Fate of erythrocytes after splenectomy

Fate of erythrocytes after splenectomy

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The spleen is considered a graveyard for red blood cells. So in case of Splenectomy (complete surgical removal of the spleen), what would be the fate of red blood cells? Would this cause Polycythemia?

The spleen is not the only organ which removes "old" erythrocytes, this happens as well in the liver and the lymph nodes. The whole process is termed Eryptosis, the Apoptosis of Erythrocytes. During the aging of erythrocytes sialic acid on their outer membrane surface is removed. This leads to the recognition by macrophages and phagocytosis of this cells by macrophages which are located in liver and spleen. For more information see this article: "Physiology and Pathophysiology of Eryptosis"

Polycythemia is the state when you have an elevated number of red blood cells, characterized by an increased Hematokrit level. This is caused by the over-production of red blood cells, which can be caused by an overproduction of hematopietic cells in the bone marrow (so called myeloproliferative syndrome, which are basically cancers), the exposure to permanently low oxygen-levels (this is what athletes exploit when the do training at high altitudes) or malignancies (usually lymphoma). It is also possible due to the mis-use of Erythropoietin (EPO) which induces the production of red blood cells. Another possibility for having too much red blood cells is the over-transfusion during a blood transfusion. For further information have a look into the Wikipedia. The article there is pretty complete and contains a number of references.

So in case of Splenectomy (complete surgical removal of the spleen), what would be the fate of red blood cells? Would this cause Polycythemia?

According to wikipedia none of the side effects are related to red blood cell count (just the quality of those cells).

As splenectomy causes an increased risk of sepsis due to encapsulated organisms (such as S. pneumoniae and Haemophilus influenzae) the patient should receive the pneumococcal conjugate vaccine (Prevnar), Hib vaccine, and the meningococcal vaccine; see asplenia. These bacteria often cause a sore throat under normal circumstances but after splenectomy, when infecting bacteria cannot be adequately opsonized, the infection becomes more severe.

An increase in blood leukocytes can occur following a splenectomy.2 The post-splenectomy platelet count may rise to abnormally high levels (thrombocytosis), leading to an increased risk of potentially fatal clot formation. There also is some conjecture that post-splenectomy patients may be at elevated risk of subsequently developing diabetes.4 Splenectomy may also lead to chronic neutrophilia. Splenectomy patients typically have Howell-Jolly bodies5 and less commonly Heinz bodies in their blood smears.7 Heinz bodies are usually found in cases of G6PD (Glucose-6-Phosphate Dehydrogenase) and chronic liver disease.8

Splenectomy can be recommended by inherited blood diseases (e.g. hereditary spherocytosis, thalassemia, etc… ) which cause anaemia due to the destruction of abnormal red blood cells in the spleen. After surgery the RBC count is around normal in these cases, so removing the spleen does not cause polycythemia. If you check wikipedia - polycythemia, splenectomy or other spleen related diseases do not cause polycythemia, while polycythemia can cause enlarged spleen which may be removed.

Erythropoiesis and so red blood cell count is regulated by EPO which is regulated by blood oxygen level, so spleen does not regulate the production of red blood cells directly. Removing the spleen cannot cause upregulation of EPO and so polycythemia. Mean erythrocyte age is increased, so in long term there can be a slight increase of RBC count because of a function loss by old cells, but that is not significant enough to cause disease.

  • 2010 - Partial splenectomy for hereditary spherocytosis: a multi-institutional review
  • 2014 - The spleen and sickle cell disease: the sick(led) spleen
  • 2013 - Red blood cell vesiculation in hereditary hemolytic anemia
  • 2007 - Erythropoietin after a century of research: younger than ever
  • 1982 - Glycosylated hemoglobins (GHb): an index of red cell survival.

To support the speculation that macrophages might also have a function in erythropoiesis in the context of disease and to further characterize their importance in erythropoiesis in vivo, Ramos and colleagues show that macrophages regulate erythroid development in polycythemia vera, β-thalassemia and anemia (Ramos et al., 2013). Chemical depletion of macrophages by clodronate liposome administration prevents mice from recovering from induced anemia, suggesting an essential function of macrophages in promoting stress erythropoiesis in vivo. Conversely, macrophage depletion not only improves the phenotype of polycythemia vera and reverses the pathological aspects of the disease, but also alleviates anemia caused by β-thalassemia. These results propose an important dual role of macrophages in physiological and pathological erythropoiesis in vivo. Both studies suggest that macrophages exert two seemingly contradictory actions on erythropoiesis. On one hand, macrophages are indispensable for stress erythropoiesis in vivo. In their absence erythroid production in the bone marrow and spleen in response to bleeding is impaired. However, macrophages can also be deleterious in the context of polycythemia vera and β-thalassemia, since depletion of macrophages leads to a decreased disease pathology. Moreover, ex vivo cultured human macrophages from polycythemia vera patients promote proliferation of human erythroblasts and diminish differentiation. This suggests a function for macrophages in disease progression since polycythemia vera is characterized by an overactive erythron and excessive erythropoiesis (Ramos et al., 2013). These findings might pave the way to future therapies implementing macrophage depletion in the treatment of erythroid disorders like polycythemia vera and β-thalassemia.

  • 2014 - Review article of macrophages and red blood cells; a complex love story

Recovery after surgery is about 4-6 weeks. Liver and lymph nodes can take over the functions of the spleen partially. Every blood cell count may be elevated (esp. platelet count). The functions of the spleen are:

  • storing iron (in the form of ferritin or bilirubin, to protect it from pathogens)
  • storing blood (for the case of blood loss e.g. injury)
  • filtering out damaged or old blood cells (by inherited diseases abnormal red blood cells are removed from blood as well)
  • filtering out phatogens, infected blood cells, etc… (to protect the body from sepsis)

And ofc. a lot of immune cells live there. Therefore in rare cases sepsis can occur after splenectomy immediately, and can cause life threatening conditions.

  • 2001 - Infectious complications in asplenic hosts
  • 1996 - Overwhelming postsplenectomy infection
  • 1975 - Immunological studies in the postsplenectomy syndrome
  • 1995 - Preservation of the spleen improves survival after radical surgery for gastric cancer.
  • 2009 - Effect of splenectomy on antitumor immune system in mice.
  • 2012 - Thrombocytosis in asplenia syndrome with congenital heart disease: a previously unrecognized risk factor for thromboembolism.
  • 2009 - Vascular complications after splenectomy for hematologic disorders
  • 2014 - Review article of macrophages and red blood cells; a complex love story

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere.

  • 2012 - SIRPα/CD172a and FHOD1 Are Unique Markers of Littoral Cells, a Recently Evolved Major Cell Population of Red Pulp of Human Spleen

  • 2011 - Splenectomy Associated Changes in IgM Memory B Cells in an Adult Spleen Registry Cohort

The spleen contains immune cells, from which macrophages phagocytose the senescent red blood cells. They degrade the hemoglobin into amino acids, bilirubin and iron. The iron is sent to the blood with transferrin, which can be captured and stored by the spleen or the liver or can be used to build new red blood cells in the red bone marrow. The amino acids can be used to build new proteins or they can be degraded by the liver or the kidney. The bilirubin is transported to the liver where it is conjugated and excreted into the bile. The other parts of the red blood cells are recycled as well. For the macrophages it is easier to do this kind of work if you have a dedicated organ which can help to filter out the cells for destruction, but it is not impossible without it, because macrophages live in other organs/tissues as well.

Red blood cells can get rid of damaged parts by creating microvesicles so they can elongate their lives. Vesiculation is facilitated by the spleen. These microvesicles can contain hemoglobin as well, and they are captured by the liver.

  • 2004 - The red cell revisited--matters of life and death.
  • 2008 - Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy
  • 2003 - Hemoglobin loss from erythrocytes in vivo results from spleen-facilitated vesiculation
  • RBC Storage
  • 2008 - RBC-derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components.

The macrophages of the spleen have a remarkable function that enables them to remove unwanted damage from the RBC membrane, leaving the RBC intact (Crosby, 1957; Schnitzer et al., 1972). Removal of these intracellular inclusions seems to occur within the open circulation where the RBC are also checked for their loss of deformability to check for age. To achieve this, RBC must pass through the endothelial slits of the sinus to reenter the blood circulation. During this course, cells that are non-deformable will be removed from the circulation by residential macrophages. In the mean-time all inclusion bodies are also being removed. In splenectomized patients or in patients with a non-functional spleen, phagocytosis of the inclusion bodies fails and results in a retention of a variety of intracellular inclusions within the RBC, such as Howell-jolly bodies (inclusions of nuclear chromatin remnants) (Wilkins and Wright, 2000), Heinz bodies (inclusions of denatured hemoglobin caused by oxidative damage) (Wilkins and Wright, 2000) siderocytes (RBC containing granules of iron that are not part of the cell's hemoglobin) (Wilkins and Wright, 2000) and Pappenheimer bodies inclusion bodies formed by phagosomes that have been engulfing excessive amounts of iron (Wilkins and Wright, 2000).

