1. No category

From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1982 59: 586-593
Dexamethasone synergistically induces chemotactic peptide receptor
expression in HL-60 cells
KM Skubitz, YS Zhen and JT August
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Dexamethasone
Synergistically
Receptor
Expression
By Keith
We
have
examined
the
of dexamethasone
human
promyelocytic
ity of surface
receptors
Dexamethasone
markedly
synergistic
ylsulfoxide.
but
f-met-Ieu-phe
peptide
binding
peptide
receptors
affinity.
The
and
f-met-leu-phe
activ-
the
the
chemotactic
acid.
in
inducing
The
increase
HL-60
due
to
cells.
an
increase
than
an
in the
ORTICOSTEROIDS
in
was
exert
an
was
clinical
treated
with
(0.01-I
receptor
mg/ml)
for the
(FMLP)
peptide.’
of
of
effect
fail
of rabbit
of
to this
neutro-
phils to l0_6_l08
M corticosteroids
for 16 hr has been
reported
to decrease
the cells chemotactic
response
to
FMLP
without
altering
FMLP
binding
to the cell,
possibly
by
altering
prostaglandin
metabolism.2
Recently,
sone have
physiologic
been reported
Fc receptors
concentrations
to decrease
on the surface
of
dexamethaexpression
the
of HL-60
cells,3
a possible
mechanism
of corticosteroids.
of the antiinflammatory
These
results,
together
earlier
showed
studies
that
that
of
activity
with
our
concentra-
tions of corticosteroids
did not alter the receptor
affinity for FMLP
in mature
human
neutrophils,
led us to
analyze
the effect of corticosteroids
on the activity
of
the chemotactic
peptide
receptor
of HL-60
cells as
they differentiate
to mature
neutrophils.
The HL-60
cell line, established
from a patient
with
acute
promyelocytic
leukemia,4
grows
in culture
pri-
From
the
Departments
Experimental
of
Medicine.
Chinese
September
Presented
in part
ofHematology
reprint
Medicine,
Johns
Wolfe
© I 982
586
and
annual
Antonio,
requests
Hopkins
Pharmacology
Hopkins
the
of
accepted
Antibiotics,
Texas,
to Keith
December
M. Skubitz,
University
Street,
Baltimore,
Md.
by Grune
& Stratton,
Inc.
School
21205.
5-8,
of
there
was
assessed
by
to
reduce
had
steroid
no reproducible
by the
for
binding
blocked
on HL-60
The
half
maximal
constant
of
cells
with
methyltestosterone
or
tested.
in HL-60
not
when
tetrazolium.
criteria
required
known
the
effect
morphology
effect
three
receptors
was
1 7a
little
cell
nitroblue
but
the
the
steroid
or progesterone.
as leukemic
promyelocytes.
to differentiate
to more
granulocytic7
foxide,
RO1
1981.
Department
Medicine,
725
cell line can be
cells of either
chemical
stimulus,
dimethylformamide,
trans-retinoic
acid,
have
and
type
as has been
dimethylsul-
phorbol
esters.
been
reported
of HL-60
cells
Gluco-
to have
in culture,10
no
but
can act to decrease
the expression
of Fc receptors
the surface
of HL-60
cells.3
Some
leukemic
cell
lines of other
species
can be induced
to differentiate
with glucocorticoid
hormones,’2”3
and with others
differentiation
in response
to inducing
agents
is blocked
by dexamethasone.7
on
article
we describe
concentrations
surface
receptor
tion,
tion
the FMLP
of HL-60
markedly
the
effect
of physiologic
of dexamethasone
on the activity
important
in immune
effector
receptor,
as well
cells in culture.
synergistic
in
of a
func-
as on the differentiaDexamethasone
was
inducing
FMLP
receptor
activity.
In contrast,
the hormone
had relatively
little
effect
on the induction
of more
differentiated
cell
morphology
or the ability
of the cells to reduce
NBT,
two other
indicators
of granulocyte
maturation.
