The Role Of Splenic Macrophage In Apoptotic Cell-Induced Immune Response

ObjectiveTo investigate the interaction between macrophages and dendritic cells after phago-cytosis of apoptotic cells. To study the contribution of splenic macrophage to apoptoticcell-triggered immune response and provide the basis for deeply understand the role of cellapoptosis in immune homeostasis and immune regulation.Methods1. Induce splenic immune cells become apoptotic cells with40Gy X-ray irradiation,examine apoptosis rate by Annexin-V-FITC and PI double staining.2. CFSE-labeled apoptotic cells were transferred(i.v.)into mice.0.5h,1h and2h later,flow cytometry was used to detect the phagocytosis of apoptotic cells by immune cell sub-sets in the spleen.3. Laser confocal microscopy was used to observe the phagocytosis of apoptotic cellsby macrophages and dendritic cells in frozen slide of spleen in mice.4. Using Clodronate liposomes (CLs) to effectively deplete mouse splenic macro-phages, Flow cytometry was used to detect the depletion rate of immune cells in spleen andperipheral blood at day1and day5after injection of CLs.5. CD4+T cells and apoptotic cells were transferred (i.v.)into the mice which had beendepleted splenic macrophages. Flow cytometry was used to study the role of macrophagesin apoptotic cell-induced antigen-specific CD4+T cell-mediated immune response.Results1. We observed cell apoptosis induced by40Gy dose of X-ray irradiation. Apoptoticcells were induced at37℃and5%CO2saturated humidity incubator, testing the apoptosisrate after incubating2hours.The results showed that early apoptosis rate was45.7%, lateapoptosis rate was12.4%.2. We found that the ability of phagocytosis of apoptotic cells by splenic immune cells in the mice. There displayed the highest efficiency (Immune cells engulfed apoptotic cellsaccounted for0.14%of all immune cells in the spleen) in the spleen after1h of apoptoticcells injection. We found that macrophages were the major cells to engulf apoptotic cells.Dendritic cells were also able to phagocytose apoptotic cells, and lymphoid dendritic cellswere3times as efficient as myloid dendritic cells.3. After1day of CLs injection, compared with no CLs group, monocytes were themajor depleted cells in the blood (p <0.001), and most F4/80+macrophages were removedfrom the spleen (p <0.001). Additionally, lymphoid dendritic cells were also severely de-pleted from spleen （p<0.001）. Five days later, the immune cells except forF4/80+macrophages had returned to the normal levels. This data showed that CLs can ef-fectively remove the spleen macrophages in mice.4. After removal of splenic macrophages, dendritic cells still had the ability to presentapoptosis-associated antigen. Apoptotic cell-induced antigen-specific CD4+T cell responsewere reduced about50%in mice that were depleted macrophage when compared with thatwith intact macrophage(p <0.001). These data suggested the involvement of macrophagesin antigen presentation.Conclution1. CLs can effectively remove the splenic macrophages in the mice. There was sig-nificant difference in phagocytosis of apoptotic cells between macrophages and dendriticcells at day5after CLs treatment.2. Macrophage was involved in apoptotic cell-induced immune response.3. These results help to deeply understand the role of macrophages in apoptoticcell-induced immune response and provide a theoretical basis for further study of the un-derlying mechanism.