Uroepithelial cells can directly respond to Mycobacterium bovis bacillus Calmette-Guérin through Toll-like receptor signalling.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Miyazaki
Jun
J
Department of Urology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-City, Ibaraki 305-8575, Japan. kontama@v004.vaio.ne.jp
Kawai
Koji
K
Oikawa
Takehiro
T
Johraku
Akira
A
Hattori
Kazunori
K
Shimazui
Toru
T
Akaza
Hideyuki
H
D000818:Animals; D001500:BCG Vaccine / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology*; D006801:Humans; D009163:Mycobacterium bovis; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D014380:Tuberculosis, Bovine / Q000382:microbiology*; D014552:Urinary Tract Infections / Q000382:microbiology*
D001500:BCG Vaccine; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4