Phagocytosis Related Molecules Expressed On B-1 B Cells

Characteristics and functions of B-1 B cells have attracted a lot attention during the last decade. It is clear now that B-1 cells are unique B cell subset. In contrast to classical B-2 cells, B-1 B cells are B220lowIgMhiIgDlowCD23-CD43+Mac-1+ and maily located in peritoneal cavity. B-1 cell play important roles in innate immunity. Before adaptive immunity is established, B-1 cell proliferation, differentiation, and antibody secretion can be accomplished within a few hours after encountering antigens , and this activation dosen't need somatic hypermutation. B-1 cells can also participate in the mucosal immunity through IgA secretion . In addition, B-1 cells are critical in autoimmunity, and participate in ischemia-reperfusion, atherosclerosis, autoimmune hemolytic anemia.During the past years, research on B-1 cell function in innate immunity mainly focused on their role as antibody producing cells and antigen presenting cells. However, B-1 cells have some unique properties such as induced proliferation when treated with phorbol esters, expressing CD11b, F4/80 and some other molecules related to phagocytosis, indicating that B-1 cells might have more functions in innate immunity. Recently, Sunyer's group demonstrated that B cells from teleost fish have potent in vitro and in vivo phagocytic activities. It revealed for the first time that primitive vertebrates B cells have active phagocytic capacities.In our previous studies on the role of natural antibodies against bacteria infection, we injected CFSE (Carboxy Fluoroscein Succinimidyl Ester, a kind of green fluorescent dye) -labeled S. Aureus into PerC of C57BL/6 mice and found that a small population of CD19 positive cells also showed CFSE staining. There are two possible explanations, one is that the SA adhere to B cells surface, the other is that B cell engulfed SA, or both. To make clear whether this staining was caused by intracellular ingestion, we further investgate this phenomenon by confocal microscopy and transmission electron microscopy. Results of confocal microscopy showed that there were green particles in the cytoplasma of CD19+ cells. Results of transmission electron microscopy also showed round bacterial were seen in the cytoplasm of B cells. These data suggested that mouse PerC B cells had phagocytic abilities to S. Aureus.The next issue to be explored is the possible mechanism involved in this process, for example, which receptor is involved in the phagocytosis. From the view of cell surface molecles related to phagocytosis, our study investgate the molecules may involved in B cell phagosytosis and do some fundermental research on mechanism of B cell phagocytosis.1. Research on phagocytosis related cell surface molecule expressing on mice PerC B-1 cellsPeritoneal cells and spleen cells obtained from normal C57BL/6 mice was isolated and purified with anti-IgM magnetic beads. Total RNA of the cells were extracted and semiquantitative RT-PCR was used to detect the expression level of cell surface molecules. Flow cytometry(FCM) were used to detect CD14 and CD36 expression on B1 and B2 cell.RT-PCR result showed that CD206,CD14,TLR-2,TLR-4,CD36,MARCO,CD32,CD11b,CD18,ABCA1 expressed on normal mice PerC B-1 cells, but their expression level is lower compared with phagocyte. Between these molecules, CD14,CD36,MARCO,CD11b expression level is higher on B-1 cell compared with that on B-2 cell.FCM results showed that low level of CD14 and CD36 expressed on B-1 and B-2 cell and it is consistent with the RT-PCR results. On the basis of these results, we use SA and LPS as model antigen to further analyze the change of these molecules'expression level after antigen stimulation.2. Expression level of phagocytosis related cell surface molecules after SA stimulationSA was injected into mice peritoneal cavity, and then peritoneal cells and spleen cells were isolated at different time points and purified by magnetic activated cell sorting. Semiquantitative RT-PCR was used to detect the expression level of cell surface molecule. RT-PCR results showed that CD14 and CD206 expression was upregulated after SA stimulation, and the expression level is higher on B-1 cell compared with B-2 cell.3. Expression level of phagocytosis related cell surface molecules after LPS stimulationLPS was injected into mice peritoneal cavity, and then peritoneal cells and spleen cells were isolated and purified at different time points by magnetic activated cell sorting. Semiquantitative RT-PCR was used to detect the expression level of cell surface molecule. RT-PCR results showed that CD14 and CD11b expression was upregulated after LPS stimulation, and the expression level is higher on B-1 cell compared with B-2 cell. 4. Effects of CD14 and CD11b blockage on PerC B cell phagocytosisAccording to the mRNA results of previous study, we choose CD14 and CD11b to see the effects of blocking,. Mice PerC cells were obtained as described before, and after 2 hours static culture the cell culture supernatant was collected. CD14 and (or) CD11b blocking antibody were added to the cells, fllowed by adding Co60 inactivated SA. After 2 hours incubation, ratio of phagocytic potencial B cells was detected by FCM. It showed no statistical difference between the blocking group and negative control group.In conclusion, our results revealed for the first time that CD206, CD14, CD36, MARCO, CD32, CD18 and ABCA1 all expressed on B-1 B cells. So many phagocytosis related molecules expressed on B-1 B cells indicated that B-1 B cells might have phagocytic capacitie. Among these molecules, espression level of CD14, CD36, MARCO and CD11b is higher on B-1 cell compared with B-2 cell. This result indicated that these molecules may involved in some unique function of B-1 cells. Further study suggested that CD206 and MARCO involved in B-1 cell respond to S. Aureus and CD11b, MARCO, CD14 involved in B-1 cell respond to LPS.