Sperber K, Goswami S K, Gollub E, Mayer L, Marom Z
Division of Clinical Immunology, Mount Sinai Medical Center, New York, N.Y. 10029.
J Allergy Clin Immunol. 1991 Feb;87(2):490-8. doi: 10.1016/0091-6749(91)90007-b.
A pulmonary macrophage-monocyte-derived mucus secretagogue (MMS) oligopeptide has been previously reported to induce mucus secretion in an in vitro model system with human airway explants and secretory epithelial cells. To understand the possible role of macrophages in the regulation of secretion of mucus, our laboratory has used a series of human macrophage hybridomas that were generated by fusing an hypoxanthine guanine phosphoribosyl transferase-deficient promonocytic line, U937, with macrophages obtaining by maturing monocytes in Teflon bags. The cell lines were proven to be true hybridomas by acquisition of donor class I antigens, additional chromosomes, as well as macrophage specific (maximum velocity) not present on the U937 parent line. One clone, clone 63, produced large amounts of an oligopeptide with an approximate molecular weight of 2000, which was identified from culture supernatants by ultrafiltration, chromatography, isoelectric focusing, and Western blot. Processed clone 63 supernatant had biologic activity causing increased secretion of radiolabeled glycoconjugate in both cultured airways and secretory epithelial cells. Immunoblot analysis with a polyclonal rabbit antisera generated against MMS was positive, and Western blot analysis produced a band at approximately 2000 daltons, consistent with the previously described MMS. MMS secretion could be stimulated by zymosan and lipopolysaccharide and inhibited by both cycloheximide and erythromycin. Dexamethasone had a different effect, appearing to stimulate MMS production intracellularly but inhibiting its release once it was synthesized. The availability of cloned hybridomas allows for study of the regulation of mucus secretagogue production as well as purification of molecular species and provides a valuable tool for the study of mucus secretion.
先前有报道称，一种源自肺巨噬细胞 - 单核细胞的黏液分泌刺激物（MMS）寡肽，能在包含人气道外植体和分泌性上皮细胞的体外模型系统中诱导黏液分泌。为了解巨噬细胞在黏液分泌调节中的可能作用，我们实验室使用了一系列人巨噬细胞杂交瘤，这些杂交瘤是通过将次黄嘌呤鸟嘌呤磷酸核糖基转移酶缺陷的前单核细胞系U937与在聚四氟乙烯袋中使单核细胞成熟而获得的巨噬细胞融合产生的。通过获取供体I类抗原、额外的染色体以及U937亲代细胞系所没有的巨噬细胞特异性（最大速度），证明这些细胞系是真正的杂交瘤。一个克隆，即克隆63，产生了大量分子量约为2000的寡肽，通过超滤、色谱、等电聚焦和蛋白质免疫印迹从培养上清液中鉴定出该寡肽。处理后的克隆63上清液具有生物活性，可导致培养的气道和分泌性上皮细胞中放射性标记糖缀合物的分泌增加。用针对MMS产生的兔多克隆抗血清进行的免疫印迹分析呈阳性，蛋白质免疫印迹分析在约2000道尔顿处产生一条带，与先前描述的MMS一致。酵母聚糖和脂多糖可刺激MMS分泌，而环己酰亚胺和红霉素均可抑制其分泌。地塞米松有不同的作用，似乎在细胞内刺激MMS产生，但在其合成后抑制其释放。克隆杂交瘤的可得性使得能够研究黏液分泌刺激物产生的调节以及分子种类的纯化，并为黏液分泌的研究提供了有价值的工具。
