Accelerated Programmed Cell Death (Apoptosis) in

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RAPID COMMUNICATION
Accelerated Programmed Cell Death (Apoptosis) in Erythroid Precursors
of Patients With Severe @-Thalassemia(Cooley’s Anemia)
By J. Yuan, E. Angelucci, G. Lucarelli, M. Aljutf, L.M. Snyder, C.R. Kiefer, L. Ma, and S.L. Schrier
The profound and life-threatening anemia in patients with
Cooley‘s anemia is ascribed primarily to intramedullaryhemolysis (ineffective erythropoiesis), the cause of which is
obscure. Based on prior morphologic data showing nuclear
abnormalities, w e hypothesized that accelerated apoptosis could occur in these erythroid precursors. The highly
successful bone marrow (BM) transplantation program for
patients with Cooley’s anemia provided us with a unique
opportunity to test this hypothesis. W e obtained pretransplantation BM aspiration samples from patients undergoing BM transplantation in Pesaro, Italy and from their allogeneic donors. The erythroid precursors were isolated
using ficoll sedimentation and then panning selecting for
CD45- cells. Cytospin and Giemsa staining showed that
the separation provided greater than 90% erythroblasts.
Five million of these erythroblasts were lysed and their
DNA was isolated. There were obvious ladder patterns of
DNA breakdown products in 8-thalassemia major samples, with less occurringin &thalassemia trait. Normal individuals showed only a slight smear of breakdown of DNA.
These results indicate there is enhanced apoptosis in the
erythroblasts in the BMs of Cooley’s anemia patients. This
finding might partially explain why most of these erythroblasts never survive to become mature erythrocytes.
0 1993 by The American Society of Hematology.
I
membrane.* Our hypothesis is that unmatched excess a-globin chain accumulation somehow causes these defects and
ultimately the death in marrow of about 80%of the affected
erythroid precursors. This hypothesis would be consistent
with our observations on the important role of specific globin accumulation in the thalassemia.’
The role of programmed cell death (apoptosis) has been
shown for many tissues and cell lines.’@’*Apoptosis is a
selective process of physiologic cell deletion. Its occurrence
plays a major role in the control of normal and abnormal
proces~es.‘~
Apoptosis has been described in erythroid precursors in in vitro s y ~ t e m s . ’ ~Although
.’~
there is presently
no evidence for programmed cell death in normal erythropoiesis, the late or orthrochromic erythroblast with a shrunken pyknotic nucleus being extruded from the cell looks
morphologically like apoptosis. In this study, we evaluated
the possibility that a-globin chain accumulation could trigger or enhance programmed cell death in erythroid progenitors of patients with 6-thalassemia major.
The successful allogeneic BM transplantation program
for patients with P-thalassemia major in Pesaro, Italy provided us with a unique opportunity to test the hypothesis
that enhanced programmed cell death may contribute to the
profound intramedullary hemolysis that occurs in the BM
of these patients.
We obtained pretransplantation marrow samples from
the P-thalassemia major patients and from their sibling allogeneic donors, who either have the &thalassemia trait or are
normal. Our study consisted of seven P-thalassemia major
patients, five P-thalassemia trait donors, and four normal
donor bone marrow samples (all shipped on ice from Italy)
plus four normal bone marrow samples locally obtained
from the Stanford University Hospital Bone Marrow Transplantation Program, according to the protocols approved by
the Stanford Institutional Review Board. We also studied
two additional “control” subjects. One patient had severe
myelodysplastic syndrome with morphologic evidence of
ineffective erythropoiesis and megaloblastoid erythroid precursors in marrow. The other patient was a child completing
an otherwise uncomplicated induction for acute lymphoblastic leukemia whose marrow aspirate showed brisk nor-
N THE SEVERE FORMS of human thalassemia, anemia and its treatment with transfusions cause morbidity
and mortality.’.’ Ineffective erythropoiesis in the bone
marrow (BM) and peripheral hemolysis of red blood cells
(RBCs) leads to anemia.3,4Peripheral hemolysis of RBCs
may contribute to the anemia more in severe a-thalassemia
(Hb-H d i s e a ~ e ) ,but
’ ~ ~intramedullary hemolysis is the major kinetic defect that leads to anemia in P-thalassemia major or Cooley’s anemia.3 As shown by Finch et al,3 the
plasma iron turnover rate in 6-thalassemia major patients,
as a measure of the onset of erythropoiesis, can be 10 times
that of normal patients. However, the delivery of thalassemic RBCs to the peripheral blood in these patients is much
reduced, thereby indicating extraordinary intramedullary
erythroid cell death. The cause of this death in the marrow
has never been fully identified, but there are morphologic
clues showing that erythroid precursors in &thalassemia
major show evidence of a-globin chain deposition: cytoplasmic vac~olization,~
and abnormalities of the nuclear
From the Divisione Ematologica di Muraglia, Centro Trapianto
di Midollo Osseo, Ospedale Di Pesaro, Italy; the Department of
Hospital Laboratories, University of Massachusetts Medical
Center, Worcester, MA; the Department ofImmunology and Microbiology, Medical College ofGeorgia, Augusta, GA; and the Division
of Hematology, Stanford University School of Medicine, Stanford,
CA.