The molecular mechanism that underlies the removal of inclusion bodies is largely unknown. In Willekens et al. (2003) presented an analogy to the removal of Heinz bodies when discussing RBC that lose hemoglobin through vesiculation. Via the process of RBC vesiculation the RBC loses aggregated hemoglobin, which is important to maintain the homeostasis of RBC, increases in density and becomes smaller (Piomelli and Seaman, 1993). It was suggested that this process is also facilitated by the macrophages of the spleen, in which older cells vesiculate more than younger ones. Clearly, macrophages play a pivotal role in the clearance of damaged content from circulating RBC (Crosby, 1957; Willekens et al., 2003) and vesiculation is an interesting and plausible mechanism to explain the efficient removal of damaged content while leaving the RBC intact (Wilson et al., 1987). The molecular mechanism by which macrophages in the spleen would be facilitating RBC vesiculation is still unknown.

  • 2014 - Review article of macrophages and red blood cells; a complex love story

Aged or abnormal red blood cells with exposed phosphatidylserine (PS-RBCs) are cleared from the circulation by splenic macrophages. In asplenic patients, other mononuclear phagocytic cells in tissues and in circulation may function in this capacity.

  • 2010 - Activation of mononuclear phagocytes and its relationship to asplenia and phosphatidylserine exposing red blood cells in hemoglobin E/β-thalassemia patients

Suicidal death of erythrocytes (eryptosis) is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. Exposed phosphatidylserine is recognized by macrophages that engulf and degrade the affected cells. Eryptosis is triggered by erythrocyte injury after several stressors, including oxidative stress.

  • 2006 - Mechanisms and significance of eryptosis.

CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor signal regulatory protein alpha (SIRPα) expressed by macrophages. Thus, the CD47-SIRPα interaction constitutes a negative signal for erythrocyte phagocytosis. However, we recently reported that CD47 does not only function as a 'don't eat me' signal for uptake but can also act as an 'eat me' signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47- SIRPα interactions.

  • 2013 - CD47 functions as a removal marker on aged erythrocytes
  • 2013 - Mechanisms tagging senescent red blood cells for clearance in healthy humans
  • 2011 - Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro
  • 2011 - Human red blood cell aging: correlative changes in surface charge and cell properties
  • 2012 - Naturally Occurring Autoantibodies in Mediating Clearance of Senescent Red Blood Cells

21.1: Sickle Cell Anemia

Genes are translated into proteins mutations often (but not always) result in changes in the sequence of amino acids in those proteins. Changes in the amino acid sequence can modify (in various ways) or even completely destroy protein function. Proteins have many functions within cells, and a change in those functions results in a change in the phenotype of that cell or organism. So a mutation as simple as a single base change in a DNA sequence can have dramatic effects on phenotype. One of the best examples of this phenomenon can be observed when mutations occur in the gene for one of the protein components of the red blood cell protein we call hemoglobin.

A major component of the erythrocytes (red blood cells) found in ver­tebrates is hemoglobin. A molecule of hemoglobin from a normal adult human contains four proteins (two identical alpha polypeptides and two identical beta polypeptides) surrounding a core of heme (complex molecule containing an atom of iron which can combine reversibly with oxygen). Thus, hemoglobin functions as the major oxygen-carrying constituent of blood. Because of hemoglobin, a given volume of blood can carry far more oxygen than could be dissolved in an equal volume of water.

In many human populations, particularly those with origins in Central Africa or the Mediterranean, there are individuals who suffer from severe anemia and whose blood contains numerous distorted, sickle-shaped erythrocytes. Hence, the disease was given the name sickle cell anemia.

Figure 1. Notice the sickle shaped cells in the image by Dr Graham Beards via Wikimedia Commons

Biochemical studies established that the gene affected in sickle-cell ane­mia has the code for an abnormal beta polypeptide, which is one of the components of the hemoglobin molecule. Therefore, there are two different forms of the hemoglobin gene that codes for the beta chain:

  • Form 1: HbA has the code for a normal beta chain
  • Form 2: HbS has the code for an abnormal beta chain

Humans are diploid organisms they have two copies of most genes. However, the two copies they possess do not have to be identical. When there are two possible alleles for a gene (such as in the gene for the beta chain of hemoglobin), a diploid individual will have one of three possible combinations of the two alleles. They can be Hb A Hb A , Hb A Hb S , or Hb S Hb S .

The set of alleles present in an individual for a given gene is known as the individual&rsquos genotype. The three combinations of two alleles above are therefore the three different genotypes. Individuals that have two copies of the same allele are called homozygous individuals with two different alleles are called heterozygous. So an individual that is Hb A Hb A is homozygous normal beta chain an individual that is Hb A Hb S is heterozygous and an individual that is Hb S Hb S is homozygous abnormal beta chain. It is the homozygous Hb S individuals that contain sickle-shaped blood cells.

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1. The erythrocytes of splenectomized dogs show increased resistance to the action of hypotonic salt solutions and to specific hemolytic immune serum. The degree of resistance appears to increase with the length of time that has elapsed after splenectomy.

2. This increased resistance of the erythrocytes is not due to an increased antihemolytic power of the animal's serum or to a diminished complementary value of the serum, but is a property depending upon the erythrocytes themselves.

3. Non-splenectomized animals receiving a single injection of specific hemolytic immune serum and developing a temporary anemia show likewise on recovery an increased resistance of the corpuscles without the presence of antihemolysin in demonstrable amount.

4. As anemia of varying grade is a characteristic result of splenectomy, it would appear that the increased resistance of the corpuscles is a concomitant of the regeneration of the red cells following such anemia and is thus analogous to the increased resistance of such cells not infrequently observed in various forms of experimental anemia.

5. There is no evidence to indicate that the anemia after splenectomy is due to the presence of hemolytic bodies, or that the increased resistance of the cells is due to antihemolytic bodies, accumulating in the serum as the result of the ablation of the spleen. It is evident therefore that the spleen in some way controls or regulates blood destruction (and regeneration ?), and in the hope of throwing light on the subject, an investigation of the bone marrow and lymph nodes of splenectomized dogs is now under way.


We thank A. J. Mirando, S. Ide, E. Hocke and K. Abramson for assistance and discussions staff at Duke Molecular Physiology Institute Molecular Genomics Core for generation and analysis of 10x Genomics scRNA-seq libraries T. Kimura for reading the manuscript and discussion G. S. Baht for the instruction of the parabiosis surgery. This research was supported by a grant from the National Institute on Aging (NIA) of the National Institutes of Health (NIH) R01 AG049745 and NIH R01 AI088100. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.


IES Model and Two-Component RBC Model.

To capture the geometry of IES in the human spleen, we construct a model that comprises four solid elements, as illustrated in Fig. 1. The two vertical bars represent annular fibers with a width of 1 μm, whereas the two horizontal bars represent endothelial cells. The thickness of the slit wall is 1.89 μm, and the width and height of the slit are 4 μm and 1.2 μm, respectively, based on the experimentally documented slit geometry (19).

Simulating RBC passage through IES. The membrane of the RBC is explicitly represented by CG particles. A: actin junctions B: spectrin particles C: glycophorin particles D: band-3 particles E: lipid particles. The width and height of the simulated slit are 4 μm and 1.2 μm, respectively. The width of the vertical bars is 1 μm, and the thickness of the slit wall is 1.89 μm.

We use the computer code OpenRBC (22) to model healthy and diseased RBCs. As shown in Fig. 1, the lipid bilayer and the cytoskeleton as well as transmembrane proteins are explicitly represented in the RBC model. This allows simulations of RBC vesiculation and of cell morphological changes induced by protein defects in blood disorders. Further details of the RBC model can be found in SI Appendix.

Pressure Gradients and S/V Ratio Determine the Passage of RBCs through IES.

