Protective role of bovine neutrophils in Pasteurella haemolytica-mediated endothelial cell damage

Abstract

The purpose of this study was to determine if Pasteurella haemolytica can directly injure bovine pulmonary endothelial cells (EC) and if neutrophils have a beneficial or detrimental role in bacterium-EC interaction. Various combinations of live P. haemolytica, heat-killed P. haemolytica, anti-P. haemolytica immune serum, polymyxin B, and bovine neutrophils were added to confluent monolayers of bovine EC. Monitoring included determination of 51Cr release from EC, phase microscopy, and transmission electron microscopy. Although toxic changes were not evident at 5 h postinoculation, both live and heat-killed P. haemolytica produced extensive EC damage by 22 h postinoculation. Damage by live P. haemolytica was prevented only when both neutrophils and immune serum were used. Polymyxin B effectively prevented the toxic effect of heat-killed P. haemolytica, suggesting that lipopolysaccharide was the major toxic factor. Morphological studies showed close apposition of P. haemolytica to EC membranes, neutrophil activation, and adherence to EC but no evidence of neutrophil-associated EC membrane damage. These studies demonstrate that neutrophils and immune serum in combination are effective in preventing EC damage mediated by live P. haemolytica.

Bovine respiratory syncytial virus infection influences the impact ofα4- andβ2-integrin-mediated adhesion of peripheral blood neutrophils

“…Isolated cells were seeded into a 25‐cm 2 flask, coated with 1% gelatin (Merck, Darmstadt, Germany), and cultured in Iscove's tissue culture medium (G ibco BRL, Paisley, UK) supplemented with 10% fetal calf serum (FCS), 50 IU/ml penicillin, 50 µ g/ml streptomycin and 2 m m l ‐glutamine (all from Sigma, Zwijndrecht, the Netherlands). After 2–3 days, single endothelial colonies were identified by typical cobblestone morphology [30] and subcultured. The subcultured endothelial cells were characterized by uptake of acetylated low‐density lipoprotein labelled with fluorescent 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (Dil‐Ac‐LDL, Sigma, Zwijndrecht, the Netherlands) and screened by fluorescence microscopy (data not shown) [31].…”

Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica

“…Inflammatory mediators, such as IL-1-beta, TNF-alpha and leukotrienes, released by the neutrophils, are thought to be involved in the tissue damage that ensues. Indeed, neutrophil function is critical to the disease process since inflammation and much of the tissue destruction is reduced in neutrophil-depleted animals (20,21).…”