J Bacteriol Virol.  2012 Mar;42(1):77-82. 10.4167/jbv.2012.42.1.77.

Anti-inflammatory Activity of Carpinus tschonoskii Leaves Extract in R848-stimulated Bone Marrow-derived Macrophages and Dendritic Cells

• Kang SH
• Koo JE
• Hong HJ
• Mathema VB
• Koh YS

• KMID: 1434776
• DOI: http://doi.org/10.4167/jbv.2012.42.1.77

Abstract

The present study aims to evaluate the anti-inflammatory effect of methanol extract from leaves of Carpinus tschonoskii (CE) on R848-stimulated primary bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Primary BMDMs and BMDCs were used for pro-inflammatory cytokine production. Human embryonic kidney cell line 293T (HEK293T) was used to access NF-kappaB activity. In all cases, R848 was used to stimulate the cells. The CE (0~150 microg/ml) was treated to BMDMs, BMDCs, and HEK293T cells. CE pre-treatment in R848-stimulated BMDMs and BMDCs showed a dose-dependent inhibitory effect on pro-inflammatory cytokine (e.g., IL-12 p40, IL-6, and TNF-alpha) production as compared to non-treated controls. In NF-kappaB reporter gene assay, the CE pre-treatment inhibited NF-kappaB-dependent luciferase activity in a dose-dependent manner. Overall, our findings suggest that CE has significant inhibitory effect on pro-inflammatory cytokine production and deserve further studies concerning potentials of CE for medicinal uses.

Keyword

Carpinus tschonoskii; Pro-inflammatory cytokine; NF-kappaB; TLR7

MeSH Terms

Betulaceae
Cell Line
Corynebacterium
Dendritic Cells
Genes, Reporter
Humans
Interleukin-12
Interleukin-6
Kidney
Luciferases
Macrophages
Methanol
NF-kappa B
Corynebacterium
Interleukin-12
Interleukin-6
Luciferases
Methanol
NF-kappa B

Figure

• 

  Figure 1 Inhibitory effects of Carpinus tschonoskii leaves extract (CE) on IL-12 p40, IL-6, and TNF-α production in R848-stimulated bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDM) were treated with CE (25~150 µg/ml) for 1 h before stimulation with R848 (1 µM). Supernatants were harvested 18 h after stimulation. Concentrations of murine IL-12 p40, IL-6, and TNF-α in the culture supernatants were determined by ELISA. Results shown are the mean ± SD of an experiment done in triplicate and are representative of three separate experiments (*p < 0.05, **p < 0.01 versus CE-untreated cells stimulated with TLR7 ligand).

• 

  Figure 2 Inhibitory effects of Carpinus tschonoskii leaves extract (CE) on IL-12 p40, IL-6, and TNF-α production in R848-stimulated bone marrow-derived dendritic cells. Bone marrow-derived dendritic cells (BMDC) were treated with CE (25~150 µg/ml) for 1 h before stimulation with R848 (1 µM). Supernatants were harvested 18 h after stimulation. Concentrations of murine IL-12 p40, IL-6, and TNF-α in the culture supernatants were determined by ELISA. Results shown are the mean ± SD of an experiment done in triplicate and are representative of three separate experiments (*p < 0.05, **p < 0.01 versus CE-untreated cells stimulated with TLR7 ligand).

• 

  Figure 3 Carpinus tschonoskii leaves extract (CE) treatment inhibited NF-κB reporter activity in HEK293T cells. HEK293T cells were transfected with empty vector (pcDNA3) or TLR7-expressing plasmid (pcDNA3-TLR7), and then treated with CE for 1 h before stimulation with R848 (1 µM). Cell lysates were prepared and assayed for firefly luciferase versus renilla activities, and results were expressed as fold increases. All results shown are the mean ± SD of an experiment done in triplicate and are representative of three separate experiments. (*p < 0.05, **p < 0.01 versus CE-untreated cells stimulated with TLR7 ligand).

Reference

1. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001. 1:135–145.

2. Lampropoulou V, Hoehlig K, Roch T, Neves P, Calderón Gómez E, Sweenie CH, et al. TLR-activated B cells suppress T cell-mediated autoimmunity. J Immunol. 2008. 180:4763–4773.

3. Efron PA, Tsujimoto H, Bahjat FR, Ungaro R, Debernardis J, Tannahill C, et al. Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha. J Endotoxin Res. 2005. 11:145–160.

4. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010. 140:805–820.

5. Koh YS. Nucleic acid recognition and signaling by Toll-like receptor 9: compartment-dependent regulation. J Bacteriol Virol. 2011. 41:131–132.

6. Yuk JM, Jo EK. Toll-like receptors and innate immunity. J Bacteriol Virol. 2011. 41:225–235.

7. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol. 2002. 3:196–200.

8. Guiducci C, Gong M, Xu Z, Gill M, Chaussabel D, Meeker T, et al. TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus. Nature. 2010. 465:937–941.

9. Rubtsov AV, Rubtsova K, Fischer A, Meehan RT, Gillis JZ, Kappler JW, et al. Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c⁺ B-cell population is important for the development of autoimmunity. Blood. 2011. 118:1305–1315.

10. Bollrath J, Phesse TJ, von Burstin VA, Putoczki T, Bennecke M, Bateman T, et al. gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell. 2009. 15:91–102.

11. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell. 2009. 15:103–113.

12. Chang CS, Chang KS. Typification of Corylopsis coreana (Hamamelidaceae) and Carpinus laxiflora var. longispica (Betulaceae). J Jpn Bot. 2010. 85:270–276.

13. Zhang R, Kang KA, Piao MJ, Park JW, Shin TK, Yoo BS, et al. Cytoprotective activity of Carpinus tschonoskii against H₂O₂ induced oxidative stress. Nat Prod Sci. 2007. 13:118–122.

14. Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu Rev Immunol. 2001. 19:163–196.

15. Bromberg J, Wang TC. Inflammation and cancer: IL-6 and STAT3 complete the link. Cancer Cell. 2009. 15:79–80.

16. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010. 11:373–384.

17. Bao L, Lindgren JU, van der Meide P, Zhu S, Ljunggren HG, Zhu J. The critical role of IL-12 p40 in initiating, enhancing, and perpetuating pathogenic events in murine experimental autoimmune neuritis. Brain Pathol. 2002. 12:420–429.

18. Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S. Host innate immune receptors and beyond: making sense of microbial infections. Cell Host Microbe. 2008. 3:352–363.

19. Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J. 1998. 334:297–314.

20. Trikha M, Corringham R, Klein B, Rossi JF. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence. Clin Cancer Res. 2003. 9:4653–4665.

21. Opal SM, DePalo VA. Anti-inflammatory cytokines. Chest. 2000. 117:1162–1172.

22. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001. 2:675–680.