Cooperative activation of CCL5 expression by TLR3 and tumor necrosis factor-alpha or interferon-gamma through nuclear factor-kappaB or STAT-1 in airway epithelial cells.
BACKGROUND
CCL5/RANTES contributes to prolonged eosinophilic inflammation and asthma exacerbation after a viral infection. We studied the mechanism of CCL5 expression using viral product double-stranded RNA (dsRNA), a ligand of Toll-like receptor 3 (TLR3), and inflammatory cytokines in airway epithelial cells.


METHODS
The airway epithelial cell line BEAS-2B was used in our in vitro study, and the levels of CCL5 mRNA and CCL5 protein expression were determined using real-time PCR and ELISA. The activity of the CCL5 promoter region and nuclear factor (NF)-kappaB was assessed by dual luciferase assay using specific luciferase reporter plasmids. We used actinomycin D to assess the stability of mRNA. Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was analyzed by Western blot.


RESULTS
Synthetic dsRNA up-regulated the expression of CCL5 mRNA and CCL5 protein. Adding TNF-alpha or IFN-gamma to dsRNA further increased the expression of CCL5. The combination of TNF-alpha and dsRNA cooperatively activated the CCL5 promoter region and the NF-kappaB-specific reporter. IFN-gamma did not activate these reporters. However, it increased the stability of CCL5 mRNA induced by dsRNA. IFN-gamma phosphorylated STAT-1, but dsRNA did not. The effects of IFN-gamma were not evident in the cells transfected with short interfering RNA for STAT-1.


CONCLUSIONS
Cross-talk between TLR3 signaling and inflammatory cytokines regulates the expression of CCL5 in airway epithelial cells. In this mechanism, TNF-alpha may activate NF-kappaB, in cooperation with TLR3 signaling. IFN-gamma may stabilize CCL5 mRNA up-regulated by TLR3. This mechanism may depend on STAT-1.
Homma
Tetsuya
T
Division of Respiratory Medicine and Allergy, Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan.
Matsukura
Satoshi
S
Hirose
Takashi
T
Ohnishi
Tsukasa
T
Kimura
Teruaki
T
Kurokawa
Masatsugu
M
Ieki
Koushi
K
Odaka
Miho
M
Suzuki
Shintaro
S
Watanabe
Shin
S
Sato
Masayuki
M
Kawaguchi
Mio
M
Schleimer
Robert P
RP
Adachi
Mitsuru
M
D002461:Cell Line, Transformed; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020871:RNA Stability / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D020545:Respiratory Mucosa / Q000166:cytology*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C483024:CCL5 protein, human; D018946:Chemokine CCL5; D016207:Cytokines; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma