Immunopharmacology and Immunotoxicology
ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: http://www.tandfonline.com/loi/iipi20
Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways Yixuan Huang, Mengrou Ren, Ying Xiong, Tong Wang, Hongyu Chen, Xuming Deng & Jing Lu To cite this article: Yixuan Huang, Mengrou Ren, Ying Xiong, Tong Wang, Hongyu Chen, Xuming Deng & Jing Lu (2015) Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways, Immunopharmacology and Immunotoxicology, 37:4, 366-374 To link to this article: http://dx.doi.org/10.3109/08923973.2015.1063644
Published online: 16 Jul 2015.
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Date: 14 September 2015, At: 01:15
http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(4): 366–374 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1063644
RESEARCH ARTICLE
Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways Yixuan Huang1*, Mengrou Ren1*, Ying Xiong2, Tong Wang1, Hongyu Chen1, Xuming Deng2, and Jing Lu1,2 1
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Laboratory of Nutrition and Function Food, College of Light Industry Economics and Management, Jilin University, Changchun, Jilin, People’s Republic of China and 2Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin, People’s Republic of China Abstract
Keywords
Tubeimoside-1 (TBMS1) is one of the important components in Bolbostemma paniculatum (Maxim.) Franque. In the study, its immunosuppressive effects on murine T lymphocyte responses were evaluated in vitro and in vivo. The data showed that TBMS1 inhibited ConAinduced T lymphocyte proliferation, decreased the ratio of CD4+/CD8+, suppressed IL-2, IFN-g, IL-4 and IL-6 production and mRNA expression, down-regulate activation of NF-kB, NFAT2 and AP-1 signal transduction pathways in vitro. In addition, administration of TBMS1 significantly inhibited T cell-mediated DTH response in vivo. These findings indicated that TBMS1 inhibits the proliferation and activation of T lymphocytes in mice.
Cytokine, NF-kB, suppression, T lymphocyte, tubeimoside-1
Introduction TBMS1, as shown in Figure 1(A), is the main component extracted Bolbostemma paniculatum (Maxim.) Franquet, which has been widely used in traditional Chinese medicine for the treatment. TBMS1 is triterpenoid saponin with a particular cyclic structure with a dicrotalic acid bridge between the oligosaccharide chains at C-3 and C-28. Previous studies have shown that TBMS1 could inhibit the growth of several human cancer cell lines and macrophages1–3. These findings indicate that TBMS1 has the potential inhibition function through its effect on cells. However, so far there is no report published about the immunosuppressive activity of TBMS1 on T cell. So in the present study, we use murine T cell as target to study immunosuppression effect of TBMS1 on T-cell activation, and explored the potential mechanism underlying this effect. T cell is the important part of immune system and participates mainly in process of cell-mediated immune responses. T cell activation is a complex process that involves the induction of gene transcription, the expression of new cell surface molecules, and the secretion of cytokines4. Many immune-related diseases, such as multiple sclerosis,
*These authors contributed equally to this work. Address for correspondence: Dr Jing Lu, Laboratory of Nutrition and Function Food, College of Light Industry Economics and Management, Jilin University, Changchun, Jilin, People’s Republic of China. E-mail: [email protected] (or) Professor Xuming Deng, Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin, People’s Republic of China. E-mail: [email protected]
History Received 23 November 2014 Revised 26 May 2015 Accepted 15 June 2015 Published online 16 July 2015
rheumatoid arthritis, contact hypersensitivity and transplantation, have been known to be related to T cell activation5. Concanavalin A (ConA) is a kind of lectin and has been widely used for many years to activate T cells. Although the precise mechanism of action of this agent is unknown, it is likely that Con A activates T cells by indirectly cross-linking the T cell receptor (TCR)6. In the study, we found that TBMS1 significantly inhibited ConA-stimulated T cell proliferation, ratio of CD4+/CD8+ and cytokine production in vitro. The data also showed that TBMS1 exhibited an immunosuppressive effect through NF-kB, NFAT2 and AP-1 pathways. Moreover, we examined the effect of TBMS1 on 2,4-dinitro-1-fluorobenzene (DNFB)induced ear swelling and inflammatory cell infiltration. The data suggested that TBMS1 had protective effects of delayed type hypersensitivity (DTH). Our study is benefit to provide some useful information on its promising therapeutic application as a kind of immunosuppressive drug.
Materials and methods Materials Tubeimoside-1 (purity 498%, C63H98O29, molecular weight 1318) was from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China) and dissolved in PBS and stored at 4 C. Roswell Park Memorial Institute (RPMI) 1640 medium and fetal calf serum (FCS) were from GIBCO BRL (Grand Island, NY). Penicillin, streptomycin, cyclophosphamide (CTX), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenylmethylsulfonyl fluouride (PMSF), ConA, 1-fluoro-2,4-dinitrobenzene (DNFB), propidium iodide (PI),
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Figure 1. (A) The structure of TBMS1. (B) Effects of TBMS1 on ConA-induced T lymphocyte proliferation. Splenocytes (5 106 cells/ml) were incubated with various concentrations of testing compounds in the presence or absence of ConA (5 mg/ml) for 48 h. Cell proliferation was assayed by a conventional MTT method. Data represent mean ± SD of five observations; ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
Tween-20, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Interferon gamma (IFN-g), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6) ELISA kits were purchased from Biolegend Inc. (San Diego, CA). BioRT cDNA first strand synthesis kit was purchased from Bioer Technology (Changchun, Jilin, China). Phospho-specific antibodies for IkB as well as antibodies against IkB, nuclear factor kB (NF-kB), nuclear factor of activated T cells 2 (NFAT2) and activator protein-1 (AP-1) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L) and peroxidase-conjugated Affinipure goat anti-rabbit IgG (H + L) were purchased from PTG (Chicago, IL). Fluorescein isothiocyanate (FITC)-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD4 antibodies were purchased from BD Pharmingen (San Diego, CA). Primers were purchased from Shennengbocai Co. (Shanghai, China).
