From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Marrow
I. Effect
Culture
in Diffusion
of Mature
Granulocytes
By Roel Willemze,
Factors
Richard
influencing
been
granulopoiesis
evaluated
implanted
using
in
rabbits.
An
chambers
I. Walker,
the
penic
by nitrogen
made
neutropenic
cavity
in hosts
made
granulocytes.
in
inhibited
RECENT
N
YEARS
in vitro
cyte
in
colonies’3
from
and
host
cells
humoral
that
that
tion
in
stimulators
or
from late
to inhibit
differentiated
lymphocyte
reported
a soluble
from
mature
granulocytes
ogous
on
to
other
the
DC
has
or
been
with
or
The
derived
from
onstrated.
and
A factor
other
analogous
or
derived
sources
have
Although
the
activity)
is
The
increased
From
the
June
Supported
by
Address
©
Blood,
for
Chapel
HL
Grant
IN-ISP
/978
Vol.
reprint
by Grune
51,
Fund,
No.
of
to
be required
role
of this
of
neutrophil
cells
in
Division
accepted
17835
August
from
from
Koningin
requests:
NC.
from
derived
while
be
having
a “chalone”
anal-
injection
into
mice
of granulocyte
inhibitory
of
bearing
production
activity
a
im-
in
associated
established.
has
mononuclear
for
factor
this
yet
cells,
growth
of
(called
activity
to
be
dem-
macrophages,
granulocytes
in
colony-stimulating
in
urine
and
serum
count.25’26
diffusion
chambers
of Hematology.
implanted
University
of North
into
Carolina
ani-
School
NC.
13. 1977.
Hill,
blood
of Medicine,
Hill,
Grant
Cancer
shown
medium
been
derived
been shown
there
has
substance
Intraperitoneal
this
suggests
factors
have
recently
to erythropoietin
concentration
the
production
Institutional
cine,
the
Department
Submitted
the Dutch
been
physiologic
with
of Medicine, Chapel
and
factors
uncertain,
inversely
not
stimulator
vitro.23’24
fluctuates
has
of
presumed
kinetics
by
to
inhibition
role
cells
and
proliferation
cells.’3
studied
granulo-
implanted
polypeptide
It is considered
to cause
been
wastes,
granulocytes
and
more
conditioned
granulocytes
A granulopoietic
isolate
inhibition
weight
physiologic
of
clearly
of
of granulocyte
feedback
skin
granulocyte
reported
chambers.20-22
that
granulocyte
for
have
nutrients,
Mature
in vitro,’0”2
cells.’319
described
chalone
planted
to
inhibits
replicating
in-
granulocytes
differentiation
(DC)
of
molecular
that
inhibitor
granulocyte
those
small
and
Analysis
subject
granulocytes.9”
proliferation
been
no effect
be
Mature
granulopoiesis
exchange
inhibitors.48
may
marked
especially
cell
growth
chambers
allowing
granulopoiesis
the
diffusion
while
a
growth,
involved
influencing
support
cell
replication
and this inhibiboth myeloid
and erythroid
elements,
although
the data
suggest
a
greater
effect on myelopoiesis.
In contrast
to the mouse,
erythropoiesis
in chambers
in rabbits
remained
prominent
for over
1 wk.
neutro-
factors
systems
G. Palmer
produced
in
of
mustard
occurs in mice
by x-ray or drug. The
intraperitoneal
injection
of leukocytes
inhibited
the growth
of cells in chambers
implanted
in rabbits.
Removal
of mature
granulocytes
from marrow
prior to cham-
I
and Jeifress
inoculation
increase
in granulopoiesis
implanted
her
chambers
peritoneal
increase
John C. Herion,
have
diffusion
Chambers
in Rabbits.
on Cell Production
19,
1977.
National
the
Wilhelmina
Richard
Institutes
A merican
Cancer
of Health,
Society.
the
Roel
Rochrock
Willemze
Research
was
Fund,
supported
by
Fonds.
I. Walker.
Department
of Medicine,
UNC
School
of
Medi-
27514.
& Stratton.
1 (January),
Inc.
1978
ISSN
0006
-4971/78/5201-0012$02.0O/0.
