Studies on the Mechanisms of Lipopolysaccharide-induced Degranulation of Hemocytes of Invertebrate Animals (Horseshoe Crabs).

用脂多糖（LPS）刺激洗净的鲎血细胞，在显微镜下观察到细胞脱颗粒。因此，血浆组分不需要用于脱粒。虽然细胞的脱粒是由钙离子载体诱导的，但即使在细胞外EDTA的存在下也观察到脱粒。因此，这表明脱粒不需要细胞外的钙流入，而是由细胞内钙池的钙释放诱导的。我们还通过使用FITC或生物素标记的LPS的流式细胞术分析了血细胞的LPS结合。血细胞显示出强的LPS结合能力，其甚至比在LPS应答哺乳动物细胞上发现的那些更强。与过量的完整LPS竞争结合，并通过细胞的蛋白酶处理降低结合。此外，观察到与来自沙门氏菌R595的LPS的结合，其缺乏O-抗原多糖。这些结果有力地表明存在特异性识别并结合脂A的蛋白质，该蛋白质具有LPS的大部分生物活性。尽管我们试图通过哺乳动物细胞上的表达克隆来克隆该蛋白质的cDNA，但我们不能获得任何真实的阳性信号。我们制备了几种抗血细胞质膜的单克隆抗体，通过筛选这些抗体来分离结合蛋白。到目前为止，我们发现一些抗体显示出抑制血细胞的LPS结合。我们目前正在研究这种结合是否是特异性的，以及它们是否抑制血细胞的脱粒。

When washed horseshoe crab hemocytes were stimulated with lipopolysaccharide (LPS), degranulation of the cells was found to occur under microscopic observation. Therefore, plasma components are not required for the degranulation. Although the degranulation of the cells is induced by a calcium ionophore, the degranulation was observed even in the presence of extracellular EDTA.Thus, it was suggested that the degranulation does not require calcium influx from outside of the cells, but is induced by calcium release from the intracellular calcium pool.We also analyzed LPS-binding of the hemocyte by using flow cytometry utilizing FITC- or biotin-labeled LPS . The hemocytes showed a strong LPS binding ability, which was even stronger than those found on the LPS -responsive mammalian cell. The binding was competed with excess amounts of intact LPS and was reduced by protease treatments of the cells. Furthermore, the binding was observed with LPS derived from Salmonella minnesota R595, which lacks the O-antigen polysaccharide. These results strongly indicated the presence of a protein(s) that specifically recognizes and bind to lipid A, which has most of the biological activity of LPS.Although we tried to clone the cDNA for this protein by expression cloning on mammalian cells, we could not obtain any real positive signals. We had prepared several monoclonal antibodies against the hemocyte plasma membrane in order to isolate the binding protein by screening these antibodies. Thus far, we found that some of the antibodies were shown to inhibit the LPS-binding of the hemocytes. We are currently examining if this binding is specific and if they inhibit the degranulation of the hemocytes.