Hemolysis in the spleen drives erythrocyte turnover
Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. A study by Gottlieb and colleagues in 2012 found that although RPM phagocytose artifically aged erythrocytes in vivo, co-incubation of isolated RPM and the same artifically aged erythrocytes in vitro did not result in phagocytosis. As such, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear.
To study in vitro erythrophagocytosis we isolated human spleen macrophages. Erythrocyte populations isolated from human spleen tissue were characterized by flow cytometry. In vivo mouse transfusion experiments were performed to assess the process of degradation. Flow experiments were performed in order to mimick in vivo sequestration.
Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule–driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs.
These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.