Over the life span of about 120 d, the cytoskeleton of the RBC stiffens and its membrane components undergo degradation, resulting in loss of surface area through release of vesicles (23 ⇓ –25). Senescent RBCs with reduced surface area become less deformable and thus are amenable to sequestration and removal in the spleen. In this section, we simulate healthy RBCs with reduced surface area traversing the splenic IES. Pressure gradients of 3, 5, 8, 10, 15, and 20 Paμm −1 are applied to drive the RBC through IES. These pressure gradients fall within the range found in in vitro experiments (17)—that is, 1 to 30 Paμm −1 . Such values were sufficient to reproduce the physiological dynamics of RBCs transiting through IES in rat spleen (14). First, we model an RBC with a surface area of 140 μm 2 and volume of 90 μm 3 . Our simulation results show that when driven by a pressure gradient of 3 Paμm −1 , the RBC is retained by IES (Fig. 2 A and B). When the pressure gradients are equal to or larger than 5 Paμm −1 , RBCs are able to pass through IES. This critical pressure gradient is on the same order of magnitude as the pressure gradient of ∼1 Paμm −1 , which was sufficient to drive RBCs of all sizes found in blood through the slits in microsphere experiments and ex vivo perfusion of human spleen (5, 15). The origins of the difference between the critical pressure gradient inferred from this work and the values reported from previous studies are discussed in detail in SI Appendix.

(A and B) An RBC with surface area of 140 μm 2 and volume of 90 μm 3 is retained by IES under a pressure gradient of 3 Paμm −1 because of an insufficient driving force. (C and D) An RBC with surface area of 110 μm 2 and volume of 90 μm 3 is retained by IES under a pressure gradient of 8 Paμm −1 due to reduced surface area. (E–H) Four sequential snapshots of the RBC with surface area of 140 μm 2 and volume of 90 μm 3 passage through IES under a pressure gradient of 20 Paμm −1 (see Movie S1). Only one half of the RBC is displayed for clarity.

Fig. 2 EH illustrate a sequence of shape changes of the RBC (surface area of 140 μm 2 and volume of 90 μm 3 ) during its deformation through IES. When the RBC moves into the slit (Fig. 2F), the portion inside the slit is being squeezed, whereas the rest of the RBC membrane is expanded to accommodate the excluded volume, thus forming a dumbbell shape. As the RBC moves toward the downstream side of IES, the downstream bulge expands, while the upstream bulge shrinks. Subsequently, the cell membrane in the slit infolds toward the cell body and creates a concave region, as shown in Fig. 2G. After crossing the IES, the deformed RBC gradually spreads out the inward-folded membrane, turning to a bullet shape (Fig. 2H). The dynamics of the RBC model passage through IES are consistent with the in vivo microscopic observations of the transilluminated rat spleen (14) and the in vitro microfluidic study of human RBCs passage through IES-like slits (17). When driven by increased pressure gradients, the RBC dynamics are similar except that the higher pressure gradients lead to faster RBC traversal.

Next we show results for cases where the pressure gradient is 5 Paμm −1 and the surface area of RBCs is reduced from 140 μm 2 to 100 μm 2 in decrements of 10 μm 2 . For the surface areas of 130 μm 2 and 120 μm 2 , RBCs are still able to pass through IES. However, in the case of the surface area of 110 μm 2 , representing about 21% surface area loss, the simulated RBC is retained by IES (Fig. 2 C and D). This result is consistent with prior ex vivo experimental observations that RBCs with more than 18% surface area loss were mostly entrapped in the spleen (5). This finding is also validated by an analytical model (19, 26) that defines the relationship between the critical surface area and volume for healthy RBCs, beyond which traversal through IES is predicated to be compromised. Given the geometry of the slit, the minimum surface area (A), below which the RBCs with fixed volume (V) are retained by the slit, is given by (19, 26) A = 4 < 2 π [ f − 1 ( V ) ] 2 − π f − 1 ( V ) g 1 ( V ) >+ 2 π L s g 2 ( V ) < D s 2 + L s 2 − L s ⁡ sin [ g 2 ( V ) ] 2 g 2 ( V ) >, [1] where V = 2 [ 4 3 π R 3 − 1 3 π h 2 ( 3 R − h ) ] + 2 π A c y c . All other symbols are defined in SI Appendix. This model predicts a critical surface area of 113.1 μm 2 for the RBC volume of 90 μm 3 , which validates the critical area obtained from our simulations. Then, we increase the pressure gradients to 8, 10, 15, and 20 Paμm −1 , respectively, and we find that RBCs with surface areas of 110 μm 2 and 100 μm 2 still cannot pass through IES when driven by these increased pressure gradients.

Traversing IES Causes Intrasplenic Vesiculation of RBCs in HS.

In HS, defects occur in the RBC membrane proteins, such as ankyrin, protein 4.2, band-3, and spectrin (8, 10). These protein deficiencies alter the RBC membrane in two distinct ways (8). In RBCs with deficiencies in the band-3 or protein 4.2, the vertical connections between the spectrin filaments and the lipid bilayer are diminished, whereas the spectrin content is normal (Fig. 3B). On the other hand, spectrin-deficient or ankyrin-deficient RBCs are characterized by depleted spectrin content and reduced numbers of actin junction complexes, while the overall structure of the membrane cytoskeleton is preserved (Fig. 3C). These alterations in the RBC membrane weaken the vertical linkages between the cytoskeleton and the lipid bilayer, causing surface area loss through release of vesicles (8, 10). HS RBCs with reduced surface area transform progressively from a biconcave shape to a near-spherical shape. The concomitant decrease in cell deformability leads to RBC retention and premature removal by the spleen (5, 27). Although the genetic basis and clinical consequences of HS are known, the mechanics of membrane loss has hitherto not been explored in detail. In this section, we simulate band-3–deficient and spectrin-deficient RBCs traveling through IES. Varying degrees of protein deficiency are examined for these two types of HS RBCs. The deformability of the HS RBCs following reduction in surface area is evaluated by stretching the RBCs, similar to the manner in which RBC deformation was induced in optical tweezers experiments (28, 29). A deformability index (DI) is computed based on the deformation of the stretched RBC. Details of the calculations of DI are given in SI Appendix.

(A) Cytoskeleton in a healthy RBC model. (B) In the band-3–deficient HS RBC model, the band-3 sites are randomly removed (highlighted by red dotted circles) to represent the effect of band-3 deficiency. (C) In the spectrin-deficient HS RBC model, the density of spectrin network is reduced to represent the effect of spectrin deficiency. (D–I) Six sequential snapshots (top views) of an HS RBC with a vertical connectivity of 60% passage through IES under a pressure gradient of 10 Paμm −1 (see Movie S2). Reduced vertical connectivity leads to the detachment of the lipid bilayer from the cytoskeleton and subsequent RBC vesiculation. The lipid CG particles (red particles) are plotted at a smaller size to visualize the cytoskeleton below (green particles).

We first simulate the band-3–deficient RBCs by reducing the connectivity between the band-3 proteins and spectrin filaments (vertical connectivity) in the RBC model. The vertical connectivity is reduced from 100% to 0% in decrements of 20%, representing elevated degrees of band-3 deficiency. The surface area and volume of the simulated RBCs are 140 μm 2 and 90 μm 3 , respectively. Fig. 3 DI show a sequence of shape changes of an HS RBC with a vertical connectivity of 60% as it passes through an IES (top views). Due to weakened cohesion between the lipid bilayer and the cytoskeleton, the lipid bilayer detaches from the cytoskeleton when the RBC traverses IES (Fig. 3F). One detachment separates from the RBC and forms a vesicle, whereas the other develops into a tubular vesicle and passes through IES behind the RBC (Fig. 3 G and H). Eventually, this tubular vesicle detaches from the RBC and reshapes into a separate spherical vesicle (Fig. 3I). Detailed discussion of this vesiculation process can be found in SI Appendix. These simulations confirm that reduced vertical connectivity compromises the cohesion between the cytoskeleton and the lipid bilayer and that surface area loss of RBCs in HS occurs when they traverse the IES, thereby clearly establishing the connection between the spleen and shape alterations in diseased RBCs.

We also examine RBCs with various vertical connectivities. At each vertical connectivity, pressure gradients of 5, 8, 10, 15, and 20 Paμm −1 are applied. The blue curve in Fig. 4A shows that RBCs shed more surface area as the vertical connectivity is reduced. In other words, the increased degree of band-3 deficiency exacerbates membrane loss of the HS RBCs. Notably, the fraction of surface area loss from the HS RBCs with a vertical connectivity of 0% is larger than the IES retention threshold (the black dashed line in Fig. 4A) inferred from ex vivo experiments (5), implying that these HS RBCs do not successfully pass through IES. The corresponding DI of the band-3–deficient RBCs (the blue curve in Fig. 4B) decreases as the degree of band-3 deficiency increases. These results illustrate the correlation between the degree of protein deficiency and clinical expressions of RBCs in HS.

