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* Department of Anesthesiology and Intensive Care Medicine and
Department of Pathology, Tübingen University Hospital, Tübingen, Germany;
Department of Anesthesia, Perioperative and Pain Medicine, Children’s Hospital, Harvard Medical School, Boston, MA 02115; and
Institute of Brain Research, Tübingen University Hospital, Tübingen, Germany
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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200,000 patients develop ALI annually in the United States, leading to 75,000 deaths and accounting for up to 3.6 million hospital days (2). The pathogenesis of ALI is characterized by the influx of a protein-rich edema fluid into the interstitial and intraalveolar spaces as a consequence of increased permeability of the alveolar-capillary barrier. The importance of endothelial injury and increased vascular permeability to the formation of pulmonary edema in this disorder has been well-established (1). Nevertheless, molecular details of how pulmonary capillary leakage is caused and maintained during ALI are largely unknown and studies linking its mechanisms with mechanical ventilation are currently areas of intense investigation (3, 4). Despite the large impact of ALI on morbidity and mortality in critically ill patients (1), many episodes of ALI are self-limiting, and resolve spontaneously through unknown mechanisms. For example, patients undergoing major surgery requiring prolonged mechanical ventilation have an overall incidence of ALI between 0.2 and 5%, depending on the kind of surgery (5, 6, 7). Based on the rare occurrence of clinically relevant ALI in patients requiring mechanical ventilation, we hypothesized the existence of innate adaptive pathways to dampen acute increases in the capillary-alveolar permeability elicited by mechanical stretch or ventilation. In fact, the present study revealed a protective role of extracellular adenosine generation via the ectoapyrase (CD39, ATP/ADP conversion to AMP) and the ecto-5'-nucleotidase (CD73, AMP conversion to adenosine) during ALI induced by mechanical ventilation.
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Materials and Methods |
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Human microvascular endothelial cells (HMEC-1) and human bronchial epithelial cells (Calu-3) were cultured as described previously (8, 9).
In vitro stretch model
To study the consequences of cyclic mechanical stretch on the release of barrier protective mediators, we adopted a previously described in vitro model resembling mechanical ventilation by applying cyclic mechanical stretch (10). In short, Calu-3 human bronchial epithelial cells were plated on BioFlex culture plates-collagen type I (BF-3001C; FlexCell International) and allowed to attach and grow to 80% confluence. The medium was changed to MEM plus 10% FBS. Plates were then placed on a FlexCell FX-4000T Tension Plus System and stretched at 20% stretch maximum, 0.7% stretch minimum, sine wave 5 s, on 5 s off. Supernatants were collected at indicated time points, flash-frozen, and stored at –80°C for further analysis. In controls, supernatants from Calu-3 human epithelia cultured under similar conditions at rest (without application of cyclic stretch) were used. In duplicate wells, cells adherent to the plates were used for transcriptional analysis (see below).
HPLC analysis
Supernatants were resolved by HPLC with a pump P680 and a UVD 170 detector on a reverse-phase column (Grom-Sil 120-ODS-ST-5µ; 150 x 3 mm; Grom) using a mobile-phase gradient from 0 to 25% acetonitril/0.3 mM KH2PO4 (pH 5) in 5 min as described previously (9, 11, 12). UV absorption spectra were measured at 260 nm. In subsets of experiments, fractions were collected, evaporated to dryness by speed-vac, reconstituted in HBSS (20-fold concentrated), and bioactivity was determined by permeability assay (see below). In additional experiments, lungs from mechanically ventilated animals (pressure controlled ventilation, inspiratory pressure 15 millibar (mbar), 100% inspired oxygen concentration) were harvested at indicated time points and flash-frozen in liquid nitrogen. Adenine nucleosides were extracted from frozen lungs using 0.4 N perchloric acid and adenosine levels were determined as described previously (13). In subsets of experiments, adenosine concentrations were determined in the bronchoalveolar lavage (BAL) fluid derived from mice exposed to mechanical ventilation over indicated time periods (inspiratory pressure of 15 mbar, 100% inspired oxygen concentration). To obtain BAL fluid, the tracheal tube was disconnected from the mechanical ventilator and the lungs were lavaged three times with 0.5 ml of PBS containing 200 µM dipyridamole and 1 µg/ml deoxycoformycin. Removed fluid was centrifuged immediately, shock-frozen with liquid nitrogen for HPLC analysis.
