channel function lead to increased intracellular levels of Ca2+ and subsequent KCNN4 (Gardos channel) activation (Cahalan et al., 2015; Danielczok et al., 2017). KCNN4 activation leads to efflux of K+ and dehydration. The relatively low intracellular levels of K+ could therefore serve as a biomarker for HX (Zarychanski et al., 2012; Gallagher, 2017). Although PIEZO1 mutations leads to KCNN4 (or Gardos channel) activation, RBCs with mutations in KCNN4 (Gardos channelopathy) exhibit a different pathology (Fermo et al., 2017a). For example, HX caused by mutations in PIEZO1 lead to severe RBC dehydration, which is also shown by a left-shift of Ohyper in the osmotic gradient curve (see **Figure 1B** and see section "Techniques to Determine RBC Deformability") while mutations in KCNN4 (or Gardos channel) may cause only slight alterations in the osmotic gradient curve (Fermo et al., 2017b). In addition, RBCs from patients with KCNN4 mutations were found to exhibit increased activity of Na-K-ATPase that is most likely triggered by the increase in the intracellular Na+ concentrations. The hyperactivated Na, K-pump compensates for the dissipation in Na/K gradients and pumps K+ back into the cells, but fails to restore normal K+ content completely (Fermo et al., 2017a). Currently, however, it is difficult to measure intracellular water content or RBC volume

directly and accurately enough, which makes it challenging to discriminate between water loss and membrane loss.

### Cytoskeletal Network

### Primary Changes of the Cytoskeletal Network

In addition to the protein defects, described above, that directly disturb the membrane and thereby the ability of the RBC to deform there are also secondary causes affecting membrane (protein) function and, thereby, RBC deformability. Examples of these are membrane protein phosphorylation, RBC density and RBC vesiculation.

The RBC membrane is built from a basic triangular network of α- and β-spectrin molecules connected to band 3, ankyrin and protein 4.1 (Perrotta et al., 2008; Salomao et al., 2008). This network provides the RBC with a certain stability and simultaneously the ability to deform. HS (HS) is characterized by defects in proteins forming the cytoskeletal network or proteins forming ankyrin and junctional complexes. This usually concerns mutations in band 3, protein 4.2, α-spectrin, or ankyrin (Perrotta et al., 2008). These mutations lead to membrane instability, membrane loss through release of hemoglobin-free vesicles, with consequent decreased surface area-to-volume ratio, increase in MCHC and density and formation of spherocytes (Perrotta et al., 2008). Spherocytes show a characteristic loss of RBC deformability, leading to premature removal of these RBCs from the circulation (Perrotta et al., 2008). Hereditary elliptocytosis (HE) is a disorder characterized by disturbances of the horizontal cytoskeletal interactions and are usually caused by mutations in α-spectrin, β-spectrin and protein 4.1R. As the name suggests, the disturbed horizontal interactions lead to the formation of elliptocytes. The majority of HE patients are clinically asymptomatic, but few patients suffer from hemolysis, jaundice and splenomegaly (Da Costa et al., 2013).

### Secondary Disturbances of the Cytoskeletal Network

The triangular spectrin network, connected through ankyrin and protein 4.1R to band 3 supports membrane stability and contributes to RBC flexibility and deformability. Tyrosine phosphorylation of in the individual proteins regulates interaction forces between the elements of this network. Tyrosine phosphorylation is regulated by phosphotyrosine kinases and by phosphotyrosine phosphatases. Under normal conditions, protein regulatory tyrosine residues are only phosphorylated to a limited degree (Terra et al., 1998; Zipser et al., 2002). In the RBC membrane, phosphorylation of protein 4.1R, for example, leads to a reduced membrane stability (Muravyov and Tikhomirova, 2013), and phosphorylation of ankyrin regulates its binding to band 3 (Muravyov and Tikhomirova, 2013). The dissociation constant (Kd) between ankyrin and band 3 influences RBC deformability without a loss in elasticity, underlining that cytoskeletal protein phosphorylation has implications on RBC deformability (Anong, 2006).

The effect of phosphorylation of band 3, the most abundant membrane protein, on RBC deformability is not entirely clear. The possible physiological relevance of band 3 protein Tyr phosphorylation state emerges from the fact that it is controlled by hormones such as insulin (Marques et al., 2000) and that it is

altered in patients with hemoglobinopathies (Terra et al., 1998). Saldanha et al. (2007) showed that dephosphorylation of band 3 Tyr residues does not affect RBC deformability. It, therefore, appears that phosphorylation of band 3 does not affect RBC deformability directly, but may alter band 3 protein aggregation and its interaction with extracellular matrix components.

Lopes de Almeida et al. (2012) showed that high concentrations of fibrinogen in combination with dephosphorylated band 3, leads to mildly increased RBC deformability at low shear stress. In contrast to these findings, Reid et al. (1984) report decreased RBC deformability in diabetic patients with hyperfibrinogenemia in absence of dephosphorylating agents (Reid et al., 1984). The decreased RBC deformability in the absence of dephosphorylating agents seems to underline that phosphorylation is not involved. However, insulin is implicated in the activation of tyrosine kinases that in turn phosphorylate band 3. In diabetes types I and II, glucose and insulin homeostasis is disturbed leading to increased protein tyrosine phosphatase activities and aberrant band 3 phosphorylation patterns (Marques et al., 2000), potentially leading to reduced RBC deformability and increased RBC turnover. This same principle may also play a role in patients with sepsis, where investigators have shown that tyrosine phosphorylation of band 3 was increased in septic mice and that this increase was accompanied by reduced RBC deformability (Condon et al., 2007).

Band 3 phosphorylation was found to be increased by a rise in intracellular Ca2+-concentrations due to dissociation of phosphotyrosine phosphatase from band 3 (Zipser et al., 2002). Interestingly, sickle cell disease is characterized by an increase in both intracellular Ca2+ concentration (Bogdanova et al., 2013) and tyrosine phosphorylation of band 3 (Terra et al., 1998), which potentially could thus influence RBC membrane stability. The importance of band 3 phosphorylation for RBC shape and deformability is highlighted by a number of in vitro experiments. For example, morphological changes of RBCs were observed after Tyr-phosphorylation of band 3 mediated by pervanadate (Bordin et al., 1995). In contrast, Ser/Thr-phosphorylation of spectrin, which was selectively induced by okadaic acid did not result in morphological changes (Bordin et al., 1995). Unfortunately, the deformability of RBCs after pervanadate and okadaic treatment was not measured in these studies, but it seems reasonable to assume that RBC deformability is affected since both membrane protein phosphorylation and RBC morphology have been associated with altered RBC deformability (Reid et al., 1984; Chabanel et al., 1987; Hasegawa et al., 1993; Saldanha et al., 2007; Lopes de Almeida et al., 2012).

### RBC Hydration Changes Associated With Cytoskeletal Defects

Although reduced RBC deformability in HS mainly results from cytoskeletal disturbances (Perrotta et al., 2008), spherocytes experience dehydration and increased leakage of K+ (De Franceschi et al., 1997; Gallagher, 2017). To compensate for this increased K+ leakage, spherocytes are also found to have increased activity of the Na-K pump and the NKCC1 (also known as the Na-K-2Cl cotransporter, coded by gene SLC12A2) (Vives Corrons and Besson, 2001). RBCs of transgenic mice deficient for ankyrin or spectrin show increased activity of the Na-K pump, but normal activities of the NKCC1 and K+-Cl− cotransporters (Peters et al., 1999). This contrasts with protein 4.2-deficient mice, where Na-K pump activity is normal, while activities of NKCC1 and K+-Cl− cotransporters are increased (Peters et al., 1999). These differences in pump activities between protein 4.2 deficient and spectrin- or ankyrin-deficient mice support the concept that membrane proteins interact with ion channels of the RBC.

The RBC dehydration in HS also involves increased intracellular Ca2+ levels (Hertz et al., 2017). Increased intracellular Ca2+ correlates with decreased RBC deformability and RBC membrane stiffening (Bogdanova et al., 2013). For example, after in vitro ATP-depletion of HS RBCs, Ca2+ influx is increased compared to healthy RBCs (Shimoda et al., 1984). The detailed mechanisms beyond this Ca2+ influx have up to now not been identified and may include PIEZO1 or NMDA receptors both of which are activated by shear stress in "stiff " less deformable cells.

In protein 4.2−/− mice, displaying the HS phenotype, KCNN4 (or known as Gardos channels in RBCs) were noted to be functionally up-regulated, or at least more KCNN4 activity was measured (Peters et al., 1999; De Franceschi et al., 2005). This can be considered as a protective mechanism that leads to an outward flux of K+ and water from RBCs. This mechanism may compensate for the decreased surface-to-area ratio. Indeed, increased lysis was observed when these RBCs were exposed to KCNN4 (or Gardos-channel) inhibitors (De Franceschi et al., 2005). These findings indicate that KCNN4 is activated in HS in order to facilitate RBC dehydration, which maintains hemoglobin in RBCs at the expense of water. Recent research confirms the finding that RBC dehydration and formation of denser RBCs may protect the RBC in HS from lysis and premature uptake. We observed that mild HS is accompanied with prolonged RBC life-span, relatively mild reductions in RBC deformability and denser RBCs when compared with RBCs from patients with moderate/severe HS (Huisjes et al., 2017a). Most likely, the mild reduction in RBC deformability in patients with mild HS avoids premature splenic uptake and thus may provide the HS RBC time to lose membrane and to become dense.

### Red Cell Metabolism

### Primary Changes of Red Cell Metabolism

Mature RBCs of healthy individuals lack mitochondria and are therefore entirely dependent on the glycolytic pathway for the production of energy in the form of ATP. In the glycolytic pathway, glucose is converted to lactate by several enzymatic steps (**Figure 3**) (van Wijk and van Solinge, 2005; Koralkova et al., 2014). Key regulatory enzymes in the glycolytic pathway are HK, phosphofructokinase and PK. Hereditary metabolic disorders, or enzymopathies, are disorders impairing cellular energy production and balance, in particular ATP production. ATP is essential in the regulation of RBC deformability, viability, regulatory cascades involving phosphorylation and activity of ion transport involving Na, K-ATPase, and Ca

ATPase (Weed et al., 1969; Tuvia et al., 1998; Martinov et al., 2000; Fischer et al., 2003; Park et al., 2010; Makhro et al., 2016).

Adenosine triphosphate plays a key role in maintenance of ion gradients and should impact cytoskeletal structure and RBC shape (Tuvia et al., 1998; Park et al., 2010). However, glycolytic enzymopathies usually do not show altered RBC morphology (Koralkova et al., 2014; Huisjes et al., 2017b). For reasons that are not yet well understood, metabolically defective RBCs are prematurely removed by the spleen, causing chronic Hereditary Non-Spherocytic Hemolytic Anemia (HNSHA) (Koralkova et al., 2014).

Several observations point to mechanisms that may be involved in the premature clearance. RBCs from a patient with pyruvate kinase deficiency (PKD) were found to leak K+ more rapidly and were found to consume ATP at an accelerated rate (Nathan et al., 1965). This most likely contributes to the decreased RBC viability in this disease and could result in reduced RBC deformability (Leblond et al., 1978). A possible explanation for increased clearance of RBCs with metabolic defects is the

imbalance in Ca2+ uptake and extrusion (Bogdanova et al., 2013; Hertz et al., 2017). In agreement with the hypothesis that ATP deprivation will cause Ca2+ overload due to the inability to actively extrude Ca2+ from the cells by the Ca2+ pumps, decreased ATP levels in RBCs with metabolic defects were associated with abnormally high intracellular Ca2+ and exposure of PS, an important cellular 'eat-me' signal (de Back et al., 2014). Alternatively, defective metabolism may impact the redox state of RBCs making them more susceptible to oxidative stress (Cappellini and Fiorelli, 2008).

Despite the intuitive feeling that intracellular ATP levels should correlate with cellular deformability, evidence for this is so far inconclusive. This may be related to the techniques used. For example, research performed by Karger et al. (2012) did not show any correlation between intracellular ATP and deformability in RBC-concentrates intended for transfusion when assessing deformability by increasing shear stress. On the other hand, studies using viscosity and filterability assays did find correlations between intracellular ATP and deformability and showed that deformability is indeed dependent on intracellular ATP levels (Weed et al., 1969; Fischer et al., 2003). Atomic force microscopy studies also revealed increased membrane stiffness after ATP depletion due to reduced spectrin phosphorylation (Picas et al., 2013). We hypothesize that the different results from these experiments are possibly caused by the fact that ATP is not required for RBCs to elongate after the application of external forces, such as shear stress but ATP might be essential in the process of RBC deformation toward different shapes, as seen in filterability measurements.

#### Secondary Changes of Metabolism

Cyclic AMP (cAMP) is a second messenger and is derived from ATP. In nucleated human cells, cyclic AMP (cAMP) is synthesized from ATP by adenylate cyclase (AC) upon stimulation by G-protein coupled receptors (GPCR) and there is evidence that this process also occurs in human RBCs (Oonishi et al., 1997; Tuvia et al., 1999; Sprague et al., 2006; Kostova et al., 2015) (see **Figure 4**).  The activation of the erythrocyte β2-adrenergic receptor by catecholamines such as epinephrine (or adrenaline) affects RBC deformability (Tuvia et al., 1999). In vitro experiments with increased concentration of adrenaline, lead to increased membrane fluctuations and increased RBC filterability (Tuvia et al., 1999).

