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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.


Morphology of dendritic cells after various treatments. (A) Bone marrow hematopoietic cells-imdendritic cells (BMHC-imDC) were grown from rat bone marrow in completed RPMI media 1640 containing 20 ng/mL recombinant rat GM-CSF and 10 ng/mL IL-4; (B) BMHC-imDC treated with 50 μg/mL NR-PS on day seven (200×); (C) A photomicrogaph of rat bone marrow hematopoietic cells (BMHC) isolated from four to seeks week old SD rats after staining with Liu’s-stain (200×); (D) BMHC-imDCs were generated from BMHC by treatment with 20 ng/mL GM-CSF and 10 ng/mL IL-4 for seven days. Note the presence of the short dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (E) Lipopolysaccharide(1 μg/mL) was used to stimulate the maturation of BMHC-imDC into BMHC-mDC. Note the presence of the long dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (F) Bone marrow hematopoietic cell-derived immature dendritic cells (BMHC-imDCs) treated with 50 μg/mL NR-PS for 48hr (Liu’s stain, 200×).
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f2-ijms-13-10722: Morphology of dendritic cells after various treatments. (A) Bone marrow hematopoietic cells-imdendritic cells (BMHC-imDC) were grown from rat bone marrow in completed RPMI media 1640 containing 20 ng/mL recombinant rat GM-CSF and 10 ng/mL IL-4; (B) BMHC-imDC treated with 50 μg/mL NR-PS on day seven (200×); (C) A photomicrogaph of rat bone marrow hematopoietic cells (BMHC) isolated from four to seeks week old SD rats after staining with Liu’s-stain (200×); (D) BMHC-imDCs were generated from BMHC by treatment with 20 ng/mL GM-CSF and 10 ng/mL IL-4 for seven days. Note the presence of the short dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (E) Lipopolysaccharide(1 μg/mL) was used to stimulate the maturation of BMHC-imDC into BMHC-mDC. Note the presence of the long dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (F) Bone marrow hematopoietic cell-derived immature dendritic cells (BMHC-imDCs) treated with 50 μg/mL NR-PS for 48hr (Liu’s stain, 200×).

Mentions: Bone marrow-derived DC (BMDC) maturation is attended by alteration of the cells’ morphological, phenotypic and functional properties [31,32]. According to Inaba et al., it was found that treatment with GM-CSF and IL-4 was able to stimulate rat BMHCs to form dendritic cells and that similar results were obtained in mice [33]. So, we used GM-CSF and IL-4 to stimulate bone marrow hematopoietic cells (BMHCs) to differentiation and become BMHC-imDCs as Talmor et al. reported [34]. In order to measure the bioactivity of NR-PS using BMHC-imDCs, the morphological changes in BMHC-imDCs were monitored after NR-PS treatment. First, using live cell observation, BMHC-imDCs were obtained by treating BMHCs with rGM-CSF and rIL-4 for nine days without NS-PS and these cells were compared with others that had been treated with a test concentration of 50 μg/mL NS-PS at the immature dendritic cell stage on the seventh day and then cultured until the ninth day. The BMHC-imDCs were activated by NR-PS, and this was shown by the presence of dendritic protrusions on the cell surface (Figure 2A,B). Figure 2 shows the cells after staining with Liu’s-stain in order to observe any morphological changes. The BHMC-imDCs can be seen to be round with polymorphic nuclei and small protrusions on the cell surface in the absence of stimulation (Figure 2C,D). However, after treated with 50 μg/mL NR-PS (Figure 2F) or 1 μg/mL LPS (positive control) for 48 h (Figure 2E), the cells are now larger, and the nuclei have become even more polymorphic. Furthermore, the dendritic protrusions on the cell surfaces have become more pronounced and elongated [34]. These findings strongly suggest that NR-PS treatment promotes dendritic cell maturation.


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)

Morphology of dendritic cells after various treatments. (A) Bone marrow hematopoietic cells-imdendritic cells (BMHC-imDC) were grown from rat bone marrow in completed RPMI media 1640 containing 20 ng/mL recombinant rat GM-CSF and 10 ng/mL IL-4; (B) BMHC-imDC treated with 50 μg/mL NR-PS on day seven (200×); (C) A photomicrogaph of rat bone marrow hematopoietic cells (BMHC) isolated from four to seeks week old SD rats after staining with Liu’s-stain (200×); (D) BMHC-imDCs were generated from BMHC by treatment with 20 ng/mL GM-CSF and 10 ng/mL IL-4 for seven days. Note the presence of the short dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (E) Lipopolysaccharide(1 μg/mL) was used to stimulate the maturation of BMHC-imDC into BMHC-mDC. Note the presence of the long dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (F) Bone marrow hematopoietic cell-derived immature dendritic cells (BMHC-imDCs) treated with 50 μg/mL NR-PS for 48hr (Liu’s stain, 200×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijms-13-10722: Morphology of dendritic cells after various treatments. (A) Bone marrow hematopoietic cells-imdendritic cells (BMHC-imDC) were grown from rat bone marrow in completed RPMI media 1640 containing 20 ng/mL recombinant rat GM-CSF and 10 ng/mL IL-4; (B) BMHC-imDC treated with 50 μg/mL NR-PS on day seven (200×); (C) A photomicrogaph of rat bone marrow hematopoietic cells (BMHC) isolated from four to seeks week old SD rats after staining with Liu’s-stain (200×); (D) BMHC-imDCs were generated from BMHC by treatment with 20 ng/mL GM-CSF and 10 ng/mL IL-4 for seven days. Note the presence of the short dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (E) Lipopolysaccharide(1 μg/mL) was used to stimulate the maturation of BMHC-imDC into BMHC-mDC. Note the presence of the long dendritic processes (arrow) associated with the plasma membrane (Liu’s staining, 200×); (F) Bone marrow hematopoietic cell-derived immature dendritic cells (BMHC-imDCs) treated with 50 μg/mL NR-PS for 48hr (Liu’s stain, 200×).
Mentions: Bone marrow-derived DC (BMDC) maturation is attended by alteration of the cells’ morphological, phenotypic and functional properties [31,32]. According to Inaba et al., it was found that treatment with GM-CSF and IL-4 was able to stimulate rat BMHCs to form dendritic cells and that similar results were obtained in mice [33]. So, we used GM-CSF and IL-4 to stimulate bone marrow hematopoietic cells (BMHCs) to differentiation and become BMHC-imDCs as Talmor et al. reported [34]. In order to measure the bioactivity of NR-PS using BMHC-imDCs, the morphological changes in BMHC-imDCs were monitored after NR-PS treatment. First, using live cell observation, BMHC-imDCs were obtained by treating BMHCs with rGM-CSF and rIL-4 for nine days without NS-PS and these cells were compared with others that had been treated with a test concentration of 50 μg/mL NS-PS at the immature dendritic cell stage on the seventh day and then cultured until the ninth day. The BMHC-imDCs were activated by NR-PS, and this was shown by the presence of dendritic protrusions on the cell surface (Figure 2A,B). Figure 2 shows the cells after staining with Liu’s-stain in order to observe any morphological changes. The BHMC-imDCs can be seen to be round with polymorphic nuclei and small protrusions on the cell surface in the absence of stimulation (Figure 2C,D). However, after treated with 50 μg/mL NR-PS (Figure 2F) or 1 μg/mL LPS (positive control) for 48 h (Figure 2E), the cells are now larger, and the nuclei have become even more polymorphic. Furthermore, the dendritic protrusions on the cell surfaces have become more pronounced and elongated [34]. These findings strongly suggest that NR-PS treatment promotes dendritic cell maturation.

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.