(A) Fractional surface area loss of HS RBCs after passage through IES. For the band-3–deficient RBCs, the surface area loss is increased with the decreased vertical connectivity. For the spectrin-deficient RBCs, the surface area loss is increased with the decreased spectrin density. The error bars are computed based on pressure gradient values of 5, 8, 10, 15, and 20 Paμm −1 . The black dashed line highlights the critical fraction of surface area loss that determines the retention of RBCs reported by ex vivo experiment (5). (B) DI of the band-3–deficient and the spectrin-deficient RBCs at different levels of HS-related protein deficiency. The brown bar graph shows DI of spectrin-deficient RBCs measured by osmotic gradient ektacytometry at a fixed osmolality of 300 mOsmol/kg (30).

Next, we reduce the spectrin density in the cytoskeleton by decreasing the number of actin junction complexes in the RBC model (Fig. 3C) to mimic spectrin-deficient RBCs. To capture the wide spectrum of clinical severity observed in HS, the spectrin density is decreased from 100% (healthy) to 40% in decrements of 20%. When the spectrin-deficient RBCs pass through IES, the lipid bilayer in the spectrin-depleted area can bud off and develop into vesicles (8). More discussion regarding the vesiculation process is given in SI Appendix. Fig. 4A (red curve) illustrates that the fractional surface area loss of spectrin-deficient RBCs increases with decreased spectrin density, in agreement with clinical evidence (8). Fig. 4B shows that reduced spectrin density leads to decreased DI of the spectrin-deficient RBCs (red curve). In particular, the values of DI are consistent with the values experimentally measured by ektacytometry at a fixed osmolality of 300 mOsmol/kg (30, 31), indicating that the trends predicted by the present results are validated by clinical observations.

The surface area and volume of HS RBCs following the formation of vesicles are plotted in Fig. 5. The blue and red symbols in Fig. 5 show that as the HS-related protein defects increase, the S/V ratios of HS RBCs gradually decrease toward the retention threshold predicted by Eq. 1 (black solid line) as well as the retention threshold reported by ex vivo experiments (black dashed line) (5). This plot implies that HS RBCs with low degrees of protein deficiency could pass IES despite reduced surface area. Therefore, these RBCs undergo a gradual decrease in their life spans, leading to mild anemia. Conversely, RBCs with high degrees of protein deficiency undergo rapid drops of S/V ratio after crossing IES. As a result, the expected life spans of these RBCs are shortened markedly, leading to severe anemia. These findings provide a mechanistic rationale for the connections among molecular defects, RBC alterations, and the broad spectrum of clinical severity found in HS.

Prediction of splenic IES retention for healthy RBCs (no protein deficiency) and HS RBCs. Healthy RBCs (green color symbols) with surface area of 140 (⧫), 130(▾), 120 (•), 110 (▴), and 100 μm 2 (■) and a fixed volume of 90 μm 3 are examined, respectively. The surface area and volume of HS RBCs after their passage through IES are plotted. Blue color symbols denote the band-3–deficient RBCs with vertical connectivities of 80% (▾), 60% (•), 40% (×), 20% (▴), and 0% (■). Red color symbols denote the spectrin-deficient RBCs with spectrin density of 80% (▾), 60% (•), and 40% (×). The error bars are computed based on pressure gradient values of 5, 8, 10, 15, and 20 Paμm −1 . The black solid and dashed lines highlight the RBC retention threshold predicted by an analytical model given by Eq. 1 (19, 26) and the threshold reported by ex vivo experiment (5), respectively. RBCs with surface area and volume above these thresholds are able to cross IES otherwise, RBCs are retained by IES.

Fig. 4A shows that as the level of HS-related protein deficiency increases, the spectrin-deficient RBCs with dispersed cytoskeleton shed more membrane surface than the band-3–deficient RBCs whose cytoskeleton is dense. Fig. 5 also shows that as the spectrin density decreases, the spectrin-deficient RBCs (red symbols) undergo a more rapid drop of S/V ratio than the band-3–deficient RBCs (blue symbols) for the same decrease in volume. These results suggest that the spectrin-deficient RBCs are more amenable to vesiculation than the band-3–deficient RBCs when traversing IES. This finding provides a compelling mechanistic rationale for the clinically documented observation that splenectomy prolongs the survival of spectrin/ankyrin-deficient RBCs but not band-3–deficient RBCs (32). The distinct clinical manifestations between these two types of HS RBCs are likely attributed to the fact that the band-3–deficient RBCs in the spleen shed surface area to the same extent as they do through other mechanisms, such as by antibody-triggered vesiculation or trogocytosis (32), while the spectrin-deficient RBCs lose membrane surface predominantly in the spleen.

Traversing IES Causes Shape Transition and Fragmentation of RBCs in HE.

In HE, defects occur in cytoskeletal proteins of RBCs, such as α-spectrin, β-spectrin, and protein 4.1R (11). The α- and β-spectrin deficiencies, which account for nearly 95% of HE cases, disrupt the self-association of spectrin dimers, whereas the protein 4.1R deficiency, responsible for ∼5% of HE cases, alters the cohesion of the spectrin–actin–protein 4.1R junctional complexes (11, 33). The characteristics of RBCs in HE are increased cell fragility and shape transition from the biconcave to the elliptical shape (9, 11). In severe forms of HE, cell fragments have been detected in patients’ blood smear (9). The prevailing hypothesis is that RBCs in HE become damaged during their transit through narrow pathways in microcirculation (33, 34), although little clinical evidence is available to support this hypothesis. Here, we examine RBCs displaying different degrees of HE-related protein deficiency as they travel through IES and explore how the traversal through IES contributes to the pathological alterations of RBCs in HE. We break the spectrin filaments in the RBC model (Fig. 6 A, Inset), mimicking the disrupted spectrin tetramers. As the severity of HE varies according to the degree of the impaired cytoskeleton (33), we reduce the percentage of the intact spectrin filaments (horizontal connectivity) from 100% to 20% in decrements of 10%. At each horizontal connectivity, pressure gradients of 5, 8, 10, 15, and 20 Paμm −1 are examined.

(A) Aspect ratios of HE RBCs after passage through IES. When the horizontal connectivity is reduced to 40% or less, HE RBCs break into fragments due to reduced cytoskeleton integrity. The error bars are computed based on pressure gradient values of 5, 8, 10, 15, and 20 Paμm −1 . Inset shows that in the spectrin-deficient HE RBC model, spectrin filaments are randomly disassociated (highlighted by red dotted circles), mimicking the disrupted spectrin tetramers. (B–D) Three sequential snapshots (top views) of an HE RBC with a horizontal connectivity of 50% crossing IES (see Movie S3). (E–G) Three sequential snapshots (top views) of an HE RBC with a horizontal connectivity of 20% breaking into fragments after crossing IES (see Movie S4).

A notable feature of HE RBCs is the elongation of RBCs after passage through IES (Fig. 6 BD). Although vesiculation of HE RBCs is also observed, it is less pronounced compared with that of HS RBCs. Here, we quantify the shape transition of HE RBCs by computing the aspect ratio of cells after their egress from IES. The aspect ratio is defined as L A / L T , where L A is the length of the RBC along its moving direction and L T is the length of the RBC perpendicular to its moving direction. As shown in Fig. 6A, RBCs with decreased horizontal connectivity undergo further elongation during their passage through IES. These elongated HE RBCs cannot fully recover their original biconcave shape due to impaired cell elasticity, leading to the progressive shape transition to elliptical shape. When the horizontal connectivity is 40% or less (30% and 20% in our simulations), the model predicts that the HE RBCs break into cell fragments. Fig. 6 EG illustrate three sequential snapshots of an RBC with a horizontal connectivity of 20% passage through IES. It is noted that the portions of RBCs protruding into the luminal sides of IES are elongated and subsequently break apart from the RBC, forming two cell fragments. These simulations provide a mechanistic rationale for RBC fragmentation in HE and for the presence of cell fragments in the blood smear of HE patients (9, 11).

Genetics of erythrocyte hydration

Genomewide association studies (GWASs) have demonstrated that a significant component of erythrocyte hydration is genetically determined (reviewed in Kim-Hellmuth and Lappalainen 115 ). Variation in indices of erythrocyte hydration, MCHC and MCV, are strongly influenced by genetic factors. 116,117 In 1 GWAS, the most significant association for MCHC was a single-nucleotide polymorphism at 16q24 linked to the PIEZO1 gene locus. In another, an enhancer of an erythrocyte calcium ATPase (ATP2B4) was linked to MCHC. 118

GWASs have also shown that in most complex diseases, common variants explain only a fraction of genetic risk, as expected from the effects of natural selection. Recent studies indicate rare, independent mutations are major contributors to phenotypic variation in complex diseases, such as hypertension, hypertriglyceridemia, and ischemic stroke. Thus rare, independent mutations may predominate, and analyses of these rare variants add to our understanding of the genetic contributions to complex disease. 119 Frequently, these variants identify critical pathways regulating the trait under study, such as rare hypertension-associated mutations in channels, transporters, and regulatory proteins involved in sodium handling by the kidney. 120

Thus, coinheritance of disease alleles that influence erythrocyte hydration (eg, HX or SCD), and common or rare alleles that influence erythrocyte hydration and disease, may worsen or ameliorate disease. 121,122 Identification of common and rare hydration-associated genetic variants will provide information on the pathways regulating erythrocyte volume homeostasis.

