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Abstract
Background
Asthma is a syndrome that is characterized by a variable degree of airflow obstruction, bronchial hyperresponsiveness, and airway inflammation. Colchicine is an inexpensive and safe medication with unique anti-inflammatory properties. IL-1Ra (Interleukin-1 receptor antagonist) mediates the anti-inflammatory effect in human inflammatory diseases, including asthma. This study examined whether IL-1Ra mediates the anti-inflammatory effect of colchicine in normal human bronchial epithelial cells (NHBE), RAW 264.7 cells (murine macrophage cell line), and a mouse lung.
Methods
NHBE, RAW 264.7 cells and BALB/c mice were stimulated with colchicine, and the increase in the IL-1Ra level was estimated by ELISA, Western analysis and RT-PCR analysis.
Results
Colchicine stimulated NHBE and RAW 264.7 cells to release IL-1Ra into the supernatant in a dose-and time-dependent manner. The major isoform of IL-1Ra in NHBE and RAW 264.7 cells is type I icIL-1Ra, and sIL-1Ra, respectively. IL-1Ra up-regulation was blocked by PD98059, a specific inhibitor in MAPK pathways. Colchicine also stimulated the secretion of IL-1Ra into the bronchoalveolar lavage (BAL) fluid of BALB/c mouse.
Figures and Tables
Figure 1
Dose kinetics of IL-1Ra elaboration in colchicine treated NHBE. (A) NHBE were incubated in various colchicine concentration media for 48 hours. The levels of immunoreactive IL-1Ra in supernatants were quantitated by ELISA. Colchicine stimulates NHBE to release IL-1Ra into supernatant with dose dependent manner. IL-1Ra elaborations in 0.1 and 0.5 µM colchicine concentrations show statistically significant differences compared to control (*p<0.05). However, in high colchicine concentrations, IL-1Ra elaborations are decreased. The noted values represent the mean ± SEM of a minimum of five assays. (B) Western blot results also show dose dependent IL-1Ra elaboration. SEM: standard error of the mean.
Figure 2
Time kinetics of IL-1Ra elaboration in colchicine treated NHBE. (A) NHBE were incubated in 0.1 µM colchicine concentration media for 2, 4, 8, 16, 24, 48 hours. The levels of immunoreactive IL-1Ra in supernatants were quantitated by ELISA. Colchicine stimulates NHBE to release IL-1Ra into supernatant with time dependent manner. IL-1Ra elaboration shows statistically significant differences compared to control after 16 hours (*p<0.05). The noted values represent the mean ± SEM of a minimum of five assays. (B) Western blot results show also time dependent IL-1Ra elaboration.
Figure 3
IL-1Ra isoform mRNA expression in colchicine treated NHBE. NHBE were incubated in various colchicine concentration media for 48 hours. The supernatant was removed, total cellular RNA was isolated, and RT-PCR was performed with primers specific for icIL-1Ra type I, icIL-1Ra type II, and β actin. icIL-1Ra I mRNA expressions are dominantly observed. However, there are no differences in IL-1Ra mRNA expression in various colchicine concentrations.
Figure 4
IL-1Ra elaboration in NHBE treated with colchicine variants. NHBE were incubated in media containing colchicine variants for 16 hours. The levels of immunoreactive IL-1Ra in supernatants were quantitated by ELISA. Colchicine variants do not stimulate IL-1Ra elaboration whereas chochicine does. The noted values represent the mean + SEM of a minimum of five assays.
Figure 5
IL-1Ra elaboration in colchicine treated mouse lung. Colchicine was administrated intraperitoneally to BALB/c mouse. The mouse was sacrificed 48 hours later, the elaborations of IL-1Ra in the total lung and bronchoalveolar lavage (BAL) fluid were quantitated by Western blot. (A) Colchicine stimulates to release IL-1Ra with dose dependent manner in mouse lung. (B) Clochicine stimulates to release IL-1Ra in BAL fluid. The lysate shows more dominant band than supernatant. rIL-1Ra (recombinant human nonglycosylated mature sIL-1Ra) and LPS treated RAW 264.7 cells (LPS Raw; 100 ng/ml for 48 hours) are used as positive contols for IL-1Ra elaboration.
Figure 6
Effect of PD98059 on IL-1Ra elaboration in colchicine treated NHBE. A. NHBE were incubated in various colchicine concentration media for 16 hours. The levels of immunoreactive IL-1Ra in supernatant were quantitated by ELISA. IL-1Ra elaboration by colchicine stimulation is inhibited by MAPK inhibitor (PD98059) with dose dependent manner. The noted values represent the mean + SEM of a minimum of five assays.
Figure 7
IL1-Ra isoform mRNA expression in colchicine/PD98059 treated RAW 264.7 cells. RAW 264.7 cells were incubated in various colchicine/PD98059 concentration media for 48 hours. The supernatant was removed, total cellular RNA was isolated, and RT-PCR was performed with primers specific for sIL-1Ra, icIL-1Ra type I. sIL-1Ra mRNA expression is dominantly observed in RAW 264.7 cell than icIL-1Ra type I, and mRNA expression by colchicine is decreased by PD98059 in dose-dependent manner.
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