Aim

To study the release mechanism of C-X-C motif chemokine 11 (CXCL11) and other chemokines after the co-cultivation of CD4⁺ and CD8⁺ T cells with the renal tubular epithelial cells (RTEC) in the process of allograft renal transplantation rejection.

Methods

The Human CD4⁺, CD8⁺ T cells were obtained from the blood of volunteers and kidney transplantation (Ktx) patients, and co-cultured with renal tubular epithelial cells (RTEC) in vitro. RT-PCR was run for detecting the mRNA transcription of CXCL11, IFN-induced protein of 10 (CXCL10), and IL-6 in cells after RTEC was stimulated with IFN-γ or co-cultured with CD4⁺ and CD8⁺ T cells. The concentration of CXCL11, CXCL10 and IL-6 in the culture medium was detected by Multiplex Assay after RTEC was stimulated with IFN-γ or co-cultured with CD4⁺ and CD8⁺ T cells. IFN-γ receptor antibody was used for interfering with the above reaction and the blocking effect was observed. Western blot was used for protein expression analysis. Finally, we applied renal biopsies from kidney transplantation patients with and without rejection to verify the results of the above experiments by using RT-PCR and Western blot.

Results

The mRNA expression of CXCL11 and CXCL10 were significantly increased after RTEC was stimulated with IFN-γ or co-cultured with CD4⁺ and CD8⁺ T cells. Multiplex Assay showed that the concentration of CXCL11 and CXCL10 in the supernatant were significantly increased in a time-dependence fashion after stimulation RTEC by IFN-γ. Anti-IFN-γ receptor1 (anti-IFN-γR1) antibody could reduce the production of CXCL11 and CXCL10 in this situation. The concentration of CXCL11 and CXCL11 in the supernatant was significantly increased with a time-dependent effect after the co-culture of CD4⁺ and CD8⁺ T cells with RTEC. The anti-IFN-γR1 blocked this effect. Our study showed that the expression levels of CXCL11 and CXCL10 were upgraded in the biopsies of patients with renal transplant rejection comparatively to pre-transplant biopsies, both at mRNA and protein levels.

Conclusions

RTEC and T cells can stimulate each other during the acute rejection of allogeneic kidney transplantation and secret CXCL11，CXCL10 and other chemokines. IFN-γ plays a key role in this process.

中文翻译：

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趋化因子 CXCL11 在肾小管上皮细胞与 CD4+ 和 CD8+ T 细胞反应中产生的机制

目标

^{研究CD4 +}、CD8 ⁺ T细胞与肾小管上皮细胞(RTEC)共培养后CXC基序趋化因子11(CXCL11)等趋化因子在同种异体肾移植排斥反应过程中的释放机制。

方法

人CD4 ⁺、CD8 ⁺ T细胞取自志愿者和肾移植(Ktx)患者的血液，并与肾小管上皮细胞(RTEC)体外共培养。RT-PCR 检测 RTEC 经 IFN-γ 刺激或与 CD4 ⁺和 CD8 ⁺ T 细胞共培养后细胞中 CXCL11、IFN 诱导的蛋白 10 (CXCL10) 和 IL-6 的 mRNA 转录。^{RTEC经IFN-γ刺激或与CD4 +}和CD8 ⁺共培养后，通过Multiplex Assay检测培养基中CXCL11、CXCL10和IL-6的浓度T细胞。用IFN-γ受体抗体干扰上述反应，观察阻断作用。蛋白质印迹用于蛋白质表达分析。最后，我们应用来自有和没有排斥的肾移植患者的肾活检，通过使用 RT-PCR 和蛋白质印迹来验证上述实验的结果。

结果

^{用IFN-γ刺激RTEC或与CD4 +}和CD8 ⁺ T细胞共培养后，CXCL11和CXCL10的mRNA表达显着增加。Multiplex Assay 表明，在 IFN-γ 刺激 RTEC 后，上清液中 CXCL11 和 CXCL10 的浓度以时间依赖性方式显着增加。在这种情况下，抗 IFN-γ 受体 1（抗 IFN-γR1）抗体可以减少 CXCL11 和 CXCL10 的产生。^{CD4 +}和CD8 ⁺共培养后，上清液中CXCL11和CXCL11的浓度显着增加，呈时间依赖性具有 RTEC 的 T 细胞。抗 IFN-γR1 阻断了这种作用。我们的研究表明，与移植前活检相比，肾移植排斥患者活检中 CXCL11 和 CXCL10 的表达水平在 mRNA 和蛋白质水平上都有所提高。

结论

RTEC和T细胞在同种异体肾移植急性排斥反应过程中可以相互刺激，分泌CXCL11、CXCL10等趋化因子。IFN-γ 在此过程中起关键作用。