Luxol-fast-blue staining of EoL-1 cells. (original magnification x200; current magnification x 130). (E) MGG staining of EoL-1 cells cultured in alkaline medium.
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1985 66: 1233-1240
Establishment and characterization of a new human eosinophilic leukemia cell line H Saito, A Bourinbaiar, M Ginsburg, K Minato, E Ceresi, K Yamada, D Machover, J Breard and G Mathe
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Establishment
and
Characterization
of a New
Leukemia By Hiroshi
Aldar
Saito,
Bourinbaiar, David
Cell
Mich#{234}leGinsburg,
Machover,
Jacqueline
A human eosinophilic leukemia cell line, designated as EoL. was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling
time
of 48
hours
for
about
one
year.
The
reactivity
of these cells was tested with a panel of monoclonal antibodies; they were found to express surface Ia antigen, myeloid antigen (IF1 0, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue
E
OSINOPHILIC 1912
disease present
leukemia
by Stillman.’
Since
(EL)
was
then,
diagnosis
remains a problem, since large numbers of immature
putative not yet lines
characteristic been clearly
have
been
features defined.25
established
granulocytic,7’#{176}
and
first
described
blasts leukemic
patients with leukemias,”2
monocytic
rare
most cases of EL do not eosinophilic cells, and
of eosinophilic Several human
from
in
of this
Keisuke
Minato,
lymphocytic,6 and they have
Enzo
Br#{233}ard, and Georges
Eosinophilic
Ceresi,
Kazuyoshi
Yamada,
Math#{233}
and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO). the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties. S 1985 by Grune & Stratton. Inc.
Cell culture and cloning. On Feb 24, 1984, the proportion of WBCs rose to 16.9 x 104/,.L, 43% eosinophils, and 54% blasts. After obtainment of the patient’s informed consent, according to institutional
have cell
Human
Line
guidelines,
30 mL
of peripheral
blood
the blast-rich fraction was purified gradient separation (50%-60%--68%
by Percoll Percoll
from
and
the
48-well
top
layer
tissue
1640
were
culture
(GIBCO,
collected
plates
Grand
(Costar,
Island,
was
cultured
at
Cambridge,
NY)
obtained,
and
discontinuous density solution). The blasts 1 x
NY)
supplemented
106/mL
using
with
in
RPMI
10%
fetal
proven to be very useful in in vitro models for the study of leukemic cell properties. To date, however, no eosinophilic cell line has been reported. Recently we were able to estab-
calf serum (FCS), 2 mmol/L of glutamine, I mmol/L of pyruvate and 1% nonessential aminoacids (Seromed, Mulhouse, France). The cultures were incubated at 37 #{176}C in a humidified atmosphere of 5%
lish a continuous
CO2 and
and characterization
culture
line ofeosinophilic
we describe of this
patient,
cells
from
an EL
in this report the establishment unique cell line, EoL.
ability
and
MATERIALS
the
D.T.,
report.
Paul-Brousse
Hospital,
of hyperleukocytosis ciency.
The
increasing
upper
history
His
that
past he
and
an
examination
In I 979,
fibrosis
was
was
out.
with
immunology,
and
(WBC:14.8
also
x
showed
hematopoietic
allergic
negative. syndrome
edema.
replacement
1984 and laboratory findings were as follows: g/dL, hematocrit 41%, RBC count 402 x l04/zL, 16.6 x 104/,.L with 50% blasts, 12% myelocytic
hemoglobin and WBC eosinophils,
12.8 count I 1%
neutrophils,
1%
basophils,
and
marked
increase
phils,
10%
elocytic
metamyelocytic
consisting obtained, lation
cerebral
Blood,
4%
and
of
steroids the
(prothrombin
and
patient time
33%
treated
blasts.
77 sec/
An
No
of disseminated
4%
EL
promy-
diagnosis
remission
1 3 5cc, fibrinogen
was
chemotherapy could
intravascular
hemorrhage.
