Theo A. Berkhout

The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10.

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.

Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN-γ and TNF-α and Is Modulated by Metalloproteinase Activity

Fractalkine/CX3C-chemokine ligand 1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). In this study we demonstrate that IFN-γ and TNF-α, but not IL-1β, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-γ and TNF-α together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.

Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59–62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24–98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 ± 0.15 nm) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 ± 1.4, 0.85–1.6, and 0.7 ± 0.2 nm respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

The role of fractalkine in the recruitment of monocytes to the endothelium.

Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.

CCR2B receptor antagonists : Conversion of a weak HTS hit to a potent lead compound

A weak HTS hit at the CCR2B receptor has been converted into a potent antagonist by array SAR studies. Selectivity over the closely related CCR5 receptor is also achieved.

Conformationally restricted indolopiperidine derivatives as potent CCR2B receptor antagonists

The preparation and biological evaluation of a series of indolopiperidine CCR2B receptor antagonists possessing a conformationally restricted C-5 linker chain in combination with a restricted piperidine ring are described. Compared to the parent compound 1, analogue 8 shows a dramatic improvement in selectivity against a range of 5-HT and dopaminergic receptors.

Selective binding of the truncated form of the chemokine CKβ8 (25–99) to CC chemokine receptor 1 (CCR1)

Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKbeta8. To obtain conclusive evidence, binding-displacement studies of 125I-CKbeta8 (25-99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The Ic50 for displacement of 125I-CKbeta8 (25-99) with CKbeta8 (25-99) was 0.22 nM. The longer forms of CKbeta8 (24-99 and 1-99) also displaced 125I-CKbeta8, with Ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKbeta8 (25-99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKbeta8. We conclude that CCR1 is a receptor for different-length CKbeta8 and that CKbeta8 (25-99) has a similar affinity for CCR1 as macrophage inflammatory protein-1alpha (MIP-1alpha). The longer variants of CKbeta8 are significantly less potent than CKbeta8 (25-99) and MIP-1a on CCR1 and monocytes (P < 0.05).