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Embo molecular medicine 5(3), 471-481 (2013). doi:10.1002/emmm.201201717, Published by Wiley-Blackwell, Chichester

Chemokines are important mediators and regulators of leukocyte trafficking, therefore, they play a crucial role in the development of inflammatory diseases. CCL5 or RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemokine of relevance to many diseases. Moreover, CCL5-induced monocyte adhesion to inflamed endothelium was shown to be improved in the presence of CXCL4 (Platelet Factor 4). Since this synergy could be attributed to heterodimer formation, the first section of the present study surveys the structural interaction of CCL5 with CXCL4. The interaction was monitored employing the 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) technique. For this purpose, 15N-enriched CCL5 was recombinantly expressed in E. coli and subsequently purified. In HSQC spectroscopy, chemical shift changes were mainly observed in the N-terminal residues, which pointed toward a CC-type rather than a CXC-type interaction. Furthermore, small peptide antagonists, inhibiting the CXCL4/CCL5 dimerization, were designed (CKEY2 and the mouse orthologue MKEY). To investigate their pharmacological potential, the influence of MKEY on leukocyte adhesion to activated endothelium was monitored using intravital microscopy. As a control Met-CCT5, a strong antagonist for CCR1 and CCR5, was cloned, expressed and purified employing FPLC and HPLC techniques. Leukocyte recruitment was severely impaired in the presence of MKEY, compared to a control peptide (sMKEY) and in a similar range of Met-CCL5 which encourages the assumption that the synergy is mediated via the receptors CCR1 and/or CCR5. Despite all similarities, CCR1 and CCR5 were shown to mediate distinct functions when bound to CCL5, CCR1 rather mediates arrest and CCR5 appears to be more responsible for transendothelial migration. To establish which domains are important for this functional selectivity, we constructed different CCR5 variants with the distinct extracellular regions of CCR1. These chimeras were stably expressed in L1.2 and HEK293 cells and we investigated their function in response to CCL5, different CCL5 mutants, or together with CXCL4 using chemotaxis and cell arrest assays under laminar flow. First of all, CCL5, CCL5 40s and CCL5-E66A were recombinantly expressed and purified employing FPLC and HPLC techniques. By implementing CCL5 mutants (e.g. CCL5-E66A) with oligomerization defects in laminar flow assays, we were able to show that all receptor variants require oligomerization of CCL5 in order to function properly. In addition, our results reveal that the 40S loop of CCL5 is important for both the CCR1- and CCR5-mediated cell arrest. The 50s loop of CCL5, however, appeared to have a strong preference for CCR5 in inducing cell arrest, since CCR1 responded normal towards CCL5 50s and CCR5 being non-responsive. When the N-terminal domain of CCR5 was exchanged for that of CCR1, the resulting chimera was fully responsive towards CCL5 50s, suggesting that the N-terminal region of CCR1 interacts with the 50s domain of CCR5. The synergistic effect of CXCL4 on CCL5 induced cell arrest was observed in cells exclusively expressing CCR1 when compared to cells expressing CCR5. When the third extracellular loop of CCR1 was engineered into CCR5, the resulting chimeric receptor showed a significant response to the CXCL4/CCL5 heterocomplex, compared to CCL5 alone. These results were confirmed by constructing CCR1-based reverse chimeras for the N-terminal domain and the third extracellular loop. Furthermore we could show the heterodimerization of CCR1 and CCR5 and the synergy of the CXCL4/CCL5 complex is in THP-1 cells mediated via Gαi. In conclusion these results indicate that the extracellular regions of CCR1 and CCR5 have distinct and defined functions in leukocyte recruitment in response to CCL5. In the third section of this thesis the role of the sialyltransferase ST3Gal-IV on CCL5 receptor interaction was investigated, by using neutrophils and monocytes isolated from ST3Gal-IV deficient and from control mice in functional assays in vitro. The results indicate that the addition of sialic acids to the terminal portions of the N- or O-linked sugar chains of the corresponding receptors of CCL5 is of a minor importance for receptor binding and activation, since the cells similarly mobilize calcium upon stimulation with CCL5. Whereas, the adhesion of neutrophils and monocytes from ST3Gal-IV-/- was significant diminished. Taken together the results obtained here rather support the importance of ST3Gal-IV on the generation of functional selectins, which is in line with previous publications.