Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes
© Tsai et al; licensee BioMed Central Ltd. 2011
Received: 14 October 2010
Accepted: 25 March 2011
Published: 25 March 2011
Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes.
Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction.
AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression.
AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.
The central event in the generation of immune responses is the activation and clonal expansion of T cells. Interaction of T cells with antigens initiates a cascade of biochemical events and gene expression that induces the resting T cells to activate and proliferate . Activation of nuclear factor of activated T cells (NF-AT) and a series of genes such as interleukin-2 (IL-2) and interferon-γ (IFN-γ) are pivotal in the growth of T lymphocytes induced by antigens [2, 3]. Thus, growth modulators or other external events affecting T cell proliferation are likely to act by controlling the expression or function of the products of these genes . The immune responses to invasive organisms, if inappropriately intense or prolonged, may paradoxically aggravate the injury or even cause death. The use of immunomodulatory medications must therefore be discreet. Regulation of T lymphocyte activation and proliferation and cytokine production is one of the action mechanisms [5, 6].
Chinese medicinal herbs are now widely acknowledged for their immunomodulatory activities . A member of the Compositae family, Arctium lappa (Niubang) is regarded as an effective Chinese medicine for alleviation of rheumatic pain and fever . Arctigenin (AC), a bioactive component of A. lappa, has various biological activities including: (1) inhibition of nitric oxide, interlukin-6 and tumor necrosis factor-α production in macrophages [8, 9]; (2) anti-proliferative activity against leukemia cells ; and (3) protective effects on hepatocytes from CCl4 injury . Definitive evidence for its effects on T cell-mediated immune responses has been scarce.
The present study aims to elucidate the effects of AC on T lymphocytes proliferation, production and gene expression of IL-2 and IFN-γ in T lymphocytes induced by anti-CD3/CD28 antibodies (Ab) and NF-AT activation.
Preparation of arctigenin (AC)
Ten healthy male participants aged between 20 and 32 years (mean 26) were selected for this study. The experimental protocol were reviewed and approved by the institutional human experimentation committee of Fu-Jen University. Written informed consent was obtained from all participants.
Preparation of primary human T lymphocytes
Heparinized human peripheral bloods (80 ml) were obtained from healthy donors. The peripheral blood was centrifuged at 850 × g (Sorvall Legend RT, Kendro, Germany) at 4°C for ten minutes to remove the plasma. The blood cells were diluted with phosphate buffered saline (PBS) and then centrifuged in a Ficoll-Hypaque discontinuous gradient (specific gravity 1.077) at 420 × g (Sorvall Legend RT, Kendro, Germany) for 30 minutes. The peripheral blood mononuclear cell (PBMC) layers were collected and washed with cold distilled water and 10× Hanks' buffer saline solution (HBSS) to remove red blood cells. T lymphocytes were separated from PBMC by nylon wool columns (Wako Chemicals, USA). Purified T lymphocytes had >87% CD3+ cells and <0.5% CD14+ or CD19+ cells. The cells were re-suspended to a concentration of 2 × 106 cells/ml in RPMI-1640 medium supplemented with 2% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin .
The lymphoproliferation test was modified from a previously described method . Briefly, the density of T lymphocytes was adjusted to 2 × 106 cells/ml before use. Cell suspension (100 μl) was applied into each well of a 96-well flat-bottomed plate (Nunc 167008, Nunclon, Denmark) with or without anti-CD3 (1 μg/ml)/CD28 (3 μg/ml) antibody (eBioscience, USA). Cyclosporin A (CsA, 2.5 μM), an immuno-suppressor, was used as a reference drug . AC was added to the cells at various concentrations (6.25, 12.5 and 25 μM). The plates were incubated in 5% CO2-air humidified atmosphere at 37°C for three days. Subsequently, tritiated thymidine (1 μCi/well, New England Nuclear, USA) was added into each well. After incubated for 16 hours, the cells were harvested on glass fiber filters by an automatic harvester (Dynatech, Multimash 2000, UK). Radioactivity (counting per minute, CPM) in the filters was measured by a scintillation counter (LS 6000IC, Beckman Instruments Inc., USA). The inhibitory activity of AC on T lymphocytes proliferation was calculated according to the following formula:
Inhibitory activity (%) = [Control group (CPM) - Experiment group (CPM)]/Control group (CPM) × 100%
Determination of IL-2 and IFN-γ production
Primary human T lymphocytes (2 × 105 cells/well) were cultured with anti-CD3/CD28 Ab alone or in combination with cyclosporin A (CsA) or various concentrations of AC for three days. The cell supernatants were then collected and assayed for IL-2 and IFN-γ concentrations by enzyme immunoassays (EIAs; R&D systems, USA).
