A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

Although acute lung injury contributes significantly to critical illness, resolution often occurs spontaneously via activation of incompletely understood pathways. We recently found that mechanical ventilation of mice increases the level of pulmonary adenosine, and that mice deficient for extracellular adenosine generation show increased pulmonary edema and inflammation after ventilator-induced lung injury (VILI). Here, we profiled the response to VILI in mice with genetic deletions of each of the 4 adenosine receptors (ARs) and found that deletion of the A2BAR gene was specifically associated with reduced survival time and increased pulmonary albumin leakage after injury. In WT mice, treatment with an A2BAR-selective antagonist resulted in enhanced pulmonary inflammation, edema, and attenuated gas exchange, while an A2BAR agonist attenuated VILI. In bone marrow–chimeric A2BAR mice, although the pulmonary inflammatory response involved A2BAR signaling from bone marrow–derived cells, A2BARs located on the lung tissue attenuated VILI-induced albumin leakage and pulmonary edema. Furthermore, measurement of alveolar fluid clearance (AFC) demonstrated that A2BAR signaling enhanced amiloride-sensitive fluid transport and elevation of pulmonary cAMP levels following VILI, suggesting that A2BAR agonist treatment protects by drying out the lungs. Similar enhancement of pulmonary cAMP and AFC were also observed after β-adrenergic stimulation, a pathway known to promote AFC. Taken together, these studies reveal a role for A2BAR signaling in attenuating VILI and implicate this receptor as a potential therapeutic target during acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening disorders that can develop in the course of different clinical conditions such as pneumonia, acid aspiration, major trauma, or prolonged mechanical ventilation, and contribute significantly to critical illness (1). Recent epidemiological studies show that each year 75,000 patients in the United States alone die from ARDS (2). The pathogenesis of these diseases is characterized by influx of a protein-rich edema fluid into the interstitial and intraalveolar spaces as a consequence of increased permeability of the alveolar-capillary barrier (1) in conjunction with excessive invasion of inflammatory cells — particularly polymorphonuclear neutrophils (3–6). At present, only little is known about how to target the alveolar-capillary barrier function or leukocyte trafficking therapeutically during ALI. In fact, to our knowledge, no such strategies have been translated into clinical practice, and we are unaware of any specific therapy currently available beyond mechanical ventilation and other supportive measures (1).

Despite the large impact of ALI on morbidity and mortality in critically ill patients (1), many episodes 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 (7–9). In a recent study to identify endogenous pathways to attenuate ventilator-induced lung injury (VILI), we found that extracellular adenosine accumulates in the supernatant of pulmonary epithelia exposed to cyclic mechanical stretch in vitro (10). Similarly, pulmonary adenosine levels were elevated during mechanical ventilation in vivo (10). In fact, mice deficient in extracellular adenosine production showed dramatic increases in pulmonary edema and pulmonary inflammation when exposed to VILI (10). However, adenosine-dependent signaling pathways of lung protection during VILI remain unknown. As such, extracellular adenosine can signal through any of 4 G protein–coupled adenosine receptors (ARs), A1AR, A2AAR, A2BAR, and A3AR, which have all been implicated in tissue protection in different models of injury or inflammation (10–28). Therefore, we designed the present study to test the hypothesis that AR signaling plays an important role in VILI. For this purpose, we assessed VILI in genetic models for each individual AR. Because these studies pointed toward a pivotal role of A2BAR signaling, we confirmed these genetic studies using pharmacological approaches with specific A2BAR agonists and antagonists. Next, we created bone marrow chimeras to study A2BAR effects on hematopoietic versus non-hematopoietic cells. Finally, we pursued the contribution of A2BAR signaling to alveolar fluid clearance (AFC) during VILI. Taken together, these studies point toward a critical role of A2BAR signaling in attenuating VILI by dampening pulmonary inflammation, attenuation of pulmonary edema, and enhancement of amiloride-sensitive fluid transport mechanisms to dry out the lungs.

VILI is selectively enhanced in gene-targeted mice for the A2BAR. Based on previous studies showing extracellular adenosine generation in lung protection during VILI (10), we hypothesized that AR deficiency would significantly enhance pulmonary edema and attenuate survival during VILI. To this end, we exposed previously characterized mice that were gene-targeted for A1AR (29), A2AAR (30), A2BAR (13), or A3AR (22) as well as corresponding age-, weight-, and gender-matched littermate controls to VILI. As shown in Figure 1, survival time during VILI or VILI-induced increases of albumin leakage into the bronchoalveolar (BAL) fluid were similar between A1AR^–/–, A2AAR^–/–, and A3AR^–/– mice and their controls (Figure 1, A, B, and D). In contrast, survival time was significantly shortened in A2BAR^–/– mice (Figure 1C; P < 0.001), while albumin leakage into the BAL fluid was dramatically increased (Figure 1C; P < 0.001). Taken together, these data indicate that specific genetic deletion of the A2BAR enhances murine VILI.

Figure 1

VILI in mice gene-targeted for individual ARs. Previously characterized A1AR^–/– (A; ref. 29), A2AAR^–/– (B; ref. 30), A2BAR^–/– (C), or A3AR^–/– mice (D; ref. 22) or corresponding littermate controls were exposed to VILI, and survival times were determined during VILI. Mechanical ventilation was applied using pressure-controlled settings (inspiratory pressure of 35 mbar, inspired oxygen concentration 100%; respiratory rate and inspiratory/expiratory ratio were adjusted to maintain normal pH) until a cardiac standstill was observed in the surface electrocardiogram. Note the significantly attenuated survival of A2BAR^–/– mice (C; P < 0.001, n = 8). Albumin concentration in the BAL fluid was determined by ELISA. For this purpose, the mice were mechanically ventilated using pressure-controlled ventilation with an inspired oxygen concentration of 100% for 180 minutes at 45 mbar. Note the significantly increased albumin concentration in the BAL fluid of A2BAR^–/– mice (C; P < 0.001, n = 6).

