[Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction].
OBJECTIVE
To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)- alpha during their induction of dendritic cell differentiation.


METHODS
PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-alpha, given before or 30 min after pre-treatment with 0.5 mug/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells.


RESULTS
PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-alpha for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-alpha.


CONCLUSIONS
Under serum-free conditions, both CI and TNF-alpha are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-alpha.
Wu
Jun
J
Department of Immunology, Southern Medical University, Guangzhou 510515, China. Juwuwang@Hotmail.com
Wang
Xiao-huai
XH
Yang
De-mao
DM
Yang
Tai-cheng
TC
Wang
Jie
J
Chen
Zheng-liang
ZL
D000001:Calcimycin / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016572:Cyclosporine / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D009000:Monocytes / Q000166:cytology*; D011994:Recombinant Proteins; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000001:Calcimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016572:Cyclosporine