ENHANCEMENT OF NEUTROPHIL-MEDIATED INJURY TO BOVINE 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 A1 was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A Cr-51 release cytotoxicity assay was used as a measure of BPAEC killing. The mechani sms 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 stimu lation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimu lated PMNs was diminished in the presence of the H2O2 inactivator, cat alase. The membrane-permeant H2O2, hydroxyl radical (HO.) scavenger 1, 3-dimethyl-2 thiourea, and the HO. scavenger dimethyl sulfoxide but no t the myeloperoxidase inhibitor sodium azide attenuated this BPAEC kil ling. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection aga inst BPAEC killing induced by leukotoxin-stimulated-PMNs. These data w ere compatible with the concept that the H2O2 generated by leukotoxin- stimulated PMNs interacts with intracellular iron in the endothelial c ell to form highly reactive HO.. We suggest that HO. may be a key fact or in BPAEC killing. Furthermore, since the elastase-specific inhibito r N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also at tenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functi oned 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 o f how leukotoxin-stimulated PMNs injure lung endothelial cells and pro vides new insight into the pathogenesis of bovine pneumonic pasteurell osis.