ABSTRACT
Objective To study whether Fcα/μ receptor(Fcα/μR)can mediate complement killing of human glomerular mesangial cells.Methods Fcα/μR cDNA contained plasmid,pcDNA3.1-Fcα/μR was transfected into a human glomerular mesangial cell(NHMC).Fcα/μR expression was detected by Western blot and laser scanning eonfocal microscopy.Binding of IgM-immune complexes(IgM-IC)to the Fcα/μR on cell membrane was detected by flowcytometry and laser scanning confocal microscopy.Killing of cells by complement was shown by Trypan blue exclusion assay.Results NHMC cells transfected with Fcα/μR could bind IgM and IgM-IC.After treatment with complement,added IgM-IC,the death rate of pcDNA3.1-Fcα/μR transfected cell was significant higher than the control groups of wild type cell,pcDNA3.1 transfected cell and the pcDNA3.1-Fcα/μR transfected cell without IgM-IC.Conclusion IgM-IC can bind to the Fcα/μR expressed NHMC cells and mediate complement killing of the cells.
ABSTRACT
Objective To explore the role and the possible molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody played and involved in pathogenesis of atherosclerosis. Methods Natural anti-oxLDL IgM monoclonal antibody 3A6 was generated by using standard hybridoma production techniques. Influence of 3A6 on formation of foam cells was observed by Oil Red O staining and affinity of Na125I-conjugated oxLDL on the naive and LPS-activated macrophages. After LPS stimulation on macrophages, anti-TLR4 neutralizing mAb, p38MAPK specific inhibitor SB203580, NF-kB specific inhibitor PDTC or RNAi targeting Fcα/μ receptor (Fcamr) were applied, respectively. Results Natural anti-oxLDL IgM monoclonal antibody 3 A6 were found specifically inhibit the binding of CuoxLDL to naive macrophages but not the binding of CuoxLDL to LPS-activated macrophages. It also promoted the formation of CuoxLDL-mediated foam macrophages. 3A6 F(ab')2 or pre-incubation with un-related IgM inhibited the binding of 3A6/CuoxLDL complex to LPS-activated macrophages. LPS up-regulated the expression of Fcamr in macrophages in a dose- and time-dependent manner, which was attenuated by treatment with anti-TLR4. LPS induced the phosphorylation of p38MAPK and translocation of NF-kB p65, contributing to the up-regulated expression of Fcα/μ receptor in macrophages. Conclusions Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to naive macrophages in vitro. However, LPS, through the Toll-like receptor (TLR)4 receptor, activated the p38MAPK and NF-kB pathways and up-regulated the expression of Fcα/μ receptor in macrophages, which promoted the binding of 3A6/CuoxLDL complex to macrophages through binding with Fc fragments and the formation of foam macrophages. Therefore, our findings provide a new explanation why bacterial infection deteriorates the pathogenesis of atherosclerosis.