A2BAR^–/– mice show increased pulmonary inflammation during VILI. To confirm our findings of enhanced VILI in A2BAR^–/– mice, we next measured additional functional parameters. Here, we found that increases in lung water content (Figure 2A; P < 0.01) and attenuated gas exchange during VILI were enhanced in A2BAR^–/– mice compared with controls (A2BAR^+/+; Figure 2B; P < 0.01). Based on a recent study showing that A2BAR signaling attenuates pulmonary inflammation during acute hypoxia (31), we studied the influence of A2BAR signaling on pulmonary inflammation. As shown in Figure 2C, increases in pulmonary neutrophil numbers (as measured by myeloperoxidase [MPO]) with VILI were dramatically enhanced in A2BAR^–/– mice (P < 0.001). Similarly, increases in proinflammatory cytokines (pulmonary TNF-α, IL-6, and keratinocyte-derived chemokine [KC, a mouse ortholog of human IL-8]) were enhanced (Figure 2, D–F; P < 0.001), while levels of the antiinflammatory cytokine IL-10 (Figure 2G; P < 0.001) were attenuated. Consistent with studies showing that pulmonary A2BAR signaling attenuates NF-κB activation via adenosine-mediated cullin-1 deneddylation (31), we pursued pulmonary NF-κB activation during VILI. Consistent with previous studies of mechanical stretch (32, 33), VILI was associated with a significant increase of pulmonary NF-κB activity (Figure 2H; P < 0.001). However, pulmonary NF-κB activity was increased at baseline (Figure 2H; P < 0.001), and VILI-induced increases of NF-κB activity were further enhanced in A2BAR^–/– mice (Figure 2H; P < 0.001). While a number of mechanisms have been suggested to mediate the endogenous inactivation of NF-κB, each of these pathways converges on changes in the inducible degradation of IκBα (34). Thus, we determined whether A2BAR signaling influences IκB activity during VILI. In fact, VILI-associated decreases in pulmonary IκB activity were accentuated in A2BAR^–/– mice (Figure 2I; P < 0.001), confirming a role of A2BAR signaling in attenuating IκB/NF-κB–dependent pulmonary inflammation during VILI. To confirm increased susceptibility of A2BAR^–/– mice to lung injury on a histological level, we next examined lungs from A2BAR^–/– and control mice after 180 minutes of ventilation at 45 mbar. As shown by VILI scores (Figure 2J) and in images from pulmonary sections (Figure 2K), histological signs of pulmonary injury associated with VILI were dramatically enhanced in A2BAR^–/– mice. Taken together, these results reveal increased pulmonary inflammation and severity of VILI associated with genetic deletion of A2BAR.

Figure 2

VILI in mice gene-targeted for the A2BAR. (A–H) A2BAR^–/– mice or littermate controls (A2BAR^+/+) were mechanically ventilated using pressure-controlled ventilation with an inspired oxygen concentration of 100% over 180 minutes at 45 mbar. (A) Following ventilation, lungs were excised en bloc and weighed. Lungs were lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Results are presented as mean ± SD (n = 6). (B) To assess pulmonary gas exchange, blood gas analyses were performed by obtaining arterial blood via cardiac puncture. Analysis was performed immediately, and the ratio of the arterial partial pressure of oxygen (PaO₂) to the fraction of inspired oxygen (FiO₂) was determined. Results are presented as mean ± SD (n = 6). (C) Pulmonary neutrophil accumulation was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD (ΔOD) over 5 minutes. Results are presented as mean ± SD (n = 6). (D–I) TNF-α, IL-6, KC, IL-10, NF-κB, and IκBα levels were evaluated in lung tissue homogenates using a mouse ELISA. Results are presented as mean ± SD (n = 6). (J) For quantification of histological tissue damage by VILI following 180 min ventilation, VILI scores were assessed in A2BAR^–/– or corresponding littermate control mice. Results are displayed as median (midline within boxes) and range (bars above and below boxes) (n = 4). (K) One of 4 representative photomicrographs (original magnification, ×200) stained with hematoxylin and eosin is displayed.

A2BAR is induced by mechanical ventilation. To further characterize A2BAR signaling in innate protection during VILI, we next pursued transcriptional consequences of mechanical ventilation on A2BAR expression patterns in vivo. For this purpose, we ventilated mice for 0 to 4 hours using pressure-controlled ventilation (100% inspired oxygen concentration, inspiratory pressure 35 mbar). After sacrificing the animals at the time points indicated in Figure 3, harvesting the lungs, and isolating RNA, we assessed transcript levels of ARs via real-time RT-PCR. Consistent with previous studies showing selective induction of the A2BAR with large-volume ventilation (35), these experiments revealed a prompt and selective induction of the A2BAR (Figure 3A; P < 0.01). In contrast, transcript levels of the A1AR or A3AR were repressed (P < 0.05; Figure 3A), while A2AAR transcript levels were unchanged with mechanical ventilation (Figure 3A). However, pharmacological studies of survival time or pulmonary albumin leakage with A1AR antagonist (DPCPX, 1 mg/kg i.p.) or A3AR antagonist (MRS1191, 1 mg/kg i.p.) revealed no functional changes in A1AR or A3AR inhibition (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34203DS1). Consistent with previous studies (36), comparative analysis of pulmonary AR transcript levels at baseline showed that A2BAR had the highest expressional levels (Figure 3B). This distribution was even further magnified following mechanical ventilation, resulting in 5.3-fold higher transcript levels of the A2BAR compared with the A2AAR (Figure 3B). Because a previous study had shown oxygen-dependent modulation of adenosine-signaling effects (37), we measured relative transcription levels of A2BAR following 4 hours of ventilation at different inspired oxygen concentrations (Supplemental Figure 2). However, we noted no transcriptional effects of the inspired oxygen concentration on A2BAR expression. Therefore, all further experiments were performed at 100% inspired oxygen concentration. Next we measured A2BAR protein by western blot analysis from whole lungs of ventilated mice and observed ventilation time–dependent increases in A2BAR protein (Figure 3C). Similarly, immunohistochemical staining confirmed A2BAR induction with mechanical ventilation on both pulmonary epithelia and endothelia (Figure 3D), while IgG controls stained negative. Based on studies demonstrating that activation of A2AAR and/or A2BAR is associated with adenylate cyclase–dependent increases in intracellular cAMP (38), leading to increased PKA activity (39), we next studied cAMP levels and PKA activity in pulmonary tissues during mechanical ventilation. As shown in Figure 4, we observed elevations of pulmonary cAMP levels (Figure 4A) and PKA activity (Figure 4B) with mechanical ventilation in WT and A2AAR^–/– mice. In contrast, mechanical ventilation–associated increases in cAMP/PKA activity were abolished in A2BAR^–/– mice. Consistent with other studies (40), we were able to confirm a protective role of pulmonary cAMP elevation during VILI. In fact, we achieved similar cAMP elevations by intratracheal application of a β2-adrenergic agonist (zinterol, 10^–7 M, 300 μl intratracheally). This treatment was associated with elevated cAMP levels, attenuated albumin leakage, and improved survival (Figure 4, C–E). Taken together, these results suggest that the A2BAR is the predominant pulmonary AR during VILI. As such, A2BAR signaling effects result in elevated pulmonary cAMP levels and attenuation of ALI.

Figure 3

Transcriptional consequences of mechanical ventilation on AR expression. (A) C57BL/6 mice were mechanically ventilated (inspiratory pressure of 35 mbar, 100% oxygen). After the indicated time periods, lungs were harvested, total RNA was isolated, and A1AR, A2AAR, A2BAR, and A3AR mRNA levels were determined by real-time RT-PCR. Data were calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 min ventilation) ± SD at each indicated time (n = 4). Note selective induction of the A2BAR gene during high-pressure ventilation (10-fold, P < 0.01; n = 4). (B) Comparative gene expression of pulmonary ARs using real-time PCR. Relative expression levels in untreated controls or in mice after 180 min mechanical ventilation (inspiratory pressure of 35 mbar, 100% oxygen) are shown. Values are expressed as mean ± SEM (n = 4). *P < 0.05 compared with A2AAR. (C) Mice were mechanically ventilated (35 mbar inspiratory pressure, 100% oxygen), and lungs were harvested at the indicated time points, shock frozen, and lysed, and proteins were resolved by SDS-PAGE. Resultant western blots were probed with anti-A2BAR antibodies. To control for loading conditions, blots were stripped and reprobed for actin expression. One representative experiment of 3 is shown. (D) To examine the influence of mechanical ventilation on pulmonary A2BAR expression patterns, C57BL/6 mice were ventilated in a pressure-controlled setting over 0 h or 3 h (35 mbar inspiratory pressure, 100% inspired oxygen concentration). Lungs were stained with antibodies for A2BAR. IgG controls were used at identical concentrations and staining conditions as the target primary antibodies (original magnification, ×400; n = 4).