Experimental animals Male BALB/c mice (6 weeks old) weighing 18–20 g were purchased from Jilin University Experimental Animal Center and acclimatized for 7 days before use. The mice were housed under standard conditions of 24 ± 1 C, 12 h/12 h light–dark cycles and fed with a standard laboratory pellet chow diet and tap water. All mice used in experiments were approved by the Ethic Committee of Jilin University. Isolation and culture of murine splenocytes Male BALB/c mice were killed by cervical dislocation at the end of the experiment and thoroughly wiped with 70% ethanol and their abdominal cavity opened with the help of sterile scissors and forceps in an aseptic environment. The spleens of the animals were removed aseptically and washed in the sterile phosphate-buffered saline (PBS). Erythrocytes were lysed with Tris–NH4Cl (0.155 M NH4Cl and 16.5 mM Tris,
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pH 7.2). A single-cell suspension was prepared; cell debris and clumps were removed. The splenocytes were isolated and cultured in RPMI-1640 medium containing penicillin/streptomycin (100 U/ml and 100 mg/ml, respectively) and 10% FCS. Purified T cells were isolated from splenocytes by positive selection using MACS CD3 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as directed by the manufacturer7. Percent purity and viability were checked by FACS and exceeded 90% alive before experiments.
cytokines secretion in vitro. Briefly, splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well plates in the present of different doses of TBMS1 (2, 4 or 6 mM) for 48 h at 37 C in a 5% CO2 atmosphere. Then the plate was centrifuged at 1500 rpm for 10 min, and culture supernatants were collected for measurement of cytokine levels. The concentration of each cytokine in the culture medium was quantified using a competitive ELISA kit (BioLegend Inc., San Diego, CA) according to the manufacturer’s instructions.
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Splenocytes cytotoxicity and proliferation assay In order to determine whether TBMS1 affects lymphocyte viability, splenocytes from BALB/c mice were exposed to various concentrations (0–12 mM) of sodium TBMS1 for 48 h. Briefly, mouse lymphocytes (5 106 cells/ml) were cultured in triplicates in a 96-well flat-bottomed plate for 48 h with TBMS1 or ConA (5 mg/ml) + TBMS1. The cells cultured with medium alone were used as controls. MTT (5 mg/ml) was added 4 h before the end of culture. After removal of MTT, the formazan precipitate was solubilized in DMSO (100 ml/well) and measured on a microplate reader (Bio-Rad Laboratories, Hercules, CA) at the absorbance wavelength of 570 nm. Cell staining for T lymphocyte populations Cell staining for phenotypic analysis was carried out as described by Lee et al8,9. Briefly, single-cell suspensions were prepared from the spleen of each treatment by gently rubbing the spleen through a nylon mesh filter. Cellular debris was removed, and cells were washed in PBS and re-suspended (107 cells/ml) in FACS buffer (PBS, 10 mM Hepes buffer, 0.01% sodium azide and 1% heat-inactivated fetal bovine serum). Cells were labeled in a 96-well plate (106 cells/well) and stained with perCp/Cy5.5-conjugated anti-CD3 antibody, PE-conjugated anti-CD4 antibody and FITC-conjugated antiCD8 antibody for 30 min at 4 C in the dark. Each sample was re-suspended in a fixing solution of 0.5 ml PBS and analyzed by BD FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA). Cell Quest Software (Becton-Dickinson) was used to identify and quantify distinct populations of cells by mean fluorescent intensity (MFI). A minimum of 10 000 cells per sample were counted.
RT-PCR analysis Purified cells were treated as described above. Total RNA were extracted using Trizol (Incitrogen, Carlsbad, CA) according to the supplier’s instructions. The extracted final RNA was re-suspended in 20 ml of DEPC water. The concentration of total RNA was determined by absorbance at 260 nm and A260 nm/A280 nm confirmed the purity of RNA. An equal amount of total RNA (1 mg) was reversetranscribed with the BioRT cDNA first strand synthesis kit (Bioer Technology, Changchun, Jilin, China). Real-time PCR was performed using SYBR Green Super mix with an iCycler1 thermal cycler (Bio-Rad Laboratories). Primers were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The specific primers, amplified cycles and annealing temperature of each gene are listed in Table 1. All primer sequences were checked in Gen Bank (National Center for Biotechnology Information, Bethesda, MD) to avoid inadvertent sequence homologies. The amplification was performed in 25 ml reactions containing 8 ml cDNA, 10 mM forward and reverse primer, 2.5 ml 10 PCR buffer containing MgCl2, 10 mM dNTP mixture, 2.5 U Taq polymerase and 11.5 ml sterile ddH2O. Thermal cycling was performed in: one cycle at 94 C, 2 min; followed by 30–40 cycle each including denaturation at 94 C 30 s, 35 s at appropriate anneal temperature, extension 1 min at 72 C and one cycle at 72 C, 5 min. Final PCR products were separated on 2% agarose gel. The size of the amplified fragments was determined by comparison with a standard DNA marker. Western blotting analysis
Determination of cytokine levels in vitro In this study, we selected IL-2, IFN-g as Th1 cytokines and IL-4, IL-6 as Th2 cytokines to test the effect of TBMS1 on
Purified T cells were treated with different concentrations (2, 4 and 6 mM) of TBMS1 for 1 h, and then incubated in the presence of ConA (5 mg/ml) for 30 min. Cells were collected
Table 1. Sequences of primers used for RT-PCR gene expression studies. Gene Actin IL-2 IFN-g IL-4 IL-6
Primer sequence 0
0
5 -ATCATGTTTGAGACCTCCAACA-3 50 -CATCTCTTGCTCGAAGTCCA-30 50 -CTACAGCGGAAGCACAGC-30 50 -TCCTCAGAAAGTCCACCA-30 50 -TCTGAGACAATGAACGCTAC-30 50 -TTCCACATCTATGCCACT-30 50 -ACGGCACAGAGCTATTGATG-30 50 -ATGGTGGCCAGTACTACGA-30 50 -TCCAGTTGCCTTCTTGGGAC-30 50 -GTGTAATTAAGCCTCCGACTTG-30
Product size (bp)
Annealing temperature ( C)
Amplified cycle
318
58
30
375
56
30
144
55
30
455
58
30
140
58
30
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1 h at room temperature. The blots were again washed three times with TBST, and then developed using an enhanced chemiluminescence (ECL) kit (Sigma, St. Louis, MO). T cell-mediated DTH response in vivo Contact hypersensitivity to DNFB was induced in BALB/c mice by two daily paintings with 0.5% DNFB dissolved in acetone/olive oil (4:1). The degree of hypersensitivity was assayed 4 days after the second painting by measuring the increments of ear thickness 36 h after challenging the ears with 0.2% DNFB. The control group was treated with the solvent without DNFB and the DTH group was using DNFB. The TBMS1-treated DTH group was intraperitoneal injected (i.p.) with TBMS1 (1, 2 and 4 mg/kg) daily for
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by centrifugation, washed twice with PBS, and suspended in an extraction lysis buffer (Beyotime, Jiangsu, China) containing protease inhibitors. The cytoplasmic protein or nucleoprotein concentration was determined using a protein assay reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of total cellular protein (60 mg) were resolved by 12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with blocking solution (5% bovine serum albumin), followed by an overnight incubation at 4 C with the appropriate primary antibody. The blots were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then incubated with a 1:7000 dilution of horseradish peroxidase-conjugated secondary antibody (PTG) for
Role of TBMS1 in mouse T lymphocytes
Figure 2. Effects of TBMS1 on CD4+ and CD8+ expression. Splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well flatbottom plates and treated with various concentrations of TBMS1. Cells were cultured 48 h and collected, then stained with PE-anti-CD4/L3T4 or FITCanti-CD8/lyt-2 for analysis by flow cytometry. The values are presented as means ± SD (n ¼ 3). The results presented are from one experiment, which is representative of two others performed. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
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5 consecutive days. The positive control group was injected (i.p.) with CTX (20 mg/kg) for every 2 days. All the mice were killed by cervical dislocation at the end of the experiment and their ears were removed. The DTH response to DNFB was evaluated by measuring the weight difference of the right and the left ear10. Meanwhile, histopathologic evaluation was performed on mice. Right ears were removed 36 h after final DNFB application and fixed with 4% paraformaldehyde in PBS (pH 7.4). Ear tissues were sectioned, embedded in paraffin and cut at 5 mm. Tissue sections were then stained with hematoxylin–eosin (HE) for general morphology.
