The Expression Of LAIR Family And Its Regulatory Effect On LPS-induced Production Of Inflammatory Cytokines By Dentritic Cells

The leukocyte-associated Ig-like receptor(LAIR) is a member of the Ig superfamily(IgSF) with a single immunoglobulin-like domain and two immunoreceptor tyrosine-based inhibitory motifs(ITIMs) in its cytoplasmic tail. In 1983,Burns prepared monoclonal antibodies(mAbs),designated 9.1C3,by immunizing mouse with large granular lymphocytes(LGL) and found 9.1C3 mAb could inhibit NK cell cytotoxicity against K562 target cells.There are two members in LAIR family,LAIR-1 and LAIR-2.In 2003,Ouyang in our group confirmed that 9.1C3 is identical to LAIR-1 molecule.LAIR-1 and LAIR-2 were assigned new CD numbers,CD305 and CD306 respectively at the eighth workshop and conference on human leucocyte differentiation antigen(HLDA8). Human LAIR genes are localized in the leukocyte receptor complex(LRC) on human chromosome 19q13.4.The LAIR-1 and LAIR-2 gene lie close together in the LRC and are transcribed in opposite direction.LAIR-1 cDNA has 1728 base pairs and four different splice variants of the LAIR-1 mRNA have been cloned.Human LAIR-1 is a type-Ⅰtransmembrane glycoprotein of 287 amino acids containing a single extracellular Ig-like domain and two immunoreceptor tyrosine-based inhibitory motifs(ITIMs) in its cytoplasmic tail. LAIR-2 gene encodes a 135 amino acids protein that shares～84%homology to human LAIR-1 and lacks a transmembrane and intracellular domain suggesting it is a secreted protein.LAIR-2 has three different isoforms.The murine LAIR-1 mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region similar with human chromosome 19q 13.4 where the LRC is located.LAIR-1 is broadly expressed on a variety of hematopoietic cells and functions as inhibitory immune receptor since crosslinking of LAIR-1 by mAb in vitro delivers a potent inhibitory signal that is capable of inhibiting cellular functions of many kinds of immune cells.Human LAIR-1 selectively recruits the tyrosine phosphatases SHP-1 and SHP-2 upon cross-linking by LAIR-1 mAb.The mLAIR-1 protein shares 40% sequence identity with human LAIR-1,and has similar structure with human LAIR-1.Mouse LAIR-1 also is broadly expressed on various immune cells and is capable of inhibiting immune response.Upon crosslinking of mLAIR-1 by specific antibody the cytoplasmic tail of this molecule can be phosphorylated, and subsequently recruited SHP-2 but not SHP-1.In 2006,Mayaard group found collagen is the functional ligand for LAIR-1 with high affinity.The interaction of this receptor and its ligand is dependent on the conserved Gly-Pro-Hyp collagen repeats.Recently,they found in vitro that both LAIR-1 and LAIR-2 can bind to the same site of the collagen and LAIR-2 can block the interaction between LAIR-1 and collagen by binding collagen with high affinity.Mouse LAIR-1 also can bind collagen base on Gly-Pro-Hyp collagen repeats. LAIR-1-collagen interaction may be regulated by secreted LAIR molecule. Soluble LAIR-1(sLAIR-1) and LAIR-2 could block the interaction between membrane LAIR-1 and transmembrane or extracellular collagen.The affinity of LAIR-2 binding to collagen is higher than that of LAIR-1 binding to the ligand. Therefore LAIR-2 may function as a potential competitive molecule of membrane LAIR-1 and subsequently regulate LAIR-1 mediated inhibitory activity.In this study,two eukaryotic expression vectors pIg/3C-LAIR-2 and pCDNA3.1-LAIR-2 were established and transfected to CHO cells.After screening with G418 and cloning the cell lines which stably expressed LAIR-2-Fc fusion protein or full-length LAIR-2 were established.LAIR-2-Fc fusion protein and full-length LAIR-2 from cell culture supernatant were purified with protein A and LAIR-2 monoclonal antibody coupled separose 4B affinity chromatography column respectively.Both LAIR-2-Fc fusion protein and full-length LAIR-2 can be recognized by three LAIR-2 mAbs which were raised by immunizing mouse with prokaryotic expressed LAIR-2 protein and worked well in flow cytometry(FCM) and immunocytochemistry staining. Using optimized ELISA kits for detecting quantitatively sLAIR-1 and LAIR-2 molecules,we examined the human serum and urine LAIR molecules.We found both LAIR-1 and LAIR-2 level in the sera of patients with infection diseases were higher than that in normal human.The concentration of LAIR-2 in the serum as well as urine from pregnant women was much higher than that in normal human.Furthermore,we found there were positive correlations between sLAIR-1 and LAIR-2 levels as well as sLAIR-1 or LAIR-2 levels in serum and urine.In additon,we stained the placenta tissue slice with LAIR-1-Fc fusion protein and LAIR-2-Fc fusion protein,and found LAIR-2 binded the placenta tissue basal lamina with higher affinity suggesting that LAIR-2 may compete with membrane LAIR-1 for binding collagen ligand to regulate LAIR-1 inhibitory function. We also examined LAIR-1 expression in nonhematopoietic tissue using immunohistochemistry staining and confocal microscopy and found LAIR-1 selectively expressed in microgliocytes of human central nerve system based on the fact that LAIR-1 was co-expressed with IBA,a microgliocyte marker. Morever,LAIR-1 expression on microgliocytes was elevated in glioma tissue. The Toll-like receptor(TLR) family plays an instructive role in innate immune response against microbial pathogens,as well as the subsequent paticipate in the induction of adaptive immune response.TLR4,expressed on mononuclear macrophages,mast cells neutraphil,intestinal tract epithelial cells and endothelial cells,recognizes LPS and introduce inflammatory response against microbial pathogens.High dose LPS can result in vigorous inflammatory response upon binding its TLR4 receptor in vivo.It is important to study on the regulation of TLR4-mediated signaling and pathology.In order to investigate the regulatory effect of LAIR-1 on TLR4 triggering cytokine production in the mouse,the model of LPS-stimulated murine DC to produce inflammatory cytokines and cross-linking mLAIR-1 by corresponding monoclonal antibody were employed. We generated three rat anti-mouse LAIR-1 monoclonal antibodies and confirmed the mAb FMU-mLAIR-1.2 could recognize the native LAIR-1 molecules on murine cell lines.Then cross-linking of LAIR-1 molecule expressed on DC2.4 cells by coated mAb,and quantitative mesurement of inflammatory cytokine frome LPS-stimulated DC2.4 cells were emplyed.We found the IL-6 and TNF-αlevels increased significantly in supernatant from DC2.4 cell cultrure in the presence of LPS when cross-linked with coated mAb FMU-mLAIR1.2.We hypothezide that the tyrosine phosphatase(s) recruited by LAIR-1 cross-linking may attenuate activity of a signal molecule which inhibit TLR4 mediated signaling.Alternatively,LAIR-1 cross-linking promotes the function of a molecule participating in TLR4 signaling pathway and the production of some inflammatory cytokine.