Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase CD39 (original) (raw)
Cd39 genotype and clinical sequelae of cerebral ischemia. Permanent cerebral ischemia was induced in CD39-deficient and WT mice with the use of a photothrombotic model of middle cerebral artery (MCA) occlusion (20). Forty-eight hours later, T2-weighted cortical MRIs were performed to assess infarct volume (Figure 1A). Infarct volumes in CD39-deficient mice were 78.7% larger than those in WT controls (Figure 1B). The larger infarct volumes in Cd39–/– mice were of functional significance, since they also demonstrated greater neurological deficits than WT mice (Figure 1C). The data obtained with this model of stroke parallel those previously reported with intraluminal MCA occlusion using a nylon monofilament — studies in which larger cerebral infarct volumes were observed in Cd39–/– mice 24 hours after ischemia (7).
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.
CD39 modulation of ischemic leukosequestration. CD39-deficient mice are characterized by enhanced platelet deposition in ischemic cerebral tissue, yet platelet activation alone cannot account for all of the sequelae of cerebral infarction. For instance, when a short-acting GPIIb/IIIa antagonist was given to mice in the setting of stroke, although platelet deposition was markedly diminished, there was reduction in, but not complete rescue from, cerebral infarction (2). We hypothesized that the ischemia susceptibility of Cd39–/– mice could be attributed not only to release by platelets of granular contents, exposure of platelet surface adhesion molecules such as P-selectin, and provision of a procoagulant phospholipid surface, but also to increased postischemic leukocyte infiltration. Histologic examination revealed that large numbers of macrophages and neutrophils are recruited to ischemic cortex and suggested that absence of CD39 in particular exaggerates macrophage recruitment into the ischemic zone (Figure 2, A–L). Using flow cytometry to quantify leukocyte infiltration, we analyzed ischemic and nonischemic hemispheres of mice 48 hours after ischemia induction. Antibodies against CD45 and LY-6G characterized the neutrophil (CD45hiLY-6G+) subpopulation of the infiltrating cells (green in Figure 2M); F4/80 positivity in combination with CD45 staining identified macrophages (CD45hiF4/80+) (blue in Figure 2N) (21, 22). The CD45loF4/80+ cells observed could represent activated microglial cells, which are known to express F4/80 (23, 24). Ischemic hemispheres of untransplanted CD39-deficient mice demonstrated 61% more total infiltrating nucleated cells as compared with the ischemic hemispheres of untransplanted WT mice. Conversely, in nonischemic hemispheres of controls, the numbers of infiltrating cells did not differ significantly between genotypes (data not shown). This implied that CD39 does not affect baseline numbers of resident leukocytes in the brain. Subpopulation analysis demonstrated a 2-fold enrichment in the number of infiltrating neutrophils and macrophages in each ischemic hemisphere of CD39-deficient versus control mice (259 × 103 ± 24 × 103 vs. 134 × 103 ± 4 × 103 neutrophils per hemisphere and 108 × 103 ± 5 × 103 vs. 46 × 103 ± 3 × 103 macrophages per hemisphere) (Figure 2, O and P).
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.