Determination of cell viability
Resting or anti-CD3/CD28 Ab-activated T lymphocytes were cultured in a medium, namely DMSO (0.1%), or various concentrations of AC (6.25, 12.5 and 25 μM) for four days. After stained by trypan blue, total, viable and non-viable cell numbers were counted with a hemocytometer under microscope. The percentage of viable cells was calculated according to the following formula:
Viability (%) = (Viable Cell Number/Total Cell Number) × 100%
Extraction of total cellular RNA
T lymphocytes (5 × 106) were activated with or without anti-CD3/CD28 Ab and co-cultured with 6.25, 12.5 or 25 μM of AC for 18 hours. T lymphocytes were collected and lysed by RNA-Bee™ (Tel-Test, USA). After centrifugation with 12000 × g (Sigma 2K15, B Braun, Germany) at 4°C for 15 min, the supernatants were extracted with a phenol-chloroform mixture. The extracted RNA was precipitated with 100% cold ethanol. The total cellular RNA was pelleted by centrifugation and re-dissolved in diethyl pyrocarbonate (DEPC)-treated water. The concentration of RNA was calculated according to its optical density at 260 nm.
Reverse transcription-polymerase chain reaction (RT-PCR)
Oligonucleotide sequences of the primers used for amplification of IL-2, IFN-γ and GAPDH mRNA in primary human T lymphocytes
Predicted size (bp)
5'-GTC ACA AAC AGT GCA CCT AC-3' 5'-GAA AGT GAA TTC TGG GTC CC-3'
5'-GCA GAG CCA AAT TGT CTC CT-3' 5'-ATG CTC TTC GAC CTC GAA AC-3'
5': TGA AGG TCG GAG TCA ACG GAT TTG GT 3': CAT GTG GGC CAT GAG GTC CAC CAC
Jurkat cells (5 × 104) were transfected by pGL4.30 (luc2P/NFAT-RE/Hygro) with Lipofectamin™ 2000 (Invitrogen, USA) for 24 hours according to the manufacturer's instructions. Then, the cells were cultured with anti-CD3 (1 μg/ml)/CD28 (3 μg/ml) Ab in the presence or absence of AC (6.25, 12.5 and 25 μM) or CsA (2.5 μM) for four hours. Total cell lysates were extracted with 1× reporter lysis buffer (Promega, USA). Total cell lysates (10 μg) were used to determine luciferase activity by the Luciferase Assay System (Promega, USA).
Data were presented as mean ± standard deviation (SD). The differences between groups were assessed with student's t test and corrected with the Bonferroni test. Correlations between AC concentration and activity parameters were calculated with Pearson product-moment correlation test. P < 0.05 was considered statistically significant.
Effects of AC on primary human T lymphocytes proliferation
Viability of primary human T lymphocytes treated with various concentrations of AC
We examined the viabilities of resting or anti-CD3/CD28 activated T lymphocytes treated with 6.25, 12.5 and 25 μM respectively for four days. AC had no cytotoxicity, ie the viabilities of resting or activated cells were not significantly decreased after treatment with various concentrations of AC for four days (Figure 2B). In comparison with the medium-treated group, neither the viability of the resting T lymphocytes nor that of the anti-CD3/CD28-activated T lymphocytes was reduced by DMSO, indicating that decreased T lymphocytes proliferation by AC was not related to direct cytotoxicity.
Effects of AC on IL-2 and IFN-γ production in primary human T lymphocytes
Inhibitory effects of AC on IL-2 and IFN-γ gene expression in primary human T lymphocytes
Inhibitory effects of AC on NF-AT activation
Effects of CsA on IL-2, IFN-γ and cell proliferation in T lymphocytes activated with anti-CD3/CD28 Ab
To determine whether AC decreased NF-AT activation, gene expression of IL-2 and IFN-γ and cell proliferation in T lymphocytes, we added CsA (2.5 μM), an NF-AT inhibitor, into T lymphocytes and analyzed gene expression of IL-2 and IFN-γ as well as cell proliferation. While IL-2 (P = 0.001) and IFN-γ mRNA (P = 0.002) were significantly induced in anti-CD3/CD28 Ab-activated T lymphocytes, CsA signigicantly blocked IL-2 (P = 0.001) and IFN-γ (P = 0.008) expression in the cells (Figures 4C and 4D). CsA also significantly reduced IL-2 (P = 0.001) and IFN-γ (P = 0.003) production in the activated cells (Figures 3A and 3B). Furthermore, the T lymphocyte proliferation stimulated by anti-CD3/CD28 Ab was significantly suppressed by CsA (Figure 2A; P = 0.003).
Several pharmacological effects were identified in A. lappa such as anti-bacterial infection, scavenging free radicals , binding platelet-activating factors  and inhibiting acute ear swelling . This study showed that AC from A. lappa had a profound inhibitory effect on the proliferation of primary human T lymphocytes stimulated by anti-CD3/CD28 Ab. The proliferation-suppressive actions of AC were not explained by a drug-induced reduction in cell viability. We observed that AC decreased production and mRNA expression of IL-2 and IFN-γ and activation of NF-AT in human T lymphocytes induced by anti-CD3/CD28 Ab.
Apart from A. lappa, AC is found in various plants such as Bardanae fructus, Saussurea medusa, Torreya nucifera and lepomea cairica. AC prevents leukocytes from recruitment into the inflamed tissue . AC blocks TNF-α production by impairments of AP-1 activation . The present study demonstrated that AC suppressed proliferation and IL-2 and IFN-γ production in primary human T lymphocytes activated by anti-CD3/CD28 Ab. AC is a potent inducer of apoptosis for HL-60 T leukemia cells, MH60 B lymphoma cells and SW480 colon cancer cells . Thus, we could not rule out the possibility that AC inhibited the proliferation of primary human T lymphocytes via the apoptosis pathway. The possible inhibitory effect of DMSO on primary human T lymphocytes was also studied in these experiments. The cell proliferation and viability were not changed by DMSO. Therefore, the inhibitory function of AC was unlikely related to DMSO.
Interaction of T lymphocytes with antigens initiates a cascade of genes expression such as IL-2 and IFN-γ mRNA inducing the resting T cells to proliferate . This study showed that AC inhibited IL-2 and IFN-γ productions in primary human T lymphocytes stimulated by anti-CD3/CD28 Ab. The impairments of IL-2 and IFN-γ production were related to the suppression of their mRNA transcriptions by AC. Since T lymphocyte proliferation is primarily mediated by IL-2, inhibition of IL-2 production is a central mechanism of action of several immunosuppressants such as CsA. This study also demonstrated that CsA inhibited IL-2 and IFN-γ gene expression and cell proliferation in primary human T lymphocytes induced by anti-CD3/CD28 Ab, suggesting that AC actions are similar to those of CsA which induces arrest activation and proliferation of T cells by inhibiting IL-2 transcription . Furthermore, the preliminary data from immunofluorescence staining indicated that AC had no effect on IL-2 receptor expression in primary human T lymphocytes activated by anti-CD3/CD28 Ab (data not shown), suggesting that the reduction of proliferation in AC-treated T lymphocytes was not caused by down-regulation of IL-2 receptor expression. Failure to produce IL-2 and IFN-γ may be the reason why primary human T lymphocytes do not proliferate.
