**4. RBC as redox modulators**

The main function of RBC is oxygen transport, for which they have evolved efficient nonenzymatic and enzymatic antioxidative systems for protection against oxidizing substances to which they are exposed. The nonenzymatic systems include reduced glutathione, thioredoxin, ascorbic acid, and vitamin E. The most important antioxidant enzymes include SOD, thioredoxin reductase/peroxiredoxin system, catalase, glutathione peroxidase, glutathione reductase, plasma membrane oxidoreductases, and the metHb reductase/NADH/glycolysis system that maintains Hb in a Fe(II)-active form [19].

Although these systems mainly serve the RBC own requirements, it seems that since they are produced in excess they can be utilized for antioxidant protection of other cells, at least under conditions of oxidative stress. This function may affect the intra- and extracellular milieus throughout the body, especially of cells in the circulation and in the perivascular tissues (endothelial cells).

Several characteristics, in addition to their extra reducing power, make RBC ideal candidates to serve as redox mediators. These include their vast number, mobility, and occurrence throughout the body. The consequence of their antioxidative activity could be oxidative damage to the RBC themself, facilitating their erythrophagocytosis, degradation, and detoxification of their oxidized constituents by macrophages in reticuloendothelial systems, mainly the spleen and the liver. These damaged/old RBC are replaced by new RBC that are continuously formed in the bone marrow.

A "bystander" effect of cells on the oxidative status of other neighboring cells has been described previously in other circumstances of oxidative stress induced by ionizing or photoradiation [20, 21].

#### **4.1 Proofs of the concept**

The concept of the RBC protective role was first introduced by Fazi et al. [22]. They showed that RBC are able to inactivate harmful xenobiotics, including 1-chloro-2,4-dinitrobenzene, by conjugation with glutathione and suggested that it may be possible to treat xenobiotic intoxication by transfusion of GSH-loaded RBC.

Richards et al. have shown that RBC can protect endothelial cells from PMNinduced damage [23]. PMN exert their antibacterial effect by generating a burst of ROS (respiratory burst) in response to toxins released by phagocytosed bacteria. These ROS not only kill the bacteria but also damage the PMN themselves and other neighboring cells (inflammation). The respiratory burst can be reproduced

**53**

**Figure 1.**

*(FACS-calibur<sup>R</sup>*

*diacetate, washed, and then incubated with 6 × 106*

*restored by iron chelation or antioxidants.*

RBC (**Figure 1**).

*Red Blood Cells as Redox Modulators in Hemolytic Anemia*

in vitro by incubating PMN with phorbol myristate acetate (PMA). In their study, 51Cr-labeled endothelial cells were incubated with PMA-triggered PMN. Damage to the endothelial cells was measured by the release of 51Cr into the incubation medium. Adding RBC to the mixture reduced the damage dose-dependently. Analyzing the RBC following the incubation, revealed reduced levels of 2,3-diphosphoglyceric acid and glutathione, and increased levels of the oxidation products malondialdehyde and metHb. These results indicated that these RBC are under oxidative stress compared with RBC incubated alone or with non-triggered PMN. The authors suggested that

We have studied the effect of RBC on the oxidative status of other cells by measuring oxidative parameters by flow cytometry. Following pulse-labeling of cells with the probe 2′7′-dichlorodihydrofluorescindiacetate, their fluorescence was proportional to their ROS content. The increase in their fluorescence after washing indicated their rate of generation of ROS. In our experiments, the labeled cells were incubated with RBC derived from either normal donors or patients with β-thalassemia. Normal RBC had a dose-dependent decrease effect on ROS genera-

It is well known that thalassemic RBC are under oxidative stress and contain more free iron load (the labile iron pool) than normal RBC [26]. To explore this condition on their AOC, RBC were exposed to agents that affect their oxidative stress or iron overload: normal RBC—to the oxidant hydrogen peroxide or to an iron source, ferric ammonium citrate, and thalassemic RBC—to the antioxidant N-acetyl cysteine or to the iron chelator, Desferal. The RBC were then mixed with the probelabeled cells, and the kinetics of ROS generated by the labeled cells was monitored during incubation. The results indicated that oxidants and iron reduced the AOC of

*The effects of iron overload and oxidative stress on the antioxidative capacity (AOC) of normal and thalassemia RBC. The human myeloid leukemia HL60 cells were labeled with 2′-7′-di-chlorofluorescein* 