### **Intracellular mechanisms that sense and control cellular energy status in RBCs**

Phosphodiesterases tightly regulate levels of cAMP in RBCs (Adderley et al., 2011). PDEs catabolize the phosphodiesters in cyclic nucleotides, such as cAMP to AMP or cGMP to GMP. By inhibiting these PDEs, cyclic nucleotides remain intact and active. The reports of cAMP effects on RBC deformability are, however, ambivalent. Recently it was found that sildenafil, which is a PDE inhibitor and well-known for its function in the treatment of erectile dysfunction and pulmonary hypertension, has beneficial effects on RBC deformability in sickle cell disease at low concentrations in vitro. Increase in nitric monoxide bioavailability was suggested to improve RBC deformability (Grau et al., 2013; Kuhn et al., 2017). However, increased concentrations of sildenafil impaired sickle RBC filterability (Hagley et al., 2011). Typically, inhibition of cGMP-specific PDE type 5 (PDE-5) by sildenafil reduces the degradation of the second messenger cGMP to GMP and increases smooth muscles relaxation. In RBCs, this PDE-5 inhibition increases not only intracellular levels of cGMP but also preserves intracellular levels of cAMP via the inhibitor activity of cGMP on PDE-3 (see **Figure 5**) (Adderley et al., 2010; Knebel et al., 2013). The effect of increased cAMP levels was also studied using a spleen-like microfiltration system. Increased retention in the microfiltration system, reflecting decreased deformability, was observed after

incubation of RBCs with sildenafil (Ramdani et al., 2015). Moreover, the production of cGMP from GTP is synthesized by guanylate cyclase (Derbyshire and Marletta, 2012). Guanylate cyclase is activated by nitric oxide (NO) and the addition of sodium nitroprusside to RBCs as an artificial nitric oxide donor does prevent Ca2+ influx and Ca2+-mediated stiffness of RBCs (Barodka et al., 2014).

In addition, the effect of cAMP on the release of ATP is still under debate. A role for cAMP in the release of ATP to its extracellular environment via PKA was proposed (Sprague et al., 2007; Adderley et al., 2011; Knebel et al., 2013) (see **Figure 4**). Others, however, have attributed the release of ATP solely to hemolysis of the RBC (Sikora et al., 2014) or to release via transporters such as Pannexin 1 (Locovei et al., 2006) or PIEZO1 (Cinar et al., 2015).

Recently AMPK was also found to be present in RBCs (Thali et al., 2010). The exact function of AMPK in RBCs is unknown, however, in other cells, AMPK is essential to balance ATP consumption and ATP production. In muscles and fat AMPK regulates glucose uptake (Mihaylova and Shaw, 2011). Likely, AMPK also regulates these processes in RBCs, as RBCs from AMPK−/− mice were less viable after in vitro glucose depletion (Föller et al., 2009). Interestingly, these AMPK−/− mice had increased reticulocytes count, decreased RBC deformability, increased osmotic fragility, increased spleen size and decreased RBC lifespan (Foretz et al., 2010; Wang et al., 2010). This indicates a significant contribution of AMPK in maintaining RBC function and viability. AMPK is an energysensing enzyme, which is activated by low AMP: ATP ratios and inhibited by high AMP: ATP ratios (Wang et al., 2003). Under low ATP concentrations, AMP and ADP can bind to AMPK and change its conformation, thereby preventing the protein from activation by phosphorylation (Mihaylova and Shaw, 2011). Chemical agents that increase intracellular levels of cAMP are forskolin and isobutyl-methyl-xanthine (IBMX). Forskolin activates AC and thus leads to increased formation cAMP from ATP, while IBMX inhibits PDEs and maintains cAMP levels. Both forskolin and IBMX lead to AMPK inhibition, which reveals a molecular interaction between cAMP and AMPK activity (Hurley et al., 2006). This interaction could play a role in altered deformability in patients suffering from glycolytic enzymopathies. The low RBC ATP concentrations in these patients would lead to low levels of cAMP and increased intracellular levels of non-phosphorylated (inactive) AMPK.

Phosphorylation of AMPK may also be involved in G6PDdeficiency. G6PD deficiency is the most common RBC enzyme deficiency with over 400 million affected people (Cappellini and Fiorelli, 2008). Deficiency of this enzyme is associated with decreased ability of the RBC to withstand oxidative stress (Cappellini and Fiorelli, 2008; Mangat et al., 2014; Tang et al., 2015). Exposure of normal and G6PD-deficient RBCs to the oxidant diamide leads to a decrease in RBC deformability of the G6PD-deficient RBCs due to the inability of the RBC to maintain the intracellular glutathione-pool (Tang et al., 2015). Depletion of glutathione also leads to activation of AMPK by phosphorylation in G6PD-deficient RBCs. The activation of AMPK in G6PDdeficient RBCs after exposure to oxidative stress is most likely a consequence of ATP-consuming compensation mechanisms after excessive glutathione loss (Tang et al., 2015).

Protein kinase C alpha (PKCα) possibly also regulates RBC deformability. PKCα is involved in the phosphorylation of proteins in the RBC, such as adducin, protein 4.1R and protein 4.9 (George et al., 2010). Moreover, PKCα is involved in stimulation of glucose uptake by phosphorylation of the glucose transporter and participates in the activity of calcium channels (Kanno et al., 1995; George et al., 2010; Wagner-Britz et al., 2013). Consequently, phosphorylation and dephosphorylation of these proteins can be altered upon depletion of glucose. Glucose depletion was found to activate PKCα and this is accompanied by an increase in intracellular Ca2+ and, consequently, exposure of the membrane phospholipid PS (Klarl et al., 2006). The phosphorylation of adducin and protein 4.1R by PKCα could change RBC behavior and could influence RBC deformability (George et al., 2010) in a way similar to what is seen, for example, after phosphorylation of band 3 (Saldanha et al., 2007). Interestingly, homology analysis between murine, rat and human showed a conserved region in both liver- and RBC-specific PK for a PKCα binding site (Kanno et al., 1995). PKCα regulates RBC membrane stability and this conserved region for PK indicates a possible role for PK in this process (Kanno et al., 1995). In addition, PK converts phosphoenolpyruvate (PEP) to pyruvate (**Figure 3**) and previously it has been recognized that phosphorylation of PK by cAMP-dependent protein kinases leads to a reduced affinity of PEP for PK, which leads to a reduced production of ATP (Marie et al., 1980). The decreased cAMP and ATP production in metabolic disorders of the RBC, such as PK-deficiency, could subsequently result in disturbed RBC deformability (Leblond et al., 1978).

### **Role of purines and pyrimidines in RBC deformability**

It has been suggested that pools of ATP are intracellular enclosed by the membrane proteins ankyrin, β-spectrin, band 3, and GAPDH, serving as substrates for both the Na+, K+-, and Ca2+ -pumps (Chu et al., 2012). This indicates an interplay between membrane proteins, energy stores and ion channel function of the RBC. Interestingly, the chemotherapeutic agent 5-FU was shown to induce changes in RBC rigidity, morphology and ion balance, most likely by altering ATP levels in the RBC (Spasojevic et al., 2005 ´ ). 5-FU is a pyrimidine antagonist (Longley et al., 2003) and since purine and pyrimidines play key roles in cellular metabolism and energy homeostasis. 5-FU could, therefore, influence RBC deformability. The activating and feedback mechanisms of purines and pyrimidines on glycolytic enzymes have previously been described by Seider and Kim (1979) and Tomoda et al. (1982). For example, in bovine RBCs incubated with glucose, adenosine stimulated ATP production compared to incubation with glucose alone. Most likely, adenine (i.e., the nucleobase of adenosine) stimulates HK activity (Seider and Kim, 1979). The effect of pyrimidines in RBCs has also been studied in two patients with a pyrimidine 50 -nucleotidase (P5N) deficiency, which lack this enzyme involved in clearance of pyrimidines from the RBC. P5N deficiency increases intracellular concentrations of pyrimidine nucleotides, which eventually leads to hemolytic anemia (Vives L Corrons, 2000). P5N-deficient patients were also found to have increased levels of reduced glutathione (Tomoda et al., 1982). At the same time, pyrimidine 5 0 -nucleotides (such as cytidine mono-, di-, and triphosphate (CMP, CDP, CTP) or uridine mono-, di-, and triphosphate (UMP, UDP, UTP)) decreased the activity of glucose-6-phosphate dehydrogenase (G6PD, **Figure 3**). As mentioned earlier G6PD activity is crucial in the anti-oxidative defense of RBCs. Indeed, RBCs from P5N-deficient patients were found to be more susceptible to oxidative stress, as reflected by increased formation of Heinz bodies (Tomoda et al., 1982), even despite increased reduced glutathione concentrations. Besides their role as a morphological marker of oxidative stress, Heinz bodies can lead to a decreased deformability by themselves (Hasegawa et al., 1993).

### Hemoglobin

#### Primary Changes of Hemoglobin

Hemoglobin is the main component of the RBC and responsible for the delivery and removal of oxygen and carbon dioxide to and from the tissues, respectively. Intracellular hemoglobin concentrations and its state (polymerization, crystallization, degradation, and oxidation) also defines cytosolic viscosity making up 19.9–22.3 mmol/L in cells of healthy humans. Hemoglobin is composed of two α- and β-hemoglobin molecules together composing a heterotetramer. Disorders of hemoglobin can be subdivided into hemoglobinopathies (e.g., sickle cell anemia) and thalassemias (α- and β-thalassemia) (Galanello and Origa, 2010; Harteveld and Higgs, 2010; Higgs et al., 2012; Ware et al., 2017).

Sickle cell anemia is caused by a single point mutation in the HBB gene at position 6 substituting glutamic acid to valine (HbS). This substitution causes formation of HbS polymers of deoxygenated hemoglobin. This transition is usually rapidly reversed upon reoxygenation, but induces progressive damage of membrane driving dehydration to the extreme to the state when HbS polymers do not dissociate as its concentration exceeds it solubility threshold. Destabilization of the membrane and decrease in deformability leads to intravascular hemolysis and to vaso-occlusive events (Ware et al., 2017). Sickle RBCs are poorly deformable (Alapan et al., 2015, 2016) and this is, in part, due to changes in hydration status of the RBC.

In α- and β-thalassemia, α- and β-hemoglobin chains are affected, respectively, which leads to an imbalance in the synthesis of globin chains and to an inability to form sufficient quantities of hemoglobin heterotetramers. The imbalanced synthesis in thalassemia leads to the formation of hemoglobin precipitates, so-called Heinz bodies. RBCs in α-thalassemia show increased hydration (Bunyaratvej et al., 1994; Chui et al., 2003), whereas the hydration of RBCs in β-thalassemia is either decreased or increased (Bunyaratvej et al., 1994; Brugnara and Mohandas, 2013). Both Heinz bodies and the altered hydration state of the RBC are known to impair RBC deformability (Clark et al., 1983; Losco et al., 2001).

### Oxygenation of Hemoglobin

The main function of RBCs is to transport oxygen to the tissues in the human body. The oxygenation state of hemoglobin is known to influence various processes of the RBC (Gibson et al., 2000; Stefanovic et al., 2013). Several ion transporters are oxygen sensitive, such as K+-Cl− cotransporter (KCC1) and the NKCC1 (Na-K-2Cl cotransporter) (Bogdanova et al., 2009). The function of band 3 seems, however, unaltered upon deoxygenation and oxygenation (Gibson et al., 2000). On the other hand, hemoglobin binds the cytosolic domain of band 3 and this binding is regulated by pH (Eisinger et al., 1982; Chétrite and Cassoly, 1985). Deoxygenation reduces the binding of ankyrin to band 3 and dissociation leads to more freely diffusible band 3 (Stefanovic et al., 2013). In addition, the binding between ankyrin and band 3 is also regulated by 2,3-disphosphoglycerate (2,3-DPG) (Moriyama et al., 1993). 2,3-DPG promotes the dissociation of oxygen from hemoglobin and promotes the release of oxygen from the RBC to the tissues (van Wijk and van Solinge, 2005). In the oxygenated state 2,3-DPG is not bound to hemoglobin and leads to less deformable and more fragile RBCs, while in the deoxygenated state 2,3-DPG is bound to hemoglobin and leads to an increase in RBC deformability and less fragile RBCs (Moriyama et al., 1993). The exact role of the reduced binding between ankyrin and band 3 after deoxygenation and binding of 2,3-DPG to hemoglobin is currently unknown, although it is hypothesized that a mild membrane weakening is beneficial in deoxygenated capillaries to facilitate smooth traveling of the RBC (Stefanovic et al., 2013).

### RBC Hydration Changes Associated With Hemoglobin Defects

#### **RBC dehydration in sickle cell anemia**

The RBC dehydration in sickle cell anemia is caused by facilitated K + loss through hyperactivated KCNN4 (also known as the

Gardos Channel in RBCs) and K+-Cl− cotransporter that is not compensated by uptake of equal amounts of Na+ (Clark et al., 1982; Izumo et al., 1987). Regardless of the ion transport pathway involved, RBC dehydration raises the HbS concentration, thereby affecting the equilibrium of HbS-polymerization and depolymerization in favor of the polymerized version (Eaton and Hofrichter, 1990; Brugnara, 1993). Na/K/ATPase was found to be more active in RBCs from sickle cell disease patients and can contribute to RBC dehydration (Izumo et al., 1987). Dehydration of RBCs in sickle cell disease contributes significantly to the decreased deformability since dehydration promotes the probability of polymerization of HbS by 20–40-fold (Eaton and Hofrichter, 1990) and result in prolonged polymerization of hemoglobin and thus prolonged sickling of RBCs (Brugnara, 1993). Sickled RBCs are far less deformable.

Two mechanisms involved in abnormally high K+ loss are presented in **Figure 6**. The first mechanism of K+ loss involves the ubiquitously expressed K-Cl co-transporter (KCC), of which the isoforms KCC1, KCC3, and KCC4 are expressed in human RBCs (Crable et al., 2005).

KCC1, is most often studied in RBCs and referred to as KCC. KCC in RBCs are known to be regulated by intracellular pH, Mg2+ concentrations as well as volume and oxygenation states of hemoglobin (Adragna et al., 2004; Khan et al., 2004). Under physiological conditions, KCC activation restores cell volume after swelling, but KCC activation may also respond to the changes in the RBC redox state (Adragna et al., 2004). In healthy RBCs, KCC is oxygen sensitive but only activated at high oxygen tension (pO2). At low oxygen tension, KCC becomes sensitive to other stimuli (Gibson et al., 1998, 2001; Muzyamba et al., 2006). In sickle cell disease, in contrast, KCC is also activated at low oxygen tension, most likely due to increased phosphorylation of KCC in sickle RBCs (Muzyamba et al., 2006). In addition, KCC activity is intrinsically higher, even in older sickle RBCs (Bize et al., 2003).

