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Immunomodulating activity of Nymphaea rubra Roxb. extracts: activation of rat dendritic cells and improvement of the T(H)1 immune response.

Cheng JH, Lee SY, Lien YY, Lee MS, Sheu SC - Int J Mol Sci (2012)

Bottom Line: CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%).In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%).In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T(H)1 cytokines.

View Article: PubMed Central - PubMed

Affiliation: The Department of Nursing, Shu Zen College of Medicine and Management, Kaohsiung 821, Taiwan; E-Mail: cjaiho@yahoo.com.tw.

ABSTRACT
Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a T(H)1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T(H)1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.

No MeSH data available.


Related in: MedlinePlus

Analysis of the surface phenotypes of BMHC-imDC using fluorescence activated cell sorting (FACS) scan. On day nine, non-adherent cells were stained with (A) fluorescein isothiocyanate (FITC) conjugated isotype control; (B) conjugated anti-rat CD11c antibody.
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f3-ijms-13-10722: Analysis of the surface phenotypes of BMHC-imDC using fluorescence activated cell sorting (FACS) scan. On day nine, non-adherent cells were stained with (A) fluorescein isothiocyanate (FITC) conjugated isotype control; (B) conjugated anti-rat CD11c antibody.

Mentions: Maturation of DCs is distinguished by a reduction in antigen-processing capacity together with an increase in cell surface expression of MHC class II molecules and the cell markers CD80, CD86, CD11c and CD40 [34,35]. The cell marker CD11c (LFA-1) was used to monitor the rat DCs [36,37]. After cytokine treatment, the BMHCs differentiated into BMHC-imDCs and these cells were shown to be 95.27% CD11c+ by fluorescence activated cell sorting (FACS) analysis (Figure 3). This process was used to set up a BMHC-imDCs assay system that measured the maturation of DCs after stimulation by NR-PS. In order to examine the activation of dendritic cells by NR-PS, the presence of the indicator molecules CD80 and CD86 as well as MHC class II expression were measured. In terms of T cell activation, MHC class II is functional during antigen presentation, while CD80 and CD86 (B7-1 and B7-2, respectively) are important co-stimulatory molecules. We used various concentrations of NR-PS (3.125, 6.25, 12.5, 25, 50 and 100 μg/mL) to treat the BMHC-imDCs for 48 h. The proportion of positive cells and the mean fluorescence intensity of the cells for CD80, CD/86 and MHCclass II were measured (Tables 2 and 3). When compared with the untreated control (65.45% ± 0.97% for positive cell ratio and 24.60% ± 3.19% for mean fluorescence intensity) and positive control treated with 1 μg/mL LPS (85.76% ± 3.06% for positive cell ratio and 49.00% ± 9.18% for mean fluorescence intensity), all NR-PS treatments showed an increase in the positive cell ratio (84.49% ± 10.86%, 84.80% ± 7.94%, 85.68% ± 18.66%, 87.16% ± 8.49%, 84.44% ± 6.35% and 84.56% ± 7.62%) (Table 2). There was a similar increase in mean fluorescence intensity to 47.23% ± 3.72%, 42.73% ± 4.20%, 45.69% ± 3.58%, 48.48% ± 4.26%, 38.03% ± 6.27% and 43.70% ± 7.61% (Table 2). Furthermore, similar results were also obtained for MHC class II expression. The untreated control was 34.87% ± 1.96% for the positive cell ratio and 46.55% ± 4.97% for mean fluorescence intensity, while the LPS positive control gave 73.20% ± 6.16% for positive cell ratio and 57.62% ± 1.87% for mean fluorescence intensity. On treatment with various concentrations of NR-PS, the positive cell ratio was increased to 45.06% ± 8.11%, 44.88% ± 5.11%, 49.18% ± 10.41%, 52.01% ± 10.11%, 49.27% ± 5.03% and 50.27% ± 5.24%, respectively (Table 3), while the mean fluorescence intensity was increased to 51.09% ± 4.73%, 51.94% ± 2.89%, 55.62% ± 8.21%, 60.74% ± 10.06%, 57.30% ± 4.76% and 57.70% ± 2.68%, respectively (Table 3). Thus treating DCs with NR-PS increases the positive cell ratio and the mean fluorescence intensity for all markers compared to the untreated control with the highest activation of the DCs cells being at 25 μg/mL NR-PS treatment (Tables 2 and 3). This confirms clearly that NR-PS is able to enhance the maturation of DCs.