Vol 66. No
a
eosino-
2% metamyelocytic
by combination
vincristine.
died
showed
2% eosinophils, neutrophils,
and
was
examination
8% promyelocytic
eosinophils,
neutrophils,
5%
marrow
precursors,
I % myelocytic
the patient and
Bone
in eosinophil
neutrophils,
neutrophils,
made,
3% lymphocytes.
eosinophils,
50
leukemic
EoL-1
granules
Leukem
Chromosome
No. of Cells Analyzed
of Cells
Name
is
ET AL
SAITO
to
In
did
basophils,
not
9
(2%) which
stain
the
I cells. cell
to induce alkaline
for seven
identify
1
7
differentiation. the
Several
maturation
medium
to nine days
culture
of myeloid
buffered
with
(pH 7.8 to 8.0),
condicells
were
7
2
1
6
2
5
18
6
7
29
1
31
22
2
1
immature
and
mature
(Fig
2E).
The
differentiated
with large
peroxidase spherical
phils.
eosinophils
EoL-l
cells
cultured
did
not
number
of
cells
increased
HEPPS
or
increased
in a similar
of
noic
(0.2
(Fig to those
with
differentiate
the number
2
cells showed
and Luxol-fast-blue granules similar
to 1.2%)
acid
1
DMSO into
positive
for
fashion
to 1.0 jzmol/L)
to about
a strong
40%
reaction
2F), and contained of normal eosinofor nine
neutrophils,
days
(0.8%
whereas
Luxol-fast-blue
the
staining
as in alkaline
medium.
Reti-
had
on EoL-1
cells.
no effect
I! 1
2
3
m,
u 6
t
5
I,’
U 8
9
t2
X
7
VI
4
‘a
I
, 10
11
ft 13
14
15
16
17
18
1
U 19 ..
21
20
‘.a’
-I-
22
V
‘2 ‘J
Fig 3. yotype
+6.
Giemsa-banded of
+8.
EoL-1
and 9q-.
cells,
stained showing
kar48
XV.
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HUMAN EOSINOPHILIC LEUKEMIA CELL LINE These
results
seem
to
are
analysis
cells
were
MY9), same
summarized positive for
and surface
IL-2
that
indicate
committed to eosinophilic Cell surface markers.
The
results
in Table Ia antigen,
T4.
The
of surface
EoL-l
of the original
cells
EoL-1
did
is already
the
marker
not
cells
46
EBNA
and
EBNA
TdT.
No
staining
the same patient was EoL-1 cells by indirect Cytogenetic
EBNA
of Eo-B
cells
was
cells.
analysis.
+6, +8, 9q-
(Fig
3). Fifty-three
cellular
cells.
A few
mality
was
population
cells
were
anti-T
have
any
phagocytic
additional
in the
present somes
and representing of original cells
modal
number
detected lines)
was
from
detected
of As
EoL
derived
EoL-l,
in the
percent
of EoL-2
analyzed
8 in all
together
had
mitochondria,
1
other rough
6
II 10
7
$1
original
GTG
of
the
and abnormal
banding.
Abnor-
of
9q-
and
extra
leukemic
cells
(Fig
4),
with
other
5, 9,
1 2, and
I 3 sometimes
being
various subclones. The chromoranged from 43 to 50 per cell, with a
of cored general ER,
Electron microscopy had round or deep folded present, it was small and
vesicles
and
cytoplasmic and
Golgi
a few
components apparatus
were
vacuoles, such seen
as (Fig
II
2
Is
18
of 48.
A number with
normal
for
the
on
presence
chromosomes
compact.
3, 48
of EoL-3
heterogeneity
containing
by
I’
It
the
Electron microscopy findings. studies revealed that EoL-l cells nuclei. When the nucleolus was
EoL-2,
shown in Table male karyotype
performed
revealed
expressed
chromosome
+ 12, + 13; 62%
9q-,
analysis cells
the
Chromosomes
xY,
blood
with
positive. No TdT immunofluorescence.
