From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Immunohistochemical
Localization
of Membrane
Plastic-Embedded
Mouse
Bone
Marrow
and Murine
Megakaryocyte
By
Using
mal
an
immunoperoxidase
antigen
have
detected
platelet
colonies
grown
polyclonal
proteins.
brand
and
have
llb
and
T
we
development
after
karyocytopoiesis,
of the
megakaryocyte
lack
of specific
markers.
megakaryocytes.
mega-
of the earliest
hampered
by
an
bone
the
enzyme
marrow,
has
been extensively
employed,
but additional
markers
would be
advantageous.
Recent
studies
of human
megakaryocytes
have demonstrated
a number
of useful
markers.
A peroxidase specific
for the megakaryocyte-platelet
lineage,
localized to the endoplasmic
of the megakaryocyte
platelet,
has
recently,
teins
demonstrated
been
limited
Using
IIb,
an alpha
alpha
thus
From
granule
the
membrane
the
Veterans
and
Research
Lung,
and
Blood
Grant
and
a Grant-in-Aid
funds
contributed
Submitted
Address
National
from
the
by
Nov
30, 1985;
reprint
requests
St. San
I 986 by Grune
31035
USPHS
in part
Service
HL
Institute,
1 13A,
& Stratton,
0006-4971/86/6803-0018$03.00/0
enzyme
acetate
esterase.
grown
same
in
platelet
ability
data
culture
bone
marrow
antibodies
glycoproteins
glycoprotemns
have
been
mdi-
agar
of
membrane
megakaryocyte
constituents
soft
as
the
esterase
These
glycoproteins
Furthermore.
human
histochemical
a-naphthyl
highly
indicates
conserved
during
cc 1986
by Grune
& Stratton,
Inc.
fibninogen
in both
megakaryocyte
strongly
suggests
antibodies
murine
that
against
platelet
bone
colonies
lineage
megakaryocytes
their
agar
available,
of the
be utilized
and
megakaryocytes
in soft
currently
antigens
can
marrow
grown
and
cultures.
This
well-characterized
human
megakaryocyte-
to identify
and
study
munine
precursors.
MATERIALS
AND
METHODS
Animals
C57BL/6i
roy, Calif),
under
mice, obtained
from Simonsen
were used for these experiments.
specific
conventional
pathogen-free
conditions
conditions.
EZH Co (Williams,
Preparation
Guinea
Laboratories,
Inc (GilThese mice were bred
and
pigs and
then
maintained
under
rats were purchased
from
Calif).
ofMurine,
Guinea
Pig,
and
Rat
Bone
Marrow
the
National
CA
Heart
April
to
Paula
Association
HL
Heart,
The
31610;
with
29, 1986.
E.
Administration
94121.
Health;
and HL
F32
affiliate.
accepted
Dr
of
/4264
Stenberg,
Hospital,
femoral
by cervical
marrow
dislocation
samples
and
were
guinea
obtained
pig and
rat
from
animals
femoral
marrow
were obtained
from animals killed by ether anesthesia.
The
femur was split lengthwise
and the fragments
of marrow
gently
pushed out from the bone into 4% paraformaldehyde
in 0.1 mol/L
phosphate
buffer, pH 7.4, at 4 #{176}C.
Specimens
of marrow were fixed
for four to six hours at 4 #{176}C.
samples
Preparation
Antonio;
Francisco.
Award
from
American
Inc.
San
Mouse
killed
Fran-
Structural
San
Service
Institutes
the Texas
CA
similar
for
markers.
megakaryocyte
and
San
and
Center,
Center,
Grants
Veterans
Francisco,
Cellular
Research
and
Pathology,
of Medicine,
Science
Medical
by National
Administration;
Veterans
and
Health
Administration
in part
factor
Medicine,
School
of Medicine
of Texas
06955
696
of morsystems
(GMP-140),
Willebrand
of Laboratory
Departments
Supported
glycoprotein
von
of California
University
of these
and
Specimens
glycoproteins
University
Biology,
Pathology
reports
culture
far.3’7’2
the Departments
cisco;
However,
in tissue
techniques
on plastic-embedantibodies
to human
platelet
constituents,
plasma
membrane
glycoproteins
lila
and
granule
Medicine,
©
More
immunohistochemical
and
Clement
‘
in vivo and in vitro.2
studies
of megakaryocytes
ded sections’3
and
we have detected
and
reticulum
and peninuclear
envelope
and the dense
tubular
system
of the
antibodies
specific
for platelet
membrane
glycoprohave been employed
for identification
of human
mega-
karyocytes
phologic
have
been
of the
murmne
these
each
evolution.
culture
of
Acetylcholinesterase,
a
chloroacetate
against
identify
in
macro-
marrow
been
many
directed
for
megakaryocytes
express
in mouse
and
and
in rodent
that
plate-
culture.
negative
megakaryocytes
positive
for
Wille-
glycoproteins
understanding
has
cate
that
of
were
demonstrated
negative
Proteins
and Jack Levin
marrow
weakly
and
days
recognition
megakaryocytes
to
von
label
models
lineage
pattern:
to
Granulocyte
morphologic
but
cells
colonies
glycoproteins
membrane
our
and
bone
megakaryocytes
demonstrated
improved
bone
days.
human
for
seven
to
have
to seven
membrane
marrow
of animal
has
three
against
the
colonies
Murine
megakaryocyte
labeling
megakaryocytes.
systems
restricted
for
bone
antibodies
marrow
HE
both
colonies
GMP-140.
bone
culture
and
in
monoclonal
murine
mouse
P. McEver,
phage
opti-
level.
in
Rodger
we
permits
constituents
prepared
fibrinogen.
