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Isoquinolinesulfonamide Protein Kinase Inhibitors H7 and H8 Enhance the Effects
of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) on Neutrophil
Function and Inhibit GM-CSF Receptor Internalization
By Asim Khwaja, Pamela J. Roberts, H. Mark Jones, Kwee Yong, Mervyn S. Jaswon, and David C. Linch
Human granulocyte-macrophage colony-stimulating factor
(GM-CSF) increases neutrophil surface expression of the
cellular adhesion molecule CD11 b and primes the respiratory burst stimulated by the bacterial peptide f-met-leuphe (FMLP). W e have examined the effects of the isoquinolinesulfonamide protein kinase inhibitors H7 and H8 on
these functions of GM-CSF using whole blood assays.
Concentrations of H7 and H8 that inhibited the 12-0tetradecanoyl-phorbol-I 3-acetate (TPA) stimulated upregulation of CD11b expression and activation of the respiratory burst, both augmented the effects of GM-CSF. H7 and
H8 enhanced the GM-CSF-stimulated increase in CD11b
expression to 21 5% k 10% (P< .05) and 233% k 45%
( P < .05), respectively, of the value obtained with GM-CSF
alone. The GM-CSF priming of the FMLP-stimulated oxidative burst was increased to 190% k 44% ( P < .01) by
preincubation with H7 and to 172% + 25% (P< .01) with
H8. Preincubation with H8 did not affect overall binding of
126
I-GM-CSF to neutrophils, but inhibited GM-CSF receptor
internalization after ligand binding ( P < .05). These data
indicate that the effects of GM-CSF are not mediated by
protein kinase C and that a phosphorylation event downmodulates the neutrophil response to GM-CSF. It suggests
that internalization of the receptor-ligand complex is not a
rate-limiting step in signal transduction, and that regulation of the rate of internalization may be an important level
of control of the activity of GM-CSF.
0 1990 by The American Society of Hematology.
G
with FMLP is greater in whole blood than in isolated
neutrophils, probably due to partial priming caused by the
purification procedures leading to an increased response in
the non-GM-CSF treated cells.’ We show here that when
added to whole blood, GM-CSF causes marked upregulation
of CAM expression, suggesting that this effect is direct and
not merely the priming of a degranulation response induced
by neutrophil purification procedures.
The mechanism by which GM-CSF primes phagocytes or
exerts any direct effect is unclear. There is increasing
evidence that the effects of GM-CSF are mediated by a
pertussis toxin sensitive G
but subsequent steps in
the activation pathway are not well-defined. Sullivan et all2
have reported the rapid release of arachidonic acid from the
membrane of purified neutrophils in response to GM-CSF.
Recently, Coffey et all3 have described stimulation of guanylate cyclase activity in neutrophils by GM-CSF. Murine
GM-CSF has been shown to cause serine phosphorylation of
68-Kd membrane and cytosolic proteins and the tyrosine
phosphorylation of a 90-Kd protein in the murine growth
factor-dependent hematopoietic cell line B6SUtAl. I 4 Phosphorylation of the membrane p68 was also produced by the
phorbol ester TPA, a direct activator of protein kinase C
(PKC). However, no evidence of PKC translocation in
response to GM-CSF stimulation in human neutrophils has
been obtained.” Gomez-Cambronero et a1” have recently
described an increase in several tyrosine phosphorylated
proteins in neutrophils stimulated by GM-CSF, but it is
unclear whether these changes are nonspecific or related to
mechanisms of signal transduction.
In this study we show that preincubation of whole blood
with the protein kinase inhibitors l-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride (H7) and N - [2-(methylamino) ethyl]-5-isoquinoline-sulfonamide dihydrochloride
(H8) inhibits the effects of TPA on both stimulation of the
neutrophil respiratory burst and CAM upregulation, but
significantly augments the effects of GM-CSF. Experiments
with ’”I-GM-CSF indicate that these kinase inhibitors do
not modulate GM-CSF binding to its specific receptors on
the cell surface, but that they do inhibit internalization of the
receptor after ligand binding.
