HEMOGLOBIN, 16(6), 447-467(1992)
SICKLE AND THALASSEMIC ERYTHROID PROGENITOR CELLS
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
ARE DIFFERENT FROM NORMAL
Liya He', Robert Acosta', Mary Ellen Knobloch1 , JoAnn C Thomson1 , Patricia Giardina2 and Rona S Weinberg1
Blanche P Alter'", 'The
Polly Annenberg Levee Hematology Center
Departments of Medicine and Pediatrics Mount Sinai School of Medicine, New York, New York 10029 and 2Department of Pediatrics New York Hospital-Cornell Medical Center, New York, New York 10021 *BPA Current Address: Division of Pediatric Hematology-Oncology, University of Texas Medical Branch, Galveston, Texas 77555 ABSTRACT Blood erythroid progenitors (BFU-E) from patients with sickle and thalassemic syndromes were compared with those from normal individuals. The day of maximal colony formation in methyl cellulose was slightly later in the cultures from the patients with hemoglobinopathies than in the normal cultures. The number of colonies/100,000 mononuclear cells was similar in all cultures on day 13, but was higher in the hemoglobinopathy cultures on the day of maximal growth. The number of BFU-E/mL o f blood was significantly higher than normal at all times in both sickle cell anemia and thalassemia. The proportional synthesis of 7 globin was twice normal in all sickle cultures, and 4 times normal in those from f thalassemia. Hemin and interleukin-3 increased the numbers of erythroid colonies in all cultures, but did not consistently alter the globin synthesis patterns. Each progenitor population has a unique pattern in terms of time course, number of BFU-E, and level of 7 globin synthesis. These features indicate distinct types of BFU-E, or differences in accessory cells, or both, which distinguish blood-borne erythropoiesis in normals and those with hemoglobinopathies.
Received
: A p h i e 22, 7992.
AccepXed : SepZe~nben 16, 1 9 9 2 .
447 Copyright 0 1992 by Marcel Dekker, Inc.
ALTER ET AL.
448 INTRODUCTION
Patients with hemolytic anemias such as sickle cell disease or #?-thalassemia major are anemic, with increased blood reticulocytes and hypercellular, erythroid bone marrows.
They
often have increased levels of Hb F, which if sufficiently high may lead to some modulation of clinical symptoms.
In vitro
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
studies may assist in our understanding of erythropoiesis in these patients and may eventually provide therapeutic insights. Previous comparisons of circulating erythroid progenitors in sickle patients and normal controls suggested an increase in relative numbers (per 100,000 mononuclear cells plated) and in absolute numbers (per mL of peripheral blood) (1, 2).
Croizat and
Nagel (1) found that increased BFU-E/100,000 mononuclear cells correlated with low levels of fetal hemoglobin (Hb F) in vivo, while this relationship was not apparent in other smaller reports (3, 4 ) .
All studies noted that the synthesis of Hb F in colonies
derived from sickle blood BFU-E was higher than in those from normal BFU-E. In one report, the level of Hb F synthesis in the sickle colonies was correlated with the level of Hb F in the circulation ( 4 ) , while this association was not found by other laboratories ( 3 ,
5 , 6).
The relations between
sickle
erythropoiesis, sickle colony Hb F synthesis, and in vivo Hb F levels are thus not entirely clear. Another feature by which sickle erythropoiesis was reported t o differ
from normal
is the in vitro
sensitivity
to
erythropoietin (Ep): sickle marrow and blood cultures were more sensitive to Ep, and this sensitivity required the presence of adherent cells (1, 7).
Croizat et a1 suggested that the adherent
cells (monocytes) from sickle patients with low Hb F produced a burst promoting activity (BPA), and that the BFU-E from this group of patients were responsive to this BPA (1, 8 ) . Several investigators reported increased numbers of BFU-E in the peripheral blood of patients with ,f3-thalassemiamajor (9, 1012).
Although it was suggested that splenectomy might be
ERYTHROID PROGENITOR CELLS
449
responsible for the higher numbers of BFU-E in the thalassemic patients, Meytes et a1 actually noted a decrease in blood BFUE/100,000 mononuclear cells after splenectomy in patients with
thalassemia, hereditary
spherocytosis, and
thrombocytopenic purpura (13).
idiopathic
In addition, Johnson et a1 found
increased BFU-E/mL of blood in thalassemics, unrelated to
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
splenectomy (11).
As might be predicted, the relative synthesis
of 7 globin was higher in culture from thalassemics than from normals ( 9 , 10, 14-17). We have examined a number of variables in our comparison of peripheral blood erythroid progenitors from patients with hemoglobinopathies with those from normal individuals.
These
included the time course o f colony growth, numbers o f progenitors, Ep sensitivity, response to hemin and interleukin-3, and globin chain synthesis.
