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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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Cytoskeleton modifications in  D1 cells after TNFα treatment. Immature (a)  and mature (b) D1 cells morphology was analyzed by confocal microscopy using reflection  interference contrast which gives the best visualization of the cell interface zone when attached to a glass support (37). Confocal laser  scanning microscopy of D1 cells stained with  anti-vinculin (c and d), phalloidin (e and f),  and anti-tubulin (g and h) was also performed.  Immature D1 cells (a, c, e, and g) appear to be  adherent (a) and characterized by: vinculin  containing adhesive structures (a and c, arrows), subcortical actin aggregates (e), and  highly organized tubulin (g). D1 treatment  with TNFα (b, d, f, and h) clearly induces  morphological modification (b). Mature D1  cells lose adherence (b), vinculin (d), and subcortical actin organization (   f   ). No differences  in the amount of vinculin protein are detectable by Western blot analysis (d, inset). Microtubules are only partially affected by TNFα  treatment (h).
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Figure 6: Cytoskeleton modifications in D1 cells after TNFα treatment. Immature (a) and mature (b) D1 cells morphology was analyzed by confocal microscopy using reflection interference contrast which gives the best visualization of the cell interface zone when attached to a glass support (37). Confocal laser scanning microscopy of D1 cells stained with anti-vinculin (c and d), phalloidin (e and f), and anti-tubulin (g and h) was also performed. Immature D1 cells (a, c, e, and g) appear to be adherent (a) and characterized by: vinculin containing adhesive structures (a and c, arrows), subcortical actin aggregates (e), and highly organized tubulin (g). D1 treatment with TNFα (b, d, f, and h) clearly induces morphological modification (b). Mature D1 cells lose adherence (b), vinculin (d), and subcortical actin organization (   f   ). No differences in the amount of vinculin protein are detectable by Western blot analysis (d, inset). Microtubules are only partially affected by TNFα treatment (h).

Mentions: Cell migration requires a coordinate series of events that include a sequential rearrangement of both surface adhesion molecules and of the actin-based cytoskeleton. Immature D1 cells are characterized by numerous membrane expansions that terminate in adhesive structures displayed in the panel by black arrows (Fig. 6 a). Highly organized vinculin can be visualized in the adhesive structures (Fig. 6 c, arrows). These structures are also characterized by the simultaneous staining for F-actin and vinculin (compare c with e). Unstimulated D1 cells shown subcortical actin aggregates, as visualized by phalloidin staining (Fig. 6 e). After TNFα treatment, D1 cells undergo a profound change in shape and lose both the large membrane expansions and adhesiveness (Fig. 6 b). Vinculin is disassembled (Fig. 6 d) and polymerized F-actin is no longer detectable (Fig. 6 f  ). However, equal levels of vinculin protein were detected in both TNFα treated and untreated D1 cells, as shown by western blot analysis (Fig. 6 d, inset). In contrast to the disassembly of the actin filaments organization, microtubules are only partially affected by TNFα treatment (Fig. 6, g and h). The centriolar organization of microtubules appear to be more prominent after TNFα treatment.


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)

Cytoskeleton modifications in  D1 cells after TNFα treatment. Immature (a)  and mature (b) D1 cells morphology was analyzed by confocal microscopy using reflection  interference contrast which gives the best visualization of the cell interface zone when attached to a glass support (37). Confocal laser  scanning microscopy of D1 cells stained with  anti-vinculin (c and d), phalloidin (e and f),  and anti-tubulin (g and h) was also performed.  Immature D1 cells (a, c, e, and g) appear to be  adherent (a) and characterized by: vinculin  containing adhesive structures (a and c, arrows), subcortical actin aggregates (e), and  highly organized tubulin (g). D1 treatment  with TNFα (b, d, f, and h) clearly induces  morphological modification (b). Mature D1  cells lose adherence (b), vinculin (d), and subcortical actin organization (   f   ). No differences  in the amount of vinculin protein are detectable by Western blot analysis (d, inset). Microtubules are only partially affected by TNFα  treatment (h).
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Figure 6: Cytoskeleton modifications in D1 cells after TNFα treatment. Immature (a) and mature (b) D1 cells morphology was analyzed by confocal microscopy using reflection interference contrast which gives the best visualization of the cell interface zone when attached to a glass support (37). Confocal laser scanning microscopy of D1 cells stained with anti-vinculin (c and d), phalloidin (e and f), and anti-tubulin (g and h) was also performed. Immature D1 cells (a, c, e, and g) appear to be adherent (a) and characterized by: vinculin containing adhesive structures (a and c, arrows), subcortical actin aggregates (e), and highly organized tubulin (g). D1 treatment with TNFα (b, d, f, and h) clearly induces morphological modification (b). Mature D1 cells lose adherence (b), vinculin (d), and subcortical actin organization (   f   ). No differences in the amount of vinculin protein are detectable by Western blot analysis (d, inset). Microtubules are only partially affected by TNFα treatment (h).
Mentions: Cell migration requires a coordinate series of events that include a sequential rearrangement of both surface adhesion molecules and of the actin-based cytoskeleton. Immature D1 cells are characterized by numerous membrane expansions that terminate in adhesive structures displayed in the panel by black arrows (Fig. 6 a). Highly organized vinculin can be visualized in the adhesive structures (Fig. 6 c, arrows). These structures are also characterized by the simultaneous staining for F-actin and vinculin (compare c with e). Unstimulated D1 cells shown subcortical actin aggregates, as visualized by phalloidin staining (Fig. 6 e). After TNFα treatment, D1 cells undergo a profound change in shape and lose both the large membrane expansions and adhesiveness (Fig. 6 b). Vinculin is disassembled (Fig. 6 d) and polymerized F-actin is no longer detectable (Fig. 6 f  ). However, equal levels of vinculin protein were detected in both TNFα treated and untreated D1 cells, as shown by western blot analysis (Fig. 6 d, inset). In contrast to the disassembly of the actin filaments organization, microtubules are only partially affected by TNFα treatment (Fig. 6, g and h). The centriolar organization of microtubules appear to be more prominent after TNFα treatment.

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