Interferon-gamma up-regulates toll-like receptor 4 and cooperates with lipopolysaccharide to produce macrophage-derived chemokine and interferon-gamma inducible protein-10 in human bladder cancer cell line RT4.
OBJECTIVE
Previously we have reported that RT4, a well differentiated human bladder cancer line, increases the expression of macrophage derived chemokine (MDC) and interferon (IFN)-gamma-inducible protein-10 (IP-10) in response to IFN-gamma and tumor necrosis factor (TNF)-alpha. In this study we examined the signal mechanisms for inducting these 2 chemokines in RT4 cells by lipopolysaccharide (LPS), IFN-gamma and TNF-alpha.


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-gamma synergized the productions of the 2 chemokines. IFN-gamma up-regulated the expression of TLR-4, which is an LPS binding receptor. Although LPS and IFN-gamma alone marginally activated nuclear factor (NF)-kappaB but not AP-1, LPS plus IFN-gamma augmented NF-kappaB and AP-1. Specific inhibition of NF-kappaB 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-gamma. TNF-alpha also activated NF-kappaB, AP-1 and ERK1/2. However, TNF-alpha plus IFN- gamma was associated with the activation of NF-kappaB but not of AP-1/ERK1/2 for the induction of MDC and IP-10.


CONCLUSIONS
IFN-gamma enhances LPS for the induction of MDC and IP-10 through up-regulation of TLR-4, and the signal pathways of NF-kappaB and AP-1/ERK1/2. This mechanism may help us understand inflammatory responses of the bladder to localized bacterial infection.
Yamada
Hiroshi
H
Department of Urology, University of Iowa, Iowa City 52242, USA.
Odonnell
Michael A
MA
Matsumoto
Tetsuro
T
Luo
Yi
Y
D045744:Cell Line, Tumor; D054422:Chemokine CCL22; D054357:Chemokine CXCL10; D019742:Chemokines, CC / Q000235:genetics*; D019743:Chemokines, CXC / Q000235:genetics*; D004357:Drug Synergism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D008070:Lipopolysaccharides / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
C515482:CCL22 protein, human; D054422:Chemokine CCL22; D054357:Chemokine CXCL10; D019742:Chemokines, CC; D019743:Chemokines, CXC; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma