Katz H R, Levine J S, Austen K F
J Immunol. 1987 Sep 1;139(5):1640-6.
The acidic glycosphingolipid, ganglioside GM1, which is the binding site for cholera toxin on many cell types, was identified by chemical and by flow cytometric analyses of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC). Ganglioside GM1 and other acidic glycosphingolipids were isolated from BMMC by chloroform/methanol extraction and chromatography on DEAE-Sephadex and were analyzed by thin layer chromatography. The presence of ganglioside GM1 in the BMMC extract was demonstrated by its co-migration with ganglioside GM1 standard in thin layer chromatography and by the binding of peroxidase-labeled cholera toxin B subunit to both molecules. As assessed by fluorescence flow cytometric analysis of the binding of fluorescein-conjugated cholera toxin B subunit, the majority of BMMC expressed ganglioside GM1 on their surface, and the total presentation per cell increased as cells progressed from the G1 to S to G2 + M phases of the cell cycle. The addition of increasing amounts of cholera toxin starting with 0.08 microgram/ml to BMMC cultured in 50% WEHI 3-conditioned medium containing IL 3 for 48 hr caused the adhesion of BMMC to the tissue culture flasks to increase in a dose-related manner, from less than 1% adherent cells in cultures without toxin to a plateau value of approximately 17% adherent in the presence of 1.25 micrograms/ml of toxin. The histamine content of BMMC increased from 26.7 +/- 3.59 ng/10(6) cells (mean +/- SD, n = 4) for control cultures to 201 +/- 17.4 ng/10(6) cells (mean +/- SD, n = 4) for nonadherent cells and to 588 +/- 89.4 ng/10(6) cells (mean +/- SD, n = 4) for adherent cells after 48 hr of culture in 0.31 microgram/ml cholera toxin, which was the optimal dose for nonadherent and adherent populations. The content of another preformed intragranular mediator, beta-hexosaminidase, did not increase appreciably in the presence of cholera toxin (n = 3). The increase in the histamine content of BMMC after the addition of 0.31 microgram/ml cholera toxin was detectable at 4 hr, plateaued by 24 to 48 hr, and gradually declined over the next 6 days. Cholera toxin also augmented the histamine content of BMMC in the presence of purified synthetic IL 3. Preincubation of whole cholera toxin with purified ganglioside GM1 inhibited the histamine-augmenting effects of cholera toxin on BMMC, indicating that the effect was not due to a contaminant, and neither the A nor B subunit of cholera toxin alone increased the histamine content of BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)
酸性糖鞘脂神经节苷脂GM1是霍乱毒素在许多细胞类型上的结合位点，通过对小鼠白细胞介素3依赖的骨髓培养来源肥大细胞（BMMC）进行化学分析和流式细胞术分析得以鉴定。通过氯仿/甲醇萃取以及在DEAE - 葡聚糖凝胶上的色谱法从BMMC中分离出神经节苷脂GM1和其他酸性糖鞘脂，并通过薄层色谱法进行分析。在薄层色谱中，BMMC提取物中的神经节苷脂GM1与神经节苷脂GM1标准品共迁移，以及过氧化物酶标记的霍乱毒素B亚基与这两种分子的结合，证明了BMMC提取物中存在神经节苷脂GM1。通过对荧光素偶联的霍乱毒素B亚基结合的荧光流式细胞术分析评估，大多数BMMC在其表面表达神经节苷脂GM1，并且随着细胞从细胞周期的G1期进展到S期再到G2 + M期，每个细胞的总表达量增加。从0.08微克/毫升开始向在含IL - 3的50% WEHI 3条件培养基中培养48小时的BMMC中添加越来越多的霍乱毒素，导致BMMC对组织培养瓶的粘附以剂量相关的方式增加，从无毒素培养物中不到1%的贴壁细胞增加到在存在1.25微克/毫升毒素时约17%的贴壁平台值。BMMC的组胺含量从对照培养物的26.7±3.59纳克/10⁶细胞（平均值±标准差，n = 4）增加到非贴壁细胞的201±17.4纳克/10⁶细胞（平均值±标准差，n = 4），以及在0.31微克/毫升霍乱毒素中培养48小时后贴壁细胞的588±89.4纳克/10⁶细胞（平均值±标准差，n = 4），这是对非贴壁和贴壁群体的最佳剂量。在霍乱毒素存在下，另一种预先形成的颗粒内介质β - 己糖胺酶的含量没有明显增加（n = 3）。添加0.31微克/毫升霍乱毒素后BMMC组胺含量的增加在4小时时可检测到，在24至48小时达到平台期，并在接下来的6天逐渐下降。在纯化的合成IL - 3存在下，霍乱毒素也增加了BMMC的组胺含量。用纯化的神经节苷脂GM1对完整霍乱毒素进行预孵育可抑制霍乱毒素对BMMC的组胺增强作用，表明该作用不是由于污染物引起的，并且霍乱毒素单独的A亚基或B亚基都不会增加BMMC的组胺含量。（摘要截断于400字）
