Immune Netw.  2010 Aug;10(4):135-143. 10.4110/in.2010.10.4.135.

Cordyceps militaris Enhances MHC-restricted Antigen Presentation via the Induced Expression of MHC Molecules and Production of Cytokines

• Shin S
• Park Y
• Kim S
• Oh HE
• Ko YW
• Han S
• Lee S
• Lee CK
• Cho K
• Kim K

• KMID: 2150672
• DOI: http://doi.org/10.4110/in.2010.10.4.135

Abstract

BACKGROUND
Cordyceps militarys water extract (CME) has been reported to exert antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not yet been elucidated. In this study, we examined the effects of CME on the antigen presenting function of antigen presenting cells (APCs).
METHODS
Dendritic cells (DCs) were cultured in the presence of CME, and then allowed to phagocytose microspheres containing ovalbumin (OVA). After washing and fixing the efficacy of OVA, peptide presentation by DCs were evaluated using CD8 and CD4 T cells. Also, we confirmed the protein levels of proinflammatory cytokines through western blot analysis.
RESULTS
CME enhanced both MHC class I and class II-restricted presentation of OVA in DCs. In addition, the expression of both MHC class I and II molecules was enhanced, but there was no changes in the phagocytic activity of exogenous OVA. Furthermore, CME induced the protein levels of iNOS, COX-2, proinflammatory cytokines, and nuclear p65 in a concentration-dependent manner, as determined by western blot.
CONCLUSION
These results provide an understanding of the mechanism of the immuno-enhancing activity of CME on the induction of MHC-restricted antigen presentation in relation to their actions on APCs.

Keyword

Cordyceps militaris; MHC-restricted antigen presentation; Proinflammatory cytokines; Antigen presenting cells

MeSH Terms

Antigen Presentation
Antigen-Presenting Cells
Blotting, Western
Cordyceps
Cytokines
Dendritic Cells
Microspheres
Ovalbumin
Ovum
T-Lymphocytes
Water
Cytokines
Ovalbumin
Water

Figure

• 

  Figure 1 Effects of CME on the cross-presentation of exogenous OVA in DCs. (A) DC2.4 cells and (B) BM-DCs were incubated with the indicated amounts of CME for 2 hrs, and then combined with OVA-microspheres. After 6 hrs incubation, the cells were washed, fixed, and the amounts of OVA peptides presented on MHC class I molecules were assessed using OVA-specific CD8 T cell hybridoma, CD8OVA1.3. The amounts of IL-2 produced from OVA-specific CD8 T cells were assayed by a commercial IL-2 ELISA kit. Data have been presented as means±S.D. of three independent experiments. *p<0.05 vs. cells only; †p<0.05, ‡p<0.01 vs. OVA only.

• 

  Figure 2 Effects of CME on the MHC class II-restricted presentation of exogenous OVA in APCs. (A) BM-DCs and (B) peritoneal macrophages were incubated with the indicated amounts of the CME for 2 hrs, and then combined with OVA-microspheres. After 6 hrs incubation, the cells were washed, fixed, and the amounts of OVA peptides presented on MHC class II molecules were assessed using OVA-specific CD4 T cell hybridoma, DOBW. The amounts of IL-2 produced from OVA-specific CD4 T cells were assayed by a commercial IL-2 ELISA kit. Data have been presented as means±S.D. of three independent experiments. *p<0.05 vs. cells only; †p<0.05, ‡p<0.01 vs. OVA only.

• 

  Figure 3 Effects of CME on the phagocytic activity. (A) DC2.4 cells and (B) BM-DCs were cultured with CME for 2 hrs, and then combined with microspheres containing both OVA and FITC (6.25µg/ml). After 2 hrs incubation, unphagocytozed microspherses were washed, and the cells were harvested by gentle pipetting, and then analyzed by flow cytometry.

• 

  Figure 4 Effects of CME on the expression of MHC molecules. (A) DC2.4 cells and (B) BM-DCs were cultured with CME for 2 hrs, and then the cells were harvested by gentle pipetting. The expression levels of class I and class II MHC molecules were assessed using anti-H-2Kb and anti-I-Ab monoclonal antibodies.

• 

  Figure 5 Effects of CME on the expression of pro-inflammatory cytokines, cytokine-related enzymes, and NF-κB protein. Cells were incubated overnight with various concentrations of CME (12.5~200 µg/ml) in the absence or presence of LPS (100 ng/ml). The expression of (A) pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, (B) cytokine-related enzymes, iNOS and COX-2, and (C) NF-κB protein was examined. Total cellular proteins (20µg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and detected with specific antibodies, as described in the Materials and Methods. β-actin was used as a control. The experiment was repeated in triplicate and similar results were obtained in all three.

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