Feldbush T L, Lafrenz D
Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri.
J Immunol. 1991 Dec 1;147(11):3690-5.
Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.
最近我们报道称，碱性磷酸酶（APase）的表达是B细胞活化的一个标志物。酶活性仅在活化的B细胞中发现，而在T细胞中未发现。通过流式细胞术我们发现，一些APase存在于细胞膜上（mAPase），并且通过功能分析表明，一些APase会自发释放到组织培养基中。在本报告中，对活化B淋巴细胞上mAPase的表达进行了更全面的表征。使用两种针对大鼠APase的单克隆抗体来测量mAPase膜表达的动力学。在活化后48小时内，通过流式细胞术可检测到mAPase，并且其与转铁蛋白受体和IL-2R协同增加。在培养的第4至5天，就阳性细胞数量和平均荧光强度而言，可检测到mAPase的最大膜表达。分别使用羟基脲和秋水仙酰胺将细胞阻滞在G1/S期和G2/M期，结果显示mAPase的初始表达发生在细胞进入细胞周期的S期时。使用Hoechst DNA染料33342和FITC标记的APase特异性单克隆抗体进行双色流式细胞术分析证实了这一点。最后，使用磷脂酰肌醇特异性磷脂酶C，我们能够证明60%至80%的mAPase通过糖基磷脂酰肌醇连接与膜相连。由此我们得出结论，mAPase可以添加到通过糖基磷脂酰肌醇连接锚定在膜上并在分化的B细胞上表达的糖蛋白不断增加的列表中。这个列表现在包括Thy-1、BLAST-1、Jlld和mAPase。
