Chen R L, Reynolds C P, Seeger R C
Division of Hematology-Oncology, MS 57, Department of Pediatrics, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90054-0700, USA.
Cancer Immunol Immunother. 2000 Feb;48(11):603-12. doi: 10.1007/s002620050008.
Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6-18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100-2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5-50:1) and mAb ch.14.18 dose (0.1-10 microg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 microg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed.
中性粒细胞和单核细胞（MNC）可介导针对癌细胞的抗体依赖性细胞毒性（ADCC）。为了研究嗜中性粒细胞和MNC与嵌合抗二唾液酸神经节苷脂（GD2）单克隆抗体（mAb）ch14.18对神经母细胞瘤细胞的细胞毒性和生长抑制作用，我们开发了数字图像显微镜扫描（DIMSCAN）测定法，该方法可在6 - 18小时（细胞毒性测定）或7天（生长测定）后测量96孔板中靶细胞的荧光。神经母细胞瘤细胞系（GD2阳性：SMS - KCN、SMS - LHN、LA - N - 1；GD2阴性：SK - N - SH）在细胞毒性测定中预先加载羧基荧光素乙酰甲酯，或在生长测定中培养7天后用二醋酸荧光素原位标记。通过DIMSCAN定量的荧光在两种测定中（每孔100 - 2000个细胞）均与神经母细胞瘤细胞数量相关。在细胞毒性试验中，嗜中性粒细胞和MNC均有效地介导了GD2阳性而非GD2阴性神经母细胞瘤细胞系的ADCC。嗜中性粒细胞和MNC的细胞毒性均随效应细胞与靶细胞（E:T）比例（5 - 50:1）和mAb ch.14.18剂量（0.1 - 10μg/ml）的增加而增加。加入粒细胞 -巨噬细胞集落刺激因子（GM - CSF）后，嗜中性粒细胞的ADCC增加，但MNC的ADCC未增加。嗜中性粒细胞，特别是与重组人GM - CSF（rhGM - CSF）一起，在高E:T比例（50:1）和mAb剂量（10μg/ml）下显著抑制GD2阳性细胞系的生长。在没有抗体的情况下，嗜中性粒细胞抑制一种细胞系（LA - N - 1）的生长，但刺激另外两种细胞系（SMS - KCN、SMS - LHN）的生长。如果神经母细胞瘤细胞不表达GD2（SK - N - SH），无论是否存在抗体，嗜中性粒细胞都会刺激其生长。嗜中性粒细胞培养上清液增加了SK - N - SH、LA - N - 1和SMS - KCN细胞的生长，而MNC培养上清液增加了SK - N - SH的生长。总之，嗜中性粒细胞可以用嵌合抗GD2抗体介导细胞毒性和生长抑制，但如果不存在抗体或不表达GD2，也可以促进肿瘤细胞生长。
