**7. The erythrocyte and purinergic signaling**

Cells of the vascular compartment communicate with each other by adenosine and its nucleotides ADP and ATP, a process called purinergic signaling. A set of 19 receptor subunits form receptors for adenosine and its nucleotides [93]. The ATPbinding P2X7 receptor is found on erythrocytes and many other cells in the vascular compartment. Binding of the ATP ligand to the P2X7 receptor of erythrocytes leads to phosphatidylserine exposure and clearance by macrophages [2]. ATP generated from erythrocytes can activate the P2X7 receptor on other cells like endothelial and myocardial cells. This can lead to inflammasome activation and may lead to pyroptosis of the cell through activation of gasdermin [94]. Structural studies of the P2X7 receptor show a "leaping dolphin"-like structure, where the head is formed by the extracellular domain and the tail by the transmembrane helices [95]. Unlike other P2X receptors, the P2X7 receptor shows a rather extended C-terminal domain described as the "cytoplasmic ballast" [96]. The cytoplasmic ballast contains one GDP-binding site, two zinc-binding sites and similarity to TNF receptor I and lipopolysaccharide binding domains [97]. When exposed to high ATP concentrations, the P2X7 receptor can form a macropore that allows passage of solutes up to 900 Da in size leading to apoptosis of the cell. The macropore size is sometimes incorrectly stated to be 900 kDa [94, 98, 99]. The macropore may be formed by the P2X7 receptor or together with some other membrane proteins like pannexin or connexin. Some evidence suggests that the P2X7 receptor is inhibited by magnesium [100]. The P2Y12 receptor is expressed on erythrocytes but is most known for its expression on platelets, where binding to ADP is part of the process of platelet activation, which is an important part of blood coagulation. Due to this, the P2Y12 receptor has been the focus of pharmaceutical development for instance leading to the antagonists clopidogrel, prasugrel, and ticagrelor. The P2Y13 receptor is activated by ADP on erythrocytes leading to diminishing of ATP export. Purinergic signaling in erythrocytes is part of the interaction between erythrocytes, platelets, and endothelial cells, both in normal physiological conditions and in pathological conditions such as diabetes [101].

Export of ATP from erythrocytes takes place through the pannexin transmembrane protein. ATP can then be metabolized in the extracellular space, for instance in plasma, to ADP and AMP by ectonucleoside triphosphate diphosphohydrolase, usually called CD39. AMP can then be further metabolized to adenosine by ecto-5′-nucleotidase, usually called CD73. CD39 and CD73 are transmembrane proteins expressed on the plasma membrane of many cell types including endothelial cells [102, 103]. Crystal structures of CD39 and CD73 show calcium and zinc ions [104, 105]. Interestingly, CD39 and CD73 collaborate with heme oxygenase-1 in heme catabolism [106]. Adenosine generated by CD73 increases heme oxygenase-1 in macrophages through stimulation of the adenosine A2A or A2B receptors. Heme and ATP can be generated from erythrocytes as a consequence of hemolysis. The intracellular pool of adenosine can be replenished by equilibrative nucleoside transporter 1 [101], which is a transporter localized in the plasma membrane of erythrocytes and other

#### *Erythrocytes as Messengers for Information and Energy Exchange between Cells DOI: http://dx.doi.org/10.5772/intechopen.108321*

cells. AMP can then be regenerated in the erythrocyte from adenosine by adenosine kinase [107], an enzyme in the purine nucleotide salvage pathway [108].

Uric acid, the result of adenosine and guanosine catabolism, is found in plasma where it may have antioxidative properties [109]. Significant correlations between uric acid levels and several erythrocyte parameters have been found, such as mean corpuscular volume, mean corpuscular hemoglobin concentration, and erythrocyte distribution width [109, 110]. However, decreased plasma uric acid levels accomplished by the recombinant urate oxidase Pegloticase did not affect the oxidative status of plasma [111]. A possible interpretation of this result is that the antioxidative properties of erythrocytes could compensate for the loss of uric acid's antioxidative capacity.

### **8. The erythrocyte, cytokine, and immune cell relation**

Erythrocytes have been found to be a reservoir of cytokines, a group of immune signaling proteins. Cytokines include chemokines, interferons, interleukins, and the hormone erythropoietin. Close to 50 different cytokines have been identified in or associated with erythrocytes [112]. Erythrocytes probably due to cytokine storage have a role in defense against pathogens, immune function, and homeostasis [2]. Erythrocytes also interact with the cellular part of the immune system (**Figure 1**). Macrophages are an important part of the cellular immune system that participate in both the birth and death of erythrocytes. Macrophages phagocytose senescent erythrocytes when they pass through liver or spleen. A phagocytosis signal is provided by phosphatidylserine exposure on erythrocytes, recognized by the TIM (T cell immunoglobulin and mucin domain containing) and CD300 receptors on macrophages. The CD47 membrane protein, which is present on erythrocytes, binds to the SIRPalfa protein on macrophages to downregulate phagocytosis [113]. Erythrocytes stored for long time periods can induce M2 macrophage polarization through the immunosuppressive interleukin-10 [114], thereby downregulating immunity. Macrophages are also necessary in erythropoiesis, the formation of new erythrocytes, where they interact with the erythroid progenitors in erythroblastic islands [115, 116]. Macrophages promote erythroblast proliferation and differentiation by secreting growth factors, providing nutrients like iron and finally phagocytosing nuclei of the nascent reticulocytes. Early-stage erythroblasts respond to growth factors like interleukin-3, stem cell factor, and erythropoietin. Several receptor-ligand pairs facilitate macrophage-erythroblast interactions. Vascular cell adhesion molecule1 of the macrophage interacts with integrin alfa4beta1 of the erythroblast and integrin alfaV of the macrophage interacts with intercellular adhesion molecule4 of the erythroblast [115]. Erythroblast macrophage protein of the macrophage interacts with erythroblast macrophage protein expressed on the surface of the erythroblast. CD163 and CD169 are expressed on macrophages and are known to be necessary for erythropoiesis, but the corresponding molecule on the erythroblast has not been identified. Finally, DNase2alfa of the macrophage is necessary for the phagocytosing of the nuclei of the nascent reticulocytes [116]. Selenium and selenoproteins are other factors necessary for erythropoiesis. Mutation of the selenocysteine-transfer-RNA, sometimes abbreviated Trsp, selenoprotein W, and glutathione peroxidase 4 genes in mice led to defective erythropoiesis [117–119]. Selenoprotein W has been suggested to be an adaptor protein to the E3 ubiquitin ligase TRIM21 [120]. Ubiquitin is a protein that delivers proteins for degradation to the proteasome, a protein complex that degrades mainly

