Unruh Tammy L, Li Haidong, Mutch Cathlin M, Shariat Neda, Grigoriou Lana, Sanyal Ratna, Brown Christopher B, Deans Julie P
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
Immunology. 2005 Oct;116(2):223-32. doi: 10.1111/j.1365-2567.2005.02213.x.
The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non-ionic detergent Triton-X-100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains. Apoptosis induced by rituximab hypercrosslinking has been shown to require src family kinases (SFK), which are enriched in rafts. In this report we provide experimental evidence that SFK-dependent apoptotic signals induced by rituximab are raft dependent. Cholesterol depletion prevented the association of hypercrosslinked CD20 with detergent-insoluble rafts, and attenuated both calcium mobilization and apoptosis induced with rituximab. CD20 cocapped with the raft-associated transmembrane adaptor LAB/NTAL after hypercrosslinking with CD20 mAbs, regardless of their ability to induce a change in the affinity of CD20 for rafts. Taken together, the data demonstrate that CD20 hypercrosslinking via rituximab activates SFKs and downstream signalling events by clustering membrane rafts in which antibody-bound CD20 is localized in a high-affinity configuration.
单克隆抗体（mAb）利妥昔单抗可使B细胞非霍奇金淋巴瘤患者和基于抗体的自身免疫性疾病患者产生客观的临床反应。利妥昔单抗介导B细胞耗竭的机制尚未完全明确，可能包括通过靶抗原CD20进行信号传导的直接作用。与大多数（但并非全部）CD20单克隆抗体一样，利妥昔单抗可使CD20蛋白在非离子去污剂Triton-X-100中的溶解度发生急剧变化，这反映出CD20对膜筏信号结构域的固有亲和力显著增加。已证明利妥昔单抗过度交联诱导的细胞凋亡需要src家族激酶（SFK），而SFK在筏中含量丰富。在本报告中，我们提供了实验证据，证明利妥昔单抗诱导的依赖SFK的凋亡信号是依赖筏的。胆固醇耗竭可阻止过度交联的CD20与去污剂不溶性筏的结合，并减弱利妥昔单抗诱导的钙动员和细胞凋亡。用CD20单克隆抗体过度交联后，CD20与筏相关跨膜衔接子LAB/NTAL共帽，无论其是否能够诱导CD20对筏的亲和力发生变化。综上所述，数据表明通过利妥昔单抗进行的CD20过度交联通过聚集膜筏激活SFK和下游信号事件，其中抗体结合的CD20以高亲和力构型定位在膜筏中。
