Targeted by Metalloproteinases Identification of Steps and Substrates during Transendothelial Migration: Monocyte ADAM17 Promotes Diapedesis

Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metal-loproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metal-loproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial-and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloprotein-ase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knock-down of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of trans-endothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium. I nflammation is a key process in disease pathogenesis, and leukocyte transendothelial migration into inflamed tissues controls both initiation and progression of acute and chronic inflammatory diseases. Transendothelial migration is a sequential, multistep process that consists of leukocyte rolling, capture, and firm adhesion to endothelium, followed by locomotion across the endothelium and diapedesis through the intact vessel into the tissue, usually at endothelial junctions (1–3). Engagement of key ligands and their counterreceptors expressed on both leukocytes and en-dothelial cells have been shown to be responsible for each step of transendothelial migration (1–3). However, very little is known about regulatory mechanisms underlying the progression through each step and transitions between steps. A molecular mechanism capable of rapidly reducing adhesion molecule interactions is their proteolytic cleavage or " ectodomain shedding " (4). In fact, soluble forms of multiple adhesion molecules involved in each step of leukocyte transendothelial migration are proteolytically shed, and elevated levels are detected in physiological fluids from various inflammatory diseases (4, 5). …