The Transmembrane CXC-Chemokine Ligand 16 Is Induced by IFN-γ and TNF-α and Shed by the Activity of the Disintegrin-Like Metalloproteinase ADAM10

Abstract

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

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

“…We and others have found that ADAM10 is responsible for most of the constitutive shedding, but not for the PMA-induced cleavage of CXCL16 (15,36). A role of ADAM17 in the latter process has been suggested by inhibitor studies (15), but needs to be confirmed by ADAM17-deficient cell lines.…”

“…The antiserum against CX3CL1 was raised in rabbits and characterized previously (39). Expression vectors for human CX3CL1 and CXCL16 cDNA were described before (15,34). For detection of C-terminal cleavage fragments (CTFs) of CX3CL1, a 2Z-tag (40) was fused to the C terminus of CX3CL1.…”

“…Quantification of CX3CL1 and CXCL16 was performed, as described before (15,34). Briefly, a 96-well plate (Microlon; Greiner Bioscience) was coated overnight with 6 g/ml mouse anti-CX3CL1 (clone 81506) in 50 mM Na 2 CO 3 (pH 9.3), subsequently blocked with PBS-T containing 2% BSA for 2 h. Samples (50 l/well) and a standard prepared as eight serial 1/2 dilutions of 3.9 nM full-size CX3CL1 in either medium or cell lysis buffer were added to the plate for 2 h. Following washing, 300 ng/ml biotinylated anti-CX3CL1 mAb (clone 51637) in PBS-T containing 1% BSA was added to each well, and the plate was incubated at room temperature for 1 h. After 1-h incubation with 100 mU/ml streptavidin-peroxidase conjugate (Roche) in PBS-T with 1% BSA, the bound enzymatic activity was quantified using a chromogenic peroxidase substrate (BM blue; Roche).…”

Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

“…1D). Since the action of endogenous metalloproteinases may facilitate the release of soluble CXCL16 into the tissue culture supernatant [4,5], chemotaxis of CXCR6-transfected L1.2 cells to varying dilutions of culture media was assayed (Fig. 1E).…”

“…Although typically produced as soluble, secreted proteins, the chemokine CXCL16 also exists as a membrane-bound form, consisting of a chemokine domain, a mucin-type stalk, a single-pass transmembrane (TM) domain and a cytoplasmic tail [2,3]. The mucin stalks serve to tether the chemokines to the cell membrane, while soluble versions of CXCL16 can be released by the action of membrane metalloproteinases [4,5].…”