Macromolecule paracellular permeability assay
Using a modification of methods previously described (14), HMEC-1 were grown on polycarbonate permeable supports (0.4-µm pore, 6.5-mm diameter; Costar) and studied 7–10 days after seeding (2–5 days after confluency). Paracellular flux rates were determined as described previously. In a subset of experiments, the nonspecific adenosine receptor antagonist 8-phenyl-theophylline (8-PT; 10 µM; Sigma-Aldrich) was added to the inserts and the supernatants, before the start of the experiment. In other experiments and as described previously, supernatants were treated with bovine adenosine deaminase at a concentration that was previously shown effective in abolishing adenosine-mediated barrier responses (0.1 nM; Sigma-Aldrich) (11).
Murine mechanical ventilation
All animal protocols were in accordance with the German guidelines for use of living animals and were approved by the Institutional Animal Care and Use Committee of the Tübingen University Hospital and the Regierungspräsidium Tübingen. Mice deficient in cd39 on the C57BL/6/129 svj strain or in cd73 on the BL6 stain were generated, validated, and characterized as described previously (15, 16). As controls, littermate mice matched according to sex, age, and weight were used. Animals were anesthetized with pentobarbital (70 mg/kg i.p. for induction; 20 mg/kg/h for maintenance) and placed on a temperature-controlled heated table with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37°C. In addition, all animals were monitored with an electrocardiogram (Hewlett Packard). Fluid replacement was performed with normal saline, 0.05 ml/h i.p. Tracheotomy was performed in a supine position. In short, the trachea was surgically exposed, horizontally incised, and a blunt polyurethane cannula (Insyte 22G; BD Biosciences) was introduced and sutured in place. The tracheal tube was connected to a mechanical ventilator (Servo 900C; Siemens, with pediatric tubing). Mice were ventilated in a pressure-controlled ventilation mode at different inspiratory pressure levels (15, 35, and 45 mbar) for different time periods (30–90 min). Respiratory rate and inspiratory-to-expiratory time ratios were adjusted based on arterial blood gas sampling obtained by cardiac puncture in control animals to maintain a carbon dioxide partial pressure between 35 and 40 mmHg and a pH between 7.35 and 7.40. All animals were ventilated with 100% inspired oxygen, expect during experiments to determine a potential influence of the inspired oxygen concentration on CD39 or CD73 transcription. In subsets of experiments, mice were treated with the ecto-5'-nucleotidase inhibitor adenosine 5'-(
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-methylene) diphosphate (APCP; Sigma-Aldrich; 100 mg/kg i.p.), soluble apyrase (Sigma-Aldrich; 10 U i.p.), soluble 5'-nucleotidase (Sigma-Aldrich; purified from Crotalus atrox venom; 10 U i.p.), the A2A adenosine receptor (AR) antagonist ZM241385 (Tocris Cookson; 1 mg/kg i.p.), or the A2BAR antagonist MRS1754 (Tocris Cookson; 1 mg/kg i.p.) 30 min before induction of anesthesia.
Transcriptional analysis
To examine the influence of mechanical ventilation on CD39 and CD73 transcript levels, C57BL/6J mice (Charles River Laboratories) were ventilated in a pressure-controlled fashion with indicated settings. Mice were euthanized at indicated time points and the remaining blood was removed from the pulmonary circulation by injection of 1 ml of PBS into the right heart. Lungs were excised and immediately frozen at –80°C until transcriptional profiling. For this purpose, total RNA was isolated using the total RNA isolation NucleoSpin RNA II kit as described previously (Macherey & Nagel) (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). The primer sets for the RT-PCR contained 1 µM sense and 1 µM antisense with SYBR Green I (Molecular Probes). 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. Primer sequences for human CD39/CD73 were 5'-AGC AGC TGA AAT ATG CTG GC-3', 5'-GAG ACA GTA TCT GCC GAA GTC C-3' and 5'-ATT GCA AAG TGG TTC AAA GTC A-3', 5'-ACA CTT GGC CAG TAA AAT AGG G-3', (sense/antisense), 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 or human -actin (sense primer, 5'-ACATTGGCATGGCTTTGTTT-3' and antisense primer, 5'-GTTTGCTCCAACCAACTGCT-3 (murine); sense primer 5'-GGT GGC TTT TAG GAT GGC AAG-3' and antisense primer 5'-ACT GGA ACG GTG AAG GTG ACA G-3' (human)) in identical reactions were used to control for the starting template. Levels and fold change in mRNA were determined as described previously (18).
