In this issue of Blood, Klei and colleagues present novel data to support the hypothesis that adhering to extracellular membranes (ECM), and not red pulp macrophages (RPMs), is the first step in clearance of senescent red blood cells (RBCs).1 

RBCs have a fixed average lifespan within a given species (ie, 120 days in humans, 55 days in mice, etc). Although multiple cellular and biochemical changes that correlate with RBC age have been documented, the specific changes that lead to clearance of senescent RBCs from circulation remain unclear. This is not due to a lack of possibilities; indeed, there have been myriad hypotheses with supporting evidence.2-5  These include loss of sialic acid, decreased CD47, increased cellular rigidity, exposure of phosphatidylserine and/or other oxidized lipids, eryptosis, and the exposure of neoepitopes called “senescent antigen” (typically alterations in Band3) to which naturally occurring immunoglobulin G (IgG) autoantibodies bind, opsonizing the RBCs and promoting clearance by phagocytes.5  However, one problem, which is common to all experimental biology, is the issue that because something can occur, does not mean that it does occur in vivo under normal conditions. For example, despite the highly repeated observation that RBCs accumulate anti-Band3 IgG on their surface as they age, and that the bound IgG can promote phagocytosis of senescent RBCs,5  RBCs have a normal lifespan in agammaglobulinemic mice also lacking C3.6  Rejecting evidence such as this is often shrugged off by advocates of the hypothesis by claiming that redundant pathways exist, thus demonstrating only that there must be additional mechanisms.

Of course, redundant pathways certainly exist in biology. That said, the logical result of evoking redundancy also renders any hypothesis unrejectable (ie, unfalsifiable). Nevertheless, biology is certainly known to be complex. In addition to redundant pathways, multiple pathways (often in opposition) can coexist and result in outcomes that are based on a balance of opposing factors, and this must be taken into account in RBC clearance biology as well.4  However, when a field fails to make significant forward progress on narrowing down hypotheses after decades of work, it may not be due to inherent redundancy and complexity, but may be due to the correct hypothesis having not yet been posited.

Senescent RBCs are cleared largely by RPMs in the spleen. Reductionist analysis of this process has proven difficult, because RPM phagocytose senescent RBCs in vivo, but not in vitro,7  at least under the conditions tested thus far. However, while analyzing RPMs taken directly from human spleens, Klei et al observed that only 3% had visibly engulfed RBCs, far fewer than the predicted 30% based on RBC and RPM numbers. Moreover, consumed RBCs had the appearance of RBC ghosts. Using a mouse model in which both the cytoplasmic and the membrane components of RBCs were labeled with different dyes, they demonstrated that senescent RBCs become trapped in the extracellular space of the spleen where they lose their cytoplasmic content while their membranes remain intact.

Klei et al present a provocative new hypothesis that resolves the discordance between RPM behavior in vitro and in vivo. The reason RPMs do not phagocytose senescent RBCs in vitro is not because they have lost the capacity but because they never had it; RPMs do not phagocytose senescent RBCs in vivo either. Rather, senescent RBCs degrade into ghosts in the extracellular space of the red pulp, and then it is the ghosts that are consumed by RPMs. In their words, “the splenic architecture is required to drive hemolysis that allows for the recognition of the erythrocyte remnant by RPM.” Klei et al go on to demonstrate that senescent RBCs bind to laminin-α5 and progressively transition into ghosts over time under shear stress. Moreover, they show that laminin-α5 is expressed in the red pulp extracellular matrix. Indeed, applying 2-photon microscopy to human spleens, RBCs with the characteristics of senescent cells were observed associated with laminin-α5.

Perhaps the most profound implications of the work of Klei et al are that changes in surface properties of intact senescent RBCs are insufficient to cause consumption by RPMs, and an additional change occurs in the process of becoming a ghost. This raises the possibility that previous in vitro work studying mechanisms and properties of phagocytosis of senescent RBCs may not be relevant, because senescent RBCs are not directly phagocytosed. Such concerns may not affect studies using in vivo clearance from circulation as a readout but may change their interpretation around what leads to adherence to the ECM, leads to becoming a ghost, or leads to consumption once an RBC is a ghost. Of course, the ghost is derived from the membrane of the RBC, and changes taking place prior to becoming a ghost may be what affects ghost consumption as well; thus, in vitro studies may still be relevant, but also require reinterpretation.

Like most new hypotheses, in addition to providing some answers to old questions, this hypothesis leads to new questions. If the model of Klei et al is correct, then why does one observe increased RBC lifespan (including irregular morphology of old RBCs) when phagocytes are depleted from rodents8 ? Is this observation a problem for the theory, or does the inability to phagocytose ghosts simply backup the system, resulting in a saturated ECM to which additional senescent RBCs can no longer adhere? A second question is why the lifespan of healthy RBCs is not increased after splenectomy (ie, excluding therapeutic effects of treating RBC abnormalities or autoimmune hemolytic anemia); indeed, early rodent studies showed that RBCs in splenectomized mice are sequestered and cleared in the liver.9  However, the liver has neither fenestrated architecture nor RPMs. Is there some ECM equivalent in the liver and is laminan-α5 expressed there, and if so, where is it in the hepatic architecture?

In aggregate, Klei et al have made a fundamental advance in our understanding of the process of clearance of senescent RBCs, which will compel a reinterpretation of existing data, and promises to significantly advance the field going forward.

Conflict-of-interest disclosure: The author declares no competing financial interests.

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