NF-AT is a major player in the control of T lymphocytes activation and proliferation . After anti-CD3/CD28 Ab stimulation, calcium-dependent phosphatase calcineurin binds to NF-AT, dephosphorylates NF-AT and causes nuclear import of NF-AT. The binding domain of NF-AT is Rel similarity domain located in numerous cytokine promoters. IL-2 and IFN-γ gene expressions in T lymphocytes are controlled by NF-AT-dependent promoters/enhancers . This study found that AC decreased NF-AT activation. NF-AT is a target for the immunosuppressants CsA and FK506 which are efficient inhibitors of T cell activation . This study also demonstrated that CsA blocked NF-AT activation, suggesting that AC inhibited IL-2 and IFN-γ production and cell proliferation in primary human T lymphocytes by modulation of NF-AT activation. Interleukin-10 is mainly produced by regulatory T lymphocytes and regulates other immune cells . We also showed that AC (25 μM) did not affect IL-10 production in primary human T lymphocytes induced by anti-CD3/CD28 Ab (453 ± 88 vs. 412 ± 75pg/ml).
AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.
reverse transcription-polymerase chain reaction
peripheral blood mononuclear cells
phosphate buffered saline
Hanks' buffer saline solution
fetal calf serum
nuclear factor of activated T cells
This study was partially supported by grants from Council of Agriculture (97-1.2.1-al-22), National Science Council (NSC96-2320-B-030-006-MY3; NSC 99-2320-B-030-004-MY3), Committee on Chinese Medicine and Pharmacy (CCMP96-RD-207) and Fu-Jen University (9991A15/10993104995-4), Taiwan.
- Kuo YC, Yang NS, Chou CJ, Lin LC, Tsai WJ: Regulation of cell proliferation, gene expression, production of cytokines and cell cycle progression in primary human T lymphocytes by piperlactam S isolated from Piper kadsura. Mol Pharmacol. 2000, 58: 1057-1066.PubMedGoogle Scholar
- Rao A, Luo C, Hogan PG: Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997, 15: 707-747. 10.1146/annurev.immunol.15.1.707.View ArticlePubMedGoogle Scholar
- Rochman Y, Spolski R, Leonard WJ: New insights into the regulation of T cells by gamma (c) family cytokines. Nat Rev Immunol. 2009, 9: 480-490. 10.1038/nri2580.PubMed CentralView ArticlePubMedGoogle Scholar
- Kuo YC, Weng SC, Chou CJ, Chang TT, Tsai WJ: Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae. Br J Pharmacol. 2003, 140: 895-906. 10.1038/sj.bjp.0705500.PubMed CentralView ArticlePubMedGoogle Scholar
- Hoyer KK, Dooms H, Barron L, Abbas AK: Interleukin- in the development and control of inflammatory disease. Immunol Rev. 2008, 226: 19-28. 10.1111/j.1600-065X.2008.00697.x.View ArticlePubMedGoogle Scholar
- Liu CP, Kuo YC, Lin YL, Liao JF, Shen CC, Chen CF, Tsai WJ: (S)-Armepavine inhibits human peripheral blood mononuclear cells activation by regulating Itk and PLCγ activation in a PI3K-dependent manner. J Leukoc Biol. 2007, 81: 1276-1286. 10.1189/jlb.0106056.View ArticlePubMedGoogle Scholar
- Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho BP: Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002, 97: 1027-1031. 10.1590/S0074-02762002000700017.View ArticlePubMedGoogle Scholar
- Zhao F, Wang L, Liu K: In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J Ethnopharmacol. 2009, 122: 457-462. 10.1016/j.jep.2009.01.038.View ArticlePubMedGoogle Scholar
- Cho MK, Jang YP, Kim YC, Kim SG: Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent KK inhibition: the role in TNF-alpha inhibition. Int Immunopharmacol. 2004, 4: 1419-1429. 10.1016/j.intimp.2004.06.011.View ArticlePubMedGoogle Scholar