Spleen structure

The body of the spleen appears red and pulpy, surrounded by a tough capsule. The red pulp consists of blood vessels (splenic sinusoids) interwoven with connective tissue (splenic cords). The red pulp filters the blood and removes old and defective blood cells. The white pulp is inside the red pulp, and consists of little lumps of lymphoid tissue.

Antibodies are made inside the white pulp. Similarly to other organs of the lymphatic system, particular immune cells (B lymphocytes and T lymphocytes) and blood cells are either made or matured inside the spleen. Blood enters the spleen via the splenic artery, which subdivides into many tiny branches. Each branch is encased in a clump of lymphocytes, which means every drop of blood is filtered for foreign particles as it enters the spleen.

How do red blood cells know when to die?

Human red blood cells (RBCs) are normally phagocytized by macrophages of splenic and hepatic sinusoids at 120 days of age. The destruction of RBCs is ultimately controlled by antagonist effects of phosphatidylserine (PS) and CD47 on the phagocytic activity of macrophages. In this work, we introduce a conceptual model that explains RBC lifespan as a consequence of the dynamics of these molecules. Specifically, we suggest that PS and CD47 define a molecular algorithm that sets the timing of RBC phagocytosis. We show that significant changes in RBC lifespan described in the literature can be explained as alternative outcomes of this algorithm when it is executed in different conditions of oxygen availability. The theoretical model introduced here provides a unified framework to understand a variety of empirical observations regarding RBC biology. It also highlights the role of RBC lifespan as a key element of RBC homeostasis.

1. Introduction

The population of red blood cells (RBCs) in the organism must remain within definite limits in order to ensure the oxygenation of body tissues and to maintain adequate values of blood pressure and viscosity. This is achieved by means of homeostatic mechanisms that control the ratio between cell production and destruction and compensate any unbalance between oxygen supply and demand by increasing or reducing the number of circulating RBCs [1,2].

The formation of new RBCs is controlled by erythropoietin (Epo), a hormone produced by fibroblasts of peritubular capillaries in the kidney that induces proliferation and differentiation of erythroid precursor cells in the bone marrow [3]. On the other hand, RBCs are removed by macrophages of the mononuclear phagocytic system (MPS) when passing through the splenic and hepatic sinusoids. Macrophages identify and phagocytize RBCs that have attained a critical age (120 days in humans and 60 days in mice) in a process known as erythrophagocytosis [4–6].

In hypoxia, fibroblasts increase the release of Epo, thus accelerating the production of new cells and boosting the population of RBCs [7,8]. Conversely, if oxygen levels rise above physiological needs (e.g. in acclimation to higher partial pressure of oxygen after descent to sea level from high altitudes), fibroblasts lower the production of Epo and the population of RBCs shrinks to a new equilibrium size [3,9,10]. Excess of oxygen supply also entails an increase in the rate of cell destruction caused by neocytolysis, a homeostatic mechanism that entails the selective removal of RBCs of only 10 or 11 days of age, and contributes to the rapid reduction of the number of cells [11–14].

The switch from 120 days to 11 days of duration in response to environmental factors indicates that lifespan is not a fixed, intrinsic feature of RBCs. This point is further evidenced by the fact that RBCs live around 40 days less in newborn humans than in adults [15]. Even if the mechanisms that regulate changes in RBC lifespan remain obscure, it is widely assumed that RBC ageing and death are ultimately caused by oxidative stress (OS) [16–18]. The continuous exposure to highly reactive oxygen radicals deteriorates the membrane and cytoplasm of the RBC, which may eventually compromise its function [19]. In fact, higher sensitivities to OS correlate with shorter lifespans [20]. This observation has been interpreted as evidence of an active mechanism that would set RBC lifespan by fine-tuning the expression of genes that confer resistance to OS in erythroid precursors [16,20]. From this approach, human RBCs would be genetically configured to show signs of OS-driven senescence around the age of 120 days. Macrophages of the MPS would then identify aged RBCs by means of these signs [21].

In our opinion, the explanation of RBC lifespan as determined exclusively by OS is incomplete. For one thing, not all aged RBCs show the typical signs of severe OS-derived damage, such as cell shrinkage and membrane blebbing [22,23]. As a matter of fact, defective RBCs of any age are not destroyed by erythrophagocytosis, but through an alternative mechanism known as eryptosis [24,25]. This suggests that normal and damaged RBCs follow different phagocytosis pathways. On the other hand, the 10-fold decrease in RBC lifespan during neocytolysis would require a substantial reduction in the resistance of RBCs to oxidative damage. This would multiply the risk of RBC malfunction, a feature that seems unlikely for a physiological homeostatic mechanism. Alternatively, neocytolysis and erythrophagocytosis might be driven by different mechanisms [3,11], implying that some aspects of RBC lifespan cannot be explained by OS alone.

We postulate in this work that OS should not be considered as the key determinant of RBC lifespan, even if it causes the destruction of a fraction of circulating cells, and certainly imposes an upper boundary to the potential duration of RBCs in the blood. We suggest that lifespan is set by means of a molecular algorithm that controls cell-to-cell interactions between RBCs and macrophages of the MPS. We will show that such an algorithm could allow to fine-tune RBC lifespan in a variety of ways, thus providing a flexible system to adapt the number of cells to the demand of oxygen in the tissues.

The view of RBC lifespan introduced here frames a theoretical foundation in which to integrate different observations regarding RBC biology, such as erythrophagocytosis, neocytolysis and the seemingly paradoxical presence of auto-antibodies against host RBCs in the organism. In particular, we will show that these phenomena emerge as alternative outcomes of the same mechanisms working under different conditions of oxygen availability.

2. A conceptual model of red blood cell lifespan determination

(E1) PS is confined to the inner layer of the cell membrane in newly formed RBCs, so it is invisible for macrophages. Such membrane asymmetry is progressively lost in ageing RBCs, which increases PS exposure in the cell surface [34–36]. Therefore, pro-phagocytic effect of PS intensifies with the age of the RBC (figure 1a).

(E2) Conversely, the anti-phagocytic activity of CD47 is higher at the birth of the RBC [37,38]. Progressively lower expression of the protein or conformational changes in its spatial structure diminish its activity as a phagocytosis inhibitor as the cell ages [39] (figure 1a).

(E3) The effects of PS and CD47 cancel out each other [40], so that the net balance between PS and CD47 in an RBC determines whether or not it is destroyed by macrophages [22]. From points E1 and E2, it follows that ‘don’t-eat-me’ signals offset ‘eat-me’ signals in the membrane of young RBCs, preventing their phagocytosis. The difference between ‘eat-me’ and ‘don’t-eat-me’ signals grows in ageing RBCs until it reaches a critical threshold that elicits their destruction by macrophages of the MPS [40,41] (figure 1b).

(E4) It has also been observed that RBCs with sufficiently low levels of CD47 are also phagocytized regardless of the amount of PS present in their surface [42,43]. In this case, young RBCs are not destroyed because of the anti-phagocytic effect of CD47 (evidence E2). Owing to progressive loss of CD47 activity in ageing RBCs ‘don’t-eat-me’ signals eventually fall below a certain level that prompts the phagocytosis of the cell (figure 1c).

Figure 1. Rationale of the conceptual model of RBC lifespan determination. (a) Time evolution of membrane signals in a RBC according to empirical evidence (see points E1 and E2). (b) A RBC is phagocytized when the difference between ‘eat-me’ and ‘don’t-eat-me’ signals in its membrane attains a critical threshold (evidence E3). (c) An RBC can also be phagocytized if its level of ‘don’t-eat-me’ signals falls below a critical threshold (E4). (d) The conditions triggering RBC phagocytosis are mutually exclusive. In this example, the phagocytosis of the RBC occurs because the expression of ‘don’t-eat-me’ signals falls below a critical threshold (condition E4). (e,f) Different dynamics of membrane signals result in different lifespans (e) or in the RBC being phagocytized because it fulfils condition E3 before condition E4 (f).

The conditions that trigger RBC phagocytosis (points E3 and E4) seem to be simultaneously fulfilled by ageing RBCs. However, since any particular RBC can only be phagocytized once, both conditions are in fact mutually exclusive. Only the first of the thresholds to be reached determines the lifespan of the RBC (figure 1df). On the other hand, both conditions seem to accomplish the same purpose, since both foster the phagocytosis of aged RBCs and the survival of young cells. This raises the question of why two apparently redundant pathways of RBC removal exist.