Immunoblotting experiments
In subsets of experiments, we determined CD39 and CD73 protein content from whole lungs. For this purpose, C57BL/6J mice (Charles River Laboratories) were ventilated with indicated ventilator settings and euthanized. Remaining blood was removed from the pulmonary circulation by injection of 1 ml of PBS into the right heart. The lungs were excised and immediately frozen at –80°C until immunoblotting. For this purpose, tissues were homogenized and lysed for 10 min in ice-cold lysis buffer (107 polymorphonuclear neutrophils (PMN)/500 µl; 150 mM NaCl, 25 mM Tris (pH 8.0), 5 mM EDTA, 2% Triton X-100, and 10% mammalian tissue protease inhibitor mixture; Sigma-Aldrich), and collected into microfuge tubes. After spinning at 14,000 x g to remove cell debris, the pellet was discarded. Proteins were solubilized in reducing Laemmli sample buffer and heated to 90°C for 5 min. Samples were resolved on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 (PBST) and 4% BSA. The membranes were incubated in 10 µg/ml CD39 goat polyclonal Ab raised against the C terminus (Santa Cruz Biotechnology) or CD73 rabbit polyclonal Ab raised against the amino acids 275–574 (Santa Cruz Biotechnology) for 1 h at room temperature, followed by 10-min washes in PBS. The membranes were incubated in 1:3,000 donkey anti-goat HRP for CD39 (Santa Biotechnology), or goat anti-rabbit HRP for CD73 (Perbio Science). The wash was repeated and proteins were detected by ECL.
Immunohistochemistry
To examine the influence of mechanical ventilation on pulmonary CD39 and CD73 expression, mice were ventilated in a pressure-controlled fashion over indicated time periods. Mice were euthanized and the lungs were perfused via the right ventricle with 5 ml of PBS. Lungs were subsequently removed and stained with CD39 goat polyclonal Ab raised against the C terminus (Santa Cruz Biotechnology) or CD73 rabbit polyclonal Ab raised against the amino acids 275–574 (Santa Cruz Biotechnology) as described previously (17). In controls, normal goat or rabbit IgG was used at identical concentrations and staining conditions as the target primary Abs.
Bronchoalveolar lavage
To obtain BAL fluid, the tracheal tube was disconnected from the mechanical ventilator and the lungs were lavaged three times with 0.5 ml of PBS. All removed fluid was centrifuged immediately and the supernatant was aliquoted for albumin or MIP-2 determination.
Measurement of albumin and MIP-2 in BAL fluid
BAL fluid samples were thawed to 4°C and albumin or MIP-2 concentrations were measured using murine quantitative ELISA systems (albumin: Bethyl; MIP-2: R&D Systems), according to the instructions given by the manufacturer. All analyses were made in triplicate and mean values were used for statistical analysis.
Myeloperoxidase assay (MPO)
Pulmonary neutrophil sequestration was quantified using a myeloperoxidase (MPO) assay as described previously (19, 20, 21). In short, animals were euthanized and lungs were perfused with 5 ml of PBS through the right ventricle. Lungs were excised, frozen in liquid nitrogen, lyophilized, mechanically homogenized, and washed in a 50 mM potassium phosphate buffer solution (pH 6.0) to remove all hemoglobin. The resulting pellet was resuspended in 1.5 ml of a solution containing hexadecyltrimethylammonium bromide (5 g/L). The solution was subjected to three cycles of freezing (on dry ice) and thawing (at room temperature), sonicated for 40 s, and centrifuged at 10,000 x g for 5 min at 4°C. The supernatant was then assayed for MPO activity using a spectrophotometric reaction with o-dianisidine hydrochloride (Sigma-Aldrich) at 450 nm. Absorbance at 450 nm was measured and reported as difference in OD (
OD) over 5 min.
Wet-to-dry ratios
Wet-to-dry ratios were measured as previously described (12, 15). In short, following ventilation with indicated settings, lungs were excised en bloc. The weight was obtained immediately to prevent evaporative fluid loss of the tissues. Lungs were than lyophilized for 48 h and the dry weight was measured. Wet-to-dry ratios were then calculated as milligrams of water per milligram of dry tissue.
Blood gas analysis
To assess pulmonary gas exchange, blood gas analyses were performed in subsets of experiments by obtaining arterial blood via cardiac puncture. In short, a lateral thoracotomy was performed to access the left heart and blood was obtained via cardiac puncture. Analysis was performed immediately after collection with the I-STAT Analyzer (Abbott) and the arterial partial pressure of oxygen (PaO2) was measured, in addition to arterial partial carbon dioxide pressure and pH values.
Histopathological evaluation of ALI
Following ventilation at indicated settings, the mice were euthanized and lungs were fixed by instillation of 10% formaldehyde solution via the tracheal cannula at a pressure of 20 mbar. Lungs were then embedded in paraffin and stained with H&E. Two random tissue sections from four different lungs in each group were examined by a pathologist who was blinded to the genetic background/treatment of the mice. ALI was scored as described previously, (22) according to the following criteria: 1) alveolar congestion, 2) hemorrhage, 3) infiltration or aggregation of neutrophils in airspace or vessel wall, and 4) thickness of the alveolar wall/hyaline membrane formation. For each subject, a 5-point scale was applied: 0, minimal (little) damage; 1+, mild damage; 2+, moderate damage; 3+, severe damage; and 4+, maximal damage. Points were added up and are expressed as median ± range (n = 4).
Data analysis
Lung injury score and survival data are given as median (range), all other data are presented as mean ± SD from four to six animals per condition. We performed statistical analysis using the Student t test (two sided, < 0.05) or ANOVA to determine group differences. Lung injury score was analyzed with the Kruskal-Wallis rank test. Kaplan Maier curves were compared using the log-rank test (Mantel-Haenszel) test. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Many episodes of ALI are self-limiting and resolve spontaneously through unknown mechanisms. Therefore, we hypothesized the existence of metabolic pathways responsible for the innate protection of the capillary-alveolar barrier function during conditions of stretch or mechanical ventilation. To pursue this hypothesis, we adopted an in vitro model of cyclic mechanical stretch (10). For this purpose, we exposed Calu-3 human bronchial epithelia over 48 h to cyclic mechanical stretch, collected the supernatants, and tested them on a previously described model of endothelial barrier function (14). Indeed, we found that paracellular endothelial flux rates were significantly attenuated using supernatant of stretched epithelia (approximately one-third of control, 48 h of cyclic mechanical stretch, Fig. 1A). Such results suggest the release of a soluble bioactive compound from pulmonary epithelia upon cyclic mechanical stretch that attenuates endothelial leakage.
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We next sought to determine the existence of soluble mediators released from Calu-3 cells undergoing cyclic mechanical stretch into their supernatant—responsible for endothelial barrier protection. To do this, we used HPLC to fractionate supernatants derived from Calu-3 exposed for 48 h to cyclic mechanical stretch and tested bioactivity of individual fractions on endothelial barrier effects. As shown in Fig. 1, B and C, only fraction 5 improved endothelial barrier function (82 ± 6.2% decrease in FITC-flux rate, p < 0.001). Based on further analysis of size, stability, UV spectroscopy, and HPLC retention time (see red dotted line, Fig. 1B), the bioactive content of fraction 5 was subsequently identified as adenosine. Moreover, treatment of fraction 5 with adenosine deaminase (ADA) (0.1 nM) (15) completely abolished its barrier protective effects (2 ± 1.1% decrease in FITC-flux rate, data not shown). Consistently, endothelial barrier effects of supernatants from Calu-3 were neutralized using the nonspecific adenosine receptor antagonist 8-PT (10 µM) or following treatment with ADA (0.1 nM, Fig. 1D). Quantification of the adenosine concentration within the supernatant by HPLC measurements confirmed increased adenosine concentrations with exposure to cyclic mechanical stretch (Fig. 1E). To confirm that the observed increases in adenosine within the supernatants are not limited to the above in vitro model of cyclic stretch, we performed mechanical ventilation in mice for 0–3 h (pressure-controlled ventilation, 100% inspired oxygen, inspiratory pressure 15 mbar), flash froze the lungs, and determined pulmonary adenosine concentrations via HPLC. Consistent with the above in vitro findings from cyclic mechanical stretch, we found time-dependent increases of pulmonary adenosine with mechanical ventilation (Fig. 1F). Similarly, pulmonary adenosine concentrations were increased in the BAL fluid, suggesting that increases in adenosine occur extracellularly (Fig. 1F). Together, these results indicate that pulmonary adenosine is increased during cyclic mechanical stretch as occurs during mechanical ventilation.