The loss of deformability caused by the formation of HbSpolymers at low oxygen tensions (Odièvre et al., 2011) is enhanced by the KCC activation and leads to additional cell shrinkage. The clinical relevance of KCC activity is emphasized by observations in individuals with mild sickle cell disease. In these patients, an increased K-Cl cotransport activity is associated with increased likelihood of hospitalization, because of acute vaso-occlusive problems caused by less deformable dehydrated RBCs (Rees et al., 2015).

One more trigger of K+ leak from RBCs is the increase in the intracellular Ca2+ levels and the activation of Ca2+ sensitive K+ channels (KCNN4 or known as the Gardos channel in RBCs) (Gallagher, 2017). These channels driving K + loss and dehydration are hyperactivated in RBCs of sickle cell disease patients secondary to the high intracellular Ca2+ levels (Bookchin and Lew, 1980; Bogdanova et al., 2013) (see **Figure 6**). KCNN4 is expressed in various cell types (Gallagher, 2013) and are inhibited by imidazole antimycotics, such as clotrimazole (McNaughton-Smith et al., 2008). In sickle cell disease, KCNN4 (or known as the Gardos channel in RBCs) is activated by two pathways. Firstly, KCNN4 can be activated by a signaling cascade initially triggered by several cytokines, such as PAF, interleukin-10 (IL-10) and endothelin 1 (ET-1) (Rivera, 2002) also involving activation of PKCα (Rivera et al., 1999; Wagner-Britz et al., 2013). In addition, PAF and ET-1 concentrations are increased in plasma from patients with sickle cell disease and are assumed to contribute to the adhesion of sickle RBCs to endothelium that is responsible for the vaso-occlusive events (Rivera, 2002). Also, these cytokines generate denser RBCs through RBC dehydration after an oxygenation/deoxygenation cycle (Rivera, 2002). Secondly, sickle cell disease patients have increased expression of the NMDAreceptor on their RBC membranes, causing pathological influx of Ca2+ after stimulation with receptor agonists (such as glycine and homocysteic acid) (Hänggi et al., 2014) which could contribute to dehydration mediated by KCNN4. Currently, a clinical trial using memantine as a NMDA-receptor antagonist is being tested in sickle cell disease patients (Bogdanova et al., 2017). In addition, the KCNN4 (or Gardos channel) blocker senicapoc (ICA-17043) has recently been tested in sickle cell disease. Senicapoc increased hemoglobin concentrations and hematocrit, and improved RBC hydration. Despite these encouraging findings, this study has been terminated prematurely because senicapoc did not meet its primary efficacy endpoint, defined as a decrease in painful crises (Ataga et al., 2011). From the clinical results obtained with senicapoc it can be hypothesized that the RBC dehydration and changes in RBC hydration after treatment with senicapoc are not causally related to the vaso-occlusive events which are often observed in sickle cell anemia.

Another pathway that increases intracellular Ca2+ levels in sickle cell disease is the mechanosensitive PIEZO1 channel (also described at section "Primary Changes of RBC Hydration"). PIEZO1 channels are widely expressed in vertebrates across various cell types and in RBC these channels are responsible for regulation of volume homeostasis upon mechanical signals (Zarychanski et al., 2012; Ge et al., 2015). Sickle RBCs show increased permeability for Ca2+ and Mg2+ upon deoxygenation. This increased permeability results in an increased deoxygenation-induced cation conductance in sickle cell disease, which can be entirely blocked by GsMTx4 (Ma et al., 2012; Cahalan et al., 2015). The gating modifier GsMTx4 blocks the mechanically sensitive part of PIEZO1 that supports a closed state of this ion channel (Bae et al., 2011), preventing Ca2+ influx (Jacques-Fricke et al., 2006).

#### **RBC hydration changes in thalassemia**

Three increasingly severe phenotypes can be distinguished in β-thalassemia, i.e., β-thalassemia minor, intermedia and major (Higgs et al., 2012). Intracellular concentrations of Na+ in RBCs from patients with β-thalassemia minor and β-thalassemia major are comparable with those in RBCs from healthy adults, but K+ levels are slightly increased (Cividalli et al., 1971). Upon in vitro incubation at 37◦C, RBCs from patients with β-thalassemia major show increased leakage of K+ and elevation of intracellular Na+ levels when compared with healthy RBCs. In RBCs from β-thalassemia minor patients, leakage of K+ and intracellular Na+ levels are comparable to the healthy RBCs after in vitro incubation at 37◦C (Cividalli et al., 1971).

Leakage of K+ from RBCs in thalassemia is possibly linked to the precipitation of excess α- or β-globin chains inside the RBCs, which is a characteristic feature of this disease. The precipitated globin chains in RBCs can lead to oxidative stress and can affect transport of Na+ and K+ in thalassemia RBCs (Olivieri et al., 1994). In both β-thalassemia and α-thalassemia, RBCs show increased efflux of K+ (Olivieri et al., 1994). Moreover, the amount of hemoglobin aggregates in RBCs is correlated with K+ efflux (Nathan and Gunn, 1966). The presence of these aggregates reduces RBC deformability (Lubin and Desforges, 1972; Hasegawa et al., 1993) thereby revealing correlation between K+ fluxes, Heinz body formation, and reduced RBC deformability. The precipitation of globin chains and leakage of K + from RBCs in thalassemia is also linked, or may even be augmented, by overload of Ca2+ (Shalev et al., 1984; Bookchin et al., 1988).

Another important factor in K+ loss from RBCs in β-thalassemia involves the K+-Cl− cotransporter, which can be affected by oxidative stress (Olivieri et al., 1994). This K+ loss by the K+-Cl− cotransporter is inhibited by increased intracellular concentrations of Mg2+ (De Franceschi et al., 1998; Adragna et al., 2004). In a trial with β-thalassemia intermedia patients, dietary Mg2+ supplementation did lead to increased intracellular concentrations of Mg2+ and to a reduction in the activities for the K+-Cl− cotransporter and Na-K pump (Na/K/ATPase). However, Mg2+ did not influence the activities of NKCC1 (also known as the Na-K-Cl cotransporter). Although dietary Mg2+ supplementation in β-thalassemia patients does not influence the hemoglobin concentration in blood, it does lead to a significant decrease in the absolute reticulocyte number. This is possibly due to improved RBC survival time (De Franceschi et al., 1998).

Little research has been performed on KCNN4 (or also known as the Gardos channel in RBCs) in thalassemia. An exception is the work on a mouse model with a homozygous deletion in the β-globin chain (de Franceschi et al., 1996). These mice were treated with clotrimazole, an antifungal imidazole derivate with KCNN4 blocking properties. During treatment with clotrimazole, hemoglobin concentrations in blood remained constant. In addition, it caused a decrease in MCHC whereas hematocrit and intracellular K+ levels increased indicating that hydration state of the RBC was restored. Combination of clotrimazole with erythropoietin administration did increase hemoglobin levels in these mice to a higher extent than erythropoietin alone, possibly by promoting proliferation and differentiation during erythropoiesis (de Franceschi et al., 1996).

### RBC Vesiculation

Intracellular levels of Ca2+ tightly regulate RBC vesiculation and elevated levels of intracellular Ca2+ are known to induce PS exposure and RBC vesiculation (Bevers et al., 1992; Nguyen et al., 2011; Fens et al., 2012; Alaarg et al., 2013) (**Figure 7**).

The RBC-vesiculation is a physiological process and leads to reduced RBC deformability. During its 120-day life span, RBC lose membrane surface and hemoglobin content through vesiculation (Werre et al., 2004; Ciana et al., 2017) a process in particular prominent in the youngest RBCs (Greenwalt and

Dumaswala, 1988). As membrane is shed, this leads to a decreased surface-to-volume ratio in the RBC. During this RBC aging, hemoglobin-free vesicles are shed which leads to RBC dehydration and older RBCs with increased MCHC (Bosch et al., 1994; Bosman, 2013). Altogether, this leads to a reduction in membrane elasticity and RBC deformability (Linderkamp et al., 1993; Bosch et al., 1994). This implies that there is an important role for vesiculation in RBC deformability.

While RBC-vesiculation is considered a physiological process, RBC vesiculation is increased in various forms of hereditary hemolytic anemia, such as HS and sickle cell disease (Perrotta et al., 2008; Alaarg et al., 2013). In HS, differences in deformability between spectrin/ankyrin-deficient and band 3-deficient RBCs were observed after splenectomy. While splenectomy prevented premature removal of young RBCs from the circulation in both groups, the loss of deformability during RBC-aging was delayed after splenectomy in spectrin/ankyrin-deficient RBCs. This contrasts with band 3-deficient RBCs, where splenectomy did not lead to a delay in the deformability decrease during RBC-aging. This could be explained by the fact that spectrin/ankyrin-deficient RBCs are more prone to shed band 3-containing vesicles. Clustered band 3 is known to induce binding of autologous IgG, which facilitates removal of RBC by macrophages (Kay et al., 1983; Kay, 1984; Willekens et al., 2008). Shedding of band 3-containing vesicles from spectrin/ankyrindeficient RBCs, therefore, would avoid IgG-opsonisation and clearance of the RBC (Reliene et al., 2002).

Red blood cell vesiculation is suggested as a mechanism to protect the cell from removal from the circulation. By vesiculation, the RBC can shed "eat-me" signals such as PS and specific band 3 cleavage products that react with senescent antigens (Willekens et al., 2008). By shedding these "eat-me" signals, the RBC may escape clearance until the reduction in RBC deformability causes trapping in the spleen. In addition, the lipid bilayer of the RBC is a complex system with various microdomains. These microdomains of the RBC, or so called 'lipid rafts' are specific membrane parts with high concentrations of cholesterol, sphingomyelin and gangliosides (Pike, 2003). These specific lipid rafts have a high abundance of the membrane proteins stomatin, flotillin-1, flottilin-2 (Salzer and Prohaska, 2001) and band 3 (Cai et al., 2012). RBC vesicles shed during storage are enriched in the lipid raft marker stomatin (Salzer et al., 2008) and indicates that specific lipid domains are shed during RBC vesiculation (Leonard et al., 2017).

The relationship between vesiculation and deformability is nicely illustrated by an experiment where chlorpromazine is added to RBCs to stabilize the RBC membrane. During overnight incubation with chlorpromazine at 37◦C in a glucose-free buffer, vesiculation is inhibited and RBC deformability is maintained. Without chlorpromazine strong vesiculation of RBCs under these conditions occur as PS is exposed on the RBC surface. The activity of chlorpromazine may be explained by the amphiphilic properties of the molecule directly affecting the RBC membrane (Bütikofer et al., 1989).

High-throughput screening with chemical compound libraries also revealed that RBC-vesiculation can be driven by drugs and chemical compounds. For example, vesiculation of RBCs is driven by certain kinase pathways, including Jak-STAT and protein kinase B. Increased RBC vesiculation is also observed after addition of paclitaxel to blood, probably through its formulation excipient containing Cremophor. Not only RBC vesiculation is influenced by the vehicle Cremophor, but the compound also increases whole blood viscosity and transforms RBC to stomatocytes (Vader et al., 2013).

### Adaptive Responses

Exercise leads to increased heart rate as a response to provide tissues with sufficient amount of oxygen. Exercise is accompanied

with several stress factors that may affect RBC deformability, such as shear stress, hyperthermia, and glucose consumption (Carlson and Mawdsley, 1986; Szygula, 1990; Smith, 1995; Mairbäurl, 2013). These stress factors can lead to mechanical rupture, stimulated erythropoiesis and can decrease the average RBC age in athletes (Mairbäurl, 2013).

In a study with 24 trained cyclists, RBC deformability was decreased directly after exercise (Oostenbrug et al., 1997). The biology beyond the decrease in RBC deformability is unknown, although increased blood flow may be involved. The change in deformability after exercise can be the results of cytoskeletal changes, such as membrane loss. In addition, decreased levels of haptoglobin and increased levels of bilirubin were observed after marathon races (Jordan et al., 1998; Simpson et al., 2006). On the other hand, RBC deformability seems to be generally increased in well-trained athletes (Mairbäurl, 2013). A study comparing RBC membrane fluidity with the RBC membrane composition in controls and runners observed increased RBC membrane fluidity in endurance runners and sprinters. Although the intake of nutrients was comparable between the running groups and the control group, endurance running was accompanied with reduced concentrations of saturated fatty acids in RBC membranes (Kamada et al., 1993). These results indicate that the RBC can adapt upon exposure to exercise and that these adapted RBCs facilitate exercise and proper delivery of oxygen to the tissues. Interestingly, Smith et al. (1999) observed a higher maximal RBC deformability in elite cyclists when compared with sedentary healthy controls. Also, RBC populations with lower RBC densities were observed in the elite cyclists, which could indicate increased RBC turnover (Smith et al., 1999). A possible explanation for the increased RBC deformability in elite cyclist could be that extreme exercise leads to membrane loss and early RBC uptake, which is likely to be compensated with relatively young and good deformable RBCs. Whether increased RBC deformability would lead to better sports performances is unknown.

Autologous erythropoietin production is stimulated upon exposure to high altitudes. Acute exposure to high altitude, however, does not affect RBC deformability and RBC rheology (Reinhart et al., 1991). The effects of chronic exposure to high altitude seems largely unexplored. RBC deformability related processes were investigated in RBC concentrates obtained from Tibetans living at high altitude and from Tibetans at living at lowland. RBC viscosity was increased, and RBCs were more osmotic fragile in Tibetians living at low altitude when compared to RBC concentrates obtained from their lowland residents (Zhong et al., 2015). The increased viscosity can, however, be affected by increased hemoglobin levels and the decreased osmotic fragility can be affected by the decreased MCV values in Tibetans living at high altitude. The effects of exercise under hypoxia on RBC properties were investigated by Mao et al. (2011). Exercise under hypoxic conditions was found to decrease KCNN4 (or Gardos-channel) modulated deformability and KCNN4 modulated volume and down-regulated the senescence markers CD47 and CD147 (Mao et al., 2011). These results indicate RBC dysfunction after exercise under hypoxia. The dysfunction of RBCs under hypoxic conditions in combination with the erythropoietin-driven increase in RBC production leads to RBCs with relative low densities (Samaja et al., 1993), possibly caused by both increased RBC turnover and increased RBC production.