Submitted February 8, 1993; accepted April 8, 1993.
Supported by National Institutes of Health Grant No. 5RO1
DKI 3682.
Presented in preliminary form at the American Society of Hematology Meeting 1992, Anaheim, CA (Blood 80:342a, 1992 [abstr,
SUPPI 11).
Address reprint requests to S.L. Schrier, MD, Division of Hematology, S-161, Stanford University School ofMedicine, 300 Pasteur
Dr, Stanford, CA 94305-5112.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology
0006-49 71/93/8203-003 9$3.00/0
374
Blood, VOI 82, NO 2 (July 15).1993:pp 374-377
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375
APOPTOSIS AND @-THALASSEMIA
Fig 1. Thalassemic BM cells before (A) and after
(B) CD45- selection. Ten milliliters of BM was collected from @-thalassemiamajor patients. The hematopoietic precursors, after the removal of RBCs by
ficoll centrifugation and lysis with 0.83mol/L NH,CI,
are shown in (A). Ten million of these cells were further treated by reacting with monoclonal anti-CD45
antibodies. After washing with phosphate-buffered
saline, these cells were loaded onto goat antimouse
19-coatedAIS Microcellectors and incubated at room
temperature for 1 hour. The nonadherent CD45cells are shown in (B).
moblastic erythroid hyperplasia (75% of all marrow cells).
Hematopoietic progenitors were isolated using ficoll centrifugation to remove mature RBCs and the remaining RBCs
were removed by lysis in ammonium chloride solution. It
was critical to work with a “pure” population of erythroid
precursors, therefore, cells were isolated by panning onto
AIS MicroCellector plates (Applied Immune Sciences,
Menlo Park, CA) and CD45- cells were selected. CD45 is a
membrane protein that is present on lymphoid and myeloid
lineages but it is absent in erythroid precursors. Flasks
coated with secondary antibody were used to remove
Table 1. Marrow Erythroid Precursor Differential Count
Normal
(%I
Proerythroblasts
Basophilic erythroblasts
Polychromatophilicerythroblasts
Orthrochromic ervthroblasts
3
9
18
70
Thal Trait
(%)
Thal Major
13
19
24
44
10
18
49
(%I
23
CD45-labeled cells. After the removal of RBCs, the marrow
suspension in Cooley’s anemia consists of a mixture of erythroid, myeloid, and lymphoid progenitors (Fig IA), but
our CD45 negative selection process yielded a preparation
that contained greater than 95% erythroblasts (Fig I B). In
addition to cytospin and differential counts, these erythroid
precursors were also analyzed by laser confocal immunofluorescence microscopy.16 To detect apoptosis. the erythroid
precursors were then counted and 5 million of these cells
from normal patients, donors, and affected patients were
lysed and centrifuged. The DNA from both supernatant and
pellet was precipitated. The pellet contains most of the intact chromosomal DNA, whereas the supernatant contains
the majority of the DNA breakdown products. The supernatant was used to evaluate apoptosis. After removal of
RNA by RNase, the DNA samples from the supernatant
were loaded onto an agarose gel and electrophoresed. Examples of the DNA pattern from supernatants are shown in Fig
2. There were obvious ‘‘ladder’’ patterns of DNA breakdown products in &thalassemia major patients and, to a
lesser extent, in /3-thalassemia trait donors. Normal individuals had the least amount of this sort of DNA breakdown.
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376
YUAN ET AL
4
Kb
2.32
1.93
1.37
1.26
0.70
1
2
3
4
Neither of the two “controls,” one representing intense erythroid hyperplasia and the other a form of dysplastic ineffective erythropoiesis, showed ‘‘ladder’’ pattern formation
in their separated erythroid precursors (data not shown).