GMP-140
in
demonstrated
megakaryocyte
Using
agar
antibodies
we
factor.
and
a-granule
Beckstead,
H.
microscope
glycoproteins
and
in soft
Jay
that
light
platelet
megakaryocytes
lIla
the
membrane
marrow
let
at
two
E. Stenberg,
technique
localization
three
Using
Paula
and a-Granule
Megakaryocytes
Colonies
Clinical
4150
of
Murine
Colony
Samples
Spleen cells were obtained
as previously
described.’4
All cultures
were performed
in an agar medium (final concentration
of agar was
0.3% in Dulbecco’s
modified Eagle’s medium).’4
Final concentration
of horse serum
was 20%. One mL of a cell suspension
in agar
medium
was
cultured
with
0. 1 mL
of pokeweed
mitogen
stimulated
spleen
cell-conditioned
medium,
and incubated
for three to seven
days in a humidified
atmosphere
of 10% CO2 in air. At various times,
colonies were either removed
from culture dishes with a finely drawn
Pasteur
pipette and placed in the fixative
solution,
or fixative was
gently poured over the entire agar disc. In the latter case, the discs
then were gently removed from the 35 mm plastic dishes and placed
in a larger
volume
of fixative.
All samples were fixed at 4 #{176}C
in 4%
paraformaldehyde
in 0.1 mol/L phosphate
buffer, pH 7.4, for four to
six hours.
Blood,
Vol 68, No
3 (September),
1986:
pp 696-702
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
MURINE
MEGAKARYOCYTE
Preparation
Plastic
ANTIGENS
ofBone
Marrow
and
697
Colony
Samples
for
Embedding
The following
steps were performed
at 4 #{176}C:
Colonies
in suspenprocessed
by allowing them to settle by gravity after each
step. After
fixation, samples were rinsed three times with 0. 1 mol/L
phosphate
buffer which contained
2% sucrose and 50 mM NH4CI.
The samples were then dehydrated
in 50% acetone
for 0.5 hour, 95%
acetone
for 0.5 hour, 100% acetone
for 0.5 hour, and 50 parts
acetone/SO
parts glycol methacrylate
monomer
for 0.5 hour followed by infiltration
with 100% glycol methacrylate
monomer
overnight.
Subsequently,
the specimens
were embedded
in a mixture
sion were
composed
ofglycol
methacrylate
monomer
(20
mL),
benzol
via
400 with N,N-dimethylaniline
(0.5
Pa). The embedded
tissue was
mm Hg) at 4 #{176}C
and the plastic
One to three um sections were then
JB-4 microtome.
The sections were
to coverslips
and air dried at room tempera-
water
blueing
ture.
and finally
Control
Histochemical
and
Routine
staining
procedures
as previously
procedure’3
Immunohistochemical
and
enzyme
The
of a biotin-avidin
described.’5”
is a modification
nohistochemical
procedures
following
carried
sequence.
out at room
on
Unless
plastic
sections
otherwise
histochemistry
were
immunohistochemical
procedure.’7
Immuwere
specified,
performed
in
temperature.
Digestion
37 #{176}C.
Incubation
We
directed
localize
specimens
cytes
Monoclonal
(512)21(1:500);
incubations
with
antibodies:
mouse
antihuman
mouse
CMF-PBS.
antihuman
glycoprotein
platelet
IIb
(Tab)22
GMP-140
(1:500).
Incubation
with the appropriate
secondary
antibody,
ie, biotinylated
goat antirabbit
IgG (Vector,
Burlingame,
Calif, I :200) or
biotinylated
horse antimouse
IgG (Vector,
1 :200) for one hour.
(e) Inhibition
of endogenous
peroxidase
by incubation
with 0.3%
in 30%
methanol
for three
the
reaction
of 0.5% CuSO4
of irrelevant
fluid for the primary
and
secondary
anti-
antibodies
reagents.
bone
from
by
three
marrow
( 1)
groups:
investigate
at early
of nonspecific
Staining
With Antibodies
megakaryoproteins
membrane
or
(3)
can
be
in platelets,
proteins,
a-granule
against
human
rodent
antigens,
localization
times.
evidence
murine
These
to
of intact
localization
plasma
proteins,
their
culture
and
cultures.
fine-structural
membrane
antibodies
proteins
the megakaryocytes
soft agar
their
and monoclonal
human
platelet
within
of rodent
classified,
(2)
matrix
apro-
platelet
proteins
we were able to
in bone marrow
cells
Control
preparations
and in cells
showed
no
staining.
minutes.
Incubation
with the avidin-biotin-peroxidase
complex
(Vector, I :80) for one hour.
