Yang Hua, Dithmar Stefan, Grossniklaus Hans E
Department of Ophthalmology, Emory University, Atlanta, Georgia 30322, USA.
Invest Ophthalmol Vis Sci. 2004 Jul;45(7):2056-64. doi: 10.1167/iovs.03-1331.
PURPOSE: To investigate in a murine model the mechanism by which micrometastatic melanoma, which spreads from the eye to the liver, is controlled by interferon (IFN)-alpha 2b. METHODS: Major histocompatibility (MHC) class I antigen (H-2, all haplotypes) expression in three murine melanoma cell lines (Queens, B16LS9, B16F10) was determined by flow cytometric immunophenotyping. The cell lines were heterotopically inoculated into the posterior compartments (PCs) of C57Bl/6 mice, and the mice were given intraperitoneal (IP) injections of IFN-alpha 2b or PBS for 1 or 4 days before enucleation at 7 days after inoculation. Groups of mice were made NK deficient or depleted with subcutaneous (s.c.) injection of anti-asialo GM1. The mice were killed at 28 days or 56 days (survival experiment) after inoculation, and the number of hepatic micrometastases was histologically determined. NK cells were isolated from the spleen and liver at necropsy, and propidium iodide labeled target-specific cytolysis was determined by flow cytometry. The micrometastases were evaluated for apoptosis and proliferation with TUNEL and MIB1 immunostaining, respectively, and TUNEL-to-MIB1 ratios were determined. Hepatic NK cells were immunostained with CD49b. RESULTS: MHC class I antigen was expressed in the three cell lines in the order of Queens < B16LS9 < B16F10. All cell lines grew, were confined to the PC, and formed hepatic micrometastases. A decrease in micrometastases, an increase in target-specific cytolysis, and an increase in survival correlated with decreased HLA class I expression by the melanoma cells. The IFN-alpha 2b treatment resulted in a boost of intrinsic hepatic NK cells, demonstrated in NK-deficient but not NK-depleted mice. The treatment effect corresponded to increased apoptosis (TUNEL)-proliferation (MIB1) ratios in the micrometastases. Immunostaining demonstrated an increased number of intrahepatic NK cells associated with the micrometastases in treated groups. CONCLUSIONS: Neoadjuvant IFN-alpha 2b results in decreased hepatic micrometastasis and increased survival time through increased intrinsic hepatic NK cell-mediated tumor apoptosis in a murine model of metastatic ocular melanoma.
目的：在小鼠模型中研究从眼部扩散至肝脏的微转移黑色素瘤受干扰素（IFN）-α 2b控制的机制。 方法：通过流式细胞术免疫表型分析确定三种小鼠黑色素瘤细胞系（Queens、B16LS9、B16F10）中主要组织相容性（MHC）I类抗原（H-2，所有单倍型）的表达。将细胞系异位接种到C57Bl/6小鼠的后房（PC）中，在接种后7天摘除眼球前1天或4天，给小鼠腹腔内（IP）注射IFN-α 2b或磷酸盐缓冲液（PBS）。通过皮下（s.c.）注射抗去唾液酸GM1使小鼠NK细胞缺陷或耗竭。在接种后28天或56天（生存实验）处死小鼠，通过组织学方法确定肝脏微转移灶的数量。在尸检时从脾脏和肝脏中分离NK细胞，通过流式细胞术测定碘化丙啶标记的靶特异性细胞溶解。分别用TUNEL和MIB1免疫染色评估微转移灶的凋亡和增殖情况，并确定TUNEL与MIB1的比值。用CD49b对肝脏NK细胞进行免疫染色。 结果：MHC I类抗原在三种细胞系中的表达顺序为Queens < B16LS9 < B16F10。所有细胞系均生长，并局限于PC，且形成肝脏微转移灶。微转移灶数量减少、靶特异性细胞溶解增加以及生存率提高与黑色素瘤细胞HLA I类表达降低相关。IFN-α 2b治疗导致肝脏固有NK细胞增强，这在NK细胞缺陷而非NK细胞耗竭的小鼠中得到证实。治疗效果与微转移灶中凋亡（TUNEL）-增殖（MIB1）比值增加相对应。免疫染色显示治疗组中与微转移灶相关的肝内NK细胞数量增加。 结论：在转移性眼黑色素瘤小鼠模型中，新辅助IFN-α 2b通过增加肝脏固有NK细胞介导的肿瘤凋亡，导致肝脏微转移灶减少和生存时间延长。
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