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The influence of Ouabain on human dendritic cells maturation.

Nascimento CR, Valente RC, Echevarria-Lima J, Fontes CF, de Araujo-Martins L, Araujo EG, Rumjanek VM - Mediators Inflamm. (2014)

Bottom Line: However, Ouabain effects on dendritic cells (DCs) were not explored so far.Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained.Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, 21 941-902 Rio de Janeiro, RJ, Brazil ; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21 941-902 Rio de Janeiro, RJ, Brazil.

ABSTRACT
Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

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Related in: MedlinePlus

Phagocytic activity of dendritic cells cultured with Ouabain. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF-α (50 ng/mL), in the presence or absence of 100 nM Oua. After this period, cells were incubated with FITC-dextran (0.5 mg/mL) for 1 h at 37°C and analyzed by flow cytometry. Data are expressed as the percentage of positive cells and MFI (mean fluorescence intensity) of positive cells (mean ± SEM of 6 independent experiments). ∗ and ∗∗ are significantly different from respective controls ((a): without TNF; (b): without Oua) (∗P < 0.05; ∗∗P < 0.01).
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fig9: Phagocytic activity of dendritic cells cultured with Ouabain. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF-α (50 ng/mL), in the presence or absence of 100 nM Oua. After this period, cells were incubated with FITC-dextran (0.5 mg/mL) for 1 h at 37°C and analyzed by flow cytometry. Data are expressed as the percentage of positive cells and MFI (mean fluorescence intensity) of positive cells (mean ± SEM of 6 independent experiments). ∗ and ∗∗ are significantly different from respective controls ((a): without TNF; (b): without Oua) (∗P < 0.05; ∗∗P < 0.01).

Mentions: FITC-dextran uptake after 7 days was measured. As depicted in Figure 9, around 75% of immature DCs captured FITC-dextran particles (Figure 9(a)). Strikingly, in the presence of 100 nM Ouabain (added in the last 48 h of culture) the percentage of DCs able to capture FITC-dextran and the phagocytic capacity of these cells decreased nearly 20% and 45%, respectively.


The influence of Ouabain on human dendritic cells maturation.

Nascimento CR, Valente RC, Echevarria-Lima J, Fontes CF, de Araujo-Martins L, Araujo EG, Rumjanek VM - Mediators Inflamm. (2014)

Phagocytic activity of dendritic cells cultured with Ouabain. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF-α (50 ng/mL), in the presence or absence of 100 nM Oua. After this period, cells were incubated with FITC-dextran (0.5 mg/mL) for 1 h at 37°C and analyzed by flow cytometry. Data are expressed as the percentage of positive cells and MFI (mean fluorescence intensity) of positive cells (mean ± SEM of 6 independent experiments). ∗ and ∗∗ are significantly different from respective controls ((a): without TNF; (b): without Oua) (∗P < 0.05; ∗∗P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4291013&req=5

fig9: Phagocytic activity of dendritic cells cultured with Ouabain. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF-α (50 ng/mL), in the presence or absence of 100 nM Oua. After this period, cells were incubated with FITC-dextran (0.5 mg/mL) for 1 h at 37°C and analyzed by flow cytometry. Data are expressed as the percentage of positive cells and MFI (mean fluorescence intensity) of positive cells (mean ± SEM of 6 independent experiments). ∗ and ∗∗ are significantly different from respective controls ((a): without TNF; (b): without Oua) (∗P < 0.05; ∗∗P < 0.01).
Mentions: FITC-dextran uptake after 7 days was measured. As depicted in Figure 9, around 75% of immature DCs captured FITC-dextran particles (Figure 9(a)). Strikingly, in the presence of 100 nM Ouabain (added in the last 48 h of culture) the percentage of DCs able to capture FITC-dextran and the phagocytic capacity of these cells decreased nearly 20% and 45%, respectively.

Bottom Line: However, Ouabain effects on dendritic cells (DCs) were not explored so far.Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained.Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, 21 941-902 Rio de Janeiro, RJ, Brazil ; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21 941-902 Rio de Janeiro, RJ, Brazil.

ABSTRACT
Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Show MeSH
Related in: MedlinePlus