The Roles Of MD-2 In LPS Transmembrane Signaling On Monocytes And Its Relationship With TLR4 And CD14

Monocyte and macrophage are the most effective cells in the innate immune system. The crucial first line defense against infection with innate immune response is the ability to sense the presence of invading microorganisms. Lipopolysaccaride (LPS), the main component of the outer membrane of Gram-negative bacteria is the most potent elicitor of innate immune response. An extremely low concentration of LPS can be sensed by monocytes and macrophages, leading to the production of cytokines and other molecules that are required to fight the invading microorganism and then activate the adaptive immune system. The innate immune response to LPS is vital to effectively combat an infection, however, in normal hosts, when an excess of inflammatory mediators produced by LPS-activated cells may result in systemic inflammation and the sepsis syndrome, a life-threatening condition that is characterized by fever, hypotension, compromised cardiac performance, coagulopathy, and multiple end-organ failure. Therefore, it is necessary to explore the mechanism of monocyte for sensing the presence of LPS.The functional membrane receptor for LPS is comprised of at least three proteins, CD14, TLR4, MD-2. CD14 had long been discovered as an LPS binding receptor, but for it is not a transmembrane protein, so it can not transfer LPS signals into cytoplasma. TLR4 has been defined as the main cellular signal transducer for LPS, although it has been shown to be a critical component for cell activation by LPS, but accumulating evidence suggest that TLR4 requires additional components to perform its fully function. Recently, a small, extracellular protein known as MD-2 was found to associate with TLR4 extracellular domain and enhance the sensibility to LPS. Mice lacking MD-2 are resistant to endotxoxin shock. MD-2 mediates the adverse effects of endotoxin equal to TLR4. Because the mature human MD-2 is a very small secreted protein just containing 160 amino acids residues, it will be more feasible to regulate LPS transmembrane signal via adjusting MD-2 expression. MD-2 is therefore a potential target for a therapy that neutralized the endotoxin. So we nowfocus our research on the MD-2 expression patterns on monocytes and its role in LPS transmembrane signal.LPS induced cell activation depends on its receptor expression patterns. Several reports have described the expression pattern of TLR4 and CD 14 on the monocytes respectively and there are very little about MD-2 expression report on the monocytes so far. Furthermore, there are no simultaneous reports about the three LPS receptor genes expression pattern in one treated sample of monocytes. While, Ribonuclease protection assay (RPA) is a highly sensitive and specific method to detect and quantitate RNAs. Two distinct advantages of the multi-probe RPA approach are its sensitivity and capacity to simultaneously quantify several mRNA species in a single sample of total RNA. So we want to adopt the method to confirm the three genes expression patterns simultaneously on monocytes stimulated by LPS. So we firstly construct the RPA template set which will be used for detecting expression patterns of MD-2, TLR4, CD 14 mRNAs in human monocyte cells.In order to provide sufficient experimental materials, elucidate the roles of MD-2 in LPS-induced monocyte activation and its signal transduction, according to an report of a point mutant MD-2C95Y losing completely its function, we construct the identical mutant with site-directed PCR method, and then we transfected the normal MD-2 expression vector and the newly constructed mutant vector into the THP1 cells to compare the different effect of MD-2 and MD-2C95y on LPS transmembrane signal.Finally, we sought to determine the mechanisms of MD-2 influencing the LPS transmembrane signal. LPS binds to CD 14 and MD-2 directly, but whether it can binds to TLR4 is currently unknown, and does MD-2 promote LPS binding to TLR4? We used transient transfections to express human TLR4, MD-2, or CD 14 alone or in different combinations in HKE 293 cells. The abili...