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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.

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Regulation of spontaneous DC death. Immediately after maturation, human monocyte-derived DCs were cultured for 4 days in the presence or absence of 100 μM z-VAD.fmk added every day (A) or culture medium supplemented every 2 days with 800 U/ml GMCSF and 100 U/ml IL-4 (B). Aliquots of cells were collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from 5 independent experiments are shown. (C) Effects of maturation factors on spontaneous DC death. DCs were induced to mature by the addition of 1 μg/ml LPS for 18 hrs or 10 μg/ml poly (I:C) or 5 μg/ml anti-CD40 mAb and then mature DCs cultured for 4 days, with an aliquot of cells collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from five independent experiments are shown.
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fig03: Regulation of spontaneous DC death. Immediately after maturation, human monocyte-derived DCs were cultured for 4 days in the presence or absence of 100 μM z-VAD.fmk added every day (A) or culture medium supplemented every 2 days with 800 U/ml GMCSF and 100 U/ml IL-4 (B). Aliquots of cells were collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from 5 independent experiments are shown. (C) Effects of maturation factors on spontaneous DC death. DCs were induced to mature by the addition of 1 μg/ml LPS for 18 hrs or 10 μg/ml poly (I:C) or 5 μg/ml anti-CD40 mAb and then mature DCs cultured for 4 days, with an aliquot of cells collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from five independent experiments are shown.

Mentions: To determine whether the caspase family of proteases is necessary for Δψm disruption and cell death, cells were incubated with z-VAD.fmk, a generalized irreversible caspase inhibitor. The z-VAD.fmk failed to prevent spontaneous Δψm disruption (Fig. 3A, left panel), confirming that caspases must act downstream of the mitochondrion. The z-VAD.fmk, at concentrations that completely block caspase activity, only delayed but did not prevent spontaneous DC death (Fig. 3A, right panel). We also asked whether the spontaneous reduction of ΔΨm and nuclear apoptosis could be reversed if a survival stimulus was applied (Fig. 3B). Thus, when the level of cytokines (GMCSF + IL-4) was maintained in the culture medium, Δψm disruption and DC death were significantly delayed during the first days of incubation, confirming the close association between the onset of death and mitochondrial dysfunction in DCs. However, this protective effect disappeared after 4 days of culture, even when cytokines were used at higher concentrations (data not shown).


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)

Regulation of spontaneous DC death. Immediately after maturation, human monocyte-derived DCs were cultured for 4 days in the presence or absence of 100 μM z-VAD.fmk added every day (A) or culture medium supplemented every 2 days with 800 U/ml GMCSF and 100 U/ml IL-4 (B). Aliquots of cells were collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from 5 independent experiments are shown. (C) Effects of maturation factors on spontaneous DC death. DCs were induced to mature by the addition of 1 μg/ml LPS for 18 hrs or 10 μg/ml poly (I:C) or 5 μg/ml anti-CD40 mAb and then mature DCs cultured for 4 days, with an aliquot of cells collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from five independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Regulation of spontaneous DC death. Immediately after maturation, human monocyte-derived DCs were cultured for 4 days in the presence or absence of 100 μM z-VAD.fmk added every day (A) or culture medium supplemented every 2 days with 800 U/ml GMCSF and 100 U/ml IL-4 (B). Aliquots of cells were collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from 5 independent experiments are shown. (C) Effects of maturation factors on spontaneous DC death. DCs were induced to mature by the addition of 1 μg/ml LPS for 18 hrs or 10 μg/ml poly (I:C) or 5 μg/ml anti-CD40 mAb and then mature DCs cultured for 4 days, with an aliquot of cells collected every day (0, 1, 2, 3 or 4 days of culture) for determination of Δψm (DiOC6(3) staining) and cell death (PI uptake). Mean ± S.D. from five independent experiments are shown.
Mentions: To determine whether the caspase family of proteases is necessary for Δψm disruption and cell death, cells were incubated with z-VAD.fmk, a generalized irreversible caspase inhibitor. The z-VAD.fmk failed to prevent spontaneous Δψm disruption (Fig. 3A, left panel), confirming that caspases must act downstream of the mitochondrion. The z-VAD.fmk, at concentrations that completely block caspase activity, only delayed but did not prevent spontaneous DC death (Fig. 3A, right panel). We also asked whether the spontaneous reduction of ΔΨm and nuclear apoptosis could be reversed if a survival stimulus was applied (Fig. 3B). Thus, when the level of cytokines (GMCSF + IL-4) was maintained in the culture medium, Δψm disruption and DC death were significantly delayed during the first days of incubation, confirming the close association between the onset of death and mitochondrial dysfunction in DCs. However, this protective effect disappeared after 4 days of culture, even when cytokines were used at higher concentrations (data not shown).

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