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Statistical analysis All values were expressed as mean ± standard deviation (SD) and statistical analysis was performed by one-way ANOVA using SPSS 13.0 (SPSS Inc., Chicago, IL) to evaluate the significance of differences between groups. Statistical significance were accepted when p50.05 or p50.01.
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Results Cell viability and cell proliferation We can see the data from Figure 1(B), there was no obvious toxicity against splenocytes at concentrations below 6 mM, whereas a dose-dependent decrease in cell viability was found at higher concentrations (8–12 mM). Simultaneously, the activity of TBMS1 was evaluated on splenocyte proliferation induced by ConA, which only stimulates T cell-mediated immunity. These results illustrated the dose-dependent inhibitory effect of TBMS1 on ConA-induced T splenocyte proliferation. The results also indicated that the immunosuppressive activities of TBMS1 in subsequent experiments observed here, at concentrations up to 6 mM, were not caused by its cytotoxicity. Effect of TBMS1 on CD4+ and CD8+ T lymphocyte populations We investigated the effects of TBMS1 on the subsets CD4+ and CD8+ T cells by flow cytometric analysis (Figure 2).
Figure 3. Effects of TBMS1 on Th1 and Th2 cytokines production and mRNA expression. Splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well plates in the present of different doses of TBMS1 (2, 4 or 6 mM) for 48 h at 37 C in a 5% CO2 atmosphere. (A) The 48 h cultured supernatants were collected for determining the contents of IL-2, IFN-g, IL-4 and IL-6 by ELISA. The absorbances of six wells per treatment were averaged. Bars represent means ± SD. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group. (B) The 48 h cultured cells were harvested for detecting these four cytokines mRNA expressions by RT-PCR. Three independent experiments were performed with similar results.
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Figure 4. Effects of TBMS1 on NF-kB, NFAT2 and AP-1 signaling pathways. Cells were purified using a separation column. The cells were pretreated with 2, 4 or 6 mM of TBMS1 for 1 h and then stimulated with ConA for another 30 min. Protein samples were extracted according to the manufacturer’s instructions and analyzed by Western blotting with specific antibodies. Quantification of each protein expression was normalized to b-actin using a densitometer (Imaging System). The data are representative of three independent experiments and expressed as mean ± SEM. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
CD4+ levels and the ratio of CD4+/CD8+ were higher in the ConA group compared with that in the control group (p50.01). There was a down-regulation of the CD4+ T cell populations after TBMS1 plus ConA treatment, but the alteration of CD8+ T cell populations was not significant, resulting in the decrease of the CD4+/CD8+ ratio. These results indicated that CD4+ T cells were more sensitive to
inhibitory effects of TBMS1. TBMS1 exhibited inhibition function by reducing the CD4+ T cell subpopulation. Effects of TBMS1 on cytokine secretion in vitro As shown in Figure 3(A), TBMS1 simultaneously inhibit Th1 and Th2 cytokines production in a dose-dependent manner.
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In order to study whether TBMS1 decrease these four cytokines expression through the regulation of gene transcription, we also tested mRNA expression of IL-2, IFN-g, IL-4 and IL-6 by RT-PCR method. The result showed the same conclusion (Figure 3B). Effects of TBMS1 on signaling pathways activation
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In our experiment, we examined the effect of TBMS1 on NF-kB p65, NFAT2 and AP-1 signal pathways in purified T cells by western blotting. As shown in Figure 4, the IkB protein decreased after ConA administration, but increased after TBMS1 administration. On the contrary, p-IkB protein showed the opposite trend in the cytoplasm. In nucleprotein, the results showed that the activation of these pathways in mouse T lymphocytes increased after ConA administration
Figure 5. Effects of TBMS1 on DNFB-induced DTH reaction. BALB/c mice were sensitized with 0.5% DNFB on Days 0 and 1 and then challenged by DNFB on Day 5. Vehicle, CTX (20 mg/kg) and TBMS1 (1, 2 and 4 mg/kg) were administered to each group by i.p. injection on 5 consecutive days (Day 1–5). Ear swelling was calculated as the increase in ear patch weight (mg) between left (DNFB-untreated) and right (DNFB-treated) ear. Results are presented as means ± SD (n ¼ 10). ##p50.01 versus control group, *p50.05 or **p50.01 versus DTH group.
