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Adenosine receptor [A.sub.3] ([A.sub.3]) regulates directed movement of polymorphonuclear cells (PMNs) to sites of inflammation and has been implicated as a relevant mediator in models of inflammatory diseases. Here, we sought to characterize the role of [A.sub.3] in a murine model of lung inflammation. Initial studies revealed that pulmonary [A.sub.3] transcript levels were elevated following LPS exposure in vivo. In addition, inhalation of LPS increased the accumulation of PMNs in wild-type and [A.sub.3.sup.-/-] mice in all lung compartments. Pretreatment with the specific [A.sub.3]-agonist CI-IB-MECA significantly decreased migration of PMNs into lung interstitium and alveolar air space of wild-type mice but not of [A.sub.3.sup.-/-] mice. Lower PMN counts were associated with reduced levels of TNF-[alpha] and IL-6 in the alveolar space of wild-type mice that received Cl-IB-MECA. In addition, Cl-IB-MECA attenuated LPS-induced microvascular permeability in wild-type mice as assessed by the extravasation of Evans blue. In pulmonary microvascular endothelial cells, Cl-IB-MECA reduced LPS-induced cytoskeletal remodeling and cell retraction, consistent with a specific role of [A.sub.3] for maintaining endothelial integrity. Migratory activity of human PMNs across an endothelial or epithelial monolayer was reduced when [A.sub.3] was activated on PMNs. Studies in chimeric mice, however, revealed that Cl-IB-MECA required [A.sub.3] on both hematopoietic and nonhematopoietic cells to reduce transmigration in vivo. Together, our results shed new light on the role of [A.sub.3] in LPS-induced PMN trafficking in the lung and suggest pharmacological modulation of [A.sub.3]-dependent pathways as a promising approach in lung inflammation. acute lung injury; polymorphonuclear leukocytes; pulmonary circulation; adenosine receptors doi: 10.1152/ajplung.00083.2010.

Acute pulmonary inflammation is still a frightening complication in intensive care units. In our previous study, we determined that heme oxygenase (HO)-1 had anti-inflammatory effects in pulmonary inflammation. Recent literature has emphasized a link between HO-1 and the nucleotide adenosine. Since adenosine A2A- and A2B-receptors play a pivotal role in pulmonary inflammation, we investigated their link to the enzyme HO-1. In a murine model of pulmonary inflammation, the activation of HO-1 by hemin significantly decreased polymorphonuclear leukocyte (PMN) migration into the lung. This anti-inflammatory reduction of PMN migration was abolished in A2A- and A2B-knockout mice. Administration of hemin significantly reduced chemokine levels in the BAL of wild-type animals but had no effects in A2A-/- and A2B-/- mice. Microvascular permeability was significantly attenuated in HO-1-stimulated wild-type mice, but not in A2A-/- and A2B-/- mice. The activity of HO-1 rose after LPS inhalation in wild-type animals and, surprisingly, also in A2A-/- and A2B-/- mice after the additional administration of hemin. Immunofluorescence images of animals revealed alveolar macrophages to be the major source of HO-1 activity in both knockout strains--in contrast to wild-type animals, where HO-1 was also significantly augmented in the lung tissue. In vitro studies on PMN migration further confirmed our in vivo findings. In conclusion, we linked the anti-inflammatory effects of HO-1 to functional A2A/A2B-receptor signaling under conditions of pulmonary inflammation. Our findings may explain why targeting HO-1 in acute pulmonary inflammation has failed to prove effective in some patients, since septic patients have altered adenosine receptor expression. [ABSTRACT FROM AUTHOR]

Adenosine receptor A3 (A3) regulates directed movement of polymorphonuclear cells (PMNs) to sites of inflammation and has been implicated as a relevant mediator in models of inflammatory diseases. Here, we sought to characterize the role of A3 in a murine model of lung inflammation. Initial studies revealed that pulmonary A3 transcript levels were elevated following LPS exposure in vivo. In addition, inhalation of LPS increased the accumulation of PMNs in wild-type and A3-/- mice in all lung compartments. Pretreatment with the specific A3-agonist Cl-IB-MECA significantly decreased migration of PMNs into lung interstitium and alveolar air space of wild-type mice but not of A3-/- mice. Lower PMN counts were associated with reduced levels of TNF-α and IL-6 in the alveolar space of wild-type mice that received Cl-IB-MECA. In addition, Cl-IB-MECA attenuated LPS-induced microvascular permeability in wild-type mice as assessed by the extravasation of Evans blue. In pulmonary microvascular endothelial cells, Cl-IB-MECA reduced LPS-induced cytoskeletal remodeling and cell retraction, consistent with a specific role of A3 for maintaining endothelial integrity. Migratory activity of human PMNs across an endothelial or epithelial monolayer was reduced when A3 was activated on PMNs. Studies in chimeric mice, however, revealed that Cl-IB-MECA required A3 on both hematopoietic and nonhematopoietic cells to reduce transmigration in vivo. Together, our results shed new light on the role of A3 in LPS-induced PMN trafficking in the lung and suggest pharmacological modulation of A3-dependent pathways as a promising approach in lung inflammation. [ABSTRACT FROM AUTHOR]

