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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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Related in: MedlinePlus

Spontaneous DC death involves caspase activation and mitochondrial dysfunction. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day (0, 1, 2, 3 or 4 days of culture) for determination of caspase activation (A) and mitochondrial dysfunction (B, C, D). (A) Caspases 3/7 activity was measured (left part) as described in Materials and Methods. When indicated, DCs were exposed for 1 hr to z-VAD-fmk (100 μM) as a positive control for caspase inhibition. Mean ± S.D. from three independent experiments are shown. Active caspase 3 was identified by Western blotting (right part). Three independent experiments gave similar results. (B) Cytosolic protein fractions were obtained and cytochrome C, AIF and Smac Diablo mitochondrial release in DCs was evaluated by Western blotting. (left part). When indicated, DCs were treated for 24 hrs with z-VAD-fmk (100 μM). Equal loading was checked by probing with anti-G3PDH antibody. Blots were also probed for cyt c oxidase to exlude mitochondria contamination in the cytosol. As a control for detection of cyt c oxidase, a total lysate was loaded. Fluorescence images (right part) of immunostaining with cytochrome c (green) or AIF (green) and nuclear Hoechst 33342 staining (blue) of DCs at day 0 (D0) or day 1 (D1) of culture. Original magnification ×630. (C) Simultaneous assessment of Δψm and ROS production performed with DiOC6(3) and HE. As a control, DCs were incubated with the protonophore mClCCP (100 μM, 15 min., 37°C) as a negative control for DiOC6(3) staining. One representative experiment of four is shown. (D and E). Mature DCs were cultured for 36 hrs under standard conditions, and cells then stained with DiOC6(3) and PI before flow cytometric analysis. Same results were obtained with CMX-ROS and YOPRO-1 staining.These flow cytometric parameters were used for sorting (D). The left square depicts the viable sorted Δψm low subpopulation and the right square represents the ΔΨm high counterparts. (E) Determination of caspase activity in Δψm-sorted populations as shown in D. Caspases -3/7, -8, and -9 activity was estimated after short-term culture of sorted viable Δψm high and low DC subpopulations (E). Mean ± S.D. from three independent experiments are shown. *Statistically significant between purified Δψm low and Δψm high subpopulations. (F) Mitochondrial calcium retention capacity. At day 0 (D0, left panel) or day 2 (D2, right panel) of culture, DCs were permeabilized with digitonin and calcium uptake measured with a calcium-sensitive electrode after the addition of calcium (each 10 μM CaCl2 pulse [arrows] was detected as a peak in calcium concentration). Three independent experiments gave similar results.
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fig02: Spontaneous DC death involves caspase activation and mitochondrial dysfunction. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day (0, 1, 2, 3 or 4 days of culture) for determination of caspase activation (A) and mitochondrial dysfunction (B, C, D). (A) Caspases 3/7 activity was measured (left part) as described in Materials and Methods. When indicated, DCs were exposed for 1 hr to z-VAD-fmk (100 μM) as a positive control for caspase inhibition. Mean ± S.D. from three independent experiments are shown. Active caspase 3 was identified by Western blotting (right part). Three independent experiments gave similar results. (B) Cytosolic protein fractions were obtained and cytochrome C, AIF and Smac Diablo mitochondrial release in DCs was evaluated by Western blotting. (left part). When indicated, DCs were treated for 24 hrs with z-VAD-fmk (100 μM). Equal loading was checked by probing with anti-G3PDH antibody. Blots were also probed for cyt c oxidase to exlude mitochondria contamination in the cytosol. As a control for detection of cyt c oxidase, a total lysate was loaded. Fluorescence images (right part) of immunostaining with cytochrome c (green) or AIF (green) and nuclear Hoechst 33342 staining (blue) of DCs at day 0 (D0) or day 1 (D1) of culture. Original magnification ×630. (C) Simultaneous assessment of Δψm and ROS production performed with DiOC6(3) and HE. As a control, DCs were incubated with the protonophore mClCCP (100 μM, 15 min., 37°C) as a negative control for DiOC6(3) staining. One representative experiment of four is shown. (D and E). Mature DCs were cultured for 36 hrs under standard conditions, and cells then stained with DiOC6(3) and PI before flow cytometric analysis. Same results were obtained with CMX-ROS and YOPRO-1 staining.These flow cytometric parameters were used for sorting (D). The left square depicts the viable sorted Δψm low subpopulation and the right square represents the ΔΨm high counterparts. (E) Determination of caspase activity in Δψm-sorted populations as shown in D. Caspases -3/7, -8, and -9 activity was estimated after short-term culture of sorted viable Δψm high and low DC subpopulations (E). Mean ± S.D. from three independent experiments are shown. *Statistically significant between purified Δψm low and Δψm high subpopulations. (F) Mitochondrial calcium retention capacity. At day 0 (D0, left panel) or day 2 (D2, right panel) of culture, DCs were permeabilized with digitonin and calcium uptake measured with a calcium-sensitive electrode after the addition of calcium (each 10 μM CaCl2 pulse [arrows] was detected as a peak in calcium concentration). Three independent experiments gave similar results.

