidase (conversion of AMP to adenosine) and assessed neutrophil accumulation into the BAL (Fig. 7A ) or capillary-alveolar leakage (Evan’s blue extravasation, Fig. 7B ). Serendipitously, treatment with soluble apyrase/nucleotidase attenuated LPS-induced lung injury, as assessed by BAL neutrophil accumulation and pulmonary edema. Taken together, these studies confirm an anti-inflammatory and tissue-protective role of extracellular adenosine production during acute lung injury induced by LPS inhalation.


Figure 7
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  		Figure 7. Effect of soluble ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) on LPS-induced PMN trafficking (A) and microvascular permeability (B). Pretreatment of wild-type mice with soluble apyrase (converts ATP/ADP to AMP) or nucleotidase (converts AMP to adenosine) significantly reduced LPS-induced PMN accumulation in the BAL and microvascular permeability. Data are means ± SD from 4 experiments. *P < 0.05 vs. negative control without LPS; #P < 0.05 vs. positive control (LPS, no pretreatment).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ALI significantly contributes to critical illness, as it occurs frequently (2) and carries a high mortality rate (1) . Moreover, the only therapeutic interventions currently available are elimination of the causative agents and supportive therapy (1) . Recent studies suggest that targeting neutrophil trafficking during ALI may represent a potential therapeutic target (5 , 6 , 24) . Here, we used LPS-induced lung injury as a model to identify endogenous mechanisms of lung protection and to test our findings in a therapeutic approach. On the basis of the hypothesis that extracellular adenosine production could influence PMN-trafficking during ALI, initial in vivo studies revealed prominent increases in pulmonary CD39 and CD73 transcript levels following LPS exposure. Since ecto-apyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate-limiting for extracellular adenosine generation, we examined their functional role in PMN trafficking and during ALI. Here, we found increased PMN trafficking into the interstitial and intra-alveolar lung compartments in conjunction with attenuated capillary-alveolar barrier function in mice with targeted gene deletion of cd39 or cd73 when exposed to LPS inhalation. Studies with a pharmacological inhibitor of extracellular nucleotide phosphohydrolysis (POM-1) suggested a crucial role of CD39 and CD73 enzymatic function in these responses. Moreover, treatment with soluble apyrase or nucleotidase resulted in significant attenuation of ALI in wild-type animals. Taken together, these studies identify CD39 and CD73 as critical control points of pulmonary PMN trafficking during acute lung injury induced by LPS inhalation.

Consistent with the present study, other investigations confirmed protective effects of adenosine on the pulmonary integrity in models of acute injury. For example, a study on CD73-mediated adenosine production found tissue protection in a model of bleomycin-induced lung injury (32) . Other studies found a protective role of CD39- and CD73-dependent adenosine production during cyclic mechanical stretch in vitro or during ventilator-induced lung injury in vivo (23) . In fact, the authors could demonstrate transcription-dependent induction of CD39 and CD73 during stretch exposure in vivo and in vitro. Moreover, treatment with soluble apyrase or nucleotidase enhanced survival time and attenuated lung inflammation during ventilator-induced lung injury (23) . Similarly, studies of hypoxia-associated lung inflammation and vascular leakage during hypoxia confirmed an anti-inflammatory role for CD73 and CD39 in these models (20 , 21 , 34) . In contrast to these findings, a recent study examined the role of CD73 in experimental autoimmune encephalomyelitis and found that cd73–/– mice are protected from encephalitis (47) . Moreover, blockade with an A2AAR-specific antagonist protected wild-type mice from encephalitis, suggesting that CD73-dependent adenosine production and signaling through A2AARs are required for the efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalitis. Possible explanation for these contrasting findings may be related to different mechanisms governing inflammatory cell trafficking during inflammation of the central nervous system or the lungs. Other differences between both models could include differences in the migratory stimulus (LPS-induced inflammation vs. autoimmune encephalitis) or differences between the subtype of inflammatory cells that were studies (neutrophils vs. lymphocytes).

