Abstract 3920: Ecto-enzymatic Regulation Of alphaMbeta2-integrin Mediated Leukocyte Trafficking
Phosphorylated nucleotides released by leukocytes induce expression of adhesion molecules and establish chemotactic gradients. These same leukocytes express the phosphohydrolytic enzymes CD39 and CD73 on their cell surface. These enzymes dissipate nucleotides and thereby reduce purinergic receptor stimulation. This led to the hypothesis that CD39, a cell surface apyrase, could regulate the adhesion and chemotaxis of leukocytes by metabolizing ambient ATP. RAW 264.7 macrophages transfected with a CD39 expression construct expressed 15 times more CD39 mRNA than vector controls, with ambient media ATP levels dissipated to 29% that of vector controls. Flow cytometry revealed that CD39-transfectants had a diminished adhesive phenotype, as alphaMbeta2-integrin mean fluorescent intensity (MFI) was reduced by 47% on CD39-overexpressing cells vs. vector-transfected controls (99 ± 6 MFI vs. 162 ± 3 MFI, p<0.001). Suppression of alphaMbeta2-integrin by CD39 was not due to CD39’s ability to promote adenosine generation, as downstream suppression of CD73 with APCP did not further alter alphaMbeta2 expression. In contrast, blockade of the P2X7 receptor with ox-ATP reduced alphaMbeta2-integrin levels in vector transfectants by 40% (p<0.01). Blockade of other P2 receptors by non-specific antagonists suramin and TNP-ATP had no effect. Not surprisingly, as CD39 dissipates ATP and hence itself should indirectly reduce P2X7 receptor stimulation, P2X7 receptor blockade had little effect on alphaMbeta2-expression in CD39 transfectants. The specific P2X7 agonist bz-ATP was able to dose-dependently induce alphaMbeta2-integrin expression in both vector transfected and CD39-overexpressing cell lines. The reduced alphaMbeta2-integrin expression in CD39 transfectants manifested as a 90% reduction in leukocyte transmigration on fibrin(ogen) coated transwells (p<0.01). AlphaM blockade revealed transmigration was alphaMbeta2-integrin dependent in both vector and CD39 transfectants. This data represents a new paradigm for leukocyte trafficking whereby cell surface enzymes control leukocytes’ own migration in a cell autonomous fashion. Further, these data could explain the heightened inflammation seen in the ischemic tissue of CD39 deficient mice.
This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).