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-γ , tumour necrosis factor-α , interleukin-2, -12, -4, -6, -10, -8 and -1 β were assayed using an enzyme-linked immunosorbent assay.
RESULTSUroepithelial 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-γ , tumour necrosis factor-α , interleukin-2, -12, -4, -10 and -1 β were not detected in the culture supernatant.
CONCLUSIONThese 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.