Figure 1.
CC10 is overexpressed in pCC10 transfected BEAS-2B cells.
CC10 expression was detected by means of (A) immunofluorescence staining and (B) western blotting. CC10 was mainly expressed in the cytoplasm of pCC10 transfected BEAS-2B cells. One representative experiment of the three independent experiments is shown. CC10, Clara cell 10-kDa protein. Proteins extracted from human inferior turbinate mucosa from 2 patients undergoing partial inferior turbinectomy were used as positive controls for CC10 expression.
Figure 2.
CC10 gene transfer inhibits IL-1β stimulated IL-8 expression in BEAS-2B cells.
(A) IL-8 mRNA and (B) protein expression in BEAS-2B cells without transfection, and mock or pCC10 transduced BEAS-2B cells stimulated with or without IL-1β at a concentration of 10 ng/mL. n = 3, *P<0.01 compared with cells without any treatment, #P<0.05 compared with mock transduced cells treated with IL-1β. CC10, Clara cell 10-kDa protein; IL, interleukin.
Figure 3.
CC10 gene transfer suppresses IL-1β induced NF-κB transcription activity in BEAS-2B cells.
BEAS-2B cells transfected with either pCC10 or mock were transiently contransfected with pGL4.32[luc2P/NF-κB-RE/Hygro] vector and pGL4.74[hRluc/TK] vector. The firefly luciferase activity was normalized to renilla luciferase activity. n = 3, *P<0.05 compared with cells without any treatment, #P<0.05 compared with mock transfected cells treated with 10 ng/mL of IL-1β. CC10, Clara cell 10-kDa protein; IL, interleukin.
Figure 4.
CC10 gene transfer prevents NF-κB nuclear translocation in BEAS-2B cells.
BEAS-2B cells transfected with either mock or pCC10 were treated with 10 ng/mL of IL-1β for 30 or 60 minutes. Total and nuclear proteins were extracted and then performed to western blot. (A) Nuclear NF-κB p65 (N-p65) and (B) total p65 (T-p65) were detected. One representative experiment of the three independent experiments is shown. (C) and (D) The semi-quantitative analysis of western-blot gels and results were presented as relative protein expression levels. n = 3, **P<0.01 and *P<0.05. CC10, Clara cell 10-kDa protein; IL, interleukin.
Figure 5.
CC10 gene transfer inhibits the phosphorylation of IκB-α but not IKKα/β in BEAS-2B cells.
BEAS-2B cells were transfected with either mock or pCC10 and treated with 10 ng/mL of IL-1β for 30 or 60 minutes. (A) Total protein was extracted, and phosphorylated IκB-α and IKKα/β were detected by means of western blot. One representative experiment of the three independent experiments is shown. (B) The semi-quantitative analysis of western-blot gels and results were presented as relative protein expression levels. n = 3, **P<0.01 and *P<0.05. CC10, Clara cell 10-kDa protein; IL, interleukin.
Figure 6.
CC10 does not interact with NF-κB p65/p50 subunits directly.
BEAS-2B cells transfected with pCC10 were stimulated with 10 ng/mL of IL-1β for 4 hours. Cellular proteins were immunoprecipitated with anti-p65 antibody. Immunoblotting was performed with anti-p65 and anti-CC10 antibodies (lane 1). Rabbit normal serum was served as a negative control for immunoprecipitation (lane 2) and human inferior turbinate mucosa were used as positive controls for CC10 and p65 expression (lane 3). One representative experiment of the three independent experiments is shown.
Figure 7.
CC10 expression levels are inversely correlated with IL-8 production evoked by IL-1β in human sinonasal mucosa.
Sinonasal mucosa obtained from chronic rhinosinusitis and control subjects were cultured in vitro and stimulated with 10 ng/mL IL-1β for 24 hours, and then the CC10 levels in tissue homogenates and IL-8 levels in supernatants were determined by means of ELISA. Partial correlations were computed on IL-8 protein and CC10 protein levels after adjustment for diagnostic groups.
Figure 8.
Induction of CC10 protein expression in CC10-knockout mice by gene transfer.
Representative photomicrographs of CC10 immunohistochemical staining of sinonasal mucosa sections of mock and pCC10 transfected CC10 knockout mice. CC10 expression was detected three days after transfection. Original magnification ×400.
Figure 9.
CC10 gene transfer suppresses IL-1β induced IL-8 expression through inhibiting NF-κB nuclear translocation in CC10-knockout mice.
(A) and (B) Representative photomicrographs of IL-8 and p65 protein immunohistochemical staining of sinonasal mucosa sections of controls mice without any treatment and mock and pCC10 transfected mice with IL-1β stimulation. Original magnification ×400. Arrows, p65 positive staining in nucleus of epithelial cells. (C) The mean optical density of IL-8 in airway epithelium. (D) The number of nuclear p65 positive cells in airway epithelium. n = 6 mice per group **P<0.01.