Immune Netw.  2010 Feb;10(1):5-14. 10.4110/in.2010.10.1.5.

Ginsan Enhances Humoral Antibody Response to Orally Delivered Antigen

• Na HS
• Lim YJ
• Yun YS
• Kweon MN
• Lee HC

• KMID: 1456192
• DOI: http://doi.org/10.4110/in.2010.10.1.5

Abstract

BACKGROUND
There have been several reports describing the capability of ginseng extracts as an adjuvant. In this study, we tested if ginsan, a polysaccharide extracted from Panax ginseng, was effective in enhancing antibody response to orally delivered Salmonella antigen. METHODS: Ginsan was treated before oral salmonella antigen administration. Salmonella specific antibody was determined by ELISA. mRNA expression was determined by RT-PCR. Cell migration was determined by confocal microscopy and flow cytometry. COX expression was detected by western blot. RESULTS: Ginsan treatment before oral Salmonella antigen delivery significantly increased both secretory and serum antibody production. Ginsan increased the expression of COX in the Peyer's patches. Various genes were screened and we found that CCL3 mRNA expression was increased in the Peyer's patch. Ginsan increased dendritic cells in the Peyer's patch and newly migrated dendritic cells were mostly found in the subepithelial dome region. When COX inhibitors were treated, the expression of CCL3 was reduced. COX inhibitor also antagonized both the migration of dendritic cells and the humoral immune response against oral Salmonella antigen. CONCLUSION: Ginsan effectively enhances the humoral immune response to orally delivered antigen, mediated by CCL3 via COX. Ginsan may serve as a potent vaccine suppliment for oral immunization.

Keyword

Panax ginseng; Ginsan; Oral Vaccine; Adjuvant; Cyclooxygenase

MeSH Terms

Antibody Formation
Blotting, Western
Cell Movement
Dendritic Cells
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Immunity, Humoral
Immunization
Microscopy, Confocal
Panax
Peyer's Patches
Polysaccharides
Prostaglandin-Endoperoxide Synthases
RNA, Messenger
Salmonella
Polysaccharides
Prostaglandin-Endoperoxide Synthases
RNA, Messenger

Figure

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  Figure 1 Ginsan induces higher humoral immune response against paraformaldehyde fixed S. typhimurium. BALB/c mice immunized with 1×109 colony forming units fixed Salmonella twice each week for two weeks. Ginsan was applied one day before oral immunization. Two weeks after the final immunization, the mice were sacrificed and saliva, vaginal wash, stool and serum were obtained. (A) Salmonella specific IgG in serum. (B) Salmonella specific IgA in saliva, vaginal flush or stool. Each group consists of five mice (*p<0.05).

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  Figure 2 Effect of ginsan on COX expression in Peyer's patches. (A) Semi-quantitative RT-PCR for COX-1 and COX-2 mRNA from the Peyer's patches. Total RNA was isolated and used for RT-PCR. PCR products were visualized on 2% agarose gel using ethidium bromide. (B) Western blotting of COX-1 and COX-2 in the Peyer's patch and ileum. Increased COX-1 expression was evident using both approaches.

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  Figure 3 Effect of ginsan on various chemokine mRNA expression. Semiquantitative RT-PCR analysis of CCL expression in the Peyer's patch, spleen and ileum were analyzed. Total RNA isolated following ginsan treatment was used for RT-PCR. PCR products were visualized on 2% agarose gel using ethidium bromide. Ginsan specifically enhanced CCL3 expression in the Peyer's patch.

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  Figure 4 Localization of CCL3 in the Peyer's patches. Frozen sections of Peyer's patches were doubly stained with antibodies against CD11c (green) in combination with anti-CCL3 (red). Panels (A) and (C) display the controls, and panels (B) and (D) display the ginsan treatment. (A, B ×600; C, D ×1,800).

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  Figure 5 Analysis of the migration of splenocytes to the Peyer's patches. (A) Ginsan treatment significantly enhanced CD11c positive cell numbrs in the Peyer's patch. (*p<0.05) (B) BALB/c mice were pretreated with ginsan for two days. GFP splenocytes (5×106) were then intraveneously introduced. To determine migration of the splenocytes, Peyer's patches were fixed and examined. Ginsan treatment greatly enhanced migration of GFP splenocytes to the subepithelial dome of Peyer's patches. (C) Newly migrating cells were mostly found near CD11c positive cells.

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  Figure 6 Effect of ginsan and COX-1 specific inhibitor (A) or non-specific COX-inhibitor (B) on the expression of CCL3 mRNA in Peyer's patches. Total RNA was isolated and used RT-PCR can was prepared by reverse transcription. The PCR products were visualized on 2% agarose gel using ethidium bromide. CCL3 expression was antagonized by SC-560 and aspirin (ASA) treatment. (Ct, PBS; Gs, Ginsan; Sc, SC-560; ASA).

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  Figure 7 Effect of ginsan and COX inhibitor on cell migration to the Peyer's patches. Cells from Peyer's patches were prepared following various treatments and GFP splenocyte transfer. Cells were stained with antibodies against CD11c. CD11c cells were increased following ginsan treatment, which was antagonized by ASA treatment (*p<0.05).

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  Figure 8 Effect of ginsan and COX inhibitor on Salmonella-specific Ig following oral immunization. Each group was orally immunized with paraformaldehyde-fixed S. typhimurium. (A) Salmonella specific IgG in serum. (B) Salmonella specific IgA in stool (*p<0.05).

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