Our
results
suggest
that steroids
augment
activity
of the
granulocyte
chemotactic
peptide
receptor
during
differentiation
induced
by other
physiologically
active
agents.
tactic
There
peptide
is no evidence
that inhibition
receptor
activity
in developing
is a mechanism
of the
of chemomyeloid
antiinflammatory
effect
of
corticosteroids.
MATERIALS
Society
This
mature
or macrophage/monocytic8’#{176}
corticoid
hormones
effect on differentiation
10, /981.
American
M.D.,
for
by the appropriate
demonstrated
with
cells
and
November
ofthe
School
China.
5T-32-CA-09243
of Health.
meeting
and
University
Institute
Beijing,
Grants
Institutes
28, 1981,
0006-4971/82/5903-0019$Ol.00/O
and
Johns
Sciences,
at the
in San
Address
North
Md.
ofMedical
in part
by
the National
Submitted
Medicine
The
Baltimore,
Academy
Supported
CAI947Ifrom
of
of
Therapeutics,
cells
alone
agents
In this
suggesting
physiologic
the
of dexamethasone
manly
induced
the
corticosteroids
to respond
peritoneal
as
was
as assessed
hormone
blocking
exhibit
a reduced
affinity
of the cell
chemotactic
tripeptide
f-met-leu-phe
and simultaneously
In vitro exposure
of
differentiation
the
antiinflammatory
in vivo and are used in a wide variety
settings
for this effect.
Human
granulocytes
concentrations
binding
spe-
action
high
ability
in
receptor
relatively
peptide
dexamethasone
concentration
synergism
correlated
with
activity
increase
of dexamethasone
nM
August
differentiation
Dexamethasone
dimeth-
trans-retinoic
for
Peptide
Cells
and J. Thomas
HL-60
and
by
Zhen,
cific
dimethylformamide
rather
effect
(HI-SO)
Chemotactic
in HL-60
of the
of 500
with
was
concentrations
at a concentration
with
not
Yong-su
of differentiation
cells
for the
binding
M. Skubitz,
of physiologic
induction
leukemia
peptide.
C
effect
on the
Induces
AND
METHODS
Chemicals
Dimethylsulfoxide
burg,
Pa.) and
cal Company,
(DMSO)
(il.
N,N-Dimethylformamide
Inc.,
modified
Eagle’s
desired
concentrations.
Milwaukee,
medium
Baker
Wisc.)
(DMEM)
Chemical
Co.,
(DMF)
(Aldrich
were
prepared
(Gibco,
Grand
I 2-0-Tetradecanoylphorbol-
Blood,
Vol.
59.
PhillipsChemi-
in Dulbecco’s
Island,
N.Y.)
at
1 3-acetate
No.
3 (March),
1982
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
EFFECT
OF
DEXAMETHASONE
(IPA)
(PL
acetone
and
ries,
San
in
ON
Biochemicals
stored
Inc.,
CarIes,
Calif.)
was
and
stored
at -70#{176}C until
then
sodium
in 500
diluted
mM
St.
(Pharmco,
Publicker
tration
of
terone
and
at
10.2
and
and
concentration
Cell
Culture
HL-60
were
kindly
in
(FCS)
DMEM
with
Sterile
mg/mI
seeded
before
viability
were
then
at 2.5-5
added
the
volume
the
were
previously
described.’
0.5
3H-FMLP
(46.4
as
culture
cells
bovine
I 5 mm.
performed
at
results
cells
were
The
once
I 06/ml.
in
Slides
containing
scraping,
expressed
balanced
salt
were
filtered
from
that
the
binding
distortion
nuclear
pattern
seen
cells
and
in
with
prepara-
were
performed
significance
was
by an appropriate
by
shown,
analysis
individual
of
variance.
comparisons
t test.
binding
of
by incubation
previously
been
3H-FMLP
to
of the cells
HL-60
with
reported.2224
cells
1 20 mM
Growth
was
DMF
of HL-60
cells in the presence
of both
120 mM DMF
and 500
nM DEX markedly
increased
3H-FMLP
binding
(Fig.
1). Induction
of 3H-FMLP
binding
by DMF
alone was
several
fold lower.
The average
per cell was 200 in the control
treated
with I 20 mM DMF,
and
in the
increase
presence
500
of 500
in 3H-FMLP
number
of receptors
cells,
16,150
in cells
64,600
in cells treated
nM DEX.
3H-FMLP
increased
in HL-60
cells
nM
DEX
binding
in the activity
not an alteration
without
can
of surface
in receptor
DMF.
be attributed
3H-FMLP
affinity
for
the
0
In
Mass.)
washed,
and
experiments
0
the
x
were
U,
0
internaliza-
binding,
defined
absence
a Shandon-Elliot
England).
U
0
as the
versus
or
of the
0
the
E
Similarly,
group
and
bizarre
patterns
that
cells
that
that
but
with
readily
into
cells
a condensed
of nuclear
segmentation.
I
In
granulocytes,
forms,
cells
U.
promyelocytes,
segmentation,
divided
have
into
.1
were
pattern
of cell
was often
quite
and
band
and
usually
5 was
cells
the
blasts
divided
with
pattern
)
(4)
1 was
centrifuge
counts
of normal
(1
promyelocytes
nucleus
chromatin
but
five groups:
Group
neutrophils
Because
microscopy
metamyelocytes,
blasts
Cytospin
Differential
in the development
into
neutrophils.
marked
tin
with
(3)
HL-60
mature
forma-
cytospin
solution
incubated
peptide-receptor
Wright’s
stained
cells.
as determined
by light
control
nucleoli.
comparisons
to an increase
receptors
and
and
Boston,
binding
as specific
Surrey,
myelocytes,
fine
at 37#{176}C.
The
blue-black
stained
0.2
Wisc.),
V
C
prepared
on
segmented
for 20 mm
reduced
on Wright’s
with
0
we classified
(2)
incubated
Milwaukee,
0.
were
different
as has
This
FMLP.
SCA-0030,
performed
differentiation
Co.,
ng of IPA
previously
at 2 x 106
Analysis
performed
grown
following
Morphology
(model
ml were
intracellular
with
120 mM
DMF
plus
binding
was not reproducibly
individual
PBS
and
a
Cell
Inducing
flask
Nuclear,
then
prevent
3H-FMLP
of 10 tM
0.2
as
suspended
(3H-FMLP)
was determinor
modifications
as
albumin
Similar
4#{176}Cto
between
with
England
counted.
are
oxal-
medium.
in Hank’s
serum
New
The
was
mg/mI
indicated
with
10
(Sigma).’8
exclusion.
culture
calf
(Sig-
x
(Sigma)
200
assayed
Receptors
Ci/mmole,
3H-FMLP
dye
acid
Briefly,
mg/mI
2
by washing
with
free as
(Corning,
approximately
tissue
of f-met-leu-[3H]phe
by Williams
et al.’9
containing
presence
twice
split
FCS,
Chemical
determined
statistical
induced
acid
0.150
insulin
blue
harvested
cells
Specific
binding
mined
as described
difference
When
(National
Logan,
reached
to
oJFMLP
tion.#{176}2’All
and
diaminetetraacetic
Determination
also
a small
Gallo
(GIBCO),
was
Of cells
RESULTS
fetal
Irypan
adhering
were
ethylene
washing
bound
by
R.C.
bovine
containing
cultures.
Utah),
U/mI
10%
(Aldrich
containing
was
NBT
their
Inc.,
concentration
in
Cells
at 37#{176}C
for
deposits
Statistical
daily
heat-inactivated
x l05/ml
cell
by IPA
10 mM
0.2
determined
experiments.
induction
(prepared
10%
pyruvate
and
the
was
agents
sodium
(Aldrich),
in PBS
of cells
Multiple
to 20 times
with
NBT
reduce
modifications.
tions.
ethanol
Systems,
0.05
were
M and
at a concen-
The cells (mycoplasma
in 25 sq cm I flasks
ma),
week,
zan
17-a-Methyltestos-
reagents
0.2%
to
minor
in DMEM
of
percent
ethanol
Pa.)
