Limits...
Apoptosis-related mitochondrial dysfunction defines human monocyte-derived dendritic cells with impaired immuno-stimulatory capacities.

Castera L, Hatzfeld-Charbonnier AS, Ballot C, Charbonnel F, Dhuiege E, Velu T, Formstecher P, Mortier L, Marchetti P - J. Cell. Mol. Med. (2008)

Bottom Line: Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated.These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC.Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

View Article: PubMed Central - PubMed

Affiliation: Inserm U837 and Plate-forme de Biothérapie, Faculté de Médecine Université de Lille II 1, Place Verdun, Lille Cedex, France.

ABSTRACT
The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

Show MeSH

Related in: MedlinePlus

Spontaneous death of freshly matured human monocyte-derived DCs. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day for cell death determination (0, 1, 2, 3 or 4 days of culture). (A) Flow cytometric analysis of Annexin V-FITC binding and PI staining in cultured DCs at different incubation times (days). The percentage of DCs in each quadrant is indicated. Similar results were obtained from five separate donors. (B) Flow cytometric determination of the percentage of sub-G1 cells obtained by cell cycle evaluation and the percentage of PI permeable cells (PI +) at different culture intervals (days) is shown. Mean ± S.D. from 5 independent experiments are shown. (C) When indicated, cytospin preparations of DCs were stained with May-Grundwald-Giemsa to evaluate general DC morphology (Ao: Apoptotic cell; Nc: Necrotic cell). Micrographs were taken under a transmission electron microscope (original magnification ×630). Two independent experiments gave similar results. (D) Agarose gel electrophoresis of DC DNA before (0) and after (1 or 2 days) culture. Two independent experiments gave similar results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4496146&req=5

fig01: Spontaneous death of freshly matured human monocyte-derived DCs. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day for cell death determination (0, 1, 2, 3 or 4 days of culture). (A) Flow cytometric analysis of Annexin V-FITC binding and PI staining in cultured DCs at different incubation times (days). The percentage of DCs in each quadrant is indicated. Similar results were obtained from five separate donors. (B) Flow cytometric determination of the percentage of sub-G1 cells obtained by cell cycle evaluation and the percentage of PI permeable cells (PI +) at different culture intervals (days) is shown. Mean ± S.D. from 5 independent experiments are shown. (C) When indicated, cytospin preparations of DCs were stained with May-Grundwald-Giemsa to evaluate general DC morphology (Ao: Apoptotic cell; Nc: Necrotic cell). Micrographs were taken under a transmission electron microscope (original magnification ×630). Two independent experiments gave similar results. (D) Agarose gel electrophoresis of DC DNA before (0) and after (1 or 2 days) culture. Two independent experiments gave similar results.

Mentions: The levels of spontaneous cell death in monocyte-derived DCs were first determined. We cultured freshly matured human mono-cyte-derived DCs for up to 4 days in complete medium (RPMI including 10% foetal calf serum) and every day an aliquot of cells was examined by dual staining flow cytometry with annexin V and the vital dye PI. Annexin V-positive and PI-negative cells were identified as apoptotic DCs and cells that were both PI-positive and annexin V-positive identified as dead DCs (secondary necrosis) (Fig. 1A). The number of apoptotic DCs increased during the first days of culture (up to 2 days). This was followed by a plateau phase, although the number of dead cells then steadily increased, and after 4 days of culture approximately 50% of DCs were dead (Fig. 1A). Kinetic studies of sub-G1 cells and PI uptake, respectively, indicated that the apoptotic percentage was lower than the percentage of dead (PI-positive) cells (Fig. 1B), confirming the results obtained with annexin V/PI (Fig. 1A). Additionally, the microscopic images of cells stained with MGG demonstrated the significant presence of apoptotic DCs (i.e. condensed nuclei and shrunken cytoplasm) occurring after 2 days of culture (Fig. 1C). This observation of apoptotic cells was confirmed by the presence of the electophoretic pattern of DNA laddering (Fig. 1D). Contrasting with the presence of apoptotic features, cellular signs of primary necrosis, such as cells with loose nuclei and swollen cytoplasm, were found in 10–20% of DCs in association with cell debris later, i.e. at day 4 of culture (Fig. 1C). Thus, freshly matured monocyte-derived DCs underwent constitutive cell death characterized by hallmarks of apoptosis, as well as necrosis.


