AbstractExcessive neutrophilic inflammation of the airways in response to infection is characteristic for patients with CF. There is also an important but not fully understood role for platelets. Previous studies established increased copper levels in the circulation and in the sputum in CF. Inhaled tobramycin was suggested to have an anti-inflammatory effect beyond eradicating Pseudomonas aeruginosa. This study tested the hypothesis that tobramycin has anti-inflammatory and anti-oxidant efficacy due to its ability to bind copper into a copper-tobramycin complex.
A copper-tobramycin complex was synthesised and the UV-VIS spectrum analysed. Neutrophil migration through a TNF-α-stimulated human lung microvascular endothelial cell layer towards thrombin-activated platelets was measured. The role of CFTRinh-172 on neutrophil transendothelial migration was assessed. Endothelial tobramycin uptake and CFTR expression were assessed using immunocytochemistry. Endothelial oxidative stress was measured using a fluorescent indicator. Neutrophils were stimulated to measure reactive oxygen species (ROS) production and neutrophil elastase (NE) activity, spectrophotometrically.
Platelet and endothelium-derived NAP-2 and IL-8, respectively, contributed to neutrophil transendothelial migration. Copper-tobramycin was shown to be more effective than tobramycin in limiting migration of neutrophils. Both, tobramycin and copper-tobramycin accumulated in endothelial cells via a heparan sulphate-dependent mechanism, decreased intracellular ROS and increased endothelial surface CFTR expression. CFTRinh-172 failed to create an inflammatory profile in endothelium. Copper-tobramycin decreased extracellular superoxide released by activated neutrophils, and displaced NE from sites of encryption, making it more susceptible to inhibition by α1-antitrypsin.
The antibiotic tobramycin was demonstrated to be a multi-potent drug with additional anti-inflammatory and anti-oxidant properties. These effects, desirable in CF treatment, are due to copper binding.
|Date of Award
|Jan Shute (Supervisor) & David Laight (Supervisor)