ml
of cells
with
dissolved
Chemical
in 200-proof
by
Md.).
grown
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
cells
200-proof
in DMEM
provided
(HY-Clone,
acid
in
to the cell
mM
acetic
(Sigma
of
The
N.Y.)
serum
All
diluted
gift
of 2 x l02
(RA)
dissolved
DMEM.
of Health,
Bethesda,
by culture)
were
Corning,
dissolved
in a small
and
Dohme)
was
was
Sharp
in DMEM.
to addition
ability
described’2
7.4
Pa.,
Philadelphia,
were
were
prior
cells
Institutes
determined
in
IPA)
Reduction
The
diluted
pH
a generous
dissolved
diluted
NBT
cells/mI
Inc.,
(Sigma)
diluted
and
final
10 mM,
saline,
acid
was
then
in
Laborato-
(DEX),
Point,
(Merck,
Industries,
progesterone
FMLP
at
to a concentration
Mo.)
dissolved
use.
Trans-retinoic
Louis,
103M,
M
except
Na2CO3
in DMEM.
Company,
was
(Peninsula
in DMSO
phosphate
Sharp,
and Dohme,
West
of PBS.
Indomethacin
dissolved
FMLP
phosphate-buffered
Dexamethasone
Merck,
volume
Wisc.),
use.
dissolved
587
CELLS
Milwaukee,
at -70#{176}Cuntil
calcium/magnesium-free
(PBS),
HL-60
00
and
(5)
resemble
often
had
retained
discernible
that
resemble
nuclear
chroma-
l50
[3H-FMLP](nM)
a
Fig. 1 .
Dexamethasone
potentiates
the induction
by DMF of
FMLP receptors
on the surface
of HL-60 cells. HL-60 cells were
cultured
for 6 days in the presence
of the indicated
inducing
agents and assayed
for surface
FMLP receptors
as described
in the
text.
The specific
H-FMLP
binding
per cell at 37CC is plotted
versus the concentration
of H-FMLP.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
588
SKUBITZ.
Fig.
tiates
face
2.
in the
AND
AUGUST
Dexamethasone
the
FMLP
HL-60
U.
ZHEN,
cells
were
presence
poten-
by DMF of suron HL-60 cells.
induction
receptors
cultured
of 1 20
mM
(#{149})
(r = 0.951 ) or without
DEX (r = 0.938).
SpecifIc
for 6 days
DMF with
(0) 500 nM
3H-FMLP
binding at 4’C is plotted
according
to
the method
of Scatchard,
as B/F =
BoI#{231},- B K.
where
B is the concentration
of
bound
peptide
cxpressed
as cpm per 5 x 1 0’ cells. F is
the
concentration
of free
peptide.
and B,, is the total
concentration
of
receptors.
The calculated
K
was
5 x 1 0’ M ‘ for both groups
of cells.
while
the number
of receptors
per
cell
B(cpm
the ligand
as shown
by
from
binding
experiments
xIO3)
Scatchard
analysis25
of data
performed
at 37#{176}C(not
or 4#{176}C
(Fig.
2). 3H-FMLP
binding
to HL-60
cells was greater
at 37#{176}Cthan
at 4#{176}C,
as has been
reported
for normal
human
neutrophils,
perhaps
representing
peptide
internalization.”26
The
increase
in
shown)
FMLP
All
binding
further
was
nearly
studies
were
maximal
by day
performed
6 (Fig.
using
was
grown
a
exposure
performing
threefold
in the
higher
presence
of the cells to the inducing
assays
of differentiation.
on the
of DEX.
agents
cells
prior
to
The relation
of the effect
of DEX
to its interaction
with the steroid
receptor
was examined.
Potentiation
of
120 mM DMF-induced
HL-60
cell differentiation
by
DEX was determined
by FMLP
binding
after removal
3).
6-day
In
0
‘C
E
U
V
C
0
0.
-J
U.
I
In
[DEXJ(nM)
Fig. 4.
t (days)
Fig. 3.
DMF in the presence
or absence
of dexamethasone
induced
a time-dependent
increase
in FMLP binding to the surface
of HL-60
cells.
Specific
3H-FMLP
binding.
expressed
as cpm bound
per 1 0 cells in the presence
of 50 nM 3H-FMLP.
is plotted
versus
time
of
exposure
of
HL-60
cells
to
either
1 20
mM
DMF
mM DMF plus 500 nM DEX (#{149}).
500 nM DEX
(0). or PBS
point
is the mean
of three
separate
determinations
duplicate
experiment
gave similar
results.
(A).
(0).
±
120
Each
SD. A
The
of DMF
potentiation
induction
of 3H-FMLP
binding
to HL-60
to HL-60
presence
presence
cells by DEX is dose dependent.
Specific
3H-FMLP
binding
cells.
expressed
as cpm
bound
per
1 0’ cells
in the
of 50 nM3H-FMLP.
is shown
for cells grown
6 days in the
(#{149})
or absence
(A) of 1 20 mM DMF
and varying
concen-
trations
of DEX as shown.
Half-maximal
potentiation
occurred
5 and 50 nMDEX.
High concentrations
of DEX alone often
between
induced
mean
similar
a small
of triplicate
results.
increase
samples
in 3H-FMLP
±
SEM.
binding.
A duplicate
Each
point
is the
experiment
gave
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
EFFECT
OF
DEXAMETHASONE
of steroid
at a DEX
half
by washing.
concentration
maximal
potency
reported
DEX,
K
ranging
cally
at
-10
.-.-4 x
nM
l0
from
HL-60
DEX
589
(Fig.
system
receptors
M.27
0.05
4).
Thus,
correlates
of HL-60
DEX
alone,
to 500,000
HL-60
cell differentiation
(Table
1) or by NBT
I 7-a-methyltestosterone
block
the effect
CELLS
The effect of DEX was maximal
of 500 nM and approximately
of DEX
in this
affinity
of steroid
tions
on
ON
with
cells
had
as assessed
reduction
metabolism,
prostaglandins
Indomethacin,
activity
are
(not
known
In contrast,
shown).
to alter
Table
1
.
had
no effect
asone
Dexameth
Potent
iates
that
study
cells.
at
RA
induced
by
division,
between
DEX
and
by cell
of H 1-60
of
by morphology
or
was
a slight,
(Fig.
6).
inducer
as the
of more
ability
to
measureable
the presence
FMLP
of RA
suppressed,
of FMLP
DMF
in a dosereceptors
(Fig.
7).
This
effect on the cells as
blue exclusion,
and
counts,
were
additive
with DMF,
as previously
and DMF
in inducing
the ability
entiation
Cells
Induced
not
signifi-
with
b y DMF
reported,7
of HL-60
1.6
x l0
or D MSO
or DEX
cells to
M TPA
But Not
of Cells
Percent
Atypical
Myelocyte
RA
Segmented
Band
Metamyelocyte
-
14.3
48
-
19.7
43.3
90mM
+DEX
13.7
-
8.7
90mM
-DEX
32.3
-
60mM
+DEX
34.7
60mM
-DEX
30mM
+DEX
Normal
20
PMN
Atypical
7.6
-
5.7
0
-
48
22.7
7
-
7.3
43.6
14.3
2.3
-
-
8
39
16.3
2
-
78.7
-
8.3
10.3
91.5
-
3.5
3
2.7
0
-
2
0
-
4.5
1
1
12.3
13.3
5.5
-
7.8
9.8
6.8
-
12.5
12.8
9.8
-
6.3
-
5.5
1.8
-
2.8
1.3
-
4.5
3.3
.3
-
30mM
-DEX
89.5
-
4
160mM
+DEX
63.7
-
5.3
160mM
-DEX
71
-
4.8
130mM
+DEX
58
-
7
130mM
-DEX
70.8
-
3.8
9.5
9.8
100mM
+DEX
78
-
5.3
9.5
100mM
-DEX
85.5
-
5
5.5
70mM
+DEX
87.8
-
4
70mM
-
-DEX
89.5
-
2.5
2.5
3.8
1.8
106M
+DEX
5
64
0.3
0.3
0
-
30.3
106M
-DEX
5.3
60.3
0
0.3
0
-
34
10’M
+DEX
16
70.7
0.7
1.7
0
-
11
10’M
-DEX
13
68.3
0.3
0.3
0
-
18
10’M
+DEX
60.3
31.3
2.7
0.3
0
-
5.3
10’M
-DEX
57
32.3
2.7
1.3
0.3
-
6.3
10’M
+DEX
87.3
6.3
2.3
1.3
0
-
2.7
10’M
-DEX
85.3
7.7
2
1.3
0
-
+DEX
93.5
-
5
1.5
0
-
-DEX
91.5
-
varying
presence
the
concentrations
is reported
in
triplicate.
