Requirements for cleavage and function of the transmembrane chemokine fractalkine/CX3CL1

Leukocytes are recruited from the blood vessel wall towards the inflamed tissue. This process occurs in multiple steps and is regulated in part by chemokines. Among the large family of chemokines that mediate directional cell migration an unusual member the CX3CL1 (fractalkine) is described existing as a transmembrane and a soluble molecule. The generation of this soluble CX3CL1 molecule occurs upon ectodomain shedding by metalloproteinases, in particular by ADAM10. By binding to the receptor CX3CR1 the transmembrane CX3CL1 mediates adhesion and transmigration of leukocytes subpopulations. Up to date, it is not clear how ADAM10 contributes to the function of mouse CX3CL1. The goal of this thesis was to characterise the molecular determinants of mouse CX3CL1 for shedding and function of this molecule. Results of this study demonstrated that mouse CX3CL1 is predominately shed by ADAM10-dependent mechanisms as shown by pharmacological inhibition and transcriptional silencing. As described for human CX3CL1, shedding of mouse CX3CL1 is enhanced by ionomycin treatment, which also involves ADAM10-activity. Deletion and replacement of the cleavage region of mouse CX3CL1 could not further abrogate its release suggesting that other domains of mouse CX3CL1 may influence this process. And indeed, release of mouse CX3CL1 was found to be further influenced by the chemokine domain and also the cytoplasmic tail. However, truncation of the cytoplasmic tail led also to impaired cellular trafficking. In fact, when wt mouse CX3CL1 and the truncated variant were sufficiently expressed on the cell surface, both were still shed. Additionally, adhesion and transmigration experiments with human PBMCs revealed that the truncation did not affect the function of the transmembrane mouse CX3CL1 as an adhesion and a transmigration molecule. This latter finding suggests that potential signalling events induced by the cytoplasmic tail of CX3CL1 are not involved in the recruitment of leukocytes to the inflamed tissue. This work indicates that multiple molecular determinants within mouse CX3CL1 influence its shedding via ADAM10, suggesting that exchange of several domains of mouse CX3CL1 cannot completely prevent its shedding. The cytoplasmic tail of mouse CX3CL1 appears to be relevant for correct trafficking of mouse CX3CL1 but neither for shedding, adhesion or transmigration. Nevertheless, shedding of CX3CL1 by ADAM10 and ADAM17 could still represent a crucial step within the leukocyte recruitment process in developing vascular diseases such as atherosclerosis