Development of a Wound Dressing Targeting Neutrophil Function

Abstract

Earlier studies show that neutrophils are virtually unable to kill Staphylococcus aureus in vitro. However, upon addition of 10 mM N-acetylcysteine (NAC) or reduced glutathione (GSH) the neutrophil bacterial killing ability becomes excellent. We want to exploit this phenomenon to develop a wound dressing material that will improve neutrophil function. To study the mechanisms behind the downregulation of neutrophil elimination of bacteria, we used different markers for neutrophil function on surface-adhering neutrophils in contact with S. aureus with or without addition of the antioxidants NAC or GSH. Analysis by scanning electron microscopy showed cell shrinkage and numerous cytoplasmic processes on surface-adhering neutrophils exposed to S. aureus. In cells exposed to S. aureus and GSH, the cells were of normal size and the cytoplasm was spread as in normal attachment. Staining for intracellular GSH, a hallmark of oxidative stress, showed little difference between the experimental groups, indicating that the cells were not damaged by traditional oxidative stress. The H₂O₂ production of neutrophils, measured by Amplex red, was correlated to bacterial exposure and was not affected by the addition of scavengers. The intracellular and extracellular production of ROS was measured by luminol-amplified chemiluminescence. The apparent ROS-production was mostly intracellular and decreased in the presence of scavengers. However, extracellular production of ROS was not affected by the addition of NAC. The production of nitric oxide (NO) was measured spectrophotometrically as the production of nitrate apparently decreased in the presence of scavengers, probably as a result of interference with the reagents in the test system. In conclusion, differences between leukocytes that were able to eliminate S. aureus and those that were not were mainly seen in the morphology of the cells and in cell viability. The morphological findings point to a difference in NO signaling in the absence and presence of ROS scavengers.