EoL-3 cell lines were analyzed. of EoL-l cells had a hyperdiploid
Cytogenetic
membrane cell
+8,
49 XY, +4, +8, 9q-, + 13 (data not cells showed mainly a normal karyotype
to express
(B cell
and 61%
XY.
original
activity.
lines.
49 XY,
karyotype The Eo-B
peripheral
had the
leukemic
karyotype
had the shown).
2. The original leukemic myeloid antigen (IF1O,
(anti-Tac).
as those
line
pathways.
A fraction of the cells was also found Fc ‘y receptors and to react weakly antibody
cell
differentiation
receptors
markers
the
1237
3
4
Iu
5
UI’ \
8
U
11
12
13
14
15
16
17
18
1$-
I.
19
20
9
-1X
_O_,
Fig 4. Giemsa-banded karyotype of fresh original philic leukemia cells. showing +8.
+9.
and 9q-.
stained eosino48 XV.
-4
B-
-4-
21
22
V
4
From bloodjournal.hematologylibrary.org by guest on June 11, 2013. For personal use only.
SAITO
1238
5A).
No viral
structure
cells
induced
by DMSO
was
asynchrony
characterized
and
mature
a more
spherical, normal lacked
(Fig
differentiated immature
5B).
mic
and
were
often
mem-
to granules
The granules, (Fig 5C).
of
however,
derived
here
from
the
suffering nating
in
surface
markers,
of the
EoL
C
cells
stained
for
eosinophils,
with
toluidine
stained
peripheral
of
man
for
and
EoL cells originated
cytogenetic
analysis
from the patient’s had
a blastic
appearance
demonstrated
leukemic without
blasts.
we
with
IL-2 cells
that
positive specific results
receptors. had
a battery
of
cell types.
The
some
myeloid
In addition, anti-T4
antibodies are consistent
not
which
metachromatic
is
when
of the EoL cells were studied. As marker for mature or immature
Fc ‘y receptors, with
Luxol-fast-blue,
were
with
granules.
blue.
used and
with
but
various
Ia antigen
Most
cytoplas-
stained
granules
reacting
case termistaining,
when
specific
were
to myelo-
(2%) of the cells,
These
eosinophils, a 33-year-old
similar
percentage which,
size and color of eosinophilic
EoL,
hypereosinophilia Cytochemical
morphologically
A small granules,
had the typical
of a EL cell line,
blood
were
MGG,
the establishment
from a six-year-long blastic transformation.
and
The surface markers there is yet no specific
DISCUSSION
We report
granules
blasts or monoblasts. however, contained
nucleus
The granules
in appearance
precursors. crystalloids
EoL-1
nuclear-cytoplasmic
electron-dense
and were similar
eosinophilic characteristic
The
showed
by a relatively
cytoplasm
homogenously
brane-bound,
found.
( 1 .0%)
ET AL
a small and
antibody. were with
some
monoclonal
EoL cells antigens,
and
positive
expressed
fraction (I 1%) of the ( I 6%) were weakly
cells
All other
negative. the notion
antibodies
were
T, B, or monocyte-
Taken together, these that EoL cells belong to
\
Fig 5. cultured
(A) Ultrastructural appearance of EoL-1 cells under usual condition (original magnification x4.800; current magnification x3.120). (B) Ultrastructural appearance of EoL-1 cells cultured in dimethyl sulfoxide (1 .0%) for seven days (original magnification x 11.000; current magnification x 7.1 50). (C) High magnification of granules in EoL-1 cells (original magnification x25.000; current magnification x 16.250).
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EOSINOPHILIC
HUMAN
the
granulocytic
EoL
cells
LEUKEMIA
lineage.
