Genetically active cells are susceptible to genetic diseases (mutations, chromosome aberration, and cancer). They are also prone to infections depending on the receptors they express; cellular receptors can be parasitized by a variety of viruses (6). The following hypothetical example illustrates the importance of enucleation of mammalian erythroblasts and the inactivation of nuclei of avian erythrocytes (7) (and other vertebrates) in evading genetic and infectious diseases. Hypothetically, if one particle of a lytic virus were to release 50 particles every hour after invading a single human ME, then all circulating MEs of an individual will be lysed and death will occur in few hours (less than 10 h post infection).

Thalassemia, sickle cell anemia, spherocytosis, PRCA, and erythroid and bone marrow cancers are major genetic diseases of progenitor cells of erythrocytes (8–10). However, none of these diseases is known to initiate in ME. Enucleation of mammalian erythroblasts has circumvented erythrocyte susceptibility to genetic disease; they are genetically epimmune. Accordingly, mature mammalian erythrocytes are protected against genetic diseases including cancer. On the other hand, non-mammalian vertebrates have Nucleated Mature Erythrocytes and they achieved a similar level of epimmunity to mME by adopting a different, yet efficient, strategy. The avian erythrocyte nucleus is genetically inactive, i.e., dormant, or metabolically inactive (11). All vertebrate erythrocyte nuclei become condensed and transcriptionally inactive (12). Avian erythrocyte nucleus is retained in an inactive transcriptional state during terminal erythropoiesis; erythrocytes show no transcription, no DNA synthesis, and no protein synthesis including hemoglobin (13). Avian erythrocytes have active mitochondria (14) that are lost together with ribosomes and cellular RNA in aging nucleated erythrocytes (13, 15). In rainbow trout fish, erythrocytes experience many changes throughout their 4- to 6-month life span including loss of mitochondria, ability to mount heat shock response, and reduction in biosynthetic processes (7).

Nuclei of avian erythrocytes are unable to synthesize detectable RNA (including globin RNA) (16). Ninety percent of RNA is degraded within 8 days in the young circulating avian embryonic erythrocytes, degradation of RNA continues to near zero over the following 10 days (17), indicating the absence of RNA synthesis as well.

Dormant hen nuclei inserted into the cytoplasm of HeLa human cell line increase in size and transform from elliptic shape to become spherical (~3 μm diameter). Then they are induced to resume RNA and DNA synthesis (18).

Heterogeneous nuclear ribonucleic acid (hnRNA) metabolism in differentiating avian erythroid cells revealed the existence of stable hnRNA derivatives (19); the function of which was not clarified. Does it behave as an antisense RNA, an inhibitor of DNA replication, and/or transcription?

This question is raised again; avian erythrocytes synthesize messenger-like RNAs via endogenous polymerase II activity, these include polyadenylated species but contain no mRNA (16). Gene repression and chromatin condensation are accomplished by histone H5 and a 42 kDa-basic non-histone protein known as Mature Erythrocyte Nuclear Termination stage-specific protein (MENT) that accumulates in erythrocyte nuclei of adult chicken (20). However, in vitro LPS-stimulated rainbow and chicken erythrocytes were able to increase levels of Toll-like receptor transcription, supporting the conclusion that nMEs are transcriptionally active and can translate TLR mRNA [St Paul et al. (21) and others] which contradicts earlier reports (11–13, 16, 18, 20, 22, 23). These conflicting reports need to be sorted out and carefully analyzed to clarify this discrepancy.

In general, differentiated cells have partially active nuclei as indicated by the many tissue types of vertebrates (i.e., hepatocyte vs myocyte or neurons) rendering them partially susceptible to phenotypic mutation (i.e., with some exceptions, mutated genes that are inactive will have no consequences on the inert gene and the phenotype of that cell). Similarly, these cells will only engage microbes through their limited surface components which is subject to modification by mutation or interaction with intrinsic immune complexes or extrinsic drugs (cefotetan, ceftriaxone, and piperacillin) as in drug-induced immune hemolytic anemia (24, 25).

Epigenetics, histone code, gene imprinting, iRNA, anti-sense RNA, and their combination are likely to play important roles in achieving tight regulation of nuclear activities, arrest cell differentiation, cell division, and inactivate nuclei of nME.

Such nuclear inactivation renders MEs refractory to genetic diseases, similar to enucleated mammalian erythrocytes.

Claims of parasitic protozoan, bacterial, fungal, and viral diseases of ME are vague and unsubstantiated as revealed by examining some of these infectious diseases of “erythrocytes.”

During final stages of enucleation of mammalian erythroblast, certain receptors are depleted from reticulocytes by way of vesicle sorting and trafficking of proteins to pyrenocytes or reticulocytes (26, 27). Transferrin CD71 receptor is completely eliminated from reticulocytes by sorting to pyrenocytes, unlike glycophorin A/TER119 receptor which sorts to reticulocytes (26). CD71⁺ erythrocytes are rare in peripheral blood (27). Accordingly, mMEs express fewer cell-surface receptors than erythroblasts.

Since viruses and bacteria can replicate in a very short time relative to ME life span (>20 days in chicken, 120 days in human, 4–6 months in fish, and >500 days in turtles), life span of ME cannot explain erythrocyte resistance to infections. On the contrary, the absence of nuclear activity (due to enucleation in mammals and inactivation in other vertebrates) offers a likely explanation.

Enucleated cells have the advantage of reduced cell-surface receptors, allowing erythrocytes to evade microbial attachment, colonization, and invasion.

In addition to their small size, ME must be flexible (deformable) in order to pass through the narrow network of capillaries (human capillaries are 3–10 µm in diameter) (12, 71). Enucleation of mammalian erythroblasts contributes to erythrocyte deformability and efficiency of oxygen-carrying capacity (26, 72, 73). Several lines of evidence show that loss of deformability results from ligating erythrocyte receptors such as complement receptor (CR1) by complement-opsonized microbes or other immune complexes. The loss of deformability plays an important role in the clearance of rigid red cells. Complement–erythrocyte interaction leads to the phosphorylation of the cytoskeletal (band 3) protein which limits erythrocyte deformability (74, 75). Rigidity of erythrocyte in cases of stomatocytosis, discocytosis, and antibody opsonization signals their removal by the phagocytic cells (76). In cases of bacterial infections, complement opsonized bacteria are captured by erythrocytes CR1 where erythrocytes become rigid causing slow microcirculation in the area of infection, eventually entering the liver and spleen where they interact with resident sinusoidal macrophages. They deliver captured bacteria and immune complexes to macrophage and carry on back to circulation (40, 74, 77).

Plasmodium falciparum evades immune clearance by maintaining gametocyte-infected red cells normally deformable; infected cells do not lose their deformability and remain in the circulation for a longer period. The drug sildenafil (viagra) induces rigidity of gametocyte-infected red cells due to protein kinase A-mediated phosphorylation leading to their removal. The phosphorylation of C-terminus serine residue of the protein SubTElomeric Variable Open Reading frame is a signal conveyed to the cytoskeleton through ankyrin, a complex that regulates red cell deformability (78). Erythrocyte deformability is subject to several factors; increased intracellular calcium ions result in rigid cells (74).

Relevant to complement–erythrocyte interaction is the human collectins, mannan-binding lectin, and surfactant protein A and D, which in the presence of an infectious agent may activate the lectin pathway (79).

Human orthochromatic erythroblasts lose the ability to divide as they exit the cell cycle (26). Human reticulocytes undergo several changes within 1–2 days after being released into the circulation, they mature into erythrocytes which lose an average of 15% of their hemoglobin over their lifespan. In vitro, reticulocytes are larger than erythrocytes and contain more RNA and hemoglobin relative to MEs (80). The number of erythrocytes is monitored and regulated by erythropoietin in response to levels of blood oxygen (81).

If hypothetical erythrocytes (HEs) had metabolically active nuclei and were capable of cell division, then new levels of homeostasis have to be attained by other organ systems. The immune system must handle excessive lysis, infections, and malignancies of HEs. Other systems also must reach homeostasis in response to HE activities. However, if HEs double their number by cell division, an increase in blood volume may lead to hypertension, increased blood viscosity, sluggish circulation, and stressed heart.

A regulatory feedback system coordinating HE numbers will be required; such a system must signal every HE to halt or commence cell division, a process that requires one or more receptors which in turn may become targets for microbial attack.

In order to flow smoothly in the circulation without interfering with physiological functions of other cells, erythrocyte cell surface receptors and structures must be limited to a few permissible types that coat erythrocyte surface such as the highly glycosylated glycophorins. Such simple structure (72) should allow for a smooth erythrocyte (blood) flow. Blood transfusion between compatible human individuals is indicative of the simple basic surface structures expressed by human erythrocytes; unlike other tissue grafts and implants, which require histocompatibility matching. Scanning electron microscopy shows smooth erythrocyte surfaces relative to surfaces of activated platelets and leukocytes (72, 82). The simple surface structure reduces erythrocyte adherence to endothelia, leukocytes, bone marrow, cardiac muscle, cardiac valves, other erythrocytes, and microbes. Meanwhile, confinement of erythrocytes to the closed circulatory compartment prohibits its contact with other tissues (e.g., alveolar parenchyma, hepatocytes, fibroblasts, and neurons).

As leukocytes respond to cytokines and signals of inflammation, they extravasate (exit the circulation) to other tissues. Since the main function of erythrocytes is in gas exchange, it is counterproductive for erythrocytes to wonder outside the circulation, they do not perform extravasation and remain trapped inside the circulation, thereby defining the closed circulatory system. Inability of red cells to respond to extravasation signals indicates the importance of enucleation (or nuclear inactivation) in the prevention of extravasation and cell division (see Other Issues Resolved by Enucleation or Nuclear Inactivation).

Signaling molecules are released in the blood at very low concentration; if MEs were to capture or non-specifically interfere with such signaling molecules, the signal will be attenuated or interrupted before reaching its target receptor. MEs are depleted from transferrin CD71 receptor (26) since they do not require transferrin, accordingly they do not compete with other cells for signals or nutrients. MEs transport glucose via GLUT1, which has low affinity to glucose, they also utilize fructose via GLUT5 (83). GLUT 5 has been reported on enterocyte, muscle, kidney, brain, and testis cells, but has not been reported on leukocytes.

In unicellular organisms and simple organisms (e.g., sponges), O₂/CO₂ exchange with the environment is direct. In arthropods including insects, oxygen transport is carried out by extracellular globins; hemerythrins and hemocyanins (84–86). Backswimmer insects (Anisops and Buenoa) hemoglobin is located intracellularly in the cells of large abdominal trachea (87). Vertebrates adopted an intracellular hemoglobin system (the red cells, erythrocytes, or RBCs) for gas exchange. This intracellular system has overcome several problems associated with extracellular globins; serum viscosity and the short half-life of extracellular globins relative to the long half-life of intracellular hemoglobin (88–90). Since vertebrates have closed circulatory systems, a third problem was resolved by confining hemoglobin and erythrocytes to the circulatory system and preventing them from leaking outside the circulation. The importance of confining hemoglobin to the closed circulation is emphasized by the plasma haptoglobin that binds serum hemoglobin and prevents it from causing renal damage (91). Excess soluble hemoglobin can deplete haptoglobin which appears diagnostically in urine (92). Leakage of hemoglobin outside the circulation is counterproductive. Whereas blood flow rate is about one cycle per minute (93), leakage of hemoglobin or ME outside the circulation will prolong the flow rate far beyond the 1 min rate. Therefore, localization of hemoglobin intracellularly is a suitable solution for closed circulatory systems. Leukocytes exit circulation via extravasation (diapedesis) (94), whereas erythrocytes are incapable of extravasation, they remain to define the closed circulation of vertebrates. While the intracellular entrapment of hemoglobin has solved viscosity, globin turnover, and hemoglobin leakage problems on one hand, it created significant problems on the other. In addition to epimmunity and deformability, three other problems that erythrocytes must contend with are discussed in the following sections.

The absence of nucleus from mMEs allows the cell to acquire the biconcave-discoid shape, hence, creating a high A/V ratio. Human biconcave RBCs have a high ratio: ~1.53/μm, whereas the ratio for the spherical shape, i.e., nucleated RBC (erythroblast) is ~0.833/μm (95).

Non-mammalian vertebrates (birds, fish, amphibians, and reptiles) possess nucleated erythrocytes that contain cytoplasmic organelles (96) and functional mitochondria (14). Avian chromatin condensation, nuclear collapse, and inactivation allow avian erythrocytes to appear as flat, thin, flexible, elliptic cells under scanning electron microscope ~(2.24 × 7 µm wide) (97) with a predicted A/V ratio of ~1.53/μm (97–100).

Uninterrupted supply of oxygen is maintained by the large numbers of circulating erythrocytes (2–3.1 × 10¹³ in adult human), timely heartbeat, and normal breathing rate (at rest human breathing rate is 12–16 times per minute) (101). The size and number of circulating human erythrocytes are subject to variation; oxygen concentration, gender, and alleles of TRIM58 gene resulting from SNPs (5).