(g) Development
of peroxidase
reaction
by preincubation
with
0.05% 3,3-diaminobenzidine
in CMF-PBS
for ten minutes,
followed
by incubation
with a CMF-PBS
solution containing
0.05% diaminobenzidine,
0.1% H2O2, 0.1 mol/L imidazole,
and 0.1 mol/L sodium
to Platelet
Plasma
Membrane
Proteins
Glycoprotein
Polyclonal
lila.
lIla
munine
labeled
bone
plasm
but
staining
was
marrow
most
of other
antisera
virtually
ing in megakaryocytes
against
megakaryocytes
distributed
intense
on the
hematopoietic
human
identi-
all morphologically
was
(Fig
1). Stain-
throughout
plasma
cells
pig and rat megakaryocytes
were also labeled
(not shown),
from
but
was
the
cyto-
membrane.
No
observed.
Guinea
bone marrow
not as strongly
specimens
as munine
megakaryocytes.
megakaryocyte
initiation
colonies,
soft agar
of
fixed and
cultures,
processed
three
were
strongly
reactive
for GP lIla (Fig 2), as were colonies
processed
days after culture
(Fig 3). Granulocyte
colonies
(Fig
macrophage
tions
were
colonies
Glycoprotein
identifiable
munine
shown).
The
with antibodies
Platelet
that
labeled
bone
antibodies
virtually
marrow
Heterologous
mouse
platelets
identifiable
shown).
The
days
after
initiation
prepara-
against
human
all morphologically
(not
to that
antiserum
(PAS)
munine
staining
of
same
megakaryocytes
obtained
with antibodies
to GP
Murine
megakaryocyte
colonies,
to seven
the
seven
4) and
lila.
pattern
was similar
lila (vide supra).
Antiserum.
morphologically
karyocytes
(not
from
for GP
Monoclonal
(Tab)
staining
to GP
against
4, inset)
unreactive
Jib.
Ilb
in rabbits
(Fig
completely
glycoprotein
(d)
(f)
substances
obtained
Murine
days
after
between
(1) Polyclonal
antibodies:
rabbit antihuman
von Willebrand
factor (Factor
VIlI-related
antigen)
(Dako,
Santa
Barbara,
Calif.
1 : 1000);
rabbit
antihuman
fibrinogen
antiserum
(Cappel,
Malvern,
Pa, 1 :1000);
rabbit
antihuman
platelet
membrane
glycoprotein
lIla” (I :2000); rabbit antihuman
a-granule
membrane
glycoprotein
(GMP-140)”
(1:2000);
rabbit
antimouse
platelet
antiserum
(1 :SOO)#{176};rabbit antirat brain acetylcholinesterase
(Dr Jean Massoulie, Paris, France,
1:100); rabbit antihuman
lysozyme
(Dako,
Santa Barbara,
Calif. 1:500).
H202
of primary
these
sections
antibody)
rinsed
substitution
or ascites
have utilized
polyclonal
against
a variety
of
fiable
were
in water,
RESULTS
glycoprotein
secondary
(2)
the
included
serum,
and the elimination
with 3% normal goat or horse serum (same species
in Dulbecco’s
Ca+ +- and Mg+ +-free
phosphate-buffered
saline (CMF-PBS)
(KC1, 3 mmol; KH2PO4,
1
mmol; NaCl, 150 mmol; Na2HPO4.7H2O,
8 mmol; pH 7.36 to 7.45)
for 30 minutes
at 37 #{176}C.
(c) Incubation
overnight
at 4 #{176}C
with one of the antisera
listed
below. All dilutions were made in CMF-PBS
with 3% normal goat or
horse serum
(same species as secondary
antibody,
vide infra)
and
(b)
as
3 times
with a solution
all incubations
with 0.25% trypsin in Saline A (NaC1, 150 mmol;
KCI, S mmol; glucose, S mmol; phenol red, 0.01 mmol; NaHCO3,
7
mmol) (Gibco,
Santa Clara,
Calif),
pH 7.6, for ten minutes
at
(a)
procedures
teins. Since these antibodies
strongly
cross-reacted
with
performed
were
drying
granule
Procedures
the
washing
with Gill’s hematoxylin
#2 for 1.5 minutes;
water (0.2% Na2CO3, 1% MgSO4 in tap water);
and mounting
with Permount.
Scott’s
preimmune
bodies,
into
Enzyme
with
After
by incubation
peroxide
(0.09 g), and polyethylene
glycol
mL; Polysciences
Inc. Warrington,
placed under vacuum
( 15 to 20
allowed to polymerize
overnight.
cut with glass knives on a Sorvall
transferred
azide for five minutes.
was further enhanced
for five minutes.
(h) Counterstaining
labeled
bone
pattern
lila (vide
fixed and
soft
agar
obtained
produced
virtually
supra).
processed
cultures,
strongly
labeled
with
PAS
(not shown).
