Thursday, 20 April 2017 11:22

Ischemia-Reperfusion Injury Following Transplantation: Implications for the Modulation of CXCL8 Chemokine Function

B. Martinez Burgo,1 S. Ali,1 J. Kirby,1 N. Sheerin,1 S. Cobb,2 D. Kashanin3

1Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, UK
2Chemistry Department, Durham University, Durham, UK
3Cellix Ltd., Dublin, Ireland

Am J Transplant. 2016; 16 (suppl 3)

 

Abstract

Ischemia-Reperfusion Injury (IRI) is a major contributor to subsequent graft dysfunction. Chemokines play a key role in leukocyte recruitment towards to the injured tissue. CXCL8 is a critical inflammatory mediator of neutrophil migration, such as in kidney transplantation. Chemokines, in addition to binding with their G protein-coupled receptor (GPCR) on the leukocyte surface, also interact with endothelial glycosaminoglycan (GAG). Therefore, their activity can be modulated by inhibiting either of these system. Our aim was to design peptides targeting the receptor, or GAG-binding region of CXCL8 to block the chemokine activity.

Methods: A range of CXCL8 peptides were synthesised to target receptor-binding domain and GAG-binding domain. Chemical synthesis was carried out by Solid-Phase Peptide Synthesis (SPPS), MS and HPLC, and Analytical HPLC. Initial characterization of neutrophils was done through flow cytometry and qRT-PCR. In vitro analysis of peptides was performed through chemotaxis and flow-based cell adhesion assays with primary neutrophils.

Results: CXCL8 peptides and peptide mutants corresponding to the GAG-binding region of CXCL8, mainly its C-terminal α-helix, were chemically synthesised and peptide purity analysed (90%). Selected peptides were studied in vitro for their role in neutrophil migration. Flow-based adhesion studies showed very significant decrease in adhesion of primary neutrophils to TNF-α-stimulated HUVECs (p<0.001) in co-incubation of CXCL8 with WT peptide or E70K peptide (glutamic acid substituted with lysine), suggesting a competitive role of peptides in displacing CXCL8 from GAG. WT showed less ability than E70K mutant to decrease adhesion, potentially because lysine binds more strongly to polyanionic GAG. However, difference was not significant. Moreover, neutrophil migration was also shown to decrease in preliminary studies of CXCL8 in co-treatment with the peptides (p<0.05). Future in vivo analysis may help to unravel modulatory roles.

Conclusion: Better understanding of chemokine function and strategies for competitive modulation could offer therapeutic opportunities to protect from neutrophil-derived damage during IRI.