CD39 and CD73 are induced by cyclic stretch or mechanical ventilation
As previous studies had shown that CD39 and CD73 are rate limiting for extracellular adenosine generation (9, 15), we next investigated transcriptional consequences of cyclic stretch on CD39 and CD73 expression patterns in vitro and in vivo. As shown in Fig. 1G, CD39 and CD73 mRNA levels are induced in Calu-3 epithelia following exposure to cyclic mechanical stretch in vitro. As next step, we performed murine mechanical ventilation to investigate transcriptional consequences on ectonucleotidase expression. For this purpose, we ventilated mice for 0–150 min using pressure-controlled ventilation (100% inspired oxygen concentration, inspiratory pressure 15 mbar). After sacrificing the animals at indicated time points, harvesting the lungs, and isolation of RNA, we assessed transcript levels of nucleotidases with real-time RT-PCR. These experiments revealed a prompt and robust induction of CD39 and CD73 transcript levels with increasing time periods of ventilation (Fig. 2A). As a previous study had shown oxygen-dependent modulation of adenosine signaling effects (3), we measured relative transcriptional levels of CD39/73 following 150 min of ventilation at different inspired oxygen concentrations (Fig. 2B). We observed no transcriptional effects of the inspired oxygen concentration on CD39/73 expression patterns. Therefore, all additional experiments were performed at 100% inspired oxygen concentration. As next step, we measured CD39/73 protein by Western blot analysis from whole lungs of ventilated mice. We found ventilation time-dependent increases in CD39/73 protein (Fig. 2C). Similarly, immunohistochemical staining confirmed CD39/CD73 induction with mechanical ventilation on both pulmonary epithelia and endothelia (Fig. 2, D and E), while IgG controls were negative. Taken together, these results reveal oxygen-independent increases of pulmonary CD39 and CD73 with mechanical ventilation.
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After having shown increased pulmonary adenosine concentrations and induction of ectoapyrase and ecto-5'-nucleotidase with mechanical ventilation, we next pursued their functional contribution to ALI induced by mechanical ventilation. For this purpose, we used previously characterized cd39–/– mice (16) or age-, gender-, and weight-matched littermate controls and induced ALI via high-pressure ventilation (90 min, 45 mbar). As shown in Fig. 3A, cd39–/– mice showed a profound increases of albumin leakage into their BAL compared with littermate controls. In addition, increases in lung water (Fig. 3B) and PMN infiltration (MPO tissue activity, Fig. 3C) were significantly enhanced in cd39–/– mice. The fact that cd39–/– mice were more prone to ALI induced by mechanical ventilation was also reflected in functional studies: cd39–/– mice had significantly lower arterial oxygen partial pressure levels than control animals (Fig. 3D). Moreover, increases of pulmonary adenosine with ALI were attenuated in cd39–/– mice (Fig. 3E). Taken together, these data reveal a functional role of CD39 in ALI induced by mechanical ventilation.