Figure 4

cAMP levels and PKA activity during VILI. (A and B) To assess cAMP levels and PKA activity in pulmonary tissues during VILI, A2BAR^–/– mice and littermate controls were mechanically ventilated using pressure-controlled ventilation with an inspired oxygen concentration of 100% over 180 minutes at 45 mbar. Animals were euthanized, and lungs were perfused with 5 ml of PBS through the right ventricle. Lungs were excised, shock-frozen utilizing liquid nitrogen, and mechanically homogenized. cAMP levels (A) and PKA activity (B) were determined by ELISA. Note that the increases in cAMP and PKA activity associated with mechanical ventilation were abolished in A2BAR^–/– mice. (C) Treatment with the specific β2-adrenergic agonist zinterol during VILI. Control C57BL/6 mice were treated with 300 μl 10^–7 M intratracheal zinterol, and baseline cAMP levels were determined in lung homogenates using a competitive immunoassay kit. (D) Control C57BL/6 mice were treated with 300 μl 10^–7 M intratracheal zinterol or vehicle control, followed by mechanical ventilation using pressure-controlled settings at an inspired oxygen concentration of 100% and 45 mbar inspiratory pressure for 0 or 180 min. Albumin concentration in the BAL fluid was determined by murine ELISA. (E) Survival times during VILI after 300 μl 10^–7 M intratracheal zinterol or vehicle treatment. Mechanical ventilation was applied using pressure-controlled settings (inspiratory pressure of 35 mbar, inspired oxygen concentration 100%, respiratory rate and inspiratory/expiratory ratio were adjusted to maintain normal pH) until a cardiac standstill was observed in the surface electrocardiogram.

Influence of A2BAR antagonist (PSB1115) and agonist (BAY 60-6583) treatment on VILI. After having shown that the A2BAR^–/– mice developed profound pulmonary inflammation and tissue damage when exposed to VILI, we next sought to confirm these findings using pharmacological approaches. We first studied the highly selective A2BAR inhibitor PSB1115 (10 mg/kg i.p.) (13) during VILI. As shown in Figure 5, A–C, PSB1115 treatment was associated with attenuated survival time in WT and A2AAR^–/– mice, but not in A2BAR^–/– mice. Moreover, pulmonary albumin leakage and lung inflammation were enhanced following PSB1115 treatment (Figure 5, D–G; P < 0.001). At the same time, pulmonary levels of the antiinflammatory cytokine IL-10 were suppressed (Figure 5H; P < 0.001), while pulmonary gas exchange was attenuated (Figure 5I; P < 0.001). Next, we treated mice with a previously described highly selective agonist of the A2BAR (BAY 60-6583, 2 mg/kg i.p.) (13, 16). As shown in Figure 6, pretreatment with BAY 60-6583 significantly improved survival and albumin leakage during VILI in WT and A2AAR^–/– mice, but not in A2BAR^–/– mice (Figure 6, A–C; P < 0.001). Similarly, pulmonary inflammation was attenuated, while pulmonary IL-10 levels were increased and gas exchange improved with BAY 60-6583 treatment (Figure 6, D–H; P < 0.001). To extend these studies into a model of endotoxin-induced ALI, we next exposed A2BAR^–/– mice and corresponding controls to aerosolized LPS and assessed pulmonary inflammation and albumin leakage into the BAL 24 hours afterward. Similar to our studies in VILI, we found enhanced pulmonary inflammation (Figure 7A) and albumin leakage (Figure 7B) in A2BAR^–/– mice compared with their controls when exposed to inhaled LPS. Moreover, treatment of WT mice with the selective A2BAR agonist BAY 60-6583 was associated with attenuated pulmonary inflammation (Figure 7C) and albumin leakage into the BAL (Figure 7D) during endotoxin-induced ALI. Taken together, these data show a similar phenotype of the A2BAR in endotoxin-induced ALI and suggest that the A2BAR may be a therapeutic target in ALI induced by mechanical ventilation or LPS inhalation.

Figure 5

A2BAR antagonist treatment PSB1115 during VILI. (A–C), A2BAR^+/+, A2AAR^–/–, and A2BAR^–/– mice and their corresponding littermate controls were treated with 10 mg/kg PSB1115 or vehicle 30 minutes prior to induction of anesthesia. Mechanical ventilation was begun, and mice were ventilated using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration) until a cardiac standstill was observed in the surface electrocardiogram (P < 0.01, n = 8). (D) Mechanical ventilation was begun, and mice were ventilated for 0 or 180 minutes using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration). Albumin concentration in the BAL fluid was determined by ELISA (n = 6). (E) Pulmonary neutrophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD over 5 minutes (n = 6). (F–H) TNF-α, NF-κB, and IL-10 levels were evaluated in lung tissue homogenates using a murine ELISA (n = 6). (I) To assess pulmonary gas exchange, blood gas analyses were performed by obtaining arterial blood via cardiac puncture. Analysis was performed immediately, and the ratio of the arterial partial pressure of oxygen to the fraction of inspired oxygen was determined. Results are presented as mean ± SD (n = 6).

Figure 6

A2BAR agonist (BAY 60-6583) treatment. (A–C) A2BAR^+/+, A2AAR^–/–, and A2BAR^–/– mice and their corresponding littermate controls were treated with 2 mg/kg BAY 60-6583 or vehicle 30 minutes prior to induction of anesthesia. Mechanical ventilation was begun, and mice were ventilated using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration) until a cardiac standstill was observed in the surface electrocardiogram (P < 0.01, n = 8). In other studies, albumin concentrations in the BAL fluid were determined by ELISA after mechanical ventilation using pressure-controlled settings with an inspired oxygen concentration of 100% for 180 minutes at 45 mbar. (D) Pulmonary neutrophil sequestration was quantified using a MPO assay (n = 6). (E–G) TNF-α, NF-κB, and IL-10 levels were evaluated in lung tissue homogenates using murine ELISA (n = 6). (H) To assess pulmonary gas exchange, blood gas analyses were performed by obtaining arterial blood via cardiac puncture. The ratio of the arterial partial pressure of oxygen to the fraction of inspired oxygen was determined. Results are presented as mean ± SD (n = 6).

Figure 7

A2BAR signaling during LPS-induced lung injury. (A and B) A2BAR^–/– and A2BAR^+/+ mice were exposed to LPS inhalation for 30 minutes. Twenty-four hours after LPS exposure, TNF-α in lung tissue homogenates and albumin concentration in the BAL fluid were determined using a murine ELISA (n = 6). (C and D) A2BAR^+/+ mice received 2 mg/kg BAY 60-6583 i.p. or were treated with vehicle 30 minutes prior to LPS inhalation. (C) TNF-α levels in lung tissues and (D) albumin concentrations in the BAL fluid (n = 6).

