Miyazaki Jun, Kawai Koji, Oikawa Takehiro, Johraku Akira, Hattori Kazunori, Shimazui Toru, Akaza Hideyuki
Department of Urology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan.
BJU Int. 2006 Apr;97(4):860-4. doi: 10.1111/j.1464-410X.2006.06026.x.
To investigate, in a human urinary tract cell line, the interaction of Toll-like receptor (TLR) signals with cytoplasmic adapter proteins MyD88 and bacillus Calmette-Guérin (BCG), and evaluate the epithelial cytokine response to BCG infection. Intravesical BCG therapy is effective against carcinoma in situ and as prophylaxis for recurrence, but although immunological mechanisms have been assumed, the mechanisms of the antitumour effects of BCG have not been completely elucidated.
The cell line was first screened for TLR expression by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from a human urinary cell line, Hu35E6E7, and cDNA synthesised. PCR was used to measure the gene expression of TLR-2, -3, -4, -5, -9, MyD88, MD-2, CD14 and interleukin-8 and -6. The Hu35E6E7 cell line was cultured in keratinocyte serum-free medium, and BCG was added to the cell culture. After Hu35E6E7 cells were stimulated by BCG for various periods, the total RNA of the cells was extracted. Quantitative real-time PCR was conducted for MyD88 using appropriate probes, and the expression of MyD88 analysed. The cell supernatant was collected, and the levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -6, -10, -8 and -1beta were assayed using an enzyme-linked immunosorbent assay.
Uroepithelial cells expressed TLR-2, -3, -4 and -9, and MyD88, MD2, CD14, interleukin-6 and -8 were also detected. At 3, 6, 9 and 12 h after adding BCG, quantitative PCR assay showed that the expression of MyD88 was maximal at 6 h. The presence of BCG stimulated the release only of interleukin-6 and -8 from Hu35E6E7 cells after 6 h. By contrast, interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -10 and -1beta were not detected in the culture supernatant.
These results show that uroepithelial cells, but not immune cells, responded directly to BCG through TLR signalling. Further investigation is needed to determine the role of cytokines released from uroepithelial cells after BCG infection.
在一种人泌尿道细胞系中，研究Toll样受体（TLR）信号与胞质衔接蛋白髓样分化因子88（MyD88）及卡介苗（BCG）之间的相互作用，并评估上皮细胞对卡介苗感染的细胞因子反应。膀胱内卡介苗治疗对原位癌有效且可预防复发，尽管推测存在免疫机制，但卡介苗抗肿瘤作用的机制尚未完全阐明。
首先通过逆转录聚合酶链反应（RT-PCR）对该细胞系进行TLR表达筛查。从人泌尿道细胞系Hu35E6E7中分离总RNA并合成cDNA。采用PCR检测TLR-2、-3、-4、-5、-9、MyD88、MD-2、CD14以及白细胞介素-8和-6的基因表达。将Hu35E6E7细胞系在无角质形成细胞血清培养基中培养，并向细胞培养物中加入卡介苗。在卡介苗刺激Hu35E6E7细胞不同时间段后，提取细胞的总RNA。使用合适的探针针对MyD88进行定量实时PCR，并分析MyD88的表达。收集细胞上清液，采用酶联免疫吸附测定法检测干扰素-γ、肿瘤坏死因子-α、白细胞介素-2、-12、-4、-6、-10、-8和-1β的水平。
尿路上皮细胞表达TLR-2、-3、-4和-9，同时也检测到MyD88、MD2、CD14、白细胞介素-6和-8。加入卡介苗后3、6、9和12小时，定量PCR检测显示MyD88在6小时时表达最高。卡介苗存在时，6小时后仅刺激Hu35E6E7细胞释放白细胞介素-6和-8。相比之下，培养上清液中未检测到干扰素-γ、肿瘤坏死因子-α、白细胞介素-2、-12、-4、-10和-1β。
这些结果表明，尿路上皮细胞而非免疫细胞通过TLR信号直接对卡介苗产生反应。需要进一步研究以确定卡介苗感染后尿路上皮细胞释放的细胞因子的作用。