Immunomodulating activity of Nymphaea rubra Roxb. extracts: activation of rat dendritic cells and improvement of the T(H)1 immune response.

Cheng JH, Lee SY, Lien YY, Lee MS, Sheu SC - Int J Mol Sci (2012)

Analysis of the surface phenotypes of BMHC-imDC using fluorescence activated cell sorting (FACS) scan. On day nine, non-adherent cells were stained with (A) fluorescein isothiocyanate (FITC) conjugated isotype control; (B) conjugated anti-rat CD11c antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472710&req=5

f3-ijms-13-10722: Analysis of the surface phenotypes of BMHC-imDC using fluorescence activated cell sorting (FACS) scan. On day nine, non-adherent cells were stained with (A) fluorescein isothiocyanate (FITC) conjugated isotype control; (B) conjugated anti-rat CD11c antibody.
Mentions: Maturation of DCs is distinguished by a reduction in antigen-processing capacity together with an increase in cell surface expression of MHC class II molecules and the cell markers CD80, CD86, CD11c and CD40 [34,35]. The cell marker CD11c (LFA-1) was used to monitor the rat DCs [36,37]. After cytokine treatment, the BMHCs differentiated into BMHC-imDCs and these cells were shown to be 95.27% CD11c+ by fluorescence activated cell sorting (FACS) analysis (Figure 3). This process was used to set up a BMHC-imDCs assay system that measured the maturation of DCs after stimulation by NR-PS. In order to examine the activation of dendritic cells by NR-PS, the presence of the indicator molecules CD80 and CD86 as well as MHC class II expression were measured. In terms of T cell activation, MHC class II is functional during antigen presentation, while CD80 and CD86 (B7-1 and B7-2, respectively) are important co-stimulatory molecules. We used various concentrations of NR-PS (3.125, 6.25, 12.5, 25, 50 and 100 μg/mL) to treat the BMHC-imDCs for 48 h. The proportion of positive cells and the mean fluorescence intensity of the cells for CD80, CD/86 and MHCclass II were measured (Tables 2 and 3). When compared with the untreated control (65.45% ± 0.97% for positive cell ratio and 24.60% ± 3.19% for mean fluorescence intensity) and positive control treated with 1 μg/mL LPS (85.76% ± 3.06% for positive cell ratio and 49.00% ± 9.18% for mean fluorescence intensity), all NR-PS treatments showed an increase in the positive cell ratio (84.49% ± 10.86%, 84.80% ± 7.94%, 85.68% ± 18.66%, 87.16% ± 8.49%, 84.44% ± 6.35% and 84.56% ± 7.62%) (Table 2). There was a similar increase in mean fluorescence intensity to 47.23% ± 3.72%, 42.73% ± 4.20%, 45.69% ± 3.58%, 48.48% ± 4.26%, 38.03% ± 6.27% and 43.70% ± 7.61% (Table 2). Furthermore, similar results were also obtained for MHC class II expression. The untreated control was 34.87% ± 1.96% for the positive cell ratio and 46.55% ± 4.97% for mean fluorescence intensity, while the LPS positive control gave 73.20% ± 6.16% for positive cell ratio and 57.62% ± 1.87% for mean fluorescence intensity. On treatment with various concentrations of NR-PS, the positive cell ratio was increased to 45.06% ± 8.11%, 44.88% ± 5.11%, 49.18% ± 10.41%, 52.01% ± 10.11%, 49.27% ± 5.03% and 50.27% ± 5.24%, respectively (Table 3), while the mean fluorescence intensity was increased to 51.09% ± 4.73%, 51.94% ± 2.89%, 55.62% ± 8.21%, 60.74% ± 10.06%, 57.30% ± 4.76% and 57.70% ± 2.68%, respectively (Table 3). Thus treating DCs with NR-PS increases the positive cell ratio and the mean fluorescence intensity for all markers compared to the untreated control with the highest activation of the DCs cells being at 25 μg/mL NR-PS treatment (Tables 2 and 3). This confirms clearly that NR-PS is able to enhance the maturation of DCs.

Bottom Line: CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%).In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%).In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T(H)1 cytokines.

View Article: PubMed Central - PubMed

Affiliation: The Department of Nursing, Shu Zen College of Medicine and Management, Kaohsiung 821, Taiwan; E-Mail: cjaiho@yahoo.com.tw.

ABSTRACT
Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a T(H)1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T(H)1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.

No MeSH data available.


Related in: MedlinePlus