Mentions: To further explore the mechanism of constitutive apoptosis in DCs, we sought to investigate the role of effector caspases as well as mitochondria, two main contributors to classical apoptosis. As previously reported [23], we observed significant caspase-3/-7-like activity as early as day 0, and which increased twofold after 1 day of culture (Fig. 2A, left part) i.e. before the appearance of nuclear apoptosis (Fig. 1). This increase was maintained for up to 4 days of culture. This profile was accompanied by enhancement of the proteolytic activation of caspase-3 (Fig. 2A, right part). The mitochondrion is a key regulator that controls cell life and death by releasing several death-promoting factors into the cytosol [24]. To examine the involvement of mito-chondria in DC death, cytosolic extracts were prepared at various times from DCs in culture and subjected to immunoblotting. The immunoblotting results indicated that the pro-mitochondrial factors cytochrome c, AIF and Smac/Diablo were substantially raised in cytosolic fractions at day 1 of culture (Fig. 2B). Sub-cellular distribution of cyt c and AIF was also monitored by immunofluo-rescence microcopy confirming the spontaneous release of proapoptotic proteins (Fig. 2B). The cytosolic accumulation of cytochrome c and AIF was not abrogated by z-VAD.fmk (Fig. 2B) at concentrations that completely block caspase activity (Fig. 2A), suggesting that caspases must act downstream of the mitochon-drial signals. However, it is to note that cytosolic accumulation of Smac/Diablo was slightly attenuated in z-VAD.fmk treated cells, a result previously observed in other cell types [25].


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 DC death involves caspase activation and mitochondrial dysfunction. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day (0, 1, 2, 3 or 4 days of culture) for determination of caspase activation (A) and mitochondrial dysfunction (B, C, D). (A) Caspases 3/7 activity was measured (left part) as described in Materials and Methods. When indicated, DCs were exposed for 1 hr to z-VAD-fmk (100 μM) as a positive control for caspase inhibition. Mean ± S.D. from three independent experiments are shown. Active caspase 3 was identified by Western blotting (right part). Three independent experiments gave similar results. (B) Cytosolic protein fractions were obtained and cytochrome C, AIF and Smac Diablo mitochondrial release in DCs was evaluated by Western blotting. (left part). When indicated, DCs were treated for 24 hrs with z-VAD-fmk (100 μM). Equal loading was checked by probing with anti-G3PDH antibody. Blots were also probed for cyt c oxidase to exlude mitochondria contamination in the cytosol. As a control for detection of cyt c oxidase, a total lysate was loaded. Fluorescence images (right part) of immunostaining with cytochrome c (green) or AIF (green) and nuclear Hoechst 33342 staining (blue) of DCs at day 0 (D0) or day 1 (D1) of culture. Original magnification ×630. (C) Simultaneous assessment of Δψm and ROS production performed with DiOC6(3) and HE. As a control, DCs were incubated with the protonophore mClCCP (100 μM, 15 min., 37°C) as a negative control for DiOC6(3) staining. One representative experiment of four is shown. (D and E). Mature DCs were cultured for 36 hrs under standard conditions, and cells then stained with DiOC6(3) and PI before flow cytometric analysis. Same results were obtained with CMX-ROS and YOPRO-1 staining.These flow cytometric parameters were used for sorting (D). The left square depicts the viable sorted Δψm low subpopulation and the right square represents the ΔΨm high counterparts. (E) Determination of caspase activity in Δψm-sorted populations as shown in D. Caspases -3/7, -8, and -9 activity was estimated after short-term culture of sorted viable Δψm high and low DC subpopulations (E). Mean ± S.D. from three independent experiments are shown. *Statistically significant between purified Δψm low and Δψm high subpopulations. (F) Mitochondrial calcium retention capacity. At day 0 (D0, left panel) or day 2 (D2, right panel) of culture, DCs were permeabilized with digitonin and calcium uptake measured with a calcium-sensitive electrode after the addition of calcium (each 10 μM CaCl2 pulse [arrows] was detected as a peak in calcium concentration). Three independent experiments gave similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496146&req=5