Granulocyte
macrophage
colonies
from the same preparation
were
beled
with
PAS
(not
shown).
all
marrow
megawas similar
to
five
were
and
unla-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
698
STENBERG
ET AL
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
MURINE
MEGAKARYOCYTE
Staining
with
Protein
ANTIGENS
Antibodies
699
to Platelet
a-Granule
Membrane
Murine
megakaryocyte
(Fig 6, inset
B) to seven
140
Both polyclonal
inset)
antibodies
(not shown)
and
developed
against
granule
membrane
protein
with
kD(GMP-140)
produced
labeling
ically
identifiable
pattern
was
bodies against
platelet
and
bone
murine
staining
is consistent
monoclonal
(512)
(Fig 5,
the human
platelet
a-
a molecular
of virtually
marrow
weight
of 140
all morpholog-
megakaryocytes.
The
similar
to that observed
with the antia-granule
matrix
proteins
(vide infra)
with
the
fine-structural
localization
of
GMP-140
in human
platelets.’9
Labeling
with the monoclonal
antibody
Sl2 was weaker
than the labeling
obtained
with the polyclonal
antibodies.
All other
hematopoietic
cells
were
negative.
Polyclonal
antibodies
karyocyte
colonies
cultures
ofsplenic
granular
negative.
label
The
produced
processed
cells
(Fig
labeling
of megakaryocytes
Granulocyte
5). Some
from
and
labeling
days
while others,
usually
monoclonal
antibody
shown).
(not
variable
seven
initiation
exhibited
the
same
a strong
preparation
colonies
(not
were
and
negative
shown).
granular
identifiable
No
initiation
fibninogen
nies
(not
were
with
Antibodies
to Platelet
a-Granule
Matrix
Von
cific
preimmune
primary
any
in
Willebrand
in
factor
virtually
Antibodies
Factor.
all
(VWF)
produced
morphologically
against human von
strong
granular
identifiable
staining
munine
bone
marrow
megakaryocytes
(Fig 6, inset A). The distribution
was similar
to that observed
with antibodies
to GMP-l40.
There
was
moderate
no
labeling
endothelial
of
other
cell
staining
hematopoietic
was
cells,
observed
ascites
ies,
fluid
Fig 1 .
Bone
is easily identified
Fig 2.
soft agar
marrow
megakaryocytes
at this magnification.
Megakaryocytes
and stained
with
and samples
antigens.
The
was
serum
when
was
was
no
for
macrophage
cob-
either
substituted
staining
for
observed
was
specific
in any
for
was
primary
no staining
substituted
antisera
produced
labeled
identifiable
shown).
human
spe-
observed
or secondary
observed.
When
monoclonal
antibod-
cells.
munine
in rabbits
many,
bone
against
but
marrow
Polycbonal
antisera
lysozyme
labeled
most
not
all,
rat
brain
morphobogi-
megakaryocytes
(not
produced
in rabbits
murine
bone marrow
against
granulo-
cytes and macrophages
and cells in granulocyte
and macrophage
colonies,
but not bone
marrow
megakaryocytes
or
megakaryocytes
grown
in soft agar culture
(not shown).
but
Both
karyocyte
murine
bone
colonies
were
of murine
megakaryocyte
brown
peroxidase
reaction
(arrows)
labeled with polyclonal
antibodies
Other cells in this field are negative
(original
marrow
obtained
positive
for
megakaryocytes
from
the
seven-day
enzyme
colonies
were
embedded
product
is present
at sites
to human GP lIla demonstrate
magnification
x 600).
from a big cell colony type (Levin et al. 1 981 ) processed
three
polyclonal
antibodies
to GP lIla. The cells are strongly
reactive
Fig 3.
Megakaryocytes
from a heterogeneous
polyclonal
antibodies
to GP lIla. The megakaryocyte
shows
a lighter.
diffuse
reaction.
The membranes
magnification
x 600).
to seven
reactive
Markers
nic cells
Specimens
of normal
murine
bone marrow
immunohistochemically
for a variety
of platelet
The cells were stained with hematoxylin.
five
strongly
and
antiserum,
eliminated,
no staining
Other
rabbit
Similarly,
were
(not
shown).
Granulocyte
polyclonal
cells.
reagents
cally
Willebrand
processed
were
(not
labeled.
Controls
acetylcholinesterase
Proteins
pro-
negative.
Polycbonal
Staining
were
colonies
shown).
fibninogen
megakaryocytes
cells
of cultures
days
of
all morphologically
marrow
megakaryocyte
after
human
hematopoietic
three
initiation
antibodies
to VWF.
were negative.
in virtually
bone
other
Murine
days
against
label
murine
shown).
processed
6) after
strongly
reactive
with
macrophage
colonies
Antibodies
a strong
When
of
smaller
in size, appeared
Si 2 produced
only weak
macrophage
were
Fibrinogen.
duced
of mega-
after
cells
cultures,
Granulocyte
colonies,
days
(Fig
and
cultures
a-naphthyl
in plastic
of antigen
brown
days after initiation
of cultures
(original
magnification
x 600).
megaof spleacetate
and tested
localization.
staining
of splenic
which
cells
in
colony
type.
processed
seven
days after
initiation
of cultures.
and stained
with
plasma
membranes
(arrows)
are heavily
labeled.
while the cytoplasm
of these cells
are heavily
stained
even
in smaller.
more
immature
megakaryocytes
(original
Fig 4.