21
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22
WILLEMZE
mals
rendered
dence
for
Since
these
in
are
could
to the
arise
locus
granulocytes
The
rabbit
not
autologous
also
the
The
studies
Male
white
ilium,
the
peritoneal
New
Zealand
suspended
cavity
Culture
of the
medium
was
Biological).
Penicillin
100 jg/ml,
respectively.
Total
cell
made
counts
were
and
blood
preserve
cell
morphology
Giemsa
stain
(blasts,
promyelocytes,
for
made
during
Marrow
containing
needle
into
normal
cytes,
9%
l-2;
row
a tube
±
1 5% fetal
Smears
myeloid
±
the
postmitotic
early
normoblasts,
ilium
The
culture
34%
elution
in
ethylene
peg
placed
rendered
in
the
were
and
mononuclear
solution
of Ficoll
virtual
marrow
was
planted
l-l
from
saline.31
peritoneal
The
and
of
bone
to
May-Gr’Unwaldas
proliferating
segmented
forms).
or
cells
were
to deliver
late
(poly-
classified
as
counts
the
the
semifluid
was
0.7%;
ml the
produced
after
in a flame.
by
kilogram
96
hr
induced
were
any
per
drug
harvested
of
killed
technique
immediately
the
each
prior
was
by
to
by
injection.30
In
intraperitoneal
about
16 hr
injection
the
least
were
DC.
with
MF-l
used. After
12 hr.
loading
Chambers
hole
was sealed
kept
in
16-30
intervals
after
ice-cold
chambers
implantation,
Cells
utilizing
of 2.5
This
dose
were
incubation
rein
of nitrogen
mg
of
experiments
quantities
after
monocytes,
a
DC.
administration
Large
25
concentration
rings
were
pentobarbital.
injection
weight.
Cell
usually
Benestad5
the
intravenous
the
at
chambers
At
described
of body
given.
for
of
of cells to each
anesthesia,
animals.
1.6%;
±
to plastic
chambers
0.1 ml. The
Filled
counts
granulocytes,
2.7%.
number
ventilate
intravenous
within
granulomonocytes,
differential
±
filters
before
with
clot
the
after
these
a
aspirated
0.9%;
±
13%
42%
desired
to
pentobarbital
in rabbits
HN2)
exudates
exudates
the
allowed
freshly
proliferating
lymphocytes,
Under
of
the
SD):
±
normoblasts,
heating
to dissolve
agranulocytosis
hr
were
25
into
a 22-gauge
proliferating
I 1%
column
syringe that delivered
animals
by
Pronase
HCI,
obtained
by
cavity
from
chambers
and
DC
of
SD):
±
aspiration
through
34% ± 1.3%.
In some
studies,
marby passing
them
through
a column
cotton
in 0.1
by
gently
lymphocytes,
(mean
late
forced
(mean
normoblasts,
granulocytes
±
and
rabbits
was
1.3%;
the
16%
1%;
±
repeating
peritoneal
(mechlorethamine
duces
Island
albumin
normoblasts)
anesthetized
distribution
implantation.
harvested
from
Neutropenia
oxide,
awaiting
covered
in
U/ml
Smears
with
categorized
Differential
±
from
following
as required
using a Hamilton
chambers
(Grand
bovine
were
distribution
0.9%
and late
poor
in mature
16%
were
were
from
placed
100
Counter.
(metamyelocytes
aspirate
medium.
following
granulocytes,
by dilution
while
use
aspirated
of
stained
basophilic
of lightly
in saline.
On
the
1.6%;
a plastic
it
to
then
serum
using
were
series
postmitotic
granulocytes,
sterilization
medium
were
calf
B Cell
DC were made
by gluing
0.45-zm
pore
size,
13-mm
Millipore
glue (No. 70.000.00). At least 3 days elapsed after construction
by
of
makes
also
were
concentration
model
Large
the
l2
made
revealed
loaded
cells
cytocentrifuge
normoblasts).
sterile
revealed
0.3#{176}/s;postmitotic
adjusted
but
Chambers
a final
and
EDTA
absorbent
l%-2%;
its size
host,
Marrow
DC.
and
to
(pronormoblasts
from
2%
containing
suspensions
was
ml
marrows
±
of sterile
13%
obtained
0.5
early
normoblasts,
suspensions
were
cell
cell
were
rabbit
effect
cells.
cells
syringe
the
the
of time.
a Coulter
in the
or
orthochromatic
macrophages/mononuclear
added
preparation.
