To determine the role of the adenosine receptor A2a in a murine model of LPS-induced lung injury, migration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lung was determined by flow cytometry, microvascular permeability was assessed by the extravasation of Evans blue, and the release of chemotactic cytokines into the alveolar airspace was determined by ELISA. Measurements were performed in wild-type and A2a gene-deficient mice (A2a−/−). To differentiate the role of A2a on hemopoietic and nonhemopoietic cells, we created chimeric mice by transfer of bone marrow (BM) between wild-type and A2a−/− mice and used mice that lacked A2a expression selectively on myeloid cells (A2aflox/flox × LysM-cre). A specific A2a receptor agonist (ATL202) was used to evaluate its potential to reduce lung injury in vivo. In wild-type mice, therapeutic treatment with ATL202 reduced LPS-induced PMN recruitment, and release of cytokines. Pretreatment, but not posttreatment, also reduced Evans blue extravasation. In the BM chimeric mice lacking A2a on BM-derived cells, PMN migration into the alveolar space was increased by ∼50%. These findings were confirmed in A2aflox/flox × LysM-cre mice. ATL202 was only effective when A2a was present on BM-derived cells. A2a agonists may be effective at curbing inflammatory lung tissue damage.

We developed a flow cytometry-based assay to simultaneously quantify multiple leukocyte populations in the marginated vascular, interstitial, and alveolar compartments of the mouse lung. An intravenous injection of a fluorescently labeled anti-CD45 antibody was used to label circulating and marginated vascular leukocytes. Following vascular flushing to remove non-adherent cells and collection of broncho-alveolar lavage (BAL) fluid, lungs were digested and a second fluorescent anti-CD45 antibody was added ex vivo to identify cells not located in the vascular space. In the naïve mouse lung, we found about 11 million CD45+ leukocytes, of which 87% (9.5 million) were in the vascular marginated compartment, consisting of 17% NK cells, 17% neutrophils, 57% mononuclear myeloid cells (monocytes, macrophage precursors and dendritic cells), and 10% T cells (CD4+, CD8+, and invariant NKT cells). Non-vascular compartments including the interstitial compartment contained 7.7 × 105 cells, consisting of 49% NK cells, 25% dendritic cells, and 16% other mononuclear myeloid cells. The alveolar compartment was overwhelmingly populated by macrophages (5.63 × 105 cells, or 93%). We next studied leukocyte margination and extravasation into the lung following acid injury, a model of gastric aspiration. At 1 hour after injury, neutrophils were markedly elevated in the blood while all other circulating leukocytes declined by an average of 79 percent. At 4 hours after injury, there was a peak in the numbers of marginated neutrophils, NK cells, CD4+ and CD8+ T cells and a peak in the number of alveolar NK cells. Most interstitial cells consisted of DCs, neutrophils, and CD4+ T cells, and most alveolar compartment cells consisted of macrophages, neutrophils, and NK cells. At 24 hours after injury, there was a decline in the number of all marginated and interstitial leukocytes and a peak in alveolar neutrophils. In sum, we have developed a novel assay to study leukocyte margination and trafficking following pulmonary inflammation and show that marginated cells comprise a large fraction of lung leukocytes that increases shortly after lung injury. This assay may be of interest in future studies to determine if leukocytes become activated upon adherence to the endothelium, and have properties that distinguish them from interstitial and circulating cells.

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Rationale: Activation of the adenosine A 2B receptor (A 2B R) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A 2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A 2B R. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A 2B R -/-mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow-derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A 2B R -/-and wild-type mice, but A 2B R -/-neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A 2B R -/-mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A 2B R signaling enhances antimicrobial activity in gram-negative bacterial pneumonia.Keywords: neutrophil; extracellular traps; pneumonia; adenosine Pneumonia is a leading cause of hospitalization in the United States and is the most common infectious cause of death (1, 2). Aerobic gram-negative bacilli are the most common cause of health care-associated pneumonia; mortality rates from gram-negative pneumonia range from 30 to 60% with antimicrobial therapy (3, 4). The emergence of multiresistant strains of gram-negative bacteria, combined with limited development of new antimicrobial therapies, has exacerbated the need for new approaches to combating these pathogens (5-7). Therapy aimed at augmenting the host response has the potential to enhance bacterial clearance and improve outcomes, and could provide a new avenue for therapeutic advances in combating gramnegative pneumonia, including infections that are caused by antibiotic-resistant pathogens.Adenosine, a breakdown product of ATP, is a potent signaling molecule released from a variety of cells to dampen inflammation, limit tissue destruction, and promote repair (8, 9). In response to cellular stress or tissue injury, the extracellular concentration of adenosine can increase 100-fold. Adenosine acts on four widely expressed G protein-coupled receptors: A 1 , A 2A , A 2B , and A 3 , with variable expression among different cells. The adenosine A 2B receptor (A 2B R) has been shown to en...

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