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alone but was significantly inhibited after TBMS1 treatment in a concentration-dependent manner. Effect of TBMS1 on DNFB-induced DTH response The inhibitory effect of TBMS1 on DNFB-induced DTH was illustrated in Figure 5. The ear swelling was significantly increased in model group compared with that in control group (p50.01), indicating that DNFB-induced DTH model had been made successfully. The positive drug CTX (20 mg/kg) could suppress DTH response (p50.01). TBMS1 at dose of 6 mg/kg also significantly reduced the ear swelling in DNFBinduced mice compared with the control group (p50.01). In addition, no signs of toxicity were observed in the TBMS1treated mice on the basis of body weight and microscopic examination of individual organs (data no shown). The dermal infiltration of inflammatory cells is an important feature of DTH. Cell types contained in infiltrates include lymphocytes and mast cells. In the present study, histologic analysis revealed that there were no notable histologic changes in control group (Figure 6A). However, topical DNFB elicited the infiltrations of inflammatory cells into ear skin lesions, and that numbers of these cells were significantly increased after DNFB challenge (Figure 6B). Furthermore, the i.p. administration of TBMS1 to mice every day relieved lesion severities. Compared with the DTH group, the tissue swelling had gone down a little in the low dose of TBMS1 (1 and 2 mg/kg) groups (Figure 6C and D). In the highest dose of TBMS1 (4 mg/kg) and the CTX group (20 mg/kg), tissue hypertrophy was reduced much obviously, and only a few inflammatory cell infiltration was present (Figure 6E and F).
Discussion Immunosuppressive agents are an important drug class that is valuable for the treatment of unwanted immune
Figure 6. Histopathologic changes of mouse ear tissues under microscope (400). Control group (A): DNFB (), microphotograph showing normal structure of the ear. DTH group (B): DNFB (+), microphotograph showing histopathologic changes (edema, infiltration of inflammatory cells) of the ear. DNFB (+) + TBMS1 (1, 2 or 4 mg/kg) (C, D and E) and DNFB (+) + CTX 20 mg/kg (F): microphotograph showing decreased histopathologic changes of the ear.
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responses, particularly those giving autoimmune disease and transplant rejection11. But most of the immunosuppressants in use have a narrow range of blood concentrations and serious adverse effects in clinic. Therefore, new and safe immunosuppressive drugs against acute and chronic rejection are eagerly awaited. In this study, we reported that a new candidate, TBMS1 which is extracted from Bolbostemma paniculatum (Maxim.) Franquet has potent immunosuppressive activity in vitro and in vivo. In the beginning of our study, we investigated the cytotoxicity of TBMS1 on splenocytes. The result assured us that the effects of TBMS1 on splenocytes were due to its immunosuppressive activity, but not due to cell death at dose of 0–6 mM. Then, we examined inhibitory activity of TBMS1 on ConA-induced T lymphocytes proliferation. These results showed that TBMS1 had a high inhibitory capacity for T lymphocyte proliferation at dose of 2–6 mM. It is well-known that CD4+ and CD8+ are T helper (Th) and T cytotoxic (Tc) lymphocytes, respectively. CD4 T cells recognize antigens presented by MHC class II and mediate both the cellular immune response through Th1 cells and the humoral immune response through Th2 cells. CD8 recognizes antigens presented by MHC class I and mediates cellular immune responses through cytotoxic T cells. The detection of CD4+ and CD8+ T lymphocytes and the ratio of CD4+/CD8+ T cells may reflect cellular immune function12. Many researches have reported that the ratio of CD4+/CD8+ was higher in autoimmune and atopic diseases13. In our study, cells were incubated with specific antibodies against T-cell surface markers and analyzed by flow cytometry. The results indicated that TBMS1 can inhibit CD4+ T lymphocytes and decrease the ratio of CD4+/CD8+ T cells. Thus, TBMS1 could inhibit the activation of T lymphocytes and suppress immune function by reducing the CD4+ T cell subpopulation. Cytokines play a key role in modulating the immune response. It was clear that Th1 cells produced IL-2, IFN-g and IL-12, and other cytokines when stimulated; and Th2 cells produced IL-4, IL-5, IL-6 and IL-10. The imbalance of Th1/ Th2 would lead to immunologic disease, such as rheumatoid arthritis, type-1 diabetes and multiple sclerosis. In addition, Trges (CD4+CD25+FOXP3+) cell and TGF-b also play important effect in immunoregulatory response. In the process of T cell activation, IL-2 is the vital growth factor responsible for cell cycle progression and proliferative characteristics of activated T-cells14. IFN-g promotes Th1 cell differentiation and activates macrophages to secrete various cytokines15. Additionally, IL-4 predominantly secreted by T-cell subsets is perhaps the most important cytokine required for Th-2 phenotype generation and humoral immune responses16. As a consequence, cytokines are responsible for the development of phenotypes and are, therefore, logical targets for therapeutic immune modulation17. In the study, the results suggested that TBMS1 may be able to simultaneously inhibit Th1 and Th2 immune responses. T cell activation is a complex process that requires multiple enzymes and adaptor proteins to mediate different intracellular signaling events18. Therefore, blocking the activity of these central transcription factors or these factors’ upstream second messengers would significantly affect T cell activation19. To better elucidate the underlying mechanism of
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TBMS1 on T cells, the activation of the NF-kB, NFAT and AP-1 signaling pathways associated with T cell activation were detected by Western blotting analysis. As shown in Figure 4, TBMS1 significantly suppressed activation of NF-kB, NFAT2 and AP-1 signal transduction pathways. The inhibitory effect of TBMS1 on T cell function in vivo was confirmed in a mouse DTH model, which is usually regarded as cell-mediated immune response and plays an essential role in the immune diseases20. To determine whether TBMS1 prevented DTH, we tested the compound’s effect on the ear swelling and histology of the mouse ear skin in a DNFB-induced DTH reaction. The experiment results suggested that TBMS1 significantly inhibited the DNFB-induced DTH reactions in mice.
Conclusion In conclusion, this study reported that a natural saponin TBMS1 exhibits immunsuppressive effects on activated murine T cell in vitro and in vivo. Our data indicated that TBMS1 inhibits ConA-induced T cell proliferation, decreased the ratio of CD4+/CD8+, suppressed IL-2, IFN-g, IL-4 and IL-6 production and mRNA expression in vitro. In addition, administration of TBMS1 significantly inhibited T cellmediated DTH response in vivo. The inhibitory mechanism of TBMS1 on T cells correlated with the suppressing activation of NF-kB, NFAT2 and AP-1 signal transduction pathways.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by the Doctor Fund (New teacher).
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16. Le Gros, G Ben-Sasson SZ, Seder R, et al. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med 1990;172:921–929. 17. Oberholzer A, Oberholzer C, Moldawer LL. Cytokine signaling – regulation of the immune response in normal and critically ill states. Crit Care Med 2000;28:N3–N12. 18. Diehn M, Alizadeh AA, Rando OJ, et al. Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation. Proc Natl Acad Sci USA 2002;99:11796–11801. 19. Nelson BH, Willerford DM. Biology of the interleukin-2 receptor. Adv Immunol 1998;70:1–81. 20. Yin Y, Gong FY, Wu XX, et al. Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita. J Ethnopharmacol 2008;120:1–6.
ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: http://www.tandfonline.com/loi/iipi20
Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways Yixuan Huang, Mengrou Ren, Ying Xiong, Tong Wang, Hongyu Chen, Xuming Deng & Jing Lu To cite this article: Yixuan Huang, Mengrou Ren, Ying Xiong, Tong Wang, Hongyu Chen, Xuming Deng & Jing Lu (2015) Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways, Immunopharmacology and Immunotoxicology, 37:4, 366-374 To link to this article: http://dx.doi.org/10.3109/08923973.2015.1063644
Published online: 16 Jul 2015.
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Date: 14 September 2015, At: 01:15
http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(4): 366–374 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1063644
RESEARCH ARTICLE
Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways Yixuan Huang1*, Mengrou Ren1*, Ying Xiong2, Tong Wang1, Hongyu Chen1, Xuming Deng2, and Jing Lu1,2 1
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Laboratory of Nutrition and Function Food, College of Light Industry Economics and Management, Jilin University, Changchun, Jilin, People’s Republic of China and 2Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin, People’s Republic of China Abstract
Keywords
Tubeimoside-1 (TBMS1) is one of the important components in Bolbostemma paniculatum (Maxim.) Franque. In the study, its immunosuppressive effects on murine T lymphocyte responses were evaluated in vitro and in vivo. The data showed that TBMS1 inhibited ConAinduced T lymphocyte proliferation, decreased the ratio of CD4+/CD8+, suppressed IL-2, IFN-g, IL-4 and IL-6 production and mRNA expression, down-regulate activation of NF-kB, NFAT2 and AP-1 signal transduction pathways in vitro. In addition, administration of TBMS1 significantly inhibited T cell-mediated DTH response in vivo. These findings indicated that TBMS1 inhibits the proliferation and activation of T lymphocytes in mice.
Cytokine, NF-kB, suppression, T lymphocyte, tubeimoside-1
Introduction TBMS1, as shown in Figure 1(A), is the main component extracted Bolbostemma paniculatum (Maxim.) Franquet, which has been widely used in traditional Chinese medicine for the treatment. TBMS1 is triterpenoid saponin with a particular cyclic structure with a dicrotalic acid bridge between the oligosaccharide chains at C-3 and C-28. Previous studies have shown that TBMS1 could inhibit the growth of several human cancer cell lines and macrophages1–3. These findings indicate that TBMS1 has the potential inhibition function through its effect on cells. However, so far there is no report published about the immunosuppressive activity of TBMS1 on T cell. So in the present study, we use murine T cell as target to study immunosuppression effect of TBMS1 on T-cell activation, and explored the potential mechanism underlying this effect. T cell is the important part of immune system and participates mainly in process of cell-mediated immune responses. T cell activation is a complex process that involves the induction of gene transcription, the expression of new cell surface molecules, and the secretion of cytokines4. Many immune-related diseases, such as multiple sclerosis,
*These authors contributed equally to this work. Address for correspondence: Dr Jing Lu, Laboratory of Nutrition and Function Food, College of Light Industry Economics and Management, Jilin University, Changchun, Jilin, People’s Republic of China. E-mail: [email protected] (or) Professor Xuming Deng, Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin, People’s Republic of China. E-mail: [email protected]
History Received 23 November 2014 Revised 26 May 2015 Accepted 15 June 2015 Published online 16 July 2015
rheumatoid arthritis, contact hypersensitivity and transplantation, have been known to be related to T cell activation5. Concanavalin A (ConA) is a kind of lectin and has been widely used for many years to activate T cells. Although the precise mechanism of action of this agent is unknown, it is likely that Con A activates T cells by indirectly cross-linking the T cell receptor (TCR)6. In the study, we found that TBMS1 significantly inhibited ConA-stimulated T cell proliferation, ratio of CD4+/CD8+ and cytokine production in vitro. The data also showed that TBMS1 exhibited an immunosuppressive effect through NF-kB, NFAT2 and AP-1 pathways. Moreover, we examined the effect of TBMS1 on 2,4-dinitro-1-fluorobenzene (DNFB)induced ear swelling and inflammatory cell infiltration. The data suggested that TBMS1 had protective effects of delayed type hypersensitivity (DTH). Our study is benefit to provide some useful information on its promising therapeutic application as a kind of immunosuppressive drug.
Materials and methods Materials Tubeimoside-1 (purity 498%, C63H98O29, molecular weight 1318) was from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China) and dissolved in PBS and stored at 4 C. Roswell Park Memorial Institute (RPMI) 1640 medium and fetal calf serum (FCS) were from GIBCO BRL (Grand Island, NY). Penicillin, streptomycin, cyclophosphamide (CTX), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenylmethylsulfonyl fluouride (PMSF), ConA, 1-fluoro-2,4-dinitrobenzene (DNFB), propidium iodide (PI),
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Figure 1. (A) The structure of TBMS1. (B) Effects of TBMS1 on ConA-induced T lymphocyte proliferation. Splenocytes (5 106 cells/ml) were incubated with various concentrations of testing compounds in the presence or absence of ConA (5 mg/ml) for 48 h. Cell proliferation was assayed by a conventional MTT method. Data represent mean ± SD of five observations; ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
Tween-20, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Interferon gamma (IFN-g), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6) ELISA kits were purchased from Biolegend Inc. (San Diego, CA). BioRT cDNA first strand synthesis kit was purchased from Bioer Technology (Changchun, Jilin, China). Phospho-specific antibodies for IkB as well as antibodies against IkB, nuclear factor kB (NF-kB), nuclear factor of activated T cells 2 (NFAT2) and activator protein-1 (AP-1) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L) and peroxidase-conjugated Affinipure goat anti-rabbit IgG (H + L) were purchased from PTG (Chicago, IL). Fluorescein isothiocyanate (FITC)-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD4 antibodies were purchased from BD Pharmingen (San Diego, CA). Primers were purchased from Shennengbocai Co. (Shanghai, China).
