Cytotoxicity of Stimulated Equine Neutrophils on Equine Endothelial Cells in Culture

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.