Cells
early
because
used.
into
periods
with
smear
as
kg were
a Shandon-Elliot
myelocytes)
classed
in
neutro-
METHODS
5A medium
were
counts.
apd
and
McCoy’s
with
production
by mature
tested
in a single
inoculated
varying
in duplicate
made
differential
chromatophilic
for
streptomycin
were
were
2.4-3.0
and
rabbit
of 85%
as evi-
granulopoiesis.
animals.
animal
AND
weighing
medium,
same
and
marrow
Normoblasts
rabbits
in culture
here
AL.
ease.
MATERIALS
the
reported
chambers
technical
cell
carried
in neutropenic
multiple
interpreted
of
increased
as the experimental
with
been
stimulator
in inhibitor
in DC
to implant
has
some
neutropenic,
production
marrow
of
to a reduction
chosen
only
or x-ray28’29
amount
chambers.
cell
was
the
due
ofthe
on
possible
by drug27’28
increase
animals
chambers
phils
neutropenic
an
ET
HN2
on
of
the
of
granulocytes
of 200
glycogen
ml
drug
0.5%
injection
mustard
predictably
animals
and
pro-
given
DC
were
were
glycogen
by
HN2,
aspiration
im-
obtained
in
sterile
and
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RABBIT
MARROW
CULTURE
IN DC
23
Table 1. Total an d Differential
Culture
Condition*
N-BM
No. of
Animals
Total
Cells
4
+
2267
PE-cells
174
±
5
3343±
Postmitotic
Granulocytes
113
403
162
4
128
4658±
HN2-host
356±
p <0.05
N-BMin
4
3247
4
4658
16.2
±
609±
176
254
±
128
356
820
48.3
1250±
15.3
1027
±
1.9
1250
85
±
581
159
36
±
285
NS
80
Late
Nornoblasts
771 ± 97
p <0.005
30.9
±
p <0.005
19
±
1579±
112
NS
p <0.01
720±
18.4
NS
±
Early
Normoblasts
p <0.025
p <0.005
1.9
on Day 9
Macrophages/
Mononuclear
Cells
<0.05
p <0.005
±
Harvested
37
±
p
p <0.005
p <0.01
N-BMin
10.2
±
p <0.005
141
of Cells
Proliferating
Granulocytes
p <0.005
N-BM
Counts
487±
18.8
1732±
p <0.025
NS
14.8
p <0.005
±
29
802
±
20.9
312
±
26
±
80
720
±
18.4
487
±
18.8
829
±
94
1732
±
14.8
HN2-host
PE-cells
+
N-BMin
HN2-host
p <0.005
p <0.005
GP-BMin
4
6342±
HN2-host
273
516±9
p <0.005
GP-BMin
4
3180±
p <0.01
1893±
p <0.005
314±
224
NS
688±
111.5
15.2
p <0.025
34.5
NS
644±
NS
870±70
p <0.005
47.4
2241
p <0.005
594±37
359±
31.9
±
p <0.005
869±
18
41.5
HN2-host
PE-cells
+
Each value
from
8-16
is the mean
SE of mean
±
x 10_2
from
number
in normal
hosts;
of animals
indicated.
The value from
an imal
each
is the
mean
chambers.
*N.BM
normal
ceived
peritoneal
lavage
with
nuclear
bone marrow
exudate
sterile
cells,
were
in chambers
cells; GP-BM,
granulocyte-poor
saline.
Exudate
washed
once
cells,
HN2-host,
consisting
in sterile
host
bone marrow
isotonic
received
nitrogen
mustard;
PE-cells,
host
re-
in chambers.
of
about
saline
and
90%
granulocytes
resuspended
and
in
25
l0#{176}/
mono-
ml
McCoy’s
5A
medium.