These ‘‘ladder’’ patterns are characteristic of programmed
cell death or ap~ptosis.”*’~
It has been suggested that endo-
Fig 3. Laser confocal immunofluorescenceshowing the deposition of a-globin chains in
&thalassemia major erythroid
precursors. The erythroid precursors were fixed on Alcian
blue-coatedcover slips and permeabilized with 0.5% Triton
X-1 00 in phosphate-buffered
saline. The cells were then
reacted with monoclonal antia-globin chain antibody for 1
hour at m m temperature and
Texas Red-labeled goat antimouse IgG for 30 minute at
mom temperature.
Fig 2. DNA fragmentation of erythrocyte precursors from a
fl-thalassemia trait donor (lane 1). a &thalassemia major patient
(lane 2).and a normal individual (lane 3).Erythrocyte precursors
from respective BMs were isolated and 5 million of these cells were
lysed in a buffer consisting of 10 mmol/L Tris, pH 7.4; 1 mmol/L
EDTA; 0.2%Triton X-1 00. After centrifugation at 1 1,OOOg for 30
minutes, the supernatant was collected and the DNA was precipitated with 50% isopropanol in 3 mol/L NaCI. After digestion for 1
hour with 1 Wg/mL RNase, these samples were loaded onto an
1.5% agarose gel and electrophoresed. The gel was stained with
ethidium bromide.
nucleases attack DNA specifically to lead to this orderly
breakdown pattern.” Thus, there appears to be greatly enhanced apoptosis in the erythroblasts of P-thalassemia major patients and considerably less apoptosis in donors with
@-thalassemictrait. This finding could partially explain why
most of the erythroblasts in P-thalassemia major never mature. We also harvested CD45’ cells from the respective
marrows. The DNA from supernatants of lymphoid and
myeloid precursors showed no sign of ladder pattern formation (data not shown).
We next considered the possibility that the &thalassemic
marrow might contain a disproportionate number of the
late-stage or orthrochromic normoblasts, with pyknotic nuclei thought to be the morphologic indicator of apoptosis.
However, the differential counts (200 to 500 cells counted)
from one shipment of these marrows samples was as shown
in Table I. There are more early- and intermediate-stage
erythroid precursors in P-thalassemia major and minor
marrow, whereas normal individuals have relatively more
late-stage erythroid precursors. Therefore, it is likely that
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APOPTOSIS AND ,&THALASSEMIA
the apoptosis seen occurs in the early-stage erythroid precursors in severe @-thalassemia.
We then proposed that the a-globin chain deposition
somehow resulted in the enhanced apoptosis in the BM of
P-thalassemia major patients. If that were the case, we
would expect to see a-globin chain deposition in the early
stages of erythropoiesis, where we believe apoptosis to occur. We used laser confocal immunofluorescent microscopy, with monoclonal antihuman a-globin as the primary
antibody and Texas Red-labeled antimouse IgG as the secondary antibody, and detected the deposition of a-globin
chains (Fig 3). The monoclonal antibody” a-1-58782
(IgG2b,k) was derived by the fusion of the myeloma cell line
FOX-NY with spleen cells from an RBF/On mouse immunized with a conjugate containing a synthetic peptide corresponding to the sequence a 17-26.20This peptide contains
the a-unique sequence gly-ala-his-ala-gly (a 17-2I), which
occurs at the bend of the A and B helices in the native chain.
The antibody reacts with a chains, either native or denatured, but does not react with non-a chains. The a-globin
chains accumulate progressively as the erythroid cell undergoes progressive differentiation. Although the late-stage precursors have the most a-globin chain accumulation, a-globin chain deposition can be seen as early as the
proerythroblast stage. The heterogeneity of a-globin chain
deposition in this thalassemic sample is fairly typical. In
contrast, in very early stages of normal erythroid progenitors, we saw no such globin chain accumulation (data not
shown). This result indicated that the a-globin chain deposition in ,&thalassemia major patients occurs early enough to
cause the enhanced programmed cell death we observed.
We thus propose that enhanced programmed cell death
may partially explain the intramedullary cell death in P-thalassemia major. Abnormal a-globin chain deposition in
,&thalassemia occurs early enough in erythropoiesis to
cause accelerated apoptosis in the affected stages. The mechanisms by which a-globin chain accumulation could lead to
apoptosis is under investigation.
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1993 82: 374-377
Accelerated programmed cell death (apoptosis) in erythroid
precursors of patients with severe beta-thalassemia (Cooley's anemia)
[see comments]
J Yuan, E Angelucci, G Lucarelli, M Aljurf, LM Snyder, CR Kiefer, L Ma and SL Schrier
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