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302 Part IV Disorders of Hematopoietic Cell Development

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SNP represents a susceptibility allele that in association with the expelled from erythroblasts, whereas expression of CD47, the “eat
JAK2V617F mutation predicts poor survival. 125 me not” signal, by interacting with SIRP1α expressed by the mac-
The morphologic alterations that occur as erythroid precursor rophages, prevents engulfment and destruction of erythroblasts and
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cells mature (see Fig. 26.2) are determined by complex biochemical reticulocytes. The enucleation process is caspase independent
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changes, which accommodate the accumulation of erythroid-specific but erythroblast macrophage protein (EMP) dependent. EMP is
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proteins and the progressive decline in proliferation. Compared expressed by both erythroblasts and macrophages, and it is necessary
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with erythroid progenitor cells, erythroid precursor cells have been for proper enucleation to occur. The fact that proper enucleation
more accessible to study, and considerable information is available requires interaction with macrophages explains the old observation
about their maturation-related biochemical changes. that erythroid differentiation in the marrow occurs in discrete sites,
The shape and deformability of the red cells are determined by the the “erythroblastic islands,” which are composed of erythroblasts
appropriate assembly of their membrane proteins with the cytoplasmic surrounding a central macrophage. Presence of molecules enhanc-
cytoskeleton. Red cells survive shear forces in the microvasculature ing adherence of erythroblasts to central macrophages are thought
because transmembrane complexes embedded in the lipid bilayer to be important mediators of the interaction of erythroblasts with
attach to the cytoskeleton, ensuring its flexibility. These complexes macrophages. Several of these molecules have been described and
contain clinically relevant blood group antigens determined by summarized in recent reviews. 146–149 Among these are the VLA4 inter-
genetic polymorphisms in proteins of these complexes. 127–129 The acting with VCAM-1, the ICAM-4 on erythroblasts interacting with
similarity between the amino acid sequence of the blood group αv and α4 on macrophages, the CD163 on macrophages serving
antigens and that of proteins present on the surface of bacteria and as the Hb-Hp binding receptor, or the Palladin on Macrophages
the increased frequency of certain blood group antigens in regions with an unclear counter receptor on erythroblasts. In addition,
with high incidence of malaria suggest that blood group antigens, in direct contact soluble factors are secreted by macrophages and these
addition to ensuring appropriate membrane structure, may facilitate may serve as either positive or negative regulators of erythroblast
development of appropriate immunoreactivity toward opportunistic proliferation. Data from in vitro models of human stress erythro-
infections. poiesis indicated that glucocorticoids may induce the generation of
Most membrane cytoskeletal proteins (spectrin, glycophorin, a “unique” macrophage that interacts with multiple erythroblasts,
band 3, band 4.1, and ankyrin) accumulate after the CFU-E stage leading to the formation of “transient erythroblast islands” that
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(i.e., within the precursor cell compartment). Specifically, expression promote erythroid proliferation instead of maturation (Falchi et al
of membrane glycoproteins such as band 3 and band 4.1 is greatly and Video 26.1).
enhanced at the later stages of erythroid maturation. 126,130,131 Like- In addition to quantitative changes that occur during maturation,
wise, the quantity of polylactosaminoglycan, a specific carbohydrate gradual switches in subunit composition of some cytoskeletal proteins
chain that carries blood group ABH and Ii antigenic determinants, is occur. For example, exclusively erythroid subunits of α- and β-spectrin
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much higher in mature erythrocytes than in erythroblasts. Whereas are displayed only in end-stage cells. Likewise, multiple transcripts
a linear, virtually unbranched polylactosamine structure is present in of ankyrin or protein 4.1 have been identified, and the ratios of these
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fetal and newborn erythroid cells (reflected by i antigenic reactivity), a transcripts change during maturation. Initial expression of many
branched polylactosaminyl structure is present in adult erythroblasts of these membrane components likely begins at the progenitor cell
(reflected by I antigenic reactivity), and branching increases further level. However, in these cells, final assembly may be discouraged
as maturation progresses. 132,133 A correctly assembled cytoskeleton is because of the higher turnover of these proteins, which minimizes
important for the deformability and dynamic plasticity of red blood mutual interactions, or because of asynchrony in protein synthesis.
cells in circulation. A recently recognized player required for actin Prevention of cytoskeletal assembly at these early stages may secure
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assembly in red cells, Rac GTPase, has been identified. Glycopho- more membrane fluidity and cell motility needed during this prolif-
rins, especially glycophorin A, are expressed fully at the CFU-E or erative phase of differentiation. Because molecular probes for many
proerythroblast level just before expression of globin, and few changes of the red cell cytoskeletal components have been developed, detailed
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occur during maturation. In contrast, the membrane glycoproteins information about the transcription and processing of most of these
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p105 and p95 decline during the later stages of maturation, and proteins is beginning to emerge. For example, band 3, the major
yet other membrane glycoproteins, such as vimentin (an intermediate anion transport protein of human erythrocytes, is a key component
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filament protein), are totally lost. Loss of vimentin expression at the of a multicomplex that also contains protein 4.2. Appropriate display
late erythroblastic stages most likely facilitates enucleation. of this protein complex on the cell membrane is dependent on
The process of erythroblast enucleation involves membrane critical interactions established between newly synthesized band 3
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remodeling, chromatin condensation to form pyknotic nuclei, and and protein 4.2 already at the proerythroblast stage. 152
formation of spindle-independent motors driving the separation of Expression of the majority of genes encoding cytoskeletal
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the reticulocyte from the pyrenocyte. Partitioning of erythroblast components is not restricted to red cells. Dissecting hemopoietic
plasma membrane components to reticulocytes is regulated by the from nonhemopoietic consequences of abnormalities in these genes
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degree of skeletal linkage and by the Coimbra domain of band 3. has been difficult, but the development of mouse models that
In fact, red cells from patients with homozygous band 3 Coimbra mimic defects found in human diseases has been helpful in this
express reduced levels of multiple cell surface antigens which are all respect. 153
rescued in vitro by forced expression of normal band 3. Chromatin Gene activity during erythroid maturation is dominated by globin
condensation may require DNA demethylation, since with matura- expression. Globin represents less than 0.1% of protein at the pro-
tion the total methylation state of the DNA greatly decreases down to erythroblast level but constitutes 95% of all protein at the reticulocyte
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barely detectable levels and is mediated by the histone deacetylase level. Globin expression has been extensively studied, and its gene
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(HDAC) 2, suggesting that impairment of HDAC2 activity may regulation is well understood in molecular terms. Major steps in
contribute to the development of anemia observed in HDAC-based globin transcription and processing are known in considerable detail
cancer treatments. and are summarized elsewhere in this text (see Chapter 33). The
Nonmuscle myosin appears to represent the motor driving the sep- globin type synthesized by adult precursors is hemoglobin A (HbA;
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aration between the reticulocyte and the pyrenocyte. Reticulocytes α 2 β 2). In addition, two other minor globin components, HbA 2 (α 2δ 2)
are released in the blood, where they undergo extensive cytoplasmic and HbF (α 2γ 2), are present. Of significant biologic interest are the
remodeling to reduce the number of ribosomes and mitochondria and low amounts of HbF that continue to be synthesized throughout life.
to become mature red cells. This process is mediated by autophagic The small amount of HbF, which is present in all normal
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machinery, whereas engulfment of pyrenocytes, and subsequent individuals, has the following characteristics. (1) It is confined
degradation by macrophages, occurs only after pyrenocytes are totally to a small fraction of red cells, called F cells, which are detected
disconnected from reticulocytes. Phosphatidylserine, the “eat me” by sensitive immunofluorescence assays or acid elution techniques
flag for apoptotic cells, is also used for engulfment of pyrenocytes and usually constitute 2% to 5% of all red cells. Within each F