연구배경 : IL-8은 강력한 화학주성인자로서 결핵감염 부위로 염증세포들을 동원함으로서 결핵균에 대한 숙주의 방어기전에 있어서 중요한 역할을 한다. IL-8의 유전자의 발현에 있어서 NF-${kappa}B$가 중요한 역할을 한다. 저자들은 결핵 감염시 폐상피세포가 NF-${kappa}B$ 의존성으로 IL-8을 분비하는지 알아보고자 하였다. 방 법 : 말초혈액단핵구에 결핵균을 감염시키고 24시간 배양 후 배양상층액(CoMTB)을 얻었다. 결핵균, CoMTB로 자극한 A549 세포주의 IL-8 분비 정도를 ELISA 방법으로 측정하였다. CoMTB로 자극한 A549 세포주의 IL-8 mRNA 의 발현 정도를 RT-PCR로, $I{kappa}B{alpha}$의 분해를 western blot 분석으로, NF-${kappa}B$의 핵이동과 DNA 결합은 electrophoretic mobility shift assay(EMSA)를 이용하여, 그리고 NF-${kappa}B$ 의존성 IL-8 유전자의 전사활성은 luciferase reporter gene assay를 이용하여 측정하였다. 결 과 : A549 세포주를 CoMTB로 24시간 자극하여 얻은 배양액의 IL-8 농도는 $46.8{pm}4.8;ng/ml$로 분비하여 결핵균으로 직접 자극하였을 때의 $6.8{pm}2.9;ng/ml$보다 높았다. CoMTB로 A549 세포주를 자극하였을 때 IL-8 mRNA의 발현이 증가하였고, $I{kappa}B{alpha}$의 분해가 일어났으며, NF-${kappa}B$의 핵이동과 DNA 결합이 일어났으며, NF-${kappa}B$ 의존성 IL-8 유전자의 전사활성이 증가하였다. 결 론 : 결핵병변에서 폐상피세포는 결핵균을 탐식한 단핵식 세포와의 상호작용에 의해 NF-${kappa}B$ 의존성으로 IL-8을 분비한다.
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 produce IL-8 in response to M. tuberculosis. IL-8 gene expression is mainly regulated on the level of transcription by NF-${kappa}B$. This study investigated whether or not A549 cells produce IL-8 in NF-${kappa}B$ dependent mechanism in response to macrophages phagocytosing M. tuberculosis. 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 $I{kappa}B{alpha}$ degradation was measured using western blot analysis. CoMTB induced nuclear translocation and DNA binding of NF-${kappa}B$ was also examined using an electrophoretic mobility shift assay(EMSA), and the CoMTB induced NF-${kappa}B$ dependent IL-8 transcriptional activity was measured using a luciferase reporter gene assay. Results : CoMTB induced IL-8 production by A549 cells($46.8{pm}4.8;ng/ml$) was higher than with direct stimulation with M. tuberculosis ($6.8{pm}2.9;ng/ml$). CoMTB induced IL-8 mRNA expression increased after 2 h of stimulation and was sustained for 24 h. $I{kappa}B{alpha}$ was degraded after 10 min of CoMTB stimulation and reappeared by 60 min. CoMTB stimulated the nuclear translocation and DNA binding of NF-${kappa}B$. The CoMTB induced NF-${kappa}B$ dependent IL-8 transcriptional activity($13.6{pm}4.3$ times control) was higher than either CoMCont($2.0{pm}0.6$ times control) or M. tuberculosis ($1.4{pm}0.6$ times control). Conclusion : A conditioned medium of peripheral blood monocytes phagocytosing M. tuberculosis stimulates NF-${kappa}B$ dependent IL-8 production by the lung epithelial cells.
