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

Analysis of apoptosis-related gene expression during constitutive DC death. Apoptosis-related gene expression was analysed either immediately after maturation of human monocyte-derived DCs (day 0, D0) or at day 1 (D1) of standard culture (A). Gene expression changes were assessed by comparative filter cDNA macroarray measurements using a representative apoptosis array of mature DCs at day 0 and day 1 of culture as the example. One representative image of three independent experiments with separate donors is shown. (B) Expression changes of one up-regulated and seven down-regulated genes in DCs cultured for 24 hrs (D1) compared to DCs harvested immediately after maturation (D0). Similar changes were seen when DCs were compared via cDNA macroarray (black bars, n= 3) and quantitative (Q-PCR) (white bars, n= 6)(see Materials and Methods for details). (C) Analysis of DC apoptosis protein expression by Western blotting immediately after maturation (D0) or at day 1 (D1) of standard culture. The expression of several Bcl-2 members (Bim, Bcl-xl, Bad, Bcl-2, Bax) and the inhibitors of apoptosis, XIAP and c-FLIP, was analysed. Actin was used as a standard for equal loadingof protein. One of four independent experiments with separate donors is represented. Four independent immunoblottings were scanned on a densitometerand the relative expression of proteins was determined. The intensity of the values obtained (mean ± S.E.M.) were expressed in arbitrary units.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4496146&req=5

fig04: Analysis of apoptosis-related gene expression during constitutive DC death. Apoptosis-related gene expression was analysed either immediately after maturation of human monocyte-derived DCs (day 0, D0) or at day 1 (D1) of standard culture (A). Gene expression changes were assessed by comparative filter cDNA macroarray measurements using a representative apoptosis array of mature DCs at day 0 and day 1 of culture as the example. One representative image of three independent experiments with separate donors is shown. (B) Expression changes of one up-regulated and seven down-regulated genes in DCs cultured for 24 hrs (D1) compared to DCs harvested immediately after maturation (D0). Similar changes were seen when DCs were compared via cDNA macroarray (black bars, n= 3) and quantitative (Q-PCR) (white bars, n= 6)(see Materials and Methods for details). (C) Analysis of DC apoptosis protein expression by Western blotting immediately after maturation (D0) or at day 1 (D1) of standard culture. The expression of several Bcl-2 members (Bim, Bcl-xl, Bad, Bcl-2, Bax) and the inhibitors of apoptosis, XIAP and c-FLIP, was analysed. Actin was used as a standard for equal loadingof protein. One of four independent experiments with separate donors is represented. Four independent immunoblottings were scanned on a densitometerand the relative expression of proteins was determined. The intensity of the values obtained (mean ± S.E.M.) were expressed in arbitrary units.

Mentions: To gain new insights into the molecular processes that regulate mature DC apoptosis, we screened 96 apoptotic genes for changes in their expression profile. Using the apoptosis macroarray, we were able to identify 9 genes that were differentially expressed (1.5-fold difference) after 1 day in culture (Fig. 4A and B). Expression of genes encoding eight molecules diminished over 24 hrs in culture (Fig. 4), whereas we found that expression of the pro-apoptotic gene BIM was significantly increased (Fig. 4A and B). Real time quantitative-PCR supported the macroarray results for c-FLIP, Mcl-1, Bcl-X, Bim, Rip-2, Fas, Gadd45 and TRAIL, with minor differences only in scale of the down-regulation and up-regulation levels observed (Fig. 4B). On the basis of these results, the levels (relative to ß-actin) of some important apoptosis-related proteins were assayed by Western blot analysis. The anti-apoptotic proteins c-FLIP and XIAP were down-regulated in DCs at day 1 of culture compared with day 0 (Fig. 4C). It is known that the susceptibility of DCs to apoptotic signals is regulated, in part, by the relative levels and competing dimerizations between Bcl-2 family members [28]. After 1 day of DC culture, the proapoptotic protein, Bim, was also markedly increased, correlating with the mRNA profile; however, no change in other Bcl-2 family protein concentrations was evident (Fig. 4C). These data correlate well with the apoptotic phenotype determined in the previous experiments (Figs. 1–3). These results indicate spontaneous modulations in the expression of apoptosis-related genes in DCs likely accounts for facilitating spontaneous mitochondrial dysfunction and cell death to occur.