Experimental animals Male BALB/c mice (6 weeks old) weighing 18–20 g were purchased from Jilin University Experimental Animal Center and acclimatized for 7 days before use. The mice were housed under standard conditions of 24 ± 1 C, 12 h/12 h light–dark cycles and fed with a standard laboratory pellet chow diet and tap water. All mice used in experiments were approved by the Ethic Committee of Jilin University. Isolation and culture of murine splenocytes Male BALB/c mice were killed by cervical dislocation at the end of the experiment and thoroughly wiped with 70% ethanol and their abdominal cavity opened with the help of sterile scissors and forceps in an aseptic environment. The spleens of the animals were removed aseptically and washed in the sterile phosphate-buffered saline (PBS). Erythrocytes were lysed with Tris–NH4Cl (0.155 M NH4Cl and 16.5 mM Tris,
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pH 7.2). A single-cell suspension was prepared; cell debris and clumps were removed. The splenocytes were isolated and cultured in RPMI-1640 medium containing penicillin/streptomycin (100 U/ml and 100 mg/ml, respectively) and 10% FCS. Purified T cells were isolated from splenocytes by positive selection using MACS CD3 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as directed by the manufacturer7. Percent purity and viability were checked by FACS and exceeded 90% alive before experiments.
cytokines secretion in vitro. Briefly, splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well plates in the present of different doses of TBMS1 (2, 4 or 6 mM) for 48 h at 37 C in a 5% CO2 atmosphere. Then the plate was centrifuged at 1500 rpm for 10 min, and culture supernatants were collected for measurement of cytokine levels. The concentration of each cytokine in the culture medium was quantified using a competitive ELISA kit (BioLegend Inc., San Diego, CA) according to the manufacturer’s instructions.
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Splenocytes cytotoxicity and proliferation assay In order to determine whether TBMS1 affects lymphocyte viability, splenocytes from BALB/c mice were exposed to various concentrations (0–12 mM) of sodium TBMS1 for 48 h. Briefly, mouse lymphocytes (5 106 cells/ml) were cultured in triplicates in a 96-well flat-bottomed plate for 48 h with TBMS1 or ConA (5 mg/ml) + TBMS1. The cells cultured with medium alone were used as controls. MTT (5 mg/ml) was added 4 h before the end of culture. After removal of MTT, the formazan precipitate was solubilized in DMSO (100 ml/well) and measured on a microplate reader (Bio-Rad Laboratories, Hercules, CA) at the absorbance wavelength of 570 nm. Cell staining for T lymphocyte populations Cell staining for phenotypic analysis was carried out as described by Lee et al8,9. Briefly, single-cell suspensions were prepared from the spleen of each treatment by gently rubbing the spleen through a nylon mesh filter. Cellular debris was removed, and cells were washed in PBS and re-suspended (107 cells/ml) in FACS buffer (PBS, 10 mM Hepes buffer, 0.01% sodium azide and 1% heat-inactivated fetal bovine serum). Cells were labeled in a 96-well plate (106 cells/well) and stained with perCp/Cy5.5-conjugated anti-CD3 antibody, PE-conjugated anti-CD4 antibody and FITC-conjugated antiCD8 antibody for 30 min at 4 C in the dark. Each sample was re-suspended in a fixing solution of 0.5 ml PBS and analyzed by BD FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA). Cell Quest Software (Becton-Dickinson) was used to identify and quantify distinct populations of cells by mean fluorescent intensity (MFI). A minimum of 10 000 cells per sample were counted.
RT-PCR analysis Purified cells were treated as described above. Total RNA were extracted using Trizol (Incitrogen, Carlsbad, CA) according to the supplier’s instructions. The extracted final RNA was re-suspended in 20 ml of DEPC water. The concentration of total RNA was determined by absorbance at 260 nm and A260 nm/A280 nm confirmed the purity of RNA. An equal amount of total RNA (1 mg) was reversetranscribed with the BioRT cDNA first strand synthesis kit (Bioer Technology, Changchun, Jilin, China). Real-time PCR was performed using SYBR Green Super mix with an iCycler1 thermal cycler (Bio-Rad Laboratories). Primers were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The specific primers, amplified cycles and annealing temperature of each gene are listed in Table 1. All primer sequences were checked in Gen Bank (National Center for Biotechnology Information, Bethesda, MD) to avoid inadvertent sequence homologies. The amplification was performed in 25 ml reactions containing 8 ml cDNA, 10 mM forward and reverse primer, 2.5 ml 10 PCR buffer containing MgCl2, 10 mM dNTP mixture, 2.5 U Taq polymerase and 11.5 ml sterile ddH2O. Thermal cycling was performed in: one cycle at 94 C, 2 min; followed by 30–40 cycle each including denaturation at 94 C 30 s, 35 s at appropriate anneal temperature, extension 1 min at 72 C and one cycle at 72 C, 5 min. Final PCR products were separated on 2% agarose gel. The size of the amplified fragments was determined by comparison with a standard DNA marker. Western blotting analysis
Determination of cytokine levels in vitro In this study, we selected IL-2, IFN-g as Th1 cytokines and IL-4, IL-6 as Th2 cytokines to test the effect of TBMS1 on
Purified T cells were treated with different concentrations (2, 4 and 6 mM) of TBMS1 for 1 h, and then incubated in the presence of ConA (5 mg/ml) for 30 min. Cells were collected
Table 1. Sequences of primers used for RT-PCR gene expression studies. Gene Actin IL-2 IFN-g IL-4 IL-6
Primer sequence 0
0
5 -ATCATGTTTGAGACCTCCAACA-3 50 -CATCTCTTGCTCGAAGTCCA-30 50 -CTACAGCGGAAGCACAGC-30 50 -TCCTCAGAAAGTCCACCA-30 50 -TCTGAGACAATGAACGCTAC-30 50 -TTCCACATCTATGCCACT-30 50 -ACGGCACAGAGCTATTGATG-30 50 -ATGGTGGCCAGTACTACGA-30 50 -TCCAGTTGCCTTCTTGGGAC-30 50 -GTGTAATTAAGCCTCCGACTTG-30
Product size (bp)
Annealing temperature ( C)
Amplified cycle
318
58
30
375
56
30
144
55
30
455
58
30
140
58
30
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1 h at room temperature. The blots were again washed three times with TBST, and then developed using an enhanced chemiluminescence (ECL) kit (Sigma, St. Louis, MO). T cell-mediated DTH response in vivo Contact hypersensitivity to DNFB was induced in BALB/c mice by two daily paintings with 0.5% DNFB dissolved in acetone/olive oil (4:1). The degree of hypersensitivity was assayed 4 days after the second painting by measuring the increments of ear thickness 36 h after challenging the ears with 0.2% DNFB. The control group was treated with the solvent without DNFB and the DTH group was using DNFB. The TBMS1-treated DTH group was intraperitoneal injected (i.p.) with TBMS1 (1, 2 and 4 mg/kg) daily for
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by centrifugation, washed twice with PBS, and suspended in an extraction lysis buffer (Beyotime, Jiangsu, China) containing protease inhibitors. The cytoplasmic protein or nucleoprotein concentration was determined using a protein assay reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of total cellular protein (60 mg) were resolved by 12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with blocking solution (5% bovine serum albumin), followed by an overnight incubation at 4 C with the appropriate primary antibody. The blots were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then incubated with a 1:7000 dilution of horseradish peroxidase-conjugated secondary antibody (PTG) for
Role of TBMS1 in mouse T lymphocytes
Figure 2. Effects of TBMS1 on CD4+ and CD8+ expression. Splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well flatbottom plates and treated with various concentrations of TBMS1. Cells were cultured 48 h and collected, then stained with PE-anti-CD4/L3T4 or FITCanti-CD8/lyt-2 for analysis by flow cytometry. The values are presented as means ± SD (n ¼ 3). The results presented are from one experiment, which is representative of two others performed. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
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5 consecutive days. The positive control group was injected (i.p.) with CTX (20 mg/kg) for every 2 days. All the mice were killed by cervical dislocation at the end of the experiment and their ears were removed. The DTH response to DNFB was evaluated by measuring the weight difference of the right and the left ear10. Meanwhile, histopathologic evaluation was performed on mice. Right ears were removed 36 h after final DNFB application and fixed with 4% paraformaldehyde in PBS (pH 7.4). Ear tissues were sectioned, embedded in paraffin and cut at 5 mm. Tissue sections were then stained with hematoxylin–eosin (HE) for general morphology.