VILI in A2BAR bone marrow–chimeric mice. Based on the above results showing expression of the A2BAR on pulmonary tissues, and other studies showing A2BAR on myeloid cells (15, 21), we generated A2BAR bone marrow–chimeric mice to study the contribution of pulmonary versus myeloid A2BARs to lung injury during VILI. As expected, A2BAR^+/+→A2BAR^+/+ chimeric mice showed a similar degree of VILI-induced increases in ALI as did wild type mice, while A2BAR^–/–→A2BAR^–/– mice showed a similar phenotype as that of A2BAR^–/– mice (Figure 8, A–F). Interestingly, A2BAR^+/+→A2BAR^–/– chimeric mice exhibited VILI-induced albumin leakage and increases in lung water, similar to A2BAR^–/– mice (Figure 8, A and B). In contrast, A2BAR^–/–→A2BAR^+/+ chimeric mice had attenuated VILI-induced albumin leakage and lung water, very similar to A2BAR^+/+ mice (Figure 8A). In contrast, inflammatory changes (increased MPO, TNF-α, IL-6, and KC) were intermediate in chimeric mice with the genetic deletion of either the myeloid A2BAR (A2BAR^–/–→A2BAR^+/+) or the pulmonary A2BAR (A2BAR^+/+→A2BAR^–/–), suggesting a combination of pulmonary and myeloid A2BAR signaling events in attenuating lung inflammation during VILI. Taken together, these studies suggest a dual role of the A2BAR during VILI in which pulmonary A2BARs mainly attenuate pulmonary edema and albumin leakage, while neutrophil accumulation and release of proinflammatory mediators are affected by both pulmonary and myeloid A2BARs.

Figure 8

VILI in A2BAR bone marrow–chimeric mice. A2BAR bone marrow–chimeric mice were subjected to VILI using mechanical ventilation for 180 minutes at an inspiratory pressure of 45 mbar and 100% inspired oxygen concentration. (A) Albumin concentration in the BAL fluid was determined by ELISA. WT/WT, A2BAR^+/+/A2BAR^+/+; KO/WT, A2BAR^–/–/A2BAR^+/+; KO/KO, A2BAR^–/–/ A2BAR^–/–; WT/KO, A2BAR^+/+/A2BAR^–/–. (B) Following ventilation at the indicated settings, lungs were excised en bloc and weighed. Lungs were lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Note the increased albumin concentration and lung water content in A2BAR^–/–/A2BAR^–/– and A2BAR^+/+/A2BAR^–/– mice compared with A2BAR^+/+/A2BAR^+/+ mice (P < 0.001). (C) Pulmonary neutrophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD over 5 minutes. (D–F) TNF-α, IL-6, and KC levels were evaluated in lung tissue homogenates using a mouse ELISA. Note the similar degree of pulmonary inflammation in A2BAR^–/–/A2BAR^+/+ and A2BAR^+/+/A2BAR^–/– mice compared with A2BAR^+/+/A2BAR^+/+ mice. n = 6.

Barrier protection of BAY 60-6583 requires pulmonary A2BARs, while antiinflammatory effects involve pulmonary and myeloid A2BARs. To further study the contribution of pulmonary and hematopoietic A2BARs to VILI, we next exposed A2BAR bone marrow–chimeric mice pretreated with BAY 60-6583 to VILI. Bone marrow–chimeric A2BAR mice expressing the A2BAR on tissues but not on their hematopoietic cells (A2BAR^–/–→A2BAR^+/+ mice) showed reduced BAL albumin leakage and lower wet/dry ratios when treated with BAY 60-6583 (2 mg/kg i.p.) compared with their vehicle-treated counterparts. In contrast, BAY 60-6583 treatment had no effect on albumin leakage or wet/dry ratios in A2BAR^+/+→A2BAR^–/– or A2BAR^–/–→A2BAR^–/– chimeric mice, suggesting that pulmonary A2BARs play a role in dampening pulmonary edema during VILI (Figure 9, A and B). In contrast, pulmonary inflammation (as assessed by pulmonary TNF-α and MPO levels) was attenuated with BAY 60-6583 pretreatment in both A2BAR^+/+→A2BAR^–/– and A2BAR^–/–→A2BAR^+/+ mice, suggesting a combination of pulmonary and hematopoietic A2BARs in attenuating lung inflammation during VILI (Figure 9, C and D). Taken together, these data suggest that signaling through pulmonary A2BARs is critical for attenuating VILI-associated increases in pulmonary edema, while myeloid and pulmonary A2BAR signaling attenuates lung inflammation induced by VILI.

Figure 9

BAY 60-6583–dependent lung protection during VILI in A2BAR bone marrow–chimeric mice. (A–D) A2BAR bone marrow–chimeric mice were treated with BAY 60-6583 (2 mg/kg) 30 minutes prior to induction of anesthesia. Mechanical ventilation was begun, and mice were ventilated using an inspiratory pressure of 45 mbar, 100% inspired oxygen concentration, for 180 minutes. Results are presented as mean ± SD. n = 6. (A) Albumin concentration in the BAL fluid was determined by ELISA. (B) Following mechanical ventilation, lungs were excised en bloc and weighed. Lungs were then lyophilized for 48 hours, and lung water content (mg lung water/mg dry tissue) was determined. Capillary-alveolar barrier protection was observed only in A2BAR^+/+/A2BAR^+/+ and A2BAR^–/–/A2BAR^+/+ mice (P < 0.001; n = 6). (C) Pulmonary neutrophil sequestration was quantified using a MPO assay. MPO activity was assessed using a spectrophotometric reaction with O-dianisidine hydrochloride. Absorbance at 450 nm was measured and reported as difference in OD over 5 minutes. (D) TNF-α levels were evaluated in lung tissue homogenates using a mouse ELISA. Note the similar levels of pulmonary inflammation in A2BAR^–/–/A2BAR^+/+ and A2BAR^+/+/A2BAR^–/– mice.

A2BAR signaling enhances amiloride-sensitive fluid transport during VILI. Based on previous studies showing a critical role of extracellular adenosine signaling in maintaining proper fluid levels in the lung alveoli (41–43) and our observation of increased pulmonary edema during VILI with genetic deletion or pharmacological inhibition of the A2BAR, we pursued the hypothesis that A2BAR signaling enhances AFC during VILI. For this purpose, we measured AFC in A2BAR^–/– mice and littermate controls (Figure 10A) during VILI. While AFC was enhanced with VILI, AFC was significantly attenuated in A2BAR^–/– mice. Intraalveolar treatment with the epithelial sodium channel (ENaC) inhibitor amiloride revealed that increases in AFC with VILI involve ENaC-dependent fluid transport. In addition, differences in AFC between A2BAR^–/– and A2BAR^+/+ mice were attenuated following amiloride treatment, suggesting that A2BAR signaling contributes to ENaC-dependent fluid transport. As shown in Figure 10B, intraalveolar treatment of WT mice with the A2BAR antagonist PSB1115 attenuated AFC, resulting in a similar phenotype to that observed in A2BAR^–/– mice. Studies in A2BAR bone marrow–chimeric mice revealed that mice with pulmonary deletion of the A2BAR had attenuated AFC during VILI, while deletion of the myeloid A2BAR had little effect on AFC (Figure 10C). Moreover, treatment of WT mice with the A2BAR-specific agonist BAY 60-6583 increased AFC (Figure 10D) and survival time during VILI (Figure 10, E and F). However, increases in AFC or survival with BAY 60-6583 were abolished when given together with amiloride (Figure 10, D–F), suggesting that A2BAR signaling enhances ENaC-dependent fluid transport. Additional studies with a specific inhibitor of the CFTR (CFTR_inh-172, 10^–6 M, 300 μl intratracheally) (41) showed no influence on AFC during VILI (Supplemental Figure 3). These findings are consistent with studies showing no changes in AFC in gene-targeted mice for the CFTR (44) and with studies using selective CFTR inhibitors (CFTR_inh-172, 1 μM) (41).