The
is interesting.
addition
to activated
hairy cell cell lines,
presence
Recent
of IL-2
studies
T cells,
leukemia, and on
1239
LINE
CELL
these
the
presence
abnormal AMMoL
were
found
in
cases
of EL that
B
such
as the
data
indicated
on
virus-transformed therefore, the
receptors are not specific of the T cell lineage thought.2224 We are not aware, however, demonstrating
in
shown
receptors
some Epstein-Barr activated B cells;
that,
receptors
have
IL-2
IL-2
receptors
on
myeloid
of the
EoL
cell
showed
cells.
Cytogenetic abnormal
analysis karyotype,
also found chromosome
and
to have an 8 and 9q-
.
the
original
lines
leukemic
abnormal In contrast,
an
cells
karyotype of the karyotype
an extra of the B
were
to 8.0)
increased
features
We
gave
established
the
of the
a leukemic tried
to induce
40%.
frequency
of cells
granules,
Luxol-fast-blue
The
addition
comparable
have
abnormalities
any
line
the
differin 7.0
staining) in the culture
In contrast,
It is interesting
culture (pH
of
eosinophilic
of DMSO
results.
effect.
with
above
cell
cells by various techniques. The buffered with HEPPS or HEPES
2% to about not
showed
of I 6.2533 All
therefore
(cytoplasmic
medium
reported
we had
nature. of these medium
did
already
or deletion
that
entiation alkaline
from
were
lacking crystalloid. The eosinophils of to be part of the leukemic process, but no
inversion
eosinophilic
as previously of any report
of
granules appeared
retinoic
acid
to compare
these
cell line obtained from the same blood sample was normal. We suggested that, during the evolution of leukemic cells, dominant clones in EoL-l (48 XY, +6, +8, 9q- , EoL-2 (49
results
xY,
the
DM50.’3 The uniform maturation of EoL-l cells in eosinophils under various culture conditions is thus in favor of a commitment of these blasts to the eosinophilic lineage. Such features have not been described in any previously reported myeloid leukemia cell lines. In this respect, EoL lines seem to
dif-
have
9q
+8,
9q-,
, + 1 3) might
-
tageously 9q
EL
extra
and
EoL-3
be derived
from
more
original
had
the
a variety
XY,
+8,
or advan-
while
keeping
of the
cases
of cytogenetic
reported
abnormalities,
chromosome.25
abnormalities
+4,
expansive
Several
Philadelphia
chromosomal
(49
subclones,
8 chromosome.
have
including ferent
+13),
modified
and
-
as
+12,
Among
described
the
in EL,
on other
sively
found
acute
nonlymphocytic
that
in
patients
MoL)
EL.
9q
anomaly
-
myeloproliferative
with
acute
is almost
disorders,
leukemias.3#{176} It
and abundant
sion
The
myelomonocytic
bone
marrow
of 16 chromosome.3t’32
The
shown
eosinophils
unique
into
neutrophils
under
influence
of
characteristics.
disease,
and therefore
few fresh
blasts can be obtained. These cell lines in vitro model for the study of malignant ties and
the
eosinophilic
differentiation
eosinophilic
could thus be a useful eosinophilic proper-
pathways.
excluin
ACKNOWLEDGMENT
recently
leukemia
eosinophils
and
EL is a rare
especially
was
medium
trisomy
of group C chromosomes is a relatively frequent finding,229 but interstitial deletions of the long arm of chromosome #9 (9q - ), which were found on EoL cell lines, have not been reported
with those with the promyelocytic leukemia cell line, which differentiates into eosinophils in alkaline
HL-60,
The authors
(AM-
Elisabeth
had an inverof AMMoL
had
Daniel helpful
gratefully
appreciate
Couv#{233}. The authors and Professor comments.
the editorial
also gratefully
G. Flandrin
(Hbpital
assistance
of Mrs
acknowledge
Dr MT.
Saint-Louis)
for their
REFERENCES I . Stillman
RG:
ofeosinophile
A case
cells.