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After having shown a protective role for CD39 in ALI, we next pursued the functional contributions of CD73 which catalyzes the final conversion of AMP to adenosine and is considered the pacemaker for extracellular adenosine generation (9, 15, 23). For this purpose, we treated BL6 mice with the CD73 inhibitor APCP (100 mg/kg i.p.). In fact, BAL-albumin leakage (1.6 ± 0.1-fold, p < 0.001), lung water content (7.3 ± 0.5 vs 8.0 ± 0.1 mg/mg dry tissue, p < 0.05), MPO (1.8- ± 0.06-fold, p < 0.0001) were increased after APCP treatment in comparison to vehicle control (90 min ventilation time, 45 mbar inspiratory pressure, data not shown). Consistent with our findings in cd39–/– mice, the PaO2 was decreased from 39 ± 3.6 to 30 ± 2.8 mmHg following APCP treatment (90 min ventilation at 45 mbar inspiratory pressure, p < 0.01, data not shown). Taken together, these studies provide pharmacological evidence that inhibition of CD73 function results in increased susceptibility to ALI from mechanical ventilation.
ALI is increased in cd73–/– mice
Based on these pharmacological studies showing increased severity of ALI with CD73 inhibition, we next pursued the effects of mechanical ventilation on ALI in previously characterized cd73–/– mice (15). Similar to cd39–/– mice, these studies revealed significant increases of albumin leakage, pulmonary edema, and tissue-MPO activity, as well as decreased PaO2, values in cd73–/– mice compared with their littermate controls (Fig. 4, A–D). Similar to cd39–/– mice, increases of pulmonary adenosine with ALI were attenuated in cd73–/– mice (Fig. 4E). Taken together, these studies provide genetic and pharmacological evidence for a protective role of extracellular adenosine generation via CD73 during ALI induced by mechanical ventilation.
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To confirm increased susceptibility of cd39–/– and cd73–/– mice to lung injury induced by mechanical ventilation, we examined lungs from cd39–/– or cd73–/– mice after 90 min of ventilation at 15 or 45 mbar in comparison to littermate controls. As shown in the macroscopic and histological images of whole lungs from ventilated littermates, edema, alveolar congestion, neutrophil infiltration, and hemorrhage are increased with high-pressure ventilation (Fig. 5, A and B). However, these findings were dramatically increased in cd39–/– or cd73–/– mice. This could also be confirmed by quantitative analysis of pulmonary histology (Fig. 5, C and D). Taken together, these results confirm increased susceptibility of cd39–/– and cd73–/– mice to ventilator-induced ALI.
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As proof of principle, we reconstituted cd39–/– and cd73–/– mice via i.p. injection with soluble apyrase or 5'-nucleotidase, respectively (10 U i.p.). In fact, increased albumin leakage, inflammation, and attenuated gas exchange of cd39–/– (Fig. 6A) or cd73–/– mice (Fig. 6B) was reversed with apyrase or 5'-nucleotidase treatment. Similar treatment of littermate controls was therapeutic, as it attenuated ALI-induced albumin leakage and inflammation while simultaneously improving gas exchange. Taken together, these studies provide strong evidence that CD39 and CD73 are critical control points for maintaining the capillary-alveolar barrier function and attenuating inflammation during episodes of noninfectious ALI.
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To further pursue the therapeutic effects of soluble apyrase or 5'-nucleotidase, we treated C57BL/6/129 mice with soluble apyrase or BL6 mice with soluble 5'-nucleotidases (10 U i.p.). As shown in Fig. 7, A–D, such treatment was associated with improved survival and attenuated histological signs of ventilator induced tissue damage. Moreover, increases of pulmonary adenosine levels with mechanical ventilation were "hyper"-elevated by treatment with soluble apyrase or 5'-nucleotidase (Fig. 7, E and F). Taken together, these data suggest treatment with soluble nucleotidases as therapeutic strategy for noninfectious ALI.
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After having shown strong protective effects of CD39- and CD73-dependent adenosine production during ALI induced by mechanical ventilation, we were interested through which individual adenosine receptor this protective effect is mediated. Based on previous studies showing attenuation of acute injury or vascular barrier dysfunction by A2AAR (24) or A2BAR signaling (9, 14, 17, 25), we used specific antagonists of these receptors. In this study, we treated BL6 mice with the A2AAR antagonist ZM241385 (Fig. 8, A and B) or the A2BAR antagonist MRS 1754 (Fig. 8, C and D). Although A2AAR antagonist treatment did not alter survival or albumin leakage during ALI, treatment with the A2BAR antagonist was associated with profound increases in albumin leakage into the BAL fluid and decreased survival. These pharmacological studies suggest a role of the A2BAR in adenosine-dependent protection of the alveolar-capillary barrier function during ALI induced by mechanical ventilation.