Our results suggest that the circulating
erythroid progenitors (and/or accessory cells) in these patients have unique characteristics; the progenitors may be less mature than those in normal individuals. MATERIALS AND METHODS Heparinized blood was obtained from normal adult volunteers, patients with sickle cell anemia, and patients with
8'- and
/J+-
thalassemia major and intermedia. All procedures were approved by the Institutional Review Boards of the Mount Sinai School of Medicine, the Albert Einstein College of Medicine, the New York Hospital-Cornell Medical Center, and the New York University College of Medicine.
The blood was centrifuged over Ficoll-Paque
(Pharmacia Fine Chemicals, Piscataway, NJ) at 450 x g for 30 min at 18OC, and the mononuclear cells recovered and cultured at 300,000 cells p e r mL.
Cultures were done in a medium without
nucleosides (Gibco Laboratories, Grand Island, NY), with 0.8% methyl cellulose (Fisher Scientific Co, P'ittsburgh,PA), 30% fetal calf serum (Armor Pharmaceutical Co), 1% deionized bovine serum albumin, 10-4M 2-mercaptoethanol, 0.1 U penicillin and 0.1 pg
ALTER ET AL. streptomycin per mL. Recombinant erythropoietin (Ep) was kindly provided by Ortho Pharmaceuticals, Raritan, NJ, and was used at several concentrations from 0.01 to 4 U/mL.
Some experiments
included 100 pM hemin (Sigma Chemical Co, St Louis MO) or 50 U/mL interleukin-3 (IL-3, Amgen Co, Thousand Oaks, CA).
Cultures were
done in triplicate in 0 . 3 mL volumes in NUNC 4-well dishes
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(InterMed, Roskilde, Denmark), and incubated in high humidity at 37OC in 4% C 0 2 .
BFU-E-derived colonies were counted on days 9 ,
13, 1 6 , and 20 using a Stereozoom dissecting microscope (Bausch & Lomb, Inc, Rochester, NY), and the results from the 3 wells on each day averaged.
Data are shown as the number
of
colonies/100,000 mononuclear cells plated, as well as the number o f BFU-E/mL o f peripheral blood ([colonies/100,000 cells] X
[number of mononuclear cells recovered from each mL of blood]). Ep 1 / 2 max is the concentration of Ep which produced 50% of the maximal number of colonies, and was determined from the Ep dose response curve for each donor.
Globin chain synthesis was
determined by the addition of 100 pC 3H-leucine to each well for
16 to 20 h r s , followed b y recovery of the cells, lysis, electrophoresis on Triton acid urea polyacrylamide gels and fluorography.
Details o f the methods for cultures and for
analysis of globin chain synthesis were published previously (18). Hb F levels in the patients were determined by alkali denaturation
(19).
Hb F 210% was considered to be "high", similar to the cut-
o f f used by Croizat et a1 (8).
There were 27 normal individuals, who were from 2 2 to 6 2 years old, and included 2 2 males and 5 females.
Studies were
done in 2 0 patients with sickle cell anemia (SS).
Thirteen had
low Hb F ( 2 were transfusion-dependent), consisting of 7 males and 6 females, ages 9, 12, 1 4 , and 1 7 to 55 years.
Hemoglobins ranged
from 6 . 6 to 12 g/dL, and Hb F from 1.4 to 8 . 8 % (mean 4 . 3 % ) . Seven S S who had high Hb
F were ages 3, 10, 14, 15, and 34 to 49 years
old, including 4 males and 3 females. Their hemoglobins were from
7 to 11.1 g/dL, and their Hb F ranged from 10 to 20% (mean 14.5%).
All S S patients had normal MCVs.
We studied 7 transfusion-
ERYTHROID PROGENITOR CELLS
45 1
m
1 -1
I
w
30
I
I
I
I
A
0 0 0
1000
o- 20
I
m
0 7
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
\
m W -
7 c
500
10
\ r
Z
0 A 0 0
0
I
I
10
15
10
20
DAY
I
I
15
20
0
DAY FIGURE 1
Time course of blood BFU-E-derivedcolony growth. A) Colonies/100,000 MNc B) BmT-E/mL of peripheral blood. A normal controls. 0 Hb SS with high Hb F o Hb SS with low H b F. 0 TM. 0 TI.
dependent patients with B-thalassemia major (TM), consisting of 1 male and 6 females, 6 , 14, and 16 to 22 years of age.
On the
study day their hemoglobins were from 9 . 3 to 11.5 g/dL.
We also
studied 8 patients with 8-thalassemia intermedia (TI), 5 males and 3 females, 19 to 38 years of age.
None had received a transfusion
within 5 months prior to study. Hemoglobins were from 6 . 8 to 10.2 g/dL, and Hb F 2 4 to 98%. splenectomized.
A l l thalassemics had been
Globin synthesis was analyzed in cultures from 4
TM and 6 TI who were B + .
There were also 2 splenectomized
controls, one a normal 62 year old male, and the other a 26 year old female with pyruvate kinase (PK) deficiency.
Not all
variables could be examined in all patients. RESULTS Time Courses of Erythroid Colony Growth:
BFU-E are usually done with Ep at 2 U / m L ,
Cultures of blood
for 1 3 or 14 days.
