Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes.
OBJECTIVE
This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro.


METHODS
PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h.


RESULTS
After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity.


CONCLUSIONS
This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.
Tang
Ling-ling
LL
Department of Infectious Disease, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. lltang72@126.com
Zhang
Zhe
Z
Zheng
Jie-sheng
JS
Sheng
Ji-fang
JF
Liu
Ke-zhou
KZ
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D010641:Phenotype; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor