**2. The erythrocyte in immunology**

Erythrocyte surface antigens play an important role in the erythrocyte interaction with pathogens and in other immune reactions. To begin with, the Duffy antigen has been found to be the entry point for *Plasmodium vivax*. This is supported by the finding that Duffy-negative individuals have some protection against *P. vivax* infection. Duffy negativity is common in the African population, although *P. vivax* may be able to infect some Duffy negative individuals [4]. Duffy was found to be a receptor of the chemokine with Cysteine-X-Cysteine (C-X-C) motif ligand 8 (CXCL8, also called interleukin-8) (**Figure 1**), and other chemokines such as chemokine with Cysteine-Cysteine (C-C) motif ligand 2 (CCL2, also called macrophage chemoattractant protein-1 (MCP-1)) were later also found to bind to Duffy [5]. The Duffy protein is a putative G-protein coupled receptor, but lacks some parts that would be necessary for signaling and is therefore regarded as a non-signaling receptor. The current hypothesis is that Duffy functions as a buffer-storage-sink for chemokines in the body. One indication for this is the reduced neutrophil counts and increased risk of acquiring human immunodeficiency virus (HIV-1) that has been found in Duffy-negative individuals [6]. However, overall neutrophil effector functions were largely unaffected in HIV-1-infected and non-infected Duffynegative individuals [7]. One explanation for this could be an increased proteolytic activity as a compensatory mechanism for lower absolute neutrophil counts [7].

Chemokines belong to a group of immune signaling proteins called cytokines. In addition to chemokines, interferons, interleukins and the hormone erythropoietin are classified as cytokines. From the initial discovery of chemokine binding to Duffy, many more cytokines have been shown to be associated with the erythrocyte [8]. A considerable step forward was taken in a study where almost 50 different cytokines were identified in erythrocyte preparations [9]. Among chemokines, several from the C-C and the C-X-C families were found. Among interleukins, the interleukin-1, interleukin-6, interleukin-12 and interleukin-17 families were present (**Figure 1**). Present were also granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3, interleukin-5, tumor necrosis factor (TNF) superfamily and platelet derived growth factor (PDGF) (**Figure 1**). Interferon type I was represented by interferon-alpha2 whereas interferon type II was represented by interferon-gamma (**Figure 1**). The study suggests that the erythrocyte has significant capacity to contribute to the cytokine pool and immune homeostasis.

#### **Figure 1.**

*Schematic figure of the erythrocyte in immunology. An erythrocyte contains 1.2 million band3 and 0.5 million glycophorin a molecules. Abbreviations are explained in the text.*

**11**

density lipoprotein.

*Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis*

In addition, cytokine and cytokine receptor gene expression has been found in both

Most of the cytokines have been localized extracellularly on the erythrocyte possibly bound to transmembrane receptors or associated to the glycocalyx. Macrophage migration inhibitory factor 1 (MIF1), D-dopachrome tautomerase (DDT) and interleukin-33 were found to have an intracellular localization [8, 12] (**Figure 1**). Interleukin-33 is a nuclear localized interleukin belonging to the interleukin-1 family [12]. Interleukin-33 is probably left over after expelling of the nucleus from the reticulocyte. MIF1 and DDT were found to exist in the erythrocyte, although the exact receptor has not been identified. In other tissues, a receptor for MIF1 and DDT is formed by CD74 and the hyaluronan receptor CD44 (**Figure 1**), which is also present on erythrocytes [13]. Intracellular as well as extracellular erythrocyte cytokines may be released by hemolysis or in proteincontaining micro-vesicles or micro-particles produced by the erythrocyte [14–16]. Toll-like receptors (TLR) are a group of trans-membrane receptors of the innate immune system. TLR9 was shown to be present on erythrocytes [17] (**Figure 1**), although not all erythrocytes express TLR9 and considerable interindividual differences in expression was found [17]. Presence of mitochondrial but not nuclear DNA was confirmed on human and mouse erythrocytes. A knock-out mouse for TLR9 lost much of the mitochondrial DNA binding capacity otherwise found on human and mouse erythrocytes. TLR9 protein was mainly localized in the erythrocyte close to the plasma membrane probably binding to band3, a 14-transmembrane helices protein also known as anion exchanger 1 (AE1 or SLC4A1). Some evidence was found for erythrocyte scavenging of mitochondrial DNA from microvascular endothelial cells [17]. The erythrocyte may also interact with macrophages through the TLR9 receptor [18] (**Figure 1**). Additional TLRs may also exist in the erythrocyte, since expression of several TLRs was found in murine erythroblasts [11]. Erythrocytes also store and export the multifunctional molecule sphingosine-

1-phosphate (S1P) (**Figure 1**). Inside the erythrocyte S1P has been shown to be involved in regulation of glycolysis [19]. S1P binds the S1P-receptor-1 on lymphocytes, antagonizing their egress from secondary lymphoid organs and the thymus. When bound to apolipoprotein M, S1P reduces lymphopoiesis by binding S1P-receptor-1 on common lymphoid progenitors in the bone marrow [20]. Erythrocytes synthesize S1P from sphingosine obtained from plasma in a reaction catalyzed by sphingosine-kinase-1. Because of lack of cellular organelles and their associated enzymes, no S1P-degrading enzyme is present in erythrocytes. These therefore effectively function as a S1P storage facility. Through site-directed mutagenesis and gene-knock-out studies, the major facilitator superfamily domain 2b (Mfsd2b) was found to be the exporter of S1P from erythrocytes [21]. Export of S1P from erythrocytes (**Figure 1**) was inhibited by an anion transport inhibitor, suggesting that also band3 may be involved in S1P export [22]. Erythrocytes are considered the major source of S1P plasma levels because of low plasma S1P levels in anemic patients. S1P plasma levels are also regulated by the lipid phosphate phosphatases, LPP1 and LPP3, on the plasma membrane of endothelial cells [23]. In addition, S1P levels in plasma may also depend on the presence and regulation of S1P carrier molecules like albumin and apolipoprotein M in complex with high-

Important for entry of *P. falciparum* into the erythrocyte are glycophorin A, band3, complement receptor 1 (CR1) and basigin [24] (**Figure 1**). Basigin is also known as the extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147. The protein glycosylation underlying the ABO blood group system, is present on erythrocyte membrane proteins like band3. The ABO glycosylation is used by *Plasmodium* to facilitate rosetting, a stage in *Plasmodium* pathogenesis [25].

*DOI: http://dx.doi.org/10.5772/intechopen.97850*

human and murine erythroblasts [10, 11].

#### *Erythrocytes as Biomarkers of Virus and Bacteria in View of Metal Ion Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.97850*

In addition, cytokine and cytokine receptor gene expression has been found in both human and murine erythroblasts [10, 11].

Most of the cytokines have been localized extracellularly on the erythrocyte possibly bound to transmembrane receptors or associated to the glycocalyx. Macrophage migration inhibitory factor 1 (MIF1), D-dopachrome tautomerase (DDT) and interleukin-33 were found to have an intracellular localization [8, 12] (**Figure 1**). Interleukin-33 is a nuclear localized interleukin belonging to the interleukin-1 family [12]. Interleukin-33 is probably left over after expelling of the nucleus from the reticulocyte. MIF1 and DDT were found to exist in the erythrocyte, although the exact receptor has not been identified. In other tissues, a receptor for MIF1 and DDT is formed by CD74 and the hyaluronan receptor CD44 (**Figure 1**), which is also present on erythrocytes [13]. Intracellular as well as extracellular erythrocyte cytokines may be released by hemolysis or in proteincontaining micro-vesicles or micro-particles produced by the erythrocyte [14–16].

Toll-like receptors (TLR) are a group of trans-membrane receptors of the innate immune system. TLR9 was shown to be present on erythrocytes [17] (**Figure 1**), although not all erythrocytes express TLR9 and considerable interindividual differences in expression was found [17]. Presence of mitochondrial but not nuclear DNA was confirmed on human and mouse erythrocytes. A knock-out mouse for TLR9 lost much of the mitochondrial DNA binding capacity otherwise found on human and mouse erythrocytes. TLR9 protein was mainly localized in the erythrocyte close to the plasma membrane probably binding to band3, a 14-transmembrane helices protein also known as anion exchanger 1 (AE1 or SLC4A1). Some evidence was found for erythrocyte scavenging of mitochondrial DNA from microvascular endothelial cells [17]. The erythrocyte may also interact with macrophages through the TLR9 receptor [18] (**Figure 1**). Additional TLRs may also exist in the erythrocyte, since expression of several TLRs was found in murine erythroblasts [11].

Erythrocytes also store and export the multifunctional molecule sphingosine-1-phosphate (S1P) (**Figure 1**). Inside the erythrocyte S1P has been shown to be involved in regulation of glycolysis [19]. S1P binds the S1P-receptor-1 on lymphocytes, antagonizing their egress from secondary lymphoid organs and the thymus. When bound to apolipoprotein M, S1P reduces lymphopoiesis by binding S1P-receptor-1 on common lymphoid progenitors in the bone marrow [20]. Erythrocytes synthesize S1P from sphingosine obtained from plasma in a reaction catalyzed by sphingosine-kinase-1. Because of lack of cellular organelles and their associated enzymes, no S1P-degrading enzyme is present in erythrocytes. These therefore effectively function as a S1P storage facility. Through site-directed mutagenesis and gene-knock-out studies, the major facilitator superfamily domain 2b (Mfsd2b) was found to be the exporter of S1P from erythrocytes [21]. Export of S1P from erythrocytes (**Figure 1**) was inhibited by an anion transport inhibitor, suggesting that also band3 may be involved in S1P export [22]. Erythrocytes are considered the major source of S1P plasma levels because of low plasma S1P levels in anemic patients. S1P plasma levels are also regulated by the lipid phosphate phosphatases, LPP1 and LPP3, on the plasma membrane of endothelial cells [23]. In addition, S1P levels in plasma may also depend on the presence and regulation of S1P carrier molecules like albumin and apolipoprotein M in complex with highdensity lipoprotein.