Between
they
1.25
were
and
injected
2 x I0
of severe
leukopenia
peritoneal
exudate
cells
recipients’
blood
neutrophil
The
growth
of intraperitoneal
Studies
rabbit
the
represented
mean
individual
means
total
marrow
cell
paired,
but
were
as plotted
To
test
centrations
delivered
2900
cells/MI
Efficiency
in a beaker
In
which
the
period
control
detectable
time
animals
effect
on
the
of
to prepare
mean
derived
data
in each
were
and
slides
in DC
were
The
effect
HN2.
cell
growth
value
were
a minimum
Thus,
in
for
DC
as
on
ratios
for
day
on
the
a
each
represented
that
day.
same
After
beginning
counting.
by application
the
of
8 and
point
a given
containing
determined
calculated
of
each
harvested
for differential
was
expressed
rabbit
chambers
counts
were
on
used.
the
chambers
the
the
cell
p values
granulocytes
and
8- 16 chambers
contents
with
studied.
point
the
marrow
by treatment
of marrow
which
t test;
granulocyte-depleted
then
from
pooled
of cell
were
done.
After
into each of five small
recovered;
no
in
to
raw
cells
data
of
the
in
Table
shown
harvested/cells
nonI,
inoculated,
graphs.
accuracy
When
cells/.tl
limited
injection30).
exudate
sample
of from
between
on
and
rabbits
done,
were
Student’s
in the
experiments
were
produced
neutropenic
was
at each
the
were
determined
the
enumerated.
(SD)
all
of difference
not
in
injections
They
peritoneal
a given
value
for
preparation
two-tailed
injection
cell
6 post-HN2
days.
rendered
animals
rabbits
mean
counts
Significance
2 and
same
isologous
containing
the
ofthe
each
Such
unmodified
neutropenic
in 4-9
16 DC
the
in hosts
of
and
done
of
for
hosts.
days
on
aspirated
and
injections
were
maximum
given
obtained
DC
counts.
hosts
in normal
were
between
were
of freshly
in normal
implanted
cells
into
(usually
curves
established
such
intraperitoneally
0.1
were
when
when
cells/MI
inoculated,
the
of harvest
was tested
medium,
±
experiments
containing
3300
cells/s.d
were
inoculated,
3784
198 cells/MI
were
by loading
and
chambers,
the cell concentration
tubes
and the number
2460
were
2830
of
culture
into
measuring
centrifuge
ml of a suspension
recovered;
3950
were
delivery
then
DC,
harvesting
±
with
four
in
a suspension,
of cells in each
cells/.d
was
different
cell
con-
replicates
were
tube
was promptly
inoculated,
3455
370
±
inoculated,
2523
±
115
cells/MI
were
110
were
recovered;
and
when
cells/MI
recovered.
sealing
them.
them,
Using
allowing
this
them
routine,
to
stand
80%-90%
for
1 hr
of
the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
WILLEMZE
24
ET
AL.
0
0
w
2.0
NUCLEATED
a-
0
WLjJ
I-F-
CELLS/DC
3xI05
#{149}
6x105
cr,<
w-J
>D
<0
IZ
.0
C/)
Oo
0.5
H
Effect of number
Fig. 1.
2
4
DAYS
6
AFTER
8
0
12
14
16
8
normal
planted
IMPLANTATION
marrow
on
cell
chambers
cells
growth
of
im-
in
in control animals.
0
0
w
a00
LU
‘-I-
LUL
10
u,4
GRANULOCYTES
ui-i
>:D
POSTMITOTIC
40
I
U)
(I)
WLUJ
‘-I-.
>->00
0
-J
05
PROLIFERATING
zz
0
44
C)
0
a-
IS
a00
LU LU
U)<
ui-I
>:D
10
a-0
<0
#{182}U)
0.5
ma
00
a0
0
zz
C-)
d
aw
a-
05
-
MACROPHAGES/MONONUCLEAR
00
CELLS
Cn
UI
>
j
a-0
<0
025
-
z
U)
WU)
(-J
<-I
Fig. 2.
inoculated
implanted
Distribution
of cell types
in
with 3 x JO5 normal
marrow
in control animals.
chambers
cells and
xw
a.0
0,j
a40
“0
2
DAYS
4
AFTER
68
10
IMPLANTATION
12
14
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RABBIT
cells
MARROW
CULTURE
computed
Ficoll-
to
Pronase
be
IN
within
solution
did
25
DC
the
not
chamber
alter
could
this
be
recovered.