ABSTRACT Extracellular adenosine has been implicated as anti-inflammatory signaling molecule during acute lung injury (ALI). The main source of extracellular adenosine stems from a coordinated two-step enzymatic conversion of precursor nucleotides via the ectoapyrase (CD39) and the ecto-5'-nucleotidase (CD73). the present study, we hypothesized a critical role CD39 and CD73 in mediating pulmonary neutrophil (PMN) transmigration during lipopolysaccharide (LPS) -induced lung injury. Initial studies revealed that pulmonary CD39 and CD73 transcript levels were elevated following LPS exposure in vivo. Moreover, LPS-induced accumulation of PMN into the lungs was enhanced in cd39-/- or cd73-/- mice, particularly into the interstitial and intra-alveolar compartment. Such increases in PMN trafficking were accompanied by corresponding changes in alveolar-capillary leakage. Similarly, inhibition of extracellular nucleotide phosphohydrolysis with the nonspecific ecto-nucleosidetriphosphate- diphosphohydrolases inhibitor POM-1 confirmed increased pulmonary PMN accumulation wild-type, but not in gene-targeted mice for cd39 or cd73. Finally, treatment with apyrase or nucleotidase was associated with attenuated pulmonary neutrophil accumulation and pulmonary edema during LPS-induced lung injury. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in attenuating PMN trafficking into the lungs during LPS-induced lung injury and suggest treatment with their soluble compounds as a therapeutic strategy. [ABSTRACT FROM AUTHOR]

Acute pulmonary inflammation is still a frightening complication in intensive care units. In our previous study, we determined that heme oxygenase (HO)-1 had anti-inflammatory effects in pulmonary inflammation. Recent literature has emphasized a link between HO-1 and the nucleotide adenosine. Since adenosine A 2A - and A 2B -receptors play a pivotal role in pulmonary inflammation, we investigated their link to the enzyme HO-1. In a murine model of pulmonary inflammation, the activation of HO-1 by hemin significantly decreased polymorphonuclear leukocyte (PMN) migration into the lung. This anti-inflammatory reduction of PMN migration was abolished in A 2A - and A 2B -knockout mice. Administration of hemin significantly reduced chemokine levels in the BAL of wild-type animals but had no effects in A 2A -/- and A 2B -/- mice. Microvascular permeability was significantly attenuated in HO-1-stimulated wild-type mice, but not in A 2A -/- and A 2B -/- mice. The activity of HO-1 rose after LPS inhalation in wild-type animals and, surprisingly, also in A 2A -/- and A 2B -/- mice after the additional administration of hemin. Immunofluorescence images of animals revealed alveolar macrophages to be the major source of HO-1 activity in both knockout strains-in contrast to wild-type animals, where HO-1 was also significantly augmented in the lung tissue. In vitro studies on PMN migration further confirmed our in vivo findings. In conclusion, we linked the anti-inflammatory effects of HO-1 to functional A 2A /A 2B -receptor signaling under conditions of pulmonary inflammation. Our findings may explain why targeting HO-1 in acute pulmonary inflammation has failed to prove effective in some patients, since septic patients have altered adenosine receptor expression.

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils into the different compartments of the lung. Recent studies showed evidence that the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 influence migration of immune cells and their activity was linked to adenosine concentrations. We investigated the particular role of CXCR4- and CXCR7-inhibition and the potential link to the adenosine A 2B -receptor, which plays an important anti-inflammatory role in the lung. After LPS-inhalation for 45 minutes, administration of the CXCR4-inhibitor (AMD3100) decreased transendothelial and transepithelial migration, whereas CXCR7-antagonism influenced epithelial migration exclusively. In A 2B -/- mice, no anti-inflammatory effects were detectible through either one of the agents. Using chimeric mice, we identified A 2B on hematopoietic cells to be crucial for these anti-inflammatory effects of CXCR4/7-inhibition. Both inhibitors decreased TNFα, IL6, CXCL1 and CXCL2/3 levels in the bronchoalveolar lavage of wild type mice, while not influencing the chemokine release in A 2B -/- mice. Inflammation augmented the expression of both receptors and their inhibition increased A 2B -levels upon inflammation. In vitro assays with human epithelium/endothelium confirmed our in vivo findings. During inflammation, inhibition of CXCR4- and CXCR7-receptors prevented microvascular permeability in wild type but not in A 2B -/- mice, highlighting the pivotal role of an active A 2B -receptor in this setting. The combination of both inhibitors had a synergistic effect in preventing capillary leakage. In conclusion, we determined the pivotal role of CXCR4- and CXCR7-inhibition in acute pulmonary inflammation, which depended on A 2B -receptor signalling.

Recruiting polymorphonuclear neutrophil granulocytes (PMNs) from circulation and bone marrow to the site of inflammation is one of the pivotal mechanisms of the innate immune system. During inflammation, the enzyme heme oxygenase 1 (HO-1) has been shown to reduce PMN migration. Although these effects have been described in various models, underlying mechanisms remain elusive. Recent studies revealed an influence of HO-1 on different cells of the bone marrow. We investigated the particular role of the bone marrow in terms of HO-1-dependent pulmonary inflammation. In a murine model of LPS inhalation, stimulation of HO-1 by cobalt (III) protoporphyrin-IX-chloride (CoPP) resulted in reduced segmented PMN migration into the alveolar space. In the CoPP group, segmented PMNs were also decreased intravascularly, and concordantly, mature and immature PMN populations were higher in the bone marrow. Inhibition of the enzyme by tin protoporphyrin-IX increased segmented and banded PMN migration into the bronchoalveolar lavage fluid with enhanced PMN release from the bone marrow and aggravated parameters of tissue inflammation. Oxidative burst activity was significantly higher in immature compared with mature PMNs. The chemokine stromal-derived factor-1 (SDF-1), which mediates homing of leukocytes into the bone marrow and is decreased in inflammation, was increased by CoPP. When SDF-1 was blocked by the specific antagonist AMD3100, HO-1 activation was no longer effective in curbing PMN trafficking to the inflamed lungs. In conclusion, we show evidence that the anti-inflammatory effects of HO-1 are largely mediated by inhibiting the release of segmented PMNs from the bone marrow rather than direct effects within the lung., (Copyright © 2014 the American Physiological Society.)