- Matsumoto T, Hosono-Nishiyama K, Yamada H: Antiproliferative and apoptotic effects of butyrolactone lignans from Arctium lappa on leukemic cells. Planta Med. 2006, 72: 276-278. 10.1055/s-2005-916174.View ArticlePubMedGoogle Scholar
- Kim SH, Jang YP, Sung SH, Kim CJ, Kim JW, Kim YC: Hepatoprotective dibenzylbutyrolactone lignans of Torreya nucifera against CCl4-induced toxicity in primary cultured rat hepatocytes. Biol Pharm Bull. 2003, 26: 1202-1205. 10.1248/bpb.26.1202.View ArticlePubMedGoogle Scholar
- Liu S, Chen K, Schliemann W, Strack D: Isolation and identification of arctiin and arctigenin in leaves of Burdock (Arcticum lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS. Phytochem Anal. 2005, 16: 86-89. 10.1002/pca.816.View ArticlePubMedGoogle Scholar
- Wu MH, Tsai WJ, Don MJ, Chen YC, Kuo YC: Tanshinlactone A from Salvia miltiorrhiza modulates interleukin-2 and interferon-γ gene expression. J Ethnopharmacol. 2007, 113: 210-217. 10.1016/j.jep.2007.05.026.View ArticlePubMedGoogle Scholar
- Schreiber SL, Crabtree GR: The mechanism of action of cyclosporin A and FK 506. Immunol Today. 1992, 13: 136-142. 10.1016/0167-5699(92)90111-J.View ArticlePubMedGoogle Scholar
- Chen YC, Tsai WJ, Wu MH, Lin LC, Kuo YC: Suberosin inhibits human peripheral blood mononuclear cells proliferation through the modulation of NF-AT and NF-κB transcription factors. Br J Pharmacol. 2007, 150: 298-312. 10.1038/sj.bjp.0706987.PubMed CentralView ArticlePubMedGoogle Scholar
- Seko Y, Cole S, Kasprzak W, Shapiro BA, Ragheb JA: The role of cytokine mRNA stability in the pathogenesis of autoimmune disease. Autoimmun Rev. 2006, 5: 299-305. 10.1016/j.autrev.2005.10.013.View ArticlePubMedGoogle Scholar
- Crabtree GR: Contingent genetic regulatory events in T lymphocyte activation. Science. 1989, 243: 355-361. 10.1126/science.2783497.View ArticlePubMedGoogle Scholar
- Lin CC, Lu JM, Yang JJ, Chuang SC, Ujiie T: Anti-inflammatory and radical scavenge effects of Articum lappa. Am J Chin Med. 1996, 24: 127-137. 10.1142/S0192415X96000177.View ArticlePubMedGoogle Scholar
- Iwakami S, Wu JB, Ebizuka Y, Sankawa U: Platelet activating factor (PAF) antagonists contained in medicinal plants: lignans and sesquiterpenes. Chem Pharm Bull (Tokyo). 1992, 40: 1196-1198.View ArticleGoogle Scholar
- Knipping K, van Esch ECAM, Wijering SC, van der Heide S, Dubois AE, Garsen J: In vitro and in vivo anti-allergic effects of Arctium lappa. Exp Biol Med. 2008, 233: 1469-1477. 10.3181/0803-RM-97.View ArticleGoogle Scholar
- Kang HS, Lee JY, Kim CJ: Anti-inflammatory activity of arctigenin from Forsythiae fructus. J Ethnopharmacol. 2008, 116: 305-312. 10.1016/j.jep.2007.11.030.View ArticlePubMedGoogle Scholar
- Yoo JH, Lee HJ, Kang K, Jho EH, Kim CY, Baturen D, Tunsag J, Nho CW: Lignans inhibit cell growth via regulation of Wnt/beta-catenin signaling. Food Chem Toxicol. 2010, 48: 2247-2252. 10.1016/j.fct.2010.05.056.View ArticlePubMedGoogle Scholar
- Arai K, Lee F, Miyajima A: Cytokines: Coordinators of immune and inflammatory response. Annu Rev Biochem. 1990, 59: 783-836. 10.1146/annurev.bi.59.070190.004031.View ArticlePubMedGoogle Scholar
- Serfling E, Berberich-Siebelt F, Chuvpilo S, Jankevics E, Klein-Hessling S, Twardzik T, Avots A: The role of NF-AT transcription factors in T cell activation and differentiation. Biochim Biophys Acta. 2000, 1498: 1-18. 10.1016/S0167-4889(00)00082-3.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.