Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase CD39

Abstract

Leukocyte and platelet accumulation at sites of cerebral ischemia exacerbate cerebral damage. The ectoenzyme CD39 on the plasmalemma of endothelial cells metabolizes ADP to suppress platelet accumulation in the ischemic brain. However, the role of leukocyte surface CD39 in regulating monocyte and neutrophil trafficking in this setting is not known. Here we have demonstrated in mice what we believe to be a novel mechanism by which CD39 on monocytes and neutrophils regulates their own sequestration into ischemic cerebral tissue, by catabolizing nucleotides released by injured cells, thereby inhibiting their chemotaxis, adhesion, and transmigration. Bone marrow reconstitution and provision of an apyrase, an enzyme that hydrolyzes nucleoside tri- and diphosphates, each normalized ischemic leukosequestration and cerebral infarction in CD39-deficient mice. Leukocytes purified from Cd39-/- mice had a markedly diminished capacity to phosphohydrolyze adenine nucleotides and regulate platelet reactivity, suggesting that leukocyte ectoapyrases modulate the ambient vascular nucleotide milieu. Dissipation of ATP by CD39 reduced P2X7 receptor stimulation and thereby suppressed baseline leukocyte alphaMbeta2-integrin expression. As alphaMbeta2-integrin blockade reversed the postischemic, inflammatory phenotype of Cd39-/- mice, these data suggest that phosphohydrolytic activity on the leukocyte surface suppresses cell-cell interactions that would otherwise promote thrombosis or inflammation. These studies indicate that CD39 on both endothelial cells and leukocytes reduces inflammatory cell trafficking and platelet reactivity, with a consequent reduction in tissue injury following cerebral ischemic challenge.

Figure 1. Effect of Cd39 genotype on resistance to cerebral ischemia 48 hours after MCA occlusion.

(A) Representative magnetic resonance images of WT and Cd39^–/– brains after cerebral ischemia. (B) Quantification of average cerebral infarct volume in ischemic WT and Cd39^–/– brains. (C) Functional effects of cerebral infarction were assessed using a neurologic deficit score, with higher scores indicating a greater deficit (50). The horizontal bars indicate the average neurologic deficit score for each group. n = 6 per group; *P < 0.03, **P < 0.01.

Figure 2. Role of CD39 in leukocyte sequestration in the ischemic cerebrum.

The ischemic brains of WT (A–C) and Cd39^–/– (D–F) mice were stained for nuclei (A and D) and neutrophils (B, C, E, and F). Scale bars: 1,000 μm (A, B, D, and E), 100 μm (C and F). Adjacent sections of WT (G–I) and Cd39^–/– (J–L) ischemic mouse brains were stained for nuclei (G and J) and macrophages (H, I, K, and L). Scale bars: 1,000 μm (G, H, J, and K), 100 μm (I and L). The white boxes in the center panels show the magnified area in the right panels. (M) Representative scattergrams of LY-6G–stained neutrophil populations (green) within the ischemic and contralateral hemispheres of WT and Cd39^–/– mice as well as isotype control. (N) Representative scattergrams of F4/80-stained macrophage populations (blue) within the ischemic and contralateral hemispheres of WT and Cd39^–/– mice as well as isotype control. The effect of CD39 on the infiltration of leukocyte subpopulations was assessed using flow cytometry: neutrophils (O) and macrophages (P). n = 6 per group in M–P; *P < 0.005, **P < 0.0001.

Figure 3. Circulating ectoapyrase activity confers resistance to cerebral ischemia.

Leukocytes purified from WT and Cd39^–/– mice were coincubated with [8-¹⁴C]ATP or [8-¹⁴C]ADP to assess the functional importance of leukocyte CD39. Metabolic products were resolved via TLC, and representative radioactivity histograms are shown for ATP (A) and ADP (B) (L, ladder comprising radiolabeled ATP, ADP, and AMP). Forty-eight hours after ischemia induction, MRI was performed to assess the therapeutic potential of soluble apyrase in diminishing cerebral infarction. Crosses (†) indicate the metabolite added to the reaction (C) Multiple strokes were scored to generate aggregate cerebral infarct volumes. Additional mice were subjected to flow cytometric analysis to determine neutrophil (D) and macrophage (E) infiltration following apyrase administration. n = 4 per group in C–E; *P < 0.04, **P < 0.02, ***P < 0.001.

Figure 4. CD39 deficiency does not impair bone marrow reconstitution.

To determine the contribution of donor and recipient cells to the neutrophil and monocyte subpopulations, we developed a quantitative PCR that measured neomycin gene dosage. (A) Mixtures of WT and Cd39^–/– peripheral blood leukocytes (x axis indicates cell number × 10,000) were used to validate this assay. Leukocyte buffy coats from bone marrow–transplanted mice were sorted by flow cytometry to isolate the neutrophil and monocyte subpopulations. The neutrophils (B) and monocytes (C) were then quantified for relative neomycin DNA. Peripheral blood from untransplanted WT (green) and Cd39^–/– (black) mice was separated into neutrophil (D) and monocyte (E) populations by flow cytometry and then examined for CD39 expression. Isotype control is shown in orange. Peripheral leukocytes from bone marrow chimeric mice were sorted into neutrophil (F) and monocyte (G) populations and stained for CD39: WT→WT (green), WT→KO (red), KO→WT (magenta), and KO→KO (black). Whole-lung homogenates from bone marrow chimeric mice were sorted for endothelial cells (H) and stained for CD39: WT→WT (green), WT→KO (red), KO→WT (magenta), and KO→KO (black). (I) Whole-lung digests from bone marrow–transplanted mice were analyzed for relative CD39 expression on endothelial, neutrophil, and macrophage subpopulations. n = 4 or 5 per group; *P < 0.001, **P < 0.01, ***P < 0.05.