26.0 13.9 10.7 13.4
13.2 9.5 4.8 5.8 5 25 8 16
20 27 20 27 20 9
ss N
36.2 23.8 20.5 21.3
17.4 16.5 30.4 574 1098 0.33 8.7 8.7 8.9 9.5
2.5 8.9 14.9 515 703 0.21
3 20 11 15
16 20 16 20 16 8
8.5 6 17 7.6 2.6 15 32.4 15 20
Mean SD
*** ** **
***
* *
***
P
vs N I
62.8 58.2 56.3 68.1
14.6 14.6 16.0 16.0
16.3 2.7 18.7 8.8 24.3 6.4 747 486 926 423 0.49 0.36
2 3
4
3
7 7 7 7 7 7
* *** *** ***
** ***
***
8 7
a
8
8
8
55.8 9.5 5 50.7 6.4 4 47.4 9.6 4
17.0 1.7 20.1 14.0 26.6 12.1 999 804 1332 531 0.72 0.6
30.7 34.2 8 8.6 1.2 8 50.9 23.4 a
3.6 2.9 f 10.0 0.9 7 36.8 10.3 7
N
The1 I n t Mean SD
That Major vs N l Mean SD N P
*** *** ***
***
***
***
P
vs N l
P 800 BFUE/mL) in 6 of 9 SS patients with low Hb F and 3 of 7 with high Hb Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
F.
Our results are more variable than those reported i n an
exclusively adult S S population by Croizat and Nagel (1). However, the inclusion of a few younger patients in our studies does not explain the variability, since the only S S low Hb F patients who had low numbers of BFU-E/mL were adults. The increased numbers of circulating BFU-E in patients with Hb SS (and particularly those with low Hb F) have several possible explanations.
Blood BFU-E may be more prevalent due to expanded
erythropoiesis because of anemia and hypoxic stress.
The degree
of anemia was similar, however, in our Hb S S patients with high and low Hb F.
BFU-E in patients with Hb SS may be more responsive
than those of normals to combinations of hematopoietic growth factors.
More sickle BFU-E may be in cycle ( 8 ) , or fewer BFU-E
may be undergoing programmed cell death, as has been described for murine progenitors (31).
The role of accessory cells from
patients with H b S S and low Hb F i n producing more "burst promoting activity" (BPA) than cells from patients with high Hb F (8) also warrants further investigation.
The Ep half-max values in the SS studies were on average lower than normal, particularly in the S S high Hb F group.
The
only category of patients in whom we have previously observed increased Ep sensitivity is those with polycythemia Vera ( 3 2 ) . Increased sensitivity to Ep has been reported by others in Hb S S in circulating BFU-E and marrow CFU-E, but those reports did not distinguish patients according to Hb F (1, 7); perhaps they included larger numbers o f patients with high Hb F.
The
progenitors from patients with Hb S S with high Hb F were noted by others to differ from those with low Hb F in their sensitivity to
462
ALTER ET AL.
IL-3 (33), and in the Ep affinity of their high affinity Ep receptors (34), supporting the suggestion that progenitor cells may differ depending on the in vivo Hb F level.
However, we
studied too few patients to address this issue in detail. The relative synthesis of 7 globin in S S cultures declined with time, similar to the decline in the normal cultures.
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
Nevertheless, the
% 7
globin was 1.5- to 2-fold higher at all
times in the SS cultures, and was not related to the
%
Hb F in
vivo (Figures 4 and 5), similar to the results in most other reports (3, 5, 6).
The age of the donor was if anything inversely
related to the relative 7 synthesis; the 3 prepubertal patients with low Hb F in vivo had 16 to 20
% -y
synthesis in their
colonies, and the 4 with high Hb F had 17 to 34 (mean 2 5 ) synthesis in culture.
% 7
In fact, there was no clear relation to
patient age. The elevated proportion of
-y
globin synthesis in the
cultures of patients with all types of sickle syndromes suggests that sickle BFU-E may differ from normal in a common manner, such as being intrinsically less mature, and/or producing colonies with erythroblasts which are less mature (25-27).
Sickle BFU-E may be
preprogrammed for high Hb F , as a manifestation of "stress erythropoiesis".
The longer time for sickle colonies to reach
peak growth is consistent with progenitor immaturity; perhaps the level of
globin would have reached normal if we had waited until
day 25.
Another possibility, however, is that sickle BFU-E-
derived cells respond to soluble factors in a manner different from normal with regard to globin synthesis patterns (30, 35).
In
addition, the accessory cells of sickle patients may produce a 7 globin regulatory factor which is different from that produced by normals. This factor would be common to all sickle syndromes, and therefore different from the BPA which increases the number of colonies in patients with Hb SS and low Hb F. There might also be 2 classes of BFU-E in sickle patients, one similar to normal, and another which has the characteristics of immaturity outlined here. Analysis of globin synthesis in single colonies from one Hb S S patient did not indicate a bimodal pattern, however (36).
ERYTHROID PROGENITOR CELLS
463
Hemin significantly increased the day 13 and the peak colony numbers (Table 11) similar to our previous observations (28); hrmin is an effective growth factor even in cultures from patients with hemolysis and presumably elevated levels of plasma hemin (37).
A s in the normals, hemin did not cause a substantial change
in the 7 globin synthesis patterns.
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
cultures from 2 S S patients.
We only added IL-3 to
The day of peak growth was
accelerated, and the number of colonies was doubled on day 13. There was no impact on 7 globin synthesis. Thalassemia:
As
in the SS cultures, colonies from
thalassemic cultures took longer than normal to reach maximal number; only 2 TM cultures (from patients ages 14 and 19) peaked by day 13.
The thalassemic progenitors might also be less mature
than normal, thus taking longer to form colonies. The number of colonies/100,000 MNC was higher than normal, but not statistically significant. BFU-E/mL were significantly increased, however, at all times, consistent with a common theme of hemolytic anemia. Four of the 7 TM patients had >800 BFU-E/mL ( 8 9 0 to 1740), as did
5 of the 8 TI patients (1000 to 2800).
The thalassemic results
resemble those seen in the S S patients with low Hb F , although the thalassemic patients had higher numbers of MNC in the circulation, suggesting greater erythroid stress.
These findings in
nontransfused TI patients are not surprising, but their presence in TM patients immediately before transfusion suggests that erythropoiesis in TM was inadequately suppressed.
Thus
thalassemic BFU-E resemble SS more than normal BFU-E. Study of relative
-y
globin chain synthesis is only
meaningful in P+-thalassemia ( 4 TM and 5 TI patients). a semblance of a temporal decline, but
-y
There was
values remained above
50%. This is much higher than in the SS cultures (and very much
higher than in the normals), and reflects more than just erythroid stress.
The specific values in each thalassemic are presumably
governed by the mutation causing the P+ status; this was not defined for all of our patients. Hemin increased the numbers of colonies in the thalassemic cultures, particularly on day 13, and thus accelerated growth more
ALTER ET AL.
464
than it increased the eventual amount of maximal growth. was a significant increase in the day 13 hemin.
% y
There
globin synthesis with
IL-3 also led to an acceleration of colony growth, with
higher numbers on day 13; there was no effect on globin synthesis. A s in the S S studies, hemin was effective in vitro even in
cultures from patients who presumably had increased levels of plasma hemin. Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
Hemoglobinopathies:
Patients with both sickle and
thalassemic disorders have an increased concentration of blood BFU-E/MNC, and increased numbers of BFU-E/mL, indicative of expanded erythropoiesis in the circulation.
Our thalassemics had
all been surgically splenectomized, and the Hb S S patients were all functionally autosplenectomized. Our normal and hemolytic anemia splenectomy controls had BFU-E numbers consistent with their primary category, not related to the splenectomy.
Others
have also indicated that circulating BFU-E are not affected by splenectomy
(11, 13).
The
increased
blood
B F U - E in
hemoglobinopathies presumably reflects the expanded erythropoiesis secondary to hemolysis; studies of peripheral blood may mirror the degree of erythroid activity in the marrow. BFU-E may be released from the marrow prematurely during erythroid stress.
The numbers
of circulating BFU-E/mL on the peak day in the S S and thalassemic studies were similar to each other, but less than the numbers we found previously in cultures from fetal and neonatal blood (4300 2 3900 and 3500 2 2300 respectively), suggesting that erythropoiesis in hemoglobinopathies is less expanded than erythropoiesis during ontogeny (20). Ep sensitivity varied widely in all groups, with both the
highest and the lowest values in sickle and thalassemic studies. On average, fetal BFU-E were more sensitive than normal adult BF'UE ( 2 0 ) , but there was wide variation there as well.
Thus Ep
sensitivity does not serve as a clear parameter for distinction of fetal, normal, sickle, or thalassemic erythropoiesis. Our cultures were deliberat,ely performed in medium containing serum, and using total mononuclear cells, in order to
ERYTHROID PROGENITOR CELLS
465
demonstrate that there are clear differences between in vitro erythropoiesis in patients with the various syndromes and in normal individuals.
These findings provide directions for the
next phase of these studies, which will be to determine whether there are unique features to the BFU-E themselves, or to the accessory cells and their products, or both, for each category of hemoglobinopathy patient or normal.
These differences vary in
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
their effect on the time of growth, numbers of colonies, sensitivities to Ep, hemin, and IL-3, and levels of y globin synthesis, and thus multiple mechanisms are anticipated. Nevertheless, it is clear that the BFU-E and/or the accessory cells confer specific constellations of features on blood-borne erythropoiesis in normals and in patients with the various hemoglobin disorders; each has a unique phenotype. ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institutes of Health (HL26132 and HL38655).