Circulating apyrase protects Cd39–/– mice from cerebral ischemia. We hypothesized that the heightened leukocyte flux seen in Cd39–/– mice was due in part to an absence of vascular wall CD39 activity, and also in part due to a loss of CD39 circulating on the leukocytes themselves. To examine this, we assessed the ability of WT and CD39-deficient leukocytes to metabolize ATP and ADP in their extracellular milieu. Purified buffy coats were incubated with either [8-14C]ADP or [8-14C]ATP, after which phosphohydrolysis was assessed by TLC (Figure 3, A and B). CD39-deficient leukocytes were deficient in ATPase and ADPase activity (Figure 3, A and B). To demonstrate that the loss of ectoapyrase activity in _Cd39_-null mice was driving their leukosequestration phenotype, a soluble apyrase was administered prior to ischemic induction. Apyrase is a functional analog of CD39 that has been shown to restore normal vascular homeostasis (mitigate platelet desensitization) in Cd39–/– mice (11). _Cd39_-null mice treated with apyrase, but not saline, were protected, with a diminished cerebral infarct size (42.8 ± 7.6 mm3 vs. 68.6 ± 2.1 mm3, P < 0.04 (Figure 3C). This finding is concordant with previous studies using solCD39, a soluble form of human CD39 (25) in murine (7) and rat (8) models of cerebral ischemia. Data also showed a concordant decrease in the number of neutrophils and macrophages in the ischemic brain of apyrase-treated mice when compared with saline controls (apyrase: 176 × 103 ± 23 × 103 neutrophils per hemisphere vs. saline: 331 × 103 ± 40 × 103 neutrophils per hemisphere, P < 0.02; apyrase: 60 × 103 ± 6 × 103 macrophages per hemisphere vs. saline: 144 × 103 ± 10 × 103 macrophages per hemisphere, P < 0.001) (Figure 3, D and E). These data show that inhibition of ischemia-driven leukocyte accumulation can be achieved through administration of a functional CD39 analog.
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.
Restoration of normal leukocyte mobilization in Cd39–/– mice by bone marrow transplantation. To determine whether CD39 on circulating cells or on vascular tissue was contributing to the increased cerebral ischemia susceptibility of CD39-deficient animals, a series of bone marrow transplantations were performed between _Cd39_-null and WT animals (26). Cd39–/– or WT bone marrow was transplanted into myeloablated Cd39–/– (KO→KO) or WT (WT→WT) mice, respectively (donor→recipient). These mice served as transplantation controls. Similarly, Cd39–/– or WT bone marrow was also transplanted into WT (KO→WT) or Cd39–/– (WT→KO) myeloablated recipients to generate chimeric mice with CD39 either on the resident tissue alone (KO→WT) or on bone marrow–derived cells (BMDCs) alone (WT→KO).
To confirm the efficiency of the marrow reconstitution, we developed a new quantitative PCR methodology with probe and primer sets designed against both neomycin (cassette used in generating the Cd39 knockout) and nerve growth factor (NGF; as an internal control). This allowed assay of the percentage of cells in a population that were CD39-deficient (containing neomycin) or WT (no neomycin) (Figure 4A). Fluorescence-activated cell sorting was used to collect individual neutrophil and monocyte populations for DNA isolation. The Neo/NGF assay was used to genotype the cells in each of our 4 chimeras and revealed that circulating neutrophils and monocytes were fully reconstituted (Figure 4, B and C). In a separate set of experiments, flow cytometry (Figure 4, D–G) was used to examine both the circulating neutrophil and monocyte protein expression of CD39. These methods demonstrated that at both the DNA and protein levels, neutrophils and monocytes were completely reconstituted in the chimeric mice. Further staining of tissue homogenates demonstrated that the resident endothelial populations retained the recipient phenotype following bone marrow transplantation (Figure 4I), with endothelial cells having approximately twice the surface CD39 expression of either the resident neutrophil or macrophage populations.
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.
Eight to ten weeks were allowed for full bone marrow reconstitution, after which the chimeras were subjected to photothrombotic MCA occlusion. MRIs of the homologously transplanted (i.e., WT→WT or KO→KO) and infarcted mice demonstrated that bone marrow transplantation does not alter the susceptibility to cerebral injury in ischemic stroke of WT and _Cd39–/–_mice. Mice without CD39 (KO→KO) had significantly larger infarct volumes than mice with CD39 (WT→WT) (65.6 ± 2.3 mm3 vs. 30.5 ± 5.1 mm3, P < 0.001) (Figure 5A). Chimeric mice with CD39-bearing BMDCs were largely rescued from infarction when compared with KO→KO mice (65.6 ± 2.3 mm3 vs. 42.2 ± 4.7 mm3, P < 0.01). Conversely, expression of CD39 on only the vascular tissue surface provided limited protection from cerebral ischemia (KO→WT, 59.0 ± 3.4 mm3). Furthermore, the increased infarct volumes of the KO→KO mice proved to be functionally important, as they had significantly greater neurologic deficits than WT→WT or WT→KO mice (data not shown). KO→WT chimeras had an intermediate neurologic deficit.
