Immune Netw.  2010 Dec;10(6):188-197. 10.4110/in.2010.10.6.188.

Dendritic Cell Activation by Glucan Isolated from Umbilicaria Esculenta

• Kim HS
• Kim JY
• Lee HK
• Kim MS
• Lee SR
• Kang JS
• Kim HM
• Lee KA
• Hong JT
• Kim Y
• Han SB

• KMID: 2150674
• DOI: http://doi.org/10.4110/in.2010.10.6.188

Abstract

BACKGROUND
Lichen-derived glucans have been known to stimulate the functions of immune cells. However, immunostimulatory activity of glucan obtained from edible lichen, Umbilicaria esculenta, has not been reported. Thus we evaluated the phenotype and functional maturation of dendritic cells (DCs) following treatment of extracted glucan (PUE).
METHODS
The phenotypic and functional maturation of PUE-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. PUE-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity. Finally we detected the activation of MAPK and NF-kappaB by immunoblot.
RESULTS
Phenotypic maturation of DCs was shown by the elevated expressions of CD40, CD80, CD86, and MHC class I/II molecules. Functional activation of DCs was proved by increased cytokine production of IL-12, IL-1beta, TNF-alpha, and IFN-alpha/beta, decreased endocytosis, and enhanced proliferation of allogenic T cells. Polymyxin B, specific inhibitor of lipopolysaccharide (LPS), did not affect PUE activity, which suggested that PUE was free of LPS contamination. As a mechanism of action, PUE increased phosphorylation of ERK, JNK, and p38 MAPKs, and enhanced nuclear translocation of NF-kappaB p50/p65 in DCs.
CONCLUSION
These results indicate that PUE induced DC maturation via MAPK and NF-kappaB signaling pathways.

Keyword

Umbilicaria esculenta; Glucan; Dendritic cells; MAPKs; NF-kappaB

MeSH Terms

Dendritic Cells
Endocytosis
Glucans
Interleukin-12
Lichens
Lymphocyte Culture Test, Mixed
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Phenotype
Phosphorylation
Polymyxin B
T-Lymphocytes
Tumor Necrosis Factor-alpha
Glucans
Interleukin-12
NF-kappa B
Polymyxin B
Tumor Necrosis Factor-alpha
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 Cell-type selectivity of PUE. RAW 264.7 macrophages were treated with PUE for 24 h and NO production was determined (A). Spleen cells were treated with PUE for 72 h and lymphoproliferation was determined (B). Immature DCs were treated with PUE for 24 h and MHC class I expression level was determined (C). LPS (1 µg/ml) was used as a general activator of DCs, RAW 264.7 cells, and B cells. Significance was determined using the ANOVA test versus chemically-untreated control groups (UN) (*p<0.01).

• 

  Figure 2 Phenotypic maturation of DCs by PUE. Immature DCs were treated with LPS (1 µg/ml) or PUE. After incubation for 24 h, DCs were stained with PE-conjugated CD11c Ab plus FITC-conjugated Abs against MHC class II (A), CD40 (B), CD80 (C), and CD86 (D). CD11c-PE was used to gate live DCs. Results are presented as mean±STD. In another experiment, PUE or LPS was pre-treated with 1,000 units/ml PMB for 2 h or left untreated, and then incubated with immature DCs. After incubation for 24 h, cells were stained with PE-conjugated CD11c Ab plus FITC-conjugated MHC class II Ab (E). Mean fluorescence intensities (MFI) of three separate experiments are shown. Significances were determined using the ANOVA test versus chemically-untreated control groups (UN) (*p<0.01) and versus PMB-untreated group (†p<0.01).

• 

  Figure 3 Effect of PUE on cytokine gene expression of DCs. (A) Immature DCs were activated with LPS or PUE for 4 h. Then, total RNA was isolated and RT-PCR was performed to examine gene expression levels of IL-12, IL-1β, TNF-α, and IFN-α/β. (B) Immature DCs were activated with LPS or PUE for 24 h and then treated with 0.7 mg/ml of dextran-FITC for 1 h at 37℃. After washing, DCs were stained with PE-conjugated anti-CD11c Ab and double-stained DCs were analyzed by flow cytometry. Parallel experiments were performed at 4℃ to confirm nonspecific binding of FITC-dextran. Mean fluorescence intensities (MFI) of three separate experiments are shown. Significances were determined using the ANOVA test versus chemically-untreated control groups (UN) (*p<0.01).

• 

  Figure 4 Allo-stimulation of T cells using PUE-treated DCs. Immature DCs were activated with LPS or PUE for 24 h, and treated with 40 µg/ml mitomycin C (MMC) for 1 h to prevent proliferation. MMC-treated DCs (300~10,000) were added to 1×105 T cells per well. After incubation for 3 (A) and 5 days (B), mixed cell populations were labeled with [3H]-thymidine and harvested using an automated cell harvester.

• 

  Figure 5 Effect of PUE on MAPK signalings. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days, and then further activated with LPS (1 µg/ml) or PUE for 15 min. Total cell extracts were isolated and blotted with anti-phospho-ERK, -JNK, and -p38 antibodies. UN, chemically-untreated control groups.

• 

  Figure 6 Effect of PUE on NF-κB signalings. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days, and then further activated with LPS (1 µg/ml) or PUE for 6 h and nuclear extracts were blotted with anti-p50 and anti-p65 antibodies. UN, chemically-untreated control groups.

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