Exploring the role of L-selectin shedding in regulating neutrophil polarity and chemotaxis

Inflammation is a biological response to injury – a fundamental part of which involves the influx of leukocytes to the affected area. Circulating neutrophils are typically the first responders to an inflammatory insult. Orchestrated interactions between neutrophils and the underlying endothelium and occurs in a process termed the “multi-step leukocyte adhesion cascade”. L-selectin, a cell adhesion molecule, expressed on all circulating neutrophils, and facilitates the initial tethering and rolling events. Previously, L-selectin on CD14-positive (“inflammatory”) monocytes was shown to undergo ectodomain “shedding”, exclusively during transendothelial migration (TEM) and not before. The consequence of this was to establish monocyte polarity within the subendothelial space. The primary aim of this project was to determine if L-selectin shedding during TEM was exclusive to monocytes or extended to neutrophils. Perfusion of primary human neutrophils over activated endothelial monolayers revealed that L-selectin shedding was also activated specifically during TEM. Ectopic expression of L-selectin tagged to green fluorescent protein (GFP) in HL-60 cells (a neutrophil-like cell line that does not express endogenous L-selectin) significantly increased their invasive potential across activated endothelial cells under flow. Blocking L-selectin shedding (either pharmacologically in primary neutrophils or genetically in HL60 cells) promoted cell elongation in fully transmigrated cells, which significantly impacted cell polarity and directional persistence in 2D and 3D collagen scaffolds. Additionally, manufacturing bespoke microfluidic flow chamber devices revealed that blocking L-selectin shedding impacted the neutrophil chemotaxis towards classic “end-stage chemoattractants”, such as fMLP. Taken together, L-selectin shedding ensures that fully transmigrated neutrophils can establish front-back polarity so that they can home optimally to their inflamed targets. As fMLP is secreted during sterile injury, the potential therapeutic targeting of L-selectin shedding in the setting of myocardial infarction is discussed.