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.
Given the susceptibility of CD39-deficient mice to ischemia-driven leukosequestration, we examined which CD39-bearing tissues confer protection to WT mice using the same marrow reconstitution strategy. The total number of cells infiltrating the contralateral, nonischemic hemisphere was similar across all chimeric strains (data not shown). Subpopulation analysis of ischemic hemispheres showed that CD39 on BMDCs strongly suppressed leukocyte recruitment to ischemic tissue. The total number of infiltrating neutrophils was significantly higher (P < 0.01) in KO→KO mouse brain (390 × 103 ± 18 × 103) compared with all other groups (WT→WT, 217 × 103 ± 16 × 103; KO→WT, 273 × 103 ± 28.6 × 103; WT→KO, 255 × 103 ± 21 × 103) (Figure 5B). The number of macrophages recruited to the ischemic brain correlated closely with the presence or absence of CD39 on BMDCs. The absolute number of infiltrating macrophages was significantly increased, almost 2-fold, in the KO→KO and KO→WT mouse brains when compared with either WT→WT or WT→KO mice (Figure 5C). This implies that although an acute rescue from CD39 deficiency can be obtained through administration of an apyrase or solCD39 analog, a permanent rescue can be obtained via bone marrow reconstitution with CD39-bearing cells.
CD39-bearing subpopulations contribute to platelet reactivity. CD39 has previously been shown to be a prime regulator of platelet activation and recruitment in vivo (7, 11, 27) and ex vivo (9, 27), yet the role of CD39 loss from a subpopulation has not been explored. We hypothesized that CD39 on the surface of leukocytes could be regulating platelet activation and recruitment, thereby contributing to the sequelae of cerebral ischemia in this fashion. To determine how a change in leukocyte phosphohydrolytic activity might modulate platelet reactivity, we employed whole-blood aggregometry. In contrast to platelet-rich plasma aggregometry, this assay mimics platelet-leukocyte interactions. In keeping with previous published observations in platelet-rich plasma aggregometry (7, 11), nontransplanted Cd39–/– mice demonstrated platelet desensitization when compared with WT control mice (Figure 5D). We sought to examine whether this was attributable to the catabolism of platelet-activating nucleotides by endothelial cell– or leukocyte-bound CD39 using bone marrow reconstitution studies. Two weeks following reconstitution, mice with either tissue CD39 alone (KO→WT) or BMDCs with CD39 (WT→KO) demonstrated only partial desensitization of platelets, demonstrating a role for both of these subpopulations in the maintenance of vascular homeostasis (Figure 5, E and F). This finding is complementary to previous work in which human CD39 was overexpressed on either the leukocyte or endothelial populations. These experiments showed that CD39 can regulate platelet activation from either the endothelial or leukocyte compartment (27).
CD39 deficiency and leukocyte αMβ2-integrin surface expression. ATP is known to upregulate expression of αMβ2-integrin (also known as MAC-1, CD11b/CD18), a critical glycoprotein adhesion receptor expressed on human neutrophils (16, 28). To discern whether a lack of CD39 resulted in stimulation of CD39-deficient leukocytes via basal nucleotide release, we examined peripheral blood monocyte and neutrophil populations by flow cytometry. Histologic analysis performed to confirm the cell gates revealed 92% and 97% purity of the sorted monocytes and neutrophils, respectively (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI36433DS1). There was a 50% increase in the number of αMβ2-integrin high-expressing monocytes (measured by the αM/CD11b subunit) in Cd39–/– mice compared with WT controls (Figure 6, A and C). In contrast, neutrophils did not display differences in cell-surface αMβ2-integrin expression at baseline (Figure 6, B and D). Treatment of Cd39–/– animals with a soluble apyrase restored expression of αMβ2-integrin on monocytes to near-WT levels (Figure 7). By comparison, treatment of WT animals with apyrase resulted in an approximately 40% reduction in the number of αMβ2-integrin high expressers relative to vehicle.