Granulocyte
colony and macrophage
from a representative
colony (inset) processed
after seven days of culture
and stained
for
the presence
of GP lIla. The cytoplasm
and plasma
membranes
of both granulocytes
and macrophage
are completely
negative.
Arrow
indicates
plasma membrane
of macrophage
(inset) (original
magnification
x 900; inset x 900).
Fig 5.
polyclonal
pattern
labeled
Megakaryocytes
from a heterogeneous
colony
type.
processed
seven
days after
initiation
of cultures.
and stained
with
antibodies
to GMP-140.
an alpha-granule
membrane
protein.
The two large cells in this field demonstrate
label in a granular
within
the cytoplasm
(arrows)
while a third. smaller
cell is unreactive.
Inset: A megakaryocyte
from a bone marrow
specimen.
with the monoclonal
antibody
512 which recognizes
the same 140 kD glycoprotein
as the polyclonal
anti-GMP-140
antibody.
The
cell cytoplasm
is positive
(original
magnification
x 600;
inset
x 600).
Fig 6.
Megakaryocytes
from
a heterogeneous
colony
type.
processed
seven
days after
initiation
of cultures.
and stained
with
polyclonal
antibodies
to VWF. Most of the cells in this field are labeled.
Inset A: A megakaryocyte
from a bone marrow
specimen.
positively
stained
for VWF. Inset B: A megakaryocyte
from a big cell colony type. processed
three days after initiation
of cultures.
positively
stained
for VWF (original
magnification
x 600; inset A x 600; inset B x 900).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
700
esterase,
but
(not
cytes
was
noted
more
weakly
positive
shown).
Some
within
the same
colonies
and
cyte
colonies
culture
macrophage
were
negative
(not
shown).
and
granulocyte
(not
of staining
specimen.
obtained
after
enzyme
samples
seven
in both
Since
days
were
bone
strongly
an
strated
the
alpha
granule
presence
and
populations
detected
for
colonies
grown
demonstrated
bone marrow
of platelet
agar
immunohistochemical
cultures.
technique.
megakaryocytes.
labeled
munine
granubocytes
megakaryocytes.
Guinea
pig
Therefore,
highly
also
only
antibody
and
mega-
obtained
from
earlier
report23
antihuman
lIla
are structurally
cally
lysozyme
antibodies
Newman24
membrane
conserved
Unexpectedly,
vitro.
in our
megakaryocytes
human
platelet
also labeled
membrane
strongly
polyclonal
as with
antibodies
were
examine
cells
may
be some
these
species.
At
been
least
types
study,
in Balb
C57BL/J6
differences
two
are
from
mouse
evaluated
specific
studies,
7 days
in
marker
Since
C
mice,
between
of munine
type)
some
we observed
mice
this
the
and
were
platelet
megakaryocyte
colonies
an increase
and
the
the
size
in average
size
of
days
cell size
as
of culture,
munine
megakaryocyte
maturation
in our
of mature
bone
a
3 to
colonies
culture
sys-
marrow
mega-
the
three
cells,
lIla
protein.
of
culture
may
relatively
and von
This is
morphologi-
day
colonies
the
only
are
first
be
in
reliably
large
acetylcholinesterase
size
has
megakaryocytes
been identified
the
antisera
Mazur
in the
in human
earliest
and
been
used
mouse.333
bone mar-
marker
against
of mega-
platelet
the
early
antigenic
detectable
in cells
clot
cultures.
in soft
membrane
our
cultures,
and
other
markers
at an early
markers
lysozyme
in munine
granulocytes
From
it
have
level
colony
contained
suggest
that
seven
days
specificity
presence
macrophages,
studies,
their
and
shown
types
ploidy
of the “big
type
approximately
in both
the
we conclude
of
but
not
that
the
antigens
in megakaryocytes,
cells in vitro, is another
indica-
has been
in different
the “heterogeneous”
membrane
The
by
and
our
different
ploidy
cells
is an
precursors
membrane
synthesize
von
glycoproteins
GMP-140.
Recently,
megakaryocytes
N/cell;
antibodies,
complex
point.
confirmed
megakaryocytes
and
factor,
fibninogen,
colonies
using an
Vain-
monoclonal
IIb/IIIa
maturation
is further
of these
various
from colony-forming
and
megakaryo-
studies
utilize
for the first time,
to platelet
membrane
glycofor identification
of munine
antibodies
of these
mean
et al,35 using
glycoprotein
However,
agar
lIb,
human
techniques
Significantly,
marker
of megakaryocyte
maturation,
at five or six days after initiation
of plasma
megakaryocytes
lIla,
et a13 identified
Vinci
that
tion that
Willebrand
current
in seven-
after
colony-forming
in culture
is
upon
for
by
et al2 and
determined
of
have
from
present
with immunofluorescence
glycoprotein
antiserum.
presence
derived
cells (often
immature
as
megakaryocytes
and
in megakaryocytes.
there
glycoproteins
time
this
peroxidase,
and
to
used
that
at this
fact,
staining
colonies
proteins
to
as
monocbonal
suggests
is composed
of more
ofwhich
are small and
(at which
time
their maximal
approximated
seven
of munine
based
platelet
chenker
marrow
part
by staining
for acetylcholinesterase,
for murine
megakaryocytes.26
In our
in culture,
day colonies
have reached
tem)
100),
after
colonies,
immature
have
antiplatelet
recognized
in soft agar
cultures.20’23#{176} One
type
is
composed
of 10 to 30 large megakaryocytes
which
mature
in appearance
(“big
cell”
type).