or absence
moorphological
differentiation
of inducing
as similar
to control
Duplicate
of DEX
agents,
were
(DMF.
observed
in
and
10
potentiates
I).
by concentrations
of RA that suppressed
(data
not shown).
In contrast,
RA was
31.3
performed
the
assessed
altered
binding
-DEX
neutrophils
differentials
or
6, Table
I ). There
reduction
was not due to a toxic
assessed
by Trypan
+DEX
0
segmented
DMF
cells
by
but was
differentiation
is a potent
as well
of HL-60
cells
the expression
reduce
NBT (not shown).
Cells induced
for 3 days
Differ
NBT
induction
manner,
120mM
Dexamethasone
with
although
RA
cell morphology
120mM
0
incubated
5, Table
on
HL-60
DMF
5 and
as assessed
during
related
cantly
FMLP
receptor
Normal
DMSO
by RA
(Figs.
potentiate
NBT,
RA did not induce
(Fig.
5). Furthermore,
Promyelocytes
DMF
not
reduce
receptors
cell
Blasts
as well
did
induced
effect of RA
cell viability,
prostaglandin
Morphological
DMSO
DEX
Interestingly,
differentiated
receptor
DEX on
on FMLP
of induction
of
with
the inducer
by FMLP
binding
(Fig.
yet significant,
effect
we used indomethacin
to study the role of
in the effect
of DEX
on HL-60
cells.
l05_108
M, alone
or in combination
with 1 20 mM DMF,
activity
(not shown).
with
HL-60
morphologi(not
shown).
which
alone
had no effect on FMLP
blocked
the effect of 5, 50, or 200 nM
FMLP
receptor
Since
steroids
observed
the
for
no effect
activity
in hepatoma
cells,28 were used to further
the mechanism
of the DEX
effect
in HL-60
Neither
1 7-a-methyltestosterone
nor progesterone
l05M,
activity,
the
at concentra-
nM,
and progesterone,
agents
DEX
on Tyr-aminotransferase
of
The enhancement
DEX
was greatest
as shown.
HL-60
DM50)
when
for
cells (normal)
or triplicate
the
2.5
6.5
of HL-60
inducer
cells
induced
6 days
(RA)
experiments
DMF
The
atypical,
‘
DMSO
0
or DM50
to assay.
prior
or markedly
was
by DMF
morphology
as described
gave
but
but
similar
not
RA.
not
3.7
-
-
those
of the
in the text.
results.
-
-
induced
by RA.
Cells
were
blasts
or promyelocytes
Values
are means
of 1 00 cell
significant
differences
Statistically
and
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
SKUBITZ.
590
I
I
U
I
ZHEN.
AND
AUGUST
I
I
In
0
‘C
E
0.
0
V
C
0
0.
-J
+DEX
U-
-DCX
+DE X
EX
0
30
[DM
Fig. 5.
DMF
and DM50
exert
60
90
0
120
effect
with
DEX
in the
120
160
induction
of FMLP
IO”
l08
lO6
[R A](M)
SOI(mM)
[DM
F](mM)
a synergistic
80
40
binding
to HL-60
cells.
while
RA does
not.