We thank all the
physicians at our institutions who provided samples from their patients, and we are grateful to Ronald Nagel MD for his critical discussions. REFERENCES
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SICKLE AND THALASSEMIC ERYTHROID PROGENITOR CELLS
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
ARE DIFFERENT FROM NORMAL
Liya He', Robert Acosta', Mary Ellen Knobloch1 , JoAnn C Thomson1 , Patricia Giardina2 and Rona S Weinberg1
Blanche P Alter'", 'The
Polly Annenberg Levee Hematology Center
Departments of Medicine and Pediatrics Mount Sinai School of Medicine, New York, New York 10029 and 2Department of Pediatrics New York Hospital-Cornell Medical Center, New York, New York 10021 *BPA Current Address: Division of Pediatric Hematology-Oncology, University of Texas Medical Branch, Galveston, Texas 77555 ABSTRACT Blood erythroid progenitors (BFU-E) from patients with sickle and thalassemic syndromes were compared with those from normal individuals. The day of maximal colony formation in methyl cellulose was slightly later in the cultures from the patients with hemoglobinopathies than in the normal cultures. The number of colonies/100,000 mononuclear cells was similar in all cultures on day 13, but was higher in the hemoglobinopathy cultures on the day of maximal growth. The number of BFU-E/mL o f blood was significantly higher than normal at all times in both sickle cell anemia and thalassemia. The proportional synthesis of 7 globin was twice normal in all sickle cultures, and 4 times normal in those from f thalassemia. Hemin and interleukin-3 increased the numbers of erythroid colonies in all cultures, but did not consistently alter the globin synthesis patterns. Each progenitor population has a unique pattern in terms of time course, number of BFU-E, and level of 7 globin synthesis. These features indicate distinct types of BFU-E, or differences in accessory cells, or both, which distinguish blood-borne erythropoiesis in normals and those with hemoglobinopathies.
Received
: A p h i e 22, 7992.
AccepXed : SepZe~nben 16, 1 9 9 2 .
447 Copyright 0 1992 by Marcel Dekker, Inc.
ALTER ET AL.
448 INTRODUCTION
Patients with hemolytic anemias such as sickle cell disease or #?-thalassemia major are anemic, with increased blood reticulocytes and hypercellular, erythroid bone marrows.
They
often have increased levels of Hb F, which if sufficiently high may lead to some modulation of clinical symptoms.
In vitro
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
studies may assist in our understanding of erythropoiesis in these patients and may eventually provide therapeutic insights. Previous comparisons of circulating erythroid progenitors in sickle patients and normal controls suggested an increase in relative numbers (per 100,000 mononuclear cells plated) and in absolute numbers (per mL of peripheral blood) (1, 2).
Croizat and
Nagel (1) found that increased BFU-E/100,000 mononuclear cells correlated with low levels of fetal hemoglobin (Hb F) in vivo, while this relationship was not apparent in other smaller reports (3, 4 ) .
All studies noted that the synthesis of Hb F in colonies
derived from sickle blood BFU-E was higher than in those from normal BFU-E. In one report, the level of Hb F synthesis in the sickle colonies was correlated with the level of Hb F in the circulation ( 4 ) , while this association was not found by other laboratories ( 3 ,
5 , 6).
The relations between
sickle
erythropoiesis, sickle colony Hb F synthesis, and in vivo Hb F levels are thus not entirely clear. Another feature by which sickle erythropoiesis was reported t o differ
from normal
is the in vitro
sensitivity
to
erythropoietin (Ep): sickle marrow and blood cultures were more sensitive to Ep, and this sensitivity required the presence of adherent cells (1, 7).
Croizat et a1 suggested that the adherent
cells (monocytes) from sickle patients with low Hb F produced a burst promoting activity (BPA), and that the BFU-E from this group of patients were responsive to this BPA (1, 8 ) . Several investigators reported increased numbers of BFU-E in the peripheral blood of patients with ,f3-thalassemiamajor (9, 1012).
Although it was suggested that splenectomy might be
ERYTHROID PROGENITOR CELLS
449
responsible for the higher numbers of BFU-E in the thalassemic patients, Meytes et a1 actually noted a decrease in blood BFUE/100,000 mononuclear cells after splenectomy in patients with
thalassemia, hereditary
spherocytosis, and
thrombocytopenic purpura (13).
idiopathic
In addition, Johnson et a1 found
increased BFU-E/mL of blood in thalassemics, unrelated to
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
splenectomy (11).
As might be predicted, the relative synthesis
of 7 globin was higher in culture from thalassemics than from normals ( 9 , 10, 14-17). We have examined a number of variables in our comparison of peripheral blood erythroid progenitors from patients with hemoglobinopathies with those from normal individuals.
These
included the time course o f colony growth, numbers o f progenitors, Ep sensitivity, response to hemin and interleukin-3, and globin chain synthesis.
Our results suggest that the circulating
erythroid progenitors (and/or accessory cells) in these patients have unique characteristics; the progenitors may be less mature than those in normal individuals. MATERIALS AND METHODS Heparinized blood was obtained from normal adult volunteers, patients with sickle cell anemia, and patients with
8'- and
/J+-
thalassemia major and intermedia. All procedures were approved by the Institutional Review Boards of the Mount Sinai School of Medicine, the Albert Einstein College of Medicine, the New York Hospital-Cornell Medical Center, and the New York University College of Medicine.
The blood was centrifuged over Ficoll-Paque
(Pharmacia Fine Chemicals, Piscataway, NJ) at 450 x g for 30 min at 18OC, and the mononuclear cells recovered and cultured at 300,000 cells p e r mL.
Cultures were done in a medium without
nucleosides (Gibco Laboratories, Grand Island, NY), with 0.8% methyl cellulose (Fisher Scientific Co, P'ittsburgh,PA), 30% fetal calf serum (Armor Pharmaceutical Co), 1% deionized bovine serum albumin, 10-4M 2-mercaptoethanol, 0.1 U penicillin and 0.1 pg
ALTER ET AL. streptomycin per mL. Recombinant erythropoietin (Ep) was kindly provided by Ortho Pharmaceuticals, Raritan, NJ, and was used at several concentrations from 0.01 to 4 U/mL.
Some experiments
included 100 pM hemin (Sigma Chemical Co, St Louis MO) or 50 U/mL interleukin-3 (IL-3, Amgen Co, Thousand Oaks, CA).
Cultures were
done in triplicate in 0 . 3 mL volumes in NUNC 4-well dishes
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
(InterMed, Roskilde, Denmark), and incubated in high humidity at 37OC in 4% C 0 2 .
BFU-E-derived colonies were counted on days 9 ,
13, 1 6 , and 20 using a Stereozoom dissecting microscope (Bausch & Lomb, Inc, Rochester, NY), and the results from the 3 wells on each day averaged.
Data are shown as the number
of
colonies/100,000 mononuclear cells plated, as well as the number o f BFU-E/mL o f peripheral blood ([colonies/100,000 cells] X
[number of mononuclear cells recovered from each mL of blood]). Ep 1 / 2 max is the concentration of Ep which produced 50% of the maximal number of colonies, and was determined from the Ep dose response curve for each donor.
Globin chain synthesis was
determined by the addition of 100 pC 3H-leucine to each well for
16 to 20 h r s , followed b y recovery of the cells, lysis, electrophoresis on Triton acid urea polyacrylamide gels and fluorography.
Details o f the methods for cultures and for
analysis of globin chain synthesis were published previously (18). Hb F levels in the patients were determined by alkali denaturation
(19).
Hb F 210% was considered to be "high", similar to the cut-
o f f used by Croizat et a1 (8).
There were 27 normal individuals, who were from 2 2 to 6 2 years old, and included 2 2 males and 5 females.
Studies were
done in 2 0 patients with sickle cell anemia (SS).
Thirteen had
low Hb F ( 2 were transfusion-dependent), consisting of 7 males and 6 females, ages 9, 12, 1 4 , and 1 7 to 55 years.
Hemoglobins ranged
from 6 . 6 to 12 g/dL, and Hb F from 1.4 to 8 . 8 % (mean 4 . 3 % ) . Seven S S who had high Hb
F were ages 3, 10, 14, 15, and 34 to 49 years
old, including 4 males and 3 females. Their hemoglobins were from
7 to 11.1 g/dL, and their Hb F ranged from 10 to 20% (mean 14.5%).
All S S patients had normal MCVs.
We studied 7 transfusion-
ERYTHROID PROGENITOR CELLS
45 1
m
1 -1
I
w
30
I
I
I
I
A
0 0 0
1000
o- 20
I
m
0 7
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
\
m W -
7 c
500
10
\ r
Z
0 A 0 0
0
I
I
10
15
10
20
DAY
I
I
15
20
0
DAY FIGURE 1
Time course of blood BFU-E-derivedcolony growth. A) Colonies/100,000 MNc B) BmT-E/mL of peripheral blood. A normal controls. 0 Hb SS with high Hb F o Hb SS with low H b F. 0 TM. 0 TI.
dependent patients with B-thalassemia major (TM), consisting of 1 male and 6 females, 6 , 14, and 16 to 22 years of age.
On the
study day their hemoglobins were from 9 . 3 to 11.5 g/dL.
We also
studied 8 patients with 8-thalassemia intermedia (TI), 5 males and 3 females, 19 to 38 years of age.
None had received a transfusion
within 5 months prior to study. Hemoglobins were from 6 . 8 to 10.2 g/dL, and Hb F 2 4 to 98%. splenectomized.
A l l thalassemics had been
Globin synthesis was analyzed in cultures from 4
TM and 6 TI who were B + .
There were also 2 splenectomized
controls, one a normal 62 year old male, and the other a 26 year old female with pyruvate kinase (PK) deficiency.
Not all
variables could be examined in all patients. RESULTS Time Courses of Erythroid Colony Growth:
BFU-E are usually done with Ep at 2 U / m L ,
Cultures of blood
for 1 3 or 14 days.