fig02: Spontaneous DC death involves caspase activation and mitochondrial dysfunction. Immediately after maturation, human monocyte-derived DCs were cultured under standard conditions for 4 days and an aliquot of cells was collected every day (0, 1, 2, 3 or 4 days of culture) for determination of caspase activation (A) and mitochondrial dysfunction (B, C, D). (A) Caspases 3/7 activity was measured (left part) as described in Materials and Methods. When indicated, DCs were exposed for 1 hr to z-VAD-fmk (100 μM) as a positive control for caspase inhibition. Mean ± S.D. from three independent experiments are shown. Active caspase 3 was identified by Western blotting (right part). Three independent experiments gave similar results. (B) Cytosolic protein fractions were obtained and cytochrome C, AIF and Smac Diablo mitochondrial release in DCs was evaluated by Western blotting. (left part). When indicated, DCs were treated for 24 hrs with z-VAD-fmk (100 μM). Equal loading was checked by probing with anti-G3PDH antibody. Blots were also probed for cyt c oxidase to exlude mitochondria contamination in the cytosol. As a control for detection of cyt c oxidase, a total lysate was loaded. Fluorescence images (right part) of immunostaining with cytochrome c (green) or AIF (green) and nuclear Hoechst 33342 staining (blue) of DCs at day 0 (D0) or day 1 (D1) of culture. Original magnification ×630. (C) Simultaneous assessment of Δψm and ROS production performed with DiOC6(3) and HE. As a control, DCs were incubated with the protonophore mClCCP (100 μM, 15 min., 37°C) as a negative control for DiOC6(3) staining. One representative experiment of four is shown. (D and E). Mature DCs were cultured for 36 hrs under standard conditions, and cells then stained with DiOC6(3) and PI before flow cytometric analysis. Same results were obtained with CMX-ROS and YOPRO-1 staining.These flow cytometric parameters were used for sorting (D). The left square depicts the viable sorted Δψm low subpopulation and the right square represents the ΔΨm high counterparts. (E) Determination of caspase activity in Δψm-sorted populations as shown in D. Caspases -3/7, -8, and -9 activity was estimated after short-term culture of sorted viable Δψm high and low DC subpopulations (E). Mean ± S.D. from three independent experiments are shown. *Statistically significant between purified Δψm low and Δψm high subpopulations. (F) Mitochondrial calcium retention capacity. At day 0 (D0, left panel) or day 2 (D2, right panel) of culture, DCs were permeabilized with digitonin and calcium uptake measured with a calcium-sensitive electrode after the addition of calcium (each 10 μM CaCl2 pulse [arrows] was detected as a peak in calcium concentration). Three independent experiments gave similar results.
Mentions: To further explore the mechanism of constitutive apoptosis in DCs, we sought to investigate the role of effector caspases as well as mitochondria, two main contributors to classical apoptosis. As previously reported [23], we observed significant caspase-3/-7-like activity as early as day 0, and which increased twofold after 1 day of culture (Fig. 2A, left part) i.e. before the appearance of nuclear apoptosis (Fig. 1). This increase was maintained for up to 4 days of culture. This profile was accompanied by enhancement of the proteolytic activation of caspase-3 (Fig. 2A, right part). The mitochondrion is a key regulator that controls cell life and death by releasing several death-promoting factors into the cytosol [24]. To examine the involvement of mito-chondria in DC death, cytosolic extracts were prepared at various times from DCs in culture and subjected to immunoblotting. The immunoblotting results indicated that the pro-mitochondrial factors cytochrome c, AIF and Smac/Diablo were substantially raised in cytosolic fractions at day 1 of culture (Fig. 2B). Sub-cellular distribution of cyt c and AIF was also monitored by immunofluo-rescence microcopy confirming the spontaneous release of proapoptotic proteins (Fig. 2B). The cytosolic accumulation of cytochrome c and AIF was not abrogated by z-VAD.fmk (Fig. 2B) at concentrations that completely block caspase activity (Fig. 2A), suggesting that caspases must act downstream of the mitochon-drial signals. However, it is to note that cytosolic accumulation of Smac/Diablo was slightly attenuated in z-VAD.fmk treated cells, a result previously observed in other cell types [25].

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