Important for entry of *P. falciparum* into the erythrocyte are glycophorin A, band3, complement receptor 1 (CR1) and basigin [24] (**Figure 1**). Basigin is also known as the extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147. The protein glycosylation underlying the ABO blood group system, is present on erythrocyte membrane proteins like band3. The ABO glycosylation is used by *Plasmodium* to facilitate rosetting, a stage in *Plasmodium* pathogenesis [25].

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

Erythrocyte surface antigens play an important role in the erythrocyte interac-

Chemokines belong to a group of immune signaling proteins called cytokines. In addition to chemokines, interferons, interleukins and the hormone erythropoietin are classified as cytokines. From the initial discovery of chemokine binding to Duffy, many more cytokines have been shown to be associated with the erythrocyte [8]. A considerable step forward was taken in a study where almost 50 different cytokines were identified in erythrocyte preparations [9]. Among chemokines, several from the C-C and the C-X-C families were found. Among interleukins, the interleukin-1, interleukin-6, interleukin-12 and interleukin-17 families were present (**Figure 1**). Present were also granulocyte-macrophage colony stimulating factor (GMCSF), interleukin-3, interleukin-5, tumor necrosis factor (TNF) superfamily and platelet derived growth factor (PDGF) (**Figure 1**). Interferon type I was represented by interferon-alpha2 whereas interferon type II was represented by interferon-gamma (**Figure 1**). The study suggests that the erythrocyte has significant capacity to contribute to the cytokine pool and immune homeostasis.

*Schematic figure of the erythrocyte in immunology. An erythrocyte contains 1.2 million band3 and 0.5 million* 

*glycophorin a molecules. Abbreviations are explained in the text.*

tion with pathogens and in other immune reactions. To begin with, the Duffy antigen has been found to be the entry point for *Plasmodium vivax*. This is supported by the finding that Duffy-negative individuals have some protection against *P. vivax* infection. Duffy negativity is common in the African population, although *P. vivax* may be able to infect some Duffy negative individuals [4]. Duffy was found to be a receptor of the chemokine with Cysteine-X-Cysteine (C-X-C) motif ligand 8 (CXCL8, also called interleukin-8) (**Figure 1**), and other chemokines such as chemokine with Cysteine-Cysteine (C-C) motif ligand 2 (CCL2, also called macrophage chemoattractant protein-1 (MCP-1)) were later also found to bind to Duffy [5]. The Duffy protein is a putative G-protein coupled receptor, but lacks some parts that would be necessary for signaling and is therefore regarded as a non-signaling receptor. The current hypothesis is that Duffy functions as a buffer-storage-sink for chemokines in the body. One indication for this is the reduced neutrophil counts and increased risk of acquiring human immunodeficiency virus (HIV-1) that has been found in Duffy-negative individuals [6]. However, overall neutrophil effector functions were largely unaffected in HIV-1-infected and non-infected Duffynegative individuals [7]. One explanation for this could be an increased proteolytic activity as a compensatory mechanism for lower absolute neutrophil counts [7].

**2. The erythrocyte in immunology**

**10**

**Figure 1.**

#### **Figure 2.**

*Schematic figure of the erythrocyte and its interactions with viruses. Adenovirus and parvovirus lack a lipid bilayer around the virus capsid. Covid-19 disease affects the erythrocyte without entry of SARS-CoV-2 into the erythrocyte. Abbreviations are explained in the text.*

Blood group O favors rosetting less than the A or B blood groups. Interestingly, the same pattern can be seen in Covid-19, where blood groups A or B are risk factors, whereas group O appears to be comparatively protective [26]. Glycophorin A is the most abundant sialo-glycoprotein on erythrocytes (**Figure 1**). Sialic acid residues are often used as receptors for invasion by bacteria and other pathogens [27] (**Figure 2**). Sequence analysis of glycophorin A from several primate species revealed high diversifying selection in the glycophorin A gene [27]. High selective pressure could be explained as a consequence of targeting of different pathogens by the glycophorin A receptor. This led to the pathogen decoy hypothesis, which states that the erythrocyte functions as a "flypaper", using glycophorin A to bind pathogens [27]. Pathogens may then be cleared by macrophages as erythrocytes pass through the spleen.
