Immune Netw.  2010 Dec;10(6):230-238. 10.4110/in.2010.10.6.230.

Activation of Macrophages by Exopolysaccharide Produced by MK1 Bacterial Strain Isolated from Neungee Mushroom, Sarcodon aspratus

• Im SA
• Wang W
• Lee CK
• Lee YN

• KMID: 2150679
• DOI: http://doi.org/10.4110/in.2010.10.6.230

Abstract

BACKGROUND
The MK1 strain, a novel bacterial isolate from soft-rotten tissue of the Neungee mushroom, produces copious amounts of exopolysaccharide (EPS) in a dextrose minimal medium. This study examined the molecular characteristics and immunomodulatory activity of MK1 EPS.
METHODS
The EPS in the culture supernatant was purified by cold ethanol precipitation, and characterized by SDS-PAGE/silver staining and Bio-HPLC. The immunomodulatory activities of the EPS were examined using the mouse monocytic cell line, RAW 264.7 cells.
RESULTS
The molecular weights of the purified EPS were rather heterogeneous, ranging from 10.6 to 55 kDa. The EPS was composed of glucose, rhamnose, mannose, galactose, and glucosamine at an approximate molar ratio of 1.00:0.8:0.71:0.29:0.21. EPS activated the RAW cells to produce cytokines, such as TNF-alpha and IL-1beta, and nitric oxide (NO). EPS also induced the expression of co-stimulatory molecules, such as B7-1, B7-2 and ICAM-1, and increased the phagocytic activity. The macrophage-activating activity of EPS was not due to endotoxin contamination because the treatment of EPS with polymyin B did not reduce the macrophage-activating activity.
CONCLUSION
The EPS produced from the MK1 strain exerts macrophage-activating activity.

Keyword

Exopolysaccharide; Neungee mushroom; Immunomodulator; Macrophage

MeSH Terms

Agaricales
Animals
Cell Line
Cold Temperature
Cytokines
Ethanol
Galactose
Glucosamine
Glucose
Intercellular Adhesion Molecule-1
Macrophages
Mannose
Mice
Molar
Molecular Weight
Nitric Oxide
Rhamnose
Sprains and Strains
Tumor Necrosis Factor-alpha
Cytokines
Ethanol
Galactose
Glucosamine
Glucose
Intercellular Adhesion Molecule-1
Mannose
Nitric Oxide
Rhamnose
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Photographs of MK1 exopolysaccharide. (A) Colony morphology of MK1 on potato dextrose agar, (B) Purified EPS (freeze-dried powder), (C) optical microscopy view of EPS (40×), (D) SEM view (6,000×).

• 

  Figure 2 EPS resolved by SDS-PAGE. Resolution of the protein markers (5 µl) and EPS (150 µg) on 0.1% SDS-15% polyacrylamide gel.

• 

  Figure 3 Sugar profile of EPS by Bio-LC. Twenty µg of the freeze-dried EPS was hydrolyzed at 100℃ for 4 hr with 2 M trifluoroacetic acid in sealed test tubes. The EPS-acid hydrolysates were analyzed on a CarboPacTM PA1 column with a HPAEC-PAD system. (A) The profile of neutral and amino sugar. (B) Profile of acidic sugar.

• 

  Figure 4 Growth inhibition of RAW cells stimulated with MK1. EPS was treated with a polymyxin B-affinity column to remove any possible endotoxin contamination. RAW cells were cultured in the presence of different MK1 EPS concentrations (0.8, 4, 20 and 100 µg/ml) for 2 days, and DNA synthesis was measured by [3H]-thymidine incorporation for the final 6 hr of the 2 day culture period. The CPM values of the untreated RAW cells served as control values in the calculation of % inhibition.

• 

  Figure 5 Phenotypic analysis of RAW cells stimulated with MK1 EPS. EPS was treated with a polymyxin B-affinity column to remove the possible contaminants of endotoxin. The RAW cells were cultured in the presence of EPS (100 µg/ml) for 2 days. The cells were collected, washed and used for immunophenotypic analysis. The levels of expression (thin line) are shown along with the isotype control (shaded line).

• 

  Figure 6 Cytokine and nitric oxide production of RAW cells stimulated with MK1 EPS. EPS was treated with a polymyxin B-affinity column to remove any possible endotoxin contamination. The RAW cells were cultured in the presence of different MK1 EPS concentrations (0.8, 4, 20 and 100 µg/ml) for 2 days, and the culture supernatants were assayed for TNF-α (A), IL-1β (B). The amounts of nitric oxide were measured using a Griess reagent (C).

• 

  Figure 7 Phagocytic activity of RAW cells stimulated with MK1 EPS. EPS was treated with a polymyxin B-affinity column to remove any possible endotoxin contamination. The RAW cells were cultured in the presence of EPS (100 µg/ml) for 2 days, followed by the addition of microspheres containing fluorescein isothiocyanate (FITC). After 2 hr, unphagocytozed microspheres were removed by washing. The cells were harvested, fixed, and analyzed by flow cytometry. The thin line histograms represent the phagocytic activity of the RAW cells stimulated with MK1 EPS and the shaded histograms represent the phagocytic activity of the untreated RAW cells.

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