Metelitsa Leonid S, Gillies Stephen D, Super Michael, Shimada Hiroyuki, Reynolds C Patrick, Seeger Robert C
Department of Pediatrics, Division of Hematology-Oncology, Children's Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles 90027, USA.
Blood. 2002 Jun 1;99(11):4166-73. doi: 10.1182/blood.v99.11.4166.
Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcgammaRII and FcgammaRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcgammaRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1-dependent PMN-target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined beta(2)-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcgammaRII and Mac-1-dependent enhanced adhesion and degranulation.
多形核白细胞（PMNs）介导抗体依赖性细胞毒性（ADCC），添加粒细胞-巨噬细胞集落刺激因子（GM-CSF）可增强这种毒性。我们试图确定添加抗体/GM-CSF融合蛋白是否也会增强PMN ADCC，以及这是否与Mac-1（CD11b/CD18）的上调和激活以及嗜天青颗粒胞吐作用有关。采用4小时的羧基荧光素乙酰氧基甲酯（calcein-AM）微细胞毒性试验、电子显微镜和多参数流式细胞术评估针对LA-N-1人神经母细胞瘤细胞的ADCC。在calcein-AM试验中，当PMN ADCC由抗二唾液酸神经节苷脂（抗-GD2）免疫细胞因子hu14.18/GM-CSF、与GM-CSF混合的单克隆抗体（mAb）hu14.18以及单独的hu14.18介导时，LA-N-1细胞存活率分别为10%、55%和75%。功能阻断单克隆抗体表明，单独使用hu14.18或与GM-CSF混合进行ADCC时需要FcγRII和FcγRIII，但使用hu14.18/GM-CSF进行ADCC时仅需要FcγRII。由hu14.18和hu14.18/GM-CSF介导的ADCC依赖于Mac-1。电子显微镜显示，hu14.18/GM-CSF导致的PMN对靶标的黏附、铺展和裂解作用最强。阻断Mac-1功能的单克隆抗体允许PMN与hu14.18/GM-CSF结合至靶标，但阻止黏附、铺展和细胞溶解。流式细胞术显示，无论有无GM-CSF的hu14.18以及hu14.18/GM-CSF均介导依赖于Mac-1的PMN-靶细胞共轭体形成，但GM-CSF是Mac-1最高表达和激活所必需的，这由mAb24定义的β2整合素激活表位所证实。Hu14.18/GM-CSF诱导最高水平的持续嗜天青颗粒胞吐作用，几乎仅在具有激活的Mac-1的PMN中出现。因此，hu14.18/GM-CSF促进PMN对神经母细胞瘤细胞的ADCC，这与FcγRII和依赖于Mac-1的增强黏附和脱颗粒作用相关。