In order to address this issue, we begin by remarking that CD47 and PS also play a major role in the control of the phagocytosis of other cell types by macrophages [44–46]. Specifically, CD47 is broadly expressed in the host and absent in foreign cells [29,47], while PS is confined to the membrane of apoptotic host cells [48,40]. These patterns of PS and CD47 expression allow for macrophages to identify CD47 + cells as self-structures [31,42]. In this case, accompanying high levels of PS are recognized as a mark of apoptosis, which triggers the phagocytosis of the cell and the release of anti-inflammatory signals that avoid autoimmunity against healthy tissues [49,50]. On the other hand, the absence of CD47 in a cell membrane reveals the presence of a potential infection [51,52]. Unlike the silent clearance of apoptotic host cells, phagocytosis of CD47 − cells is followed by the activation of the macrophage [53], and the secretion of pro-inflammatory signals that may lead to an innate immune response [54,55].

We postulate that the role of PS and CD47 in the phagocytosis of RBCs (as described in points E3 and E4) follows this general pattern. Young RBCs, like non-apoptotic host cells show high levels of CD47 and low levels of PS, which prevents their phagocytosis by macrophages. Among aged RBCs, those with high PS and low CD47 expression are comparable to apoptotic host cells, while those expressing very low levels of CD47 can be likened to foreign cells. Bearing these analogies in mind, we hypothesize the existence of two alternative pathways of RBC phagocytosis that entail different macrophage reactions. Specifically, we suggest that the pathway controlled by the balance between PS and CD47 (E3) is similar to the removal of apoptotic host cells. In particular, it does not trigger any immune response. By contrast, the phagocytosis of RBCs with very low CD47 expression (E4) might be analogous to the destruction of non-self agents by macrophages, and could provoke autoimmune reactions against host RBCs. In the remainder of this article, we will refer to both phagocytosis pathways as silent and immune, respectively.

The existence of an ad hoc mechanism to provoke autoimmunity may seem paradoxical. However, it has long been observed that auto-antibodies targeting host RBCs are usually present in the organism [56–58]. Anti-RBC antibodies are natural antibodies produced by B-1 cells [59,58]. Unlike antibodies from other B cell subsets, B-1 antibodies have anti-inflammatory effects, which minimize potential collateral damage to host tissues [60,61]. This might explain why anti-RBC auto-antibodies are usually innocuous [62] and only occasionally cause clinical disorders, known under the general term of autoimmune haemolytic anaemia [62]. On the other hand, natural antibodies are spontaneously produced in the absence of foreign antigens [56] so their specificity for RBCs cannot be explained as due to cross-reactivity of RBC epitopes and non-self structures encountered in previous infections. This raises the question of how these auto-antibodies are produced.

Our assumption of an immune pathway of RBC phagocytosis suggests a possible answer for this question. Macrophages of the MPS express MHC molecules, and can therefore act as antigen presenting cells [63,64]. We suggest that after phagocytizing RBCs with very low CD47 expression they would initiate an adaptive immune response, much like they do after phagocytizing foreign cells. As a matter of fact, it has been recently observed that removal of CD47 from self-RBCs suffices indeed to trigger immune responses in mice [65]. Nevertheless, since RBCs are not pathogens, macrophages of the MPS would recruit B-1 cells instead of more aggressive B cell types, leading to the production of non-inflammatory antibodies against host RBCs. The functional role of these anti-RBC auto-antibodies remains to be explained. In this respect, we will show in the following sections that anti-RBC autoimmunity, together with erythrophagocytosis and neocytolysis fit into a global, coherent model of RBC homeostasis. In order to do that, we will next state the previous conceptual model in mathematical terms.

2.1. Mathematical formalization of the conceptual model

(A1) The expression of ‘eat-me’ signals in the outer membrane of the RBC increases at a constant rate β.

(A2) The number of ‘don’t-eat-me’ signals decreases at a constant rate α. This results in an exponential decay, a behaviour that has been described for other RBC membrane proteins (e.g. [66,67]).

(A3) Two independent thresholds exist, denoted by Ts and Ti, that trigger silent and immune phagocytosis pathways, respectively.

Assumptions A1 and A2 do not intend to account for the molecular mechanisms underlying the time evolution of membrane signals. Instead, they have been chosen for the sake of simplicity in order to show the relevance of signal dynamics in RBC homeostasis. Nevertheless, new data about PS and CD47 dynamics could be easily included in this approach by modifying assumptions A1 and A2. We will discuss the implications of this particular choice of assumptions in the last section of this article.

Denoting by E(t) and D(t) the number of ‘eat-me’ and ‘don’t-eat-me’ signals at time t, respectively, assumptions A1 and A2 can be stated in mathematical terms as follows:

Integrating equations (2.1) we get an explicit expression for the dynamics of ‘eat-me’ and ‘don’t-eat-me’ signals:

From assumption A3, it follows that conditions E(ts)−D(ts)=Ts and D(ti)=Ti define the times ts and ti at which the RBC is removed through the silent and immune phagocytosis pathways, respectively. Introducing the expressions of E(t) and D(t) given by equations (2.2) in these conditions, we get the following values for ti and ts:

Equations (2.3) define the conditions that dictate the fate of RBC. The cell is cleared through the silent pathway if ts<ti and through the immune pathway otherwise. From equations (2.3), it follows that the timing of RBC phagocytosis, and hence its lifespan, is given by L = min ( t i , t s ) .

3. A theoretical framework for red blood cell homeostasis

From equations (2.1) to (2.3), ‘eat-me’ and ‘don’t-eat-me’ signals can be viewed as defining a cellular algorithm whose execution in the membrane of each RBC determines both its fate (i.e. if it is removed through the silent or the immune pathway) and its lifespan. In this section, we will show that this algorithm provides a coherent, integrative view of RBC homeostasis. According to equations (2.3), RBC fate and lifespan are unambiguously defined by the specific values of six parameters. Roughly speaking, these parameters represent the amount of membrane signals at the birth of the cell (D0 and E0), the rates of change of these signals (α and β), and the thresholds that elicit the phagocytosis of RBCs (Ts and Ti). Variations in any of these features result in changes in either the lifespan of the cell or the phagocytosis pathway leading to its destruction (figure 2). Bearing this fact in mind, we will next enumerate a series of biological mechanisms that could be used by the organism to modulate RBC lifespan, and discuss their consequences on RBC homeostasis.

Figure 2. Results of the mathematical model of RBC lifespan determination. (a) The dynamics of membrane signals as defined by equations (2.1)–(2.3) satisfy the qualitative constraints imposed by empirical evidence (E1–E4). Both the lifespan of the cell and how it is phagocytized (i.e. through the silent or the immune pathway) depend on the particular values of the model parameters. In this case, the difference between ‘eat-me’ and ‘don’t-eat-me’ signals is the first to reach its critical threshold (Ts), so that this cell is destroyed through the silent pathway at time ts (which sets its lifespan). (b) Changing the silent threshold (parameter Ts in the model) shortens the lifespan of the cell, but not the phagocytosis pathway. (c) By contrast, lower CD47 expression at the birth of the cell (parameter D0) both shortens the lifespan of the cell and changes the condition that triggers its phagocytosis (from silent to immune).

3.1. Effects of oxidative stress on red blood cell lifespan

As we noted above, OS causes the accumulation of defects in the cytosol and membrane of RBCs, increasing the probability of malfunction and even of cell lysis in the blood. In extreme cases, this may induce a severe clinical condition known as haemolysis [68]. RBCs showing signs of oxidative damage should therefore be removed from the circulation in order to minimize the risk of haemolysis. It has been suggested that the level of PS expression is one of those signs, since higher levels of OS are accompanied by higher rates of PS externalization [34,35,69,70].

PS exposure in response to OS is not a passive process. Instead, it seems to be mediated by cytoplasmic RBC proteins [27], suggesting that RBCs are able to accelerate the rate of PS externalization in case of oxidative damage. From this observation, we can deduce that higher values of parameter β (rate of PS externalization) correspond to RBCs exposed to higher levels of OS (see equations (2.1)). In agreement with empirical observations, this condition shortens RBC lifespan [20,70] (figure 3). From the perspective of our model, accelerated PS exposure in response to OS can be interpreted as an active mechanism to minimize the risk of RBC lysis in the blood. By increasing the rate of PS translocation, an RBC would hasten its phagocytosis through the silent pathway. The cell would therefore be removed from the circulation before attaining a critical level of oxidative damage that might compromise its function or even its physical viability.

