The CX3C chemokine fractalkine (CX3CL1) exists as a membrane-expressed protein promoting cell-cell adhesion and as a soluble molecule inducing chemotaxis. Transmembrane CX3CL1 is converted into its soluble form by defined proteolytic cleavage (shedding), which can be enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). PMA-induced CX3CL1 shedding has been shown to involve the tumor necrosis factor-␣-converting enzyme (TACE), whereas the constitutive cleavage in unstimulated cells remains elusive. Here we demonstrate a role of the closely related disintegrin-like metalloproteinase 10 (ADAM10) in the constitutive CX3CL1 cleavage. The hydroxamate GW280264X, capable of blocking TACE as well as ADAM10, proved to be an effective inhibitor of the constitutive and the PMA-inducible CX3CL1 cleavage in CX3CL1-expressing ECV-304 cells (CX3CL1-ECV-304), whereas GI254023X, preferentially blocking ADAM10 but not TACE, reduced the constitutive cleavage only. Overexpression of ADAM10 in COS-7 cells enhanced constitutive cleavage of CX3CL1 and, more importantly, in murine fibroblasts deficient of ADAM10 constitutive CX3CL1 cleavage was markedly reduced. Thus, ADAM10 contributes to the constitutive shedding of CX3CL1 in un- IntroductionLeukocyte recruitment to inflammatory sites involves a sequence of adhesive events that are mediated by different classes of adhesion molecules expressed on the endothelium and the leukocytes. 1 Whereas adhesion molecules of the selectin family usually contribute to the rolling of leukocytes under flow, members of the integrin family are involved in establishing a stable shear-resistant cell adhesion. Chemokines are thought to play a role in modulating cell adhesion by inducing shedding of L-selectin and by increasing functional integrins on the leukocyte surface. Thus, besides acting as chemoattractants in the tissue, chemokines can promote the transition from an early to a late adhesion type in the course of leukocyte recruitment.Within the chemokine family a transmembrane molecule termed CX3C chemokine ligand 1 (CX3CL1), or fractalkine, has been identified that by itself induces adhesion. 2 CX3CL1 is encoded as a 95-kDa multidomain molecule consisting of a chemokine domain linked to a transmembrane domain via a mucin-rich stalk. The chemokine is expressed on endothelial cells, 2 epithelial cells, 3,4 smooth muscle cells, 5,6 dendritic cells, 7,8 neurons, 9,10 and macrophages. 11 In vitro, CX3CL1 induces cell adhesion by interaction with its receptor CX3CR1 expressed on monocytes, T cells, mast cells, and natural killer cells. 2,[12][13][14] This adhesion does not require signaling of the receptor, is resistant to physiologic shear flow, and is independent of extracellular calcium. 2,15,16 Besides its activity as an adhesion molecule, CX3CL1 can be cleaved from the cell membrane to produce a soluble 80-kDa molecule that induces chemotaxis of CX3CR1-expressing leukocytes. 2 In vivo, upregulation of CX3CL1 has been found in atherosclerotic blood vessels, 6,11 rejected transplants, 1...