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. 2013 May 29:12:80.
doi: 10.1186/1475-2840-12-80.

High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells

Hao Lu, Kang Yao, Dong Huang, Aijun Sun, Yunzeng Zou, Juying Qian, Junbo Ge

High glucose induces upregulation of scavenger receptors and promotes maturation of dendritic cells

Hao Lu et al. Cardiovasc Diabetol. .

Abstract

Background: Both hyperglycaemia and dendritic cells (DCs) play causative roles in atherosclerosis. However, whether they interact in atherosclerosis remains uncertain. Therefore, we examined whether high glucose could regulate the expression of scavenger receptors responsible for oxidised low-density lipoprotein (oxLDL) uptake in DCs, a critical step in atherogenesis. In addition, we investigated the impact of glucose on DC maturation regarding changes in phenotype and cytokine secretion.

Methods: Immature DCs were cultured with different concentrations of glucose (5.5 mmol/L, 15 mmol/L, 30 mmol/L) in the absence or presence of N-acetylcysteine (NAC), SB203580 or Bay11-7082 for 24 hours. We used 30 mmol/L mannitol as a high-osmolarity control treatment. The expression of the scavenger receptors SR-A, CD36 and LOX-1 was determined by real-time PCR and western blot analysis. Furthermore, DCs were incubated with DiI-labelled oxLDL. The DiI-oxLDL-incorporated fraction was investigated by flow cytometry analysis. The intracellular production of ROS in DCs was measured by dichlorodihydrofluorescein (DCF) fluorescence using confocal microscopy. Finally, flow cytometry analysis was used to investigate immunophenotypic protein expression (CD83 and CD86). Supernatant cytokine measurements were used for immune function assays.

Results: The incubation of DCs with glucose enhanced, in a dose-dependent manner, the gene and protein expression of SR-A, CD36 and LOX-1. This effect was partially abolished by NAC, SB203580 and Bay11-7082. Incubation of DCs with mannitol (30 mmol/L) did not enhance these scavenger receptors' expression. High glucose upregulated the production of ROS and expression of p38 MAPK in DCs. NAC partially reversed p38 MAPK upregulation. High glucose increased the oxLDL-uptake capacity of DCs. Blockage of the scavenger receptors SR-A and CD36 reduced oxLDL uptake, but blockage of LOX-1 did not. Furthermore, high-glucose (15 mmol/L or 30 mmol/L) treatment increased CD86 and CD83 in DCs. High glucose also increased IL-6 and IL-12 secretion and decreased IL-10 secretion.

Conclusion: High glucose can increase the expression of the scavenger receptors SR-A, CD36 and LOX-1, which can increase the oxLDL-uptake capacity of DCs. High glucose induces a proinflammatory cytokine profile in human DCs, leading to DC maturation. These results support the hypothesis that atherosclerosis is aggravated by hyperglycaemia-induced DC activation and oxLDL uptake.

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Figures

Figure 1
Figure 1
Effects of glucose on the mRNA expression of SR-A, CD36 and LOX-1 in DCs. SR-A, CD36 and LOX-1 mRNAs were analysed by real-time quantitative RT-PCR. Representative examples of the dose-dependent expression of SR-A, CD36 and LOX-1 on DCs. Mean ± SEM. n = 3. *p < 0.05 vs. 5.5 mmol/L glucose, **p < 0.01 vs. 5.5 mmol/L glucose.
Figure 2
Figure 2
Effects of glucose on SR-A, CD36 and LOX-1 protein expression in DCs. SR-A, CD36 and LOX-1 proteins were analysed by western blotting. Densitometric analysis showed significantly increased expression of SR-A, CD36 and LOX-1 after prestimulation by 30 mmol/L glucose. Mean ± SEM. n = 3. *p < 0.05 vs. 5.5 mmol/L glucose, **p < 0.01 vs. 5.5 mmol/L glucose.
Figure 3
Figure 3
Effects of glucose on the uptake of DiI-oxLDL by DCs. Representative example of the effects of 5.5 mM Glu and 30 mM Glu on the uptake of DiI-oxLDL by DCs at 37°C.
Figure 4
Figure 4
Blocking the uptake of DiI-oxLDL by DCs. Representative histograms of blocking DiI-oxLDL uptake using antibodies against SR-A, CD36 and LOX-1 at 4°C. 30 mM Glu: 30 mmol/L glucose.
Figure 5
Figure 5
Effect of high glucose on reactive oxygen species production in DCs. Reactive oxygen species production was visualised by DCF fluorescence confocal laser microscopy. A, Representative microscopic scan. B, Quantification of reactive oxygen species production. Data analysis of 4 separate experiments, expressed as relative fluorescence. Mean ± SEM, *p < 0.01 vs. 5.5 mmol/L glucose. DIC: differential interference contrast microscopy.
Figure 6
Figure 6
Effects of p38 MAPK inhibitor, NF-κB inhibitor and NAC on glucose-induced scavenger receptors mRNA levels. DCs were pretreated for 2 hours with SB203580, NAC or BAY 11–7082 before exposure to high glucose. SR-A, CD36 and LOX-1 mRNAs were quantified by real-time PCR. Mean ± SEM. n = 3. *p < 0.05 vs. 5.5 mmol/L glucose, #p < 0.05 vs. 30 mmol/L glucose.
Figure 7
Figure 7
Effects of p38 MAPK inhibitor, NF-κB inhibitor and NAC on glucose-induced scavenger receptors protein levels. DCs were pretreated for 2 hours with SB203580, NAC or BAY 11–7082 before exposure to high glucose. SR-A, CD36 and LOX-1 protein levels were quantified by western blotting. Mean ± SEM. n = 3. *p < 0.05 vs. 5.5 mmol/L glucose, #p < 0.05 vs. 30 mmol/L glucose.
Figure 8
Figure 8
Inhibitory effects of NAC and SB203580 on p38 MAPK phosphorylation in glucose-stimulated DCs. DCs were pretreated for 2 hours with SB203580 or NAC before exposure to high glucose. Then, cells were harvested and assayed for the phosphorylation of p38 MAPK by western blots. Mean ± SEM. n = 3. *p < 0.05 vs. 5.5 mmol/L glucose, #p < 0.05 vs. 30 mmol/L glucose.
Figure 9
Figure 9
The immunophenotypic expression of DCs exposed to different concentrations of glucose. Flow cytometric analysis was performed to estimate cell-surface CD83 and CD86 expression. 5.5 mM Glu: 5.5 mmol/L glucose; 15 mM Glu: 15 mmol/L glucose; 30 mM Glu: 30 mmol/L glucose; mannitol: 30 mmol/L mannitol.
Figure 10
Figure 10
Cytokine secretion in high glucose-treated DCs. DCs of different groups were harvested, and the supernatants were collected; IL-6, IL-10, IL-12p70 and TNF-α were determined using commercially available ELISAs. *p < 0.05 vs. 5.5 mmol/L glucose. Mean ± SEM. n = 3.
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