The
other
(“heterogeneous”
greater
than
of markers,
immunohistochemical
of the cells.
by
cyte
peripheral
bone
identified,
peroxidase,
typically
are
different
preparations
and platelet
membrane
glycoproteins.5’34
Furthermore,
Vainchenker
et al7 identified
human
megakaryocytes
in plasma
clot cultures
by the presence
of platelet
species).
with monocbonal
antibodies
glycoproteins,
although
not
antibodies.
produced
from
since
that
from
appearance
megakaryocyte
karyocytes,”2
in which
they
glycoproteins
in chicken
munine
row
to glyco-
these
cells
in
Previously,
blood thrombocytes.
They showed
this by indirect
immunofluorescence,
using
polyclonal
and monoclonal
antibodies,
and
two-dimensional
polyacrylamide
gel electrophoresis,
which
revealed
the
incorporation
of 355 methionine
by
chicken
thrombocytes
into glycoprotein
IIb and lila analogs
in
demonstrate
originates
level
cultures
since
to identify
Megakaryoblasts
and macrophages,
and rat bone marrow
against
agar
immature;
refractility
with
we have concluded
that these glycoproteins
conserved.
Support
for this conclusion
has come
and
platelet
significant
mega-
cross-reacted
with
tested
and
marrow
antihuman
reactive
a recent
study
by Kunicki
demonstrated
that human
lib
in both,
of different
labeled
for both platelet
membrane
glycoprotein
Willebrand
factor,
an alpha
granule
matrix
visually
In contrast,
were
(the
the
microscope
in agar
progeny
data
probably
megakaryocyte
of soft
glycoproteins
in human
the same antibodies
and
All
murine
lIla
light
munine
which
strongly
antibodies
but not
protein
was
simultaneously
the
Our
type
of precursors,
is similar
days
we
In an
of these
using
antibodies
megakaryocytes
membrane
bone
megakaryocytes
the presence
megakaryocytes
karyocyte/platelet
appear
represent
colony
by
analysis,
that
in munine
munine
in soft
types
precursors.26’
each
marrow
polycbonal
antihuman
antibodtechnique,
we have demon-
of a variety
in
production
of platelet
the majority
of cells in both “big cell” and “heterogeneous”
colonies
labeled
for all proteins
tested.
We also demonstrated
glycoproteins
karyocytes
evidence
probably
of
although
esterase
reactive
no
colony
they
populations
of
DISCUSSION
ies and
both
cultures,
shown).
monocbonal
and
immunoperoxidase
Utilizing
However,
ET AL
observed.
Granulo-
chloroacetate
neutrophils
colony
this enzyme
marrow
karyocytes.
megakaryo-
were negative.
Munine
bone
macrophages
and megakaryo-
for the
In contrast,
human
in the degree
bone
cyte and macrophage
marrow
megakaryocytes
and
than
variability
STENBERG
2N
cells
at
that
the
of murine
populations
megakaryocyte
distributions.20’26’28’29’36
cell”type
was
The
1 6.8N/cell
while
had a mean pboidy level of only 6.8
half the cells in the latter
type of
or
low
4N
levels
ploidy
of
levels
DNA.26
Our
expressed
studies
platelet
and alpha granule
glycoproteins,
since almost
colony
types were reactive
for these markers
or
earlier
in
of
culture.
Previous
studies37’38
all
by
have
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
MURINE
MEGAKARYOCYTE
shown
that
8N
1 28N
to
megakaryocytes
can
consistent
is initiated
701
with
achieve
ploidy
cytoplasmic
with
Paulus’
in immature
after completion
unknown
whether
altered
ANTIGENS
levels
observation
that
megakaryocytes
of DNA
synthesis
(ie,
the pattern
of protein
by the maturational
ranging
state
from
the
case,
for
example,
are
granules
organelle
formation
and reaches
a peak
specific
granules
Studies
are therefore
endomitosis).37
expression
tion and pboidy
with the pattern
maturity.
Our
data
It is
may be
of the megakaryocyte,
as is
are
formed
in neutrophils,
only
are
during
in which
the
formed
during
in progress
azurophil
promyelocyte
stage
the
myelocyte
to further
correlate
levels of the cells
of appearance
and
stage.39
matura-
of megakaryocyte
of these
various
colonies
glycopro-
teins.
REFERENCES
1 Breton-Gorius
J, Guichard
.
activity
peroxidase
Path66:277,
2.
in human
i: Ultrastructural
platelets
and
localization
of
megakaryocytes.
Am
55:386, 1980
i,
histochemistry
W,
Titeux
M, Breton-Gorius
platelet
antibodies
Deschamps
staining
colonies
patients.
