Repulsive guidance molecule-A (RGM-A) inhibits leukocyte migration and mitigates inflammation.

Directed cell migration is a prerequisite not only for the development of the central nervous system, but also for topically restricted, appropriate immune responses. This is crucial for host defense and immune surveillance. Attracting environmental cues guiding leukocyte cell traffic are likely to be complemented by repulsive cues, which actively abolish cell migration. One such a paradigm exists in the developing nervous system, where neuronal migration and axonal path finding is balanced by chemoattractive and chemorepulsive cues, such as the neuronal repulsive guidance molecule-A (RGM-A). As expressed at the inflammatory site, the role of RGM-A within the immune response remains unclear. Here we report that RGM-A (i) is expressed by epithelium and leukocytes (granulocytes, monocytes, and T/B lymphocytes); (ii) inhibits leukocyte migration by contact repulsion and chemorepulsion, depending on dosage, through its receptor neogenin; and (iii) suppresses the inflammatory response in a model of zymosan-A-induced peritonitis. Systemic application of RGM-A attenuates the humoral proinflammatory response (TNF-α, IL-6, and macrophage inflammatory protein 1α), infiltration of inflammatory cell traffic, and edema formation. In contrast, the demonstrated anti-inflammatory effect of RGM-A is absent in mice homozygous for a gene trap mutation in the neo1 locus (encoding neogenin). Thus, our results suggest that RGM-A is a unique endogenous inhibitor of leukocyte chemotaxis that limits inflammatory leukocyte traffic and creates opportunities to better understand and treat pathologies caused by exacerbated or misdirected inflammatory responses.

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 beta-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.

Netrin-1 dampens pulmonary inflammation during acute lung injury.

RATIONALE: Acute lung injury (ALI) is an inflammatory disorder characterized by hypoxemia and diffuse infiltration of neutrophils into the alveolar space. The migration and extravasation of neutrophils is guided through positive guidance cues, such as chemokines. Recent work has identified the neuronal guidance protein netrin-1 to be a negative guidance cue for leukocyte migration and to hold antiinflammatory potential. OBJECTIVES: To test the role of pulmonary netrin-1 during ALI. METHODS: Pulmonary netrin-1 expression was evaluated during acute inflammation in vitro and in vivo; the netrin-1 promoter was studied using pGL4 luciferase reporter. ALI was induced through LPS inhalation and mechanical ventilation in wild-type, Ntn1(+/-), and A2BAR(-/-) animals. Exogenous netrin-1 was used to evaluate its impact on pulmonary inflammation. MEASUREMENTS AND MAIN RESULTS: Wild-type animals demonstrated repression of pulmonary netrin-1 after LPS inhalation. In vitro studies confirmed the repression of netrin-1. Studies in the putative netrin-1 promoter identified a nuclear factor-kappaB-dependent mechanism to be involved in this repression. Ntn1(+/-) animals demonstrated increased inflammatory changes after LPS inhalation compared with Ntn1(+/+) animals. Reconstitution with netrin-1 dampened the infiltration of neutrophils and cytokine production in the alveolar space. This effect was dependent on the adenosine 2b receptor. The importance of netrin-1 for the control of pulmonary inflammation could be corroborated in a model of ventilator-induced lung injury. CONCLUSIONS: Pulmonary netrin-1 levels are repressed during ALI. This results in pronounced pulmonary damage, an increased infiltration of neutrophils, and increased pulmonary inflammation. Exogenous netrin-1 significantly dampens the extent of ALI through the adenosine 2B receptor.

CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury.

BACKGROUND: Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. METHODS AND RESULTS: As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor (Na6[H2W12O40]). Polyoxometalate 1 treatment increased infarct sizes and abolished beneficial effects of preconditioning. To define relative contributions of distinct E-NTPDases, we investigated transcriptional responses of E-NTPDases 1 to 3 and 8 to preconditioning. We noted robust and selective induction of E-NTPDase 1 (CD39) transcript and protein. Histological analysis of preconditioned myocardium localized CD39 induction to endothelia and myocytes. Cd39-/- mice exhibited larger infarct sizes with ischemia (cd39+/+ 43.0+/-3.3% versus cd39-/- 52%+/-1.8; P<0.05), and cardioprotection was abrogated by preconditioning (cd39+/+ 13.3%+/-1.5 versus cd39-/- 50.5%+/-2.8; P<0.01). Heightened levels of injury after myocardial ischemia and negligible preconditioning benefits in cd39-/- mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43+/-4.2% infarct size reduction (P<0.01). CONCLUSIONS: Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

A2B adenosine receptor dampens hypoxia-induced vascular leak.

Extracellular adenosine has been implicated in adaptation to hypoxia and previous studies demonstrated a central role in vascular responses. Here, we examined the contribution of individual adenosine receptors (ARs: A1AR/A2AAR/A2BAR/A3AR) to vascular leak induced by hypoxia. Initial profiling studies revealed that siRNA-mediated repression of the A2BAR selectively increased endothelial leak in response to hypoxia in vitro. In parallel, vascular permeability was significantly increased in vascular organs of A2BAR(-/-)-mice subjected to ambient hypoxia (8% oxygen, 4 hours; eg, lung: 2.1 +/- 0.12-fold increase). By contrast, hypoxia-induced vascular leak was not accentuated in A1AR(-/-)-, A2AAR(-/-)-, or A3AR(-/-)-deficient mice, suggesting a degree of specificity for the A2BAR. Further studies in wild type mice revealed that the selective A2BAR antagonist PSB1115 resulted in profound increases in hypoxia-associated vascular leakage while A2BAR agonist (BAY60-6583 [2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)-. phenyl]pyridin-2-ylsulfanyl]acetamide]) treatment was associated with almost complete reversal of hypoxia-induced vascular leakage (eg, lung: 2.0 +/- 0.21-fold reduction). Studies in bone marrow chimeric A2BAR mice suggested a predominant role of vascular A2BARs in this response, while hypoxia-associated increases in tissue neutrophils were, at least in part, mediated by A2BAR expressing hematopoietic cells. Taken together, these studies provide pharmacologic and genetic evidence for vascular A2BAR signaling as central control point of hypoxia-associated vascular leak.