Inhibitory effects of bone marrow mesenchymal stem cells combined with leflunomide on the proliferation of mouse lymphocytes

Qiu Ying-ying1, Li Jing1, Yin Yu-jun1, Tang Yu1, You Hai-yan1, Zhu Wei2, Jiao Zhi-jun1

1Department of Rheumatology, Affiliated Hospital of Jiangsu University, Zhenjiang   212001, Jiangsu Province, China; 2Laboratory of Hematology, College of Medical Technology, Jiangsu University, Zhenjiang   212000, Jiangsu Province, China

Qiu Ying-ying★, Studying for master’s degree, Attending physician, Department of Rheumatology, Affiliated Hospital of Jiangsu University, Zhenjiang   212001, Jiangsu Province, China
lily_qiu9999@ 163.com

Correspondence to: Li Jing, Professor, Tutor of master, Department of Rheumatology, Affiliated Hospital of Jiangsu University, Zhenjiang   212001, Jiangsu Province, China
zjlijing@public.zj.js.cn

Supported by: the Social Development Program of Science and Technology Department of Jiangsu Province, No. BS2005043

Received:2007-11-30
Accepted:2008-06-17

Abstract
BACKGROUND: Leflunomide is a new immunodepressant. Its active product A771726 can inhibit the activities of dihydroorate dehydrogenase and the proliferation of activated lymphocytes in vitro.
OBJECTIVE: To investigate the inhibitory effects of bone mesenchymal stem cells (BMSCs) combined with leflunomide (LEF) on lymphocyte proliferation in mice in vitro.
DESIGN, TIME AND SETTING: The randomized control cytology immunoregulation experiment was performed at the Central Laboratory, Affiliated Hospital of Jiangsu University from June to November 2007.
MATERIALS: Forty-two BALB/c female mice aged 5-6 weeks were used in this study. Eighteen of them were used to prepare BMSCs. At the third passage, BMSCs were used in this study. Twenty-four of them were used to prepare splenic lymphocytes. LEF activated metabolic product A771726 was presented by Doctor Xiao from Cinkate, USA. Non-specific mitogen ConA and lipopolysaccharide were obtained from Sigma, USA.
METHODS：T and B lymphocyte suspension was divided into BMSCs group, LEF group, BMSCs+LEF group. The concentration of BMSCs was 2×108 L-1. Number of BMSCs: number of lymphocytes = 1: 30. The final concentration of LEF was 15 mg/L. In addition, 5 mg/L non-specific mitogen ConA and 25 mg/L lipopolysaccharide were used in each group.
MAIN OUTCOME MEASURES: MTT was used to detect the inhibitory rate of T and B lymphocyte proliferation. Activation of T and B lymphocytes was measured by surface markers. Flow cytometry was applied to determine the apoptosis rate of T lymphocytes. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine interferonγ, interleukin 10 and interleukin 4 mRNA expression of T lymphocytes.
RESULTS: Compared with the positive control, the inhibitory rate of T lymphocyte proliferation was significantly increased in the BMSCs group, LEF group, BMSCs+LEF group (P < 0.05), and the changes was greater in the BMSCs+LEF group compared with that in the BMSCs group and LEF group (P < 0.05). The inhibitory rate of B lymphocyte proliferation was similar to above-mentioned results. Compared with the positive control, CD3+CD69+ and CD3+CD28+ expression of T lymphocytes and CD19+CD69+，CD19+CD86+ expression of B lymphocytes were similar in each group. The apoptosis rates of T lymphocytes in the BMSCs group and BMSCs+LEF group significantly reduced (P < 0.05). Compared with the positive control, BMSCs combined with LEF promoted the mRNA expression of interferonγand inhibited interleukin 10 levels of T lymphocytes (P < 0.05), but had no effects on interleukin-4 mRNA expression. Changes in interferon γ and interleukin 10 were significantly greater compared with the BMSCs group and LEF group (P < 0.05).
CONCLUSION: BMSCs combined with LEF in vitro show some collaboratively inhibitory effects on the proliferation of T and B-lymphocytes, interleukin-10 mRNA expression and apoptosis of T lymphocytes, but promote the mRNA expression of interferonγ, and had no effects on activation of T and B lymphocytes or mRNA expression of interleukin-4.

Qiu YY, Li J, Yin YJ, Tang Y, You HY, Zhu W, Jiao ZJ.Inhibitory effects of bone marrow mesenchymal stem cells combined with leflunomide on the proliferation of mouse lymphocytes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):5658-5662 [www.zglckf.com/zglckf/ejournal/upfiles/08-29/29k-5658(ps).pdf]