Figure 3. Potential mechanisms of RBC lifespan modulation. (a) Higher levels of oxidative stress are associated with higher rates of PS externalization. In agreement with empirical data, the model predicts an inverse correlation between the degree of OS and RBC lifespan. If the rate of PS externalization is above a critical value (β*) the curve of ts (time to reach the silent threshold) is below the curve of ti (time to attain the immune threshold). This implies that for high values of OS (β>β*) RBCs are phagocytized through the silent pathway. Only if β<β* are RBCs destroyed through the immune pathway, which can lead to anti-RBC autoimmunity. (b) Time evolution of the difference between ‘eat-me’ and ‘don’t-eat-me’ signals in the membrane of an RBC formed at time t1. The difference between membrane signals should reach the silent threshold at time t2, thereby causing the phagocytosis of the cell. Increasing the silent threshold delays the phagocytosis of the RBC until t3, thus extending its lifespan. (c) Neocytolysis. The figure shows the dynamics of the difference between ‘eat-me’ and ‘don’t-eat-me’ signals in the membrane of two RBCs that differ in the expression of membrane signals at birth. The first cell, formed at time t1, is phagocytized at time t4 after a normal lifespan. The second cell, born at time t2>t1 with a larger difference between ‘eat-me’ and ‘don’t-eat-me’ signals in its membrane attains the silent threshold much faster, so it is destroyed at time t3, before the first cell and after a much shorter lifespan. (d) According to our model, the lifespan of each RBC is directly correlated with the level of CD47 expressed in its membrane when it is formed. Low values of CD47 expression could explain short lifespans observed during neocytolysis. Furthermore, if the initial amount of CD47 falls below a critical level (the autoimmunity threshold), the immune phagocytosis occurs before the silent pathway (ti<ts). In this case, macrophages of the MPS phagocytize RBCs after very short lifespans and initiate anti-RBC autoimmune responses.

3.2. Recovery of red blood cell homeostasis after haemorrhages

According to equations (2.3), tuning the silent phagocytosis threshold provides another mechanism to modulate RBC lifespan. This threshold is defined as the difference between ‘eat-me’ and ‘don’t-eat-me’ signals that triggers the silent phagocytosis pathway in macrophages of the MPS. Hence, from a mechanistic point of view, tuning this parameter amounts to modulating the sensitivity of macrophages to RBC signals. Increasing the silent phagocytosis threshold delays phagocytosis and extends RBC lifespan (equations (2.1) and figure 3b). Each day added to mean RBC lifespan prevents the destruction of 10 11 cells (around 1% of the total population), which is equivalent to the daily production of RBCs in normal conditions.

A significant fall in the number of RBCs after a haemorrhage may produce a deficit of oxygen in the tissues. The subsequent rise in the levels of Epo in the blood [71] eventually restores the population of RBCs and the equilibrium of oxygen. However, given that this process involves the differentiation of precursor cells it can take a few days to take the population back to its original size. Increasing the silent threshold could buffer cell loss and help to maintain the supply of oxygen until Epo-mediated recovery of the population is completed. In this regard, we remark that empirical evidence suggests that phagocytosis of macrophages of the MPS is indeed suppressed after haemorrhages [72]. Furthermore, macrophages are equipped with Epo receptors [73], implying that the tuning of the silent phagocytosis threshold might be directly controlled by the levels of plasma Epo. Once the equilibrium of oxygen is recovered, the levels of Epo would return to normal values, restoring both the silent threshold and RBC lifespan.

3.3. Neocytolysis and erythrophagocytosis

Neocytolysis and erythrophagocytosis are currently considered as alternative mechanisms of RBC removal [3,13]. In particular, it is implicitly assumed that erythrophagocytosis is the default pathway of destruction of senescent RBCs during normal homeostasis, while neocytolysis is somehow triggered by decreased levels of Epo [9,11]. Such drops of Epo occur, in particular, whenever oxygen availability in the tissues is above physiological needs. For instance, people descending to sea level after a period of acclimation to high altitudes move from lower to higher partial pressures of atmospheric oxygen. In this situation, the population of RBCs is larger than needed to ensure the supply of oxygen to the tissues, and contracts to a new equilibrium size through the selective destruction of younger RBCs [74]. The mechanisms underlying the switch from erythrophagocytosis to neocytolysis remain poorly understood [9,70].

In this work, we suggest that neocytolysis and erythrophagocytosis should not be considered as independent mechanisms, but as alternative outcomes of the algorithm of RBC lifespan determination. Specifically, both processes can be explained as caused by different patterns of PS and CD47 expression in the membrane of newly formed RBCs. Figure 3c compares the lifespan of two RBCs that differ in the amount of membrane signals at birth. The RBC with the bigger difference between PS and CD47 expression is the first one to reach the silent phagocytosis threshold, even if it is born later. Moreover, this cell is destroyed after a short lifespan, while the other is spared and will only be removed after reaching the usual RBC lifespan. These features are precisely what defines neocytolysis. Therefore, according to our model, neocytolysis occurs if RBCs formed under lower levels of Epo are born with more PS or less CD47 in their outer membrane. Empirical evidence points to the latter, since young RBCs show lower levels of CD47 and similar levels of PS (when compared with older cells) in people descending to sea level after acclimation to high altitude [12].

This result suggests that the transition from erythrophagocytosis to neocytolysis does not require a switch between alternative mechanisms of RBC destruction. Instead, the lifespan of RBCs can vary in a continuum that ranges from 10 days during neocytolysis, to 80 days in newborns and 120 days in adult humans, depending on the level of PS and/or CD47 at the birth of the cells. In order to illustrate the main point of this work, and for the sake of simplicity, we will continue our discussion assuming that Epo only affects CD47 expression in newly formed RBCs (figure 3d). Similar arguments could be drawn if Epo also determined initial PS levels.

3.4. Autoimmune responses in red blood cell homeostasis

Neocytolysis reduces the number of RBCs when oxygen supply exceeds the demands of body tissues [9,11]. We suggest that autoimmunity could provide a complementary mechanism to accelerate the contraction of the RBC population in such circumstances. According to the model, anti-RBC autoimmune responses emerge from the same process that leads to neocytolysis, namely, the reduction in CD47 expression in newly formed RBCs. If the population of RBCs is still larger than required after neocytolysis-driven contraction, the levels of Epo continue to drop. In consequence, newly formed RBCs express progressively less CD47 in their membranes (figure 3d). RBCs whose initial levels of CD47 expression falls beyond a critical point (labelled as the autoimmune threshold) are phagocytized through the immune pathway (figure 3d). The ensuing production of natural auto-antibodies would foster the death of other RBCs, further contracting the population.

The view of anti-RBC autoimmune responses as a homeostatic mechanism is supported by the fact that natural antibodies do not target all circulating RBCs, which might result in a massive and uncontrolled loss of cells. Instead, they are directed against specific epitopes usually expressed in aged RBCs and absent in young cells [57]. Moreover, RBCs of any age are also protected from the action of auto-antibodies by CD47, which is known to inhibit the phagocytosis of opsonized cells [75,28]. On the other hand, the protection provided by CD47 is dose-dependent [75], implying that the destruction of an individual RBC through this antibody-mediated pathway depends on both its levels of CD47 and the concentration of antibodies present in the blood. For this reason, only those RBCs with high CD47 expression survive in the course of more aggressive responses. Therefore, the intensity of the autoimmune response (i.e. the amount of auto-antibodies produced) determines the cohorts of RBC that are destroyed, and hence the extent of the reduction in the number of cells.

In normal conditions, autoimmunity-driven contraction of the population should eventually restore physiological levels of oxygen. Under the assumptions of our model, the subsequent rise in Epo would increase CD47 expression in new RBCs, arresting the production of anti-RBC antibodies (figure 3d). Further increases of initial CD47 would also interrupt neocytolysis and restore RBC lifespan to normal values observed in erythrophagocytosis. Therefore, Epo-dependent regulation of CD47 in new RBCs creates a switch between silent and immune phagocytosis and makes both neocytolysis and homeostatic autoimmunity reversible processes.

Our model also suggests that anti-RBC responses are only triggered if levels of OS are sufficiently low (figure 3a). Assuming the homeostatic nature of autoimmunity, this result can be understood as preventing the production of auto-antibodies in conditions of severe OS. Under these circumstances, oxidative damage can cause the abnormal destruction of many RBCs, making unlikely the need for anti-RBCs antibodies to remove an excess of cells.

