Immune Netw.  2010 Apr;10(2):55-63. 10.4110/in.2010.10.2.55.

Immunostimulatory Effects of Cordyceps militaris on Macrophages through the Enhanced Production of Cytokines via the Activation of NF-kappaB

• Shin S
• Kwon J
• Lee S
• Kong H
• Lee S
• Lee CK
• Cho K
• Ha NJ
• Kim K

• KMID: 2150660
• DOI: http://doi.org/10.4110/in.2010.10.2.55

Abstract

BACKGROUND: Cordyceps militaris has been used in traditional medicine to treat numerous diseases and has been reported to possess both antitumor and immunomodulatory activities in vitro and in vivo. However, the pharmacological and biochemical mechanisms of Cordyceps militaris extract (CME) on macrophages have not been clearly elucidated. In the present study, we examined how CME induces the production of proinflammatory cytokines, transcription factor, and the expression of co-stimulatory molecules.
METHODS
We confirmed the mRNA and protein levels of proinflammatory cytokines through RT-PCR and western blot analysis, followed by a FACS analysis for surface molecules.
RESULTS
CME dose dependently increased the production of NO and proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, and PGE(2), and it induced the protein levels of iNOS, COX-2, and proinflammatory cytokines in a concentration-dependent manner, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 was also enhanced by CME. Furthermore, the activation of the nuclear transcription factor, NF-kappaB in macrophages was stimulated by CME.
CONCLUSION
Based on these observations, CME increased proinflammatory cytokines through the activation of NF-kappaB, further suggesting that CME may prove useful as an immune-enhancing agent in the treatment of immunological disease.

Keyword

Cordyceps militaris; Proinflammatory cytokines; Macrophages

MeSH Terms

Blotting, Western
Cordyceps
Cytokines
Immune System Diseases
Intercellular Adhesion Molecule-1
Interleukin-6
Macrophages
Medicine, Traditional
NF-kappa B
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha
Cytokines
Intercellular Adhesion Molecule-1
Interleukin-6
NF-kappa B
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Effects of CME on NO and PGE2 production in macrophages. (A) RAW 264.7 cells and (B) peritoneal macrophages were treated with various concentrations (12.5, 25, 50, 100, 200 µg/ml) of CME or LPS (10 ng/ml), then incubated overnight. Nitrite levels in culture media were determined using Griess reagent and were presumed to reflect NO levels. (C) RAW 264.7 cells and (D) peritoneal macrophages were treated with 12.5~200 µg/ml of CME for 48 hrs. Levels of PGE2 in culture media were quantified using EIA kits. Data are presented as means±S.D. of three independent experiments. †p<0.01 vs. cells only; *p<0.05, **p<0.01 vs. cells only.

• 

  Figure 2 Effects of CME on the expression of iNOS and COX-2 protein and mRNA in macrophages. (A) RAW 264.7 cells were treated with various concentrations (12.5, 25, 50, 100, 200 µg/ml) of CME or LPS (100 ng/ml) for 24 hrs. Total cellular proteins (20 µg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and detected with specific antibodies, as described in Materials and Methods. (B) Cells were treated as described above. Total RNA was prepared for the RT-PCR analysis of the expression of iNOS and COX-2 in macrophages treated with various concentrations (12.5, 25, 50, 100, 200 µg/ml) of CME or LPS (100 ng/ml) for overnight. iNOS-specific sequences and COX-2-specific sequences were detected by agarose gel electrophoresis, as described in Materials and Methods. PCR for β-actin was performed to verify that the initial cDNA contents of the samples were similar. This was repeated in triplicate and similar results were obtained in all three.

• 

  Figure 3 Effect of CME on cytokine production in RAW 264.7 cells. Effects of CME on IL-1β (A), IL-6 (B), and TNF-α (C) production in macrophages. Cultures were treated with various concentrations of CME (12.5, 25, 50, 100, 200 µg/ml). The supernatants were collected, and the extracellular levels of cytokines were measured in culture media using cytokine ELISA kits. The results are reported as mean±S.D. of three independent experiments. †p<0.01 vs. cells only; *p<0.05, **p<0.01 vs. cells only.

• 

  Figure 4 Effects of CME on the expression of pro-inflammatory cytokines proteins and mRNA in macrophages. (A) RAW 264. 7 cells were treated with various concentrations (12.5, 25, 50, 100, 200 µg/ml) of CME or LPS (100 ng/ml) for 24 hrs. Total cellular proteins (20 µg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and detected with specific antibodies, as described in the Materials and Methods. (B) Cells were treated as described above. Total RNA was prepared for the RT-PCR analysis of the expression of IL-1β, IL-6, and TNF-α in macrophages treated with various concentrations (12.5, 25, 50, 100, 200 µg/ml) of CME or LPS (100 ng/ml) for overnight. IL-1β-specific sequences, IL-6-specific sequences, and TNF-α-specific sequences were detected by agarose gel electrophoresis, as described in Materials and Methods. PCR for β-actin was performed to verify that the initial cDNA contents of the samples were similar. The experiment was repeated in triplicate and similar results were obtained in all three.

• 

  Figure 5 Effects of CME on the expression of co-stimulatory molecules in RAW 264.7 cells. Cells were cultured with various concentrations of CME (12.5, 50, 200 µg/ml) or LPS (100 ng/ml) for 24 hrs. Surface ICAM-1 (A), B7-1 (B), and B7-2 (C) molecules were labeled with either anti-ICAM-1 or anti-B7-1/-2.

• 

  Figure 6 Effects of CME on NF-κB activation in RAW 264.7 cells. Cells were incubated overnight with various concentrations of CME (12.5~200 µg/ml) in the absence or presence of LPS (100 ng/ml). Protein (20 µg) from each sample was resolved in 12% SDS-PAGE and then analyzed by western blotting. β-actin was used as a control.

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