The pro-inflammatory role of copper in chemokine multimerisation and the potential of copper chelators as anti-inflammatory agents
Student thesis: Doctoral Thesis
During inflammatory reactions, leukocytes migrate from the circulation into extravascular tissues. Chemokines, 7-10 KDa proteins, immobilised on the endothelial cell surface bound to glycosaminoglycans, such as heparan sulphate, on proteoglycans have been implicated in this process. Evidence suggests that chemokines are functional in multimeric form bound to heparan sulphate. In this form, endothelial bound RANTES is a T-cell, monocyte and eosinophil chemoattractant. T-cell accumulation in respiratory tissue is a feature of asthma, cystic fibrosis and chronic obstructive pulmonary disease where T-cell derived cytokines orchestrate the inflammatory response. Central to inflammation, platelets migrate into tissues, augmenting the inflammatory response by degranulating and releasing their contents, including RANTES on activation. Since both copper and dityrosine links have been implicated in the multimerisation of the amyloid protein in Alzheimer's disease, chemokines including RANTES, IL-8 and ENA-78 were investigated for the possibility of copper-induced dityrosine formation within chemokine multimers. The addition of CuC12 and H202 to human recombinant RANTES induces multimerisation and dityrosine cross-linking, confirmed by fluorimetry, liquid chromatography mass spectroscopy and staining Western blots with a dityrosine specific monoclonal antibody. In addition, RANTES multimers actively induce chemotaxis in Boyden chambers. This finding led to the investigation of the Tcell response to endothelial and platelet derived RANTES in the absence and presence of copper chelators as potential anti-inflammatory agents in transendothelial migration assays, a physiological model of the vascular endothelium. Migration of activated Tcells across monolayers of human lung microvascular endothelial cells was RANTES-dependent and RANTES derived from thrombin stimulated platelets is active as a T-cell chemoattractant in this model of the lung microvascular endothelium. The copper chelators neocuproine, bathocuproine, D-penicillamine and tobramycin significantly inhibited T-cell migration indicating a pro-inflammatory role for copper and suggesting the use of copper chelators as potential anti-inflammatory agents.
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