Effects Of Natural IgM Monoclonal Antibodies Against Oxidized Low-density Lipoprotein On Pathogenesis Of Atherosclerosis And Its Mechanism

BackgroundAtherosclerosis is now widely recognized as a chronic inflammatory disease. In condition of high blood lipids, plasma Low-density lipoprotein (LDL) infiltrates into the artery wall and becomes oxidized LDL (oxLDL); oxLDL is uptaken by macrophages and macrophages transform into foam cells, which can cause dramatic atherosclerotic effect. A number of in vitro studies have suggested that these IgM antibodies blocked the uptake of oxLDL by macrophages and thus could prevent foam cells formation in vivo. Clinic researches proved that chronic infection (serum high level lipopolysaccharide) had close relationship with atherosclerosis pathogenesis, but the mechanisms were not clear.No data were released of whether the normal mice fed with high fat diet produced natural antibodies against oxLDL to influence development of atherosclerosis and whether these natural antibodies played protective roles when applied in vitro or in vivo, and whether these auto-antibodies were involved during course of increased mortality of atherosclerosis induced by increased infection. Based on the above, we designed and fulfilled the following experiments.Objective:1. To Generate and identify the production of natural mouse IgM monoclonal antibodies(mAbs) against LDL and oxLDL with high specificity and activity and establish foundation for the research on LDL and oxidized oxLDL and natural antibody in atherosclerosis progression.2. To explore the role and the exact molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody played and involved in pathogenesis of atherosclerosis underlying the action of IgM antibody in the binding of oxLDL to macrophages.3. By applying apoE gene knock-out mice as the animal model ,artherosclerosis was induced by high fat food.Experiments were executed on purpose to study the effects of natural anti-oxLDL antibody 3A6 on serum lipids, blood plasma oxLDL and lesion of apoE gene knock-out mice.4. To explore the role and the exact molecular mechanisms of natural anti-oxLDL IgM monoclonal antibody on pathogenesis of atherosclerosis underlying the action of bacterial lipopolysaccharide (LPS) activation in the binding of oxLDL to macrophages.Methods:1. BALB/c mice were breed in specific pathogen free conditions and fed with high cholesterol diet; the splenocytes from these mice were directly fused with Sp2/0 myeloma cells by using standard hybridoma production techniques. Hybridomas were selected on the basis of the supernatant's ability to bind with native LDL and oxLDL in ELISA. Selected hybridomas were identified by Western blot, Immunoblotting and ELISA.2. Murine macrophage cell line RAW-264.7 was cultured in vitro. OxLDL specific monoclonal antibody 3A6 with IgM isotype was purified and utilized to form complex with Na125I-conjugated oxLDL. Influence of 3A6 on formation of foam cells was observed by Oil Red O staining and the affinity of Na125I-conjugated oxLDL.3. 18 apoE gene knock-out mice at the age of 8 weeks were randomly divided into 3 groups:the control group (PBS was intraperitoneal injected 2ml once per-week); 5G8 group(5G8 was intraperitoneal injected 10μg/g weight,2ml per-week) and 3A6 group(3A6 was intraperitoneal injected 10μg/g weight,2ml per-week),then all of the animal were euthanized after fed by high fat food for 16 weeks. Blood was sampled from the eye.The serum lipids and blood plasma oxLDL was detected.Tissue was fixed by in situ liquid flow.Thoracic aorta harvested and fixed in 10% formalin,followed by paraffin imbedding,serial sections,and HE staining. The area of aortic AS plaque in every sectioned was analyzed.4. After LPS stimulation on macrophages, anti-TLR4 neutralizing mAb, p38MAPK specific inhibitor SB203580,NF-κB specific inhibitor PDTC or RNAi targeting Fcα/μreceptor were applied, respectively. The mRNA transcription and protein expression of Fcα/μreceptor in macrophages were studied by real-time RT-PCR and flow cytometry. Similarly, influence of 3A6 on formation of foam cells was observed by Oil Red O staining and the affinity of Na125I-conjugated oxLDL.Results:1. Resulting hybridomas producing anti-LDL and anti-oxLDL antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. As a result, two hybridoma cell lines, named 5G8 and 2H7 were developed, which could screte anti-LDL MAbs stably, and one hybridoma cell lines, named 3A6 was developed, which could screte anti-oxLDL MAbs stably. All of them belonged to IgM subclass, no cross reactions were found between these MAbs. The specificity of MAb was determined based on its activity in Western blot, Immunoblotting and ELISA analysis.2. Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to na?ve macrophages in vitro.3. After the apoE gene knock-out mice being fed with high fat diet for 16 weeks,there was no significant difference in serum lipids and blood plasma oxLDL among 3 groups, but obvious atherosclerotic lesion was found in their aortic root. The HE staining indicated that the thoracic aorta plaque area and correct area of the apoE-/-+3A6 group were significant reduced than the apoE-/-+PBS group and the apoE-/-+5G8 group.4. 3A6 failed to inhibit the binding of CuoxLDL to LPS-activated macrophages and promoted the formation of CuoxLDL-mediated foam macrophages. Furthermore, 3A6 F (ab′) 2 or pre-incubation with un-related IgM inhibited the binding of 3A6/CuoxLDL complex to LPS-activated macrophages, suggesting that the Fcα/μreceptor may be responsible for the binding of 3A6/CuoxLDL complex to LPS-activated macrophages. Indeed, LPS up-regulated the expression of Fcα/μreceptor in macrophages in a dose- and time-dependent manner, which was diminished by treatment with anti-TLR4 neutralizing mAb. In addition, LPS induced the phosphorylation of p38MAPK and translocation of NF-κB p65, contributing to the up-regulated expression of Fcα/μreceptor in macrophages as treatment with specific inhibitor for p38MAPK (SB203580) or NF-κB (PDTC) attenuated the up-regulation of Fcα/μreceptor expression induced by LPS in macrophages.Conclusions:1. The production of natural mouse IgM monoclonal antibodies (MAbs) anti-LDL and anti-oxLDL with high specificity and activity were generated by using standard hybridoma production techniques, which could provide a potential value for the research on lipid metabolisms and atherosclerosis progression.2. Natural anti-oxLDL IgM monoclonal antibody 3A6 specifically inhibited the binding of CuoxLDL to na?ve macrophages and foam cell formation in vitro.3. Natural anti-oxLDL antibody 3A6 has no effect on serum lipids and blood plasma oxLDL of apoE gene knock-out mice, but inhibits formation of the thoracic aorta atherosclerosisas plaque.4. LPS, through the TLR4 receptor, activated the p38MAPK and NF-κB pathways and up-regulated the expression of Fcα/μreceptor in macrophages, which promoted the binding of 3A6/CuoxLDL complex to macrophages through binding with Fc fragments and the formation of foam macrophages.5. Our findings provide a new explanation why increased LPS concentration deteriorates the pathogenesis of atherosclerosis.