Kepley C L, Wilson B S, Oliver J M
University of New Mexico School of Medicine, Department of Pathology, Asthma Research Center, Albuquerque, USA.
J Allergy Clin Immunol. 1998 Aug;102(2):304-15. doi: 10.1016/s0091-6749(98)70100-9.
In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production.
The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion).
Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.
在人血嗜碱性粒细胞中，用多价抗原交联高亲和力IgE受体FcεRI可激活一条信号通路，导致钙离子动员、肌动蛋白聚合、形态改变、分泌及细胞因子产生。
采用通过Percoll梯度离心分离，随后用抗体包被的磁珠进行阴性和/或阳性选择得到的人嗜碱性粒细胞，探讨酪氨酸激酶在人FcεRI信号传导中的作用。纯度超过95%的嗜碱性粒细胞制剂的FcεRI交联可激活蛋白酪氨酸激酶Lyn和Syk（此前与FcεRI偶联的啮齿动物肥大细胞活化有关）以及Zap-70（此前与T细胞受体信号传导有关），并导致多种蛋白质的酪氨酸磷酸化。通过对高度纯化的嗜碱性粒细胞裂解物进行蛋白质印迹法，以及通过用激酶特异性抗体和嗜碱性粒细胞特异性抗体2D7同时标记的细胞进行共聚焦荧光显微镜检查，独立证实了嗜碱性粒细胞中Lyn、Syk和Zap-70的存在。在嗜碱性粒细胞和B细胞中发现了相当数量的Lyn和Syk，而T细胞中的Zap-70含量似乎比嗜碱性粒细胞更多。酪氨酸激酶抑制剂白皮杉醇不影响IgE介导的Lyn活化，但抑制IgE诱导的Syk和Zap-70活化以及总体蛋白质酪氨酸磷酸化和分泌。在低至最佳分泌浓度范围内的一系列抗IgE浓度下，总体蛋白质酪氨酸磷酸化稳步增加。然而，在引起极少分泌的高抗IgE浓度下（分泌的特征性高剂量抑制），酪氨酸磷酸化仍继续增加。
我们的数据表明，包括Zap-70以及Lyn和Syk在内的一系列酪氨酸激酶介导的抗IgE刺激的蛋白质酪氨酸磷酸化，与人嗜碱性粒细胞中FcεRI介导的信号传导起始相关。