Figure 10

Contribution of A2BAR signaling to AFC during VILI. (A) To determine whether A2BAR signaling affects pulmonary fluid transport, we measured AFC using a mechanically ventilated live mouse model. A2BAR^–/– mice and littermate controls were mechanically ventilated in a pressure-controlled setting at 45 mbar for 0 to 180 minutes. AFC was measured by instilling 300 μl of iso-osmolar 0.9% NaCl solution with 5% BSA. Mechanical ventilation was continued for 30 minutes, and AFC was measured in the presence or absence of the ENaC inhibitor amiloride (1 mM). *P < 0.01 compared with no amiloride. n = 8. (B) Influence of A2BAR antagonist PSB1115 on AFC during VILI. Following induction of VILI, control mice received PSB1115 (1 μM) alone or in combination with amiloride (1 mM), and AFC was determined. *P < 0.01 compared with no amiloride. n = 8. (C) VILI was induced in A2BAR bone marrow–chimeric mice, and AFC was determined. *P < 0.001 compared with A2BAR^+/+/A2BAR^+/+. n = 8. (D) Influence of A2BAR agonist BAY 60-6583 on AFC during VILI. Following induction of VILI, control mice received BAY 60-6583 (1 μM) alone or in combination with amiloride (1 mM), and AFC was determined *P < 0.01 compared with no amiloride. n = 8. (E and F) Control mice were treated with intratracheal BAY 60-6583 (1 μM, 100 μl) and/or amiloride (1 mM) or vehicle following tracheotomy and initiation of mechanical ventilation. Mice were ventilated using pressure-controlled settings (inspiratory pressure of 45 mbar, 100% inspired oxygen concentration) until a cardiac standstill was observed in the surface electrocardiogram. Note that the longer survival time during VILI with A2BAR agonist treatment was abolished following amiloride treatment (n = 8).

Based on previous studies showing that elevated endogenous epinephrine can markedly upregulate AFC under pathological conditions such as sepsis or pneumonia (45, 46), we studied plasma epinephrine levels during VILI. Consistent with these studies (45, 46), plasma epinephrine levels were elevated during VILI (Figure 11A). However, no differences between A2BAR^–/– mice or their corresponding control mice were observed, suggesting that A2BAR signaling is not involved in VILI-associated increases in plasma epinephrine (Figure 11B). Similarly, increases in AFC with BAY 60-6583 treatment were not blocked by pretreatment with propanolol, a nonselective β-adrenergic receptor antagonist (Figure 11C). However, as shown earlier, treatment with the β2-adrenergic agonist zinterol (10^–7 M, 300 μl intratracheally) was associated with elevated cAMP levels, attenuated albumin leakage, and improved survival (Figure 4, C–E). Simultaneous treatment with zinterol and BAY 60-6583 were associated with increased elevations of cAMP and enhancement of AFC. In fact, ANOVA testing followed by Bonferroni correction revealed a statistically significant increase with the combination of zinterol and BAY 60-6583 (mice treated with zinterol alone had an AFC of 58.56% following high-pressure ventilation; mice treated with zinterol and BAY 60-6583 had an AFC of 64.3%; P < 0.01). While statistical analysis of these data determined that the groups were different, these differences were very small and may not be biologically significant (Figure 11D). Thus, segregation of the 2 pathways is complicated. The stimulation of A2BAR increases cAMP and PKA, as does β-agonist stimulation. While adenosine signaling resulted in Gs protein–coupled and cAMP-elevating A2BAR activation, β2-adrenergic agonist stimulation converged on a similar signaling cascade, thereby resulting in enhancement of AFC. Taken together, these studies provide evidence for a role of A2BAR in counterbalancing increases in pulmonary edema during VILI via activation of amiloride-sensitive fluid transport mechanisms.

Figure 11

Influence of β2-adrenergic and/or A2BAR signaling on AFC during VILI. (A) Epinephrine plasma levels in A2BAR^–/– and A2BAR^+/+ mice that were mechanically ventilated in a pressure-controlled setting at 45 mbar over 180 minutes. (B) Basal cAMP levels in lung tissue from A2BAR^+/+ mice that were treated with zinterol and/or BAY 60-6583. (C and D) To determine β2-adrenergic and A2BAR signaling effects on pulmonary fluid transport, A2BAR^–/– and A2BAR^+/+ mice were mechanically ventilated in a pressure-controlled setting at 45 mbar for 0 to 180 minutes. AFC was measured by instilling 300 μl of iso-osmolar 0.9% NaCl solution with 5% BSA. Mechanical ventilation was continued for 30 minutes, and AFC was measured in the presence or absence of the nonselective β-adrenergic receptor antagonist propranolol (intratracheal instillation of 10^–4 M propranolol combined with 3 mg/kg i.p.) with or without BAY 60-6583 (10^–3 M to the instilled fluid) or in the presence or absence of the β-adrenergic agonist zinterol (intratracheal, 10^–7 M). *P < 0.01 compared with no propranolol (C) or zinterol (D), by ANOVA with Bonferroni post-hoc test. n = 8. In subsets of experiments, either propranolol or zinterol were added together with BAY 60-6583 10^–3 M to the instilled fluid. ^§P < 0.01 compared with propranolol alone (C) or zinterol alone (D), by ANOVA with Bonferroni post-hoc test. n = 8.

ALI contributes substantially 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). Nevertheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Here, we used VILI as a model for ALI to study the role of extracellular adenosine signaling pathways in endogenous lung protection. Our results point toward a pivotal role of the A2BAR in attenuating VILI-induced increases in pulmonary edema and inflammation. Moreover, our in vivo studies of A2BAR bone marrow–chimeric mice suggest an important contribution in attenuating lung injury by pulmonary A2BARs by maintaining the capillary-alveolar barrier during VILI. In addition, studies of alveolar fluid transport suggest that A2BAR signaling attenuates pulmonary edema formation by enhancing amiloride-sensitive fluid transport mechanisms. These findings correlate with elevations of plasma epinephrine levels and elevations of cAMP, roughly equivalent to the effects of stimulation with a β2-adrenergic agonist. Taken together, these findings identify a surprising role for pulmonary A2BAR signaling in attenuating ALI induced by mechanical ventilation or LPS exposure, suggesting that the A2BAR protects the lungs by attenuating acute inflammation and increasing AFC.

