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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: J Thorac Cardiovasc Surg. 2009 Nov 11;139(2):474. doi: 10.1016/j.jtcvs.2009.08.033

Adenosine A2A receptor activation on CD4+ T lymphocytes and neutrophils attenuates lung ischemia-reperfusion injury

Ashish K Sharma a, Victor E Laubach a, Susan I Ramos b, Yunge Zhao a, George Stukenborg c, Joel Linden b, Irving L Kron a, Zequan Yang a
PMCID: PMC2813368  NIHMSID: NIHMS140703  PMID: 19909990

Abstract

Objective:

Adenosine A2A receptor activation potently attenuates lung ischemia-reperfusion injury. This study tests the hypothesis that adenosine A2A receptor activation attenuates ischemia-reperfusion injury by inhibiting CD4+ T cell activation and subsequent neutrophil infiltration.

Methods:

An in vivo model of lung ischemia-reperfusion injury was used. C57BL/6 mice were assigned to either sham group (left thoracotomy) or seven study groups which underwent ischemia-reperfusion (1hr left hilar occlusion plus 2hrs reperfusion). ATL313, a selective adenosine A2A receptor agonist, was administered five minutes before reperfusion with or without antibody depletion of neutrophils or CD4+ T cells. After reperfusion, the following was measured: pulmonary function using an isolated, buffer-perfused lung system, T cell infiltration by immunohistochemistry, myeloperoxidase and proinflammatory cytokine/chemokine levels in bronchoalveolar lavage fluid, lung wet/dry weight, and microvascular permeability.

Results:

ATL313 significantly improved pulmonary function and reduced edema and microvascular permeability after ischemia-reperfusion compared to control. Immunohistochemistry and myeloperoxidase content demonstrated significantly reduced infiltration of neutrophils and CD4+ T cells after ischemia-reperfusion in ATL313-treated mice. Although CD4+ T cell-depleted and neutrophil-depleted mice displayed significantly reduced lung injury, no additional protection occurred when ATL313 was administered to these mice. Expression of TNF-α, IL-17, KC, MCP-1, MIP-1, and RANTES were significantly reduced in neutrophil- and CD4+ T cell-depleted mice and reduced further by ATL313 only in neutrophil-depleted mice.

Conclusions:

These results demonstrate that CD4+ T cells play a key role in mediating lung inflammation after ischemia-reperfusion. ATL313 likely exerts its protective effect largely through activation of adenosine A2A receptors on CD4+ T cells and neutrophils.

INTRODUCTION

Ischemia-reperfusion (IR) injury entails enhanced inflammatory responses during reperfusion and remains a major cause of respiratory failure and other complications after lung transplantation (1). We have previously shown that pulmonary macrophages and neutrophils contribute importantly to lung IR injury, with macrophages serving as triggers and neutrophils as end effectors of tissue injury (2-4). Alveolar epithelial cells also interact with alveolar macrophages to augment the inflammatory response after IR (5). Although recent studies suggest that lymphocytes may play an important role in lung IR injury (6, 7), the exact role of specific subtypes of lymphocytes and their cross-talk with macrophages and neutrophils in the context of lung IR injury remain to be elucidated.

Multiple lines of evidence suggest that the adenosine A2A receptor (A2AAR) is critical for adenosine-mediated protection from IR injury. A2AAR-mediated inhibition of IR injury has been documented in various organ systems including liver, lung, kidney, and heart (8-11); however, the precise mechanisms responsible for A2AAR-mediated protection remain unknown. A2AARs are predominantly expressed on leukocyte populations and mediate a variety of physiological responses. A2AARs couple to G proteins and activate adenylyl cyclase leading to increased cellular cAMP levels (12, 13). The mechanisms of protection mediated by A2AAR activation may include inhibition of leukocyte-mediated inflammatory responses (13), vasodilation (14), and direct effects on organ parenchymal cells (12). We have previously demonstrated a T lymphocyte-mediated mechanism for A2AAR activation after myocardial IR in mice (11).

Our previous studies have shown that administration of a selective A2AAR agonist during reperfusion significantly reduces lung and heart IR injury in both in vivo and ex vivo animal models (8, 11, 15). In a recent study, we have also demonstrated a key role of CD4+ T cells in the initiation and progression of lung IR injury and that activation of CD4+ T cells lies upstream of neutrophil activation and infiltration (16). Although in vivo experiments have demonstrated the protective effect of A2AAR activation, these experiments have not identified the major cellular target(s) responsible for the salutary effects of A2AAR activation in lung IR injury. In the present study, we utilized a potent and selective A2AAR agonist, ATL313, in an in vivo mouse model of lung IR injury. By using specific antibodies to deplete CD4+ T cells or neutrophils, we demonstrate a primary role of A2AAR activation on CD4+ T lymphocytes in protecting the lung against IR injury.