Specific
binding of H-FMLP
to HL-60 cells in the presence
of 50 nM 3H-FMLP
is shown for cells grown
6 days in the presence
(#{149})
or absence
(#{244})
of
500 nM DEX and varying
concentrations
of DMF. DMSO
or RA. as shown.
Each value. expressed
as cpm bound
per 1 0’ cells. is the mean of
triplicate
samples
± SEM.
A duplicate
experiment
gave similar results.
The increases
in FMLP binding seen with DEX alone as well as that
seen with
DEX plus DMF.
DMSO.
or RA were
all statistically
significant
(p < 0.01 -0.001
). The
higher
concentrations
of RA tested
decreased
the slight increase
in FMLP
binding
induced
by DEX (p - 0.003=0.007).
The highest
concentration
of ethanol
present
in
experiments
using
RA, 0.1 % ethanol.
had no effect
when added alone on differentiation
as assessed
by the three
assay systems
used.
the
presence
or
absence
of
500
nM
DEX
became
adherent
to the culture
flask as described8’9
but did not
exhibit
significant
FMLP
binding
or NBT
reduction
(not shown).
(far
in excess
of the
binding
ologic
was sometimes
concentrations
Corticosteroids
exert
a variety
of effects
in diverse
tissues.
In man,
the administration
of corticosteroids
produces
neutrophilia29’3#{176} and
has been
reported
to
activity
either
of surface
of the polar
inhibit
myeloid
colony
formation
in bone
marrow
cultures.31’32
The differentiation
of some murine
leukemia cells
to more
mature
cells
may
be induced
by
corticosteroids,’2’13
while
in other
cases
the hormone
effect.
required
may
block
agents.7
be mediated
This
remains
DISCUSSION
the
differentiation
A widely
studied
HL-60
promyelocytic
mature
elements
costeroids
have
reported
leukemia
of the granulocytic
been found
to have
Nevertheless,
to contain
high
I
in response
system
is the
to inducing
induction
of
cells
to
more
series;
here, cortino effect on differ-
HL-60
cells
have
been
affinity
corticosteroid
recep-
tors (- 1 5,600
receptors/cell
with
nM),27
and physiologic
concentrations
been found
to decrease
the expression
on the surface
of HL-60
cells.3
We
DEX
alone,
even at a concentration
K
for DEX
-4
of DEX
have
of Fc receptors
also found
that
of 500,000
nM
level
that
saturates
the
steroid
receptors
of the HL-60
cells),
had no effect
on cell
differentiation
as determined
morphologically
or by
NBT
reduction,
although
a slight
increase
in FMLP
We
tration
receptors
have
observed.
Nevertheless,
physiof DEX
markedly
increased
the
FMLP
inducers,
examined
the
receptors
DMF
possible
when
added
or DMSO.
mechanisms
with
of this
A correlation
between
the DEX
concentration
for half-maximal
synergism
and the concenrequired
for half
saturation
in HL-60
cells suggested
that
of the steroid
this effect may
through
the steroid
receptor
mechanism.
a strong
possibility;
however,
further
support
for this mechanism
with 1 7-a-methyltestosterone
was
not provided
or progesterone,
by studies
steroid
blocking
agents
in other
cell types.
Steroids
have also
been shown
to inhibit
prostaglandin
production,2’33
and
it is possible
that the observed
synergism
results
from
an alteration
of prostaglandin
Bonser
et al. recently
reported
arachadonate
release
during
maturation.34
In our studies,
been
shown
to
inhibit
metabolism.
Moreover,
that DMSO
stimulates
induction
of HL-60
cell
indomethacin,
which
has
prostaglandin
production
in
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
EFFECT OF DEXAMETHASONE
ON HL-60
Fig.
6.
Dexamethasone
had
relatively
little
effect
on
the induction
of HL-60
cell differentiation
as assessed
by the
ability
to reduce
NBT.
DMF.