*each). Prior to incubation with cells, the RBC had been treated for 30 min: normal RBC with ferric ammonium citrate (FAC), 1 mM, and thalassemic RBC—with the iron chelator, Desferal, 5 mM—thus increasing and decreasing iron overload, respectively. Alternatively, normal RBC had been treated with the oxidant H2O2, 5 mM, and thalassemic RBCs—with the anti-oxidant N-acetyl cysteine (NAC), 5 mM—thus increasing and decreasing oxidative stress, respectively. The cells were then analyzed by a flow cytometer* 

*(mean fluorescence channel) cellular green fluorescence (FL-1), reversely indicating the AOC, of 40,000 HL60 cells, was determined. In the analysis, RBC were excluded from HL60 cells by gating based on forward - and side-light scatter and fluorescence. The results indicate that the AOC of thalassemia RBC was significantly lower than that of normal RBC, and that iron and oxidants further decreased it, but it could be* 

*; Becton-Dickinson, Immunofluorometry systems, Mountain View, CA, USA). The average* 

*/ml RBC from normal or β-thalassemia donors (N = 6* 

RBC can provide antioxidant protection to other tissues in vivo [24].

tion, while thalassemic RBC had a much inferior effect [25].

*DOI: http://dx.doi.org/10.5772/intechopen.84498*

#### *Red Blood Cells as Redox Modulators in Hemolytic Anemia DOI: http://dx.doi.org/10.5772/intechopen.84498*

*Erythrocyte*

**4. RBC as redox modulators**

Fe(II)-active form [19].

the bone marrow.

ionizing or photoradiation [20, 21].

**4.1 Proofs of the concept**

and eventually to release of heme and iron. (II) Iron overload caused by frequent blood transfusions and increased iron uptake [15]. Usually, iron uptake in the gut as well as its mobilization from storage cells, regulated by hepcidin, is downregulated by iron excess [16]. It these diseases, where the body attempts to compensate for the anemia by over production of new RBC ("ineffective erythropoiesis"), iron is in high demand. To ensure sufficient iron uptake, the developing erythroid progenitors produce factors that inhibit hepcidin production, thus overriding the regulating effect of hepcidin. (III) Iron-containing compounds (Hb and hemin) which are released by intravascular hemolysis can also add to the iron load and further aggravate the hemolysis [17]. In the absence of specific mechanisms for disposal of excess iron, under these conditions iron accumulates. Iron overload increases ROS generation by catalyzing the Haber-Weiss/Fenton biochemical reactions [3, 18].

The main function of RBC is oxygen transport, for which they have evolved efficient nonenzymatic and enzymatic antioxidative systems for protection against oxidizing substances to which they are exposed. The nonenzymatic systems include reduced glutathione, thioredoxin, ascorbic acid, and vitamin E. The most important antioxidant enzymes include SOD, thioredoxin reductase/peroxiredoxin system, catalase, glutathione peroxidase, glutathione reductase, plasma membrane oxidoreductases, and the metHb reductase/NADH/glycolysis system that maintains Hb in a

Although these systems mainly serve the RBC own requirements, it seems that since they are produced in excess they can be utilized for antioxidant protection of other cells, at least under conditions of oxidative stress. This function may affect the intra- and extracellular milieus throughout the body, especially of cells in the

Several characteristics, in addition to their extra reducing power, make RBC ideal candidates to serve as redox mediators. These include their vast number, mobility, and occurrence throughout the body. The consequence of their antioxidative activity could be oxidative damage to the RBC themself, facilitating their erythrophagocytosis, degradation, and detoxification of their oxidized constituents by macrophages in reticuloendothelial systems, mainly the spleen and the liver. These damaged/old RBC are replaced by new RBC that are continuously formed in

A "bystander" effect of cells on the oxidative status of other neighboring cells has been described previously in other circumstances of oxidative stress induced by

The concept of the RBC protective role was first introduced by Fazi et al. [22]. They showed that RBC are able to inactivate harmful xenobiotics, including 1-chloro-2,4-dinitrobenzene, by conjugation with glutathione and suggested that it may be possible to treat xenobiotic intoxication by transfusion of GSH-loaded RBC. Richards et al. have shown that RBC can protect endothelial cells from PMNinduced damage [23]. PMN exert their antibacterial effect by generating a burst of ROS (respiratory burst) in response to toxins released by phagocytosed bacteria. These ROS not only kill the bacteria but also damage the PMN themselves and other neighboring cells (inflammation). The respiratory burst can be reproduced

circulation and in the perivascular tissues (endothelial cells).

**52**

in vitro by incubating PMN with phorbol myristate acetate (PMA). In their study, 51Cr-labeled endothelial cells were incubated with PMA-triggered PMN. Damage to the endothelial cells was measured by the release of 51Cr into the incubation medium. Adding RBC to the mixture reduced the damage dose-dependently. Analyzing the RBC following the incubation, revealed reduced levels of 2,3-diphosphoglyceric acid and glutathione, and increased levels of the oxidation products malondialdehyde and metHb. These results indicated that these RBC are under oxidative stress compared with RBC incubated alone or with non-triggered PMN. The authors suggested that RBC can provide antioxidant protection to other tissues in vivo [24].

We have studied the effect of RBC on the oxidative status of other cells by measuring oxidative parameters by flow cytometry. Following pulse-labeling of cells with the probe 2′7′-dichlorodihydrofluorescindiacetate, their fluorescence was proportional to their ROS content. The increase in their fluorescence after washing indicated their rate of generation of ROS. In our experiments, the labeled cells were incubated with RBC derived from either normal donors or patients with β-thalassemia. Normal RBC had a dose-dependent decrease effect on ROS generation, while thalassemic RBC had a much inferior effect [25].

It is well known that thalassemic RBC are under oxidative stress and contain more free iron load (the labile iron pool) than normal RBC [26]. To explore this condition on their AOC, RBC were exposed to agents that affect their oxidative stress or iron overload: normal RBC—to the oxidant hydrogen peroxide or to an iron source, ferric ammonium citrate, and thalassemic RBC—to the antioxidant N-acetyl cysteine or to the iron chelator, Desferal. The RBC were then mixed with the probelabeled cells, and the kinetics of ROS generated by the labeled cells was monitored during incubation. The results indicated that oxidants and iron reduced the AOC of RBC (**Figure 1**).

#### **Figure 1.**

*The effects of iron overload and oxidative stress on the antioxidative capacity (AOC) of normal and thalassemia RBC. The human myeloid leukemia HL60 cells were labeled with 2′-7′-di-chlorofluorescein diacetate, washed, and then incubated with 6 × 106 /ml RBC from normal or β-thalassemia donors (N = 6 each). Prior to incubation with cells, the RBC had been treated for 30 min: normal RBC with ferric ammonium citrate (FAC), 1 mM, and thalassemic RBC—with the iron chelator, Desferal, 5 mM—thus increasing and decreasing iron overload, respectively. Alternatively, normal RBC had been treated with the oxidant H2O2, 5 mM, and thalassemic RBCs—with the anti-oxidant N-acetyl cysteine (NAC), 5 mM—thus increasing and decreasing oxidative stress, respectively. The cells were then analyzed by a flow cytometer (FACS-calibur<sup>R</sup> ; Becton-Dickinson, Immunofluorometry systems, Mountain View, CA, USA). The average (mean fluorescence channel) cellular green fluorescence (FL-1), reversely indicating the AOC, of 40,000 HL60 cells, was determined. In the analysis, RBC were excluded from HL60 cells by gating based on forward - and side-light scatter and fluorescence. The results indicate that the AOC of thalassemia RBC was significantly lower than that of normal RBC, and that iron and oxidants further decreased it, but it could be restored by iron chelation or antioxidants.*

#### **4.2 RBC as oxidants**

Oxidative stress, being a common feature of many diseases, affects most cells of the body, including the RBC. These diseases involve RBC directly (e.g., thalassemia) or indirectly (e.g., diabetes) [27]. We have shown oxidative stress in RBC in all the hemolytic anemias [15]. Oxidative stress in RBC diminishing their own AOC, resulting in their short survival, but also reduces their ability to protect other cells. Under extreme conditions, this situation may turn the RBC into oxidative agents, rather than antioxidative agents.

#### **4.3 Probable mechanisms involved in redox protection of RBC**

Using artificial vesicles, it has been shown that while hydrogen peroxide readily crosses biological membranes, superoxide does it very slowly [28]. Vesicles made of RBC membranes allow superoxide to cross through and oxidize cytochrome c in the suspending medium within a time-frame consistent with its half-life time [29]. This transfer probably occurs via an anion channel since it was inhibited by stilbenes, which inhibit the exchange of anions across the membrane. Whether such outward flux actually occurs in intact RBC is doubtful. Since RBC contain a large amount of SOD [30], it seems unlikely that superoxide made within the RBC would escape both the spontaneous and enzymatic dismutations and diffuse across the membrane. In contrast, an inward flux could occur. The plasma contains comparatively little SOD [31], and superoxides generated outside the RBC might diffuse inward to be scavenged by the RBC-SOD. In this fashion, RBC might limit the damage inflicted by superoxides produced by blood phagocytes and vascular endothelial cells.

Similarly, RBC provide a mechanism for inactivation of free NO [32]. NO liberated from endothelium may be taken up by RBC and inactivated by oxyHb that in turn is converted to metHb, while the NO is converted to nitrate to be secreted by the kidneys.

Another RBC redox protective mechanism involves ascorbic acid (AA) (vitamin C) [33]. In humans, AA dietary intake is essential for maintaining plasma and tissue reductive capacity. It primarily functions to scavenge superoxide anion and singlet oxygen, but it also removes other ROS generated by protein-bound redox metals and xanthine oxidase. AA itself is oxidized to an AA radical and dehydro AA. Human RBC were suggested to possess a two-layered system of redox recycling of low concentrations of the AA radical under minimal oxidative stress and a backup system of recycling of large quantities of dehydro AA under increased oxidative stress. RBC accumulation of dehydro AA as a result of prooxidative conditions originates in part outside of the RBC during the two-electron oxidation of AA, which is subsequently transported reversibly in competition with glucose by the type 1 glucose transporters spanning the RBC membrane. Alternatively, dehydro AA may be lost altogether by degradation, removing a pool of potentially reversible oxidized AA. Experimental evidence suggests that recycling of AA by the RBC significantly add to the AOC of the blood [34].

Still another potential RBC redox protective mechanism is through the release of antioxidants and antioxidative enzymes (e.g., SOD and catalase) following hemolysis. We have found that hemolysate inhibits ROS generation by cells (unpublished observation). This could also occur following shedding of membrane-bound vesicles during the maturation of erythroid precursors in the bone marrow and senescence of RBC in the circulation. Both processes are enhanced in hemolytic anemias by oxidative stress [35].

Except for direct effects, RBC may affect other cells indirectly. For example, diet-derived antioxidant polyamines tend to attach to RBC membranes, resulting in a synergistic enhancement of their antioxidative activity [36].

**55**

dylserine [35].

*Red Blood Cells as Redox Modulators in Hemolytic Anemia*

**5. Pathological significance of RBC as redox modulators**

The redox-modulating activity of RBC could affect cells and their function throughout the body. We have studied their effect on platelets. Hemolytic anemia, such as thalassemia, is often associated with high incidence of thromboembolic complications (e.g., venous thrombosis and stroke) due to platelet hyperactivation and plasma hypercoagulation [37]. Platelet functioning depends on their redox state. They have an inherent ability to produce ROS by various pathways—as a by-product of the mitochondrial respiratory chain [38] and by the NADH/NADPH oxidase [for a review see [39]] produced mainly in the pentose cycle [40]. ROS, along with NO, adenosine, and prostacyclins, may play a profound role in the regulation of platelet activities [41]. Many studies demonstrated that their functioning during clot formation involves ROS; for example, platelet activators, such as thrombin, increase

Oxidative stress in platelets may give rise to two pathological outcomes: (I) toxicity, resulting in thrombocytopenia and bleeding and (II) hyperactivation resulting in excess clot formation leading to thromboembolic complications. Exemplifying the latter is hydrogen peroxide that stimulates their oxidative stress [45], and affects their various functions: activation by: (I) arachidonic acid and collagen [46]; (II) thrombin and ADP [47–49]; (III) tyrosine phosphorylation of the platelet αIIbβ3, an independent platelet activation pathway, thereby enhancing their aggregation [50], as well as (IV) through scavenging of the platelet- or endothelium-derived NO—thereby decreasing its aggregationinhibiting effect [51]. Superoxide can also contribute to late clot growth by increasing the bioavailability of ADP and subsequently recruiting additional

Since platelets do not carry known specific inherent redox pathology, it is reasonable to attribute their oxidative stress, at least in part, to continuous exposure to oxidative insults from extra platelet sources, such as their environment, i.e., the blood plasma, and neighboring cells - blood cells and the vascular endothelium. We have shown that incubation of normal platelets with plasma from thalassemia patients, rather than with normal plasma, resulted in their oxidative stress and activation [52]. Potential plasma oxidants are iron-containing compounds such as non-transferrin-bound iron, ferritin, heme, or Hb, all of which are increased in thalassemia patients [47, 53]. Incubation of platelets with iron (ferric ammonium citrate), heme (hemin or heme arginate), or Hb stimulated their oxidative stress. Moreover, addition of the iron-chelator deferoxamine to thalassemic plasma