Incubation
of
chambers
in
the
yield.
RESU ITS
Cell
Proliferation
in DC
in Normal
Rabbits
Figure
1 shows
that
optimum
growth
occurred
in chambers
containing
3 x 10 cells.
In each instance
cell numbers
fell for 4 or 5 days
and rose to a
maximum
on the ninth
or tenth
day after
implantation.
After
this time
cell
numbers
declined
gradually
for at least the next 8 or 9 days.
Not shown
is the
observation
that
cell growth
with an inoculum
of 2 x i0 cells did not differ
significantly
from that found
with 3 x l0 cells, but the use of smaller
numbers
of cells created
problems
with
quantitation
of cell counts.
Thus
3 x iO was
selected
as the number
of cells for routine
use.
Figure
2 shows
the distribution
were
implanted
in each
chamber.
plateaued
from
phages
and
day
7 at about
unidentified
essentially
precursors
Effect
of HN2
Figure
cytes
fell rapidly
thereafter
Treatment
3 shows
the
Blood
to reach
to slightly
throughout
by day
of Cells
of rabbits.
the
cells,
on day 13. Cells
week of culture.
constant
increased
Composition
one-half
mononuclear
40
of the cells harvested
gradually
during
the first
mained
of cell types
Proliferating
1.5
of the Hosts
From
effect
of 2.5 mg
a minimum
above
of
on day
while
increased
macroto
about
identified
as lymphocytes
diminished
The number
of early
normoblasts
period
of culture,
times
Implanted
were
3 x l0 cells
granulocytes
inoculated,
scarce,
while
the number
on the Number
Harvested
granulocytes
number
initially
the
9 to about
harvested
when
and
postmitotic
late
re-
red
cell
implanted.
and
DC
HN2/kg
on
maintained
the
for
4. Circulating
circulating
granulo-
2 days
about
granulocytes
and
rose
then
rapidly
normal.
0
0
0
8
LL
0
w
F-
7
6
TOTAL
LEUKOCYTES
-J
5
uJ
a-
4
0)
2
C/)
Fig. 3.
Effect
of HN2
injected intravenously
in rabbits
on circulating
leukocytes
and
granulocytes.
Each point represents the mean
of the leukocyte or granulocyte
counts
of
at least 20 rabbits.
w
I>-
0
0
0
0
U
-J
DAYS
2
4
AFTER
HN2
6
8
ADMINISTRATION
I0
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
WILLEMZE
26
ET
AL.
C)
0
aUI
a.
UI
‘-I-.
U)4
UI
>
a-C)
40
U)
U)
uJw
>->00
00
-i-i
zz
CONTROL
44
HOST
a-a-
O EARLY
00
e LATE PRECURSORS
PRECURSORS
HN2 -TREATED
U
o
0
aUi
a.
EARLY
#{149}
LATE
IS
HOST
PRECURSORS
PRECURSORS
00
UJW
(“4
UI-i
10
05
<<
a
a
00
NORMOBLASTS
a-a00
zz
0-
U
0
aU
0.5
a.
MACROPHAGES/MONONUCLEAR
00
UI
UI
CELLS
I-I(“4
J-i
A HNHOST
A
CONTROL
Fig. 4.
Comparison
of cell
distribution
in
chambers
in
control and HN2-treated
hosts;
3 x 1O cells were inoculated
per chamber.
(Early
granulocytes
are
proliferating
cells,
and
late
granulocytes
are
postmitotic
cells.)
HOST
0.25
0
<0
U)
UI
U)
0-i
4-i
‘UI
“0
0-i
a-4
2
4
DAYS
<0
6
8
AFTER
10
12
14
IMPLANTATION
Chambers
harvested
from animals
treated
with HN2 contained
more
cells
after the fifth day then those harvested
from control
animals.
By day 9 chambers from HN2-treated
hosts contained
1.5 times
the number
inoculated,
while
chambers
from control
animals
contained
the same number
as in the inoculum
(Table
1). The means
of absolute
values
(Table
I) obtained
on day 9 in five
control
and four HN2-treated
animals
were significantly
different
(p < 0.01).