RANULOCYTE-MACROPHAGE colony-stimulating factor (GM-CSF) enhances many of the functional
responses of mature phagocytes and augments phagocyte
superoxide generation to a variety of
Because
GM-CSF does not directly stimulate the respiratory burst,
this property is referred to as priming. GM-CSF also has
other actions on mature neutrophils, some of which may be
direct effects. There is a rapid shape change‘ and increased
expression of several surface receptors, including the cellular
adhesion molecules (CAMS) recognized by CD1 l b and
CDl8 antib~dies.~.~
Studies examining the effects of GM-CSF have largely
been performed using partially purified or isolated neutrophils, and it is well-recognized that isolation procedures may
themselves modulate receptor expression and cellular
Therefore, it is difficult to determine whether an
effect of GM-CSF is direct or an augmentation of the
changes brought about by purification procedures. Therefore, we have used whole blood assays that involve minimal in
vitro manipulation to examine the effects of GM-CSF on
neutrophil function. We have previously shown that in
unmanipulated neutrophils GM-CSF primes both f-met-leuphe (FMLP) and 12-0-tetradecanoyl-phorbol13-acetate
(TPA) induced respiratory burst activity.’ The magnitude of
the difference between primed and unprimed cells stimulated
From the Department of Haematology. University College and
Middlesex School of Medicine, London, UK.
Submitted November 27, 1989; accepted April 27, 1990.
Supported in pari by the Kay Kendall Leukaemia Fund. A.K. was
supported by Hoechst. UK; K.Y. by the Medical Research Council:
and D.C.L. by the Wellcome Trust.
Address reprint requests to D.C. Linch. Department of Haematology. University College and Middlesex School of Medicine. 98
Chenies Mews, London WCl E 6HX, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7605-0002$3.00/0
996
Blood, Vol 76,No 5 (September 1). 1990:pp 996-1003
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KINASE INHIBITORS ENHANCE GM-CSF EFFECTS
997
MATERIALS AND METHODS
experiments examining the effect of protein kinase inhibitors included a 10-minute preincubation at 37OC at this stage. Cells were
incubated with '"I-GM-CSF at 37"C, and assessment of nonspecific
binding was made by incubation with greater than 100-fold excess
cold GM-CSF. At specified time points, total cell-bound activity was
measured after centrifugation through 20% glycerol at 7,OOOg for 60
seconds and freezing the pellet in the liquid phase of liquid nitrogen.
Measurement of internalization of receptor bound '2SI-GM-CSFwas
performed by subjecting an equivalent number of cells to a low pH
wash in 3% acetic acid to dissociate membrane-bound '*'I-GM-CSF
and counting the cell pellet-associated activity as above. All points
were assessed in duplicate.
Materials. Purified recombinant human GM-CSF was supplied
by Hoechst/Behringwerke (Hounslow, UK/Marburg, W. Germany) and was dissolved in RPMI (GIBCO, Paisley, Scotland),
stored at -20°C in aliquots of 1 pg/mL, and thawed on the day of
use. TPA (Sigma, Poole, UK) was dissolved in DMSO to a stock
concentration of 1 mg/mL and kept at -20°C until the day of use.
FMLP (Sigma) was dissolved in DMSO to a stock concentration of 5
mg/mL and kept at -20°C until the day of use. Escherichia coli
lipopolysaccharide preparation of endotoxin (Sigma) was dissolved
in RPMI and kept at - 20°C until the day of use. The protein kinase
inhibitor H7 was obtained from Sigma, and H8 and HA1004 from
Seikagaku ( St Petersburg, FL), reconstituted as per the manufacturers' instructions to stock 10 mmol/L solutions, and stored at 4°C.
2'7'dichlorofluorescein diacetate (DCF-DA) was obtained from
Molecular Probes (Eugene, OR), dissolved in DMSO to 100
mmol/L concentration, and kept at -20°C. A C D l l b monoclonal
antibody (antibody 44)16 was kindly donated by Dr Nancy Hogg
(Imperial Cancer Research Fund, London, UK). Radiolabeled
"'I-GM-CSF, with a specific activity of 60 to 120 pCi/pg protein,
was obtained from Amersham International (Bucks, UK).
Neutrophil CDZZb expression. Whole blood samples were obtained from healthy adult volunteers. Fresh heparinized blood was
taken into polystyrene tubes and incubated with TPA (100 ng/mL),
GM-CSF (1 ng/mL), or diluent control for 30 minutes at 37°C.