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)

Analysis of apoptosis-related gene expression during constitutive DC death. Apoptosis-related gene expression was analysed either immediately after maturation of human monocyte-derived DCs (day 0, D0) or at day 1 (D1) of standard culture (A). Gene expression changes were assessed by comparative filter cDNA macroarray measurements using a representative apoptosis array of mature DCs at day 0 and day 1 of culture as the example. One representative image of three independent experiments with separate donors is shown. (B) Expression changes of one up-regulated and seven down-regulated genes in DCs cultured for 24 hrs (D1) compared to DCs harvested immediately after maturation (D0). Similar changes were seen when DCs were compared via cDNA macroarray (black bars, n= 3) and quantitative (Q-PCR) (white bars, n= 6)(see Materials and Methods for details). (C) Analysis of DC apoptosis protein expression by Western blotting immediately after maturation (D0) or at day 1 (D1) of standard culture. The expression of several Bcl-2 members (Bim, Bcl-xl, Bad, Bcl-2, Bax) and the inhibitors of apoptosis, XIAP and c-FLIP, was analysed. Actin was used as a standard for equal loadingof protein. One of four independent experiments with separate donors is represented. Four independent immunoblottings were scanned on a densitometerand the relative expression of proteins was determined. The intensity of the values obtained (mean ± S.E.M.) were expressed in arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Analysis of apoptosis-related gene expression during constitutive DC death. Apoptosis-related gene expression was analysed either immediately after maturation of human monocyte-derived DCs (day 0, D0) or at day 1 (D1) of standard culture (A). Gene expression changes were assessed by comparative filter cDNA macroarray measurements using a representative apoptosis array of mature DCs at day 0 and day 1 of culture as the example. One representative image of three independent experiments with separate donors is shown. (B) Expression changes of one up-regulated and seven down-regulated genes in DCs cultured for 24 hrs (D1) compared to DCs harvested immediately after maturation (D0). Similar changes were seen when DCs were compared via cDNA macroarray (black bars, n= 3) and quantitative (Q-PCR) (white bars, n= 6)(see Materials and Methods for details). (C) Analysis of DC apoptosis protein expression by Western blotting immediately after maturation (D0) or at day 1 (D1) of standard culture. The expression of several Bcl-2 members (Bim, Bcl-xl, Bad, Bcl-2, Bax) and the inhibitors of apoptosis, XIAP and c-FLIP, was analysed. Actin was used as a standard for equal loadingof protein. One of four independent experiments with separate donors is represented. Four independent immunoblottings were scanned on a densitometerand the relative expression of proteins was determined. The intensity of the values obtained (mean ± S.E.M.) were expressed in arbitrary units.
Mentions: To gain new insights into the molecular processes that regulate mature DC apoptosis, we screened 96 apoptotic genes for changes in their expression profile. Using the apoptosis macroarray, we were able to identify 9 genes that were differentially expressed (1.5-fold difference) after 1 day in culture (Fig. 4A and B). Expression of genes encoding eight molecules diminished over 24 hrs in culture (Fig. 4), whereas we found that expression of the pro-apoptotic gene BIM was significantly increased (Fig. 4A and B). Real time quantitative-PCR supported the macroarray results for c-FLIP, Mcl-1, Bcl-X, Bim, Rip-2, Fas, Gadd45 and TRAIL, with minor differences only in scale of the down-regulation and up-regulation levels observed (Fig. 4B). On the basis of these results, the levels (relative to ß-actin) of some important apoptosis-related proteins were assayed by Western blot analysis. The anti-apoptotic proteins c-FLIP and XIAP were down-regulated in DCs at day 1 of culture compared with day 0 (Fig. 4C). It is known that the susceptibility of DCs to apoptotic signals is regulated, in part, by the relative levels and competing dimerizations between Bcl-2 family members [28]. After 1 day of DC culture, the proapoptotic protein, Bim, was also markedly increased, correlating with the mRNA profile; however, no change in other Bcl-2 family protein concentrations was evident (Fig. 4C). These data correlate well with the apoptotic phenotype determined in the previous experiments (Figs. 1–3). These results indicate spontaneous modulations in the expression of apoptosis-related genes in DCs likely accounts for facilitating spontaneous mitochondrial dysfunction and cell death to occur.

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