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Discussion |
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The observation of increased capillary-alveolar leakage with targeting CD39 or CD73 during mechanical ventilation suggests a protective role of extracellular adenosine signaling for maintaining the pulmonary barrier function. This is consistent with previous studies showing barrier protective effects of extracellular adenosine signaling during acute inflammation or hypoxia (26). Previous studies found different adenosine receptors responsible for tissue protection under such conditions (9, 17, 25, 27). For example, a study in mice deficient in the A2AAR found increased inflammation-associated tissue damage (27, 28) providing evidence for A2AAR signaling as a mechanism for regulating acute inflammatory responses in vivo. As previous studies have found an important contribution of leukocyte recruitment and neutrophil chemoattractants (such as MIP-2) in ventilator-induced lung injury (22), adenosine-dependent attenuation of neutrophil responses via A2AAR may be important for lung protection during mechanical ventilation. However, based on pharmacological approaches, the present studies suggest a protective role of the A2BAR during ALI induced by mechanical ventilation. This is consistent with studies on endothelial permeability (9, 14, 29), attenuated neutrophil responses (25), and myocardial protection from ischemia and reperfusion injury via A2BAR signaling.
At present, the exact source from which extracellular adenosine is generated during mechanical ventilation remains unclear. Some very carefully executed studies have shown that pulmonary epithelia and endothelia release ATP in different models of injury (30, 31, 32). For example, exposure of pulmonary endothelial cells to oxidative stress or to hyperoxia resulted in rapid but transient ATP release (31). As such, extracellular ATP can either signal directly to ATP receptors, or may function as a metabolic substrate for extracellular generation of adenosine via CD39 and CD73. In addition, extracellular ATP can also be released from inflammatory cells (e.g., PMN) or platelets (33). As such, a recent study demonstrated a role of the connexin 43 hemichannels in PMN-dependent ATP release during inflammation (34).
Consistent with the present study, other investigations confirmed protective effects of adenosine on the pulmonary integrity. For example, an excellent study on CD73-mediated adenosine production found tissue protective in a model of bleomycin-induced lung injury (35). Moreover, LPS-induced ALI was aggravated in A2AAR–/– mice (3). This very thorough study revealed that hypoxia-elicited pathways resulting in increased adenosine-signaling effects are protective during LPS-induced lung injury. In fact, the administration of high oxygen concentration further aggravated lung injury in this study (3). The authors found that this was due to inhibition of hypoxia-elicited lung protective mechanisms (27). In contrast, the present study did not find transcriptional repression of CD39 or CD73 by hyperoxia. In fact, it seems likely from the present data that during mechanical ventilation (in contrast to LPS-induced lung injury) mechanisms other than tissue oxygen availability (e.g., stretch or sheer forces) are responsible for increased adenosine production and transcriptional induction of nucleotidases.
In contrast to the present study of acute injury, studies investigating chronic pulmonary disease have identified a detrimental role of elevated adenosine levels (36, 37, 38). For example, levels of adenosine are chronically increased in the lungs of asthmatics (39), and correlate with the degree of inflammatory insult (40), suggesting a provocative role of adenosine in asthma or chronic obstructive pulmonary disease (41). In addition, 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 (42) 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 (36, 37).
In summary, the present study identifies endogenous adenosine production and signaling through the A2BAR as part of innate metabolic adaptation during ALI. In fact, genetically targeted mice with defects in the major extracellular pathway of adenosine generation (CD39 or CD73) show increased susceptibility to ALI during mechanical ventilation. In addition, treatment with their soluble compounds improves symptoms and survival during high-pressure ventilation.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Fortune Grant 1416-0-0, Interdisziplinäres Zentrum für Klinische Forschung Verbundprojekt 1597-0-0, and Deutsche Forschungsgemeinschaft Grant EL274/2-2 (to H.K.E.) and Interdiziplinäres Zentrum für Klinisehe Forschung Nachwuchsgruppe 1605-0-0 (to T.E.). 
