Effects Of Aging On The Structure And Function Of Lipid Rafts In Mouse B Lymphocytes

Objective: Lipid rafts are cholesterol- and sphingolipid-rich microdomains within the lipid bilayer membrane. Lipid rafts are involved in signal transduction, receptor-mediated endocytosis and cholesterol metabolism and transport. Recent studies have found that lipid rafts are involved in the process of B lymphocyte maturation and activation. We compared lipid rafts expression in B lymphocytes of young and aged mice and studied the impact of aging and high-cholesterol diet on the structure and function of lipid rafts in B lymphocytes. We also established an efficient method to purify and culture murine splenic B-lymphocytes in vitro.Methods: Two to four-month old BALB/c mice were randomly divided into young mice group and young mice plus cholesterol group. Eighteen to twenty-two month-old BALB/c mice were randomly divided into aged group and aged plus cholesterol group. Cholesterol groups of young and aged mice were given high-cholesterol diet. Control groups were given normal diet. Serum cholesterol levels were measured by ELISA. Spleens of mice were minced into single cell suspension for flow cytometry to study the changes in B lymphocyte subsets and the expression of lipid rafts and Ca2 + influx level. We also used sheep red blood cells to immunize mice to study antibody response. Furthermore we established in vitro culture conditions for mouse splenic B lymphocytes. The spleen cells were treated with IL-4, CD3 monoclonal antibody(m Ab), LPS, IL-4 + CD3 m Ab, IL-4 + LPS or CD3 m Ab + LPS. Control cells were not treated. Flow cytometry was used to detect the changes in the number and proportion of subpopulations of T lymphocytes and B lymphocytes.Results: 1. Flow cytometry results: ⑴ The effects of aging on the B lymphocyte subsets: compared with young mice group, the number of total peripheral B cells in aged mice was unchanged; the proportions of transitional(TR) B cell subsets T1 and T2 were significantly reduced(P<0.01), and that of T3 subset was increased(P<0.01); The percentage of B1 cells and FO B cells did not change significantly(P>0.05), but that of MZ B cells was decreased(P<0.01). ⑵ The effects of aging on the expression of B cell membrane rafts: compared with the young mice group, cholesterolexpression in old mice T1, T2 subsets were significantly increased(P<0.0001), but was significantly reduced in T3 subsets(P<0.0001); cholesterol expression in B1 subsets was increased in aged mice(P<0.001), but no difference was observed in FO subsets(P>0.05); MZ subset from the aged mice has significantly decreased cholesterol levels(P<0.0001). All B220+ B cells expressed GM1. Compared with the young mice, GM1 expression in TR, B1, FO, MZ subsets from olde mice was significantly reduced(P<0.0001). ⑶ Effects of aging on the B cell membrane Ca2 + influx: compared with the young mice, B cells from the aged mice showed reduced Ca2 + influx. 2. ELISA results: ⑴ aging on serum cholesterol levels: compared with the young mice, serum cholesterol levels in the aged mice and the young mice fed with high-cholesterol diet were significantly increased(P<0.05); There were no significant difference in the aged control mice and the aged mice fed with high cholesterol diet(P>0.05). Similarly, there was no significant difference in the aged mice and young mice fed with high-cholesterol diet(P>0.05). 3. The results of culturing splenocytes in vitro: the number of lymphocytes peaked after 3-5 days after addition of IL-4. In the LPS group, the number of lymphocytes began to increase after 3 days, and then peaked after 5 days. T lymphocytes disappeared after addition of CD3 monoclonal antibody(m Ab), so relatively pure B lymphocytes could be obtained after 2 days and the number of B lymphocytes reached peak levels after 3 days. The number of mature B lymphocytes(B220+Ig D+) increased significantly after addition of CD3 m Ab. In all the conditions we tested, TR B cell subset(B220+CD93+) disappeared completely after 24 hours of culture.Conclusions: The structure of lipid rafts on B cell surface changes in aged mice, resulting in lipid rafts signal transduction function limiting. We also showed that CD3 monoclonal antibody and IL-4 can be used to remove T lymphocytes from spleen cell culture and obtain mature B lymphocytes.