Interestingly, thalassemic RBC also increased normal platelet oxidative status. In contrast, normal RBC, unless treated with oxidants, decreased it [54]. These results suggest that thalassemic RBC, having a higher than normal ROS level, mediate oxidative stress in platelets directly, probably by contact or close proximity [25]. These results are compatible with studies showing that platelets could be activated by ROS generated by neighboring cells such as RBC, neutrophils [55, 56], fibroblasts, and

RBC might also affect platelets indirectly by a variety of mechanisms: (I) Release of iron-containing oxidants into the plasma [46, 57, 58], as mentioned above. (II) Release of ROS, e.g., superoxide anions, causing oxidation of low-density lipoprotein [59], which, in turn, might activate platelets [60]. (III) Exposure or shedding of phosphatidylserine moieties, which act as a procoagulant that amplifies the generation of thrombin and thus initiates platelet activation [61]. Thalassemic RBC have been shown to carry and shed higher than normal levels of external phosphati-

*DOI: http://dx.doi.org/10.5772/intechopen.84498*

ROS generation [42–44].

platelets [49].

reduced its effect on the platelets' ROS [52].

vascular endothelial and smooth muscle cells [39].

*Erythrocyte*

**4.2 RBC as oxidants**

antioxidative agents.

Oxidative stress, being a common feature of many diseases, affects most cells of the body, including the RBC. These diseases involve RBC directly (e.g., thalassemia) or indirectly (e.g., diabetes) [27]. We have shown oxidative stress in RBC in all the hemolytic anemias [15]. Oxidative stress in RBC diminishing their own AOC, resulting in their short survival, but also reduces their ability to protect other cells. Under extreme conditions, this situation may turn the RBC into oxidative agents, rather than

Using artificial vesicles, it has been shown that while hydrogen peroxide readily crosses biological membranes, superoxide does it very slowly [28]. Vesicles made of RBC membranes allow superoxide to cross through and oxidize cytochrome c in the suspending medium within a time-frame consistent with its half-life time [29]. This transfer probably occurs via an anion channel since it was inhibited by stilbenes, which inhibit the exchange of anions across the membrane. Whether such outward flux actually occurs in intact RBC is doubtful. Since RBC contain a large amount of SOD [30], it seems unlikely that superoxide made within the RBC would escape both the spontaneous and enzymatic dismutations and diffuse across the membrane. In contrast, an inward flux could occur. The plasma contains comparatively little SOD [31], and superoxides generated outside the RBC might diffuse inward to be scavenged by the RBC-SOD. In this fashion, RBC might limit the damage inflicted by superoxides produced by blood phagocytes and vascular endothelial cells.

Similarly, RBC provide a mechanism for inactivation of free NO [32]. NO liberated from endothelium may be taken up by RBC and inactivated by oxyHb that in turn is converted to metHb, while the NO is converted to nitrate to be secreted by the kidneys. Another RBC redox protective mechanism involves ascorbic acid (AA) (vitamin

Still another potential RBC redox protective mechanism is through the release of antioxidants and antioxidative enzymes (e.g., SOD and catalase) following hemolysis. We have found that hemolysate inhibits ROS generation by cells (unpublished observation). This could also occur following shedding of membrane-bound vesicles during the maturation of erythroid precursors in the bone marrow and senescence of RBC in the circulation. Both processes are enhanced in hemolytic

Except for direct effects, RBC may affect other cells indirectly. For example, diet-derived antioxidant polyamines tend to attach to RBC membranes, resulting in

a synergistic enhancement of their antioxidative activity [36].

C) [33]. In humans, AA dietary intake is essential for maintaining plasma and tissue reductive capacity. It primarily functions to scavenge superoxide anion and singlet oxygen, but it also removes other ROS generated by protein-bound redox metals and xanthine oxidase. AA itself is oxidized to an AA radical and dehydro AA. Human RBC were suggested to possess a two-layered system of redox recycling of low concentrations of the AA radical under minimal oxidative stress and a backup system of recycling of large quantities of dehydro AA under increased oxidative stress. RBC accumulation of dehydro AA as a result of prooxidative conditions originates in part outside of the RBC during the two-electron oxidation of AA, which is subsequently transported reversibly in competition with glucose by the type 1 glucose transporters spanning the RBC membrane. Alternatively, dehydro AA may be lost altogether by degradation, removing a pool of potentially reversible oxidized AA. Experimental evidence suggests that recycling of AA by the RBC

significantly add to the AOC of the blood [34].

anemias by oxidative stress [35].

**4.3 Probable mechanisms involved in redox protection of RBC**

**54**
