Feasibility to generate monocyte-derived dendritic cell from coculture with melanoma tumor cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Kim
Young T
YT
Department of Obstetrics and Gynecology, BK21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea. ytkchoi@yumc.yonsei.ac.kr
Hersh
Evan M
EM
Trevor
Katrina T
KT
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D019070:Cell Lineage; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016130:Immunophenotyping; D015847:Interleukin-4 / Q000378:metabolism* / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D008858:Microscopy, Phase-Contrast; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018950:Lipopolysaccharide Receptors; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor