Evidence for the Up-Regulation of Immunosuppressive A_2AR Expression on In Vivo-Activated Granulocytes Isolated from Inflamed Lungs

Abstract

<div><p>(A) The selective A_2AR agonist CGS21680 inhibited the fMLP-stimulated hydrogen peroxide production by granulocytes in blood of healthy mice to only a small degree, reflecting low levels of expression of A_2AR on naïve blood granulocytes. In contrast, granulocytes recovered by BAL from inflamed lungs 48 h after IT LPS injection were much more inhibited by CGS21680, demonstrating functional up-regulation of A_2AR on in vivo-activated cells.</p> <p>(B) CGS21680 induces cAMP accumulation in in vivo-activated granulocytes isolated from lungs 48 h after IT LPS injection. No effects of the A_2AR agonist were observed in naïve granulocytes obtained from bone marrow of healthy mice (left graph) or in in vivo-activated granulocytes recovered from inflamed lungs of <i>A_2AR</i> gene-deficient mice (right graph). The CGS21680-stimulated cAMP production observed in lung granulocytes obtained from wild-type mice could also be antagonized by the selective A_2AR antagonist ZM241385. Naïve bone marrow granulocytes were used for cAMP measurements, since it was impossible to isolate naïve cells from blood of healthy mice in sufficient numbers.</p> <p>(C) Higher levels of A_2AR-specific mRNA in in vivo-activated granulocytes. In parallel with the much stronger A_2AR agonist-induced inhibition of hydrogen peroxide production and accumulation of cAMP in in vivo-activated granulocytes, the relative levels of A_2AR-specific mRNA were much higher in in vivo-activated granulocytes obtained from inflamed lungs 48 h after IT LPS injection, as compared with naïve granulocytes isolated from the bone marrow of healthy mice (left graph). Up-regulation of A_2AR mRNA in in vivo-activated granulocytes was confirmed in another set of experimental animals breathing 21% oxygen, but was increased to a much lesser extent in animals subjected to 100% oxygen (right graph). Levels of A₁R mRNA did not change much in inflammatory lung granulocytes from animals breathing normal atmosphere, but were clearly increased in mice exposed to 100% O₂. In the two sets of experiments (left and right graphs), granulocytes were pooled from five and six mice per treatment, respectively. Taken together, the results demonstrate that granulocytes recovered from alveolar spaces of inflamed lungs did, indeed, up-regulate their A_2AR expression during these in vivo lung injury assays, thereby confirming and extending previous findings in other inflammation models [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-b11" target="_blank">11</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-b53" target="_blank">53</a>]</p></div