Toll-Like Receptors in Normal and Malignant Human Bladders

Purpose: Toll-like receptors have a major role in the innate immune response. They are expressed by immune cells and some epithelial cells. To study the role of urothelial cells in the intrabladder innate immune response we analyzed toll-like receptor expression and functionality in normal and malignant urothelial cells. Materials and Methods: Toll-like receptor 1 to 10 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction in 4 primary cultures of normal urothelium and 15 bladder cancer cell lines. Immunohistochemistry was used to detect toll-like receptor expression in 11 normal urothelial samples and 26 bladder tumors. Proinflammatory cytokine secretion by toll-like receptor agonist or bacillus Calmette-Guerin treated cultured cells was assessed by enzyme-linked immunosorbent assay. Mitogen-activated protein kinase phosphorylation was analyzed by Western blot and nuclear factor-kappa B localization was assessed by confocal microscopy. Results: Expression of most toll-like receptor mRNA was detected in cultured normal or tumor urothelial cells. Expression of toll-like receptors 2 to 4, 5, 7 and 9 protein was detected in all normal urothelial samples and most nonmuscle invasive tumors, although its intensity was decreased in the latter. Expression was markedly decreased in muscle invasive tumors. Treatment with toll-like receptor 2 and 3 agonists showed the strongest inflammatory response in 2 primary cultures of normal urothelial cells and 3 bladder cancer cell lines. Toll-like receptor 2 and 3 functionality was confirmed by the nuclear translocation of nuclear factor-kappa B and the induction of phosphorylated c-Jun N-terminal kinase mitogen-activated protein-kinase. Conclusions: Toll-like receptors are expressed in normal urothelium and nonmuscle invasive bladder tumors. In cultured urothelial cells agonist inducible toll-like receptor 2 or constitutively expressed toll-like receptor 3 is functional. These data suggest the potential use of toll-like receptor agonists for antitumor immunotherapy of nonmuscle invasive tumors.