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%0 Thesis
%A Schwarz, Nicole
%T Requirements for leukocyte transendothelial migration via the transmembrane chemokines CX3CL1 and CXCL16
%C Aachen
%I Publikationsserver der RWTH Aachen University
%M RWTH-CONV-114023
%P 135 S. : graph. Darst.
%D 2010
%Z Aachen, Techn. Hochsch., Diss., 2010
%X The chemokines CX3CL1 and CXCL16 and their receptors CX3CR1 and CXCR6 are described in vascular inflammation and inflammatory cell recruitment. CX3CL1 and CXCL16 are transmembrane surface proteins on endothelial cells inducing firm adhesion of leukocytes via the interaction with their receptors. After shedding from the cell surface by the metalloproteinases ADAM10 and ADAM17, they act as soluble chemoattractants for CX3CR1- and CXCR6-expressing leukocytes, respectively. Here, it was demonstrated that expression of transmembrane CX3CL1 on endothelial cells promotes leukocyte transendothelial migration, and details of the underlying mechanisms using mutated CX3CR1 variants were elucidated. The DRY motif required for Gi-protein-coupling was mutated to DNY, which abolished the intracellular calcium release in response to CX3CL1, but did neither affect CX3CL1 binding nor uptake. Truncation of the C-terminus reduced ligand uptake, but not ligand binding and calcium responses. Both variants effectively mediated firm cell adhesion, but not chemotaxis towards soluble CX3CL1. Furthermore, they failed to induce transmigration, but mediated retention of leukocytes on the CX3CL1-expressing cell layer. Pharmacologic and transcriptional inhibition of ADAM10 led to reduced shedding of transmembrane CX3CL1, which was associated with an almost complete suppression of transmigration in response to transmembrane CX3CL1. These results indicate a multistep process of leukocyte recruitment by transmembrane CX3CL1 involving adhesion, signaling, initiation of transmigration, and finally proteolytic release of the transmigrating leukocytes. In contrast, transmembrane CXCL16 did not promote adhesion of CXCR6-expressing cells, while soluble CXCL16 mediated chemotaxis. CXCR6 bears a DRF instead of the DRY motif. Since this mutation is implicated in the constitutive activity of other receptors, the DRF of CXCR6 was changed into DRY and DNF. Reconstitution of the DRY motif did not affect ligand binding and resulted in a slight decrease in calcium signaling, whereas the mutation into DNF abolished calcium signaling. Both mutated receptors still failed to induce adhesion to CXCL16-expressing cells. Signaling seemed to depend on the arginine residue but not on the tyrosine/phenylalanine residue in DRY/F. These results indicate that CXCL16 predominantly functions as a soluble chemokine. Furthermore, cell recruitment by transmembrane chemokines differs. While CX3CL1 induces signaling-independent adhesion and signaling-dependent transmigration, CXCL16 does not induce adhesion, but chemotaxis.
%K Leukozyt (SWD)
%K Chemotaxis (SWD)
%K Chemokine (SWD)
%K Adhäsion (SWD)
%K Metalloproteinasen (SWD)
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%U https://publications.rwth-aachen.de/record/51766