Broaddus V C, Boylan A M, Hoeffel J M, Kim K J, Sadick M, Chuntharapai A, Hébert C A
Department of Medicine, San Francisco General Hospital, CA 94110.
J Immunol. 1994 Mar 15;152(6):2960-7.
Although the potent neutrophil chemotaxin, IL-8, is a known product of endotoxin-stimulated cells in vitro, the contribution of IL-8 to neutrophil recruitment in Gram-negative endotoxin inflammation in vivo is unknown. To determine whether neutralization of IL-8 would decrease endotoxin-induced neutrophil influx, we generated neutralizing mAbs to rabbit rIL-8 for use in our rabbit model of endotoxin-induced pleurisy. One mAb, ARIL8.2, specifically inhibited both rabbit rIL-8-induced chemotactic activity and activation of the rabbit IL-8 receptor transfected in 293 cells. Anesthetized rabbits with in-dwelling pleural catheters received either neutralizing mAb (ARIL8.2; 1 mg/kg) or irrelevant isotype-matched mAb (anti-HIV gp120) i.v. 1 h before as well as intrapleurally (20 micrograms/ml) at the time of intrapleural instillation of Escherichia coli endotoxin (200 ng bilaterally). ARIL8.2 blocked 77% of endotoxin-induced neutrophil influx (21 +/- 2 (SE) x 10(6) (ARIL8.2) vs 91 +/- 15 x 10(6) (anti-gp120) (p < 0.0001)). By Western analysis, a band corresponding to rabbit IL-8 was detected in the pleural liquid of rabbits in both groups. By ELISA, however, the concentration of free, unbound IL-8 in the pleural liquid was significantly less in the ARIL8.2 group than in the anti-gp120 group for at least 4 h, confirming that ARIL8.2 bound the IL-8 generated in vivo during that time. We conclude that neutralization of IL-8 profoundly inhibits neutrophil recruitment in endotoxin-induced pleurisy indicating that IL-8 is a major chemotactic factor in this model of acute inflammation.
尽管强效中性粒细胞趋化因子白细胞介素-8（IL-8）是体外内毒素刺激细胞的已知产物，但IL-8在体内革兰氏阴性内毒素炎症中对中性粒细胞募集的作用尚不清楚。为了确定中和IL-8是否会减少内毒素诱导的中性粒细胞流入，我们制备了针对兔rIL-8的中和单克隆抗体，用于我们的内毒素诱导胸膜炎兔模型。一种单克隆抗体ARIL8.2特异性抑制兔rIL-8诱导的趋化活性以及转染到293细胞中的兔IL-8受体的激活。将带有胸腔导管的麻醉兔静脉注射中和单克隆抗体（ARIL8.2；1mg/kg）或无关的同型匹配单克隆抗体（抗HIV gp120），在胸腔内注入大肠杆菌内毒素（双侧200ng）前1小时以及注入时胸腔内注射（20μg/ml）。ARIL8.2阻断了77%的内毒素诱导的中性粒细胞流入（ARIL8.2组为21±2（SE）×10⁶，抗gp120组为91±15×10⁶，p<0.0001）。通过蛋白质印迹分析，在两组兔的胸腔积液中均检测到一条与兔IL-8相对应的条带。然而，通过酶联免疫吸附测定法，ARIL8.2组胸腔积液中游离的、未结合的IL-8浓度在至少4小时内明显低于抗gp120组，证实ARIL8.2在那段时间内结合了体内产生的IL-8。我们得出结论，中和IL-8可显著抑制内毒素诱导胸膜炎中的中性粒细胞募集，表明IL-8是这种急性炎症模型中的主要趋化因子。
