Pulmonary Endothelial Cells by Pasteurella haemolytica Leukotoxin

In this study, we used an in vitro coculture system to determine which virulence factor from Pasteurella haemolytica Al was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A 5"Cr release cytotoxicity assay was used as a measure of BPAEC killing. The mechanisms associated with this BPAEC killing were also studied. Our results demonstrated that the leukotoxin and not the lipopolysaccharide from P. haemolytica was responsible for augmenting the PMN-mediated killing of BPAEC. Furthermore, this augmented killing was related to the stimulation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimulated PMNs was diminished in the presence of the H202 inactivator, catalase. The membrane-permeant H202, hydroxyl radical (HO-) scavenger 1,3-dimethyl-2 thiourea, and the HOscavenger dimethyl sulfoxide but not the myeloperoxidase inhibitor sodium azide attenuated this BPAEC killing. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection against BPAEC killing induced by leukotoxin-stimulated PMNs. These data were compatible with the concept that the H202 generated by leukotoxin-stimulated PMNs interacts with intracellular iron in the endothelial cell to form highly reactive HO. We suggest that HO' may be a key factor in BPAEC killing. Furthermore, since the elastase-specific inhibitor N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also attenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functioned additively in protecting against BPAEC killing, we conclude that both HO and elastase may jointly contribute to BPAEC killing induced by leukotoxin-stimulated PMNs. This study broadens our understanding of how leukotoxin-stimulated PMNs injure lung endothelial cells and provides new insight into the pathogenesis of bovine pneumonic pasteurellosis.