The Development And Study Of Stable Reporter Cell Lines For Fast And Sensitive Detection Of Pyrogen

Pyrogens are a class of heterogeneous compounds that cause fever and induce inflammatory responses in the host.In clinical practice,pyrogen refers to a substance that causes a pyrogen response in a mammal.Pyrogens are classified into exogenous pyrogens and endogenous pyrogens according to their sources.Exogenous pyrogens mainly originate from the outside of the body,which are microbes(such as bacteria,viruses,fungi)and non-microorganisms(such as antigens and antineoplastic agents).Endogenous pyrogens mainly include hormones(such as steroids and prostaglandins)and cytokines(such as tumor necrosis factor,interferon,growth factor,and interleukin)in the body.The use of pyrogen-contaminated medicines and medical devices can cause systemic inflammation and,in the worst case,septic shock.Thus,accurate and fast quantification of pyrogens is crucial for drug safety.So far,there are limits for detection of pyrogens using the rabbit pyrogen test(RPT),the limulus amoebocyte lysate assasy(LAL)and monocyte activation test(MAT).In the present study,we aimed to develop a sensitive and reliable method for rapid detection of pyrogens using luciferase reporter assay.Previous reports have shown that bacterial flagellin can stimulate lung epithelial cells to produce cytokine IL-8 through Toll-like receptor TLR5.However,how other pyrogens induce activation of lung epithelial cells has not been fully clarified.Lipopolysaccharides(LPS,also known as endotoxin)is as the major pyrogen and one of the most potent bacterial inducers of cytokines.During infectious processes,pyrogens stimulate human monocytes to synthesize or release inflammatory cytokines including interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor a(TNF-a).Nuclear factor-kappa B(NF-?B)is a major transcription factor that regulates genes encoding these pro inflammatory cytokines.Meanwhile,human beta interferon(IFN-?)dramatically augments the febrile response to endotoxin in normal rabbits by the enhancement of TNF-dependent TNF production,and restores the normal febrile response to endotoxin in pryogen-tolerant rabbits.In view of this,we inserted the luciferase gene containing the NF-?B response element or the IFN-?promoter into a lentiviral vector,stably transfected into A549 cells,followed by flow cytometry to sort single cell clone,and finally made use of luciferase reporter system to identify monoclonal cell activity,thereby constructing a stable human A549 luciferase reporter cell line.Our results showed that several monoclonal stable NF-?B cell clones responded to 0.1 EU/mL endotoxin,which was less than human fever threshold at 0.3 EU/mL of endotoxin.We further used a LAL with a sensitivity of 0.125 EU/mL,which is the sensitivity of the limulus reagent widely used in medicine,compared with the reporter cells in order to evaluate pyrogens under equal conditions,and the results showed that the reporter cells reached the same level as sensitivity of LAL.In addition,we further investigated reporter cells and found that compared with the original A549,the reporter cell clones were activated at low concentrations of endotoxin stimulation and up-regulated TLR4 expression.These results indicate that reporter experiments based on human alveolar epithelial cells are promising and feasible for the further development of alternative pyrogenic assays.Therefore,the stable luciferase reporter cells have the potential to further develop into a sensitive and fast pyrogen evaluation method.