26.0 13.9 10.7 13.4
13.2 9.5 4.8 5.8 5 25 8 16
20 27 20 27 20 9
ss N
36.2 23.8 20.5 21.3
17.4 16.5 30.4 574 1098 0.33 8.7 8.7 8.9 9.5
2.5 8.9 14.9 515 703 0.21
3 20 11 15
16 20 16 20 16 8
8.5 6 17 7.6 2.6 15 32.4 15 20
Mean SD
*** ** **
***
* *
***
P
vs N I
62.8 58.2 56.3 68.1
14.6 14.6 16.0 16.0
16.3 2.7 18.7 8.8 24.3 6.4 747 486 926 423 0.49 0.36
2 3
4
3
7 7 7 7 7 7
* *** *** ***
** ***
***
8 7
a
8
8
8
55.8 9.5 5 50.7 6.4 4 47.4 9.6 4
17.0 1.7 20.1 14.0 26.6 12.1 999 804 1332 531 0.72 0.6
30.7 34.2 8 8.6 1.2 8 50.9 23.4 a
3.6 2.9 f 10.0 0.9 7 36.8 10.3 7
N
The1 I n t Mean SD
That Major vs N l Mean SD N P
*** *** ***
***
***
***
P
vs N l
P 800 BFUE/mL) in 6 of 9 SS patients with low Hb F and 3 of 7 with high Hb Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
F.
Our results are more variable than those reported i n an
exclusively adult S S population by Croizat and Nagel (1). However, the inclusion of a few younger patients in our studies does not explain the variability, since the only S S low Hb F patients who had low numbers of BFU-E/mL were adults. The increased numbers of circulating BFU-E in patients with Hb SS (and particularly those with low Hb F) have several possible explanations.
Blood BFU-E may be more prevalent due to expanded
erythropoiesis because of anemia and hypoxic stress.
The degree
of anemia was similar, however, in our Hb S S patients with high and low Hb F.
BFU-E in patients with Hb SS may be more responsive
than those of normals to combinations of hematopoietic growth factors.
More sickle BFU-E may be in cycle ( 8 ) , or fewer BFU-E
may be undergoing programmed cell death, as has been described for murine progenitors (31).
The role of accessory cells from
patients with H b S S and low Hb F i n producing more "burst promoting activity" (BPA) than cells from patients with high Hb F (8) also warrants further investigation.
The Ep half-max values in the SS studies were on average lower than normal, particularly in the S S high Hb F group.
The
only category of patients in whom we have previously observed increased Ep sensitivity is those with polycythemia Vera ( 3 2 ) . Increased sensitivity to Ep has been reported by others in Hb S S in circulating BFU-E and marrow CFU-E, but those reports did not distinguish patients according to Hb F (1, 7); perhaps they included larger numbers o f patients with high Hb F.
The
progenitors from patients with Hb S S with high Hb F were noted by others to differ from those with low Hb F in their sensitivity to
462
ALTER ET AL.
IL-3 (33), and in the Ep affinity of their high affinity Ep receptors (34), supporting the suggestion that progenitor cells may differ depending on the in vivo Hb F level.
However, we
studied too few patients to address this issue in detail. The relative synthesis of 7 globin in S S cultures declined with time, similar to the decline in the normal cultures.
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
Nevertheless, the
% 7
globin was 1.5- to 2-fold higher at all
times in the SS cultures, and was not related to the
%
Hb F in
vivo (Figures 4 and 5), similar to the results in most other reports (3, 5, 6).
The age of the donor was if anything inversely
related to the relative 7 synthesis; the 3 prepubertal patients with low Hb F in vivo had 16 to 20
% -y
synthesis in their
colonies, and the 4 with high Hb F had 17 to 34 (mean 2 5 ) synthesis in culture.
% 7
In fact, there was no clear relation to
patient age. The elevated proportion of
-y
globin synthesis in the
cultures of patients with all types of sickle syndromes suggests that sickle BFU-E may differ from normal in a common manner, such as being intrinsically less mature, and/or producing colonies with erythroblasts which are less mature (25-27).
Sickle BFU-E may be
preprogrammed for high Hb F , as a manifestation of "stress erythropoiesis".
The longer time for sickle colonies to reach
peak growth is consistent with progenitor immaturity; perhaps the level of
globin would have reached normal if we had waited until
day 25.
Another possibility, however, is that sickle BFU-E-
derived cells respond to soluble factors in a manner different from normal with regard to globin synthesis patterns (30, 35).
In
addition, the accessory cells of sickle patients may produce a 7 globin regulatory factor which is different from that produced by normals. This factor would be common to all sickle syndromes, and therefore different from the BPA which increases the number of colonies in patients with Hb SS and low Hb F. There might also be 2 classes of BFU-E in sickle patients, one similar to normal, and another which has the characteristics of immaturity outlined here. Analysis of globin synthesis in single colonies from one Hb S S patient did not indicate a bimodal pattern, however (36).
