Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. 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.

Differential interaction between DARC and SDF-1 on erythrocytes and their precursors.

The Duffy Antigen Receptor for Chemokines (DARC) is expressed on erythrocytes and on endothelium of postcapillary venules and splenic sinusoids. Absence of DARC on erythrocytes, but not on endothelium, is referred to as the Duffy negative phenotype and is associated with neutropenia. Here we provide evidence that stromal cell-derived factor 1 (SDF-1), the chemokine that restricts neutrophil precursors to the bone marrow, binds to erythrocyte progenitors in a DARC-dependent manner. Furthermore, we show that SDF-1 binding to DARC is dependent on the conformation of DARC, which gradually changes during erythroid development, resulting in the absence of SDF-1 binding to mature erythrocytes. However, SDF-1 binding to erythrocytes was found to be inducible by pre-treating erythrocytes with IL-8 or with antibodies recognizing specific epitopes on DARC. Taken together, these novel findings identify DARC on erythrocyte precursors as a receptor for SDF-1, which may be of interest in beginning to understand the development of neutropenia in situations where DARC expression is limited.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.