2 Address correspondence and reprint requests to Dr. Holger K. Eltzschig, Department of Anesthesiology and Intensive Care Medicine, Center for Biomedical Research, Tübingen University Hospital, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany. E-mail address: heltzschig{at}partners.org
3 Abbreviations used in this paper: ALI, acute lung injury; HMEC, human microvascular endothelial cell; BAL, bronchoalveolar lavage; 8-PT, 8-phenyl-theophylline; APCP, adenosine 5'-(,-methylene) diphosphate; AR, adenosine receptor; PMN, polymorphonuclear neutrophil; ADA, adenosine deaminase; MPO, myeloperoxidase; PaO2, arterial partial pressure of oxygen; mbar, millibar.
Received for publication December 8, 2006. Accepted for publication April 9, 2007.
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 H. K. Eltzschig, D. Kohler, T. Eckle, T. Kong, S. C. Robson, and S. P. Colgan Central role of Sp1-regulated CD39 in hypoxia/ischemia protection Blood, January 1, 2009; 113(1): 224 - 232. [Abstract] [Full Text] [PDF]
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T. Eckle, L. Fullbier, A. Grenz, and H. K. Eltzschig Usefulness of pressure-controlled ventilation at high inspiratory pressures to induce acute lung injury in mice Am J Physiol Lung Cell Mol Physiol, October 1, 2008; 295(4): L718 - L724. [Abstract] [Full Text] [PDF]
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M. L. Hart, M. Henn, D. Kohler, D. Kloor, M. Mittelbronn, I. C. Gorzolla, G. L. Stahl, and H. K. Eltzschig Role of extracellular nucleotide phosphohydrolysis in intestinal ischemia-reperfusion injury FASEB J, August 1, 2008; 22(8): 2784 - 2797. [Abstract] [Full Text] [PDF]
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 M. L. Hart, D. Kohler, T. Eckle, D. Kloor, G. L. Stahl, and H. K. Eltzschig Direct Treatment of Mouse or Human Blood With Soluble 5'-Nucleotidase Inhibits Platelet Aggregation Arterioscler Thromb Vasc Biol, August 1, 2008; 28(8): 1477 - 1483. [Abstract] [Full Text] [PDF]
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A. Mikhailov, A. Sokolovskaya, G. G. Yegutkin, H. Amdahl, A. West, H. Yagita, R. Lahesmaa, L. F. Thompson, S. Jalkanen, D. Blokhin, et al. CD73 Participates in Cellular Multiresistance Program and Protects against TRAIL-Induced Apoptosis J. Immunol., July 1, 2008; 181(1): 464 - 475. [Abstract] [Full Text] [PDF]
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 R. A. Fowler, N. K. J. Adhikari, D. C. Scales, W. L. Lee, and G. D. Rubenfeld Update in Critical Care 2007 Am. J. Respir. Crit. Care Med., April 15, 2008; 177(8): 808 - 819. [Full Text] [PDF]
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 F. Martinon Detection of immune danger signals by NALP3 J. Leukoc. Biol., March 1, 2008; 83(3): 507 - 511. [Abstract] [Full Text] [PDF]
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T. Eckle, M. Faigle, A. Grenz, S. Laucher, L. F. Thompson, and H. K. Eltzschig A2B adenosine receptor dampens hypoxia-induced vascular leak Blood, February 15, 2008; 111(4): 2024 - 2035. [Abstract] [Full Text] [PDF]
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S. Jalkanen and M. Salmi VAP-1 and CD73, Endothelial Cell Surface Enzymes in Leukocyte Extravasation Arterioscler Thromb Vasc Biol, January 1, 2008; 28(1): 18 - 26. [Abstract] [Full Text] [PDF]
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 D. Kohler, T. Eckle, M. Faigle, A. Grenz, M. Mittelbronn, S. Laucher, M. L. Hart, S. C. Robson, C. E. Muller, and H. K. Eltzschig CD39/Ectonucleoside Triphosphate Diphosphohydrolase 1 Provides Myocardial Protection During Cardiac Ischemia/Reperfusion Injury Circulation, October 16, 2007; 116(16): 1784 - 1794. [Abstract] [Full Text] [PDF]
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