Med
of myeloid Rec
2. Benvenisti DS, Ultman iE: five cases and review ofliterature. 3.
Antenius
Acute
A, Audebert
eosinophilic
leukemia
81:594,
human
predominance
leukemia.
Report
of
Ann Intern
Med 71:731, 1969 M, Debray i, Robineaux
Krulic
Virchows
Arch
B Cell
Pathol
4. Stavem
P, Ly B, Blichfeld
Scand
leukemia.
i Haematol
21:355,
5. NIH Conference: Clinical,
R:
the
The idiopathic and
Intern Med 97:78, 1982 6. Minowada i, Tatsumi K, Zgoda
of
study,
in
Schlossman
Bernard
leukemias.”
L,
A,
Boumsell
Ann
7.
Lozzio line
CB, with
Reber Ti: Eosinophilic
of
leukemia
L,
on the marker profiles of The results of workDausset
i,
Berlin,
Springer
Milstein
differentiation
K, Sonobe
of a new
Blood
63:254,
H,
Ohtsuki
Y,
peroxidase-positive
1984
C,
Verlag,
and characterization
of a human
differentiation
acute
26:171, 1980 JR. Misawa 5,
of the human
promyelocytic
eosinophil
colony
by Luxol-fast-blue
staining.
Exp
Hematol
8:549,
I980 Lozzio positive
Si,
Science
cell line, HL-60. i Exp Med 160:179, 1984 14. Aglietta M, Camussi G, Piacibello W: Detection of basophils growing in semisolid agar culture. Exp Hematol 9:95, 1981 I 5. iohnson GR, Metcalf D: Detection of a new type of mouse
BB:
Human
chronic
Philadelphia
myelogenous
chromosome.
leukemia Blood
16.
45:321,
Gallo of human
RC, Gallagher myeloid
ture. Nature 270:347, 1977 9. Koeffier HP, Golde DW:
RE: Continuous
leukemic
cells
growth
in suspension
Yamada
Myeloid IF1O.
Collins
P.21.
K: Establishment
1,
Typing.
activity.
monocytic leukemia cell line (THP-1). Int i Cancer I 3. Fischkoff SA, Pollak A, Gleich Gi, Testa
A scheme
197S 8.
line,
Sugimoyo
1984, pp 519-527 cell
cell
L, Gould
D:
K, Niiya
I: Establishment
K, Lok MS.
Kover
Leukocyte
I, Machida
myeloid
T, Tada
syndrome:
considerations.
E, Sagawa
L, Prestine
SF (eds):
eosinophilo-
hypereosinophilic
therapeutic
human hematopoietic differentiation based the cultured and fresh leukemia-lymphomas: shop
P: Acute
“in-between
Kubonishi
K, Miyoshi
human
to colony-stimulating
1978
I I . Ben Bassat H, Korkesh A, Voss R, Leizerowitz R, Polliack A: Establishment and characterization of a new permanent cell line (GDM-l) from a patient with myelomonoblastic leukemia. Leuk Res 6:743, 1982 I 2. Tsuchiya 5, Yamabe M, Tamaguchi Y, Kobayashi Y, Konno
27:237,
1978
pathophysiologic
Minato
P. Andreassen One
10.
Iwata
I978 myelomonocytic
cell line responsive
200:1153,
Eosinophilic
AA,
leukemia.
with
1912
and cul-
Am
17. mia
and
i Hematol
Foon
myelogenous
leukemia.
A
Okabe 15:181,
cells.
Y, Uchiyama
i: Characterization
N, antigen
KA, Schroff
lymphoma
18. Wano Acute
K,
differentiation
RW, Recent
Saito
H,
defined
Suzuki
R,
Kumagai
by a monoclonal
K:
antibody
1983
Gale
RP: Surface
advances.
T, Fukui
K, Maeda
of human interleukin
markers
Blood
60:1,
on leuke1982
M, Uchino
2 receptor
H, Yodoi
(Tac antigen)
in
From bloodjournal.hematologylibrary.org by guest on June 11, 2013. For personal use only.