The
distribution
Fig.
4.
HN2-treated
trols after
in the two
in
the
day
Effect
ofcells
Proliferating
found
and
in the
postmitotic
chambers
granulocytes
animals
were significantly
higher
day 5, while
there
was no difference
kinds
of hosts.
Early
normoblasts
HN2-treated
hosts
and
late
in these
animals
is presented
in
in diffusion
chambers
from
in comparison
with those
of conin the number
of macrophages
were significantly
higher
at day 9
normoblasts
were
higher
in
this
group
11.
of Granulocyte
Removal
on Subsequent
Growth
Marrow
from
through
a cotton
which
postmitotic
granulocytes
column
had a greater
proportion
had
tion
unmodified
for
than
did
the
marrow.
To
adjust
in DC
been removed
of cells capable
this
increase
by passage
of replicain potentially
at
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RABBIT
MARROW
CULTURE
27
IN DC
IS
0
0
Cs
aUI
a.
05
0
t:,
<0
Cn
U)
t;
2.0
IS
zz
10
0.5
aUI
a.
00
WW
MACROPHAGES/MONONUCLEAR
Fig. 5.
Comparison
of red cell precursors and
macrophages
in
normal
and
granulocyte-poor
marrow
in HN2-treated
hosts; 3 x JO5 normal
and
2.4
x
1O
granulocyte-poor
marrow
cells
were
inoculated
per chamber.
proliferative
inoculated
cells,
per
20%
fewer
cells
than
the
chamber
control. Preliminary
studies
2 x iO and 3 x i0 unmodified
The
removal
sequent
growth
samples
being
implanted
of
mature
over
obtained
in that
number
the
after
0
-i
0
same
2
of proliferating
4
DAYS
6
8
AFTER
marrow
iO unmodified
(2.4
marrow
0
12
from
marrow
x 10)
cells used
animal
greatly
marrow,
prior
administration
both
granulocytes,
were
for the
when
enhanced
kinds
of
submarrow
to HN2
treatment
and
(Table
1). The
distribution
Effect
dramatically
of A ddition
from
day
of Peritoneal
Four experiments
were
16 chambers
with normal
7 (Fig.
Exudate
then
day 9, marrow
from
implantation
had
an
postmitotic
granulocytes,
late normoblasts.
There was no significant difference between
the number
macrophages
or early normoblasts.
An increase
in proliferating
granulocytes
was apparent
in the first few days of culture,
while
postmitotic
granulocytes
creased
14
IMPLANTATION
no difference between
cell yield
cells were inoculated in the
DC.
unmodified
HN2
CELLS,,...._O
is shown
in Figs. 5 and 6. On
had been
removed
prior
to
of cells in these chambers
which
mature
granulocytes
increased
showed
marrow
of the
from
animal
0
25
of granulocyte-poor
3 x
granulocytes
that
!
and
of
in-
6).
Cells
performed
in which HN2-pretreated
and 16 chambers
with granulocyte-poor
rabbits bearing
marrow
re-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
WILLEMZE
28
ET
AL.
PROLIFERATING
GRANULOCYTES
Cs
0
aUi
ci.
0
LU
-I
C
:3
0
0
0
LU z
IU)
UI
>
aI
U)
UI
U)
UI
>0
0
-I
:3
I-
z
>0
a-
0
-I
(0
z
z
(0
a-
I-
(0
aUI
U.
A
GRANULOCYTEPOOR
MAF
-I
0
a.
0
O
2
DAYS
ceived
exudate
trol
4
6
AFTER
8
0
2
4
IMPLANTATION
daily intraperitoneal
cells
rabbits
granulocytes
were
Fig. 6.
Comparison
of granulocytes
from normal
and
granulocyte-poor
marrow
grown
in HN2-treated
hosts;
3 x JO5 normal
and 2.4 x JO5 granulocyte-poor
marrow
cells were inoculated
per chamber.
NORMAL
MARROW
a-
determined
on
days
bearing
on
the
daily
2-6,
injections
chambers
same
and
days
after
were
not
chamber
altered
2
Fig. 7.