The present studies are consistent with previous work on extracellular adenosine signaling in alveolar fluid transport (41, 42). In fact, recent studies demonstrate that adenosine helps maintain proper fluid levels in the lung alveoli, and such findings have implications for treatment of fluid buildup in the lung (42). In an elegant study, Factor et al. (41) showed that levels of extracellular adenosine regulate the activities of 2 ion channels, the ENaC and the chloride channel CFTR. Adenosine regulation of the activities of these 2 channels establishes the balance between fluid secretion into the alveolus and fluid absorption from it. Under conditions of alveolar filling — such as occurs during pulmonary edema or VILI — adenosine concentrations may decrease, resulting in loss of A1AR-mediated pathways and activation of A2AAR-mediated pathways (41, 42). A2AAR-mediated pathways increase ENaC activity, increasing sodium absorption from the alveolus. Chloride follows sodium out of the cell to maintain electroneutrality, and, as a result of this sodium and chloride flux, fluid leaves the alveolus (i.e., AFC increases) (41, 42). It is important to point out that during large-volume ventilation, the A2BAR is transcriptionally induced (an almost 10-fold induction was observed in the present study, consistent with previous studies of nucleoside receptors during large-volume ventilation; ref. 35) and becomes the predominant AR in the lungs. Such findings may explain our results for the role of A2BAR signaling in activation of AFC during VILI. While A2AAR and A2BAR signaling pathways both converge to increase pulmonary cAMP levels (43), a combined agonist of the A2AAR and A2BAR may represent the most effective pharmacological approach to utilizing adenosine pathways to dry out the lungs during pulmonary edema or VILI.

The observation of increased capillary-alveolar leakage with targeting of the A2BAR during VILI suggests a protective role of extracellular adenosine signaling for maintaining the pulmonary barrier function. This is consistent with previous studies showing barrier protection and antiinflammatory effects of extracellular adenosine signaling in models of acute injury (47), inflammation (21), and hypoxia (11, 48). Previous studies found different ARs responsible for tissue protection under such conditions (11–13, 16). For example, an elegant study in mice deficient in the A2AAR showed increased inflammation-associated tissue damage (18), providing evidence for A2AAR signaling as a mechanism for regulating acute inflammatory responses in vivo. In this study, a subthreshold dose of an inflammatory stimulus was associated with minimal tissue damage in control animals. A similar stimulus was sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death in A2AAR^–/– mice. Similar observations were made using other models of inflammation, liver damage, or bacterial endotoxin–induced septic shock, suggesting a role for A2AAR in the limitation and termination of tissue-specific and systemic inflammatory responses (18). Other studies of LPS-induced ALI confirmed a critical role for signaling through A2AARs in attenuating LPS-induced lung inflammation (4, 37). As such, another study revealed that hypoxia increased adenosine signaling, the effects of which are protective during LPS-induced lung injury (37). In fact, the administration of high oxygen concentration further aggravated lung injury in that study. The authors found that this was due to inhibition of hypoxia-elicited lung protective mechanisms (37). Along the same lines, a recent study has suggested a role for hematopoietic A2AAR signaling in attenuating LPS-induced lung injury (4). Consistent with the present work, in vitro studies have indicated a critical role of signaling through the A2BAR in the re-sealing of endothelia during transendothelial migration of neutrophils (49), particularly during conditions of limited oxygen availability (14, 15, 27, 50). Moreover, pharmacological inhibition or genetic deletion of the A2BAR during hypoxia exposure is associated with increased pulmonary edema and vascular leakage (11, 51). Interestingly, another study suggested protective roles of HIF activators in respiratory distress syndrome and bronchopulmonary dysplasia (52). Serendipitously, the pacemaker enzyme of extracellular adenosine generation, CD73 (53), and the A2BAR (54) are both selectively induced by HIF-1 and were both shown to be central to innate lung protection during VILI (10). The seemingly contradictory findings that the A2AAR appears to be critical to mediating LPS-induced lung injury (18, 37) but that the A2BAR plays a pivotal role in VILI might be explained by differences between LPS- and VILI-induced ALIs and in AR expression patterns during such conditions.

The present studies in bone marrow–chimeric mice suggest that mice genetically targeted for myeloid A2BARs exhibit no significant increase in albumin leak or lung water. On the other hand, the mice had a significant degree of pulmonary inflammation (Figure 8), as determined by increased levels of cytokines and MPO. These data indicate that increased levels of cytokines or neutrophils do not contribute to lung injury. This is consistent with previous studies showing that neutropenia does not prevent significant injury to the alveolar epithelium during ALI induced by hypoxia (55, 56). Studies of ambient hypoxia exposure (8% oxygen over 4 hours) of A2BAR bone marrow–chimeric mice revealed that hypoxia-associated vascular leakage was attenuated by vascular A2BAR signaling effects (11). In contrast, hypoxia-associated inflammation mainly involved myeloid A2BAR signaling (11).

In contrast to the present study of acute injury, studies investigating chronic pulmonary disease have identified a potential detrimental role of elevated adenosine levels (57–63). For example, levels of adenosine are chronically increased in the lungs of asthmatics (61) and correlate with the degree of inflammatory insult (62), suggesting a provocative role of adenosine in asthma or chronic obstructive pulmonary disease (63). In addition, adenosine deaminase–deficient (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 (64), and studies have suggested 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 (59, 64). Nevertheless, the present studies revealed that A2BAR-dependent lung protection was not limited to VILI but also played a role in LPS-induced lung injury in which pulmonary inflammation was measured 24 hours following exposure to endotoxin.

In summary, the present study has combined pharmacological and genetic approaches to identify extracellular adenosine signaling through the A2BAR as part of innate protection from VILI. While inflammatory aspects of VILI are mediated through myeloid and pulmonary A2BAR signaling, VILI-induced defects in the alveolar-capillary barrier are attenuated by pulmonary A2BARs. In addition, A2BAR signaling contributes to enhancing alveolar fluid transport during VILI, suggesting that A2BAR agonist treatment is a “pulmonary diuretic” and protects the lungs during VILI by enhancing AFC.

View Supplemental data

This work was supported by grants from the German Research Foundation (DFG; EL274/2-2, to H.K. Eltzschig), the Foundation for Anesthesia Research and Education (to H.K. Eltzschig and T. Eckle), and Interdisciplinary Centre for Clinical Research (IZKF) Nachwuchsgruppe 1605-0-0 (to T. Eckle) and by a DFG Research Fellowship (GR2121/1–1, to A. Grenz). The authors wish to acknowledge Cathrine Ledent, Marlene Jacobson, and Jürgen Schnermann for kindly providing us with gene-targeted mice and Ines Kuhlicke, Stephanie Zug, Marion Faigle, and Katherine Hartmann for technical assistance.

Address correspondence to: Holger K. Eltzschig, Mucosal Inflammation Program, Department of Anesthesiology and Perioperative Medicine, Biochemistry Research Building (BRB), Room 852, 4200 E. 9th Avenue, Mailstop B112, Denver, Colorado 80262, USA. Phone: (303) 315-7320; Fax: (303) 315-0369; E-mail: holger.eltzschig@uchsc.edu.

