E-selectin ligands as mechanosensitive receptors on neutrophils in health and disease

Abstract

Application of mechanical force to bonds between selectins and their ligands is a requirement for these adhesion receptors to optimally perform functions that include leukocyte tethering and activation of stable adhesion. Although all three selectins are reported to signal from the outside-in subsequent to ligand binding, E-selectin is unique in its capacity to bind multiple sialyl Lewis x presenting ligands and mediate slow rolling on the order of a micron per second. A diverse set of ligands are recognized by E-selectin in the mouse, including ESL-1, CD44 (HCELL), and PSGL-1 which are critical in transition from slow rolling to arrest and for efficient transendothelial migration. The molecular recognition process is different in humans as L-selectin is a major ligand, which along with glycolipids constitute more than half of the E-selectin receptors on human polymorphonuclear neutrophils (PMN). In addition, E-selectin is most efficient at raising the affinity and avidity of CD18 integrins that supports PMN deceleration and trafficking to sites of acute inflammation. The mechanism is only partially understood but known to involve a rise in cytosolic calcium and tyrosine phosphorylation that activates p38 MAP kinase and Syk kinase, both of which transduce signals from clustered E-selectin ligands. In this review we highlight the molecular recognition and mechanical requirements of this process to reveal how E-selectin confers selectivity and efficiency of signaling for extravasation at sites of inflammation and the mechanism of action of a new glycomimetic antagonist targeted to the lectin domain that has shown efficacy in blocking neutrophil activation and adhesion on inflamed endothelium.

Figure 1. Dynamic imaging of adhesive and signaling events that support neutrophil recruitment and assembly of the inflammatory synapse

Membrane tethers during rolling on a substrate of E-selectin is observed by phase contrast microscopy as cells flux calcium, detected as a red fluorescence using the intracellular reporter Fluoro-5. E-selectin ligands are clustered in lipid rafts as detected by DiI-C18, a fluorescent indocarbocyanine dye that preferentially partitions into lipid ordered domains (Red fluorescence) and antibody to L-selectin (green fluorescence). The inflammatory synapse is imaged with antibodies to L-selectin (green fluorescence) and PSGL-1 (red fluorescence) as previously reported⁵⁰.

Figure 2

PMN rolling on E-selectin and arrested in response to infusion of IL-8 at the indicated concentration. Neutrophils were loaded with Fluo-4 and perfused over monolayers expressing E-selectin at a shear stress of 2 dynes/cm², then exposed to a dose range of IL-8 following 2 min of shear interaction. (a) Individual neutrophils that have rolled to arrest rapidly increase their intracellular calcium in response to IL-8 (0.1 nM) resulting in an increase in Fluo-4 emission. (b) On average, neutrophils exhibited a rapid increase in Fluo-4 emission indicative of calcium flux in response to IL-8 concentrations of 0.1 nM or higher, but did not significantly increase calcium in unstimulated or at low IL-8 of 0.01 nM. Plot is representative of 4 independent experiments with measurements from at least 60 neutrophils at each labeled concentration of IL-8. c) Calcium concentration was measured by ratiometric imaging of neutrophils that sedimented onto the monolayer (Static) or rolling on the monolayer under shear stress (2 dynes/cm²) following exposure to a dose range of IL-8 from 0.001 to 5 nM. The average calcium concentration in all neutrophils in a field of view was measured over time, and the peak value was recorded. Calcium concentration was measured by ratiometric imaging in neutrophils sedimented onto the monolayer (Static) or rolling on the monolayer under shear stress (2 dynes/cm2) following exposure to a dose range of IL-8 from 0.001 to 5 nM. The average calcium concentration in all neutrophils in a field of view was measured over time, and the peak value was recorded. (adapted from Schaff et al., 2008. ABME)

Figure 3. Schematic of ESL ligation and signaling of Mac-1 dependent VOC

Following white blood cell tethering by E-selectin a signal is transduced resulting in a conformational upshift in Mac-1 to high affinity that facilitates binding to specific ligands on platelets captured from the blood stream.