Effect
of intraperitoneal
injections
of peritoneal
exudate
(PE) cells on the number and distribution
of cells in
chambers
implanted
in HN2treated
hosts; 3 x
normal
marrow
cell and 2.4
x JO5
granulocyte-poor
marrow
cells
were inoculated
per chamber.
Normal
controls
are included
for comparison.
Macrophage
values:
macrophages
harvested,
divided
by total
cells
inoculated.
Normoblast
and
granilocyte
values:
number
of
the specific cell type harvested,
divided
by the number
of the
specific
cell type
inoculated.
All results
obtained
on day 9
after DC implantation.
1-2
x
10 heterologous
they were
normal
Blood
peritoneal
BONE
()
C...)
BONE
Normal
0
fl
granulo-
MARROW
H
3
congiven
granulocytes
exudate
GRANULOCYTE-POOR
U)
peritoneal
leukopenic.
Normal
marrow
were
also
implantation.
by the
NORMAL
4
C/)
of
period
when
containing
the
[J
MARROW
host
HN2-treated
host
HN2-treated
host,
given RE. cells
UIUI
UI
Total
Cells
Post-mitotic
Gronulocytes
Prolif erating
Granulocytes
Mocrophoges
Early
Normoblosts
Late
Normoblosts
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RABBIT
MARROW
CULTURE
cyte injections.
in Table
1 and
peritoneal
cells
marrow.
not
and
The
significantly
penic
hosts
crease
of
exudate
29
significantly
granulocytes,
normal
not
reduced
both
early
numbers
of
different
from
injected
with
macrophages
9. Results
of these
studies
are
hosts
and the normal
control
the
total
late
macrophages
in
number,
in
cells.
the
the
postmitotic
of these
exudate
chambers
cell
normoblasts
and
numbers
peritoneal
in
the
and
control
shown
hosts,
pro-
unmodified
granulocytes
cells
in HN2
There
was
hosts
given
leuko-
a slight
in-
peritoneal
cells.
Using
marrow
modified
that
injection
of
except
macrophages.
cells
DC
DC were harvested
on day
Fig. 7. In these
leukopenic
exudate
liferating
were
IN
had
no
effect
HN2-treated
by
removal
of
peritoneal
exudate
Intraperitoneal
on
total
recipients
and
at day
mature
granulocytes,
cells
significantly
injection
ofculture
differential
counts
it was
reduced
medium
of
observed
all cell
containing
chambers
types
no
harvested
from
9.
DISCUSSION
Previous
mice
studies
and
animal
rats.
of
The
is suitable
for
ability
to use more
the same
animals
tologous
than
or
in the
studies
culating
cells
Cells
Cells
more
in
such
isologous
been
may
and
a phenomenon
may in part
the apparent
possibility
loss of red
late
late
advantage
normoblasts
of
activity
the
intact,
occurred
has
death
and
noted
cell
of
A net
the
by
damage
and late
live
first
rabbits.32
unchanged.
to
in
cells,
total
week
others.
for
cir-
presumably
decrease
during
been
greater
from
bled
or
change,
line.
the
from
au-
is suitable
derived
morphologic
cell
size,
is much
in severely
remain
this
marrow
either
technique
implanted
or
larger
ability
to utilize
In DC containing
thus
in
that
after
While
this
DC
effect
caused
by manipulation,
normoblasts
suggests
polymorphonuclear
erythrocytes
have
a
the
neutrophils
and
been enumerated
not
studies.
and
an
increase
regularly
later
to an increase
in the number
of granulocytes,
occurred.
Although
the total
declined,
maturation
by others,
bers of granulocytes
implanted
into the
the
in DC.
peritoneal
in a humoral
by
substances
substances
represent
proportion
appeared
neutropenia
Mature
granulocytes
have
during
the past 10 yr evidence
these
in chambers
largely
indicates
Erythropoiesis
disintegrate
of maturation
with
cell nuclei.
