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Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, Adorini L, Zimmermann VS, Davoust J, Ricciardi-Castagnoli P - J. Exp. Med. (1997)

Bottom Line: Antigen uptake and presentation of native protein antigen was reduced.In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation.This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

View Article: PubMed Central - PubMed

Affiliation: CNR Centre of Cellular and Molecular Pharmacology, Milan, Italy.

ABSTRACT
The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

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Related in: MedlinePlus

Dot plot double-color FACS® analysis of MHC class II and  B7.2 molecules of D1 bulk culture.
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Figure 3: Dot plot double-color FACS® analysis of MHC class II and B7.2 molecules of D1 bulk culture.

Mentions: Surface markers expressed in D1 cultures included CD11c (N418), FcγII/III receptor (2.4G2), and F4/80; NLDC145 was expressed only in ∼10% of the cells (Fig. 2). The majority of the cells expressed moderate levels of membrane class II molecules (I-Aint), but 20% of the cells expressed high levels (I-Abright) of class II molecules. The same staining pattern, albeit at lower fluorescence intensity, was observed for B7.2. Double-color flow cytometric analysis of unstimulated cells showed that I-Abright cells were those expressing higher levels of B7.2 (Fig. 3). D1 were positive for all class I antigens (H-2 K, D), and for the costimulatory CD40 and B7.1 molecules (Fig. 2). Adhesion and homing receptors such as CD11a (LFA-1), CD11b (Mac-1), CD54 (ICAM-1), and VLA-4 were highly expressed, as well as the heat stable antigen (HSA/J11d). In contrast, granulocytes, T and B cell markers were negative (Fig. 2), whereas c-kit and Fas ligand were expressed, although at low levels. Interestingly, the M-CSF receptor c-fms (CD115) was also expressed but experiments aimed at converting the D1 cells to a macrophage phenotype by adding rM-CSF in culture, showed that substitution of rGM-CSF (or R1 supernatants) with M-CSF (or M-CSF supplemented with fibroblast supernatant) in the culture medium, reduced the D1 cell viability to 15%, in 3 d. Growth arrest was observed in the remaining viable cells, which survived only for a few weeks. Altogether these results indicate that mouse spleen DC, grown in the culture conditions listed above, preserve the phenotype characteristic of immature DC.


Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, Adorini L, Zimmermann VS, Davoust J, Ricciardi-Castagnoli P - J. Exp. Med. (1997)

Dot plot double-color FACS® analysis of MHC class II and  B7.2 molecules of D1 bulk culture.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196118&req=5

Figure 3: Dot plot double-color FACS® analysis of MHC class II and B7.2 molecules of D1 bulk culture.
Mentions: Surface markers expressed in D1 cultures included CD11c (N418), FcγII/III receptor (2.4G2), and F4/80; NLDC145 was expressed only in ∼10% of the cells (Fig. 2). The majority of the cells expressed moderate levels of membrane class II molecules (I-Aint), but 20% of the cells expressed high levels (I-Abright) of class II molecules. The same staining pattern, albeit at lower fluorescence intensity, was observed for B7.2. Double-color flow cytometric analysis of unstimulated cells showed that I-Abright cells were those expressing higher levels of B7.2 (Fig. 3). D1 were positive for all class I antigens (H-2 K, D), and for the costimulatory CD40 and B7.1 molecules (Fig. 2). Adhesion and homing receptors such as CD11a (LFA-1), CD11b (Mac-1), CD54 (ICAM-1), and VLA-4 were highly expressed, as well as the heat stable antigen (HSA/J11d). In contrast, granulocytes, T and B cell markers were negative (Fig. 2), whereas c-kit and Fas ligand were expressed, although at low levels. Interestingly, the M-CSF receptor c-fms (CD115) was also expressed but experiments aimed at converting the D1 cells to a macrophage phenotype by adding rM-CSF in culture, showed that substitution of rGM-CSF (or R1 supernatants) with M-CSF (or M-CSF supplemented with fibroblast supernatant) in the culture medium, reduced the D1 cell viability to 15%, in 3 d. Growth arrest was observed in the remaining viable cells, which survived only for a few weeks. Altogether these results indicate that mouse spleen DC, grown in the culture conditions listed above, preserve the phenotype characteristic of immature DC.

Bottom Line: Antigen uptake and presentation of native protein antigen was reduced.In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation.This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

View Article: PubMed Central - PubMed

Affiliation: CNR Centre of Cellular and Molecular Pharmacology, Milan, Italy.

ABSTRACT
The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Show MeSH
Related in: MedlinePlus