Ectodomain shedding of L-selectin by ADAM17 in canine neutrophils

Abstract

The adhesion protein L-selectin (CD62L) is expressed at high levels by circulating neutrophils and has a critical role in initiating their recruitment at sites of inflammation. L-selectin expression is rapidly downregulated upon neutrophil activation by various stimuli through a proteolytic process referred to as ectodomain shedding, which regulates L-selectins binding avidity and neutrophil recruitment. In humans and mice, L-selectin shedding is primarily mediated by ADAM17 (a disintegrin and metalloproteinase 17). L-selectin expression is also rapidly downregulated by canine neutrophils upon their activation; however, the role of ADAM17 in this process has not been previously investigated. We show that a highly selective inhibitor of ADAM17, but not an inhibitor of its most closely related family member ADAM10, effectively blocked L-selectin downregulation from the surface of canine neutrophils following their activation. The ADAM17 inhibitors did not block the rapid upregulation of the CD18 integrin Mac-1 (CD11b/CD18), showing that they did not broadly impair neutrophil activation. To directly examine the expression of ADAM17, we used several anti-human ADAM17 mAbs. Many did not stain canine neutrophils; however, the ADAM17 function-blocking mAbs MEDI3622 and D1(A12) did stain and also blocked L-selectin downregulation. Taken together, our findings provide the first direct evidence that ADAM17 is a primary sheddase of canine L-selectin, which may serve as an important therapeutic target to prevent neutrophil dysfunction during conditions of excessive inflammation, such as sepsis.