J-F,
Bastin
Blood
of human
Guichard
megakaryocyte
and platelet
and in in vivo
59:514,
JM,
AJ: Two monoclonal
i, McMichael
as markers
Immunofluorescent
leukemic
anti-
Rabellino
peroxidase
cells from
detection
normal
17.
in
and
EM,
Levene
RB,
Vainchenker
W,
Guichard
human megakaryocyte
adult peripheral
blood
5:25,
8.
Leung
LLK,
Nachman
RL:
of
J,
Breton-Gorius
colonies in culture
cells: Ultrastructural
from
i:
Growth
fetal, neonatal,
analysis.
Blood
of
and
Cells
Vainchenker
W,
Breton-Gorius
megakaryocyte
their
colonies
maturation,
in Evatt
i,
Chapman
in vitro
BL,
Induction
and ultrastructural
RL,
Levine
Biology and Precursors:
New York, Elsevier-North
J:
Williams
of
aspects
NT
9. Fauser
macrophages,
containing
AA, Messner
HA: Identification
of megakaryocytes,
and eosinophils
in colonies of human bone marrow
neutrophilic
granulocytes
and erythroblasts.
Blood
1979
10. van Noord Mi, Blansjaar
N, Nakeff A: The processing
of
mammalian
haemopoietic
cells in thin layer agar cultures
for
electron microscopy.
Stain Tech 48:239,
1973
1 1 . Weinstein
R, Stemerman
MB, Maclntyre
DE, Steinberg
HN,
Maciag T: The morphological
and biochemical
characterization ofa line ofrat
promegakaryoblasts.
Blood 58:1 10, 1981
12.
hamster
Choudhury
C, Katsnelson
study
spleen.
Blood
Beckstead
iH:
using
aldehyde-fixed
chem
33:954,
of
I, Arnold
megakaryocytopoiesis
65: 1 282,
Optimal
E: Morphological
and
in liquid
cultures
of
198
antigen
plastic-embedded
localization
sections.
in human
i Histochem
in mice:
Sci
of bone
Chem
Rosa
iP,
Use
of avidin-biotin-peroxidase
techniques:
A comparison
(PAP) procedures.
J Histo-
Lingappa
83:1480,
VR,
Kan
YW,
McEver
RP,
1986
1985
Holland,
22.
RP,
McEver
1981,
glycoprotein
259:9799,
McEver
pp
Martin
brane
Chem
binds
23.
RP,
Beckstead
Bennett
iH,
DF:
tic-embedded
EM,
Stenberg
in human
platelets.
Martin
bone-marrow
SA,
of murine
MN:
to a
i
Identification
PE,
McEver
RP,
localization
Biol
of two
biopsy
Adamson
Shuman
Application
specimens.
memi Biol
MA,
of membrane
megakaryocytes:
Kunicki
Ti, Newman
platelet
membrane
glycoprotein
eral blood thrombocytes.
Proc
Burstein
antibody
activated
distinct sites on human platelet
using monoclonal
antibodies.
24.
25.
to
Immunohistochemical
proteins
acteristics
A monoclonal
only
1984
and functionally
glycoprotein
JIb-lIla
258:5269,
1983
Bainton
179-190
MN:
structurally
Blood
and
to plas-
67:285,
1986
Pi: Synthesis
of analogs
of human
lIb-IIIa
complex by chicken periphNatl
Acad Sci USA 82:7319,
1985
iW,
Thorning
megakaryocyte
D, Harker
colonies
in vitro.
LA:
Blood
Char-
4:169,
1979
26.
Levin
ploidy
Cyto-
57:287,
murine
i, Levin
distribution
With
studies
FC,
Penington
DG,
in
megakaryocyte
of
the
effects
Metcalf
D: Measurement
colonies
of
obtained
thrombocytopenia.
from
Blood
1981
Williams
N,
megakaryocyte
Jackson
H:
progenitor
Regulation
of the
cells by cell cycle.
proliferation
Blood
of
52:163,
I978
28.
numbers
JH, Bainton DF: Enzyme
histochemistry
on bone
Reactions
useful in the differential
diagnosis
of
H:
Levin
membrane
27.
Studies
L, Fanger
USA
Elsevier-North
culture:
1980
I 5. Beckstead
marrow
biopsies:
Blood
J, Levin FC, Metcalf
D, Penington
DG: The effects of
acute thrombocytopenia
upon megakaryocyte-CFC
and granulocyte-macrophage-CFC
in the bone marrow and spleen of mice: With
studies of ploidy distribution
in megakaryocyte
colonies,
in Evatt
BL, Levine RF, Williams
NT (eds): Megakaryocyte
Biology and
Precursors:
In Vitro Cloning
and Cellular
Properties.
New York,
20.
tissues
D: The effects of acute thromboand granulocyte-macrophagemarrow
and spleen.
Blood 56:274,
Raine
PS,
Acad
101:880,
of
1985
14. Levin J, Levin FC, Metcalf
cytopenia
on megakaryocyte-CFC
CFC
sections.