MATERIALS AND METHODS

Study Design

This study utilized 8-12 week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) which were assigned to a Sham group or one of seven experimental groups that underwent IR (1 hr left lung ischemia followed by 2 hrs reperfusion). The duration of 1 hr ischemia and 2 hr reperfusion was chosen based on our previous study which determined that this timeframe resulted in maximum pulmonary dysfunction after ischemia (16). A total of eight groups were utilized in the current study (n=5-9/group) which are summarized as follows: 1) Sham (sham thoracotomy with no ischemia, 2) IR (1 hr ischemia followed by 2 hr reperfusion), 3) IR+ATL313 (mice undergoing IR with inclusion of ATL313 in the reperfusion buffer), 4) IgG (mice pretreated with control IgG Ab which undergo IR), 5) neutrophil depletion (neutrophil-depleted mice which undergo IR), 6) neutrophil depletion+ATL313 (same as group 5 with inclusion of ATL313 in the reperfusion buffer), 7) CD4+ T cell depletion (CD4+ T cell-depleted mice which undergo IR) and 8) CD4+ T cell depletion+ATL313 (same as group 7 with inclusion of ATL313 in the reperfusion buffer). Note that all groups underwent left lung IR except the Sham group. The A2AAR agonist, ATL313 (gift of Adenosine Therapeutics, LLC, Charlottesville, VA), was administered via intravenous injection at a dose of 3μg/kg 5 minutes before reperfusion. This dose was used based upon previous studies which have shown that 3μg/kg ATL313 potently attenuates IR injury without significant hemodynamic effects (8). This study was performed concurrently with a previously reported study (16), and thus much of the data from the antibody-treated, control groups of animals in the current study (IgG, NE dep, and CD4 dep) were shared with the previous study. This study conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85-23, revised 1985) and was conducted under protocols approved by the University of Virginia's Institutional Animal Care and Use Committee.

Hemodynamic Study

The effect of ATL313 or vehicle (saline) on hemodynamics was evaluated in C57BL/6 mice. Mice were anesthetized with 1% (vol) isoflurane. The right common carotid artery was exposed and cannulated with a 1.4F Millar microtip catheter (Millar Instruments, Inc). After peripheral arterial blood pressures were acquired, the catheter tip was advanced into the left ventricular (LV) chamber. After bolus injection of 3 μg/kg ATL313 or saline (2μl/g) via left external jugular vein, LV pressures (end-systolic and end-diastolic pressures), developed pressures (dP/dt+ and dP/dt−), as well as aortic arterial pressure were continuously recorded for 30 min.

Depletion of Neutrophils and T lymphocytes

Depletion of neutrophils and CD4+ T cells was achieved by using selective antibodies as described previously (16). Rat anti-Gr-1 mAb was used to deplete neutrophils. Briefly, 10μg anti-Gr-1 mAb (eBioscience, San Diego, CA) was injected via tail vein 24 hr before undergoing study. To deplete CD4+ T cells, anti-CD4 mAb (GK1.5, eBioscience, San Diego, CA) was injected intraperitoneally on two consecutive days at a dose of 0.2 mg/mouse/day. Two days after the second injection, these animals underwent study. We have previously documented specific depletion of CD4+ T cells by anti-CD4 mAb in mice by flow cytometry of peripheral blood and spleen cells (11). Perioperatively, blood (30-40 μl) was obtained by puncturing the left external jugular vein. Circulating blood leukocyte counts were performed with a HemaVet Hematology System (CDC Technologies, Oxford, CT).