damaged proteins marked by ubiquitin. The 20S proteasome and ubiquitin have been detected in reticulocytes and mature erythrocytes. Erythrocytes from patients with Alzheimer's or Parkinson's disease show decreased 20S proteasome activity [121, 122].

Erythrocytes also interact with other cells of the immune system. Erythrocytes have been shown to inhibit T cell activation or activation-induced apoptosis presumably via reactive oxygen species-dependent pathways [123, 124]. Erythrocytes treated with a cancer cell line stimulated T cells to more proliferation and other cytokine secretion profile than if treated with control erythrocytes [125]. The C-C chemokine RANTES (regulated on activation, normal, T cell expressed, and secreted) guides transendothelial migration of eosinophils, an immune cell that is responsible for interleukin-5 production and largely involved in asthma and allergy. Erythrocytes regulate this process by scavenging RANTES in the vascular compartment [126].

### **9. Erythrocytes and function of the vascular compartment**

The erythrocyte is important for several aspects of the vascular compartment, in particular vascular tone and vascular integrity. One aspect is the signaling based on nitric oxide, a gaseous molecule produced in the vascular compartment by erythrocytes, platelets, and endothelial cells by endothelial nitric oxide synthase (eNOS). Erythrocytes both produce and release nitric oxide [127] but can also scavenge nitric oxide by hemoglobin [128]. The synthesis of nitric oxide proceeds from arginine and oxygen-generating nitric oxide and citrulline. Regulation of nitric oxide synthesis is performed by arginase I by degrading the substrate arginine to ornithine and urea. Nitric oxide synthesis can also take place by deoxy-hemoglobin acting as nitrite reductase in hypoxia.

Nitric oxide is a vasodilator based on its effects on the vascular smooth muscle cells that surround the vascular compartment and contribute to vascular tone. The vascular smooth muscle cells contain the nitric oxide receptor soluble guanylate cyclase that produces cyclic guanosine monophosphate (cGMP). Protein kinase G is a further downstream signaling component, but the steps leading to vasodilation seem to be unknown in exact detail [129], although the last step probably involves changes in myosin phosphorylation, performed by Rho-associated kinase, zipper interacting kinase [130], myosin light chain kinase, or myosin phosphatase [131]. Some of these enzymes are also regulated by calmodulin and may therefore also be indirectly regulated by magnesium [79]. Soluble guanylate cyclase and protein kinase G are also present in erythrocytes and platelets, although the precise function of this pathway in erythrocytes is unknown [132]. Nitric oxide has an inhibitory effect on platelet activation, possibly through phosphorylation of the thromboxane receptor [133]. The effects of nitric oxide also include S-nitrosation of proteins and regulation of cGMP-gated ion channels by cGMP [128]. For instance, nitric oxide protects against myocardial infarction through S-nitrosation of the mitochondrial permeability transition pore regulator cyclophilin D [134]. Nitrosation of cysteine-93 of the beta-chain of hemoglobin has been suggested as a transport mechanism for nitric oxide in the erythrocyte. This hypothesis is not currently favored as a result of mutation studies [127]. A magnesium-dependent nitrosation of glutathione, yielding S-nitrosoglutathione, has been reported and could potentially occur in erythrocytes [135]. The physiological significance of S-nitrososglutathione is however unclear.

The relative contribution of different cell types to nitric oxide production in the vascular compartment is not yet fully elucidated. Erythrocytes both produce and

#### *Erythrocytes as Messengers for Information and Energy Exchange between Cells DOI: http://dx.doi.org/10.5772/intechopen.108321*

scavenge nitric oxide, which complicates the interpretation their contribution. Several evidences from pathological conditions point to the importance of erythrocytes for generation of nitric oxide bioactivity. Increased expression of erythrocyte arginase in diabetes leads to less nitric oxide production from erythrocytes. ENOS can be monomerized in erythrocytes of type 2 diabetes patients, usually referred to as uncoupling. The monomers then produce superoxide, which is also produced by nicontinamide adenine dinucleotide oxidase (NOX), both in erythrocytes and in endothelial cells. This results in dysfunction of endothelial cells [136], a common phenomenon in type 2 diabetes. As a further example, studies of anemic patients and a mouse model of anemia show that anemia is associated with erythrocyte dysfunction and reduced nitric oxide bioactivity. ENOS activity is then increased in the vascular wall and heart as compensatory mechanisms. If anemia is combined with endothelial dysfunction, the compensatory nitric oxide bioactivity may not be sufficient and could lead to adverse outcomes in myocardial infarction [137, 138].