Figure 5. Role of CD39-bearing subpopulations in resistance to cerebral ischemia and regulation of platelet reactivity.

WT and Cd39^–/– mice underwent bone marrow reconstitution to generate chimeric mice, as a means to explore selective ablation of CD39 in endothelial and leukocyte subpopulations. (A) Quantification of average cerebral infarct volume determined by MRI in ischemic chimera brains. The contribution of CD39 on endothelium and leukocytes to leukosequestration of neutrophils (B) and macrophages (C) was examined in chimeric mice. (D) Whole-blood platelet aggregometry with ADP stimulation was performed on WT and Cd39^–/– mice. Marrow-reconstituted mice were used to explore platelet reactivity following selective ablation of CD39 in endothelial and leukocyte subpopulations. (E) Representative whole-blood platelet aggregation profiles of each of the 4 chimeric subpopulations 2 weeks after transplantation. (F) Quantification of average platelet aggregation profiles. n = 5 or 6 per group in A–D; n = 4 or 5 per group in E and F. *P < 0.01; **P < 0.001; ***P < 0.005; ^†P < 0.01 versus all other columns; ^‡P < 0.02; ^§P < 0.001 versus all other groups.

Figure 6. CD39 modulates circulating leukocyte α_Mβ₂-integrin expression.

Unstimulated whole blood of WT (black) and Cd39^–/– (gray) mice was examined for α_Mβ₂-integrin expression by staining the α_M subunit in the monocyte (A) and neutrophil (B) populations. The relative expression of α_M on the monocyte (C) and neutrophil (D) populations in WT and Cd39^–/– mice is shown. n = 9 per group; *P < 0.01.

Figure 7. Apyrase treatment modulates monocyte α_Mβ₂-integrin expression.

WT and Cd39^–/– mice were treated with soluble apyrase before examination of monocyte α_Mβ₂-integrin expression. (A) Representative histogram shifts can be seen between vehicle-treated and apyrase-treated monocytes in WT and Cd39^–/– mice. The aggregate effect of apyrase treatment on monocyte α_M expression can be seen in B. n = 4 per group; *P < 0.01 versus all other columns.

Figure 8. Regulation of α_Mβ₂-integrin in RAW 264.7 macrophages.

(A) Relative murine Cd39 mRNA expression was measured using RT-PCR in empty vector–transfected and mCD39-overexpression vector–transfected macrophages. A representative immunoblot of membrane protein is shown. (B) Free ATP was measured in the medium of each cell line to assess the effect of altered CD39 expression on ambient ATP. (C) Representative histograms of α_M expression in empty vector–transfected (black overlay), mCD39-transfected (red) macrophages, and isotype control (orange overlay). (D) Empty vector– and mCD39 vector–transfected macrophages were modulated pharmacologically to determine the contribution of various P2 receptors and adenosine formation in the regulation of α_Mβ₂-integrin. (E) bzATP, a specific P2X₇ receptor agonist, was used to treat macrophages to determine modulation of α_Mβ₂-integrin. (F) Relative P2X₇ receptor mRNA was measured by quantitative PCR in macrophage cell lines that expressed either vector or mCD39 as well as either control shRNA or shRNA targeting the P2X₇ receptor. A representative P2X₇ receptor immunoblot of membrane protein is shown. (G) α_M expression following modulation of CD39 expression, P2X₇ receptor expression, or both. n = 6 per group; *P < 0.01 versus all other groups, **P < 0.001 versus all other groups, ***P < 0.001.

Figure 9. CD39 regulates leukocyte trafficking via α_Mβ₂-integrin in vitro and in vivo.

(A) Transmigration of WT or Cd39^–/– peritoneal macrophages on fibrin(ogen)-coated Transwells. (B) Representative microscope field of transmigrated primary macrophages stained with F4/80 acquired with a 20× objective (0.325 μm/pixel). (C) Transmigration on fibrin(ogen)-coated Transwells was assessed using RAW 264.7 macrophages transfected with mCD39 or control vector. Some wells were treated with α_M functional blocking antibody or an isotype control. Migration was quantified relative to vehicle-treated, vector-transfected macrophages. In vivo studies examined leukocyte sequestration in WT and Cd39^–/– mice treated with an α_Mβ₂-integrin functional blocking antibody 48 hours after induction of cerebral ischemia. Flow cytometry was used to assess neutrophil (D) and macrophage (E) infiltration. (F) Quantification of MRI of infarcted ischemic hemispheres of mice treated with isotype control antibody or α_M-integrin functional blocking antibody. n = 3 per group in A and B, n = 9 per group in C, n = 7 per group in D–F. *P < 0.001 between indicated groups; **P < 0.001 versus all other groups; ***P < 0.05 between indicated groups.