Endotoxin induced expression of tumour necrosis factor, tissue factor and plasminogen activator inhibitor activity by peritoneal macrophages.

Abstract: Peritoneal fluid was collected aseptically from 30 healthy adult horses and 115 horses with acute gastrointestinal disease and supernatant was separated from cells by centrifugation followed by freezing until assayed for endotoxin and tumour necrosis factor activity. Peritoneal macrophages obtained from healthy horses were incubated in vitro for 3, 6, 12 or 24 h in the absence (media control) or presence of Escherichia coli 055:B5 endotoxin (final concentrations of 1, 10, 100 or 1000 ng/ml). Macrophages obtained from horses with acute gastrointestinal disease were incubated for 12 h in the absence (media control) or presence of 100 ng endotoxin/ml. At the conclusion of the incubation, macrophage supernatants were collected and frozen at -70 degrees C until analysed for tumour necrosis factor activity. Macrophage membranes were lysed and frozen at -70 degrees C until assayed for tissue factor and plasminogen activator inhibitor type 2 activity. Compared to cells incubated with media, incubation of macrophages, obtained from healthy horses, with endotoxin significantly increased tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity. These increases were dependent on the endotoxin concentration and the duration of incubation. Compared to cells incubated with media alone, incubation of macrophages, obtained from horses with acute gastrointestinal disease with endotoxin, significantly increased tumour necrosis factor and tissue factor activity. Endotoxin induced tumour necrosis factor activity in vitro was significantly less for macrophages from horses with acute gastrointestinal disease, as compared to that produced by similarly treated cells obtained from healthy horses. For those horses with acute gastrointestinal disease, macrophages obtained from horses with either endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant had significantly less endotoxin induced tumour necrosis factor in vitro, as compared to similarly treated cells obtained from horses without endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant. The results of this study indicate that exposure of equine peritoneal macrophages to endotoxin results in a significant increase in tumour necrosis factor, tissue factor and plasminogen activator inhibitor type 2 activity. After in vitro exposure to endotoxin, there is significant down-regulation of inflammatory mediator production by peritoneal macrophages obtained from endotoxaemic horses. These results suggest that these macrophages may exhibit early endotoxin tolerance.