Knox K A, Gordon J
Department of Immunology, University of Birmingham, GB.
Eur J Immunol. 1993 Oct;23(10):2578-84. doi: 10.1002/eji.1830231030.
Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross-linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose-dependent fashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti-CD40 was remarkably similar to that triggered by polyvalent anti-Ig. By contrast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca2+. Pre-incubation with the PTK inhibitor herbimycin A (5 microM) abrogated anti-CD40-mediated rescue of GC B cells from apoptosis, while genistein (40 microM) and the tyrphostins AG490 (10 microM) and AG814 (25 microM) significantly inhibited this process. Consistent with these results, herbimycin A (5 microM) abolished the expression of the 26 kDa bcl-2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti-CD40. The Ca2+ chelators BAPTA and EGTA did not significantly affect CD40-promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40-mediated rescue of GC B cells from apoptosis.
生发中心（GC）B细胞中的自发凋亡可通过使细胞表面免疫球蛋白（Ig）与固定化配体结合而被阻止，或者更有效地通过用针对CD40的可溶性单克隆抗体（mAb）处理来阻止。本研究考察了在表面CD40被连接后，GC B细胞中产生从自发凋亡中挽救的细胞内信号转导途径。用mAb使GC B细胞表面的CD40交联，始终导致多种不同底物上酪氨酸磷酸化增强：通过用选择性蛋白酪氨酸激酶（PTK）抑制剂赫曲霉素A预处理GC B细胞，这一过程能够以剂量依赖的方式被阻断。此外，用抗CD40处理后观察到的酪氨酸磷酸化模式与多价抗Ig引发的模式非常相似。相比之下，抗CD40未能刺激在表面Ig被连接后GC B细胞和静止B淋巴细胞中观察到的肌醇1,4,5 -三磷酸和胞质游离钙的增加。通过使用PTK以及细胞外和细胞内Ca2 +的选择性抑制剂，研究了在将GC B细胞从凋亡中挽救出来过程中产生的信号转导途径的参与情况。用PTK抑制剂赫曲霉素A（5 microM）预孵育消除了抗CD40介导的将GC B细胞从凋亡中挽救出来的作用，而染料木黄酮（40 microM）以及酪氨酸磷酸化抑制剂AG490（10 microM）和AG814（25 microM）显著抑制了这一过程。与这些结果一致，赫曲霉素A（5 microM）消除了通常在用抗CD40培养后观察到的赋予抗凋亡能力的26 kDa bcl - 2原癌基因产物的表达。Ca2 +螯合剂BAPTA和EGTA并未显著影响CD40促进的挽救作用。综上所述，这些结果表明，GC B细胞的CD40与功能性PTK偶联，但不与磷脂酰肌醇信号转导途径偶联，并且酪氨酸磷酸化对于CD40介导的将GC B细胞从凋亡中挽救出来是必需的。
