Selectins and their ligands mediate leukocyte rolling allowing interactions with chemokines that lead to integrin activation and arrest. Here, we demonstrate that E-selectin is critical to induce a secondary wave of activating signals transduced specifically by E-selectin ligand-1, that induces polarized, activated αMβ2 integrin clusters at the leading edge of crawling neutrophils, allowing the capture of circulating erythrocytes or platelets. In a humanized model of sickle cell disease (SCD), the capture of erythrocytes by αMβ2 microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produces vascular damage and lung injury. Inactivation of E-selectin or αMβ2 prevented tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thrombo-inflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

Leukocyte adhesion in the microvasculature influences blood rheology and plays a key role in vaso-occlusive manifestations of sickle cell disease. Notably, polymorphonuclear neutrophils (PMNs) can capture circulating sickle red blood cells (sRBCs) in inflamed venules, leading to critical reduction in blood flow and vasoocclusion. Recent studies have suggested that E-selectin expression by endothelial cells plays a key role by sending activating signals that lead to the activation of Mac-1 at the leading edge of PMNs, thereby allowing RBC capture. Thus, the inhibition of E-selectin may represent a valuable target in this disease. Here, we have tested the biologic properties of a novel synthetic pan-selectin inhibitor, GMI-1070, with in vitro assays and in a humanized model of sickle cell vasoocclusion analyzed by intravital microscopy. We have found that GMI-1070 predominantly inhibited E-selectin-mediated adhesion and dramatically inhibited sRBC-leukocyte interactions, leading to improved microcirculatory blood flow and improved survival. These results suggest that GMI-1070 may represent a valuable novel therapeutic intervention for acute sickle cell crises that should be further evaluated in a clinical trial. (Blood. 2010; 116(10):1779-1786) IntroductionSickle cell disease (SCD), one of the most common inherited blood disorders in the United States, 1 results from a single amino acid substitution in the gene encoding the ␤-globin subunit. 2 The ␤-globin subunit polymerizes in deoxygenation, producing less deformable sickle red blood cells (sRBCs) that can obstruct blood vessels. 3 Recurrent vaso-occlusive episodes cause irreversible organ damage and contribute to morbidity and mortality in patients with sickle cell disease due to acute pain crises, chronic inflammation, and ischemic end-organ damage, such as pulmonary hypertension, renal failure, and cerebrovascular injury. 4 Although the molecular basis of SCD has been well characterized, the complex cellular and molecular mechanisms underlying vaso-occlusion (VOC) have not been fully elucidated.Recent studies have suggested that VOC is a complex cascade that involves multiple blood cells, adhesion, and signaling molecules. 5 Intravital microscopy analyses in a SCD mouse model expressing exclusively human globin genes 6 indicate that sRBCs interact primarily with adherent leukocytes (white blood cells [WBCs]) in postcapillary and collecting venules of cremasteric muscle and leading to vascular obstruction. 7 The key role for leukocyte adhesion in sickle cell vascular occlusions has been suggested by the amelioration of flow abnormalities in sickle transgenic mice by anti-inflammatory therapies directed at nuclear factor-B activation, reactive oxygen species, or endothelial adhesion molecules such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), or the selectins. [7][8][9] The selectins comprise a family of 3 members that mediate adhesion events between blood cells and the endothelium. Lselectin is constitutively expressed on le...

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF–differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, α(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand–1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A–injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.

We present a simple method to identify the recruitment of leukocyte subsets and determine concurrent surface-receptor clustering in live mice. We show that CD45+ F4/80- Gr-1+ neutrophils are robustly recruited in surgery-activated cremasteric venules, whereas adherent CD45+ B220+ B lymphocytes were dominant in bone marrow venules. Most adherent Gr-1+ leukocytes are not firmly stationary but actively migrate on TNF-alpha-activated cremasteric venular endothelium and exhibit marked polarization of surface PSGL-1, but not LFA-1, to the trailing edge.

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