Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

Selectins mediate leukocyte rolling, trigger ␤ 2 -integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosinebased activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) ␥2-and phosphoinositide 3-kinase (PI3K) ␥-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP 3 ), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished G␣ i -independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. G␣ i -independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk ؊/؊ and Plcg2 ؊/؊ mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by Eselectin engagement. (Blood. 2010;115(15): 3118-3127) IntroductionLeukocyte recruitment into inflamed tissue is required for host defense and proceeds in a coordinated sequence of different steps. The first contact of neutrophils with the endothelium is mediated by selectins and their counter-receptors, followed by rolling and integrin-mediated arrest. While rolling, neutrophils collect different inflammatory signals that can activate several pathways and mediate integrin activation, arrest, crawling, and extravasation of leukocytes into inflamed tissue. 1 E-selectin is expressed on inflamed endothelial cells and can bind to different glycosylated ligands on leukocytes, including CD44, 2 P-selectin glycoprotein ligand-1 (PSGL-1), 3 CD43, 4 Eselectin ligand-1 (ESL-1), 5 macrophage antigen-1 (Mac-1; ␣ M ␤ 2 ), 6 and other unknown ligands. E-selectin engagement induces the activation of a receptor-proximal Src family immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor proteinSyk signaling pathway, which induces lymphocyte functionassociated antigen-1 (LFA-1)-dependent slow rolling in vitro and in vivo. 7,8 Selplg Ϫ/Ϫ and Syk Ϫ/Ϫ neutrophils cannot reduce their rolling velocity when rolling on E-selectin and intercellular adhesion molecule-1 (ICAM-1) in flow chamber experiments. 8 In neutrophils from Tyrobp Ϫ/Ϫ Fcrg Ϫ/Ϫ (DAP12-and FcR␥-deficient) mice, E-selectin engagement fails to phosphorylate Syk and does not induce slow rolling. 7 Binding of neutrophils to E-selectin under shear induces the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), 7 and blocking of p38 MAPK elevates the rolling velocity on E-selectin and ICAM-1 compare...

Leukocyte recruitment to sites of infection or tissue damage plays a crucial role for the innate immune response. Chemokine-dependent signaling in immune cells is a very important mechanism leading to integrin activation and leukocyte recruitment. CXC chemokine receptor 2 (CXCR2) is a prominent chemokine receptor on neutrophils. During the last years, several studies were performed investigating the role of CXCR2 in different diseases. Until now, many CXCR2 inhibitors are tested in animal models and clinical trials and promising results were obtained. This review gives an overview of the structure of CXCR2 and the signaling pathways that are activated following CXCR2 stimulation. We discuss in detail the role of this chemokine receptor in different disease models including acute lung injury, COPD, sepsis, and ischemia-reperfusion-injury. Furthermore, this review summarizes the results of clinical trials which used CXCR2 inhibitors.

Endothelial basement membranes constitute barriers to extravasating leukocytes during inflammation, a process where laminin isoforms define sites of leukocyte exit; however, how this occurs is poorly understood. In addition to a direct effect on leukocyte transmigration, we show that laminin 511 affects endothelial barrier function by stabilizing VE-cadherin at junctions and downregulating expression of CD99L2, correlating with reduced neutrophil extravasation. Binding of endothelial cells to laminin 511, but not laminin 411 or non-endothelial laminin 111, enhanced transendothelial cell electrical resistance (TEER) and inhibited neutrophil transmigration. Data suggest that endothelial adhesion to laminin 511 via β1 and β3 integrins mediates RhoA-induced VE-cadherin localization to cell-cell borders, and while CD99L2 downregulation requires integrin β1, it is RhoA-independent. Our data demonstrate that molecular information provided by basement membrane laminin 511 affects leukocyte extravasation both directly and indirectly by modulating endothelial barrier properties.

Rolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophil rolling on E-selectin induces integrin αLβ2-mediated slow rolling on intercellular adhesion molecule-1 by activating a phospholipase C (PLC)γ2- and a separate phosphoinositide-3-kinase (PI3K)γ-dependent pathway. E-selectin-signaling cooperates with chemokine signaling to recruit neutrophils into inflamed tissues. However, the distal signaling pathway linking PLCγ2 (Plcg2) to αLβ2-activation is unknown. To identify this pathway, we used different TAT-fusion-mutants and gene-deficient mice in intravital microscopy, autoperfused flow chamber, peritonitis, and biochemical studies. We found that the small GTPase Rap1 is activated following E-selectin engagement and that blocking Rap1a in Pik3cg−/− mice by a dominant-negative TAT-fusion mutant completely abolished E-selectin mediated slow rolling. We identified CalDAG-GEFI (Rasgrp2) and p38 MAPK as key signaling intermediates between PLCγ2 and Rap1a. Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster muscle were completely abolished in Rasgrp2−/− mice. The physiologic importance of CalDAG-GEFI in E-selectin-dependent integrin activation is shown by complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Rasgrp2−/− leukocytes treated with pertussis toxin to block Gαi-signaling. Our data demonstrate that Rap1a activation by p38 MAPK and CalDAG-GEFI is involved in E-selectin-dependent slow rolling and leukocyte recruitment.

Leukocyte recruitment to the kidney during acute injury is mediated by E-selectin–mediated rolling and requires SLP-76 and the adaptor protein ADAP.

A subset of PSGL-1 is constitutively associated with L-selectin and signals through Src family kinases to activate LFA-1, which regulates neutrophil slow rolling and recruitment.