Page 368 - Hematology_ Basic Principles and Practice ( PDFDrive )

304 Part IV Disorders of Hematopoietic Cell Development

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hemochromatosis. The most prevalent form of hereditary hemo- generated in a stochastic fashion from pluripotent stem cells.
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chromatosis (type 1; HFE1) involves mutations in HFE. Most Neither EPO nor other lineage-restricted regulators play any role
families with juvenile hemochromatosis (HFE2) have mutations in in determining lineage commitment. According to this model, EPO
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the HJV gene. Homozygous nonsense and single-point muta- influences erythroid differentiation by rescuing (from apoptosis) cells
tions causing methionine→lysine substitution at position 172 of the that express EPOR and amplifying them further. Whether EPORs are
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protein M172K have been detected in the gene encoding TfR2 in present on all BFU-E (detectable only in a subset of BFU-E) is not
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patients with familial hemochromatosis HFE3. Autosomal dominant clear. Thus whether the presence of EPOR in BFU-E is synchronous
iron overload is associated with previously unrecognized ferroportin with the initial commitment event or follows it, is not known. In
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1 mutations (p.R88T and p.I180T) and with mutations in the addition to the permissive role of EPO ascribed by the stochastic
divalent metal transporter 1 gene DMT1, which mediates apical theory, experiments in vivo, in anemic states, or after pharmacologic
iron uptake in duodenal enterocytes and iron transfer from the TfR doses of EPO suggest that high levels of EPO hasten the transition
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endosomal cycle into the cytosol in erythroid cells. The observation from BFU-E to hemoglobin-synthesizing cells by decreasing either
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that targeted deletions of any of these genes (including TfR2) induce the number of divisions required for this transition or the resting
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a hemochromatosis-like syndrome in mice provides proof of direct periods between cell divisions. Autoradiographic studies of purified
involvement of the mutations in disease development. 191–194 On the BFU-E populations indicate that EPORs increase as BFU-E mature
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other hand, the finding that hepcidin is a gene modifier of the HEF to CFU-E, with the highest level observed at the CFU-E/proeryth-
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mouse model of hemochromatosis suggests that heterogeneity at the roblast boundary. That the transition from BFU-E to CFU-E
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hepcidin locus mediates the low penetrance of the genetic disease. occurs under the influence of EPO suggests ligand (EPO)-induced
In addition to its role in determining the pathobiology of hereditary receptor upregulation. Whether the magnitude of such upregulation
hemochromatosis, hepcidin plays an important role in determining is dependent on EPO dose and whether it can modulate the rate of
the anemia of chronic diseases. Based on the central involvement of entry of these cells into the maturing compartment is unclear.
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hepcidin in iron regulation and its pathologic conditions, a hepcidin BFU-E and CFU-E can be generated in vitro and in vivo, in
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assay has been proposed as a useful tool for diagnosing iron disorders the absence of EPO or EPOR (in EPO or EPOR mice), but their
and monitoring their treatment. On the other hand, development of survival and terminal maturation normally are dependent on EPO.
hepcidin agonists and antagonists may provide useful therapeutics for For CFU-E, EPO seems to stimulate all the biochemical processes
treatment of iron disorders. 196 characterizing erythroid cells (i.e., heme synthesis, globin synthesis,
The patterns of TfR and glycophorin A expression during erythroid and synthesis of cytoskeletal proteins). However, the necessity of
maturation have been exploited to define flow cytometric criteria EPO in these processes is not absolute. In vitro experiments showing
that distinguish the different populations of erythroid precursors in complete maturation of BFU-E in the absence of EPO suggest that
mice and men. By coupling size and forward scatter (both progres- other factors or combinations of factors can influence red cell matura-
sively reduced) with CD71 (TfR) and TER-119, murine erythroid tion. Activation of the gp130 signaling pathway by use of soluble
precursors were divided into the classes TER-119 med CD71 high , TER- IL-6 receptor and IL-6 leads to full terminal erythroid maturation
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119 high CD71 high , TER-119 high CD71 med , and TER-119 high CD71 , (in the presence of stem cell factor and IL-3 but in the absence of
which correspond to proerythroblasts and basophilic, chromatophilic, EPO), suggesting some form of cross-circuiting in signaling pathways
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and orthochromatophilic erythroblasts, respectively. However, among hematopoietic growth factor receptors. 207,208 Furthermore,
such distinction is not conserved in all mouse strains. For example, stimulation by TPO of erythroid colony formation from yolk sac cells
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in C57Bl/6 mice, CD71 expression levels remain constant during in the absence of EPOR (in EPOR embryos) can be explained
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maturation. CD44/glycophorin A expression provides a better flow by the same reasoning and the finding of a very high proportion of
cytometric definition of the maturation stage of murine erythro- bipotent erythroid/megakaryocytic progenitors in yolk sac carrying
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blasts. Although reduced levels of CD44 do not correlate with both EPO and TPO receptors (c-Mpl) compared to adult bone
the maturation of human erythroblasts, it marks loss of proliferation marrow. 63,210,211
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potential of these cells. Double CD71/glycophorin A staining is Whatever the precise mode of EPO action, it directly affects the
therefore still used as criteria to define human erythroblast precursors number of CFU-E and the maturation of their progeny. This control
by flow cytometry. However, the pattern of CD71 expression during is achieved by influencing CFU-E survival and not their cycling
erythroid maturation presents a high level of donor variability. Given status. 212 CFU-E are irrevocably lost after one cycle of DNA synthesis
that downmodulation of CD36 expression during erythroid matura- if EPO is not present. 213
tion is relatively independent of genetic variability, an alternative With the availability of radiolabeled recombinant EPO and
flow cytometric definition of erythroblast subclasses is proposed by purified or enriched populations of progenitors and precursors, has
the phenotype CD36 high /glycophorin A medium , CD36 high /glycophorin come information about the characteristics of EPORs in erythroid
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A high , and CD36 /glycophorin A high , corresponding to basophilic, cells. Direct binding studies have shown that a progressive decrease
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polychromatic, and orthochromatic erythroblasts, respectively. in the number of EPORs occurs as CFU-E and proerythroblasts
Flow cytometric criteria for reticulocytes and red cells are instead mature to reticulocytes. 68,69,173 Pure reticulocyte populations show
provided by size (reticulocytes and red cells are distinctively smaller no detectable binding to EPO. The maturation-associated decline in
than erythroblasts) and by lack of reactivity for DNA (both reticulo- the number of EPORs parallels the declining influence of EPO on
cytes and red cells) and RNA (reticulocyte only) staining. erythroid cells during the terminal phase of maturation. The exquisite
role of EPO in determining red cell numbers in the circulation has
been clearly established by direct correlations between hematocrit
ERYTHROPOIETIN AND EPOR and EPO plasma concentrations in individuals exposed to hypoxia
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and in patients with compromised kidney functions. However,
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EPO, a 35-kDa glycoprotein, is the physiologically obligatory the variabilities around the mean of hematocrit and EPO plasma
growth factor for erythroid development. It is produced mainly in levels found in normal individuals under steady-state conditions are
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the kidney by peritubular cells. A heme-containing protein senses not correlated, indicating that other factors (sex and age) cooperate
oxygen need and then triggers the synthesis of EPO and its release with EPO in determining the fluctuations in red cell mass under
into the bloodstream. 202,203 Through the interaction of EPO with steady-state hematopoiesis. 215
receptor-bearing cells within the bone marrow, physiologic oxygen Cloning and expression of EPOR has allowed for a better
demands are translated into increased red cell production. Thus understanding of the role of EPO in the regulation of erythroid
EPO is a true hormone, manufactured at one anatomic site and development. The EPOR polypeptide is a 66-kDa membrane
transported through the bloodstream to the site of activity. protein that is a member of the cytokine receptor superfamily. 216,217
According to the prevailing model of hematopoiesis, progeni- Many of the structural features of the cell surface EPOR have been
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tor cells committed to erythroid differentiation (i.e., BFU-E) are previously reviewed. Like other members of the cytokine receptor