Experiments using protein kinase inhibitors included a IO-minute
preincubation at 37°C with 1 mmol/L H7, 100 pmol/L H8, or 1
mmol/L HA1004. After stimulation with the appropriate agonist,
samples were immediately placed on ice and incubated for 30
minutes with saturating doses of CDI l b antibody. Initial experiments used UCHTl as an irrelevant immunoglobulin G1 (IgG1)
antibody control; as no significant difference was seen between
samples treated in this way and those incubated with diluent
(RPMI), subsequent experiments were conducted with diluent
control only. After three washes the cells were further incubated
with fluorescein-conjugatedrabbit anti-mouse antibody (Dako, Bucks,
UK) for 30 minutes at 4°C. Samples were then processed using the
Coulter Immunoprep Workstation (Coulter, Hialeah, FL), which
lyses red blood cells (RBCs) and fixes white blood cells. Analysis was
performed by flow cytometry using a Coulter Epics-C machine, the
neutrophils being selectively gated by virtue of their light-scattering
properties. Assessment of CDI l b expression was based on mean cell
fluorescence using a linear scale.
Neutrophil H,O, production. This was measured using a modification of the method of Bass et all' as previously described.' In
brief, freshly drawn whole blood was incubated with 100 pmol/L
DCF-DA for 15 minutes at 37°C. Experiments examining the effect
of protein kinase inhibitors on the respiratory burst included a
lominute incubation at 37°C with the respective inhibitor or diluent
control after this step. This was followed by incubation with 1
ng/mL GM-CSF for 45 minutes at 37"C, followed by the final
agonist (TPA/FMLP) for 10 minutes. The reaction was stopped by
placing the samples on ice, and they were then prepared for analysis
by flow cytometry as previously described. An estimate of total H,O,
production was made for a given sample by multiplying the percent
of responding cells by their mean cell fluorescence.
GM-CSF receptor assay. Purified neutrophils were obtained by
first preparing leukocyte-rich plasma by dextran sedimentation of
whole blood followed by layering onto a double-density gradient
composed of Histopaque 11 19 (Sigma) and Lymphoprep 1077
(Nycomed, Oslo, Norway), and centrifuging at 700gfor 30 minutes.'*
Neutrophils were harvested from the Lymphoprep/Histopaque
interface, washed twice, and resuspended in RPMI with 2% fetal calf
serum (FCS). Neutrophils, 5 x IO6, were used per point and
RESULTS
Effect of CM-CSF and TPA on neutrophil CDI 1b expression. Both TPA and GM-CSF are known to increase t h e
surface expression of neutrophil CAMs.19 In initial experiments, a direct comparison of the responses to TPA (100
n g / m L ) and GM-CSF (1 ng/mL) was made between
purified leukocytes (depleted of RBCs by dextran sedimentation) and whole blood from three normal individuals. Results
from these experiments (expressed as percent of control
response) give mean (kSEM) values for the response to TPA
of 236 2 4 3 and 146 k 25, a n d for the response to GM-CSF
of 151 10 a n d 138 40 for whole blood and separated
cells, respectively. This indicates that t h e effects of GM-CSF
and TPA on CD1 l b expression are at least as great in whole
blood as in cells t h a t have been partially purified.
In 12 experiments, GM-CSF a t a concentration of 1
n g / m L (70 pmol/L) was incubated with whole blood for 30
minutes a t 37OC. Neutrophil CD11 b expression measured by
mean cell fluorescence increased to a mean of 2 13% 28% of
control. T h e d a t a in Fig 1 show t h a t GM-CSF concentrations
as low as 250 p g / m L (17 pmol/L) caused an increase in
C D l l b expression, with a maximal response at 1 ng/mL.
T h e effect of T P A (100 ng/mL) on CDI Ib expression was
also examined. Results from 16 experiments show a n increase in C D 1 1b expression t o a mean of 274% k 43% of
control. A time course experiment showed t h a t the response
t o stimulation by GM-CSF in whole blood was rapid, with a
measurable increase in CDI 1b expression by 5 minutes, and
near maximal response by 30 minutes (Fig 2). There was
only a small increase in C D l l b expression with time in t h e
control group incubated with diluent alone, in contrast to a
more marked rise reported in experiments using separated
cek6
The question of endotoxin contamination leading directly
t o upregulation of CDI 1b expression or enhancing the effect
of GM-CSF was also addressed. Varying concentrations of
endotoxin were added to whole blood with and without
GM-CSF, and incubated for 60 minutes a t 37OC: no increase
in CDI 1 b expression (compared with control samples without endotoxin) was seen in either group at concentrations of
endotoxin u p t o a n d including 100 ng/mL. T h e endotoxin
contamination of the preparation of GM-CSF used as
measured by the limulus amebocyte assay was 0.3 pg/ng.
