Kim Young T, Hersh Evan M, Trevor Katrina T
Department of Obstetrics and Gynecology, BK21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea.
Am J Reprod Immunol. 2003 Apr;49(4):230-8. doi: 10.1034/j.1600-0897.2003.01200.x.
We investigated how melanoma cells and membrane-bound granulocyte/macrophage colony stimulating factor (mbGM-CSF) melanoma cell lines affect the differentiation of dendritic cells (DC) from CD14+ monocytes.
The malignant melanoma cell lines (Conley and Jorp) and mbGM-CSF-positive cell lines (Conley B-F8 and Jorp C-E6) were cultured and cell-free supernatants were collected from each cell line cultures to assess the GM-CSF level. Adherent monocytes were cocultured for 6-7 days with irradiated mbGM-CSF and wild type melanoma cells (50 Gy) at each 1:1 and 0.1:1 ratio in six-well culture plates in ex vivo culture medium containing interleukin (IL)-4. Immunophenotyping was performed by triple color immunofluorescence staining with flow cytometry analysis.
GM-CSF was detected at low levels in the culture supernatants of Conley B-F8 (0.48 ng/10(6) cells/24 hr), whereas there was no detectable GM-CSF in that of Conley melanoma cell line. Monocytes cultured with GM-CSF/IL-4 generated the expression of high levels of HLA DR, CD1a and CD86, while the expression of CD14 and CD83 were in low amounts. However, the addition of GM-CSF to these cultures resulted in an increased expression of these markers and decreased that of CD14. Monocytes cocultured with Jorp C-E6 illustrated similar expression pattern of CD1a, HLA DR and CD14 in the presence or absence of GM-CSF as Conley B-F8 melanoma cell line. Monocytes cocultured with Conley B-F8 melanoma cells at 1:1 and 0.1:1 ratio showed no significant difference in expression of CD1a, CD14 and CD83 between the two ratios.
Our results illustrate the feasibility to generate monocyte-derived DC from coculture with melanoma tumor cells in the presence of GM-CSF and IL-4. However, mbGM-CSF tumor cells did not significantly enhance the DC differentiation through juxtacrine stimulation unless soluble GM-CSF was added in the culture media.
我们研究了黑色素瘤细胞和膜结合型粒细胞/巨噬细胞集落刺激因子（mbGM-CSF）黑色素瘤细胞系如何影响CD14+单核细胞向树突状细胞（DC）的分化。
培养恶性黑色素瘤细胞系（Conley和Jorp）以及mbGM-CSF阳性细胞系（Conley B-F8和Jorp C-E6），从每个细胞系培养物中收集无细胞上清液以评估GM-CSF水平。在含有白细胞介素（IL）-4的体外培养基中，将贴壁单核细胞与经辐照的mbGM-CSF和野生型黑色素瘤细胞（50 Gy）以1:1和0.1:1的比例在六孔培养板中共培养6-7天。通过三色免疫荧光染色和流式细胞术分析进行免疫表型分析。
在Conley B-F8的培养上清液中检测到低水平的GM-CSF（0.48 ng/10(6)个细胞/24小时），而在Conley黑色素瘤细胞系的培养上清液中未检测到GM-CSF。用GM-CSF/IL-4培养的单核细胞产生高水平的HLA DR、CD1a和CD86表达，而CD14和CD83的表达量较低。然而，向这些培养物中添加GM-CSF导致这些标志物的表达增加，CD14的表达减少。与Jorp C-E6共培养的单核细胞在有或没有GM-CSF的情况下，其CD1a、HLA DR和CD14的表达模式与Conley B-F8黑色素瘤细胞系相似。与Conley B-F8黑色素瘤细胞以1:1和0.1:1比例共培养的单核细胞在两种比例下CD1a、CD14和CD83的表达无显著差异。
我们的结果表明，在GM-CSF和IL-4存在的情况下，与黑色素瘤肿瘤细胞共培养产生单核细胞来源的DC具有可行性。然而，除非在培养基中添加可溶性GM-CSF，mbGM-CSF肿瘤细胞不会通过旁分泌刺激显著增强DC分化。