CD39 modulates circulating leukocyte αMβ2-integrin expression. Unstimulated whole blood of WT (black) and Cd39–/– (gray) mice was examined for αMβ2-integrin expression by staining the αM subunit in the monocyte (A) and neutrophil (B) populations. The relative expression of αM on the monocyte (C) and neutrophil (D) populations in WT and Cd39–/– mice is shown. n = 9 per group; *P < 0.01.
Apyrase treatment modulates monocyte αMβ2-integrin expression. WT and Cd39–/– mice were treated with soluble apyrase before examination of monocyte αMβ2-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 αM expression can be seen in B. n = 4 per group; *P < 0.01 versus all other columns.
CD39 overexpression reduces cell-surface αMβ2-integrin expression. We transfected RAW 264.7 murine macrophages with either empty pCDNA3.1 vector or pCDNA3.1 containing murine CD39 to explore the relationship between CD39 and αMβ2-integrin in vitro. Stable CD39 transfectants expressed 15-fold more Cd39 mRNA than vector control cells, with a concurrent increase in membrane CD39 protein (Figure 8A). After media exchange with serum-free media, RAW cells overexpressing CD39 were found to have 71% less ATP in their media when compared with vector transfectants, likely reflecting the difference in CD39 protein expression (Figure 8B). The source of this ATP was presumed to be leakage or release from the macrophages themselves. When the CD39-overexpressing cells were analyzed by flow cytometry, they maintained a resting state with 40% less cell surface αMβ2-integrin than empty vector transfectants (Figure 8C). We hypothesized that basal ATP released from cells was metabolized more efficiently in CD39-overexpressing cells. This implies that the reduced αMβ2-integrin expression phenotype results from either reduced stimulation of P2 receptors or increased adenosine generation. To distinguish between these two possibilities, we used adenosine 5′-(α,β-methylene)diphosphate (APCP), a specific CD73 inhibitor, to block conversion of CD39-generated AMP into adenosine by CD73 (19, 29). In the presence of APCP, only a small, insignificant increase in the expression of αMβ2-integrin was observed, implying that this phenotype is not dependent upon the generation of adenosine (Figure 8D).
Regulation of αMβ2-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 αM 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 αMβ2-integrin. (E) bzATP, a specific P2X7 receptor agonist, was used to treat macrophages to determine modulation of αMβ2-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) αM 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.
As the suppression of αMβ2-integrin expression by CD39 appears to be independent of adenosine generation, experiments were next directed toward elucidating a role for ambient ATP. ATP binds to and activates purinergic receptors. To investigate a direct role for purinergic receptor engagement by adenine nucleotide phosphates, we blocked purinergic signaling using the inhibitors suramin (which inhibits P2X1–3,5 and P2Y1,11) (4, 30–32), 2′,3′-_O_-(2,4,6-trinitrophenyl) ATP (TNP-ATP; which inhibits P2X1–4) (33, 34), or periodate oxidized ATP (ox-ATP; which inhibits P2X7) (35, 36). Little change in αMβ2-integrin expression was seen in either CD39-overexpressing or vector-transfected cells when treated with suramin or TNP-ATP. However, strong suppression of αMβ2-integrin expression was conferred by the specific P2X7 inhibitor ox-ATP (Figure 8D). Blockade of the P2X7 receptor in vector-transfected macrophages brought αMβ2-integrin expression to levels lower than those of CD39-overexpressing macrophages. Not surprisingly, as CD39 dissipates ATP and hence itself should indirectly reduce P2X7 receptor stimulation, P2X7 receptor blockade had a significant but smaller effect on αMβ2-expression in mCD39 transfectants. Finally, the specific P2X7 receptor agonist 2′(3′)-_O_-(4-benzoylbenzoyl)ATP (bzATP) dose-dependently induced αMβ2-integrin expression in both cell lines (Figure 8E). This suggested that suppression of αMβ2-integrin expression in CD39 transfectants is due to diminished P2X7 receptor stimulation in CD39-overexpressing cells.