Efect of CM-CSF on the neutrophil respiratory burst.
W e have recently shown, using t h e whole blood fluorimetric
technique, t h a t stimulation by
mol/L FMLP alone
causes only a modest increase in neutrophil H 2 0 2production.'
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KHWAJA ET AL
loo0
0
1
T
Fig 1. Dose-response curve of GM-CSF-stimulated neutrophil C D l l b expression (mean cell fluorescence; mean of two experiments) and GM-CSF
priming of the FMLP-stimulated neutrophil respiratory burst (product of percent positive cells and
their mean cell fluorescence; mean f SEM of three
separate experiments).
I
10
100
concentration
1000
GM-CSF
10000
(pg/ml)
Results from 26 separate experiments show a mean response
to FMLP stimulation of 559 2 108 versus 232 2 36 in
unstimulated cells (expressed as product of percent positive
cells and their mean cell fluorescence). This response can be
markedly enhanced to 3,550 2 662 by preincubation with 1
ng/mL GM-CSF for 45 minutes a t 37OC. The dose-response
curve for the GM-CSF priming of the FMLP-induced
respiratory burst is similar to that obtained for direct
stimulation of CD1 l b expression, with a maximal effect at 1
ng/mL (70 pmol/L) GM-CSF (Fig 1).
Effect of protein kinase inhibitors on neutrophil responses
to TPA. Prior incubation of whole blood with the kinase
inhibitors H7 and H8 (10 minutes at 3 7 T ) caused significant inhibition of both TPA (100 ng/mL) induced upregulation of neutrophil CD 11b expression (Fig 3A) and the TPA
(1 pg/mL) stimulated respiratory burst (Fig 3B). H7, 1
mmol/L, inhibited the TPA-stimulated increase in CD1 l b
expression by a mean of 65% 2 8.5% (n = 8, P < .01 by
paired t-test) and TPA-stimulated H,O, production by
94% 2 4.9% (n = 4, P < .05). H8, 100 pmol/L, inhibited
CD1 lbexpression by 38% * 16% (n = 5, P < .01) and H,O,
production by 72% 2 14% (n = 4, P < .05). Preincubation
with 1 mmol/L HA1004 had no significant effect on either
TPA-mediated C D l l b expression or respiratory burst
(n = 4).
In three experiments examining the effect of H7 and H8 on
the TPA-mediated increase in C D l l b expression, a lower
dose of TPA (10 ng/mL) was used. A more marked
inhibition of the upregulation of C D l l b was observed with
H7 inhibiting the TPA response by 76% 2 16% and H8 by
62% 2 14%.
Effect of protein kinase inhibitors on neutrophil responses
to GM-CSF. Prior incubation of whole blood with H7 and
H8 caused significant enhancement of both GM-CSFinduced neutrophil CD1 l b expression (Fig 4A) and the
GM-CSF-primed FMLP-stimulated respiratory burst (Fig
4B). Preincubation with H7 gave results of 215 * 10
(expressed as percent of response seen without H7) for the
2000
1500
1000
500
Fig 2. Time-course of GM-CSF-stimulated neutrophil C D l l b expression in whole blood. In these
experiments the C D l l b expression relative to
control a t 30 minutes was 396% f 175%
(mean f SEM of three separate experiments),
which compares with 213% f 28% in a larger
number of experiments (n = 12).
"
I
0
60
30
time
(minutes)
90
120
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999
KINASE INHIBITORS ENHANCE GMCSF EFFECTS
c
-
A
z
140 1
T
1204
Kinase inhibitor
-
H7
H8
HA
1004
TPA
+
+
+
+
C
0
The effect of H7 on GM-CSF-stimulated CDl 1b expression was also examined in partially purified neutrophils
prepared by dextran sedimentation of whole blood. Results
from these experiments were consistent with findings using
whole blood techniques: preincubation with 1 mmol/L H7
inhibited TPA-stimulated CDl 1b expression completely
(n = 2) and enhanced the GM-CSF-stimulated increase to
254% 2 8% of the response seen without H7 (n = 3).