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Statistical analysis All values were expressed as mean ± standard deviation (SD) and statistical analysis was performed by one-way ANOVA using SPSS 13.0 (SPSS Inc., Chicago, IL) to evaluate the significance of differences between groups. Statistical significance were accepted when p50.05 or p50.01.
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Results Cell viability and cell proliferation We can see the data from Figure 1(B), there was no obvious toxicity against splenocytes at concentrations below 6 mM, whereas a dose-dependent decrease in cell viability was found at higher concentrations (8–12 mM). Simultaneously, the activity of TBMS1 was evaluated on splenocyte proliferation induced by ConA, which only stimulates T cell-mediated immunity. These results illustrated the dose-dependent inhibitory effect of TBMS1 on ConA-induced T splenocyte proliferation. The results also indicated that the immunosuppressive activities of TBMS1 in subsequent experiments observed here, at concentrations up to 6 mM, were not caused by its cytotoxicity. Effect of TBMS1 on CD4+ and CD8+ T lymphocyte populations We investigated the effects of TBMS1 on the subsets CD4+ and CD8+ T cells by flow cytometric analysis (Figure 2).
Figure 3. Effects of TBMS1 on Th1 and Th2 cytokines production and mRNA expression. Splenocytes (5 106 cells/ml) were stimulated with ConA (5 mg/ml) in 24-well plates in the present of different doses of TBMS1 (2, 4 or 6 mM) for 48 h at 37 C in a 5% CO2 atmosphere. (A) The 48 h cultured supernatants were collected for determining the contents of IL-2, IFN-g, IL-4 and IL-6 by ELISA. The absorbances of six wells per treatment were averaged. Bars represent means ± SD. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group. (B) The 48 h cultured cells were harvested for detecting these four cytokines mRNA expressions by RT-PCR. Three independent experiments were performed with similar results.
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Figure 4. Effects of TBMS1 on NF-kB, NFAT2 and AP-1 signaling pathways. Cells were purified using a separation column. The cells were pretreated with 2, 4 or 6 mM of TBMS1 for 1 h and then stimulated with ConA for another 30 min. Protein samples were extracted according to the manufacturer’s instructions and analyzed by Western blotting with specific antibodies. Quantification of each protein expression was normalized to b-actin using a densitometer (Imaging System). The data are representative of three independent experiments and expressed as mean ± SEM. ##p50.01 versus control group, *p50.05 or **p50.01 versus ConA group.
CD4+ levels and the ratio of CD4+/CD8+ were higher in the ConA group compared with that in the control group (p50.01). There was a down-regulation of the CD4+ T cell populations after TBMS1 plus ConA treatment, but the alteration of CD8+ T cell populations was not significant, resulting in the decrease of the CD4+/CD8+ ratio. These results indicated that CD4+ T cells were more sensitive to
inhibitory effects of TBMS1. TBMS1 exhibited inhibition function by reducing the CD4+ T cell subpopulation. Effects of TBMS1 on cytokine secretion in vitro As shown in Figure 3(A), TBMS1 simultaneously inhibit Th1 and Th2 cytokines production in a dose-dependent manner.
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In order to study whether TBMS1 decrease these four cytokines expression through the regulation of gene transcription, we also tested mRNA expression of IL-2, IFN-g, IL-4 and IL-6 by RT-PCR method. The result showed the same conclusion (Figure 3B). Effects of TBMS1 on signaling pathways activation
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In our experiment, we examined the effect of TBMS1 on NF-kB p65, NFAT2 and AP-1 signal pathways in purified T cells by western blotting. As shown in Figure 4, the IkB protein decreased after ConA administration, but increased after TBMS1 administration. On the contrary, p-IkB protein showed the opposite trend in the cytoplasm. In nucleprotein, the results showed that the activation of these pathways in mouse T lymphocytes increased after ConA administration
Figure 5. Effects of TBMS1 on DNFB-induced DTH reaction. BALB/c mice were sensitized with 0.5% DNFB on Days 0 and 1 and then challenged by DNFB on Day 5. Vehicle, CTX (20 mg/kg) and TBMS1 (1, 2 and 4 mg/kg) were administered to each group by i.p. injection on 5 consecutive days (Day 1–5). Ear swelling was calculated as the increase in ear patch weight (mg) between left (DNFB-untreated) and right (DNFB-treated) ear. Results are presented as means ± SD (n ¼ 10). ##p50.01 versus control group, *p50.05 or **p50.01 versus DTH group.