To confirm that the changes in integrin expression were not due to pharmacologically induced off-target effects, we employed a gene-silencing approach. By use of an shRNA that targeted the P2X7 receptor as well as an empty vector control, the P2X7 receptor was silenced at mRNA and protein levels in both control and CD39-overexpressing macrophage cell lines (Figure 8F). These cell lines were subsequently examined by flow cytometry for αMβ2-integrin expression. The cells each carried 2 plasmids, one containing mCD39 or corresponding control vector; the other, P2X7 shRNA or corresponding control vector. These experiments confirmed the earlier transfection experiments shown in Figure 8C; i.e., when the single transfectants were additionally transduced with a control shRNA, overexpression of mCD39 still resulted in a significant diminution of αM-integrin expression. In contrast, transduction with a P2X7-silencing shRNA completely abolished the difference in integrin expression seen between CD39-overexpressing and control cells (Figure 8G). In essence, P2X7 suppression uncouples CD39 levels from regulation of αM-integrin levels. This implies that CD39 can regulate the expression of αMβ2-integrin by dissipating ATP that would otherwise activate the P2X7 receptor.
CD39 regulates leukocyte trafficking via αMβ2-integrin in vitro and in vivo. Our in vitro studies suggest that the amplified leukocyte recruitment in _Cd39_-null mice is a consequence of upregulated αMβ2-integrin expression in _Cd39_-null leukocytes. To further explore this, we performed leukocyte transmigration assays on fibrin(ogen)-coated Transwells. Fibrin(ogen) is a known cognate binding partner for αMβ2-integrin. In this assay, Cd39–/– peritoneal macrophages migrated 300% more than WT controls (Figure 9, A and B). Further, CD39-overexpressing macrophages exhibited a greater than 90% reduction in transmigration compared with control cells (Figure 9C). Antibody blockade of the αM subunit demonstrated that the enhanced leukocyte migration seen in vector-transfected cells was dependent upon αMβ2-integrin. We further sought to determine whether αMβ2-integrin might affect leukosequestration in our cerebral ischemia studies. _Cd39_-null and WT mice were treated with αM-blocking or isotype control antibody. Isotype-treated Cd39–/– mice had 61% more infiltrating neutrophils and 104% more infiltrating macrophages than isotype-treated WT mice, similar to our prior data (Figure 2) showing increased cerebral leukosequestration after ischemia in untreated Cd39–/– mice. This demonstrates that the leukocyte infiltration phenotype was not affected by an isotype-matched antibody (P < 0.001; Figure 9, D and E). In sharp contrast, treatment of ischemic Cd39–/– mice with αM-blocking antibody yielded a striking decrease in the number of infiltrating macrophages and neutrophils when compared with ischemic Cd39–/– mice treated with isotype control antibody (Figure 9, D and E). Furthermore, antibody blockade in Cd39–/– mice restored levels of leukocyte trafficking to those seen in isotype antibody–treated WT mice. Analysis of peripheral blood showed that this abrogated leukocyte trafficking was not due to pan-leukodepletion following intravenous antibody administration (data not shown). When antibody-treated mice were examined by MRI, both WT and CD39-deficient mice were significantly protected by treatment with an αM-integrin blocking antibody (Figure 9F). The fact that αM blockade was incompletely protective in Cd39–/– mice (i.e., αM blockade did not reduce infarct volumes to the extent seen in αM-antibody–treated WT mice) suggests that injury mechanisms other than αM-dependent leukosequestration are also at play.
CD39 regulates leukocyte trafficking via αMβ2-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 αM 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 αMβ2-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 αM-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.
To investigate whether heterotypic monocyte-platelet interactions could be contributing, we measured monocyte-platelet aggregates; there were no significant differences in monocyte-platelet aggregates between WT and Cd39–/– mice, though there was a trend for there to be more in the CD39-deficient mice (Supplemental Figure 2). Together, these data demonstrate a critical role for αMβ2-integrin–dependent leukosequestration in the tissue injury in ischemic stroke, especially in Cd39–/– mice.