In vivo Model of Lung Ischemia-Reperfusion

An in vivo hilar clamp model of IR was used as previously described (16). Mice undergoing IR were subjected to 1 hr ischemia (left lung hilar occlusion) followed by 2 hrs of reperfusion. Briefly, mice were anesthetized with inhalation isoflurane, intubated with PE-60 tubing and connected to a pressure-controlled ventilator (Harvard Apparatus Co, South Natick, MA). Mechanical ventilation with room air was set at 150 strokes/min, 1.0 cc stroke volume, and peak inspiratory pressure less than 20 cm H2O. Heparin (20 U/kg) was given via external jugular injection to minimize thrombosis in the pulmonary vasculature during ischemia. Left thoracotomy was performed by cutting the left 4th rib, and the left hilum was exposed. A 6-0 prolene suture was placed around the left hilum facilitated by a tip-curved (22G) gavage needle. Both ends of the suture were threaded through a 5-mm long PE-50 tube. Hilar occlusion was achieved by pulling up on the suture and thus pressing the tube against the hilum to initiate ischemia. A small surgical clip was applied to the suture above the tube to maintain tension against the hilum. The thoracotomy was then suture-closed and the mouse was extubated and allowed to recover during the 1 hr hilar occlusion period. The average time on the ventilator for each animal was 10 min. Five minutes before reperfusion, the mouse was re-anesthetized and re-intubated. Reperfusion was achieved by removing the clip, tube and suture. Again, the chest was suture-closed. The mouse was extubated and placed back in the cage during the 2-hr reperfusion period. Temperature was monitored during surgery by an anal probe and maintained between 36.5–37.5°C. Sham animals received only thoracotomy without hilar occlusion. Confirmation of cessation of blood flow during ischemia was conducted in separate mice using Evans blue dye which was injected intravenously immediately after hilar clamping, and the left lungs were harvested 1 hr later. We consistently found no detectable levels of Evans blue dye in these lungs, thereby confirming ischemia.

Measurement of Pulmonary Function

At the end of scheduled reperfusion, pulmonary function was evaluated using an isolated, buffer-perfused mouse lung system (Hugo Sachs Elektronik, March-Huggstetten, Germany) as previously described (16). Once properly perfused and ventilated, the isolated lungs were maintained on the system for a 5-min equilibration period before data was recorded for an additional 10 min. Hemodynamic and pulmonary parameters were recorded by the PULMODYN data acquisition system (Hugo Sachs Elektronik). The values during the 10-min data acquisition period remained very constant in all animals, and the pulmonary function data reported in this study reflect the final values at the end of this period.

Bronchoalveolar Lavage

After pulmonary function measurement, left lungs were lavaged with 0.4 ml PBS. The BAL fluid was centrifuged at 4°C (500 g, 5 min), and the supernatant was collected and stored at −80°C until further analysis.

Lung Wet/Dry Weight Ratio

Using separate groups of animals (n=5/group), the left lung was harvested, weighed, and then placed in a vacuum oven (at 54°C) until a stable dry weight was achieved. The lung wet weight/dry weight ratio was then calculated.

Pulmonary Microvascular Permeability

Using separate groups of animals (n=5/group), lung microvascular permeability was estimated using the Evans blue dye extravasation technique (16, 17) which is an index of change in protein permeability. Evans blue (20 mg/kg, Sigma-Aldrich) was injected intravenously 30 min before euthanasia. The pulmonary vasculature was then perfused for 10 min with PBS to remove intravascular dye. Lungs were then homogenized in PBS to extract the Evans blue and centrifuged. The absorption of Evans blue was measured in the supernatant at 620 nm and corrected for the presence of heme pigments: A620 (corrected) = A620 − (1.426 × A740 + 0.030). The concentration of Evans blue was determined according to a standard curve and expressed as μg/gram wet lung weight.

Immunohistochemistry of Pulmonary CD4+ T cells

Lung tissue was fixed with 1% paraformaldehyde at 4°C for 24 hours and then embedded in O.C.T compound before storage at −80°C. Immunostaining was performed with rat anti-mouse CD4 antibody (GK1.5) (Santa Cruz Biotechnology) using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) as described previously (16).

Measurement of Myeloperoxidase (MPO)

MPO levels were measured in BAL fluid using a commercially-available mouse MPO ELISA kit (Cell Sciences, Canton, MA).

Measurement of Cytokines/Chemokines

Cytokine and chemokine protein levels in BAL fluid were quantified using the Bioplex Bead Array technique with a multiplex cytokine panel assay (Bio-Rad Laboratories, Hercules, CA) as described previously (5, 16).

Statistical Analysis

Mean values for each group are plotted in the accompanying figures, along with bars indicating the SEM (standard error of mean). Tests of the statistical significance of the observed differences in mean values between groups were assessed using one-way analysis of variance and the Satterthwaite t-test, which provides an adjustment for unequal between group variance, and statistical significance is reported at the p < 0.05 level.

RESULTS

Effect of ATL313 on Hemodynamics

Potential changes in hemodynamics were assessed after administration of ATL313 (3μg/kg) or vehicle (saline) (n=4/group). Heart rate and mean arterial pressure remained stable in vehicle-treated mice. There was a small but insignificant increase in heart rate of approximately 14% in ATL313-treated mice compared to vehicle treatment during the first 25 min (Figure 1, A). There was a small but insignificant decrease (<20%) in mean arterial blood pressure after ATL313 administration which returned to baseline level within 10 min (Figure 1, B). LV pressures and LV developed pressures had a similar pattern of changes as arterial pressures (data not shown).

FIGURE 1.

