Hemolysis in the Spleen Drives Erythrocyte Turnover

Abstract

Erythrocytes circulate for an average of 120 days before they are removed from the circulation. Various processes and factors have been identified that may contribute to degradation of senescent erythrocytes, but this complex process is still not completely understood. Accumulation of removal signals such as phosphatidylserine exposure, changes in CD47 expression and oxidation of proteins and lipids that render them susceptible to complement deposition, may contribute to recognition and degradation by red pulp macrophages (RPM) of the spleen. However, many questions remain on the exact mechanisms that determine the fate of aged erythrocytes. This is well exemplified in a mouse study in which physiologically aged erythrocytes were found to undergo phagocytosis by RPM in vivo but not in vitro. This finding suggested that the splenic architecture may play an important role in facilitating erythrocyte turnover. Loss of membrane deformability may lead to the initial trapping of aged or damaged erythrocytes in the spleen, an event that precedes their degradation by macrophages. Loss of deformability can explain why certain genetic diseases that affect erythrocyte membrane deformability, such as is the case in sickle cell disease and spherocytosis, result in trapping in the spleen, giving rise to anaemia. Next to loss of deformability, activation of adhesion molecules, such as Lu/BCAM and CD44, specifically on aged erythrocytes has been proposed to contribute to retention of erythrocytes within the spleen, leading to their turnover. In this study we provide evidence that the splenic environment is of key importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPM we noted that only a small proportion of the macrophages were in the process of phagocytosing intact erythrocytes. Based on a range of variables, including the number of erythrocytes that are cleared daily, the number of RPM present in the spleen, the degradation rate of erythrocytes as well as differential contribution of spleen and liver to erythrocyte turnover, conservative estimates approximate that at least a 30-fold fewer erythrophagocytic events are observed in RPM than anticipated. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of a large population of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By in vivo imaging of the spleen and transfusion experiments we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis, thereby forming erythrocyte ghosts. Of note, we found that the levels of haptoglobin and hemopexin, two plasma proteins that are involved in scavenging of haemoglobin and heme, respectively, correlate well with the rate of hemolysis that was observed in the spleen. Additionally, we show that the erythrocyte adhesion molecules which are specifically activated on aged erythrocytes, Lu/BCAM and CD44, cause senescent erythrocytes to interact with the extracellular matrix of the spleen. This adhesion molecule-driven retention of senescent erythrocytes, under low shear conditions, was found to result in steady shrinkage of the erythrocytes and ultimately resulted in hemolysis and ghost formation. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghosts were found to be immediately recognized and rapidly degraded (1-3 hours) by RPM, thereby explaining the lack of phagocytosis of intact erythrocytes in the spleen. Together, these data identify hemolysis and ghost formation as key events in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated. Disclosures No relevant conflicts of interest to declare.

Macrophage‐fibroblast circuits in the spleen

“… 44,46,47 These macrophages control iron homeostasis via the recycling of iron contained in aged erythrocytes. RPM express high levels of F4/80, VCAM‐1, CD68, as well as scavenger receptors (CD163, CD91, signal regulatory protein alpha (SIRPα), CD36, complement receptor 3, Fc receptors), which allow them to clear free hemoglobin, free heme and senescent erythrocytes retained in the reticular network of the RP 4,5,48,49 . The mechanisms by which senescent erythrocytes are recognized and degraded by RPMs are still ill‐defined, since erythro‐phagocytic events are rarely observed in vivo in RPM 48,50 .…”

“…RPM express high levels of F4/80, VCAM‐1, CD68, as well as scavenger receptors (CD163, CD91, signal regulatory protein alpha (SIRPα), CD36, complement receptor 3, Fc receptors), which allow them to clear free hemoglobin, free heme and senescent erythrocytes retained in the reticular network of the RP 4,5,48,49 . The mechanisms by which senescent erythrocytes are recognized and degraded by RPMs are still ill‐defined, since erythro‐phagocytic events are rarely observed in vivo in RPM 48,50 . In vivo imaging and transfusion experiments in mice showed that senescent erythrocytes interact with the extracellular matrix of the splenic architecture and ultimately release their cytoplasmic content through rupture of their cell membrane, a process known as hemolysis 48 .…”

“…The mechanisms by which senescent erythrocytes are recognized and degraded by RPMs are still ill‐defined, since erythro‐phagocytic events are rarely observed in vivo in RPM 48,50 . In vivo imaging and transfusion experiments in mice showed that senescent erythrocytes interact with the extracellular matrix of the splenic architecture and ultimately release their cytoplasmic content through rupture of their cell membrane, a process known as hemolysis 48 . It is thought that RPM recognize and phagocytose the remnant erythrocyte “ghost shells,” now devoid of hemoglobin, along with scavenger‐associated heme and hemoglobin.…”

“…Transcriptomic comparison between splenic monocytes, pre‐RPM, and RPM from WT and IL1RL1‐deficient mice identified the IL33‐inducible transcription factor GATA2 as a master regulator of the pre‐RPM to RPM transition. The extracellular matrix of the RP exposes laminin‐α5, which facilitates trapping and hemolysis of senescent erythrocytes specifically expressing activated Lu/BCAM (Lutheran blood group and basal cell adhesion molecule) 48 . It is therefore tempting to speculate that imprinting of RPM by senescent erythrocytes is both direct, via the intake of IL33 and heme, as well as indirect, through stable positioning of RPM and erythrocyte “ghosts” within the cord.…”

TAM-ing the CIA—Tumor-Associated Macrophages and Their Potential Role in Unintended Side Effects of Therapeutics for Cancer-Induced Anemia

“…In brief, engulfed RBC are degraded and their heme-iron is recycled to the systemic circulation by FPN1-mediated iron export ( Figure 3 ). Alternatively, a novel study suggests that RBC may undergo physiologic hemolysis in the spleen and that their remnants are taken up by RPM for rapid turn-over ( 60 ). In situations when this capacity of RPM to take up damaged RBC (dRBC) and to recycle iron is overwhelmed, such as in massive hemolysis, Kupffer cells (KC) in the liver take over RBC uptake and degradation as a back-up mechanism.…”