Nonstandard abbreviations used: AFC, alveolar fluid clearance; ALI, acute lung injury; AR, adenosine receptor; BAL, bronchoalveolar; ENaC, epithelial sodium channel; KC, keratinocyte-derived chemokine; MPO, myeloperoxidase; VILI, ventilator-induced lung injury.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:3301–3315 (2008). doi:10.1172/JCI34203

T. Eckle and A. Grenz contributed equally to this work.

1. Ware, L.B., Matthay, M.A. 2000. The acute respiratory distress syndrome. N. Engl. J. Med. 342:1334-1349.
   View this article via: CrossRef PubMed Google Scholar
2. Rubenfeld, G.D., et al. 2005. Incidence and outcomes of acute lung injury. N. Engl. J. Med. 353:1685-1693.
   View this article via: CrossRef PubMed Google Scholar
3. Belperio, J.A., et al. 2002. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J. Clin. Invest. 110:1703-1716.
   View this article via: JCI PubMed Google Scholar
4. Reutershan, J., Cagnina, R.E., Chang, D., Linden, J., Ley, K. 2007. Therapeutic anti-inflammatory effects of myeloid cell adenosine receptor A2a stimulation in lipopolysaccharide-induced lung injury. J. Immunol. 179:1254-1263.
   View this article via: PubMed Google Scholar
5. Reutershan, J., et al. 2006. Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J. Clin. Invest. 116:695-702.
   View this article via: JCI CrossRef PubMed Google Scholar
6. Martin, T.R. 2002. Neutrophils and lung injury: getting it right. J. Clin. Invest. 110:1603-1605.
   View this article via: JCI PubMed Google Scholar
7. Licker, M., et al. 2003. Risk factors for acute lung injury after thoracic surgery for lung cancer. Anesth. Analg. 97:1558-1565.
   View this article via: PubMed Google Scholar
8. Milot, J., et al. 2001. Incidence and predictors of ARDS after cardiac surgery. Chest. 119:884-888.
   View this article via: CrossRef PubMed Google Scholar
9. Shorr, A.F., Abbott, K.C., Agadoa, L.Y. 2003. Acute respiratory distress syndrome after kidney transplantation: epidemiology, risk factors, and outcomes. Crit. Care Med. 31:1325-1330.
   View this article via: CrossRef PubMed Google Scholar
10. Eckle, T., et al. 2007. Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury. J. Immunol. 178:8127-8137.
    View this article via: PubMed Google Scholar
11. Eckle, T., et al. 2008. A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood. 111:2024-2035.
    View this article via: CrossRef PubMed Google Scholar
12. Eckle, T., et al. 2008. Hypoxia-inducible factor-1 is central to cardioprotection: a new paradigm for ischemic preconditioning. Circulation. 118:166-175.
    View this article via: CrossRef PubMed Google Scholar
13. Eckle, T., et al. 2007. Cardioprotection by ecto-5′-nucleotidase (CD73) and A2B adenosine receptors. Circulation. 115:1581-1590.
    View this article via: CrossRef PubMed Google Scholar
14. Eltzschig, H.K., et al. 2003. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J. Exp. Med. 198:783-796.
    View this article via: CrossRef PubMed Google Scholar
15. Eltzschig, H.K., et al. 2004. Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism. Blood. 104:3986-3992.
    View this article via: CrossRef PubMed Google Scholar
16. Grenz, A., et al. 2008. The reno-vascular A2B adenosine receptor protects the kidney from ischemia. PLoS Med. 5:e137.
17. Fredholm, B.B. 2007. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ. 14:1315-1323.
    View this article via: CrossRef PubMed Google Scholar
18. Ohta, A., Sitkovsky, M. 2001. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 414:916-920.
    View this article via: CrossRef PubMed Google Scholar
19. Lankford, A.R., et al. 2006. Effect of modulating cardiac A1 adenosine receptor expression on protection with ischemic preconditioning. Am. J. Physiol. Heart Circ. Physiol. 290:H1469-H1473.
    View this article via: CrossRef PubMed Google Scholar
20. Yang, Z., et al. 2005. Infarct-sparing effect of A2A-adenosine receptor activation is due primarily to its action on lymphocytes. Circulation. 111:2190-2197.
    View this article via: CrossRef PubMed Google Scholar
21. Yang, D., et al. 2006. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J. Clin. Invest. 116:1913-1923.
    View this article via: JCI CrossRef PubMed Google Scholar
22. Salvatore, C.A., et al. 2000. Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J. Biol. Chem. 275:4429-4434.
    View this article via: CrossRef PubMed Google Scholar
23. Lappas, C.M., Day, Y.J., Marshall, M.A., Engelhard, V.H., Linden, J. 2006. Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation. J. Exp. Med. 203:2639-2648.
    View this article via: CrossRef PubMed Google Scholar
24. Linden, J. 2005. Adenosine in tissue protection and tissue regeneration. Mol. Pharmacol. 67:1385-1387.
    View this article via: CrossRef PubMed Google Scholar
25. Sitkovsky, M., Lukashev, D. 2005. Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors. Nat. Rev. Immunol. 5:712-721.
    View this article via: CrossRef PubMed Google Scholar
26. Sitkovsky, M.V., et al. 2004. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Ann. Rev. Immunol. 22:657-682.
    View this article via: CrossRef Google Scholar
27. Eltzschig, H.K., et al. 2005. HIF-1-dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia. J. Exp. Med. 202:1493-1505.
    View this article via: CrossRef PubMed Google Scholar
28. Mi, T., et al. 2008. Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling. J. Clin. Invest. 118:1491-1501.
    View this article via: JCI CrossRef PubMed Google Scholar
29. Sun, D., et al. 2001. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc. Natl. Acad. Sci. U. S. A. 98:9983-9988.
    View this article via: CrossRef PubMed Google Scholar
30. Ledent, C., et al. 1997. Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor. Nature. 388:674-678.
    View this article via: CrossRef PubMed Google Scholar
31. Khoury, J., Ibla, J.C., Neish, A.S., Colgan, S.P. 2007. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation. J. Clin. Invest. 117:703-711.
    View this article via: JCI CrossRef PubMed Google Scholar
32. Kumar, A., et al. 2003. Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma. FASEB J. 17:1800-1811.
    View this article via: CrossRef PubMed Google Scholar
33. Li, L.F., et al. 2003. Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-kappaB-inducing kinases. Am. J. Physiol. Lung Cell Mol. Physiol. 285:L464-L475.
    View this article via: PubMed Google Scholar
34. Chen, Z.J. 2005. Ubiquitin signalling in the NF-kappaB pathway. Nat. Cell Biol. 7:758-765.
    View this article via: CrossRef PubMed Google Scholar
35. Douillet, C.D., et al. 2005. Mechanical ventilation alters airway nucleotides and purinoceptors in lung and extrapulmonary organs. Am. J. Respir. Cell Mol. Biol. 32:52-58.
    View this article via: CrossRef PubMed Google Scholar
36. Lukashev, D.E., et al. 2003. Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, A1, and A3 adenosine receptors in lymphoid organs. Biochem. Pharmacol. 65:2081-2090.