FIGURE 1

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Effect of ATL313 on mouse hemodynamics and complete blood cell counts. A, Heart rate remained stable in vehicle (saline)-treated mice (n=4/group). Heart rate was mildly elevated during the first 25 min after intravenous injection of 3μg/kg ATL313 compared to vehicle-treated mice. B, Mean arterial pressure remained stable in vehicle-treated mice (n=4/group). Small but insignificant changes in mean arterial pressure occurred after injection of ATL313 during the first 5 minutes. The time of injection of vehicle and ATL313 is indicated by an arrow. C, Complete blood cell counts after reperfusion are presented as the percentage of corresponding pre-operative baseline counts. Intra-group comparison showed a 3.5-fold increase in circulating neutrophils in both IR and IR+ATL313 groups and a >50% reduction in circulating lymphocytes after IR. Comparisons between the three groups revealed no significant differences in total WBC counts. Circulating neutrophils were significantly elevated in the IR and IR+ATL313 groups. Circulating lymphocytes were significantly reduced in the IR group (*p<0.05 vs. sham; #p<0.05 vs. other groups; n=5/group).

Effect of ATL313 on Blood Cell Counts

Blood was sampled for complete counts of circulating leukocytes prior to surgery and two hours after sham thoracotomy (Sham) or at the end of IR (IR or ATL313-treated IR animals) (n=5/group). The percent change in cell counts measured after reperfusion versus cell counts prior to ischemia (baseline) is shown (Figure 1, C). All pre-surgery and post-reperfusion measurements were performed on the same mouse to eliminate animal-to-animal variability. The numbers of pre-operative, circulating white blood cells (WBC) (baseline = 4333 ± 799/μl), neutrophils (baseline = 330 ± 83/μl), lymphocytes (baseline = 3642 ± 675/μl) and monocytes (baseline = 368 ± 116/μl) did not significantly change after sham thoracotomy. In the IR group, IR had no effect on WBC counts but caused a significant increase in neutrophils by 3.5-fold and a significant decrease in lymphocytes by 57% when compared to corresponding pre-ischemic baseline counts (p<0.05). In the ATL313-treated IR group there was a significant increase in neutrophils (2.8-fold) over pre-ischemic counts with no effect on WBC, lymphocytes or monocytes (Figure 1, C). Inter-group analysis of the changes after 2-hours reperfusion showed no significant difference in WBC or monocyte counts among the three groups. Circulating neutrophils were significantly elevated in the IR and IR+ATL313 groups versus Sham, and lymphocytes were significantly lower in the IR group versus the Sham and IR+ATL313 groups (Figure 1, C).

Pulmonary Function after IR is Improved by ATL313

Pulmonary function, evaluated by airway resistance (AR), lung compliance (LC) and pulmonary arterial pressure (PAP), was significantly impaired after IR (Figure 2, A). AR, LC, and PAP were significantly improved in ATL313-treated mice after IR. Moreover, AR, LC and PAP after IR were partially but significantly improved in neutrophil-depleted and CD4+ T cell-depleted mice compared to IgG control (Figure 2, B). No additional improvement of lung function occurred after IR when neutrophil-or CD4+ T cell-depleted mice were treated with ATL313 (Figure 2, B).

FIGURE 2.

FIGURE 2

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Pulmonary function. A, Airway resistance and pulmonary artery pressure were significantly increased while lung compliance was significantly reduced after IR compared to Sham. Pulmonary function was significantly improved after IR by ATL313 treatment (IR+ATL313) (*p<0.05 vs. Sham, #p<0.05 vs. IR, n=10/group). B, Pulmonary function in antibody-treated mice after IR. Compared to IgG-treated mice, airway resistance and pulmonary artery pressure were significantly decreased, and lung compliance was significantly increased in neutrophil-depleted (NE dep) and CD4+ T cell-depleted (CD4 dep) mice. No additional protection occurred after ATL313 treatment (ATL) (§p<0.05 vs. IgG, n=9/group).

ATL313 Inhibits Infiltration of T cells and Neutrophils

Immunostaining of peripheral lung tissue demonstrated significant infiltration of CD4+ T cells after IR compared to Sham (Figure 3). ATL313 significantly reduced infiltration of CD4+ T cells after IR (Figure 3). MPO levels in BAL fluid, used as a biochemical marker of neutrophil infiltration into alveolar air space, were also significantly increased after IR and significantly reduced by ATL313 treatment (Figure 4, A).

FIGURE 3.

