Mouse Langerhans cells do not express the high-affinity receptor for IgE
Mouse Langerhans cells do not express the high-affinity receptor for IgE
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DOI：
10.1007/s004030050401
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发表时间：
1999-04-01
影响因子：
3
通讯作者：
Ra, C
中科院分区：
文献类型：
--
作者：
Hayashi, S;Matsuda, H;Ra, C
使用FL-2检测器检测PE标记的阳性抗IA抗体染色（IA+）细胞。这些IA+细胞占PI阴性培养表皮细胞的10-20%（图1）。新鲜分离的表皮细胞含有约5-10%的IA+细胞（日期未显示）。门控这些PI阴性和IA+细胞后，评价其FL-1强度（抗小鼠FcεRI+ FITC抗仓鼠IgG或FITC小鼠IgE）。图2显示了新鲜分离的表皮细胞中IA+细胞的FL-1强度。与同型对照（仓鼠IgG）染色相比，这些IA+细胞在细胞表面上未表达任何可检测的FcεRI，我们也无法检测到FITC标记的小鼠IgE与IA+细胞的结合。图3显示了培养的表皮细胞中IA+细胞的FL-1强度。在培养的表皮细胞悬液中未检测到表达FcεRI的IA+细胞。IL-4的加入不能诱导IA+细胞上的FcεRI。在蛋白质水平上，我们在朗格汉斯细胞表面检测不到FcεRI的表达，接下来我们尝试在表皮细胞中检测FcεRI的mRNA。如前所述制备表皮细胞的总RNA。根据制造商的说明书，使用上标RT试剂盒（Gibco BRL）对总RNA（5 mg）进行逆转录。通过聚合酶链反应用DNA热循环仪（Perkin Elmer Cetus）进行cDNA的扩增。
PE-labeled positively anti-IA antibody stained (IA+) cells were detected using an FL-2 detector. These IA+ cells were 10–20% of the PI-negative cultured epidermal cells (Fig. 1). Freshly isolated epidermal cells contained about 5–10% of IA+ cells (date not shown). After gating out these PI-negative and IA+ cells, their FL-1 intensity (antimouse FcεRI+ FITC antihamster IgG or FITC mouse IgE) was evaluated. Figure 2 shows the FL-1 intensity of IA+ cells among the freshly isolated epidermal cells. These IA+ cells did not express any detectable FcεRI on the cell surface as compared with isotypic control (hamster IgG) staining, nor could we detect binding of FITC-labeled mouse IgE to the IA+ cells. Figure 3 shows the FL-1 intensity of IA+ cells among the cultured epidermal cells. We could not detect FcεRI expressing IA+ cells in cultured epidermal cell suspensions. The addition of IL-4 failed to induce FcεRI on the IA+ cells. At the protein level, we could not detect FcεRI on the cell surface of Langerhans cells.Next, we tried to detect FcεRI mRNA in epidermal cells. Total RNA of epidermal cells was prepared as previously described. The total RNA (5 mg) was subjected to reverse transcription using a superscript RT kit (Gibco BRL) according to the manufacturer’s instruction. Amplification of the cDNA was performed by polymerase chain reaction with a DNA thermal cycler (Perkin Elmer Cetus).