The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after strok

Transcriptional profiling of human monocytes identifies the inhibitory receptor CD300a as regulator of transendothelial migration.

Local inflammatory responses are characterized by the recruitment of circulating leukocytes from the blood to sites of inflammation, a process requiring the directed migration of leukocytes across the vessel wall and hence a penetration of the endothelial lining. To identify underlying signalling events and novel factors involved in these processes we screened for genes differentially expressed in human monocytes following their adhesion to and passage through an endothelial monolayer. Functional annotation clustering of the genes identified revealed an overrepresentation of those associated with inflammation/immune response, in particular early monocyte to macrophage differentiation. Among the gene products so far not implicated in monocyte transendothelial migration was the inhibitory immune receptor CD300a. CD300a mRNA and protein levels were upregulated following transmigration and engagement of the receptor by anti-CD300a antibodies markedly reduced monocyte transendothelial migration. In contrast, siRNA mediated downregulation of CD300a in human monocytes increased their rate of migration. CD300a colocalized and cosedimented with actin filaments and, when activated, caused F-actin cytoskeleton alterations. Thus, monocyte transendothelial migration is accompanied by an elevation of CD300a which serves an inhibitory function possibly required for termination of the actual transmigration

CD300a activation by antibody-engagement on the cell surface of monocytes results in a significant reduction of transendothelial migration.

<p><b>A</b>. Cell surface expression of CD300a and p58.2 antigens on human monocytes was analysed by FACS using anti-CD300a and anti-p58.2 antibodies. Control cells were incubated without primary and secondary (control 1) or only with secondary antibodies (control 2). <b>B</b>. Freshly isolated human monocytes (2×10⁶) were incubated with mouse monoclonal anti-CD300a (E59.126) or control antibodies (anti-cmyc and anti-p58.2), respectively, and then subjected to transendothelial migration assays in a two-chamber set-up. Migrated cells were collected from the lower chamber and counted using a cell culture analyzer. The number of transmigrated cells is given on the y-axis. Three independent experiments were performed and results are presented as average +/− SEM. Statistical significance was evaluated using student's t-test. * <i>P</i><0.05.</p

Primers used for real time RT-PCR.

<p>Primers used for real time RT-PCR.</p

Changes in gene expression of human monocytes induced after transendothelial migration analyzed by realtime RT-PCR.

<p>Changes in gene expression of human monocytes induced after transendothelial migration analyzed by realtime RT-PCR.</p

Monocyte genes associated with metabolism found in DAVID functional annotation clustering of the genes differentially expressed following transendothelial migration.

<p>Monocyte genes associated with metabolism found in DAVID functional annotation clustering of the genes differentially expressed following transendothelial migration.</p

CD300a associates with F-actin in COS-7 cells.

<p><b>A</b>. COS-7 cells were transfected with a CD300a expression construct and the subcellular localization of CD300a was then revealed by staining with anti-CD300a antibodies. TRITC-phalloidin was used to label the actin cytoskeleton. The bottom row of images shows cells treated with cytochalasin D (4 μM) to disrupt actin filaments. <b>B.</b> COS-7 cells ectopically expressing CD300a were incubated with monoclonal anti-CD300a antibodies to activate cell surface receptors. Cells were then left untreated or treated with 4 μM cytochalasin D followed by fixation and staining with anti-CD300a antibodies and TRITC-phalloidin. Note the localization of CD300a to actin-rich cell protrusions that is increased following the anti-CD300a antibody treatment (arrowhead). Scale bars, 10 μm <b>C.</b> Coprecipitation of CD300a with actin filaments. Triton lysates of control (non-transfected) or CD300a expressing COS-7 cells (transfected) were treated with nocodazole to depolymerize microtubules and then subjected to high speed centrifugation to pellet actin filaments. CD300a protein was detected in the actin pellet fraction suggesting a direct or an indirect association of the protein with F-actin. Input shows protein levels in the cell lysates prior to high speed centrifugation.</p

CD300a is upregulated in transmigrated monocytes.

<p>Freshly prepared human monocytes were allowed to transmigrate across a monolayer of HMEC1 cells for 4 hours at 37°C, 5% CO₂. <b>A</b>. Mean fluorescence intensity of cell surface CD300a expression obtained by FACS analysis of anti-CD300a antibody stained cells revealing CD300a on the surface of monocytes before and after transendothelial migration. Data presented are from three independent experiments. Statistical significance was evaluated using student's t-test and ratio paired t-test * <i>P</i><0.05. <b>B</b>. Representative FACS analysis revealing total CD300a levels before and after transendothelial migration. Control cells were incubated without primary and secondary (control 1) or only with secondary antibodies (control 2).</p

siRNA-mediated downregulation of CD300a increases monocyte transendothelial migration.

<p><b>A</b>. FACS analysis of cell surface CD300a expression in monocytes transfected with control or CD300a siRNAs, respectively, employing mouse monoclonal anti-CD300a and appropriately labelled secondary antibodies. Control cells were incubated without primary and secondary (control 1) or only with secondary antibodies (control 2). <b>B</b>. Freshly isolated monocytes from human buffy coats were transfected with control or CD300a siRNAs, respectively, followed by transendothelial migration analysis. The number of transmigrated cells is given on the y-axis. Three independent experiments were performed and results are presented as average +/− SEM. Statistical significance was evaluated using student's t-test. * <i>P</i><0.05.</p