Tissue-resident ecto-5' nucleotidase (CD73) regulates leukocyte trafficking in the ischemic brain

Abstract

Ectoenzymes expressed on the surface of vascular cells and leukocytes modulate the ambient nucleotide milieu. CD73 is an ecto-5' nucleotidase that catalyzes the terminal phosphohydrolysis of AMP and resides in the brain on glial cells, cells of the choroid plexus, and leukocytes. Though CD73 tightens epithelial barriers, its role in the ischemic brain remains undefined. When subjected to photothrombotic arterial occlusion, CD73(-/-) mice exhibited significantly larger (49%) cerebral infarct volumes than wild-type mice, with concordant increases in local accumulation of leukocyte subsets (neutrophils, T lymphocytes, macrophages, and microglia). CD73(-/-) mice were rescued from ischemic neurologic injury by soluble 5'-nucleotidase. In situ, CD73(-/-) macrophages upregulated expression of costimulatory molecules far more than wild-type macrophages, with a sharp increase of the CD80/CD86 ratio. To define the CD73-bearing cells responsible for ischemic cerebroprotection, mice were subjected to irradiative myeloablation, marrow reconstitution, and then stroke following engraftment. Chimeric mice lacking CD73 in tissue had larger cerebral infarct volumes and more tissue leukosequestration than did mice lacking CD73 on circulating cells. These data show a cardinal role for CD73 in suppressing ischemic tissue leukosequestration. This underscores a critical role for CD73 as a modulator of brain inflammation and immune function.

Figure 1

Effect of CD73^−/− genotype on cerebral infarct volume and functional outcome. A) MR images from representative mice (genotype indicated) 48 hours following photothrombotic MCA occlusion. B) Quantitative analysis of cerebral infarct volumes by MR at 48 hrs after MCA occlusion, with genotype indicated (n=6 per group). C) Neurologic deficit scores shown for individual animals of the indicated genotype. All 6 animals from the (A) and (B) panels are included, as well as data from another 4 animals which did not undergo infarct volume analysis by MR (n=10 per group). D) Brain water content 48 h after induction of photothrombotic MCA in contralateral (C) and ischemic (I) hemispheres in WT and CD73 null mice (n=5 per group). ***P<0.001.

Figure 2

Role of CD73 in leukocyte sequestration in the ischemic brain 48 hrs after MCA occlusion: A) Absolute number of leukocyte subpopulations (i.e., microglia, macrophages and neutrophils) in contralateral (C) and ischemic (I) hemispheres in WT and CD73 null mice (n=6 per group; ***P<0.001 B) Relative contribution of microglia, macrophages and neutrophils in contralateral (C) and ischemic (I) hemispheres in WT and CD73 null mice 48 hrs after induction of brain ischemia (n=6 per group). C) Representative dot-plot scatter analysis of leukocytes isolated from contralateral (C) and ischemic (I) hemispheres within ischemic brains of WT and CD73 null mice; double staining for CD45 and F4/80 allowed the identification of 2 different populations: CD45^low F4/80⁺ (microglia), CD45^hi F4/80⁺ (macrophages). D) Mean fluorescent intensity of macrophages expressing CD80 and CD86 molecules isolated from contralateral (C) and ischemic (I) hemispheres of CD73−/− and WT mice (n=4 per group). E) Overlay histograms illustrate the difference in mean fluorescent intensity of macrophages expressing CD80 and CD86 molecules, between ischemic hemispheres of CD73^−/− and WT mice. F) Representative scattergrams of CD45 and LY-6G stained leukocytes isolated from contralateral and ischemic hemispheres of WT and CD73 null mice. Strong positivity for both markers indicates infiltrating neutrophil population.

Figure 3

Infiltration of T cell subpopulations into the contralateral (C) and ipsilateral (I) hemispheres of wild-type and CD73^−/− mice at forty-eight hours post stroke. A) Absolute numbers of CD45+/CD4+ (helper) T cell populations. B) Absolute numbers of CD45+/CD8+ (cytotoxic) T cell populations.

Figure 4

Role of CD73 on cytokine and adhesion molecule expression. mRNA levels were estimated using semiquantitative (RT)-PCR and normalized against β-actin mRNA. Shown are plots of expression of IL-1β (A); IL-6 (B); TNF-α (C); KC (D); VCAM-1 (E) and IL-10 (F) mRNAs in contralateral (C) and ischemic (I) hemispheres of WT and CD73−/− mice (n=4 per group). ***P<0.001, ns=not significant.

Figure 5

In order to assess the therapeutic potential of soluble 5’ nucleotidase (CD73 analog) in preventing cerebral infarction, experiments were performed in a different cohort of mice. A) Average cerebral infarct volume was calculated 48 h after induction of ischemia in WT and CD73 null mice treated with soluble 5’ nucleotidase (5'NT) or vehicle (PBS) (n=6 per group), and B) Neurological deficit was measured using a 5-tired grading system in the same animals. Cells from contralateral (C) and ischemic (I) hemispheres of these mice were subjected to flow cytometric analysis to determine the absolute number of macrophages (C), microglia (F), and neutrophils (G) (n=6 per group). Similarly, mean fluorescent intensity was measured in macrophages isolated from ischemic (I) hemispheres of WT and CD73 null mice treated with soluble 5’nucleotidase or vehicle and labeled with antibodies against CD80 (D) and CD86 (E) (n=4 per group). *P<0.05; **P<0.01; ***P<0.001.

Figure 6

Selective inactivation of CD73 molecule on tissue only attenuates MCAO induced brain ischemia. A) Quantitative analyses of infarct volumes in marrow-reconstituted mice (n=4 per group). B) Locomotor activity determined by neurological deficit score shown for individual animals across the genotype 48 hrs after induction of brain injury. (n=4 per group). The contribution of CD73 on brain resident tissue and leukocytes to leukosequestration of microglia (C), neutrophils (D) and macrophages (E) was examined using bone-marrow reconstitution studies. (n=4 per group). Panels (F) and (G) show mean fluorescent intensity of macrophages expressing CD80 and CD86 molecules isolated from ischemic hemispheres of bone-marrow reconstituted mice (n=4 per group). Thirty minutes prior to induction of brain ischemia, a different cohort of WT and CD73^−/− mice were treated intraperitoneally with vehicle alone or the CD73 inhibitor AOPCP, (panel H) and 48 h later infarct volumes were calculated. (n=5 per group except n=8 for WT AOPCP). *P<0.05; **P<0.01; ***P<0.001.

Figure 7

CD73 and von Willebrand factor (vWF) expression in the brain. CD73^−/− mice (upper row) and wild-type mice (lower row) stained for CD73 in the first column and vWF in the second column. A) A lack of CD73 staining is shown in the knockout mice. B) vWF staining defines the vessel wall and together with the CD73 staining confirms the absence of CD73 in the vasculature. C) CD73 expression in the wild-type mouse can be observed in the choroid plexus. D) vWF staining of choroid plexus reveals surrounding vessels and the absence of CD73 expression.