Heme oxygenase-1 attenuates acute pulmonary inflammation by decreasing the release of segmented neutrophils from the bone marrow.

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.

Protective effects of pentoxifylline in pulmonary inflammation are adenosine receptor A2A dependent.

Pentoxifylline (PTX) has been shown to exert anti-inflammatory effects in experimental acute lung injury. However, results in humans were controversial. Recent in vitro studies suggested that the adenosine receptor A2A may be required for PTX to be effective. Therefore, we studied the association between A2A and PTX in a murine model of LPS-induced pulmonary inflammation. PTX treatment (10 mg/kg) reduced cellular influx (by 40%), microvascular permeability (30%), and the release of chemotactic cytokines into the alveolar space (TNF-α 60%, IL-6 60%, and CXCL2/3 53%, respectively). These protective effects were abolished completely in A2A(-/-) mice and in wild-type mice that had been treated with the selective A2A antagonist (1 mg/kg), but effects were not different in mice with altered adenosine levels. In vitro transmigration assays revealed a pivotal role of the endothelium in PTX-mediated PMN migration, with a reduction of 50% (2 mM PTX). This effect was also A2A dependent. Further, oxidative burst of human PMNs was A2A-dependently reduced by 53% after PTX treatment. In summary, PTX exhibits its anti-inflammatory effects in LPS-induced lung injury through an A2A-dependent pathway. These results will help to better understand previous conflicting data on PTX in inflammation and will direct further studies to consider the predominant role of A2A.

Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium.

Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.

Adenosine receptor A1 regulates polymorphonuclear cell trafficking and microvascular permeability in lipopolysaccharide-induced lung injury.

Extracellular adenosine and adenosine receptors are critically involved in various inflammatory pathways. Adenosine receptor A1 (A1AR) has been implicated in mediating transmigration of leukocytes to sites of inflammation. This study was designed to characterize the role of A1AR in a murine model of LPS-induced lung injury. LPS-induced transmigration of polymorphonuclear cells (PMNs) and microvascular permeability was elevated in A1AR(-/-) mice. Pretreatment of wild-type mice with the specific A1AR agonist 2'Me-2-chloro-N6-cyclopentyladenosine attenuated PMN accumulation in the interstitium and alveolar space as well as microvascular permeability. Lower PMN counts in the lungs of pretreated wild-type mice were associated with reduced amounts of the chemotactic cytokines TNF-α, IL-6, and CXCL2/3 in the bronchoalveolar lavage. Pretreatment was only effective when A1AR was expressed on hematopoietic cells as demonstrated in chimeric mice. These findings were confirmed by in vitro transmigration assays demonstrating that chemokine-induced transmigration of PMNs was reduced when PMNs but not when pulmonary endothelial or alveolar epithelial cells were pretreated. 2'Me-2-chloro-N6-cyclopentyladenosine prevented pulmonary endothelial but not epithelial cells from LPS-induced cellular remodeling and cell retraction. Our data reveal what we believe to be a previously unrecognized distinct role of A1AR for PMN trafficking and endothelial integrity in a model of acute lung injury.

Adenosine receptor A3 is a critical mediator in LPS-induced pulmonary inflammation.

Adenosine receptor A(3) (A(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(3) in a murine model of lung inflammation. Initial studies revealed that pulmonary A(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(3)(-/-) mice in all lung compartments. Pretreatment with the specific A(3)-agonist Cl-IB-MECA significantly decreased migration of PMNs into lung interstitium and alveolar air space of wild-type mice but not of A(3)(-/-) 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 A(3) for maintaining endothelial integrity. Migratory activity of human PMNs across an endothelial or epithelial monolayer was reduced when A(3) was activated on PMNs. Studies in chimeric mice, however, revealed that Cl-IB-MECA required A(3) on both hematopoietic and nonhematopoietic cells to reduce transmigration in vivo. Together, our results shed new light on the role of A(3) in LPS-induced PMN trafficking in the lung and suggest pharmacological modulation of A(3)-dependent pathways as a promising approach in lung inflammation.

Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs.

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 ecto-apyrase (CD39) and the ecto-5'-nucleotidase (CD73). In the present study, we hypothesized a critical role of 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-nucleoside-triphosphate-diphosphohydrolases inhibitor POM-1 confirmed increased pulmonary PMN accumulation in 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.