ENDOTOXIN-INDUCED EXPRESSION OF TUMOR-NECROSIS-FACTOR, TISSUE FACTOR AND PLASMINOGEN-ACTIVATOR INHIBITOR ACTIVITY BY PERITONEAL-MACROPHAGES

Peritoneal fluid was collected aseptically from 30 healthy adult horse s and 115 horses with acute gastrointestinal disease and supernatant w as separated from cells by centrifugation followed by freezing until a ssayed for endotoxin and tumour necrosis factor activity, Peritoneal m acrophages obtained from healthy horses were incubated in vitro for 3, 6, 12 or 24 h in the absence (media control) or presence of Escherich ia coli O55:B5 endotoxin (final concentrations of 1, 10, 100 or 1000 n g/ml), Macrophages obtained from horses with acute gastrointestinal di sease were incubated for 12 h in the absence (media control) or presen ce of 100 ng endotoxin/ml, At the conclusion of the incubation, macrop hage supernatants were collected and frozen at -70 degrees C until ana lysed for tumour necrosis factor activity, Macrophage membranes were l ysed and frozen at -70 degrees C until assayed for tissue factor and p lasminogen activator inhibitor type 2 activity. Compared to cells incu bated with media, incubation of macrophages, obtained from healthy hor ses, with endotoxin significantly increased tumour necrosis factor, ti ssue factor and plasminogen activator inhibitor type 2 activity, These increases were dependent on the endotoxin concentration and the durat ion of incubation. Compared to cells incubated with media alone, incub ation of macrophages, obtained from horses with acute gastrointestinal disease with endotoxin, significantly increased tumour necrosis facto r 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 s imilarly 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 perito neal fluid supernatant had significantly less endotoxin induced tumour necrosis factor in vitro, as compared to similarly treated cells obta ined from horses without endotoxin or tumour necrosis factor activity in the peritoneal fluid supernatant. The results of this study indicat e 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 expos ure to endotoxin, there is significant downregulation of inflammatory mediator production by peritoneal macrophages obtained from endotoxaem ic horses, These results suggest that these macrophages may exhibit ea rly endotoxin tolerance.