The Role Of SIGIRR In LPS-Induced Acute Inflammatory Response In Human Type â…¡ Alveolar Epithelial Cells (A549)

【Objective】LPS-TLR4 singnal pathway plays a central role in the nosogenesisof acute lung injury(ALI) and other acute pulmonary inflammatory diseases. It isimportant to attenuate ALI if this signa is inhabited. The single immunoglobulinIL-1 receptor-related protein (SIGIRR) is one of the molecules that inhibit TLR4.SIGIRR can interact with TLR4 and inhibit LPS signal. Being still unknown ofthe expression and function of SIGIRR in the human alveolar epithelial cells,in this study, we examined the expression of SIGIRR in normal human lungtissue and the roles of SIGIRR in the acute inflammatory responses in alveolarepithelial cells.【Methods】In this study, we examined the expression of SIGIRRin human normal alveolar epithelial cells and in A549, an immortal human lungadenocarcinoma cell line derived from human type II alveolar epithelial cells,atlevels of tissue, mRNA and protein, using immunohistochemistry, reversetranscription-PCR (RT-PCR) and Western blot. Then A549 cells were treatedwith LPS. The dynamic changes of SIGIRR mRNA levels and proteinexpression were evaluated by real-time PCR and quantitive Western blot, respectively. The eukaryotic expression vector that contained full-length cDNAof SIGIRR was constructed and transfected A549 cells to make SIGIRRoverexpresed. The impact of SIGIRR on LPS-induced activation of NF-κBwas studied through the dual-luciferase reporter assay system. The role ofSIGIRR on the cytokines release from A549 cells on response to LPS wasstudied with ELISA. And MTT was used to detect the growth and survival ofA549 cells overexpresed SIGIRR after LPS stimulation.【ResultResults】Theexpression of SIGIRR was detected in alveolar epithelial cells from all 20samples of normal lung tissue by immunohistochemistry. The expression ofSIGIRR mRNA (433 bp) was detected by RT-PCR in all 20 samples of normalhuman lung tissues and in A549 cells. The SIGIRR protein expression wasdetected by Western blot in all 20 samples of normal human lung tissues andA549 cells. The A549-derived SIGIRR protein was the same molecular weight asthe normal lung tissue-derived protein. The SIGIRR mRNA levels wereevaluated by real-time PCR. SIGIRR mRNA levels were significantly lower at 3h, 6 h and 12 h post-LPS than 0 h; however, there was no significant differencebetween mRNA levels at 0 h and 24 h. By quantitive Western blot, it was foundthat there were no statistically significant differences in the SIGIRR proteinlevels at 0h, 3h, 6h and 12h after LPS treatment, but at 24 h post-LPS exposure,SIGIRR protein levels were significantly lower than previous time points. Thetranscriptional activity of NF-κB after LPS stimulation was siginificantly lowerin A549 cells overexpressed SIGIRR than that in controls. The levels ofcytokines IL-1β, TNF-αand IL-6 was also siginificantly lower in A549 cellsoverexpressed SIGIRR than in controls. In cells that overexpressed SIGIRR, theIR was significantly lower than that of cells transfected with the empty vector ornon-transfected cells.【Conclutions】SIGIRR is expressed in hunman normal alveolar epitheial cells. It is involved in the LPS-induced acute inflammatoryresponse in alveolar epitheial cells and plays a negative regulative rloe, whichfacilitates the growth and survival of these cells.