Apoptosis-related mitochondrial dysfunction defines human monocyte-derived dendritic cells with impaired immuno-stimulatory capacities.

Castera L, Hatzfeld-Charbonnier AS, Ballot C, Charbonnel F, Dhuiege E, Velu T, Formstecher P, Mortier L, Marchetti P - J. Cell. Mol. Med. (2008)

Spontaneous death of freshly matured human monocyte-derived DCs. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day for cell death determination (0, 1, 2, 3 or 4 days of culture). (A) Flow cytometric analysis of Annexin V-FITC binding and PI staining in cultured DCs at different incubation times (days). The percentage of DCs in each quadrant is indicated. Similar results were obtained from five separate donors. (B) Flow cytometric determination of the percentage of sub-G1 cells obtained by cell cycle evaluation and the percentage of PI permeable cells (PI +) at different culture intervals (days) is shown. Mean ± S.D. from 5 independent experiments are shown. (C) When indicated, cytospin preparations of DCs were stained with May-Grundwald-Giemsa to evaluate general DC morphology (Ao: Apoptotic cell; Nc: Necrotic cell). Micrographs were taken under a transmission electron microscope (original magnification ×630). Two independent experiments gave similar results. (D) Agarose gel electrophoresis of DC DNA before (0) and after (1 or 2 days) culture. Two independent experiments gave similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496146&req=5

fig01: Spontaneous death of freshly matured human monocyte-derived DCs. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day for cell death determination (0, 1, 2, 3 or 4 days of culture). (A) Flow cytometric analysis of Annexin V-FITC binding and PI staining in cultured DCs at different incubation times (days). The percentage of DCs in each quadrant is indicated. Similar results were obtained from five separate donors. (B) Flow cytometric determination of the percentage of sub-G1 cells obtained by cell cycle evaluation and the percentage of PI permeable cells (PI +) at different culture intervals (days) is shown. Mean ± S.D. from 5 independent experiments are shown. (C) When indicated, cytospin preparations of DCs were stained with May-Grundwald-Giemsa to evaluate general DC morphology (Ao: Apoptotic cell; Nc: Necrotic cell). Micrographs were taken under a transmission electron microscope (original magnification ×630). Two independent experiments gave similar results. (D) Agarose gel electrophoresis of DC DNA before (0) and after (1 or 2 days) culture. Two independent experiments gave similar results.
Mentions: The levels of spontaneous cell death in monocyte-derived DCs were first determined. We cultured freshly matured human mono-cyte-derived DCs for up to 4 days in complete medium (RPMI including 10% foetal calf serum) and every day an aliquot of cells was examined by dual staining flow cytometry with annexin V and the vital dye PI. Annexin V-positive and PI-negative cells were identified as apoptotic DCs and cells that were both PI-positive and annexin V-positive identified as dead DCs (secondary necrosis) (Fig. 1A). The number of apoptotic DCs increased during the first days of culture (up to 2 days). This was followed by a plateau phase, although the number of dead cells then steadily increased, and after 4 days of culture approximately 50% of DCs were dead (Fig. 1A). Kinetic studies of sub-G1 cells and PI uptake, respectively, indicated that the apoptotic percentage was lower than the percentage of dead (PI-positive) cells (Fig. 1B), confirming the results obtained with annexin V/PI (Fig. 1A). Additionally, the microscopic images of cells stained with MGG demonstrated the significant presence of apoptotic DCs (i.e. condensed nuclei and shrunken cytoplasm) occurring after 2 days of culture (Fig. 1C). This observation of apoptotic cells was confirmed by the presence of the electophoretic pattern of DNA laddering (Fig. 1D). Contrasting with the presence of apoptotic features, cellular signs of primary necrosis, such as cells with loose nuclei and swollen cytoplasm, were found in 10–20% of DCs in association with cell debris later, i.e. at day 4 of culture (Fig. 1C). Thus, freshly matured monocyte-derived DCs underwent constitutive cell death characterized by hallmarks of apoptosis, as well as necrosis.

Bottom Line: Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated.These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC.Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

View Article: PubMed Central - PubMed

Affiliation: Inserm U837 and Plate-forme de Biothérapie, Faculté de Médecine Université de Lille II 1, Place Verdun, Lille Cedex, France.

ABSTRACT
The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

Show MeSH
Related in: MedlinePlus