Reticulocytes
division
and
As reported
poiesis
and
performed
here
erythropoietic
marrow,
that
been
the
and the
placed.
be due to disintegration
from
selective
loss of granulocytes
After
6 days
normoblasts
slightly
offers
may also
divide and/or
undergo
differentiated
form
of the same
implantation,
have
reported
erythropoiesis.
and
granulocytes,
in these
and
marrow,
reported
die
DC
rabbits
per animal,
the DC are
mouse
affecting
has
DC
studies
rabbit
with
factors
in
with
chambers
in which
studies
of
hematopoiesis
experience
derived
of different
to contribute
in the
long
has
from
specific
cells
of the
animals
of granulopoiesis
been
known
accumulated
or
with
and
rose
Both
observed.
to increased
granulocytes
has been
in these
to inhibit
indicating
associated
inhibitors
changed.
to the changes
host animal
led
The increased
growth
cavity
of neutropenic
stimulator
macrophages,
cell
number
cell
num-
in DC
ascribed
animals.
cell replication,
and
inhibition
of granulogranulocytes.
of granulopoiesis
and
Whether
what
their
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
30
physiologic
role
might
medium
incubated,
in which
when
containing
normal
be
remain
to
be
peritoneal
exudate
injected
intraperitoneally
marrow,
reduces
determined.
granulocytes
into
Material
and
neutropenic
granulocyte
growth
WILLEMZE
ET
derived
from
monocytes
mice
within
had
bearing
the
of granulopoiesis
when
mature
cytes
cells
from
human
to
blood
containing
studies,
neutropenic
those
animals
animals,
the
the
time
of neutropenia
measured
on
were
not
syngeneic
mouse
bone marrow
to determine
would
ninth
days
day
detectably
dude
the presence
humoral
stimulator
cell
2-6)
after
altered
of
1-2
x
l0
added
DC
cell
does
by
intraperitoneal
injection
of exudate
A marked
increase
in cell production
of mature
granulocytes
prior
removal
cell
production
erythroid
had
its
in these
most
striking
effect
not
in increasing
the
suggests
granulopoiesis
that
more
than
granulocytes,
from
number
but
inoculated
of
also
marrow
postmitotic
granulo-
of macrophage
production
was not observed
in
growth
in DC caused
by intraperitoneally
injected
increase
in growth
resulting
when
mature
granulosuggest
that
mature
granulocytes
coninhibitory
effect
involved
both
the myof a specific
inhibitor
of granulopoiesis
with mature
granulocytes
could
the striking
increase
in granulopoiesis
granulocytes
only
granulocytes
cytes were
removed
from
the inoculum
tain inhibitors
of cell replication.
This
eloid and erythroid
cells. The existence
associated
although
could
occur
with their
was also
granulocytes.
involved
of mature
cytes
produced.
Stimulation
any of the studies.
Decreased
granulocytes
and the striking
ex-
occurred
in marrows
modified
by the
to the inoculation
of the DC.
Increased
experiments
removal
cells. The
not
of a circulating
that at least
some
of the increased
growth
of marrow
cells implanted
in DC
due to the inability
ofthe
neutropenic
animal
to deposit
granulocytes
inhibitory
factor(s)
in the locus
of the chambers.
Red cell production
inhibited
growth
neutrophil
finding
in neutropenic
animals
of increased
amounts
of granulopoiesis,
but it raises
the possibility
in
during
Blood
This
the
implanted
inhibited
injections.
to
granulocytes
implantation.
by these
and
granulo-
intraperitoneally
in
substantially
chamber
were
given
production
injection
(usually
the
peritoneal
cells.
if granulocytes
decrease
intraperitoneal
as
counts
or
mouse
designed
been
DC
chambers.’9’2#{176}
Boyum
et al.33 recently
reported
a significant
depression
stimulation
of macrophage
formation
in 7-day
cultures
chambers
In these
AL.
the
not
inhibitory
be established
from
produced
by removal
effect
of
the
this study,
of mature
granulocyte
involves
erythropoiesis.
ACKNOWLEDGMENT
The
authors
are
indebted
to Marva
Dowdy
for her
excellent
technical
assistance.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1978 51: 21-31
Marrow culture in diffusion chambers in rabbits. I. Effect of mature
granulocytes on cell production
R Willemze, RI Walker, JC Herion and JG Palmer
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