A MECHANISM OF INFILTRATION OF POLYMORPHONUCLEAR CELLS AND KUPFFER CELLS IN ALCOHOLIC LIVER INJURY : PRODUCTION OF CHEMOTACTIC FACTOR BY HEPATOCYTES EXPOSED TO ETHANOL

乙醇诱导急性肝脏炎症，其组织学特征为多形核细胞（PMN）浸润的肝细胞局灶性坏死。为了了解酒精性肝损伤中细胞浸润的机制和发病机制，我们研究了一种新的由乙醇暴露的肝细胞产生的趋化因子。通过灌注和胶原酶消化，然后在甲泛葡胺梯度上差速离心，从大鼠肝脏分离肝细胞和枯否细胞。用葡聚糖沉淀法和Hypaque-Ficoll梯度离心法从大鼠血液中分离出中性粒细胞（PMNs），用30- 90 mM乙醇培养正常大鼠肝细胞，发现培养上清对大鼠PMNs和枯否细胞具有趋化活性。当从长期乙醇喂养的大鼠中分离的肝细胞在体外培养时，获得了类似的结果。培养液的冻融或透析对趋化活性影响不大，而胰蛋白酶消化和加热（56 ℃，30 min）则显著降低趋化活性。向培养基中添加转录或蛋白质合成抑制剂可降低肝细胞趋化因子的产生。通过Sephadex G-150凝胶层析，在分子量为20-25 k道尔顿和40-50 k道尔顿的两个组分中显示出趋化活性。趋化因子可促进中性粒细胞和枯否细胞产生超氧阴离子，提示趋化因子可诱导中性粒细胞和枯否细胞在肝内聚集和活化，可能参与了酒精性肝损伤的发病机制。

Ethanol induces acute hepatic inflammation histologically characterized by focal necrosis of hepatocytes infiltrated with polymorphonuclear cells (PMNs). To know the mechanism and pathogenesis of cellular infiltration in alcoholic liver injury, we investigated a novel chemotactic factor produced by hepatocytes exposed to ethanol. Hepatocytes and Kupffer cells were isolated from rat liver by perfusion and digestion with collagenase followed by differential centrifugation on metrizamide gradient. Rat PMNs were prepared from blood by dextran sedimentation and Hypaque-Ficoll gradient centrifugation.When hepatocytes isolated from normal rat liver were cultured with ethanol (30-90mM), chemotactic activity for rat PMNs and Kupffer cells was demonstrated in the culture supernatant. Similar results were obtained when hepatocytes isolated from chronically ethanol-fed rats were cultured in vitro. Freeze-thawing or dialysis of the culture medium gave little effect on chemotactic activity, while trypsin digestion and heat (56ﾟC, 30min) markedly reduced the activity. Addition of inhibitor of transcription or protein synthesis to the culture medium decreased production of the chemotactic factor from hepatocytes. Chemotactic activity was revealed in two fractions at molecular weight of 20-25 k daltons and 40-50 k daltons by use of gel chromatography with Sephadex G-150. Production of superoxide anion by PMNs and Kupffer cells was enhanced in the presence of the chemotactic factor.These results suggested that accumulation and activation of PMNs and Kupffer cells in the liver were induced by the chemotactic factor and that this chemotactic factor might contribute to the pathogenesis of alcoholic liver injury.