MA: Biogenesis
of the platelet
receptor
for fibrinogen:
for separate
precursors
for glycoproteins
llb and lIla. Proc
a-granule
ultrastructural
plastic
19. Stenberg
PE, McEver RP, Shuman
MA, Jacques
YV, Bainton DF: A platelet alpha-granule
membrane
protein (GMP-140)
is
expressed
on the plasma
membrane
after activation.
J Cell Biol
(eds):
In Vitro Cloning and CelluHolland,
1981, pp 139-
I 60
53:1023,
Bray
21.
Megakaryocyte
lar Properties.
I 3.
18.
NatI
5,
(ABC)
in immunoperoxidase
ABC and unlabeled
antibody
Cytochem
29:577, 1981
Shuman
Evidence
1979
human
Hsu
complex
between
chem
1982
Human megakaryocytes
II. Expression
of platelet proteins in early
marrow megakaryocytes.
J Exp Med 154:88, 1981
6. Koike T: Megakaryoblastic
leukemia:
The characterization
and identification
of megakaryoblasts.
Blood 64:683, 1984
7.
to 2-micron
JH, Halverson
PS, Ries CA, Bainton DF: Enzyme
and immunohistochemistry
on biopsy specimens
of
human bone marrow.
Blood 57:1088,
1981
pathological
maturation:
3. Mazur
EM, Hoffman
R, Chasis
i, Marchesi
5, Bruno E:
Immunofluorescent
identification
of human megakaryocyte
colonies
using an antiplatelet
glycoprotein
antiserum.
Blood 57:277, 1981
4. Rabellino
EM, Nachman
RL, Williams
N, Winchester
RH,
Ross
GO: Human megakaryocytes
I. Characterization
of the membrane and cytoplasmic
components
of isolated marrow megakaryocytes. J Exp Med 149:1273,
1979
5.
applied
16. Beckstead
1972
Vainchenker
megakaryocyte
and lymphoma
leukemia
i
marrow
29.
N, Jackson
H: Kinetic
and ploidy levels in developing
cells. Cell Tissue Kinet 15:483,
Williams
Lieschke
Gi:
Kinetics
analysis
colonies
1982
of the formation
of megakaryocyte
from mouse bone
of pure and heteroge-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
STENBERG
702
neous
megakaryocyte
colonies
Exp Hematol
30. Long MW,
vitro.
diesters
stimulate
The
cell.
by megakaryocyte
10:78, 1982
Gragowski
LL, Heffner
the development
burst-forming
progenitor
cells
in
G, Tabilio
A, Deschamps
i-F, Van Haeke D, Henri A,
J, Tetteroo
P. Lansdorp
PM, Hercend T, Vainchenker
W,
Breton-Gorius
J: Immunological
study of in vitro maturation
of
Vinci
35.
Guichard
CH,
Boxer
of an early
unit-megakaryocyte.
LA: Phorbol
murine
J Clin
progenitor
Invest
human
76:431,
36.
for early
mental
I 985
megakaryocytes.
cells
ofthe
Cholinesterase
megakaryocyte
Long
32.
mouse:
CW:
MW,
and
Blood 58:1032,
ing.
33.
in the
Long
mouse:
responsiveness
MW,
series.
Williams
Morphology
as a possible
Blood
42:413,
N: Immature
quantitation
marker
1973
megakaryocytes
in the
stain-
1981
Williams
Physical
N,
Ebbe
characteristics,
to thrombopoietic
5: Immature
cell cycle
stimulatory
megakaryocytes
status,
factor.
37.
diographic,
DNA
metabolism
1984
i-F, Henry-Amar
M:
from a multicompart-
Proc
NatI
Acad
Sci
in vitro
38.
karyocytes
Dupont
separation
H,
mentation:
Physico-chemical,
characterizations.
1985
Golgi
Bainton
USA
study.
35:298,
1970
H, Boisseau
M-R:
M-A,
in developing
Blood
Bricaud
classes
by
and
ultrastructural
Biol Cell 49:137,
Two
of organdIes
ultrastructural,
in homogeneous
DF, Farquhar
leukocytes.
complex
Dupont
and development
A combined
and cytophotometric
59:569,
phonuclear
progenitors.
megakaryocytes:
and
39.
56:589,
M, Deschamps
develop randomly
of committed
J-M:
Paulus
in guinea-pig
Levene RB, Lamaziere
i-MD,
Broxmeyer
HE, Lu L, RabelEM: Human
megakaryocytes
V. Changes
in the phenotypic
profile
of differentiating
megakaryocytes.
J Exp Med 161:457,
34.
i Haem
1982
Blood
1982
lino
Prenant
megakaryocytes
system
79:4410,
by acetylcholinesterase
Brit
J-M,
Paulus
Polyploid
3 1 . Jackson
ET AL
MG:
types
unit
autora-
Mega-
gravity
sedi-
cytophotometric
1983
Origin
derived
granulocytes.
of granules
from
opposite
in polymorfaces
J Cell Biol 28:277,
of the
1966
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1986 68: 696-702
Immunohistochemical localization of membrane and alpha-granule proteins
in plastic-embedded mouse bone marrow megakaryocytes and murine
megakaryocyte colonies
PE Stenberg, JH Beckstead, RP McEver and J Levin
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