Incubation of blood with FMLP increases neutrophil
C D l l b expression with a maximal effect at lo-' mol/L
(data not shown). The response is present at lo-* mol/L but
is suboptimal (approximately 80% of maximum). To assess
whether protein kinase inhibition with H7 was having a
nonspecific effect on neutrophil degranulation induced by
surface receptor bound agonists, we examined the effect of
preincubation with H7 on this response. In contrast to the
findings with GM-CSF, 1 mmol/L H7 did not significantly
1
h
d)
Kinase inhibitor
B
TPA
-
H7
+
+
3001
HA
H8 1004
+
+
Fig 3. (A) Effect of protein kinase inhibitors on TPA-stimulated
neutrophilC D l l b expression. Mean SEM of 8,6. and 4 separate
experiments for H7, H8, and HA1004, respectively. ( 6 )Effect of
protein kinase inhibitors on the TPA-stimulated neutrophil respiratory burst. Mean SEM of 4,4, and 3 separate experiments for
H7, H8, and HA1004, respectively.
*
A
-O
Y
Klnase Inhibitor
-
H7
H8
GM-CSF
+
+
+
I
m
C
*
GM-CSF-stimulated increase in CDl l b expression (n = 6,
P c .01) and 190 2 44 for the GM-CSF-primed FMLP
respiratory burst response (n = 8, P c .01). The enhancement of GM-CSF priming of the respiratory burst by H7 was
due to an increase in both the percent of responding cells
(from 56 2 11 to 72 2 7) and their mean cell fluorescence
(from 54 2 15 to 74 f 28). Typical flow cytometric histograms of the effects of H7 on GM-CSF-stimulated CDl 1b
expression and on the GM-CSF-primed FMLP respiratory
burst are shown in Fig 5. Similar results were seen with H8,
which enhanced the GM-CSF-mediated increase in CDl 1b
expression to 233 * 45 (n = 4, P < .05) and the respiratory
burst tp 172 2 25 (n = 8, P c .01). Neither H7 nor H8 alone
had any significant effect on C D l l b expression; neither
agent caused detectable H202production or had any significant effect on the unprimed FMLP-stimulated respiratory
burst. Control experiments (n = 5 ) with 1 mmol/L HA1004
showed no significant effect on GM-CSF-induced CDl 1b
expression or on the GM-CSF primed respiratory burst.
004
+
T
Y
B
HA
Kinase inhibitor
-
H7
H8
GM-CSF
+FMLP
+
+
+
HA
oo4
+
Fig 4. (A) Effect of protein kinase inhibitors on the GM-CSFstimulated increase in neutrophil C D l l b expression. Mean f SEM
of 6, 4. and 4 separate experiments for H7. H8, and HA1004,
respectively. ( 6 )Effect of protein kinase inhibitors on the GM-CSFprimed FMLP-stimulated neutrophil respiratory burst. Mean f
SEM of 9.8, and 5 separate experiments for H7, H8, and HA1004.
respectively.
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1000
KHWAJA ET AL
lB
lA
'"I-GM-CSF internalization (66% internalization at 30
minutes). Preincubation with 100 pmol/L HA1004 did not
reduce receptor internalization.
DISCUSSION
N
ID
ij =
E
R
FLUORESCENCE I " s W
Fig 5. Flow cytometric histograms from a representative
experiment showing the effect of H7 on GM-CSF-stimulated
neutrophil C D l l b expression examined in whole blood. (A),
Control: (B). H7 alone; (C), GM-CSF; (D), H7 followed by GM-CSF.
Flow cytometric histograms from a representative experiment
showing the effect of H7 on the GM-CSF-primed, FMLPstimulated neutrophil respiratory burst examined in whole blood.
(E), Control; (F), FMLP alone: (GI, GM-CSF
FMLP; (HI, H7
followed by GM-CSF
FMLP.
+
+
affect the upregulation of C D l l b induced by lo-* mol/L
FMLP (mean of 108% 2 13% of response caused by FMLP
alone, n = 3).
Effect of protein kinase inhibitors on GM-CSF receptor
kinetics. The enhancement of the effects of GM-CSF on
neutrophil functions by protein kinase inhibition may be
mediated by changes in receptor expression, ligand-receptor
interaction, receptor internalization, or at later stages in the
signal transduction pathway. Therefore, studies on neutrophil GM-CSF receptor expression and internalization were
performed using I2'I-GM-CSF.