3.5. The role of Epo in red blood cell lifespan determination

The role of Epo in RBC production and its relationship to oxygen homeostasis are well established in the literature [3]. It has been hypothesized that Epo could also control the onset of neocytolysis by modulating the interaction between macrophages of the MPS and young circulating RBCs [11]. The theoretical model presented in this work supports this hypothesis by suggesting an explicit mechanism that links Epo to RBC lifespan determination. Moreover, this model suggests that neocytolysis can be understood as a particular manifestation of a more general function of Epo as determinant of RBC destruction. This function would consist in setting RBC lifespan by adjusting the phagocytosis thresholds and the levels of CD47 expression in newly formed cells. If proven correct, this model would explain a variety of RBC responses to changes in oxygen supply to the tissues.

For instance, people descending to sea level after high-altitude acclimation show sharp fluctuations of Epo owing to altitude-related changes in the partial pressure of oxygen. Epo increases during acclimation to higher altitudes, and falls after returning to sea levels, attaining lower values than those found before altitude acclimation [12,70] (figure 4a). Similar Epo dynamics have been described in malaria patients. The destruction of RBCs by Plasmodium parasites during the first stages of malaria causes a deficit of oxygen in the tissues, and a subsequent increase of Epo [76,77]. By contrast, later stages of the infection are usually associated with insufficient production of Epo [77–79]. Epo also falls if partial pressure of oxygen increases, e.g. during spaceflights or in the return to sea level after high-altitude acclimation [80,9].

Figure 4. A theoretical model for the relationship between RBC lifespan and oxygen homeostasis. (a) Acclimation to environments with different partial pressures of oxygen, or clinical conditions that involve massive RBC loss such as malaria entail sharp fluctuations in the levels of plasma Epo (see text for references). (b) We hypothesize that Epo controls CD47 expression in newly formed RBCs, which in turn sets their expected lifespan (see equations (2.3)). In normal conditions both Epo and oxygen levels are at equilibrium, and mean RBC lifespan is around 120 days (0). Any variation in Epo, independently of its cause, changes the amount of CD47 in newly formed RBCs and hence its lifespan. From this perspective, a pronounced decrease in Epo suffices to account for the onset of neocytolysis observed in people returning to sea level after high-altitude acclimation or in malaria patients (labelled as −1 in the figure). Further drops of Epo can lead to autoimmunity (labelled as −2), which could explain the presence of auto-antibodies against host RBCs in malaria patients or in astronauts after space flights. See the text for further details.

We postulate that any drop in Epo is expected to exert similar effects on RBC lifespan independently of its cause. Within the framework of our model, these effects range from neocytolysis to the initiation of homeostatic autoimmunity (figure 4b). As a matter of fact, both neocytolysis and strong anti-RBC responses have been described in astronauts after space flights [9,74]. As for malaria infections, both Plasmodium falciparum and P. vivax infections cause the abnormal removal of an important number of non-parasitized cells (npRBCs) [81,82]. In some patients of severe malaria-derived anaemia, the destruction of npRBCs can even continue long after the infection has been cleared (see [83] and references therein). Therefore, malarial anaemia cannot be explained simply by the direct destruction of infected RBCs alone. Both selective death of young non-parasitized RBCs [84–86] and the presence of anti-RBC antibodies [87] suggest that neocytolysis and homeostatic autoimmunity might play a major role in the development of anaemia during malaria. In this situation, the anomalous drop of Epo that characterizes latter stages of malaria infection would be erroneously perceived by the organism as caused by an excess of circulating RBCs. The ensuing normal homeostatic mechanisms (neocytolysis and homeostatic autoimmunity) triggered in abnormal conditions of oxygen availability would lead to anomalous reductions in the population of RBCs.

4. Discussion

The consumption of oxygen by the organism is highly variable owing to factors such as circadian metabolic rhythms, the intensity of physical activity or even fluctuations in ambient temperature [88,89]. In consequence, homeostatic mechanisms must continuously adjust the balance between RBC production and destruction to maintain an appropriate number of RBCs. The control of RBC production by Epo is well described in the literature [3]. By contrast, many questions about RBC destruction remain largely unanswered. In particular, no universally accepted explanation of the mechanisms underlying changes in RBC lifespan is available as yet.

A substantial body of evidence points to PS and CD47 as key determinants of RBC phagocytosis [26–31]. In this work, we postulate that quantitate aspects of these dynamics explain how RBC lifespan variations are related to oxygen homeostasis. This statement is based on two main assumptions. First, that the pattern of PS and CD47 expression changes during the life of the cell, as evidenced by differences between young and aged RBCs [34–38]. Second, that the conditions that trigger RBC phagocytosis as described in the literature (see points E3 and E4 above) differ in the subsequent behaviour they elicit on macrophages of the MPS. Specifically, we postulate that the phagocytosis of RBCs with very low levels of CD47 provokes immune responses against host RBCs.

The nature of this work is necessarily speculative owing to the lack of published data about the actual dynamics of CD47 and PS in the membrane of RBCs. We have modelled plausible dynamics that satisfy the constraints imposed by available evidence. The exponential decay proposed for CD47 has actually been described for other molecules present in RBCs [66,67]. As for the increase of PS externalization observed in ageing cells, we have assumed that it occurs at a constant rate for the sake of simplicity. A different mathematical formalization of the model would involve a different set of parameters, suggesting perhaps other mechanisms of RBC lifespan modulation. In any case, the conceptual model that emerges from published evidence (outlined in figure 1) is independent of any particular mathematical formulation. From this conceptual model, PS and CD47 constitute a molecular clock that sets the timing of RBC phagocytosis. RBC lifespan should be determined by the time it takes for these signals to satisfy one of the two conditions that trigger the phagocytosis of the cell.

A mathematical version of this conceptual model suggests several mechanisms that might modulate RBC lifespan. First, changes in CD47 expression in newly formed RBCs could account for differences in lifespan observed in erythrophagocytosis and neocytolysis, as well as for the origin and function of anti-RBC autoimmunity. We remark that none of these processes is explicitly implemented in the equations of the model. Instead, they emerge as alternative outcomes of the same algorithm of lifespan determination for different values of initial CD47 expression at the birth of the cell. Second, by controlling macrophage phagocytic activity, Epo levels might continuously adjust the value of the phagocytosis thresholds, thus fine-tuning the lifespan of circulating RBCs. Finally, higher levels of OS might shorten RBC lifespan by accelerating the rate of PS exposure in the outer membrane of the cell. These mechanisms are independent and might be acting simultaneously to determine RBC lifespan. In this respect, it has been recently suggested that hypoxia-induced factors (HIFs) might be involved in shortening RBC lifespan during neocytolysis [70]. The effect of HIF would be related to lower catalase activity in young RBCs formed in hypoxia. Under this assumption, such young RBCs would be more susceptible to OS in case of a rise in oxygen availability, which would translate into higher rates of PS externalization. From the perspective of our model, this would imply that parameter β takes higher values in RBCs formed during hypoxia. At the same time, the amount of CD47 in new RBCs could be modulated by Epo depending on the levels of oxygen. The combined effects of accelerated PS expression and lower CD47 expression would result in shortened RBC lifespan and, eventually, in the production of anti-RBC auto-antibodies. In turn, autoimmunity and neocytolysis would rapidly contract the population whenever oxygen supply is above physiological needs.

Further research is needed to unveil all the mechanisms underlying RBC lifespan determination. However, irrespectively of their ultimate causes, variations in RBC lifespan play a central role in the ability of the organism to modulate the rate of RBC destruction. Specifically, if all human RBCs lived 120 days, then the temporal pattern of cell destruction would just reproduce the pattern of formation of new RBCs with a delay of 120 days. Extending mean lifespan beyond 120 days lowers the rate of cell destruction and enlarges the number of RBCs in the blood. Conversely, the phagocytosis of RBCs under 120 days of age contracts the population by increasing the rate of cell destruction. Therefore, it is clear that any theory intending to explain RBC homeostasis should explicitly address the question of how RBC lifespan is determined. The conceptual model introduced in this work constitutes a first step towards the development of such a theory. We believe that this model will improve our understanding of how RBC homeostasis is maintained in normal circumstances and how its imbalance can lead to pathology.

Authors' contributions

Both authors conceived this work, collaborated in finding and reviewing available literature on this field, contributed equally to the development of the theoretical model presented in this work, collaborated in writing the manuscript and gave final approval for publication.

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References: Biomechanics of red blood cells in human spleen and consequences for physiology and disease. Pivkin IV, Peng Z, Karniadakis GE, Buffet PA, Dao M, Suresh S. Proc Natl Acad Sci U S A. 2016 Jun 27. pii: 201606751. [Epub ahead of print]. PMID: 27354532.

Funding: NIH’s National Heart, Lung, and Blood Institute (NHLBI) National Science Foundation US Department of Energy Swiss Platform for Advanced Scientific Computing and Singapore-MIT Alliance for Research and Technology.