Abstract 135: CD73 Regulates Macrophage Trafficking In The Ischemic Brain
The 5′ ectonucleotidase CD73 is a cell surface enzyme which breaks down extracellular adenosine 5′ monophosphate (AMP) to generate the anti-inflammatory and tissue protective molecule adenosine. Mice with endogenus deletion of CD73 have limited inflammatory and prothrombotic responses, which have been attributed to attenuated leukocyte adhesion and platelet accumulation. We sought to determine whether provision of soluble CD73 (5′nucleotidase) could act to limit the influx of leukocytes and damage of brain tissue in stroke. A photochemical method of generating endothelial damage was used to promote middle cerebral arterial (MCA) thrombi which are relevant to the clinical setting of ischemic stroke. Intravenous Rose bengal was given followed by a neon laser pulse applied over the exposed MCA. CD73−/− and WT mice were injected intraperitoneally, with 7.5 U of soluble 5′nucleotidase from Crotalus atrox venom given 30 minutes before induction of brain ischemia, while their controls were injected with the same amount of saline. Infarct volumes were determined by MRI 48 hours after stroke, at which time leukocyte influx was assessed by flow cytometry. In ischemic hemispheres of CD 73−/− mice (n=3) treated with soluble CD73, the percentage of infiltrating macrophages (defined by high expression of CD45 and F4/80) was decreased in comparison with the percentage of macrophages in CD73−/− animals treated with PBS (10.2% vs. 15.3% p<0.05) . The number of CD45hi F4/80 hi macrophages in CD73−/− treated with 5′nucleotidase decrease more than twofold compared to vehicle treated CD73−/− mice (6 x104 vs. 13.5x 104, p<0.01). However, soluble 5′ nucleotidase attenuates macrophages infiltration almost twofold in WT animals in comparison with their corresponding controls (5.3x104 vs. 3x104, p<0.01). This was confirmed by a 30% decrease in infarct volume in CD73−/− animals treated with 5′ nucleotidase compared with vehicle- treated CD73−/− mice. There was a 15% reduction in infarct volume in WT mice treated with CD73 compared with saline- treated control WT mice. These data show that soluble CD73 can mitigate the inflammatory phenotype of CD73−/− and WT mice in the setting of stroke. Terminal phosphohydrolysis of AMP by CD73 may represent a new paradigm for stroke therapy.