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

乙醇诱导急性肝炎症的组织学特征是肝细胞浸润多形核细胞（PMNs）的局灶性坏死。为了了解酒精性肝损伤的细胞浸润机制和发病机制，我们研究了暴露于乙醇的肝细胞产生的一种新的趋化因子。采用胶原酶灌注消化法分离大鼠肝脏肝细胞和库普弗细胞，并在甲咪唑胺梯度下差速离心。采用葡聚糖沉降法和Hypaque-Ficoll梯度离心法制备大鼠外周血pmn。从正常大鼠肝脏分离的肝细胞用乙醇（30-90mM）培养，在培养上清中显示出对大鼠PMNs和Kupffer细胞的趋化活性。从长期乙醇喂养的大鼠中分离的肝细胞进行体外培养也得到了类似的结果。冻融或透析对趋化活性影响不大，而胰酶消化和加热（56℃，30min）显著降低了趋化活性。在培养基中加入转录或蛋白质合成抑制剂可降低肝细胞趋化因子的产生。用Sephadex G-150凝胶层析法测定了其在分子量为20-25 k道尔顿和40-50 k道尔顿两个组分的趋化活性。趋化因子的存在增强了pmn和Kupffer细胞产生超氧阴离子的能力。这些结果提示，趋化因子可诱导pmn和Kupffer细胞在肝脏内的积累和活化，该趋化因子可能参与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.