TABLE 2 | Summary of primary and secondary changes that lead to reduced RBC deformability in hereditary hemolytic anemia.

Reduced RBC deformability is noted in the table with ↓.

### SUMMARY

fphys-09-00656 June 1, 2018 Time: 12:28 # 16

Deformability is an important parameter that regulates RBC rheology, its longevity, and efficacy of O2 transport. Altered deformability is a characteristic feature of multiple forms of hereditary hemolytic anemias and is likely related to the severity of the disease. Factors regulating deformability at the cellular level are dehydration, membrane protein phosphorylation, cytoskeletal integrity, metabolism and the integrity of hemoglobin. Interaction of these factors makes RBCs more or less deformable. Measuring RBC deformability in research and diagnostic laboratories can be challenging as there are many techniques available with all their specific advantages and disadvantages.

Measuring RBC deformability is important from both a diagnostic and research point of view. RBC deformability (i) could provide information about the patient's disease and clinical severity, and (ii) could be a target for pharmacological intervention or predict the toxicity of drugs for patients with hemolytic anemias.

Reduced RBC deformability leads to an inability of the RBC to pass the splenic circulation and leads to premature removal of RBCs from the blood. Altered RBC deformability can be attributed to primary and secondary changes of RBC deformability. Primary changes of RBC deformability are directly related to the disease, such as the membrane weakening in HS or the formation of poorly deformable sickle cells in sickle cell disease. Secondary changes of RBC deformability, such as altered ion fluxes, aberrant membrane protein phosphorylation or RBC vesiculation, are not directly related to the cause of disease. Thus, both primary and secondary causes of RBC deformability can contribute to premature uptake of RBCs in the spleen. In this review and in **Table 2**, we have summarized the current knowledge on primary and secondary mechanisms of RBC

### REFERENCES

deformability in sickle cell anemia, thalassemia, HS, hereditary xerocytosis, and a number of metabolic disorders of the RBC. We have addressed and discussed the effects of ion regulation, ion channels and RBC (de)hydration on RBC deformability. In addition, we discuss the role and current knowledge of the adaptive responses on RBC deformability, intracellular energy-sensing molecules, membrane protein phosphorylation, hemoglobin deoxygenation and RBC vesiculation on RBC deformability. Knowledge about these processes in the RBC will lead to better understanding of the secondary processes involved in premature removal, and could lead to the discovery of new targets for pharmacological treatment. Furthermore, we postulate that a number of currently undiagnosed, patients with hereditary hemolytic anemia may have (genetic) defects in the here discussed secondary pathways or secondary processes that regulate RBC deformability. This review helps to understand the molecular mechanisms that maintain RBC deformability in healthy and diseased individuals, and enumerates the molecular mechanisms that are altered in RBC disorders leading to hereditary hemolytic anemia.

### AUTHOR CONTRIBUTIONS

RvW, RS, and WvS encouraged RH to investigate the properties of RBC deformability. RH and RvW wrote the manuscript with support from RS, AB, LK, and WvS.

### FUNDING

This research has received funding from the European Seventh Framework Program under grant agreement number 602121 (CoMMiTMenT).

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complement regulatory proteins CD55 (DAF) and CD59 (MACIF) on red blood cells. Eur. J. Appl. Physiol. 99, 201–204. doi: 10.1007/s00421-006-0326-2

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Huisjes, Bogdanova, van Solinge, Schiffelers, Kaestner and van Wijk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease

Joames K. F. Leal, Merel J. W. Adjobo-Hermans and Giel J. C. G. M. Bosman\*

Department of Biochemistry, Radboud University Medical Center, Nijmegen, Netherlands

Red blood cells (RBCs) generate microvesicles to remove damaged cell constituents such as oxidized hemoglobin and damaged membrane constituents, and thereby prolong their lifespan. Damage to hemoglobin, in combination with altered phosphorylation of membrane proteins such as band 3, lead to a weakening of the binding between the lipid bilayer and the cytoskeleton, and thereby to membrane budding and microparticle shedding. Microvesicle generation is disturbed in patients with RBC-centered diseases, such as sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, spherocytosis or malaria. A disturbance of the membranecytoskeleton interaction is likely to be the main underlying mechanism, as is supported by data obtained from RBCs stored in blood bank conditions. A detailed proteomic, lipidomic and immunogenic comparison of microvesicles derived from different sources is essential in the identification of the processes that trigger vesicle generation. The contribution of RBC-derived microvesicles to inflammation, thrombosis and autoimmune reactions emphasizes the need for a better understanding of the mechanisms and consequences of microvesicle generation.

#### Edited by:

Lars Kaestner, Saarland University, Germany

### Reviewed by:

Richard Van Wijk, Utrecht University, Netherlands Michel Prudent, Transfusion Interrégionale CRS SA, Switzerland Pablo Martín-Vasallo, Universidad de La Laguna, Spain

> \*Correspondence: Giel J. C. G. M. Bosman<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

Received: 21 February 2018 Accepted: 22 May 2018 Published: 08 June 2018

#### Citation:

Leal JKF, Adjobo-Hermans MJW and Bosman GJCGM (2018) Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease. Front. Physiol. 9:703. doi: 10.3389/fphys.2018.00703 Keywords: red blood cell, microvesicles, membrane, aging, inflammation, autoimmunity, hemoglobin, oxidation

## INTRODUCTION

Generation of microvesicles, i.e., extracellular vesicles that are shed from the plasma membrane (Raposo and Stoorvogel, 2013), constitutes an integral part of red blood cell (RBC) homeostasis, and is responsible for the loss of 20% of the hemoglobin and the cell membrane during physiological RBC aging in vivo, and the accompanying decrease in cell volume and increase in cell density (Willekens et al., 2003, 2008). The blood of healthy subjects contains approximately 1000 RBC-derived vesicles per microliter of plasma (Berckmans et al., 2001; Hron et al., 2007; Westerman et al., 2008; Willekens et al., 2008). The microvesicle hemoglobin composition suggests an enrichment of the irreversibly modified hemoglobins HbA1c and HbA1e2. Microvesicles contain various immunological recognition and removal signals (Willekens et al., 2008), that are responsible for a rapid elimination – probably within minutes – from the circulation (Willekens et al., 2005, 2008). Shedding of damaged cellular components by vesiculation prevents untimely removal of otherwise functional RBCs, as well as unwanted reactions of the hemostasis and immune systems. Thus, vesiculation couples general aging

processes such as oxidation and glycation to organismal homeostasis. On the other hand, RBC-centered hemoglobinopathies such as sickle cell disease and thalassemia are accompanied by a substantial increase in microvesicle levels (Ferru et al., 2014; Camus et al., 2015). Also, infection of RBC with the malaria parasite Plasmodium induces vesicle formation. However, the underlying mechanism is likely to be strongly influenced by parasite-derived proteins, and therefore beyond the scope of this review (e.g., Mantel et al., 2013). Increased vesiculation is associated with systemic inflammation, which may be directly responsible for hemolysis and anemia (Dinkla et al., 2012b, 2016). Thus, microvesicles are part of the complex of interactions between RBCs and the organism (Bosman, 2016a,b). In this review, we aim to integrate the newest data on microvesicle composition and production in various conditions, in order to obtain more insight into the basic mechanisms underlying microvesicle generation, and the involvement of RBC-derived microvesicles in pathophysiology.

### MICROVESICLES IN VIVO

Immunochemical analyses together with a proteomic inventory of RBC-derived microvesicles from the plasma of healthy individuals show the almost exclusive presence of the membrane proteins band 3 and actin (Bosman et al., 2012). Microvesicles are enriched in enzymes involved in redox homeostasis, i.e., glutathione S-transferase, thioredoxin and peroxiredoxin-1 and peroxiredoxin-2, and in ubiquitin. The hemoglobin composition of microvesicles isolated from plasma resembles that of the oldest cells, with an enrichment in irreversibly modified hemoglobins HbA1c and HbA1e2 (Willekens et al., 1997). Microvesicles display removal signals such as phosphatidylserine in the outer membrane layer and senescent cell-specific band 3 epitopes that are mainly found in the oldest RBCs (Willekens et al., 2008). Also, microvesicles contain the glycosylphosphatidylinositol (GPI)-anchored, complement-inhibiting proteins CD55 and CD59. These may not be in a functional configuration, as recently shown for the, similarly GPI-anchored enzyme acetylcholinesterase (Willekens et al., 2008; Freitas Leal et al., 2017). The striking absence of spectrin and ankyrin in microvesicles, together with immunoblot and proteomic patterns indicating extensive protein degradation in the oldest RBCs, as well as the aging-related increase in membrane-associated proteasome components (Bosman et al., 2012), all support a role for proteolytic breakdown of the band 3-ankyrin connection between the cytoskeleton and the lipid bilayer in the vesiculation process. However, the absence of especially spectrin in microvesicles isolated from the blood has led to alternative explanations for the aging-associated changes in RBC cell volume and density (Ciana et al., 2017a,b). The breakage of the band 3-ankyrin binding is predicted to cause a relaxation of the cytoskeletal spring and thereby spontaneous buckling of the lipid bilayer, resulting in evagination and vesiculation (Sens and Gov, 2007; Bosman et al., 2012) (**Figure 1**). Proteomic analysis of aging RBCs and RBC-derived microvesicles purified from the plasma support this model (Bosman et al., 2012), but do not provide many clues for the upstream processes, or for the relatively high concentration of actin in the microvesicles. The hemoglobin data, together with the accumulation of redox status-regulating enzymes, indicate that damage to hemoglobin may be such an upstream process, if not the primary trigger for vesiculation. A role for damaged hemoglobin in vesiculation is supported by the finding that the concentration of RBC-derived microvesicles is increased in the blood of patients with hemoglobinopathies (Westerman et al., 2008). The microvesicles from the blood of thalassemia patients contain high concentrations of oxidized, denatured alpha globin chains (Ferru et al., 2014). Also, these microvesicles contain enzymes involved in the maintenance of redox status such as catalase and peroxiredoxin-2, as well as large amounts of complement proteins and immunoglobulins (Ferru et al., 2014).

The protein composition of microvesicles from patients with membranopathies, i.e., abnormal RBCs such as elliptocytes and stomatocytes due to genetic aberrations in membrane proteins, is likely to differ from those of control RBCs. Actual data are lacking, but this prediction can be deduced from the effects of splenectomy on the membrane protein composition of spectrin/ankyrin-deficient and band 3-deficient spherocytes (Reliene et al., 2002; Alaarg et al., 2013). In RBCs from patients with thalassemia intermedia, hemoglobin damage may induce the formation of band 3 polymers, associated with increased phosphorylation that leads to a weakening of the band 3-ankyrin connection, resulting in microvesicle formation (Ferru et al., 2014). These observations, together with the vesiculation-reducing effect of p72Syk kinase inhibitors, not only support a central role for the binding of modified hemoglobin, possibly especially to oxidized and/or proteolytically degraded band 3 (Arashiki et al., 2013; Lange et al., 2014) in the vesiculation process (**Figure 1**), but also show the involvement of phosphorylation networks in RBC homeostasis. The involvement of various signaling pathways in RBC vesiculation was supported by the relative large numbers of signaling proteins in microvesicles obtained from the plasma of a healthy donor (Bosman et al., 2012), and in a pharmacological screening in vitro (Kostova et al., 2015).

### THE INVOLVEMENT OF MICROVESICLES IN COAGULATION AND INFLAMMATION

Most RBC-derived microvesicles from healthy donors as well as from various patients expose phosphatidylserine, which promotes not only phagocytosis but also coagulation.

In vitro, thrombin generation through the intrinsic pathway has been shown to be induced by RBC-derived microvesicles derived from sickle cell patients, storage units, or after treatment with a calcium ionophore (van Beers et al., 2009; van der Meijden et al., 2012; Rubin et al., 2013). In addition, correlations have been reported between the number of phosphatidylserine-exposing, RBC-derived microvesicles, and thrombin generation in sickle cell patients (van Beers et al., 2009; Gerotziafas et al., 2012). Microvesicles may also disturb anticoagulation reactions of the protein C system, possibly through binding of protein S (e.g.,

Koshiar et al., 2014). Increases in RBC-derived microvesicles in sickle cell disease and thalassemia patients is often accompanied by a decrease in deformability and hemolysis, which may as such constitute a risk factor for thrombosis.

Phagocytosis-triggered monocyte activation may induce proinflammatory and procoagulant endothelial cell responses (Straat et al., 2016). Thrombin may promote inflammation by activation of the complement system, e.g., by acting as C3 or C5 convertase (Zecher et al., 2014). RBCs of patients with paroxysmal nocturnal hemoglobinuria (PNH) lack the GPI-anchored proteins CD55 and CD59 that protect against complement activation-associated hemolysis. GPI-anchored proteins may be involved in raft formation (Salzer and Prohaska, 2001), and their absence may be directly responsible for the release of relatively high numbers of RBC-derived microvesicles with procoagulant activity in PNH patients in vitro (Hugel et al., 1999; Kozuma et al., 2011; Devalet et al., 2014). In addition, microvesicles scavenge NO almost as fast as free hemoglobin and much faster than RBCs, which may impair vasodilation (Donadee et al., 2011). This effect is already detectable with the number of microvesicles present in one transfusion unit (Liu et al., 2013).

### THE ROLE OF THE SPLEEN

The spleen facilitates vesiculation, as apparent from the retention of microvesicles in RBCs in asplenic individuals. In these individuals, the normal aging-related decrease in total RBC hemoglobin is absent, due to an increase in HbA1c (Willekens et al., 2003). In patients with spherocytosis, splenectomy increased RBC deformability in vitro, probably by inhibiting spleen-mediated microvesicle shedding (Reliene et al., 2002). The molecular mechanism underlying vesiculation in the spleen is unknown, but may involve a combination of biochemical and biophysical stress. Recent model simulations support the involvement of degraded hemoglobin in reducing the cytoskeleton/membrane connection, thereby promoting microvesicle shedding during splenic flow (Zhu et al., 2017). Thus, the mechanical and biochemical circumstances in the spleen, together with the presence of specialized macrophages, may make the spleen a microvesicle-based quality control and repair system. This emphasizes the importance of establishing the functionality of the spleen, especially in the study of diabetic control (Willekens et al., 2003). Also, the notable paucity of data for microvesicles generated in vivo warrants a more detailed investigation on the fundamental and clinical relationship between splenectomy or functional asplenia, RBCderived microvesicles and RBC homeostasis (Wernick et al., 2017).

