Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.
Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.
Ross
R
R
Clinical Research Unit, Department of Dermatology, Johannes Gutenberg University, Mainz, Germany.
Jonuleit
H
H
Bros
M
M
Ross
X L
XL
Yamashiro
S
S
Matsumura
F
F
Enk
A H
AH
Knop
J
J
Reske-Kunz
A B
AB
D000199:Actins / Q000378:metabolism; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000097:blood; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008562:Membrane Glycoproteins / Q000032:analysis; D008840:Microfilament Proteins / Q000096:biosynthesis*; D013997:Time Factors; D000097802:CD83 Antigen
D000199:Actins; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D002352:Carrier Proteins; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D000097802:CD83 Antigen; C020324:fascin