FIGURE 3

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Pulmonary infiltration of T cells. The average number of CD4+ T cells per high power field (HPF) was assessed via immunohistochemical staining of peripheral lung sections. The quantitative analysis is shown in the graph, and examples of immunohistochemical staining are shown for each group above the corresponding bars. CD4+ T cell numbers were significantly increased after IR compared to Sham and were significantly reduced after IR by ATL313 treatment (IR+ATL313) (*p<0.05 vs. Sham, #p<0.05 vs. IR, n=5/group).

FIGURE 4.

FIGURE 4

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Neutrophil infiltration and lung injury after reperfusion. Neutrophil infiltration was assessed by measuring MPO levels in BAL fluid (top). Lung injury was assessed by measuring pulmonary edema (wet/dry weight) (middle) and microvascular leak (Evans blue content) (bottom). A, Non-antibody-treated mice. B, Antibody-treated mice for IgG-treated control (IgG) and depletion of neutrophils (NE dep) or CD4+ T cells (CD4 dep). All antibody-treated mice underwent lung IR. ATL = ATL313 treatment (*p<0.05 vs. Sham, #p<0.05 vs. IR, §p<0.05 vs. IgG control, n=5/group).

Lung IR Injury is Reduced by ATL313

Lung injury after IR was significantly reduced in ATL313-treated mice as determined by reduced microvascular permeability (via Evans blue content) and pulmonary edema (wet/dry weight) (Figure 4, A). Lung injury (measured by microvascular permeability and pulmonary edema) and neutrophil infiltration (measured by MPO level in BAL fluid) was significantly reduced after IR in CD4+ T cell-depleted and neutrophil-depleted mice compared to IgG control (Figure 4, B). There was no further improvement in lung injury when ATL313 was administered to neutrophil-depleted or CD4+ T cell-depleted mice (Figure 4, B).

Cytokine/Chemokine Expression in ATL313-Treated Mice

Expression of TNF-α, IL-17, MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5), and KC (CXCL1) were significantly increased in BAL fluid after IR, and ATL313 treatment significantly reduced the expression of each of these cytokines/chemokines (Figure 5, A). In neutrophil-depleted mice, expression of TNF-α, MCP-1, MIP-1, and KC remained elevated after IR; however, IL-17 and RANTES were significantly reduced compared to IgG control (Figure 5, B). ATL313 treatment significantly reduced expression of MIP-1, RANTES and KC in neutrophil-depleted mice. A noticeable trend in reduction of TNF-α, IL-17 and MCP-1 production was also observed by ATL313 treatment in neutrophil-depleted mice (Figure 5, B). The expression of IL-17, MCP-1, MIP-1, RANTES and KC were all significantly reduced in CD4+ T cell-depleted mice versus IgG control but were not reduced further by ATL313 treatment (Figure 5, B). A noticeable trend in reduction of TNF-α production was observed in CD4+ T cell-depleted mice with or without ATL313 treatment.

FIGURE 5.

FIGURE 5

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Production of cytokines/chemokines after IR. Expression of TNF-α, IL-17, MCP-1, MIP-1, RANTES, and KC were measured in BAL fluid after reperfusion. Two separate statistical comparisons are shown: (A) non-antibody-treated mice, and (B) antibody-treated mice for depletion of neutrophils (NE dep) or CD4+ T cells (CD4 dep). All antibody-treated animals underwent lung IR. ATL = ATL313 treatment (*p<0.05 vs. Sham and IR+ATL, #p<0.05 vs. IgG control , §p<0.05 vs. NE dep, n=5/group).

DISCUSSION

Using antibody depletion methods, we have previously demonstrated a key role of CD4+ T lymphocytes and neutrophils in lung IR injury wherein the role of neutrophils as end effectors of lung injury is secondary to CD4+ T cell activation (16). In the current study we also utilized antibody depletion methods to investigate the effects of A2AAR activation on lung IR injury via its role on CD4+ T cells and neutrophils. We observed that depletion of CD4+ T cells significantly attenuated lung injury, neutrophil infiltration, and induction of proinflammatory cytokines/chemokines after lung IR. Similarly, neutrophil-depleted mice also displayed significantly reduced lung IR injury. The current study showed that ATL313 exerts significant protection against lung IR injury in control mice but offered no additional protection in lung function or injury when administered to CD4+ T cell-depleted or neutrophil-depleted mice. Moreover, A2AAR activation further decreased TNF-α, IL-17, KC, MCP-1, MIP-1 and RANTES production in neutrophil-depleted mice after IR, but did not offer any additional protection in CD4+ T cell-depleted mice. The attenuation of neutrophil infiltration by ATL313 likely occurs by two means: 1) directly via activation of A2AAR on neutrophils to prevent activation, and 2) indirectly via activation of A2AAR on CD4+ T cells to attenuate release of potent chemokines. The data from the current study indicates that the effect of A2AAR activation on CD4+ T cells appears to be more prominent than on neutrophils. A2AAR activation on CD4+ T cells provides protection from lung IR injury by attenuating neutrophil activation and infiltration as demonstrated by the decrease in MPO levels and significant decreases in IL-17, KC and MIP-2 (potent neutrophil chemotactic cytokines). Taken together, the results demonstrate that the protective effect of A2AAR activation against lung IR injury is due to its action on CD4+ T cells as well as neutrophils, and suggest that protection may be largely due to A2AAR activation on CD4+ T cells.

A2AARs are widely distributed largely in leukocyte cell populations. The A2AAR agonist, ATL313, binds to recombinant mouse adenosine receptor subtypes with Ki values of (nM): A2A, 2.3 < A3, 43 < A1, 241 < A2B > 1000 (18). We have previously used A2AAR knockout mice to demonstrate that ATL313 reduces lung IR injury in mice via the specific activation of A2AAR (15). The identity of the cell type(s) responsible for mediating the protective effects of A2AAR activation against lung IR injury remains unknown and thus was a focus of the current study. A2AAR activation mediates immunosuppression by inhibiting the activation of T lymphocytes and neutrophils, predominantly by a cAMP-dependent pathway. The immunosuppressive effects of A2AAR activation have been confirmed by studies showing that genetic inactivation of A2AARs increases the intensity and prolongs the duration of T lymphocyte–dependent cytokine accumulation and tissue damage (19, 20). Previous studies in lung injury and other IR models have demonstrated that A2AARs on bone marrow-derived cells are required for A2AAR-mediated protection from reperfusion injury (17, 21-23). In lung IR injury, it is postulated that A2AAR activation on CD4+ T lymphocytes and neutrophils attenuates downstream intercellular signaling cascade events involving other cell type such as alveolar macrophages, epithelial and endothelial cells.

A2AAR activation was found to inhibit the re-distribution of circulating lymphocytes after IR as characterized by elevated circulating lymphocytes and reduced lung T cell infiltration (Figures 1 and 3). ATL313 was administered shortly before the onset of reperfusion, and its mild hemodynamic effect should not have induced any preconditioning effects against lung IR injury. Activation of A2AAR resulted in reduced neutrophil infiltration as demonstrated by reduced MPO levels (Figure 4). The reduced inflammatory response as a result of A2AAR activation was also documented by significantly reduced levels of CD4+ T cell-related cytokines/chemokines (Figure 5). Activated CD4+ T cells are known to amplify the inflammatory response by producing IL-17, RANTES, IFN-γ, GM-CSF as well as MCP-1, MIP-1 and TNF-α. The results showed that expression of IL-17, RANTES, KC, MCP-1, MIP-1 and TNF-α were significantly increased after IR. ATL313 significantly abrogated the induction of all measured cytokines and chemokines. These findings suggest that ATL313 may act on CD4+ T cells to inhibit proinflammatory responses after IR.

Our results suggest that both neutrophils and CD4+ T cells are involved in the same cellular signaling pathway that leads to lung IR injury. Neutrophils are end-effectors which cause lung injury, and injury correlated with BAL MPO levels (neutrophil infiltration) in our study. CD4+ T cell depletion also significantly attenuated MPO levels, thereby implicating a role of the CD4+ T cell/neutrophil axis in lung IR injury. Both T cells and neutrophils express A2AARs, and activation of A2AARs on both of these cell types has been shown to inhibit inflammatory responses (13, 19). It is plausible that lack of further protection by ATL313 in neutrophil-depleted mice is simply due to the deficiency of neutrophils; however, somewhat better protection by ATL313 occurred in these mice in terms of reduced pulmonary artery pressure and reduced expression of cytokines. However, in CD4+ T cell-depleted mice, there was no evidence that ATL313 provided any additional protection, which may be because CD4+ T cell depletion alone is enough to prevent neutrophil activation and infiltration after IR. The attenuation of neutrophil infiltration by CD4+ T cell depletion not only signifies the role of the CD4+ T cell/neutrophil axis as the major signaling pathway but also implicates CD4+ T cells as a primary target for A2AAR-dependent attenuation of lung IR injury. Furthermore, the spectrum of changes in cytokines/chemokines is comparable between ATL313-treated mice and CD4+ T cell-depleted mice, but not comparable to neutrophil-depleted mice (Figure 5, B). This suggests that activation of A2AARs on CD4+ T cells is sufficient to inhibit IR-induced inflammatory responses and reduce lung IR injury.

