Barton M H, Collatos C, Moore J N
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Equine Vet J. 1996 Sep;28(5):382-9. doi: 10.1111/j.2042-3306.1996.tb03109.x.
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.
从30匹健康成年马和115匹患有急性胃肠疾病的马中无菌采集腹腔液，通过离心从细胞中分离出上清液，然后冷冻直至检测内毒素和肿瘤坏死因子活性。从健康马获取的腹腔巨噬细胞在无（培养基对照）或有大肠杆菌055:B5内毒素（终浓度为1、10、100或1000 ng/ml）的情况下体外培养3、6、12或24小时。从患有急性胃肠疾病的马获取的巨噬细胞在无（培养基对照）或有100 ng/ml内毒素的情况下培养12小时。培养结束时，收集巨噬细胞上清液并在-70℃冷冻直至分析肿瘤坏死因子活性。裂解巨噬细胞膜并在-70℃冷冻直至检测组织因子和纤溶酶原激活物抑制剂2型活性。与用培养基培养的细胞相比，从健康马获取的巨噬细胞与内毒素一起培养显著增加了肿瘤坏死因子、组织因子和纤溶酶原激活物抑制剂2型活性。这些增加取决于内毒素浓度和培养持续时间。与仅用培养基培养的细胞相比，从患有急性胃肠疾病的马获取的巨噬细胞与内毒素一起培养显著增加了肿瘤坏死因子和组织因子活性。与从健康马获取的经类似处理的细胞相比，从患有急性胃肠疾病的马获取的巨噬细胞在体外由内毒素诱导的肿瘤坏死因子活性显著降低。对于那些患有急性胃肠疾病的马，与从腹腔液上清液中无内毒素或肿瘤坏死因子活性的马获取的经类似处理的细胞相比，从腹腔液上清液中有内毒素或肿瘤坏死因子活性的马获取的巨噬细胞在体外由内毒素诱导的肿瘤坏死因子显著减少。本研究结果表明，马腹腔巨噬细胞暴露于内毒素会导致肿瘤坏死因子、组织因子和纤溶酶原激活物抑制剂2型活性显著增加。在体外暴露于内毒素后，从内毒素血症马获取的腹腔巨噬细胞产生的炎症介质有显著下调。这些结果表明这些巨噬细胞可能表现出早期内毒素耐受性。
