Interferon-γ up-regulates toll-like receptor 4 and cooperates with lipopolysaccharide to produce macrophage-derived chemokine and interferon-γ inducible protein-10 in human bladder cancer cell line RT4

Abstract

Purpose: Previously we have reported that RT4, a well differentiated human bladder cancer line, increases the expression of macrophage derived chemokine (MDC) and interferon (IFN)-γ-inducible protein-10 (IP-10) in response to IFN-γ and tumor necrosis factor (TNF)-α. In this study we examined the signal mechanisms for inducting these 2 chemokines in RT4 cells by lipopolysaccharide (LPS), IFN-γ and TNF-α. Materials and Methods: MDC and IP-10 expression was evaluated by sandwich enzyme-linked immunosorbent assay. Signal molecule activation was examined by Western blotting and electrophoretic mobility shift assay. The expression of toll-like receptor (TLR)-4 was analyzed by reverse transcriptase-polymerase chain reaction and flow cytometry. Results: LPS did not induce RT4 cells to produce IP-10 and MDC. However, LPS plus IFN-γ synergized the productions of the 2 chemokines. IFN-γ up-regulated the expression of TLR-4, which is an LPS binding receptor. Although LPS and IFN-γ alone marginally activated nuclear factor (NF)-κB but not AP-1, LPS plus IFN-γ augmented NF-κB and AP-1. Specific inhibition of NF-κB and AP-1 pathways decreased the production of MDC and IP-10. Extracellular regulated kinase (ERK) 1/2, an upstream signal of AP-1, was also responsive to LPS and/or IFN-γ. TNF-α also activated NF-κB, AP-1 and ERK1/2. However, TNF-α plus IFN-γ was associated with the activation of NF-κB but not of AP-1/ERK1/2 for the induction of MDC and IP-10. Conclusions: IFN-γ enhances LPS for the induction of MDC and IP-10 through up-regulation of TLR-4, and the signal pathways of NF-κB and AP-1/ERK1/2. This mechanism may help us understand inflammatory responses of the bladder to localized bacterial infection. Copyright © 2005 by American Urological Association.