An interesting observation in this study was that IR-induced IL-17 production was attenuated by ATL313. IL-17, produced largely by CD4+ NKT and Th17 cells, is a key cytokine for the recruitment, activation and migration of neutrophils (24). Thus, reduced levels of KC (a potent neutrophil chemokine) and MPO (i.e. neutrophil infiltration) in the ATL313-treated groups are likely secondary to the inactivation of CD4+ T cells. There was also a significant reduction of IL-17 in neutrophil-depleted mice which was not as robust as in CD4+ T cell-depleted mice (Figure 5, B). This supports prior studies suggesting that neutrophils may also be a source of IL-17 (25). However, in the setting of lung IR, the release of IL-17 by neutrophils appears to be regulated by CD4+ T cells via abrogation of neutrophil chemotactic factors such as KC, IL-17 and MIP-2. These results further indicate that the anti-inflammatory effect of ATL313 may largely be due to its action on A2AARs on CD4+ T cells.

In summary, the current study aimed to define the cellular sources whereby A2AAR activation exerts protection from lung IR injury. Although we found that CD4+ T cells mediate neutrophil activation during reperfusion, and the protective effect of ATL313 is largely due to its action on CD4+ T cells, a role for ATL313 in directly inhibiting neutrophil activation or infiltration could not be excluded. Activation of A2AARs by ATL313 protects the lung against IR injury. This protection is comparable to that found in CD4+ T cell-depleted mice. However, no additive effects occur when ATL313 is applied to CD4+ T cell-depleted mice. Collectively, these results suggest that CD4+ T cells mediate acute lung IR injury and that the protective effect of A2AAR activation may be largely due to its actions on CD4+ T lymphocytes.

Acknowledgments

Sources of funding: This study was funded by NIH/NHLBI grants RO1 HL077301 (VEL) and PO1 HL073361 (JL).

Glossary

Abbreviations

BAL

bronchoalveolar lavage

IgG

immunoglobulin

IR

ischemia-reperfusion

MPO

myeloperoxidase

A2AAR

adenosine A2A receptor

LC

lung compliance

PAP

pulmonary artery pressure

AR

airway resistance

PBS

phosphate-buffered saline

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of Interest: Drs. Linden and Kron were shareholders in Adenosine Therapeutics, LLC, the corporation that provided ATL313, during the time of this study.