ERYTHROID PROGENITOR CELLS
463
Hemin significantly increased the day 13 and the peak colony numbers (Table 11) similar to our previous observations (28); hrmin is an effective growth factor even in cultures from patients with hemolysis and presumably elevated levels of plasma hemin (37).
A s in the normals, hemin did not cause a substantial change
in the 7 globin synthesis patterns.
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
cultures from 2 S S patients.
We only added IL-3 to
The day of peak growth was
accelerated, and the number of colonies was doubled on day 13. There was no impact on 7 globin synthesis. Thalassemia:
As
in the SS cultures, colonies from
thalassemic cultures took longer than normal to reach maximal number; only 2 TM cultures (from patients ages 14 and 19) peaked by day 13.
The thalassemic progenitors might also be less mature
than normal, thus taking longer to form colonies. The number of colonies/100,000 MNC was higher than normal, but not statistically significant. BFU-E/mL were significantly increased, however, at all times, consistent with a common theme of hemolytic anemia. Four of the 7 TM patients had >800 BFU-E/mL ( 8 9 0 to 1740), as did
5 of the 8 TI patients (1000 to 2800).
The thalassemic results
resemble those seen in the S S patients with low Hb F , although the thalassemic patients had higher numbers of MNC in the circulation, suggesting greater erythroid stress.
These findings in
nontransfused TI patients are not surprising, but their presence in TM patients immediately before transfusion suggests that erythropoiesis in TM was inadequately suppressed.
Thus
thalassemic BFU-E resemble SS more than normal BFU-E. Study of relative
-y
globin chain synthesis is only
meaningful in P+-thalassemia ( 4 TM and 5 TI patients). a semblance of a temporal decline, but
-y
There was
values remained above
50%. This is much higher than in the SS cultures (and very much
higher than in the normals), and reflects more than just erythroid stress.
The specific values in each thalassemic are presumably
governed by the mutation causing the P+ status; this was not defined for all of our patients. Hemin increased the numbers of colonies in the thalassemic cultures, particularly on day 13, and thus accelerated growth more
ALTER ET AL.
464
than it increased the eventual amount of maximal growth. was a significant increase in the day 13 hemin.
% y
There
globin synthesis with
IL-3 also led to an acceleration of colony growth, with
higher numbers on day 13; there was no effect on globin synthesis. A s in the S S studies, hemin was effective in vitro even in
cultures from patients who presumably had increased levels of plasma hemin. Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
Hemoglobinopathies:
Patients with both sickle and
thalassemic disorders have an increased concentration of blood BFU-E/MNC, and increased numbers of BFU-E/mL, indicative of expanded erythropoiesis in the circulation.
Our thalassemics had
all been surgically splenectomized, and the Hb S S patients were all functionally autosplenectomized. Our normal and hemolytic anemia splenectomy controls had BFU-E numbers consistent with their primary category, not related to the splenectomy.
Others
have also indicated that circulating BFU-E are not affected by splenectomy
(11, 13).
The
increased
blood
B F U - E in
hemoglobinopathies presumably reflects the expanded erythropoiesis secondary to hemolysis; studies of peripheral blood may mirror the degree of erythroid activity in the marrow. BFU-E may be released from the marrow prematurely during erythroid stress.
The numbers
of circulating BFU-E/mL on the peak day in the S S and thalassemic studies were similar to each other, but less than the numbers we found previously in cultures from fetal and neonatal blood (4300 2 3900 and 3500 2 2300 respectively), suggesting that erythropoiesis in hemoglobinopathies is less expanded than erythropoiesis during ontogeny (20). Ep sensitivity varied widely in all groups, with both the
highest and the lowest values in sickle and thalassemic studies. On average, fetal BFU-E were more sensitive than normal adult BF'UE ( 2 0 ) , but there was wide variation there as well.
Thus Ep
sensitivity does not serve as a clear parameter for distinction of fetal, normal, sickle, or thalassemic erythropoiesis. Our cultures were deliberat,ely performed in medium containing serum, and using total mononuclear cells, in order to
ERYTHROID PROGENITOR CELLS
465
demonstrate that there are clear differences between in vitro erythropoiesis in patients with the various syndromes and in normal individuals.
These findings provide directions for the
next phase of these studies, which will be to determine whether there are unique features to the BFU-E themselves, or to the accessory cells and their products, or both, for each category of hemoglobinopathy patient or normal.
These differences vary in
Hemoglobin Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/21/14 For personal use only.
their effect on the time of growth, numbers of colonies, sensitivities to Ep, hemin, and IL-3, and levels of y globin synthesis, and thus multiple mechanisms are anticipated. Nevertheless, it is clear that the BFU-E and/or the accessory cells confer specific constellations of features on blood-borne erythropoiesis in normals and in patients with the various hemoglobin disorders; each has a unique phenotype. ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institutes of Health (HL26132 and HL38655).
We thank all the
physicians at our institutions who provided samples from their patients, and we are grateful to Ronald Nagel MD for his critical discussions. REFERENCES
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