1240
SAITO
normal and leukemic receptors on HUT-102 19.
A
Griffin
iD,
monoclonal
myeloid 20.
T cells: Co-expression of normal and aberrant cells. J Immunol 132:3005, 1984
Linch
D, Sabbath
antibody
progenitor
reactive
cells.
Reedman
BM,
Leuk
Klein
K, Larcom with
normal
Res 8:521,
P, Schlossman and
SF:
leukemic
human
1984
G: Cellular
of an
Epstein-
antigen in producer Int J Cancer 1 1:499,
I973 21
.
Humains.
Dutrillaux B: Sur Ia Nature Monographie des Annales
Scientifique 22.
Francaise,
LM, Marshall
Si, Jensen
iP, Bakhshi
TA: Rearrangement expression
mia.
Proc
23.
Robb
Immunol 24.
Natl
Ri:
Today Tsudo
Leukemia.
Sci
USA
5:203,
normal
Sandberg New
York,
The
Depper
antigen
JM,
of immu-
in hairy
cell
leuke-
The
F, Beckman
G, Laurell
H, Nordenson
I:
of the eosinophilic bone marrow pool in and eosinophilic leukemia. Acta Med Scand
1977
Huang
CS,
Chromosomes
Gomez
and
GA,
Kohno
causation
5, Sokal
of human
iE,
Sandberg
cancer
and
AA:
leukemia.
XXXIV, a case of “hypereosinophilic syndromes” with unusual cytogenetic findings in a chloroma, terminating in blastic transformation and CNS leukemia. Cancer 44:1284, 1979 29.
Goldman
chromosome
JM,
analysis
Najfeld
V,
Th’ng
ofeosinophilic
KH:
leukemia.
Agar
culture
i Clin Pathol
and
28:956,
30.
Mecucci
12:309,
molecule
and
its
function.
B cells.
H: Expression i Exp
Chromosomes
Elsevier,
1981,
Med in
160:612, Human
ofTac
antigen
Cancer
and
pp 227-230
26. Weinfeld A, Westin i, Swolin B: Ph’-negative eosinophilic leukemia with trisomy 8. Case report and review of cytogenetic studies. Scand i Haematol 18:413, 1977
DC, Bloomfield
of chromosome 32.
Griffin
1984
K, Kulling
G, Michaux
iL,
Noens
L,
1984
phocytic T, Uchino
C, Vermaelen
Hove WV, Tricot G, Louwagie A, Berghe HVD: Interstitial 9qdeletions in hematologic malignancies. Cancer Genet Cytogenet 3 I . Arthur
1983
1984
human AA:
2:
WJ,
and expression
of Tac 80:4522,
Interleukin
M, Uchiyama
on activated 25.
Acad
i, Hsu SM, Neckers
A, Leonard
noglobulin
and
L, Mitelman
composition eosinophilia
197S
WC, Cossman
Jaffe ES, Waldmann genes
des Chromosomes Paris, Expansion
1975
Si, Greene
Korsemeyer
et l’Origine de Genetique.
Brandt
201:177, 28.
localization
Barr virus (EBV)-associated complement-fixing and non-producer lymphoblastoid cell lines.
27.
Different reactive
ET AL
16 and
leukemia:
A new
Tantravahi with
CD: Partial marrow M,
Blood Henkle
Hi: A pericentric
dysplastic
marrow
deletion
of the long arm
eosinophilia
association.
R, Schwenn
iD, Weinstein
is associated
bone
in acute
61 :994, C, Nell
inversion eosinophils
nonlym-
1983 M,
Leavitt
of chromosome in acute
ocytic leukemia. Blood 63:800, 1984 33. Rowley iD: Biological implications ofconsistent rearrangements in leukemia and lymphoma. Cancer I 984
PR, I6
myelomon-
chromosome Res 44:3 159,