[小鼠树突状细胞上配对免疫球蛋白样受体B的表达及其与免疫耐受关系的研究]

[Study of paired immunoglobin-like receptor B expression on dendritic cells and its relationship with immune tolerance in mouse].

作者信息

Liu Zheng-Rong, Zhang Min, Li Wei-Ming, Zhou Hao, Zou Ping

机构信息

Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2007 Oct;28(10):689-93.

PMID:18399176

Abstract

OBJECTIVE

To investigate the expression of paired immunoglobin-like receptor B (PIR-B) on dendritic cells (DCs) and its relationship with tolerogenic DCs (T-DCs) in mouse.

METHODS

DC2.4 cells, an immature dendritic cell line derived from C57BL/6 mouse, were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (mDC) and cultured respectively with the recombined mouse interleukin-10 (rmIL-10) or recombined human transforming growth factor beta1 (rhTGF-beta1) to develop the tolerogenic dendritic cells (T-DC). Special small interference RNA (siRNA) molecular of PIR-B was chemically synthesized and transfected into DC2.4 cells (si-DC) by lip2000. The expression of PIR-B on DC2.4 cells was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The allogeneic lymphocyte proliferative capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR was analyzed by ELISA.

RESULTS

Semi-quantitative RT-PCR and Western blot showed that PIR-B mRNA and protein were expressed on DC2.4 cells. RmIL-10 and rhTGF-beta1 induced the higher PIR-B mRNA and protein level on T-DCs (Relative values were 0.51 +/- 0.08 and 0.58 +/- 0.23; 0.85 +/- 0.07 and 0.87 +/- 0.14; 0.79 +/- 0.10 and 0.85 +/- 0.34, respectively) (P < 0.05). LPS down-regulated the PIR-B expression on mDC (0.35 +/- 0.10 and 0.32 +/- 0.04) (P < 0.05). The PIR-B mRNA and protein expression were inhibited by siRNA transfection (decreased by 78.9% and 74.2% respectively after 48 h interference) (P < 0.05). DC2.4 cells stimulated the proliferation of BALB/c mouse allo-genetic spleen cell. The mDC enhanced alloreactivity in MLR and the IFN-gamma secretion in supernatants. The T-DCs alleviated the allo-genetic spleen cell proliferation (P < 0.05) and IFN-gamma secretion in MLR (P < 0.05). Silence of the PIR-B expression (si-DC) also promoted of alloreactivity and enhanced the IFN-gamma secretion in MLR (P < 0.05).

CONCLUSION

High expression of immune inhibition receptor PIR-B is one of the general features and molecular mechanism of dendritic cells to acquire immune tolerance in mouse.

摘要

目的

研究配对免疫球蛋白样受体B（PIR-B）在小鼠树突状细胞（DCs）上的表达及其与致耐受性DCs（T-DCs）的关系。

方法

用脂多糖（LPS）刺激源自C57BL/6小鼠的未成熟树突状细胞系DC2.4细胞48小时以诱导成熟树突状细胞（mDC），并分别与重组小鼠白细胞介素-10（rmIL-10）或重组人转化生长因子β1（rhTGF-β1）共培养以培养致耐受性树突状细胞（T-DC）。化学合成PIR-B的特异性小干扰RNA（siRNA）分子，并通过lip2000转染到DC2.4细胞中（si-DC）。通过半定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测DC2.4细胞上PIR-B的表达。采用3H-胸腺嘧啶核苷掺入试验，通过混合淋巴细胞反应（MLR）检测DCs的同种异体淋巴细胞增殖能力。通过酶联免疫吸附测定（ELISA）分析MLR上清液中IFN-γ的浓度。

结果

半定量RT-PCR和蛋白质免疫印迹法显示DC2.4细胞上表达PIR-B mRNA和蛋白。RmIL-10和rhTGF-β1诱导T-DCs上更高的PIR-B mRNA和蛋白水平（相对值分别为0.51±0.08和0.58±0.23；0.85±0.07和0.87±0.14；0.79±0.10和0.85±0.34）（P<0.05）。LPS下调mDC上的PIR-B表达（0.35±0.10和0.32±0.04）（P<0.05）。siRNA转染抑制了PIR-B mRNA和蛋白表达（48小时干扰后分别降低78.9%和74.2%）（P<0.05）。DC2.4细胞刺激BALB/c小鼠同种异体脾细胞增殖。mDC增强了MLR中的同种异体反应性以及上清液中IFN-γ的分泌。T-DCs减轻了MLR中同种异体脾细胞增殖（P<0.05）和IFN-γ分泌（P<0.05）。PIR-B表达沉默（si-DC）也促进了同种异体反应性并增强了MLR中IFN-γ的分泌（P<0.05）。