Other studies have addressed the contribution of different ARs in attenuating PMN trafficking during acute lung injury. For example, some studies of LPS-induced lung injury showed a more severe phenotype in gene-targeted mice for the A2AAR (10 , 24) . In addition, a recent study investigated the role of different ARs in acute lung injury by combining ventilator or LPS-induced lung injury (22) . These studies found that specific deletion of the A2BAR was associated with a more severe phenotype during lung injury, while treatment with an A2BAR-specific agonist resulted in attenuation of pulmonary inflammation and capillary-alveolar leakage, in association with improved survival (22) .

In addition, it remains an important question whether the reduction of PMN accumulation during LPS-induced ALI is due to diminished adhesion molecule expression and function or involves a reduction in chemokine production. On the basis of studies in A2BAR bone marrow chimeric mice that either express the adenosine A2BAR on myeloid cells or on pulmonary tissues, a recent study found a contribution of both myeloid and pulmonary adenosine receptors in mediating acute lung injury (22) . While defects in the alveolar-capillary barrier function observed in A2BAR–/– mice mainly involved pulmonary A2BARs, the increased production of cytokines and lung inflammation involved both pulmonary and myeloid adenosine receptors. Taking together, such studies suggest a combination of pulmonary (e.g., via diminished adhesion molecule expression) and myeloid ARs (e.g., by reduction in chemokine production) in attenuating inflammatory cell trafficking during ALI.

At present, it is unclear whether the observed induction of CD73 and CD39 results from a transcription-dependent induction of CD39 and CD73 on the pulmonary tissues, or from an accumulation of inflammatory cells that express these molecules. The dominant inflammatory cell type that accumulates in pulmonary tissues early after LPS exposure (within the first 24 h) are neutrophils (5) . In fact, a study of PMN-dependent ATP release and metabolism to adenosine used flow cytometry to study CD39 and CD73 expression on neutrophils (48) . These studies revealed that while neutrophils express high levels of CD39, almost no CD73 is expressed on human or murine PMNs (48) . Therefore, it appears unlikely that increases of CD73 expression during LPS-induced lung injury result from neutrophils invading the lungs. Other studies used immunohistochemistry to study CD39 and CD73 expression during acute lung injury (23) . In this study, mice were exposed to pressure-controlled ventilation at high inspiratory pressure levels (45 mbar) to induce lung injury (49) . These studies found increased expression of CD39 and CD73 on pulmonary epithelia and endothelia (23) . Moreover, previous studies found transcription-dependent increases of CD73 and CD39 expression with exposure to ambient hypoxia (20 , 50) . Taken together, such studies suggest that both CD39 and CD73 are highly transcriptionally regulated during acute injury.

In contrast to the present study of acute injury, studies investigating chronic pulmonary disease have identified a detrimental role of elevated adenosine levels (51 52 53 54 55) . For example, levels of adenosine are chronically increased in the lungs of asthmatics (56) and correlate with the degree of inflammatory insult (57) , suggesting a provocative role of adenosine in asthma or chronic obstructive pulmonary disease (58) . In addition, adenosine-deaminase (ADA)-deficient mice develop signs of chronic pulmonary injury in association with chronically elevated pulmonary adenosine levels. In fact, ADA-deficient mice die within weeks after birth from severe respiratory distress (59) , and recent studies suggest that attenuation of adenosine signaling may reverse the severe pulmonary phenotypes in ADA-deficient mice, suggesting that chronic adenosine elevation can affect signaling pathways that mediate aspects of chronic lung disease (55 , 59) .

Taken together, the present studies implicate CD39- and CD73-dependent nucleotide phosphohydrolysis in attenuating neutrophil trafficking and capillary-alveolar barrier function during ALI. Moreover, these studies suggest soluble compounds that increase nucleotide conversion to adenosine in the treatment of endotoxin-induced lung injury. Further challenges will involve the translation of these findings from mice to man and the characterization of potential side effects of such therapeutic approaches (for example, on chronic lung diseases or platelet function) (52 , 55 , 60) .


   ACKNOWLEDGMENTS
 
We thank Linda F. Thompson and Simon C. Robson for kindly providing us with gene-targeted mice, and Christa E. Mueller for pharmacological compounds. This study was supported by a German Research Foundation grant (RE 1683/3-1 to J.R.) and a Foundation for Anesthesia Education and Research grant to H.K.E.

Received for publication August 12, 2008. Accepted for publication September 11, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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