Preliminary Research Of The Mechanism Of Cytokines Regulation In THP1/CD14 Cells By Mannan-binding Lectin

Although studies for innate immunity have been developing rapidly in the past few years,its essence remains to be revealed and elucidated.Accompany to the hypothesis that innate immunity initiates acquired immune response and controls the types of response through pattern-recognition put forward by Janeway CA et al, pattern recognition molecules have become one of the hot topics of immunologic researches.As the important molecules in innate immune,pattern recognition molecules can be divided into two categories based on their exsiting forms,including the dissolvability pattern recognition molecules in the humor and pattern recognition receptors expressed in the cell surface.At present,it has been shown that among the dissolvability pattern recognition molecules surfactant proteins A and D(SP-A and SP-D),which belong to the collectin subgroup of the C-type lectin superfamily,can bridge innate immunity and adaptive immunity even regulate adaptive immunity. Among the pattern recognition receptors,macrophage mannose recetor(MMR),a member of C-type lectin receptors(CLRs) family,as well as Toll-like receptors (TLRs) family can also play these functions.Accordingly,innate immunity pattern-recognition is pressing on towards the central issue of immune response, initiation of response as well as its control.Mannan-binding lectin(MBL),a member of the collectin superfamily(C-type lectin with a collagen-like domain),along with SP-A and SP-D together,is a plasma glycoprotein mainly secreted by liver cells.MBL is a key molecule in innate immume system.MBL insufficiency is one of the most common immunodeficiencies. However,the mechanisms is still not clear.MBL,which has a total structure with C1q is a special molecule.Classical theory believes that the chief biological effect of MBL is that it forms compound with two MBL-associated serine proteases (MASP-1,MASP-2) through its collagen-like region(CLR) and then activates the complement cascade via lectin pathway to play the function of anti-infection in innate immune in the present of Ca²⁺.In the past few years,it has been reported that MBL plays important functions in immunological regulation.Correlated researches abroad demostrated that MBL could inhibit T cell response mediated by DC-SIGN,regulate the cytokines production of peripheral blood monoeuclear cell(PBMC).Previously, our research team found that MBL could inhibit of the maturation of LPS stimulated dendritic cells(DC) and inhibit the production of TNF-αand IL-12 in hunman monocyte line THP1/CD14 cells in different differentiation states.These findings also confirmed the functions of MBL in immunological regulation.However,the mechanisms of MBL in immunological regulation has not been reported yet at home and abroad.In order to explore the the mechanisms of the functions of MBL in immunological regulation,we selected the hunman monocyte line THP1/CD14 cells as our cell model.We investigated the binding of MBL to THP1/CD14 cells,the characteristics of the binding and the interaction of MBL with TLRs.This research can be divide into three parts as follow.Partâ… Detection the binding of MBL to hunman monocyte line THP1/CD14 cellsIn this part,We investigated whether MBL can bind to THP1/CD14 cells by Cell-ELISA and flow cytometry(FCM).The result of Cell-ELISA revealed that absorptance of 450nm depended on the concentration of MBL in experiment group. The result of FCM showed that MBL could hardly bind to THP1/CD14 cells in the absence of Ca²⁺ or in the presence of EDTA as a substitute of CaCl₂,but it could bind to THP1/CD14 cells in the buffers which contain 5 mmol/L or 10 mmol/L CaCl₂,and the binding capability in the buffer containing 10 mmol/L CaCl₂ is slightly increased compared to in the buffer containing 5 mmol/L CaCl₂,suggesting that the binding of them depended on the Ca²⁺ concentration.These results above indicated that natural MBL purified from plasma could actually bind to THP1/CD14 cells in physiological conditions,and this binding was evidently dependent on Ca²⁺ concentation.Thus there may exist MBL receptors or binding proteins in the cellular membrane surface of THP1/CD14 cells,and the binding of MBL to THP1/CD14 cells needs the participation of Ca²⁺.Partâ…¡Study on the characteristics of MBL binding to THP1/CD14 cellsAccording to the Ca²⁺ dependent characteristic of MBL binding to