### MICROVESICLES IN VITRO

Vesiculation also occurs during storage of RBCs in the blood bank. Storage microvesicles contain removal signals such as phosphatidylserine in the outer layer of their membrane and degraded as well as aggregated band 3 molecules, similar to microvesicles in the circulation (Bosman et al., 2008; Willekens et al., 2008). In blood bank microvesicles, the number of carbonyl groups is increased relative to RBC membranes, possibly due to the accumulation of oxidized membrane proteins band 3, actin and protein 4.1 (Bosman et al., 2008; Kriebardis et al., 2008; Delobel et al., 2016). This is accompanied by a correlation between oxidized cell constituents and vesiculation during storage (D'Alessandro et al., 2017). Blood bank microvesicles are immunologically active, as they contain immunoglobulins and complement factors, derived from the plasma fraction of the storage fluid (Bosman et al., 2008; Kriebardis et al., 2008). Also, storage microvesicles are readily recognized by pathological autoantibodies from patients with autoimmune hemolytic anemia (Dinkla et al., 2012a). These data indicate that the coupling of removal of damaged components from the RBC to their removal from the circulation is a general phenomenon for RBC-derived microvesicles. The enrichment of the GPI-anchored proteins acetylcholinesterase and CD55, as well as raft-associated forms of stomatin and the flotillins in storage microvesicles, indicates that lipid-related changes in membrane organization are involved in vesiculation during storage (Bosman et al., 2008; Salzer et al., 2008). The underlying mechanism has been proposed to be revolving around membrane budding and fission. This could be triggered by the loss of binding between cytoskeletal and membrane proteins, followed by large-scale separation of various lipid phases that may be formed by membrane protein-stabilized microdomains (Salzer et al., 2008). The loss of interaction between the cytoskeleton and cell membrane may be triggered by oxidized hemoglobin, similar to what may happen in vivo. Indeed, accumulation of oxidized hemoglobin residues during storage is accompanied by their enrichment in microvesicles (Wither et al., 2016). This role of hemoglobin in microvesicle formation is supported by the observation that, in the early phase of storage, a significant amount of hemoglobin is associated with the lipid bilayer in microvesicles (Szigyártó et al., 2018). There is a shortage of detailed quantitative and qualitative information on the primary triggers driving microvesicle production in vitro. The available data, albeit mostly showing associations, support a role of phosphorylation and rearrangement of band 3. For example, inhibition of tyrosine dephosphorylation not only induces RBC shapes such as echinocytes, which indicates a loss of interaction between the cytoskeleton and the lipid bilayer but also stimulates microvesicle production in vitro (Ferru et al., 2011, 2014; Cluitmans et al., 2016). In the misshapen cells found in patients with neuroacanthocytosis, disturbed phosphorylation and altered cell morphology are accompanied by disturbed microvesicle generation (Bosman and De Franceschi, 2008). Phosphorylation of band 3 is associated with clustering and correlates with microvesicle formation during storage and in the RBCs of patients with thalassemia intermedia (Ferru et al., 2014; Azouzi et al., 2018). Similar effects are observed upon treatment of RBCs with agents that induce aggregation of band 3 (Ferru et al., 2011; Cluitmans et al., 2016). A well-known stimulus for microvesicle formation in vitro is an artificial increase in intracellular calcium concentration. However, the protein composition of calcium-induced microvesicles differs

from storage or blood microvesicles, e.g., the content of membrane proteins, the presence of band 3 aggregates and breakdown products, and of raft-associated proteins (Bosman et al., 2008; Salzer et al., 2008; Prudent et al., 2015). This indicates that alterations in intracellular calcium concentrations are not primary factors in microvesicle generation in vivo, nor in the blood bank.

### MECHANISMS OF VESICULATION: INVOLVEMENT OF LIPIDS

The few data that are available indicate that disturbances of the organization of the lipid part of the cell membrane may play a role in the vesiculation process. The RBC membrane contains sphingomyelin/cholesterol-enriched as well as cholesterol-enriched domains that are associated with highcurvature areas. Since these domains become associated with budding membrane areas during storage at 4◦C, they have been speculated to be specific sites of microvesicle generation (Leonard et al., 2017). However, RBCs and microvesicles obtained during storage in blood bank conditions showed no significant differences in the main phospholipid classes (Laurén et al., 2018). This included the lack of enrichment of the raft-associated lipids cholesterol and sphingomyelin. Thus, lipid-involving reorganizations in the RBC membrane may be instrumental in microvesicle generation, but they do not seem to result in significant alterations in microvesicle lipid composition. Changes in membrane lipid organization, such as an increase in exposure of phosphatidylserine and/or phosphatidylethanolamine, may promote vesiculation during storage (Verhoeven et al., 2006; Larson et al., 2017). However, severe disruptions of the protein-protein interactions, that are associated with altered RBC morphology, may induce increased microvesicle generation, but are not always accompanied by increased phosphatidylserine exposure (Cluitmans et al., 2016) (**Figure 1**). In this context, it should be emphasized that not all RBC-derived microvesicles expose detectable amounts of phosphatidylserine (Willekens et al., 2008; Nielsen et al., 2014). Disruption of the lipid bilayer, e.g., by treating RBCs with sphingomyelinase, strongly catalysed microvesicle generation in vitro. This process was accompanied by the appearance of CD59 and stomatin clusters in the RBCs, supporting a role for lipid rearrangement in microvesicle formation. The sphingomyelinase-induced microvesicles were much more heterogeneous in phosphatidylserine exposure and glycophorin

A content than the microvesicles generated by spontaneous vesiculation, indicating the involvement of different mechanisms (Dinkla et al., 2012b). Thus, changes in lipid organization may facilitate microvesicle formation, but may not constitute the primary mechanism in most physiological conditions.

### MECHANISMS OF VESICULATION: COMPARISON WITH EXOSOME FORMATION

All reports on RBC-derived microvesicle composition, especially in combination with the aging-associated changes in the RBC membrane proteome, indicate the involvement of proteins that are involved in the release of exosomes as well (Raposo and Stoorvogel, 2013). This includes small GTPases, lipid raftassociated proteins such as acetylcholinesterase and flotillins, and annexins (Bosman et al., 2012; Raposo and Stoorvogel, 2013; Prudent et al., 2015). Although it is not clear how cytosolic components end up in exosomes, the mechanisms by which cytosolic molecules are recruited into RBC-derived microvesicles may be similar to those involved in exosome generation, as indicated by the presence of various chaperone proteins (Bosman et al., 2012; Raposo and Stoorvogel, 2013). The molecular details of the mechanisms underlying microvesicle generation in other cell types are largely unknown. Incorporation of the available data on RBC-derived microvesicles into the catalog 'Vesiclepedia' (Kalra et al., 2012) may be a worthwhile first step toward further elucidation of the mechanism of microvesicle in RBCs, as well as in other cell types. A comparison of RBC microvesicle data with the already available data on RBC exosomes that are shed by reticulocytes from human cord blood (Chu et al., 2018) will facilitate the identification of the molecular mechanisms involved in various types of vesiculation in vivo. Already, endocytosis and autophagy have been involved in the disappearance of CD71 and other membrane proteins during reticulocyte maturation in vitro (Griffiths et al., 2012). Such an approach profits from the possibility that RBCs create to study exosome as well as microvesicle formation during differentiation and aging in the same cell, that has a relatively homogeneous and well-charted membrane system.

### FROM MECHANISM TO MARKER TO MEDICINE

Red blood cells form microvesicles in response to a variety of physiological and pathological triggers. Although the inventory

### REFERENCES

of the composition of microvesicles generated in different circumstances is far from complete, the available data indicate that they all are enriched in damaged RBC components, depending on the various stimuli (**Figure 1**). This suggests that an exhaustive study of RBC-derived microvesicles will offer insights into the molecular mechanisms of their generation in vivo, and thereby into the physiological and pathological triggers. Also, RBC-derived microvesicles may constitute a model for the study of the biological, biophysical and clinical properties of microvesicles in general. This model will benefit from the comparison of the composition and characteristics of RBCderived microvesicles with microvesicles generated by other cell types, and with exosomes. RBC-derived microvesicles are potentially sensitive and specific biomarkers for the clinical severity of RBC-centered diseases such as sickle cell disease, thalassemia or spherocytosis (Reliene et al., 2002; Hebbel and Key, 2016; Kittivorapart et al., 2018). Also, microvesicles may reveal the activity as well as the clinical consequences, such as anemia or thrombosis, of systemic processes, such as inflammation. In addition, RBC-derived microvesicles may be useful in the transfer of surface proteins, as has been shown in the 'painting' of RBCs of PNH patients with the complementprotecting proteins CD55 and CD59 (Sloand et al., 2004).

Where RBC-derived microvesicles may be actively involved in pathology, e.g., by their procoagulant, proinflammatory or autoimmune activity (Sadallah et al., 2008), pharmacological prevention of the formation of harmful microvesicles may become of clinical importance. The recent finding that inhibition of sphingomyelinase attenuated lung inflammation caused by infusion of stored RBC-derived microvesicles (Hoehn et al., 2017), supports this notion. Thus, on one hand, microvesicle shedding may prevent the untimely removal of functional RBCs in physiological conditions. On the other hand, in pathological conditions, prevention of vesiculation following splenectomy may have beneficial effects, or prevent a pathological immune reaction, for example after massive or frequent RBC transfusion in compromised, transfusion-dependent patients.

### AUTHOR CONTRIBUTIONS

GB conceived the topic and wrote the final version. GB, JL, and MA-H wrote parts of the manuscript.

### FUNDING

JL was supported by the National Council for Scientific and Technological Development - Brazil.

band 3 clustering: mechanistic insights into human erythrocyte senescence. Biochemistry 52, 5760–5769. doi: 10.1021/bi400405p

and support low grade thrombin generation. Thromb. Haemost. 85, 639–646. doi: 10.1055/s-0037-1615646

of erythrocyte band 3. Blood 117, 5998–6006. doi: 10.1182/blood-2010-11- 317024

fphys-09-00703 June 6, 2018 Time: 16:19 # 6

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Leal, Adjobo-Hermans and Bosman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neocytolysis: How to Get Rid of the Extra Erythrocytes Formed by Stress Erythropoiesis Upon Descent From High Altitude

#### Heimo Mairbäurl\*

Medical Clinic VII, Sports Medicine, Translational Lung Research Center, German Center for Lung Research, University Hospital Heidelberg, Heidelberg, Germany

Edited by:

Lars Kaestner, Saarland University, Germany

#### Reviewed by:

Ingolf Bernhardt, Saarland University, Germany Angela Risso, University of Udine, Italy

> \*Correspondence: Heimo Mairbäurl heimo.mairbaeurl@ med.uni-heidelberg.de

#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 07 November 2017 Accepted: 20 March 2018 Published: 05 April 2018

#### Citation:

Mairbäurl H (2018) Neocytolysis: How to Get Rid of the Extra Erythrocytes Formed by Stress Erythropoiesis Upon Descent From High Altitude. Front. Physiol. 9:345. doi: 10.3389/fphys.2018.00345 Neocytolysis is the selective destruction of those erythrocytes that had been formed during stress-erythropoiesis in hypoxia in order to increase the oxygen transport capacity of blood. Neocytolysis likely aims at decreasing this excess amount of erythrocytes and hemoglobin (Hb) when it is not required anymore and to decrease blood viscosity. Neocytolysis seems to occur upon descent from high altitude. Similar processes seem to occur in microgravity, and are also discussed to mediate the replacement of erythrocytes containing fetal hemoglobin (HbF) with those having adult hemoglobin (HbA) after birth. This review will focus on hypoxia at high altitude. Hemoglobin concentration and total hemoglobin in blood increase by 20–50% depending on the altitude (i.e., the degree of hypoxia) and the duration of the sojourn. Upon return to normoxia hemoglobin concentration, hematocrit, and reticulocyte counts decrease faster than expected from inhibition of stress-erythropoiesis and normal erythrocyte destruction rates. In parallel, an increase in haptoglobin, bilirubin, and ferritin is observed, which serve as indirect markers of hemolysis and hemoglobin-breakdown. At the same time markers of progressing erythrocyte senescence appear even on reticulocytes. Unexpectedly, reticulocytes from hypoxic mice show decreased levels of the hypoxia-inducible factor HIF-1α and decreased activity of the BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), which results in elevated mitochondrial activity in these cells. Furthermore, hypoxia increases the expression of miR-21, which inhibits the expression of catalase and thus decreases one of the most important mechanisms protecting against oxygen free radicals in erythrocytes. This unleashes a series of events which likely explain neocytolysis, because upon re-oxygenation systemic and mitochondrial oxygen radical formation increases and causes the selective destruction of those erythrocytes having impaired anti-oxidant capacity.

Keywords: high altitude medicine, erythropoiesis, reticulocytes, hemoglobins, total hemoglobin mass, antioxidant capacity, hypoxia

### INTRODUCTION

Adjusting amount and function of erythrocytes is required in a variety of situations, the most obvious being the decrease in Hb after birth and the slow replacement of erythrocytes containing HbF with ones with HbA after birth (Terrenato et al., 1981). Altered blood distribution among compartments seems to induce a reduction in the total mass of erythrocytes in microgravity (Alfrey et al., 1996). Many diseases are associated with anemia by removing damaged erythrocytes (e.g., Rifkind, 1966). Although destruction likely occurs by different mechanisms, all might be classified into the non-specific term "erythrolysis." Yet another mechanism might explain the destruction of erythrocytes when highly polycythemic high altitude natives descent from high altitude (Merino, 1950). This review addresses targeted destruction of erythrocytes upon return to sea level, which lowlanders form during staying at high altitude for a limited time. Because in particular these newly formed cells seem to be removed, their destruction is called "neocytolysis" (Alfrey et al., 1997). For better understanding selectivity of destruction, biochemical characteristics, and mechanisms of cell destruction of erythrocytes formed under normal and stress conditions will be summarized.

### ADJUSTMENTS OF OXYGEN TRANSPORT TO HYPOXIA AT HIGH ALTITUDE

Oxygen supply to tissues is impaired at high altitude because decreased oxygen content of inspired air resulting in a mild decrease in arterial oxygen saturation (SaO2) at moderate altitudes (∼92% at 2,500 m) but much more pronounced at higher altitudes (∼83% at 4,500 m) because of the sigmoidal shape of the oxygen dissociation curve (Mairbäurl and Weber, 2012).

Arterial oxygen content (CaO2) varies during a sojourn at high altitude (Rasmussen et al., 2013): CaO2 is decreased upon acute ascent to high altitude, increases slightly within a few hours because of ventilatory acclimatization and respiratory alkalosis, increases further within days because of decreased plasma volume and increased hematocrit, and increases even further within weeks to months at high altitude because stimulated erythropoiesis increases total hemoglobin (tHb) reaching even pre-altitude values (Calbet et al., 2003).

Importantly, only CaO2 gets normalized but not PaO2, which is the main driving-force for oxygen diffusion to tissues, which therefore remain to some extent oxygen-limited. This seems to be well tolerated because at some point during a long-term stay at high altitude tHb reaches a stably-elevated value (Hurtado et al., 1945; Merino, 1950; Reynafarje et al., 1959). Performance is not improved equally because of impaired O2-diffusion (Calbet et al., 2003; Bärtsch and Swenson, 2014).

Stimulation of erythropoiesis at high altitude depends on HIF-2α and erythropoietin (EPO) (Haase, 2013) and adjustments of iron metabolism (Hentze et al., 2010). EPO increases rapidly upon exposure to hypoxia; the magnitude depends on the degree of hypoxia (Eckardt et al., 1989) following a semi-logarithmic function (Wenger and Kurtz, 2011). After this initial increase EPO decreases significantly to reach a steady state level that remains significantly above normoxic values, while one is still exposed to hypoxia (Wenger and Kurtz, 2011). Yet, the rate of formation of new erythrocytes stays elevated provided there is sufficient supply with iron. Iron uptake seems to peak after approximately 4 days at high altitude, reticulocytes have their highest values after approximately 7 days in hypoxia (Siri et al., 1966). This pattern of changes has been named the "EPOparadox" (Milledge and Bärtsch, 2014). It is likely caused by a shift from hematopoietic stem cells toward erythroid progenitor cells associated with up-regulation of the erythroid transcription factor GATA-1, which stimulates EPO-receptor expression (Li et al., 2011), resulting in an increased EPO-sensitivity and sustained erythropoiesis even at lower EPO. EPO receptors on progenitor cells are gradually lost during differentiation, and EPO-receptor density is very low in circulating mouse erythrocytes (Mihov et al., 2009).

The rapid increase in reticulocyte count, which is commonly observed, is likely caused by increased bone marrow blood flow and immature release of red cells (Aoki and Tavassoli, 1981). These immature cells differ in properties from reticulocytes produced under stress-free conditions, similar to what has been found in thalassemia (Rivella, 2009).

At moderate altitudes 2 weeks do not result in an increase in Hb in athletes (Friedmann et al., 1999), and stays longer than 3 weeks seem to increase total red cell volume by 60–250 ml/week (Sawka et al., 2000). Natives to high altitudes in the Andes have an increased total Hb, and their blood volume in increased by approximately 20% (Hurtado, 1964; Sánchez et al., 1970). Similar values are observed in sojourners after weeks to months at high altitude.

### MATURATION OF ERYTHROID PRECURSORS

The viability of committed erythroid progenitor cells downstream of the erythroid differentiation depends on the presence of EPO by supporting survival of erythroid precursors, which stimulates proliferation (Kimura et al., 2000). This process depends on cKit and EPO-receptors on the progenitor cells (Fisher et al., 1994; Wenger and Kurtz, 2011) and other growth factors all of which cause tyrosine-phosphorylation by Janus-kinase JAK2 (Klingmüller, 1997). JAK2 phosphorylates the EPO-receptor and the signal transducer and activator of transcription 5 (STAT5), which blocks apoptosis by inhibiting the Forkhead-Box-Protein O3 (FOXO3)-dependent pro-apoptotic pathways (Wojchowski et al., 2006). There is also an inhibition of the death receptor Fas and its ligand FasL in splenic erythroid cells, which promotes survival (Koulnis et al., 2011). The BNIP3-ligand (BNIP3L; Nix) controls mitochondrial autophagy (Sandoval et al., 2008). Thus, by the action of EPO, dividing progenitor cells are not destroyed resulting in increased production of normoblasts and release of reticulocytes into circulation (Wenger and Kurtz, 2011). In addition, phosphorylation of STAT3 increases the expression of antioxidant enzymes such as superoxide dismutase (SOD) and glutamin-cystein antiporter xCT while at the same time decreases the expression of proteins of the mitochondrial electron transfer chain (Linher-Melville and Singh, 2017). Together these two processes protect from hypoxia-induced mitochondrial oxygen radicals (ROS) (Chandel and Schumacker, 2000), which might impair erythropoiesis. Hypoxia also causes a HIF-1α and BNIP3-dependent decrease in mitochondrial activity, which further reduces ROS formation (Semenza, 2008). Therefore, during acute hypoxia, when mitochondrial adjustments have not yet been established, hypoxia likely increases mitochondrial ROS production, whereas hypoxiaadapted cells seem to produce less ROS (Chandel et al., 1997). However, there is also evidence that hypoxia might downregulate the expression of anti-oxidant enzymes such as catalase (Song et al., 2015). If this were the case, then one might speculate on a decreased anti-oxidant activity in hypoxia-adjusted cells and increased vulnerability to oxidant damage. This issue requires clarification.

### STRESS-ERYTHROPOIESIS

While normal steady-state erythropoiesis produces cells at a nearly constant rate, situations of acute tissue hypoxia such as blood loss, hemolysis, and elevated erythropoietin dramatically increase the rate of erythrocyte production and the rapid appearance of newly formed cells in circulation ("stress-erythropoiesis"). Most experimental evidence comes from work on mice, whereas evidence for human equivalents is sparse. It has to be noted that the mouse model most often used is phenylhydrazineinduced lipid-peroxydation and hemolytic anemia (Jain and Subrahmanyam, 1978), a quite "un-physiological" system.

In the acutely anemic mouse some of the EPO-induced progenitors migrate to the spleen (there may also be resident, self-renewing ones), with much enhanced maturation (Paulson et al., 2011). This is based on the finding that erythroid burst forming units (BFU-E) from the spleen differ from bone marrow BFU-E in that they form larger colonies and that the only growth factor required is EPO, whereas in the bone marrow additional burst-promoting factors are required (Valtieri et al., 1989). Bone morphogenic protein-4 (BMP4), who's expression depends on HIF-2α (Wu and Paulson, 2010), induces the expansion of BFU-E in the spleen to produce specialized resident stress erythroid progenitors (Paulson et al., 2011). It is interesting to note that hypoxia strongly stimulates the expansion of spleen-derived BFU-E (Perry et al., 2007). There is only indirect evidence for similarities between murine and human stress erythropoiesis.  Hypoxia of cultured progenitor cells from patients with sickle cell disease and thalassemia also induces the production of HbF cells. A moderate increase in HbFcontaining erythrocytes has also been found in humans after a 17-day stay at altitudes above 3,100 m (Risso et al., 2012). Thus it was speculated that this type of stress progenitors resemble the spleen-derived stress BFU-Es of the mouse model.

The marrow transit time of 59Fe was shortened in mice made anemic by phlebotomy, and there was an inverse relation between transit time and the degree of anemia (Hillman, 1969). Cells appear to be released immaturely indicated by larger size, increased reticulum content, iron uptake, membrane ion transporter activity, and increased density of transferrin receptors (TfR; CD71) on the plasma membrane (summary in Rhodes et al., 2016). TfR could be detected on circulating erythrocytes longer than during normal erythropoiesis. This indicates that the rate of maturation is similar in stress and normal erythropoiesis, but that maturation of circulating stress reticulocytes takes longer because of their immature release (Al-Huniti et al., 2005; Rhodes et al., 2016).

### NEOCYTOLYSIS

Neocytolysis is the selective destruction of the youngest population of erythrocytes in blood, just after they had left the bone marrow, and it is thought to bring an elevated erythrocyte mass (e.g., after a stay at high altitude) back to normal (Alfrey et al., 1997). It seems to occur in a variety of different situations (Alfrey et al., 1997; Harris and Epstein, 2001; Song et al., 2017). Neocytolysis appears to be caused not simply by discontinuing stress erythropoiesis but by "controlled" processes (for a recent review see Risso et al., 2014). However, indications for neocytolysis after return from high altitude hypoxia are weak.

Merino noted disappearance of polycythemia within a few days after return from high altitude and explained it by reduced erythropoiesis (Merino, 1950), which has been indicated experimentally by decreased iron incorporation in high altitude natives on traveling to low altitude (Huff et al., 1951). Also the reticulocyte number decreased. Greater "blood destruction" was indicated by increased plasma bilirubin and urobilinogen excretion (Merino, 1950).

Pace et al. based the discussion of neocytolysis (Pace et al., 1956) on a normal erythrocyte disappearance rate of 0.0083 per day (experimentally determined from the rate of removal of transfused erythrocytes; Callender et al., 1945). Erythrocyte count and Hb decreased at a rate of 0.011 per day upon return to sea level after an expedition to the Himalayas (Pace et al., 1956). This was interpreted to be caused by decreased rate of erythropoiesis and increased "erythrolysis," but also by restoring the decrease in plasma volume that occurs at high altitude (Siebenmann et al., 2017).

Rice et al. studied polycythemic residents of Cerre de Pasco (4,380 m) upon travel to sea level and found a decrease by 9% in erythrocyte mass within 3–7 days after descent, a rapid decrease in EPO, and increased bilirubin. Changes did not occur in three subjects receiving EPO upon descent (Rice et al., 2001).

Polycythemia by itself shortens erythrocyte survival (Bogdanova et al., 2007). Risso et al. (Risso et al., 2007) separated age-fractions of erythrocytes by density gradient centrifugation from blood of mountaineers 1 day after return from a mountaineering expedition and found that the youngest fraction had disappeared. Cells had acquired a "senescent phenotype" indicated by decreased levels of expression of the integrin associated protein (CD47), of the complement decay accelerating factor (DAF; CD55), and of protectin (membrane inhibitor of reactive lysis; CD59), which may be an indication of increased susceptibility to phagocytosis (Risso et al., 2007). However, only one time-point had been studied, and results may be flawed by the fact that the descent itself had lasted almost 1 week.

### Mechanisms Causing Neocytolysis

Results on three subjects (Rice et al., 2001) suggest that the withdrawal of EPO might be responsible for the destruction of erythrocytes produced in hypoxia. It is thought that increased EPO favors the expression of CD55 and CD59 such as observed in EPO-treated patients with renal anemia (Ohi et al., 2003), which protects from destruction by macrophages, whereas EPO withdrawal decreases expression and coincides with reappearance of anemia (Ohi et al., 2003), resulting in a picture comparable to that observed with EPO-withdrawal and treatment upon descent (Trial and Rice, 2004; Risso et al., 2014). This seems to be in line with the dependency of erythrocyte destruction on EPO and EPO-receptors in polycythemia of a gain-of-function mutation of the EPO-receptor (Divoky et al., 2016).

Another line of evidence suggests a role for altered antioxidant capacity. Erythrocytes exposed to anoxia have a decreased antioxidant capacity, indicated by decreased glutathione, nicotine-adenine dinucleotide (phosphate) (GSH, NADPH, and NADH, resp.) redox couples and reduced membrane thiols (Rogers et al., 2009). This has been explained with a re-direction of glycolytic flux toward the synthesis of 2,3-diphosphoglycerate (2,3-DPG) and binding to deoxygenated Hb, as well as altered glycolytic activity caused by competition of binding between deoxygenated Hb and glycolytic enzymes to band 3 (Weber et al., 2004), which also results in decreased NADPH and GSH formation (Rogers et al., 2009). Though this mechanism might adversely affect erythrocyte survival during high altitude hypoxia, it should rapidly be reversed upon descent thus protecting mature erythrocytes from oxidative stress. Erythropoiesis in hypoxic mice results in erythrocytes with a decreased anti-oxidative capacity because of decreased expression of catalase due to elevated levels of the micro-RNA miR-21 (Song et al., 2015). Anti-oxidative enzymes are typically high in young erythrocytes and decrease rapidly with aging (Bartosz and Bartkowiak, 1981). In the mouse model hypoxia during maturation increases mitochondrial activity in erythroid precursors due to suppression of HIF-1α and decreased BNIP3L. It was argued that mitochondrial ROS production would increase in normoxia and cause neocyte destruction (Song et al., 2015). This is in sharp contradiction to results showing HIF-1α-induced increase in mitophagy and reduction of mitochondrial mass in a variety of cell types thought to protect from increased ROS formation and cell destruction (Zhang et al., 2008) because hypoxic mitochondria produce more ROS than normoxic ones (Chandel et al., 1998; Chandel and Schumacker, 2000; Levraut et al., 2003). This issue awaits clarification.

Interestingly, it was found that treating mice with polyethylene glycol-conjugated catalase, which is not taken up by reticulocytes and mature erythrocytes, as well as treatment with the anti-oxidant N-acety-cysteine increased reticulocyte half-life and prevented the reduction in hematocrit (Song et al., 2015). This result contradicts the above described argument for a role for elevated mitochondrial but argues for systemic elevation of ROS which damage hypoxia-derived neocytes, a mechanism similar to re-oxygenation injury known from many organs, e.g., the lung (Pak et al., 2017).

