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Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

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Abstract

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.

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Abbreviations

AM:

Acetoxymethylester

ADAM:

A disintegrin and a metalloproteinase

CCL2:

CC-chemokine ligand 2

CX3CL1:

CX3C-chemokine ligand 1

CX3CR1:

CX3C-chemokine receptor 1

ELISA:

Enzyme-linked immunosorbent assay

FCS:

Fetal calf serum

FITC:

Fluorescein isothiocyanate

Gi protein:

Inhibitory G protein

GPCR:

G protein-coupled receptor

hFc:

Human IgG1-Fc-fragment

HUVEC:

Human umbilical vein endothelial cells

ICAM:

Intercellular adhesion molecule

IFN:

Interferon

MMP:

Matrix metalloproteinase

PBMC:

Peripheral blood mononuclear cells

PBS:

Phosphate-buffered saline

PE:

Phycoerythrin

TNF:

Tumor necrosis factor

shRNA:

Short hairpin RNA

wt:

Wild-type

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Acknowledgments

We thank Tanja Kogel and Melanie Esser for expert technical assistance. This work was supported in part by the IZKF Aachen, and by the Deutsche Forschungsgemeinschaft (SFB542, A12, and FOR809, P5).

Author information

Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany

    Nicole Schwarz, Jessica Pruessmeyer, Franz M. Hess, Daniela Dreymueller, Stefan Uhlig & Andreas Ludwig

  2. Interdisciplinary Center for Clinical Research, RWTH Aachen University, 52074, Aachen, Germany

    Jessica Pruessmeyer, Daniela Dreymueller & Andreas Ludwig

  3. Institute of Physiology, RWTH Aachen University, 52074, Aachen, Germany

    Elena Pantaler

  4. Institute for Molecular and Cellular Anatomy, RWTH Aachen University, 52074, Aachen, Germany

    Anne Koelsch & Reinhard Windoffer

  5. Institute for Immunology, Christian-Albrechts University, 24118, Kiel, Germany

    Matthias Voss

  6. Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany

    Alisina Sarabi & Christian Weber

  7. Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University, 52074, Aachen, Germany

    Antonio S. Sechi

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  1. Nicole Schwarz
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Correspondence to Andreas Ludwig.

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SUPPLEMENTAL FIGURE 1:

HUVEC were stimulated with IFN-γ and TNF-α (both 10 ng/ml) to induce endogenous CX3CL1 expression or left unstimulated for 16 h. Subsequently, cells were incubated in the absence or presence of exogenous soluble CX3CL1 (10 nM) for 1 h to investigate potential surface binding of the chemokine. Subsequently, cells were fixed, permeabilized, and stained for CX3CL1 (red) as described [20]. Nuclei were stained in blue. Scale bars represent 20 µm (PPT 3790 kb)

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Schwarz, N., Pruessmeyer, J., Hess, F.M. et al. Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1. Cell. Mol. Life Sci. 67, 4233–4248 (2010). https://doi.org/10.1007/s00018-010-0433-4

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  • DOI: https://doi.org/10.1007/s00018-010-0433-4

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