Preincubation of neutrophils with 100 pmol/L H8 did not
affect overall binding of '251-GM-CSF (Fig 6). However,
internalization of '2SI-GM-CSFwas inhibited a t all time
points examined (Fig 7), and after 30 minutes of incubation
at 37OC cells pretreated with H8 internalized only 44% 9%
'251-GM-CSF of control cells (n = 3, P < .05). A single
experiment with 100 pmol/L H7 showed similar inhibition of
In this study we used whole blood assays to examine the
effects of GM-CSF on neutrophil function and describe the
modulation of these effects by protein kinase inhibitors. We
used these techniques because there is increasing evidence
from work done in our laboratory and others that neutrophil
isolation procedures may themselves alter phagocyte
responses.6-*The production of H,O, as detected by DCF-DA
is dependent on respiratory burst activity, although the
response is augmented by myeloperoxidase (MPO), and
there is potential competition for the H 2 0 2produced between
DCF-DA and cellular reducing agents such as catalase and
glutathione.'' In the whole blood system, FMLP causes
detectable H 2 0 2 production in only a small proportion of
neutrophils, but the total response is increased six- to
sevenfold by preincubation with GM-CSF.9 In experiments
where we studied purified neutrophils, nearly all respond to
FMLP (as assessed by the DCF-DA detection of H 2 0 2
production and by NBT reduction), and the total respiratory
burst activity measured by the superoxide dismutaseinhibitable reduction of ferricytochrome c is increased approximately threefold by preincubation with GM-CSF.2' Using
the latter assay, the extracellular superoxide measured is not
modulated to the same degree by intracellular MPO and
reducing systems. This suggests that GM-CSF augments the
respiratory burst itself, although the effects of GM-CSF on
MPO and glutathione levels have not been determined. The
apparently greater effect of GM-CSF in whole blood is
probably due to the partial priming of the neutrophils
induced by the purification procedures, with an increase in
the baseline values. The effects of GM-CSF on the upregulation of CD1 l b in whole blood are similarly at least as
great as when partially purified leukocytes are used, and this
supports the idea that the upregulation of CAMS is a direct
effect of GM-CSF. The fact that neutrophil CAM expression
does not increase during a 2-hour incubation of whole blood
at 37OC without GM-CSF indicates that the process of
venesection and incubation per se does not activate the
neutrophils. This contrasts with the finding in partially
purified neutrophils where in vitro incubation at 37OC causes
a progressive rise in CAM expression.6
To examine the possible mechanisms of GM-CSF signal
transduction in neutrophils, we have looked at the effects of
protein kinase inhibitors on GM-CSF stimulation of neutrophil functions in whole blood. The isoquinolinesulfonamides
are a group of compounds that inhibit a range of protein
kinases with varying degrees of specificity.22H7, H8, and
HA1004 are all potent inhibitors of CAMP-dependent protein kinase; in addition they inhibit PKC to varying degrees,
with H7 and H8 having high activity and HA1004 relatively
little. Our results provide indirect evidence of the ability of
H7 and H8 to inhibit PKC by significantly reducing the
effects of TPA, a direct activator of PKC," on both neutrophil CAM expression and respiratory burst. In keeping with
its reported activity, HA1004 had no significant effect on
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1001
KINASE INHIBITORS ENHANCE GM-CSF EFFECTS
4000
LL
u)
3000
-
2000
H8
1000
Fig 6. Specific binding of
I-GM-CSF to purified neutrophils (cpm/lOs cells) c H8.
Mean c SEM of three separate
experiments (the data points at
60 minutes are the mean of two
experiments).