THP1/CD14 cells in part one,we presumed that MBL might bind to certain glycoproteins or other glycosylated structure in cellular membrane surface through its carbohydrate-recognition domain(CRD).Classical theory believes MBL binds to C1q receptor (C1qR) in cellular membrane surface of mononuclear macrophage through its collagen-like region(CLR).Previously,our research team have successfully expressed prokaryotic MBL-CRD protein and MBL-CLR protein and prepared cell line E8 which can secrete mouse anti-human C1qR monoclonal antibody(mAb).So,in this part,we firstly resused the cell line E8,immunized mouse to obtain abdominal dropsy,and then purificated the abdominal dropsy obtained this mAb.Secondly,we prepared MBL-CLR protein by Ni²⁺-NTA agarose chromatography.Subsequently,we focused on the functional domains of MBL binding to THP1/CD14 cells,we detected the inhibitory effect of MBL binding to THP1/CD14 cells,using sugars,prokaryotic rh(recombinant human) MBL-CRD,prokaryotic rhMBL-CLR protein,C1q and mouse anti-C1q mAb as potential inhibitors,by FCM.The results showed that their binding can be partly inhibit by some sugars,rhMBL-CRD alone and rhMBL-CLR alone.And the inhibitory effect was obviously when rhMBL-CRD and rhMBL-CLR were added together.However,C1q and mouse anti-human C1qR mAb had no inhibitory action.These data showed that THP1/CD14 cells can express sugar-sensitive MBL receptors or binding proteins,including two specific categories of receptors against CLR and CRD,and these receptors were unrelated with C1qR. Therefore,this findings reveals that MBL may binding to THP1/CD14 cells through more than one way.Partâ…¢Preliminary study of the relationship between MBL and TLRs Among mammalian TLRs,TLR-2 is expressed very extensively,and mediates recogonition of a wide range of microbial products.TLR-2 is one of the predominant TLRs expressed in mononuclear macrophage.In this part,we focused on whether MBL can directly bind to TLR-2 by ELISA and Western blot.The results of the two methods both showed that MBL can directly bind to the extracellular domain of TLR-2 protein(sTLR-2).In the research of LPS-stimulated THP1/CD14 cells,we investigated the binding capability of LPS-stimulated THP1/CD14 cells with MBL by ELISA and FCM.It was found that the binding capability of LPS(100ng/L) -stimulated THP1/CD14 cells with MBL was strengthened compared to untreated THP1/CD14 cells,statistical analysis showed the difference was significant.The results of FCM also showed the the binding capability of LPS-stimulated THP1/CD14 cells with MBL was strengthened,and the strengthen was in a LPS concentration-dependent manner.The higher concentration of LPS,the more stronger of the binding capability.Subseqently,we analyzed the impact of the stimulation of LPS to the expression of TLR-2 and TLR-4 mRNA in THP1/CD14 cells.RT-PCR analysis showed that the expression of TLR-2 and TLR-4 mRNA in LPS-stimulated THP1/CD14 cells was significantly increased after 2h compared to the non-LPS-stimulated cells,and it was dropped to the approximate level of non-LPS-stimulated cells after 24h.Eventually,we studied the signal transduction. The nulear proteins of THP1/CD14 cells,which were untreated,LPS treated,MBL and LPS treated,were extracted.Then the extracted nulear proteins were essaied by Western bolt.The result of Western bolt showed MBL can inhibit the transposition of transcription factor NF-κB induced by LPS,suggesting the mechanism of MBL in the regulation of cytokines secretion.The results above indicated that THP1/CD14 cells might epress Ca²⁺-dependent, sugar-sensitive MBL receptors or binding proteins,including both CLR-specific and CRD-specific,which have nothing to do with C1qR.MBL can directly bind to sTLR-2.The binding capability of LPS-stimulated THP1/CD14 cells was strengthened,and was consistent with the expression of TLR-2 and TLR-4 by RT-PCR in LPS-stimulated THP1/CD14 cells.The binding can be partly inhibit by sTLR-2.MBL can suppress the transposition of transcription factor NF-κB induced by LPS in THP1/CD14 cells.Therefore,we presumed that there might be two aspects of mechanisms about the inhibition of the proinflammatory cytokines produced by LPS-stimulated THP1/CD14 cells,the one is the enhanced expression of TLR-2 and TLR-4 mRNA induced by LPS,which resulted in stronger binding capability and more binding capacity;the other is the suppression of the transposition of transcription factor NF-κB induced by MBL.