### DESTRUCTION OF SENESCENT ERYTHROCYTES

The average life-span of mature erythrocytes is 100–130 days. The destruction rate amounts to approximately 1% per day. Senescent erythrocytes are removed by the reticulo-endothelial system, mainly in the spleen, where cells are tested for functionality and are sequestered "if they don't pass the test" (Rifkind, 1966). Random hemolysis of pre-senescent erythrocytes is negligible in humans but amounts to 0.5–1% per day in mice and rat (Landaw, 1988). Loss of erythrocytes is balanced by a production rate of ∼160 × 106 erythrocytes per minute in humans.

Interestingly, erythrocytes produced in hypoxic stress appear to have a shortened life span in some species, which is caused by increased random hemolysis and accelerated senescence (Fryers and Berlin, 1952). Every doubling of the erythrocyte production rate resulted in a 3.5% reduction in survival in rat (Landaw, 1988). Mechanisms seem to be intrinsic to erythrocytes, because cross-transfusion of newly formed cells into normoxic animals did not improve survival (Landaw, 1988). This is in line with reports on elevated levels of breakdown-products of heme in high altitude residents (Merino, 1950). It is further supported by an approximately 25% increase in the 59Fe disappearance rate from blood in Peruvian high altitude relative to sea-level residents (Huff et al., 1951) indicating increased production due to accelerated sequestration to maintain stably elevated Hb.

### CONSEQUENCES OF NEOCYTOLYSIS AND ACCELERATED SENESCENCE

If in fact erythrocytes produced by hypoxic marrow have characteristics limiting their survival upon return to normoxia, then hemolysis will affect different age-cohorts of erythrocytes depending on the duration of the sojourn and on the altitude (Rasmussen et al., 2013). After a sojourn at moderately high altitude for a few weeks as done by athletes for continuous or intermittent altitude training with the goal of increasing oxygen transport capacity and sea-level performance (Levine and Stray-Gundersen, 1997; Stray-Gundersen and Levine, 2008), only a small fraction of newly released erythrocytes will be found in circulation (Rasmussen et al., 2013; Garvican-Lewis et al., 2016). In fact, athletes had an elevated fraction of immature reticulocytes with high expression of TfR after returning from training at 1,905 m, which fell to sub-baseline values by day 9, and returned to baseline on day 16 after the training camp (Nadarajan et al., 2010). Similarly, increased ferritin levels were found post-altitude-training (Garvican et al., 2012). These changes are consistent with neocytolysis. However, there were no or only minor changes in tHb within 1 or 2 weeks after return from training at 2,300 m, during which total Hb had increased by ∼8% (Prommer et al., 2010; Garvican et al., 2012; Wachsmuth et al., 2013a,b), which may indicate decrease in erythrocyte mass by decreasing erythropoietic activity and by random loss, but a minor contribution of neocytolysis. In contrast, polycythemia after a stay at 5,260 m was reverted already within 1 week after descent indicating neocytolysis (Ryan et al., 2014). This may indicate that neocytolysis plays a minor role after a stay at moderate altitude, and that athletes performing altitude training may take advantage of a slightly increased oxygen carrying capacity at low altitudes for several weeks.

The situation is different for long-term sojourners and high altitude residents returning to low altitudes, because all their erythrocytes in circulation may have a decreased antioxidant capacity. Thus, elevated ROS will not only lyze young erythrocytes, but also random destruction might be increased and shorten erythrocyte life span. Hemolysis will progress until all erythrocytes with high altitude characteristics will be replaced by normoxic ones having increased antioxidant capacity. This process might cause a severe hemolytic strain, which might be

### REFERENCES

even more pronounced in polycythemic individuals with chronic mountain sickness.

### CONCLUSION AND PERSPECTIVES

There are indications of destruction of erythrocytes formed during exposure to hypoxia upon return to normoxia. Mechanisms are unclear and might include withdrawal of EPO and impaired defense against increased ROS. Experiments are needed to better define the mechanisms causing the fast and selective removal of neocytes formed in situations of stress erythropoiesis and to clearly distinguish between selective destruction and random loss.

Another aspect to be clarified concerns the use of the term "neocytolysis," which implies the destruction selectively of erythrocytes formed in an acute and transient situation of stress erythropoiesis. Thus this term cannot be used to describe the destruction of a fraction of erythrocytes produced during longterm or even life-long exposure to such a stress environment such as fetal life and being an altitude native. In those situations it needs to be sorted out whether just erythrocytes newly released from the marrow are removed, or whether all circulating cells are more susceptible to random loss, and why.

### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

training in elite endurance cyclists. Scand. J. Med. Sci. Sports 22, 95–103. doi: 10.1111/j.1600-0838.2010.01145.x

(ISA3600). Br. J. Sports Med. 47(Suppl. 1), i93–i99. doi: 10.1136/bjsports-2013-0 92761

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer IB and handling Editor declared their shared affiliation.

Copyright © 2018 Mairbäurl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro

#### Pedro J. Romero\* and Concepción Hernández-Chinea

Laboratory of Membrane Physiology, Faculty of Sciences, Institute of Experimental Biology, Central University of Venezuela, Caracas, Venezuela

In the present work we have studied in vitro the effect of increasing red cell Ca2+ ions on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages. In addition, the relative contribution to phagocytosis of phosphatidylserine exposure, autologous IgG binding, complement deposition and Gárdos channel activity was also investigated. Monocytes were obtained after ficoll-hypaque fractionation and induced to transform by adherence to glass coverslips, for 24 h at 37◦C in a RPMI medium, containing 10% fetal calf serum. Red blood cells (RBC) were loaded with Ca2+ using 10µM A23187 and 1 mM Ca-EGTA buffers, in the absence of Mg2+. Ca2+-loaded cells were transferred to above coverslips and incubated for 2 h at 37◦C under various experimental conditions, after which phagocytosis was assessed by light microscopy. Confirming earlier findings, phagocytosis depended on internal Ca2+. Accordingly; it was linearly raised from about 2–15% by increasing the free Ca2+ content of the loading solution from 0.5 to 20µM, respectively. Such a linear increase was virtually doubled by the presence of 40% autologous serum. At 7µM Ca2+, the phagocytosis degree attained with serum was practically equal to that obtained with either 2 mg/ml affinity-purified IgG or 40% IgG-depleted serum. However, phagocytosis was reduced to levels found with Ca2+ alone when IgG-depleted serum was inactivated by heat, implying an involvement of complement. On the other hand, phagocytosis in the absence of serum was markedly reduced by preincubating macrophages with phosphatidylserine-containing liposomes. In contrast, a similar incubation in the presence of serum affected it partially whereas employing liposomes made only of phosphatidylcholine essentially had no effect. Significantly, the Gárdos channel inhibitors clotrimazole (2µM) and TRAM-34 (100 nM) fully blocked serum-dependent phagocytosis. These findings show that a raised internal Ca2+ promotes erythrophagocytosis by independently triggering phosphatidylserine externalization, complement deposition and IgG binding. Serum appeared to stimulate phagocytosis in a way dependent on Gárdos activity. It seems likely that Ca2+ promoted IgG-binding to erythrocytes via Gárdos channel activation. This can be an important signal for clearance of senescent human erythrocytes under physiological conditions.

### Edited by:

Lars Kaestner, Saarland University, Germany

#### Reviewed by:

Giampaolo Minetti, University of Pavia, Italy Asya Makhro, University of Zurich, Switzerland

> \*Correspondence: Pedro J. Romero<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 10 September 2017 Accepted: 21 November 2017 Published: 04 December 2017

#### Citation:

Romero PJ and Hernández-Chinea C (2017) The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro. Front. Physiol. 8:1008. doi: 10.3389/fphys.2017.01008

Keywords: erythrophagocytosis, internal calcium, autologous serum, red blood cells, Gárdos channel

## INTRODUCTION

The normal human red blood cell (RBC) ages in the blood stream while circulating ceaselessly for a finite lifespan of nearly 120 days (Berlin and Berk, 1975). Aging appears as a continuous process of multifactorial origin, becoming abruptly interrupted by splenic retention of the senescent RBC. The sequestered cell is immediately recognized and phagocytosed by resident macrophages (Rifkind, 1966; Mebius and Kraal, 2005). Although many different hypotheses have accumulated over the years, the mechanisms responsible for trapping, recognition and destruction of aged cells are not completely elucidated (Clark, 1988; Bratosin et al., 1998; Antonelou et al., 2010; Lutz and Bogdanova, 2013). Suggested age-related changes involved in these mechanisms include dehydration with augmented cell density, decreased size, vesiculation, increased oxidative stress, band 3 clustering, band 3 phosphorylation, increased membrane IgG content and loss of membrane phospholipid asymmetry (Low et al., 1985; Lutz et al., 1987; Ciana et al., 2004; Willekens et al., 2008; Franco et al., 2013; Lutz and Bogdanova, 2013).

One of the current hypotheses that has great consensus is based upon IgG attachment to the RBC membrane and its recognition for macrophage clearance via Fc receptor interaction (Arese et al., 2005; Bosman et al., 2005; Lutz and Bogdanova, 2013). It is well-known that IgG accumulates on the outer membrane surface as the RBC ages (Lutz and Stringaro-Wipf, 1983). A small fraction of IgG binds significantly to α-galactosyl residues of presumably membrane glycolipids in aged RBCs (Galili et al., 1984). Another fraction is bound to a neo-antigenic region located on band 3 protein, which becomes progressively expressed during cell aging. This region corresponds to a distinct interdimeric band 3 epitope exposing binding sites for lowaffinity autologous IgG, which is formed upon oligomerization and clustering of band 3 aggregates during RBC senescence (Lutz et al., 1984; Kannan et al., 1991; Lutz, 2012). Additionally, it has been shown that bivalent IgG binding to interdimeric band 3 potentiates the opsonizing IgG action on RBCs by recruiting C3b, a critical complement component, thus compensating for its low-affinity binding characteristic (Lutz, 2012).

The proportion of natural IgG antibodies bound to band 3 was found about a half of that associated to α-galactosyl residues, whereas the rest constituting about 70% of the total showed an unknown specificity (Sorette et al., 1991).

Another proposed hypothesis that originally had a great impact for involving a classical apoptosis hallmark, relates to the asymmetrical distribution of membrane phospholipids, a "sine qua non" characteristic of normal living cells. Of particular importance in this context is phosphatidylserine (PS), which accumulates in the inner membrane leaflet by action of an aminophospholipid translocase (APLT), a P4-type ATPase (Bitbol et al., 1987; Morrot et al., 1990). Externalization of PS is a widely accepted signal for macrophage recognition and phagocytosis of apoptotic and effete cells (Schroit et al., 1985; McEvoy et al., 1986; Fadok et al., 1992; Kiefer and Snyder, 2000; Segawa and Nagata, 2015). The specific recognition of surface exposed PS occurs by direct interactions with membrane macrophage receptors such as stabilin-2 (Park et al., 2008) or Tim-1/Tim-4 (Kobayashi et al., 2007) and indirect ones, via serum proteins that act as bridging opsonins such as lactadherin (Hanayama et al., 2002), thrombospondin-1, growth arrestspecific 6 (Gas-6) and protein S to αvβ3/5 integrins and TAM tyrosine kinase receptors on macrophages (de Back et al., 2014; Segawa and Nagata, 2015).

In addition to the already mentioned exposure of natural antibodies-binding neoantigenic regions and surface PS, another unique signal for RBC removal has been proposed. This involves CD47, a transmembrane RBC glycoprotein that interacts with signal regulatory protein α (SIRP α) on macrophages blocking erythrophagocytosis (Oldenborg et al., 2000). However, in "artificially aged" RBCs (oxidatively treated) phagocytosis was stimulated by F(ab)2 anti-CD47, which prevented binding of oxidized CD47 to SIRPα. A similar effect seemed to be elicited by thrombospondin-1(TSP1), a known CD47 natural physiological ligand. It was therefore proposed that CD47 could act like a switch promoting or inhibiting phagocytosis, depending on whether TSP1 becomes bound or not (Oldenborg et al., 2000; Burger et al., 2012). It has been suggested that TSP1 binding to CD47 may be responsible forin vivo clearance of senescent RBCs.

An interesting hypothesis has been raised recently; drawing attention on the likely possibility that removal tagging signals on circulating RBCs may pass undetected because of their rapid dismissal. It was shown that the aging RBC decreases its membrane content of spectrin and flotillin-2, a lipid raft marker (Ciana et al., 2017). It was also found that vesicles induced by Ca2+-A23187 treatment were depleted of flotillin-2. It was proposed by above authors, that vesicles would contain a balanced lipid-bilayer/cytoskeletal protein ratio so that their release should occur without affecting the biconcave-disk shape of the cell. The hypothesis has been put forward that the continuous removal of vesicles by resident macrophages and the "pitting" splenic action during RBC aging, would reduce the cell size down to a minimum with a consequent increased rigidity (Ciana et al., 2017). This would lead to sequestration at the narrow splenic slits, recognition of accumulated tagging signals and finally clearance by phagocytosis.

On the other hand, earlier works stressed the importance of an elevated internal free Ca2+ as possible triggering signal for the events leading to clearance of senescent RBCs (Romero, 1978; Romero and Romero, 1999a; Bosman et al., 2005; Bogdanova et al., 2013). This idea finds support first, on the raised internal Ca2+ occurring during RBC aging as result of a steadily increased entry into cells having a progressive pumping deficiency (Romero and Romero, 1997, 1999b; Lew et al., 2007). Secondly, such a Ca2+ rise appears as common denominator of most of above mentioned age-related changes (Elgsaeter et al., 1976; Allan and Michell, 1977; Turrini et al., 1991; Kiefer and Snyder, 2000; Lang K. S. et al., 2003; Bogdanova et al., 2013).

