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Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes¹

Christian Hundhausen^2,*, Alexander Schulte^2,*, Beate Schulz^*, Michael G. Andrzejewski, Nicole Schwarz, Philipp von Hundelshausen, Ulrike Winter^*, Krzysztof Paliga^*, Karina Reiss^*, Paul Saftig^*, Christian Weber and Andreas Ludwig^3,

^* Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany; and Institute for Molecular Cardiovascular Research, Rheinisch-Westfälische Technische Hochschule University Hospital, Aachen, Germany

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by Deutsche Forschungsgemeinschaft Grant LU 869/1-3.

² C.H. and A.S. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Andreas Ludwig, Institute for Molecular Cardiovascular Research, University Hospital, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. E-mail address: aludwig{at}ukaachen.de

⁴ Abbreviations used in this paper: ADAM, disintegrin and metalloproteinase; CTF, C-terminal cleavage fragment; FN, fibronectin; mCD, methyl--cyclodextrin; MEF, mouse embryonic fibroblast; SLO, streptolysin O.

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