DMSO.
and RA all exert
a slight
synergistic
effect
with
DEX in
inducing
the ability
of HL-60
cells to reduce
NBT. The percent
of HL-60
cells
reducing
NBT is shown
for cells grown
6
days
in the
presence
(#{149})
or
absence
(0) of 500 nM DEX and
varying
concentrations
of DMF.
DMSO.
or RA. as shown.
Each
value
is the mean
±
SD of 9
experiments.
() Denotes
a statistically
significant
(p < 0.05)
increase
in NBT reduction
by
DEX.
HL-60
when
591
a2
I-.
z
a,
C
0
V
0
a:
‘I,
0
0
[DMF](mM)
cells,34 had no effect on FMLP
receptor
added
alone or with I 20 mM DMF
(not
Another
possible
mechanism
effect is corticosteroid-induced
face receptors
for trophic
not
CELLS
tested
this
hypothesis
of
been
endocrine
known
to
an effect
and
of steroid
studied
such
[RA](M)
documented
mechanism
DEX
system,
observed
has
modulation
of cell surhormones.
Although
we have
in this
the
activity
shown).
[DMSO](mM)
systems
have
‘C
or
induction
of the
ability
induce
presence
on
of
more
seen
more
activity
of
or absence
HL-60
NBT
differentiation
surface
of DEX,
than
either
is measured
NBT,
RA did
by
not
FMLP
receptors
in the
and actually
suppressed
the induction
of receptors
caused
by DMF
in the
presence
or absence
of DEX.
Similarly,
TPA
induced
prominent
changes
in morphology
but had no effect on
FMLP
receptor
activity
(not shown).
The differential
-I
U.
I
In
effect
of these
agents
in
activity,
a unique
myeloid
inducer
the
action.
tiation
markers
suggests
[RA] (M)
Fig. 7.
Retinoic
acid blocks induction
by DMF of FMLP receptors on HL-60
cells.
Specific
3H-FMLP
binding to HL-60 cells in the
presence
of 50 nMH-FMLP
is shown for cells grown 6 days in 120
mM DMF in the presence
(#{149})
or absence
(0) of 500 nM DEX and
varying
concentrations
of RA as shown.
Each point is the mean
of
triplicate
samples
±
SEM. A duplicate
experiment
gave similar
results.
are
effects
was
DMF
or DMSO
when differentiation
morphology
or the ability
to reduce
0.
inducer
to reduce
potent
V
C
a
well
inducer
than the increase
Although
RA is a far
0
C
as
other
corticosteroids
synergistic
E
0.
suggested
in several
target
tissues.3539
DEX
had a slight
synergistic
effect on induction
of
more
mature
HL-60
cell morphology
and a marked
effect
on FMLP
receptor
activity
when
DMF
or
DMSO
was the inducing
agent
but not when
the
inducer
was RA. In contrast,
potentiation
by DEX of
the
0
been
where
permissive
striking
with RA as the
with
DMF
or DMSO.
In
also
synergism
inducing
feature
FMLP
receptor
of HL-60
cells,
presence
of different
mechanisms
of
Such differential
induction
of differen-
systems.”#{176}2
The
phenomenon
has
recently
of
a
been
permissive
effect of physiologic
concentrations
on
HL-60
differentiation
provides
tem
for studying
the mechanism
observed
or
in other
synergistic
of glucocorticords
a new in vitro sysof corticosteroid
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
SKUBITZ.
592
action
as well
as the
role
of corticosteroids
poiesis.
The data suggest
mechanism
than the polar
in
stimulating
differentiation
that the activity
of FMLP
same
control
system
in myelo-
erties,
that RA acts by a different
inducers
DMF
and DMSO
of HL-60
and therefore
receptors
is not under
the
as the
other
differentiation
NBT
reduction
and
ZHEN.
morphology.
AND
The
tion by DEX of expression
of differentiation
such as the FMLP
receptor,
and the variable
by different
inducers,
may also be useful
tion and characterization
of these antigens.
AUGUST
potentiaantigens,
induction
in purifica-
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