Cellular and Molecular Life Sciences

, Volume 67, Issue 24, pp 4233–4248 | Cite as

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

  • Nicole Schwarz
  • Jessica Pruessmeyer
  • Franz M. Hess
  • Daniela Dreymueller
  • Elena Pantaler
  • Anne Koelsch
  • Reinhard Windoffer
  • Matthias Voss
  • Alisina Sarabi
  • Christian Weber
  • Antonio S. Sechi
  • Stefan Uhlig
  • Andreas Ludwig
Research Article

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.

Keywords

Leukocytes Endothelial cells Transmigration Adhesion Chemokine Fractalkine Metalloproteinase Shedding 

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

Supplementary material

18_2010_433_MOESM1_ESM.ppt (3.7 mb)
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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Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Nicole Schwarz
    • 1
  • Jessica Pruessmeyer
    • 1
    • 2
  • Franz M. Hess
    • 1
  • Daniela Dreymueller
    • 1
    • 2
  • Elena Pantaler
    • 3
  • Anne Koelsch
    • 4
  • Reinhard Windoffer
    • 4
  • Matthias Voss
    • 5
  • Alisina Sarabi
    • 6
  • Christian Weber
    • 6
  • Antonio S. Sechi
    • 7
  • Stefan Uhlig
    • 1
  • Andreas Ludwig
    • 1
    • 2
  1. 1.Institute of Pharmacology and ToxicologyRWTH Aachen UniversityAachenGermany
  2. 2.Interdisciplinary Center for Clinical ResearchRWTH Aachen UniversityAachenGermany
  3. 3.Institute of PhysiologyRWTH Aachen UniversityAachenGermany
  4. 4.Institute for Molecular and Cellular AnatomyRWTH Aachen UniversityAachenGermany
  5. 5.Institute for ImmunologyChristian-Albrechts UniversityKielGermany
  6. 6.Institute for Molecular Cardiovascular ResearchRWTH Aachen UniversityAachenGermany
  7. 7.Institute for Biomedical Engineering–Cell BiologyRWTH Aachen UniversityAachenGermany

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