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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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IL-12 p75 production by D1 cells pretreated or not with  TNFα upon presentation of OVA protein to the specific hybridoma  BO97.10 (striped bars or in the absence of the hybridoma open bars). IL-12  p75 was measured using an ELISA as described in Material and Methods.  Il-12 p75 was quantified from two to three titration points using standard  curves generated by purified recombinant mouse IL-12 and results expressed as cytokine concentration in pg/ml. Detection limit was 10 pg/ml.
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Figure 9: IL-12 p75 production by D1 cells pretreated or not with TNFα upon presentation of OVA protein to the specific hybridoma BO97.10 (striped bars or in the absence of the hybridoma open bars). IL-12 p75 was measured using an ELISA as described in Material and Methods. Il-12 p75 was quantified from two to three titration points using standard curves generated by purified recombinant mouse IL-12 and results expressed as cytokine concentration in pg/ml. Detection limit was 10 pg/ml.

Mentions: Bioactive IL-12 p75 is the regulatory cytokine which drives the development of Th1 cells and promotes cell-mediated immunity. IL-12 p75 production by TNFα-treated or untreated D1 cells was measured in a presentation assay in the presence of OVA-specific T cell hybridoma (BO97.9). Immature D1 cells fail to produce IL-12 p75 constitutively or upon stimulation with heattreated S. aureus and IFNγ (data not shown) or in the presence of the BO97.10 hybridoma (Fig. 9). In contrast, IL-12 p75 secretion was readily detectable in TNFα-matured D1 cells after interaction with antigen-specific T cell hybridoma. This result indicates that the feedback between T cells and DC is mediated by TCR-peptide/class II interaction. In order to exert this property, DC need to reach a stage of functional maturation which is not met by 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)

IL-12 p75 production by D1 cells pretreated or not with  TNFα upon presentation of OVA protein to the specific hybridoma  BO97.10 (striped bars or in the absence of the hybridoma open bars). IL-12  p75 was measured using an ELISA as described in Material and Methods.  Il-12 p75 was quantified from two to three titration points using standard  curves generated by purified recombinant mouse IL-12 and results expressed as cytokine concentration in pg/ml. Detection limit was 10 pg/ml.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: IL-12 p75 production by D1 cells pretreated or not with TNFα upon presentation of OVA protein to the specific hybridoma BO97.10 (striped bars or in the absence of the hybridoma open bars). IL-12 p75 was measured using an ELISA as described in Material and Methods. Il-12 p75 was quantified from two to three titration points using standard curves generated by purified recombinant mouse IL-12 and results expressed as cytokine concentration in pg/ml. Detection limit was 10 pg/ml.
Mentions: Bioactive IL-12 p75 is the regulatory cytokine which drives the development of Th1 cells and promotes cell-mediated immunity. IL-12 p75 production by TNFα-treated or untreated D1 cells was measured in a presentation assay in the presence of OVA-specific T cell hybridoma (BO97.9). Immature D1 cells fail to produce IL-12 p75 constitutively or upon stimulation with heattreated S. aureus and IFNγ (data not shown) or in the presence of the BO97.10 hybridoma (Fig. 9). In contrast, IL-12 p75 secretion was readily detectable in TNFα-matured D1 cells after interaction with antigen-specific T cell hybridoma. This result indicates that the feedback between T cells and DC is mediated by TCR-peptide/class II interaction. In order to exert this property, DC need to reach a stage of functional maturation which is not met by 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