125
0
0
15
time
neutrophil responses to TPA. We show that H7 and H8 do
not inhibit either the direct effect of GM-CSF on CAM
expression or GM-CSF priming of the FMLP-stimulated
respiratory burst, indicating that the effects of this cytokine
are not mediated by PKC and confirming the recent results of
Mege et al." Preincubation with HA1004 did not affect
neutrophil responses to GM-CSF. We have shown further
that preincubation with H7 and H8 causes significant enhancement of GM-CSF-stimulated neutrophil responses
and that this augmentation is not seen with HA1004. This
suggests that a protein kinase-mediated phosphorylation step
is important in the down-regulation of neutrophil responses
50
S
40
45
30
60
(minutes)
to GM-CSF. Other studies have reported protein phosphorylations in response to stimulation by this growth f a c t ~ r , ' ~ ~ ' ~
and it has been suggested that these may play a role in
stimulating further responses relevant to cell survival and
proliferation. The data presented here suggest that protein
phosphorylation in response to GM-CSF may not only be a
stimulatory signal but may play a part in the downregulation of the cellular response to this growth factor. The
lack of specificity of H7 and H8 do not allow conclusions to
be made as to what type of kinase activity is down-regulating
the GM-CSF response. It appears that GM-CSF does not
activate PKC, and the lack of effect of HA1004 suggests that
1
control
t
c
E
30
E E
20
10
0
0
15
30
time
(minutes)
45
60
Fig 7. Internalization of
neutrophil receptor bound '"IGM-CSF t H8. Mean + SEM of
three separate experiments
(the data points at 60 minutes
are the mean of two experiments).
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1002
KHWAJA ET AL
t h e effects of GM-CSF are not mediated by CAMPdependent protein kinases. Further studies will be required t o
address this issue.
T h e enhancement of t h e effects of GM-CSF on neutrophil
function by protein kinase inhibition was further investigated
by examining the actions of these inhibitors on GM-CSF
receptor expression and internalization after ligand binding.
The results show t h a t preincubation with H8 or H7 did not
alter overall binding of '"I-GM-CSF t o its receptors on t h e
cell surface, but t h a t they reduced markedly t h e rate and
degree of receptor internalization following '251-GM-CSF
binding. In conjunction with our observation t h a t these
protein kinase inhibitors enhance t h e functional effects of
GM-CSF, we can conclude t h a t internalization of receptorbound GM-CSF is not a rate-limiting step for t h e action of
this cytokine. Receptor-mediated endocytosis is a mechanism by which many different cell types internalize macromolecules, including several growth factors.24T h e biologic role
of receptor-mediated internalization is varied and systemspecific: it appears t o be a prerequisite for the action of t h e
cytokine interleukin-1 ( I L - l ) , which after internalization is
transported to the cell nucleus,25and variant cells unable t o
internalize IL- I are unresponsive to
In contrast, the binding of several growth factors, including epidermal growth factor (EGF)27 and M-CSF,28is followed by
internalization and intracellular degradation and acts as a
mechanism by which their effects a r e down-regulated.
It is increasingly evident t h a t phosphorylation and dephosphorylation of cell surface receptors is a n important mechanism in t h e regulation of signal t r a n s d ~ c t i o n . ~I 't has been
shown t h a t both ligand binding a n d PKC activation by
phorbol esters lead to phosphorylation of threonine 654of the
EGF receptor a n d promote i n t e r n a l i ~ a t i o n .In
~ ~ addition,
ligand binding t o the EGF receptor promotes internalization
by a non-PKC-dependent mechanism independent of threonine 654 phosphorylation. Down-regulation of the M-CSF
receptor is also brought about by phorbol esters, and in this
case it is due to the activation of a protease that cleaves the
receptor with loss of t h e extra-cellular domain.28 Phorbol
esters have been reported to down-regulate GM-CSF
receptors,29but the mechanism for this is unknown. Detailed
understanding of t h e means by which phosphorylation events
down-regulate t h e GM-CSF receptor will probably await the
cloning of this gene.
In summary, our d a t a show t h a t protein kinase inhibition
augments the effects of GM-CSF, suggesting t h a t a phosphorylation event after GM-CSF binding to its receptor is
involved in t h e down-regulation of the cellular response. T h e
finding t h a t protein kinase inhibitors block GM-CSF receptor internalization indicates that internalization is not a
rate-limiting step in the signal transduction pathway, and
raises t h e possibility t h a t regulation of t h e rate of GM-CSF
receptor internalization may be a n important mechanism by
which the effects of this cytokine are modulated.
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Isoquinolinesulfonamide protein kinase inhibitors H7 and H8 enhance
the effects of granulocyte-macrophage colony-stimulating factor
(GM-CSE) on neutrophil function and inhibit GM-CSF receptor
internalization
A Khwaja, PJ Roberts, HM Jones, K Yong, MS Jaswon and DC Linch
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