REFERENCES

  • 1.Trulock EP, Edwards LB, Taylor DO, Boucek MM, Keck BM, Hertz MI. Registry of the International Society for Heart and Lung Transplantation: twenty-third official adult lung and heart-lung transplantation report--2006. J Heart Lung Transplant. 2006;25:880–92. doi: 10.1016/j.healun.2006.06.001. [DOI] [PubMed] [Google Scholar]
  • 2.Fiser SM, Tribble CG, Long SM, Kaza AK, Cope JT, Laubach VE, et al. Lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response. J Thorac Cardiovasc Surg. 2001;121:1069–75. doi: 10.1067/mtc.2001.113603. [DOI] [PubMed] [Google Scholar]
  • 3.Gazoni LM, Tribble CG, Zhao MQ, Unger EB, Farrar RA, Ellman PI, et al. Pulmonary macrophage inhibition and inhaled nitric oxide attenuate lung ischemia-reperfusion injury. Ann Thorac Surg. 2007;84:247–53. doi: 10.1016/j.athoracsur.2007.02.036. [DOI] [PubMed] [Google Scholar]
  • 4.Zhao M, Fernandez LG, Doctor A, Sharma AK, Zarbock A, Tribble CG, et al. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2006;291:L1018–26. doi: 10.1152/ajplung.00086.2006. [DOI] [PubMed] [Google Scholar]
  • 5.Sharma AK, Fernandez LG, Awad AS, Kron IL, Laubach VE. Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2007;293:L105–13. doi: 10.1152/ajplung.00470.2006. [DOI] [PubMed] [Google Scholar]
  • 6.Geudens N, Vanaudenaerde BM, Neyrinck AP, Van De Wauwer C, Vos R, Verleden GM, et al. The importance of lymphocytes in lung ischemia-reperfusion injury. Transplant Proc. 2007;39:2659–62. doi: 10.1016/j.transproceed.2007.08.001. [DOI] [PubMed] [Google Scholar]
  • 7.de Perrot M, Young K, Imai Y, Liu M, Waddell TK, Fischer S, et al. Recipient T cells mediate reperfusion injury after lung transplantation in the rat. J Immunol. 2003;171:4995–5002. doi: 10.4049/jimmunol.171.10.4995. [DOI] [PubMed] [Google Scholar]
  • 8.Gazoni LM, Laubach VE, Mulloy DP, Bellizzi A, Unger EB, Linden J, et al. Additive protection against lung ischemia-reperfusion injury by adenosine A2A receptor activation before procurement and during reperfusion. J Thorac Cardiovasc Surg. 2008;135:156–65. doi: 10.1016/j.jtcvs.2007.08.041. [DOI] [PubMed] [Google Scholar]
  • 9.Harada N, Okajima K, Murakami K, Usune S, Sato C, Ohshima K, et al. Adenosine and selective A(2A) receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation. J Pharmacol Exp Ther. 2000;294:1034–42. [PubMed] [Google Scholar]
  • 10.Okusa MD, Linden J, Macdonald T, Huang L. Selective A2A adenosine receptor activation reduces ischemia-reperfusion injury in rat kidney. Am J Physiol. 1999;277:F404–12. doi: 10.1152/ajprenal.1999.277.3.F404. [DOI] [PubMed] [Google Scholar]
  • 11.Yang Z, Day YJ, Toufektsian MC, Xu Y, Ramos SI, Marshall MA, et al. Myocardial infarct-sparing effect of adenosine A2A receptor activation is due to its action on CD4+ T lymphocytes. Circulation. 2006;114:2056–64. doi: 10.1161/CIRCULATIONAHA.106.649244. [DOI] [PubMed] [Google Scholar]
  • 12.Dobson JG, Jr., Fenton RA. Adenosine A2 receptor function in rat ventricular myocytes. Cardiovasc Res. 1997;34:337–47. doi: 10.1016/s0008-6363(97)00023-0. [DOI] [PubMed] [Google Scholar]
  • 13.Sullivan GW, Rieger JM, Scheld WM, Macdonald TL, Linden J. Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists. Br J Pharmacol. 2001;132:1017–26. doi: 10.1038/sj.bjp.0703893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Shryock JC, Snowdy S, Baraldi PG, Cacciari B, Spalluto G, Monopoli A, et al. A2A-adenosine receptor reserve for coronary vasodilation. Circulation. 1998;98:711–8. doi: 10.1161/01.cir.98.7.711. [DOI] [PubMed] [Google Scholar]
  • 15.Sharma AK, Yang Z, Linden J, Kron IL, Laubach VE. Attenuation of ischemia-reperfusion injury by adenosine A2A receptor activation in a buffer-perfused mouse lung model. Am J Resp Crit Care Med. 2008;177:A80. [Google Scholar]
  • 16.Yang Z, Sharma AK, Linden J, Kron IL, Laubach VE. CD4+ T lymphocytes mediate acute pulmonary ischemia-reperfusion injury. J Thorac Cardiovasc Surg. 2009;137:695–702. doi: 10.1016/j.jtcvs.2008.10.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Reutershan J, Cagnina RE, Chang D, Linden J, Ley K. Therapeutic anti-inflammatory effects of myeloid cell adenosine receptor A2a stimulation in lipopolysaccharide-induced lung injury. J Immunol. 2007;179:1254–63. doi: 10.4049/jimmunol.179.2.1254. [DOI] [PubMed] [Google Scholar]
  • 18.Lappas CM, Rieger JM, Linden J. A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells. J Immunol. 2005;174:1073–80. doi: 10.4049/jimmunol.174.2.1073. [DOI] [PubMed] [Google Scholar]
  • 19.Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 2001;414:916–20. doi: 10.1038/414916a. [DOI] [PubMed] [Google Scholar]
  • 20.Koshiba M, Kojima H, Huang S, Apasov S, Sitkovsky MV. Memory of extracellular adenosine A2A purinergic receptor-mediated signaling in murine T cells. J Biol Chem. 1997;272:25881–9. doi: 10.1074/jbc.272.41.25881. [DOI] [PubMed] [Google Scholar]
  • 21.Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov. 2008;7:759–70. doi: 10.1038/nrd2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Day YJ, Li Y, Rieger JM, Ramos SI, Okusa MD, Linden J. A2A adenosine receptors on bone marrow-derived cells protect liver from ischemia-reperfusion injury. J Immunol. 2005;174:5040–6. doi: 10.4049/jimmunol.174.8.5040. [DOI] [PubMed] [Google Scholar]
  • 23.Day YJ, Huang L, McDuffie MJ, Rosin DL, Ye H, Chen JF, et al. Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells. J Clin Invest. 2003;112:883–91. doi: 10.1172/JCI15483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity. 2004;21:467–76. doi: 10.1016/j.immuni.2004.08.018. [DOI] [PubMed] [Google Scholar]
  • 25.Ferretti S, Bonneau O, Dubois GR, Jones CE, Trifilieff A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J Immunol. 2003;170:2106–12. doi: 10.4049/jimmunol.170.4.2106. [DOI] [PubMed] [Google Scholar]

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