Contrary to what would be expected from an abrupt clearance process, tagging signals steadily accumulate during the RBC lifespan. It is generally assumed that they trigger cell removal after reaching a threshold, as suggested for IgG binding where a few hundred molecules seem required (Bosman et al., 2005). In contrast with this view, previous work proposed a key role for the Gárdos channel (also known as KCNN4, KCa3.1, IKCa1) in the earlier events of senescent RBC clearance (Romero and Romero, 1999a). Accordingly, the channel would act as a molecular transducer between a monotonic signal (steadily rise in free internal Ca) and an "all-or-none" change (abrupt, selfgenerated Ca2+ increase, caused by membrane hyperpolarization due to channel opening) required for both a time-dependent sequestration and recognition of the aged cell. Essential to this view is the existent factual relationship between an increased Ca2+ content, activity of the Gárdos channel and cellular dehydration, referred to recently as the central paradigm of erythrocyte volume homeostasis (Martinac and Cox, 2017). Hence, a circulation-dependent raised cell Ca2+ above threshold levels (caused by pressure or shear stress) promotes erythrocyte dehydration by activating the Gárdos channel.

The purpose of the present study is to address the role that a raised free internal Ca2+ might possibly have on the clearance of aged cells, by studying in vitro its action on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages under various experimental conditions. A complex action of Ca2+ was revealed, presumably involving phosphatidylserine externalization, IgG binding, complement activation and Gárdos channel activity.

### MATERIALS AND METHODS

All reagents of the best quality available were purchased mainly from Sigma-Aldrich Corp., St Louis, USA. Protein A-sepharose CL-4B was obtained from GE Healthcare, Uppsala, Sweden. Fetal calf serum (FCS) was from GIBCO BRL, New York, USA. The pH of all solutions was adjusted at room temperature (RT) to ± 0.02 units. Fresh human blood (mainly 0+) was used, obtained from healthy subjects of both sexes, equally represented and mostly with ages between 35 and 55 years old.

### Isolation of Peripheral Blood Monocytes

Blood (10 ml) was collected by venipuncture in the presence of citrate (3.8%) and mixed with equal vol of phosphatebuffered saline (PBS) (310 mOsm/kg H2O, 150 mM NaCl + 20 mM (Na/K) phosphate buffer, pH 7.4), supplemented with 2 mM EDTA-Na2 and 10 mM glucose. The diluted blood was deposited on top of a ficoll-hypaque (FH) layer (density = 1.0775 g/ml) at a vol ratio blood/FH of 1:0.5 and spun down at 900 g for 30 min at RT. The cells banding at the serum-FH interphase were removed carefully and washed by resuspension in PBS medium supplemented as above and centrifuging at a sequentially decreasing low force (300, 200 and twice at 100 g). The fraction obtained consisted mainly of mononuclear cells (MNC) (>90%).

MNCs were resuspended in RPMI 1640-medium containing 0.03% glutamine and supplemented with 10% (vol/vol) heatinactivated FCS plus antibiotics (100 U/ml penicillin G and 100µg/ml streptomycin sulfate). Cells were enumerated in a Neubauer chamber and adjusted to a density of 3 × 106 cells/ml with supplemented-RPMI medium (MNC suspension). Viable cells were routinely determined by trypan blue exclusion (Freshney, 1987). Viability always exceeded 98%.

To promote cell adherence and induce macrophage transformation, aliquots of the above MNC suspension (70µl) were seeded on glass coverslips, deposited inside 6-holes culture dishes and placed in an air-CO2 incubator. After 1 h at 37◦C under a 5% CO2-humidified air atmosphere, 2 ml of warm RPMI medium supplemented as above were added to each well and incubation further continued for up to 24 h.

## Loading Erythrocytes with Ca2+

Erythrocytes from above FH fractionation (approx. 200µl from the bottom of RBCs pellet) were separated and stored overnight in PBS plus 10 mM glucose and antibiotics. They were then washed once with PBS and thrice with a choline medium, containing (mM): choline Cl, 109; KCl, 5; Tris-HCl, 20 (pH 7.4). RBCs were loaded with Ca2+ by incubating in choline medium (1% hematocrit) for 30 min at 37◦C under moderate shaking, in the presence of 10µM A23187 for uniform Ca2+ permeabilization (Dagher and Lew, 1988) and 1 mM Ca-EGTA buffers, set at different Ca/EGTA ratios to obtain 0–20µM ionized Ca (Fabiato and Fabiato, 1979). After loading, the ionophore was removed by washing once with 100 vols of choline medium containing 2 mg bovine serum albumin (BSA)/ml and twice with a similar medium but having 0.5 mg BSA/ml. RBCs were further washed twice with PBS to remove the remnant BSA and finally suspended in RPMI. Erythrocytes were enumerated in a Neubauer chamber and diluted with RPMI to about 50 times the number of monocytes/coverslip.

Since Mg2+ is also transported by A23187 (Reed and Lardy, 1972), this ion was omitted from the Ca2+-loading media in order to inactivate the Ca2+ pump. As a reference control, in some experiments Mg2+ was added to the loading medium at 0.15 mM, a concentration set at equilibrium with a −12 mV membrane potential (Flatman and Lew, 1980). Thereby, no net Mg2+ movements were expected during RBCs loading.

### Obtention of Autologous Serum (AS)

A parallel blood sample (10 ml) was collected without anticoagulant and allowed to clot at 37◦C for 2 h. Thereafter, it was centrifuged at 3,000 g for 20 min at RT and kept at 4◦C for 1 h before serum withdrawal. The AS was stored at −20◦C until use on the following day.

### Affinity Purification of IgG

Sepharose-coupled protein A was treated as recommended by the manufacturers and further used as described next. Briefly, it was washed several times with 50 mM Tris-HCl buffer (pH 7.0) by centrifuging 3 min at 500 g. One ml of this slurry sediment was packed at 15,000 g for 15 min. The supernatant was replaced by 500µl of AS, the suspension mixed and left shaking for 15 min at RT. After packing as just described, the supernatant consisting of IgG-depleted serum was recovered, supplemented with antibiotics and preserved at −20◦C until use. The total protein content of supernatants was usually 27–30 mg/ml.

The remaining sediment was washed five times with 50 mM Tris-HCl buffer (pH 7.0) and packed at 15,000 g as described before. To elute bound IgG, it was added 500µl of 0.1 M citric acid (pH 3.0); the suspension mixed and left shaking for 15 min at RT. After which, the mixture was packed as above, the supernatant containing eluted IgG was recovered and neutralized to pH 7.0-7.5 by adding appropriate amounts of 1M Tris-HCl solution (pH 9.0). The IgG solution obtained was supplemented with (mM): NaCl, 150; CaCl2, 2; MgCl2, 1; plus antibiotics, and finally stored at −20◦C until use. This solution generally contained 8–9 mg protein/ml. The efficiency of the process was checked by SDS-PAGE, using 10% polyacrylamide gels in the presence of 1% (vol/vol) 2-mercaptoethanol (Laemmli, 1970). Protein concentration was estimated using the Lowry method (Lowry et al., 1951). Equal amounts (20µg) of protein were loaded per track for electrophoretic assays.

### Preparation of Liposomes

Small unilamellar liposomes were prepared by sonication essentially as described by Fadok et al. (1992). The lipids, PS (L-αphosphatidyl-L-serine) and L-α-dimyristoyl phosphatidylcholine (PC) were dissolved in chloroform at a molar ratio PS/PC of 3:7 and roto-evaporated at 50–60◦C under reduced pressure. The dry material was resuspended in minimal medium of composition to be described later and vigorously shaken. The mixture was subsequently sonicated for two 10 min-cycles at 1◦C, in a Braun-Sonic 2000 sonicator (Fisher Scientific Products, Pittsburgh PA). The clear liposomal suspension (2 mM total lipid) was stored at −20◦C until use. Liposomes containing only PC were used as control.

### Erythrophagocytosis Assays

After adherence, coverslips were washed five times with warm RPMI medium to remove non-adherent cells. The RBCs (loaded or not with Ca2+) were suspended in RPMI medium supplemented or not with 40% (vol/vol) AS or other additions. This suspension was deposited on macrophagesattached coverslips and incubated for 2 h at 37◦C in a 5% CO2 humidified-air incubator. Thereafter, non-adherent RBCs were removed from coverslips by washing thrice with PBS and the attached cells were fixed with 2.5% glutaraldehyde in 0.15 M (Na/K) phosphate buffer (pH 7) for 15 min at RT. Subsequently, they were Wright-stained using conventional methods and observed under a light microscope (Nikon Eclipse E400).

In some experiments, macrophages were preincubated with PC- or mixed (PS + PC)-liposomes (200µM total lipid content), for 30 min at 37◦C in RPMI medium, in a 5% CO2 humidifiedair incubator. After washing five times with warm RPMI medium, macrophages were challenged with 7µM Ca2+-loaded RBCs and phagocytosis then assayed in a similar medium in the presence and absence of 40% AS, as described above.

Erythrophagocytosis was quantified (in percent) by scoring the number of macrophages having not only ingested but also attached RBCs, the latter considered an early step of the phagocytic process (Elliott and Ravichandran, 2010). At least 10 3 macrophages counts were accumulated for each condition to minimize counting error, except for experiments with liposomes where 300 cells were scored.

In some assays, the RMPI medium employed for phagocytosis was replaced by a minimal medium containing (mM): NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 1; Hepes (pH 7.5), 10; glucose, 10; penicillin G, 100 U/ml and streptomycin, 100µg/ml, leading to no significantly different results (not shown).

### Statistical Analysis

Data were analyzed using GraphPad Prism Version 5.01 software for Windows. Statistical analysis was performed by unpaired twotailed t-test when 2 groups were compared or 1-way ANOVA with Bonferroni post-tests when comparing more than 2 groups. Statistical significance of the data was defined as follows: P > 0.05 (n.s), P ≤ 0.05 (∗ ), P ≤ 0.01 (∗∗), P ≤ 0.001 (∗∗∗). Each experiment presented corresponded to a different donor.

### ETHICS

The present study was approved by the Bioethics Committee of the Faculty of Sciences, Central University of Venezuela. Investigations were carried out in accordance with the principles of the 2013 Declaration of Helsinki. Written informed consent was obtained from all blood donors participating in this study.

### RESULTS

### Effects of Ca2+ and AS on Erythrophagocytosis

Early work showed the requirement of both internal Ca2+ and presence of AS for phagocytosis of human RBCs by leukocytes in vitro (Romero and Romero, 1999a). This study, however, employed ghosts from the two cell types involved, a condition that may have obscured in part the conclusions drawn. With the interest of confirming these observations under more physiological conditions, intact RBCs were loaded with Ca2+ by means of the ionophore A23187 and exposed to monocytederived autologous macrophages. In addition, to assess a possible effect of Mg2+, phagocytosis was studied on cells treated or not with this ion during loading.

As reported in **Table 1**, with RBCs loaded in the virtual absence of both Ca2+ and Mg2+, the phagocytosis degree was about 2%, and tended to become reduced to a half when 0.15 mM Mg2+ was also added with the ionophore. Phagocytosis of these cells was significantly stimulated 2.5-fold by adding 40% AS to the assay medium whereas it remained unaltered if Mg2+ was present during loading.

In contrast, after loading with 20µM Ca2+ and no Mg2+, phagocytosis increased high significantly by nearly 900% and became additionally stimulated 2-fold by adding serum (**Table 1**). On the other hand, with 100µM Ca2+ and Mg2+ present during loading, phagocytosis was only enhanced by about 400% and serum almost trebled this effect. The extent of phagocytosis attained without AS in these cells was not significantly different from that of cells loaded in the nominal absence of divalent cations, thus suggesting they contain almost a comparable verylow ionized Ca concentration.

These results show first, that free internal Ca2+ is required for phagocytosis and that serum enhances its effect, thus confirming previous findings. Secondly, cells loaded in the nominal absence of both Ca2+ and Mg2+are more prone to become phagocytosed with serum present. This increased propensity to phagocytosis is

RBCs were loaded with and without Ca in choline medium, in the absence and presence of Mg. When Ca was present, it was added at 20 and 100µM free concentrations in solutions lacking or not Mg, respectively. Loaded cells were then assayed for phagocytosis in RPMI medium to which autologous serum (AS) was added or not. The phagocytosis degree (in percent) is presented as mean value ± SD of mean from the number of experiments shown in parenthesis. The probability (P) derived from statistical comparison of the data indicated with letters is also given. The degree of significance rises with increasing number of asterisks and (n.s) denotes not significant. See the text for further details.

also attained by omitting Mg2+ during Ca2+ loading. Thirdly, phagocytosis is markedly diminished when loading with high ionic Ca2+ in the presence of Mg2+.

Subsequent experiments were performed only on cells loaded with Ca2+ in the absence of Mg2+.

### Dependence of Phagocytosis on Intracellular Ca2+

With the interest of determining the dependence of phagocytosis on internal Ca2+, RBCs were loaded with 0, 0.5, 7, and 20µM ionized Ca2+ concentrations. The process was then assayed in RPMI medium, to which 40% AS was added or not.

The extent of phagocytosis followed a strict linear relationship (correlation coefficient r2 = 0.994) with the free Ca2+ content of cells (**Figure 1**). Accordingly, it was increased from about 2 to nearly 15% by raising Ca2+ from 0 to 20µM, respectively. Addition of serum further stimulated phagocytosis by almost 100%, while keeping the relationship linear (r 2 = 0.997). These results clearly indicate that phagocytosis depends monotonically on the ionic cell Ca2+ content, whether in absence or presence of serum.

### Effects of Preliminary Incubation of Macrophages with Liposomes

The results presented above undoubtedly show that phagocytosis is stimulated by a rise in the free Ca2+ content of erythrocytes. This ion is a well-known modulator of PS externalization in

in a choline medium without Mg. The extent of phagocytosis (in percent) after incubation without (circles) or with 40% AS (squares) is given as mean values of the number of experiments shown within parenthesis. Vertical bars represent ± 1SD of mean. Collected results from different experiments are given. The curves drawn correspond to linear regression lines, whose determination coefficients (r2 ) are 0.997 and 0.994 for data from cells incubated with and without serum, respectively.

human RBCs (Bitbol et al., 1987; Williamson et al., 1992), which in turn constitutes an important signal for macrophage recognition and erythrocyte removal (Schroit et al., 1985). To investigate if the Ca2+ action is related to PS exposure, macrophages were preincubated with PS-containing liposomes in RPMI medium. As control, macrophages were similarly exposed to PC liposomes. After washing, macrophages were challenged with 7µM Ca2+-loaded RBCs, in the presence and absence of 40% AS.