Gardner C R, Laskin J D, Laskin D L
Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08855-0789.
J Leukoc Biol. 1995 Feb;57(2):269-74. doi: 10.1002/jlb.57.2.269.
Acute endotoxemia is associated with activation of hepatic macrophages and endothelial cells. These cells release a variety of inflammatory mediators that have been implicated in tissue injury. In the present studies, we analyzed the biochemical responses of these cells to platelet-activating factor (PAF), a lipid autacoid released during hepatic inflammatory responses. To induce acute endotoxemia, rats were injected intravenously with lipopolysaccharide (LPS). Using the calcium sensitive fluorescent indicator dye Indo-1, we found that PAF induced a rapid and transient increase in intracellular calcium in both hepatic macrophages and endothelial cells. Induction of acute endotoxemia resulted in an increase in the amount of calcium mobilized by both cell types. Although endothelial cells from control rats were less responsive to PAF than macrophages, these cells were more sensitive to in vivo endotoxin. PAF was also found to cause a rapid decrease in intracellular pH in hepatic macrophages that was quantified by fluorescence image analysis using the pH sensitive dye SNAFL-calcein. This decrease occurred more rapidly in macrophages from endotoxemic rats. In cells from both control and endotoxemic rats, the effects of PAF on intracellular pH were inhibited by the specific PAF antagonist triazolam. In contrast to hepatic macrophages, PAF had no effect on intracellular pH in endothelial cells from either control or endotoxemic rats. Ligand binding studies demonstrated that both hepatic macrophages and endothelial cells possess high affinity binding sites for PAF. Macrophages expressed 6- to 7-fold more binding sites/cell than endothelial cells and exhibited a higher Kd. Whereas treatment of rats with LPS had no effect on the Kd for PAF binding to macrophages or on the number of binding sites, a significant increase in both of these receptor characteristics was observed in endothelial cells. Taken together, the present data suggest that the biochemical responses of endothelial cells and macrophages to PAF are distinct. Furthermore, cellular activation induced by PAF in endothelial cells appears to be independent of changes in intracellular pH.
急性内毒素血症与肝巨噬细胞和内皮细胞的激活有关。这些细胞释放多种与组织损伤有关的炎症介质。在本研究中，我们分析了这些细胞对血小板活化因子（PAF）的生化反应，PAF是肝脏炎症反应期间释放的一种类脂自分泌物质。为诱导急性内毒素血症，给大鼠静脉注射脂多糖（LPS）。使用钙敏感荧光指示剂染料Indo-1，我们发现PAF可诱导肝巨噬细胞和内皮细胞内的钙快速短暂升高。急性内毒素血症的诱导导致两种细胞类型动员的钙量增加。虽然对照大鼠的内皮细胞对PAF的反应比巨噬细胞弱，但这些细胞对体内内毒素更敏感。还发现PAF可导致肝巨噬细胞内pH值迅速下降，这是通过使用pH敏感染料SNAFL-钙黄绿素的荧光图像分析来定量的。这种下降在内毒素血症大鼠的巨噬细胞中发生得更快。在对照大鼠和内毒素血症大鼠的细胞中，PAF对细胞内pH值的影响均被特异性PAF拮抗剂三唑仑抑制。与肝巨噬细胞不同，PAF对对照或内毒素血症大鼠的内皮细胞内pH值均无影响。配体结合研究表明，肝巨噬细胞和内皮细胞均具有PAF的高亲和力结合位点。巨噬细胞表达的结合位点/细胞比内皮细胞多6至7倍，且表现出更高的解离常数（Kd）。用LPS处理大鼠对PAF与巨噬细胞结合的Kd或结合位点数量没有影响，但在内皮细胞中观察到这两种受体特性均显著增加。综上所述，目前的数据表明内皮细胞和巨噬细胞对PAF的生化反应是不同的。此外，PAF在内皮细胞中诱导的细胞活化似乎与细胞内pH值的变化无关。