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alone but was significantly inhibited after TBMS1 treatment in a concentration-dependent manner. Effect of TBMS1 on DNFB-induced DTH response The inhibitory effect of TBMS1 on DNFB-induced DTH was illustrated in Figure 5. The ear swelling was significantly increased in model group compared with that in control group (p50.01), indicating that DNFB-induced DTH model had been made successfully. The positive drug CTX (20 mg/kg) could suppress DTH response (p50.01). TBMS1 at dose of 6 mg/kg also significantly reduced the ear swelling in DNFBinduced mice compared with the control group (p50.01). In addition, no signs of toxicity were observed in the TBMS1treated mice on the basis of body weight and microscopic examination of individual organs (data no shown). The dermal infiltration of inflammatory cells is an important feature of DTH. Cell types contained in infiltrates include lymphocytes and mast cells. In the present study, histologic analysis revealed that there were no notable histologic changes in control group (Figure 6A). However, topical DNFB elicited the infiltrations of inflammatory cells into ear skin lesions, and that numbers of these cells were significantly increased after DNFB challenge (Figure 6B). Furthermore, the i.p. administration of TBMS1 to mice every day relieved lesion severities. Compared with the DTH group, the tissue swelling had gone down a little in the low dose of TBMS1 (1 and 2 mg/kg) groups (Figure 6C and D). In the highest dose of TBMS1 (4 mg/kg) and the CTX group (20 mg/kg), tissue hypertrophy was reduced much obviously, and only a few inflammatory cell infiltration was present (Figure 6E and F).
Discussion Immunosuppressive agents are an important drug class that is valuable for the treatment of unwanted immune
Figure 6. Histopathologic changes of mouse ear tissues under microscope (400). Control group (A): DNFB (), microphotograph showing normal structure of the ear. DTH group (B): DNFB (+), microphotograph showing histopathologic changes (edema, infiltration of inflammatory cells) of the ear. DNFB (+) + TBMS1 (1, 2 or 4 mg/kg) (C, D and E) and DNFB (+) + CTX 20 mg/kg (F): microphotograph showing decreased histopathologic changes of the ear.
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responses, particularly those giving autoimmune disease and transplant rejection11. But most of the immunosuppressants in use have a narrow range of blood concentrations and serious adverse effects in clinic. Therefore, new and safe immunosuppressive drugs against acute and chronic rejection are eagerly awaited. In this study, we reported that a new candidate, TBMS1 which is extracted from Bolbostemma paniculatum (Maxim.) Franquet has potent immunosuppressive activity in vitro and in vivo. In the beginning of our study, we investigated the cytotoxicity of TBMS1 on splenocytes. The result assured us that the effects of TBMS1 on splenocytes were due to its immunosuppressive activity, but not due to cell death at dose of 0–6 mM. Then, we examined inhibitory activity of TBMS1 on ConA-induced T lymphocytes proliferation. These results showed that TBMS1 had a high inhibitory capacity for T lymphocyte proliferation at dose of 2–6 mM. It is well-known that CD4+ and CD8+ are T helper (Th) and T cytotoxic (Tc) lymphocytes, respectively. CD4 T cells recognize antigens presented by MHC class II and mediate both the cellular immune response through Th1 cells and the humoral immune response through Th2 cells. CD8 recognizes antigens presented by MHC class I and mediates cellular immune responses through cytotoxic T cells. The detection of CD4+ and CD8+ T lymphocytes and the ratio of CD4+/CD8+ T cells may reflect cellular immune function12. Many researches have reported that the ratio of CD4+/CD8+ was higher in autoimmune and atopic diseases13. In our study, cells were incubated with specific antibodies against T-cell surface markers and analyzed by flow cytometry. The results indicated that TBMS1 can inhibit CD4+ T lymphocytes and decrease the ratio of CD4+/CD8+ T cells. Thus, TBMS1 could inhibit the activation of T lymphocytes and suppress immune function by reducing the CD4+ T cell subpopulation. Cytokines play a key role in modulating the immune response. It was clear that Th1 cells produced IL-2, IFN-g and IL-12, and other cytokines when stimulated; and Th2 cells produced IL-4, IL-5, IL-6 and IL-10. The imbalance of Th1/ Th2 would lead to immunologic disease, such as rheumatoid arthritis, type-1 diabetes and multiple sclerosis. In addition, Trges (CD4+CD25+FOXP3+) cell and TGF-b also play important effect in immunoregulatory response. In the process of T cell activation, IL-2 is the vital growth factor responsible for cell cycle progression and proliferative characteristics of activated T-cells14. IFN-g promotes Th1 cell differentiation and activates macrophages to secrete various cytokines15. Additionally, IL-4 predominantly secreted by T-cell subsets is perhaps the most important cytokine required for Th-2 phenotype generation and humoral immune responses16. As a consequence, cytokines are responsible for the development of phenotypes and are, therefore, logical targets for therapeutic immune modulation17. In the study, the results suggested that TBMS1 may be able to simultaneously inhibit Th1 and Th2 immune responses. T cell activation is a complex process that requires multiple enzymes and adaptor proteins to mediate different intracellular signaling events18. Therefore, blocking the activity of these central transcription factors or these factors’ upstream second messengers would significantly affect T cell activation19. To better elucidate the underlying mechanism of
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TBMS1 on T cells, the activation of the NF-kB, NFAT and AP-1 signaling pathways associated with T cell activation were detected by Western blotting analysis. As shown in Figure 4, TBMS1 significantly suppressed activation of NF-kB, NFAT2 and AP-1 signal transduction pathways. The inhibitory effect of TBMS1 on T cell function in vivo was confirmed in a mouse DTH model, which is usually regarded as cell-mediated immune response and plays an essential role in the immune diseases20. To determine whether TBMS1 prevented DTH, we tested the compound’s effect on the ear swelling and histology of the mouse ear skin in a DNFB-induced DTH reaction. The experiment results suggested that TBMS1 significantly inhibited the DNFB-induced DTH reactions in mice.
Conclusion In conclusion, this study reported that a natural saponin TBMS1 exhibits immunsuppressive effects on activated murine T cell in vitro and in vivo. Our data indicated that TBMS1 inhibits ConA-induced T cell proliferation, decreased the ratio of CD4+/CD8+, suppressed IL-2, IFN-g, IL-4 and IL-6 production and mRNA expression in vitro. In addition, administration of TBMS1 significantly inhibited T cellmediated DTH response in vivo. The inhibitory mechanism of TBMS1 on T cells correlated with the suppressing activation of NF-kB, NFAT2 and AP-1 signal transduction pathways.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by the Doctor Fund (New teacher).
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