    View this article via: CrossRef PubMed Google Scholar
37. Thiel, M., et al. 2005. Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury. PLoS Biol. 3:e174.
38. Fredholm, B.B., et al. 1994. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46:143-156.
    View this article via: PubMed Google Scholar
39. Comerford, K.M., Lawrence, D.W., Synnestvedt, K., Levi, B.P., Colgan, S.P. 2002. Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability. FASEB J. 16:583-585.
    View this article via: PubMed Google Scholar
40. McAuley, D.F., Frank, J.A., Fang, X., Matthay, M.A. 2004. Clinically relevant concentrations of beta2-adrenergic agonists stimulate maximal cyclic adenosine monophosphate-dependent airspace fluid clearance and decrease pulmonary edema in experimental acid-induced lung injury. Crit. Care Med. 32:1470-1476.
    View this article via: CrossRef PubMed Google Scholar
41. Factor, P., et al. 2007. Adenosine regulation of alveolar fluid clearance. Proc. Natl. Acad. Sci. U. S. A. 104:4083-4088.
    View this article via: CrossRef PubMed Google Scholar
42. Kreindler, J.L., Shapiro, S.D. 2007. Lung turns to AA (adenosine analogues) to dry out. Nat. Med. 13:406-408.
    View this article via: CrossRef PubMed Google Scholar
43. Tarran, R., Button, B., Boucher, R.C. 2006. Regulation of normal and cystic fibrosis airway surface liquid volume by phasic shear stress. Annu. Rev. Physiol. 68:543-561.
    View this article via: CrossRef PubMed Google Scholar
44. Mutlu, G.M., et al. 2005. Interdependency of beta-adrenergic receptors and CFTR in regulation of alveolar active Na+ transport. Circ. Res. 96:999-1005.
    View this article via: CrossRef PubMed Google Scholar
45. Pittet, J.F., et al. 1994. Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats. J. Clin. Invest. 94:663-671.
    View this article via: JCI CrossRef PubMed Google Scholar
46. Su, X., Robriquet, L., Folkesson, H.G., Matthay, M.A. 2006. Protective effect of endogenous beta-adrenergic tone on lung fluid balance in acute bacterial pneumonia in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 290:L769-L776.
    View this article via: CrossRef PubMed Google Scholar
47. Yang, D., et al. 2008. The A2b adenosine receptor protects against vascular injury. Proc. Natl. Acad. Sci. U. S. A. 105:792-796.
    View this article via: CrossRef PubMed Google Scholar
48. Hasko, G., Cronstein, B.N. 2004. Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 25:33-39.
    View this article via: CrossRef PubMed Google Scholar
49. Lennon, P.F., Taylor, C.T., Stahl, G.L., Colgan, S.P. 1998. Neutrophil-derived 5′-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A2B receptor activation. J. Exp. Med. 188:1433-1443.
    View this article via: CrossRef PubMed Google Scholar
50. Eltzschig, H.K., Macmanus, C.F., Colgan, S.P. 2008. Neutrophils as sources of extracellular nucleotides: functional consequences at the vascular interface. Trends Cardiovasc. Med. 18:103-107.
    View this article via: CrossRef PubMed Google Scholar
51. Thompson, L.F., et al. 2004. Crucial role for ecto-5′-nucleotidase (CD73) in vascular leakage during hypoxia. J. Exp. Med. 200:1395-1405.
    View this article via: CrossRef PubMed Google Scholar
52. Asikainen, T.M., et al. 2005. Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells and explants of primate lung. Proc. Natl. Acad. Sci. U. S. A. 102:10212-10217.
    View this article via: CrossRef PubMed Google Scholar
53. Synnestvedt, K., et al. 2002. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J. Clin. Invest. 110:993-1002.
    View this article via: JCI PubMed Google Scholar
54. Kong, T., Westerman, K.A., Faigle, M., Eltzschig, H.K., Colgan, S.P. 2006. HIF-dependent induction of adenosine A2B receptor in hypoxia. FASEB J. 20:2242-2250.
    View this article via: CrossRef PubMed Google Scholar
55. Laughlin, M.J., Wild, L., Nickerson, P.A., Matalon, S. 1986. Effects of hyperoxia on alveolar permeability of neutropenic rabbits. J. Appl. Physiol. 61:1126-1131.
    View this article via: PubMed Google Scholar
56. Raj, J.U., Hazinski, T.A., Bland, R.D. 1985. Oxygen-induced lung microvascular injury in neutropenic rabbits and lambs. J. Appl. Physiol. 58:921-927.
    View this article via: PubMed Google Scholar
57. Blackburn, M.R. 2003. Too much of a good thing: adenosine overload in adenosine-deaminase-deficient mice. Trends Pharmacol. Sci. 24:66-70.
    View this article via: CrossRef PubMed Google Scholar
58. Chunn, J.L., et al. 2005. Adenosine-dependent pulmonary fibrosis in adenosine deaminase-deficient mice. J. Immunol. 175:1937-1946.
    View this article via: PubMed Google Scholar
59. Sun, C.X., et al. 2006. Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury. J. Clin. Invest. 116:2173-2182.
    View this article via: JCI CrossRef PubMed Google Scholar
60. Sun, C.-X., et al. 2005. A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury. J. Clin. Invest. 115:35-43.
    View this article via: JCI PubMed Google Scholar
61. Driver, A.G., Kukoly, C.A., Ali, S., Mustafa, S.J. 1993. Adenosine in bronchoalveolar lavage fluid in asthma. Am. Rev. Respir. Dis. 148:91-97.
    View this article via: PubMed Google Scholar
62. Huszar, E., et al. 2002. Adenosine in exhaled breath condensate in healthy volunteers and in patients with asthma. Eur. Respir. J. 20:1393-1398.
    View this article via: CrossRef PubMed Google Scholar
63. Fozard, J.R., Hannon, J.P. 1999. Adenosine receptor ligands: potential as therapeutic agents in asthma and COPD. Pulm. Pharmacol. Ther. 12:111-114.
    View this article via: CrossRef PubMed Google Scholar
64. Blackburn, M.R., et al. 2000. Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction. J. Exp. Med. 192:159-170.
    View this article via: CrossRef PubMed Google Scholar
65. Kohler, D., et al. 2007. CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury. Circulation. 116:1784-1794.
    View this article via: CrossRef PubMed Google Scholar
66. Speyer, C.L., et al. 2005. Regulatory effects of estrogen on acute lung inflammation in mice. Am. J. Physiol. Cell Physiol. 288:C881-C890.
    View this article via: PubMed Google Scholar
67. Graff, G., Gamache, D.A., Brady, M.T., Spellman, J.M., Yanni, J.M. 1998. Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis. J. Pharmacol. Toxicol. Methods. 39:169-178.
    View this article via: CrossRef PubMed Google Scholar
68. Schneider, T., Issekutz, A.C. 1996. Quantitation of eosinophil and neutrophil infiltration into rat lung by specific assays for eosinophil peroxidase and myeloperoxidase. Application in a Brown Norway rat model of allergic pulmonary inflammation. J. Immunol. Methods. 198:1-14.
    View this article via: CrossRef PubMed Google Scholar
69. Mutlu, G.M., et al. 2004. Upregulation of alveolar epithelial active Na+ transport is dependent on beta2-adrenergic receptor signaling. Circ. Res. 94:1091-1100.
    View this article via: CrossRef PubMed Google Scholar