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

乙醇诱导的急性肝脏炎症的组织学特征是肝细胞局灶性坏死，并伴有中性粒细胞(PMN)。为了了解酒精性肝损伤中细胞浸润的机制和发病机制，我们研究了一种由乙醇诱导的肝细胞产生的新的趋化因子。采用胶原酶灌流消化，甲硝胺梯度差速离心法分离大鼠肝细胞和Kupffer细胞。用葡聚糖沉淀法和Hypaque-Ficoll梯度离心法从大鼠血中提取中性粒细胞，用30-90 mM乙醇培养正常大鼠肝细胞，培养上清液对大鼠中性粒细胞和枯否细胞具有趋化活性。从慢性酒精喂养的大鼠分离的肝细胞在体外培养时也得到了类似的结果。冻融或透析液对趋化活性影响不大，而胰酶消化和加热(56ﾟC，30min)可显著降低趋化活性。在培养基中加入转录抑制物或蛋白质合成抑制物可减少肝细胞产生趋化因子。Sephadex G-150凝胶层析显示两个组分具有趋化活性，分子量分别为20-25k道尔顿和40-50k道尔顿。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肝脏中性粒细胞和枯否细胞的积聚和活化，提示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.