Tuberc Respir Dis.  2001 Oct;51(4):315-324. 10.4046/trd.2001.51.4.315.

NF-κB Dependent IL-8 Secretion from Lung Epithelial Cells Induced by Peripheral Blood Monocytes Phagocytosing Mycobacterium Tuberculosis

Abstract

BACKGROUND
IL-8 is a potent chemotactic cytokine that plays an important role in the host defense mechanism against M. tuberculosis by recruiting inflammatory cells to the site of the infection. Lung epithelial cells, as well as alveolar macrophages are known to preduce IL-8 in response to M. tuberculosis. IL-8 gene expression is mainly regulated on the level of transcription by NF-κB. This study investigated whether or not A549 cells produce IL-8 in NF-κB dependent mechanism in response to macrophages phagocytosing M. tubersulosis.
METHODS
Peripheral blood monocytes that were obtained from healthy donors were cultured for 24 h with M. tuberculosis and a conditioned medium(CoMTB) was obtained. As a negative control, the conditioned medium without M. tuberculosis (CoMCont) was used. A549 cells were stimulated with M. tuberculosis, CoMCont and CoMTB and the IL-8 concentration in the culture media was measured by ELISA. The CoMTB induced IL-8 mRNA expression in the A549 cells was evaluated using RT-PCR, and CoMTB induced IκBα degradation was measured using western blot analysis. CoMTB induced nuclear translocation and DNA binding of NF-κB was also examined using an electrophoretic mobility shift assay(EMSA), and the CoMTB induced NF-κB dependent IL-8 transcriptional activity was measured using a luciferase reporter gene assay.
RESULTS
CoMTB induced IL-8 production by A549 cells(46.8±4.8 ng/ml) was higher than with direct stimulation with M. tuberculosis (6.8±2.9 ng/ml). CoMTB induced IL-8 mRNA expression increased after 2 h of stimulation and was sustained for 24 h. IκBα was degraded after 10 min of CoMTB stimulation and reappeared by 60 min. CoMTB stimulated the nuclear translocation and DNA binding of NF-κB. The CoMTB induced NF-κB dependent IL-8 transcriptional activity(13.6±4.3 times control) was higher than either CoMCont(2.0±0.6 times control) or M. tuberculosis (1.4±0.6 times control).
CONCLUSION
A conditioned medium of peripheral blood monocytes phagocytosing M. tuberculosis stimulates NF-κB dependent IL-8 production by the lung epithelial cells.

Keyword

IL-8; Mycobacterium tuberculosis; NF-κB; Conditioned medium
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