Department of Pulmonary Diseases, G03.550, University Medical Centre Utrecht, The Netherlands
Correspondence: Dr. Paul J. Coffer, Dept. Pulmonary Diseases, G03.550, University Medical Centre, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: P.Coffer{at}hli.azu.nl
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: signal transduction • protein kinase • cellular activation
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A consequence of activation of neutrophils is the ability to cause tissue damage during inflammatory reactions, and therefore, the activity of neutrophils must be tightly controlled. For this reason, the activation of neutrophils occurs in a multistep process. Resting neutrophils in the peripheral blood respond poorly to many activators including naturally occurring formyl peptides such as fMet-Leu-Phe (fMLP). However, when these cells are exposed to several preactivating or "priming" agents such as cytokines, effector functions like the respiratory burst, phagocytosis, and degranulation are greatly enhanced [4 , 5 ]. To understand the mechanism by which specific agents activate or prime granulocytes, it is important to define components of signaling pathways responsible for the activation of effector functions in granulocytes.
Recently, several intracellular signal-transduction cascades have been found to be activated in human neutrophils in response to cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as G-protein-coupled receptor (GPCR) agonists such as platelet-activating factor (PAF) and fMLP. One family of proteins that has been demonstrated to be activated by many priming agents is the mitogen-activated protein kinases or MAP kinases [6 7 8 9 10 ]. There are three distinct groups of MAP kinases: extracellular signal-related protein kinases (ERKs) activated by a large variety of agonist, stress-activated protein kinases (JNK/SAPKs), and the p38 MAPK family. Another signal-transduction pathway that appears to play a critical role in priming and activation of granulocyte-effector functions involves the lipid kinase phosphatidylinositol 3-kinase (PI3K). Using specific inhibitors of PI3K, wortmannin, and LY294002, we and others have recently demonstrated that this kinase plays a critical role in several neutrophil-effector functions including the respiratory burst and migration [7 , 11 12 13 ].
Despite the fact that in neutrophils cytokine and G-protein-coupled receptors can activate the signaling cascades mentioned above, little is known about the specific mechanism by which these receptors initiate these cascades. During the last few years, increasing evidence shows that tyrosine kinases are involved in transducing signals from cytokine and G-protein-coupled receptors to downstream signal cascades [14 ]. In granulocytes, it has been shown that tyrosine kinases are involved in cytokine and G-protein-coupled receptor-agonist signaling [15 16 17 18 ]. In neutrophils, several protein tyrosine kinases have been identified, including Janus kinases (JAKs) and the Src-kinase family. The JAKs, which are activated by cytokine receptors but not by G-protein-coupled receptors, phosphorylate the signal transducer and activator of transcription (STAT) transcription factors [19 20 21 ]. This pathway is important for linking many cytokine receptors to gene regulation but is not involved in activation of cytotoxic mechanisms. Of the family of Src kinases, several members such as Lyn, Hck, and Fgr are expressed in neutrophils [22 , 23 ]. These Src-kinase members have also been shown to be activated by several cytokine and G-protein-coupled receptors [18 , 24 , 25 ]. Data from cell lines and knock-out mice show that Src kinases are involved in many cellular processes including differentiation, adhesion/spreading, migration, apoptosis, gene transcription, and cell cycle [26 , 27 ].
The development of a pharmacological Src-family-selective tyrosine-kinase inhibitor pyrazolpyrimidine (PP1) has allowed the investigation of the role of this tyrosine kinase family [28 ]. In this study, we have used this inhibitor to evaluate the role of Src kinases in cytokine and chemoattractant signaling regulating neutrophil functions. Our results demonstrate that Src kinases are involved in protein kinase B (PKB) but not in MAPK activation in neutrophils. Furthermore, we show that Src kinases play a role in fMLP-induced superoxide production, migration, actin polymerization, and cytokine-mediated survival.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Isolation of human neutrophils
Blood was obtained from healthy volunteers. Mixed granulocytes
were isolated from 50–100 ml blood, which was anticoagulated with
0.32% sodium citrate as described previously [7
]. Blood
was diluted 1.4 times with phosphate-buffered saline (PBS) containing
0.32% sodium citrate and 10% human pasteurized plasma-protein
solution (40 g/L). Mononuclear cells were removed by centrifugation
over Ficoll-Pague for 20 min at 2000 rpm. The erythrocytes were lysed
in isotonic ice-cold NH4CL solution followed by
centrifugation at 4°C. Granulocytes were allowed to recover for 30
min at 37°C in Hepes-buffered RPMI 1640 medium technologies,
supplemented with L-glutamine and 0.5% HSA. All
preparations contained >97% neutrophils. Before stimulation,
neutrophils were resuspended in incubation buffer [20 mM Hepes, pH
7.4, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM
KH2PO4, 5 mM glucose, 1 mM CaCl2,
and 0.5% (v/v) HSA] for 15–30 min at 37°C.
Lyn, Hck, and Fgr kinase activity
Neutrophils were isolated as described above and incubated at
37°C for 30 min. After stimulation with fMLP, cells
(5x106) were lysed in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl,
50 mM NaF, 5 mM ethylenediaminetetraacetate (EDTA), 0.1% sodium
dodecyl sulfate (SDS), 0.5% sodium deoxycholaat, 1 mM
Na3VO4, 1% Nonidet P-40 (NP-40), 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride
(PMSF), and 1 mM benzamidine. Lysates were precleared for 30 min at
4°C with protein G-sepharose, and subsequently, Lyn, Hck, and Fgr
kinase was immunoprecipitated with 1 µg Lyn, Hck, or Fgr antibody for
1 h at 4°C on a rotating wheel. To immunoprecipitate Lyn
monoclonal antibody (mAb), protein G-sepharose was added for a further
1 h at 4°C, and protein A sepharose was used to
immunoprecipitate Hck and Fgr antibodies. After washing twice with
lysis buffer and twice with wash buffer (25 mM Tris, pH 7.5, 150 mM
NaCl, and 0.1 mM Na3VO4), precipitates were
incubated in 30 µl kinase buffer (20 mM Hepes, pH 7.5, 10 mM
MnCl2, 1 µM recombinant adenosine
5'-triphosphate (rATP), and 0.3 µCi [
32P]ATP) with
increasing concentrations of PP1 for 2 min at room temperature. The Hck
and Fgr kinase reactions contained 10 µg enolase. Reaction was
stopped by the addition of 5x Laemmli sample buffer and boiled for 5
min. Samples were separated by electrophoresis on 15%
SDS-polyacrylamide gels. Kinase activity was detected by
autoradiography.
MAP kinase activity in vitro
MAP kinase activity was measured as described previously
[7
]. In short, neutrophils were isolated as described
above and incubated at 37°C for 30 min. After pretreatment with PP1
or PD98059 and stimulation with fMLP or GM-CSF, cells
(5x106) were lysed in 50 mM Tris-Cl, pH 7.5, 100 mM NaCl,
50 mM NaF, 5 mM EDTA, 40 mM ß-glycerophosphate, 1 mM
Na3VO4, 1% Triton X-100, 10 µg/ml aprotinin,
10 µg/ml leupeptin, 1 mM PMSF, and 1 mM benzamidine. Lysates were
precleared for 30 min at 4°C with protein A-sepharose, and
subsequently, MAP kinase was immunoprecipitated with 1 µg ERK-1/ERK-2
or p38 polyclonal antisera for 1 h at 4°C on a rotating wheel.
Protein A-sepharose was then added for a further 1 h at 4°C.
After washing twice with lysis buffer, samples were washed twice with
kinase buffer (30 mM Tris-Cl, pH 8.0, 20 mM MgCl2, 2 mM
MnCl2, 10 µM rATP, 10 µg myelin basic
protein (MBP), and 0.3 µCi [32P]ATP) without ATP and
MBP. Precipitates were then incubated in 25 µl kinase buffer for 20
min at room temperature. Reaction was stopped by the addition of 5x
Laemmli sample buffer. Samples were separated by electrophoresis on
15% SDS-polyacrylamide gels. MBP phosphorylation was detected by
autoradiography.
Western blotting
Neutrophils (106 cells) were preincubated with
several concentrations of inhibitor for 20 min followed by stimulation
for the indicated time points, washed in ice-cold PBS, and immediately
lysed in Laemmli sample buffer. Total cell lysates were boiled for 5
min at 95°C and analyzed on 10% SDS-polyacrylamide gels. Proteins
were transferred to Immobilon-P. The blots for hybridization with
phospho-specific antibodies were blocked in hybridization buffer (10 mM
Tris, 150 mM NaCl, and 0.3% Tween-20) containing 3% bovine serum
albumin (BSA) for 1 h followed by incubation with phospho-specific
antibody (1/1000) in hybridization buffer with 1% BSA for 2 h at
room temperature. Second antibody was incubated in hybridization buffer
for 1 h. The Western blots for hybridization with all other
antibodies were blocked in hybridization buffer (10 mM Tris, 150 mM
NaCl, and 0.3% Tween-20) containing 5% nonfat milk for 1 h
followed by incubation with antibody (1/1000) in hybridization buffer
with 0.5% nonfat milk for 2 h at room temperature. Second
antibody was incubated in hybridization buffer containing 0.5% nonfat
milk for 1 h. Detection of all Western blots was performed by
enhanced chemiluminescence (Amersham, Little Chalfont, UK).
Filamentous actin (F-actin) measurement
Fluorescent F-actin staining was performed as described
previously [30
]. In short, neutrophils
(2.5x106 cells/ ml) were stimulated for the indicated time
points and subsequently fixed and permeabilized with ice-cold 3%
formaldehyde in PBS containing 100 µg/ml lysophosphatidylcholine for
10 min at room temperature. F-actin was stained with 30 U/ml
7-nitrobenz-2-oxa-1,3'-diazol-4-yl (NBD)-phalloidin for 30 min
at room temperature. The intracellular fluorescence was determined by
fluorescein-activated cell sorter (FACS) analysis (FACSvantage,
Becton Dickinson, San Jose, CA) by measuring a total cell count of 5000
cells per sample.
Measurement of neutrophil migration
Neutrophil migration was measured using a modification of the
method according to Boyden as described previously [7
],
using a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD).
Chemoattractants or incubation buffer (30 µl) were added to the lower
compartments. Two filters were placed between the lower and upper
compartments. The lower filter had a pore width of 0.45 µM
(Millipore, Bedford, MA), and the upper filter (cellulose nitrate) had
a pore width of 8 µM (thickness, 150 µM; Sartorius, Gottingen,
Germany). Before use, the filters were soaked in incubation buffer.
Neutrophils were placed in the upper compartment (25 µl
2x106 cells/ml). The chambers were subsequently incubated
for 1.5 h at 37°C. The upper filters were removed, fixed in
butanol/ethanol (20/80% v/v) for 10 min, and stained with Weigert
solution (composition: 1% v/v haematoxylin in ethanol mixed with a 70
mM acidic FeCl3 solution at 1:1 ratio). The filters were
dehydrated with ethanol, made transparent with xylene, and fixed upside
down. All migratory responses were quantified with an image analysis
system (Quantimet 570C, Leica Cambridge Ltd., Weitzlar, Germany) using
Quantimet 570 Control Software (QUIC version 2.02) together with
custom-made software. An automated microscope, Leitz DMRXE (Leica), was
used to step through the filters in the Z direction with 16 intervals
of 10 µM. Neutrophils were counted at each level, and the total
migration to each level was calculated. The results are expressed as
migration index, which is calculated by the cumulative migration of all
intervals (µm) divided by the total number of cells multiplied by the
amount of migrated cells. The mean of four randomly chosen points on
each filterspot was calculated.
Measurement of NADPH-oxidase activation
Superoxide was measured by cytochrome c reduction according to a
modified method described by Pick and Mizel [31
]. In
short, neutrophils (4x106 cells/ml) were preincubated for
5 min at 37°C in incubation buffer. Subsequently, neutrophils were
preincubated with inhibitors and/or cytokines for the indicated periods
of time. Hereafter, the cells (200 µl) were transferred to a
microtitre plate in a thermostat-controlled microtitre plate reader
(340 ATTC; SLT LabInstruments, Austria) and mixed with
cytochrome c (75 µM), and the incubation was continued for 5 min; the
plates were shaken every 3 s. The cells were then stimulated with
fMLP (1 µM), and cytochrome c reduction was measured every 12 s
as an increase in absorbance at 550 nm.
Oxygen consumption was measured as described previously [32 ]. In short, granulocytes were resuspended (3x106 cells/ml) in the incubation buffer and preincubated with GM-CSF (10-10M) for 30 min. After incubation, cells were brought in a stirred and thermostated airtight vessel, and inhibitor was added for 5 min. Subsequently, fMLP (1 µM) was added to activate the respiratory burst, and oxygen consumption was continually measured with an oxygen probe (Yellow Springs Instrument, Yellow Springs, OH) for several minutes.
Measurement of neutrophil apoptosis
Apoptosis of neutrophils was measured by analyzing Annexin
V-fluorescein isothiocyanate (FITC)-binding (Alexis; Kordia bv, The
Netherlands). In short, freshly isolated neutrophils
(0.5x106/ml) were resuspended in Hepes-buffered RPMI
medium supplemented with 8% Hyclone serum. After treatment with
inhibitors/cytokines, cells were incubated for the indicated times at
37°C. At the end of incubation, cells were stored at 4°C until the
last incubation time point had been reached. Subsequently, cells were
washed with ice-cold PBS and resuspended in binding buffer (10 mM
Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Annexin
V-FITC (1/40) was added to the cells and incubated for 10 min at room
temperature. After washing, cells were resuspended in binding buffer
containing 1 µg/ml propidium iodide. The fluorescence was determined
by FACS analysis (FACSvantage, Becton Dickinson) by measuring a total
cell count of 10,000 cells per sample.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (38K): [in a new window] |
Figure 1. Src-kinase inhibitor PP1 inhibits Lyn and Hck kinase activity in vitro
but not STAT5 phosphorylation in human neutrophils. Neutrophils were
stimulated with fMLP (10-6 M) for 2 min,
lysed, and immunoprecipitated with a Lyn or Hck antibody. Kinase assays
were performed in the presence of increasing concentrations of PP1 as
indicated. (A) Lyn kinase activity was determined by
autophosphorylation of Lyn for 2 min at room temperature.
Autophosphorylation of Lyn was detected by autoradiography. (B) Kinase
activity of Hck was measured for 10 min at room temperature using
enolase as substrate. Phosphorylation of enolase was detected by
autoradiography. (C) Neutrophils were pretreated with dimethyl
sulfoxide (DMSO) or increasing amounts of PP1 before stimulated GM-CSF
(10-10 M) and lysed, and samples were analyzed
by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western
blotting with antiphospho-STAT5. Data are representative of three
independent experiments.
|
Activation of MAPKs by GM-CSF or fMLP is not inhibited by PP1
Stimuli such as GM-CSF and fMLP have been described to transiently
activate MAPKs in neutrophils [6
7
8
9
10
, 37
].
Whereas Src kinases have been proposed to be important regulators of
MAPKs in several cell types [38
39
40
41
], very little is
known about the role of Src kinases in neutrophils. To determine
whether fMLP and GM-CSF activate MAPKs via Src kinase, we performed two
different assays to measure the activation state of MAPKs, an in vitro
kinase assay (Fig. 2A
and C) and Western blotting using phospho-specific
antibodies to ERK1/2 and p38 (Fig. 2B
and 2D
, respectively). Before
stimulation, neutrophils were preincubated with increasing
concentrations of PP1 for 20 min. As shown in Figure 2
, neither ERK1/2
nor p38 kinase activity was inhibited by the highest concentration of
50 µM PP1 in the in vitro kinase assay as well as the Western blots
using activation-specific phospho-antibodies against p38 and ERK1/2.
However a specific inhibitor of MEK (PD098059), the upstream activator
of ERK1/2, completely blocks ERK1/2 kinase activity. Thus, in
neutrophils, neither the GM-CSF nor fMLP receptor requires Src kinases
in the regulation of ERK1/2 and p38 MAPK activation.
 View larger version (49K): [in a new window] |
Figure 2. Activation of ERK1/2 and p38 MAPK by fMLP or GM-CSF does not require
Src kinases. Freshly isolated neutrophils were preincubated with DMSO,
PD-98059 (50 µM), or increasing concentrations of PP1 for 20 min and
subsequently stimulated with GM-CSF (10-10 M)
or fMLP (10-6 M) for 10 and 2 min,
respectively. (A and C) After stimulation, 5 x 106
cells were lysed, and 1/25th was used for Western blotting with
anti-ERK1/2 and anti-p38 MAPK antibodies to confirm equal amounts of
protein. The remainder of the sample was used for immunoprecipitation
with a mixture of ERK1 and ERK2 (1:1) antibodies or with p38 MAPK
antibody. Kinase activity of ERK1/2 and p38 MAPK was measured for 20
min at room temperature using MBP as substrate. Phosphorylation of MBP
was detected by autoradiography. (B and D) Neutrophils (106
cells) were treated as above and immediately lysed in Laemmli sample
buffer. Proteins were analyzed by SDS-PAGE, followed by Western
blotting with indicated antibodies. Data are representative of three
independent experiments.
|
 View larger version (53K): [in a new window] |
Figure 3. Src kinases are critical for fMLP and GM-CSF-induced PKB activation.
(A) Isolated neutrophils were incubated with GM-CSF
(10-10 M) or fMLP
(10-6 M) for the times indicated. After
stimulation, cells were lysed immediately in Laemmli sample buffer, and
samples were analyzed by SDS-PAGE followed by Western blotting with
antiphospho-PKB (ser 473) or with anti-PKB. (B) Neutrophils were
pretreated with DMSO, increasing concentrations of PP1, or 20 µM
LY294002 followed by stimulation with GM-CSF
(10-10 M) or fMLP
(10-6 M) for 10 or 2 min, respectively. All
samples were analyzed as described previously. Data are representative
of three independent experiments.
|
As shown in Figure 4 A , GM-CSF and fMLP individually do not activate the respiratory burst in neutrophils, whereas neutrophils treated with GM-CSF prior to fMLP activation show a large increase in superoxide production, which is markedly decreased by 50 µM PP1 and 20 µM LY294002. Neutrophils incubated with increasing concentrations of PP1 show that the maximum decrease of superoxide production is already reached at 5 µM PP1 (Fig. 4B) . The same decrease in fMLP-induced respiratory burst was observed in cells first primed with GM-CSF, followed by treatment with different concentrations of PP1 before finally being activated with fMLP (Fig. 4C) . This demonstrates that indeed Src kinases are involved in superoxide production, but it is not apparent whether Src kinases are critical for GM-CSF-mediated neutrophil priming. It is interesting that phorbol 12-myristate 13-acetate (PMA), which strongly induces the respiratory burst independently of priming, is not inhibited by 50 µM PP1 (Fig. 4D) . This demonstrates that under certain conditions, activation of the respiratory burst can occur independently of Src kinases.
 View larger version (38K): [in a new window] |
Figure 4. PP1 inhibits fMLP-induced respiratory burst but not PMA-stimulated
respiratory burst. (A and B) Neutrophils were preincubated with DMSO,
increasing amounts of PP1, or 20 µM LY294002 for 20 min before
incubation of GM-CSF (10-10 M) for 30 min
followed by stimulation with fMLP (10-6). (C)
Neutrophils were incubated with GM-CSF (10-10
M) for 30 min before treatment with DMSO or increasing amounts of PP1
for 20 min followed by stimulation with fMLP
(10-6). (D) Neutrophils were preincubated with
DMSO or with 50 µM PP1 for 20 min followed by stimulation with PMA
(0.1 µg/ml). Superoxide production was monitored continuously by
measurement of cytochrome c reduction. Results are expressed as the
optical density (OD) at a wavelength of 550 nm and are representative
of four independent experiments.
|
 View larger version (14K): [in a new window] |
Figure 5. Src kinases are not involved in fMLP-induced neutrophil migration.
Neutrophil migration was monitored in microchemotaxis Boyden chambers
in response to buffer or fMLP (10-8 M) as
described in Materials and Methods. Stimulus was placed in the lower
compartment, and cells were left to migrate for 1.5 h at 37°C.
Neutrophils were preincubated with DMSO (A), increasing amounts of PP1
(B), or 20 µM LY294002 (B). Results are expressed as migration
index ± SE (n=3).
|
PP1 and LY294002 prevent GM-CSF-mediated neutrophil survival
The lifespan of circulating neutrophils is relatively short
compared with other leukocytes and cannot be dramatically extended
[49
]. However, several stimuli can delay apoptosis
including GM-CSF and LPS [50
], whereas other stimuli,
such as TNF-
, accelerate neutrophil apoptosis [51
,
52
]. In several cell lines, it has been shown that the
PI3K-PKB pathway plays an important role in cell survival
[53
]. Because we have demonstrated in this study that
Src kinases regulate PI3K-PKB activity, it is reasonable to conclude
that Src kinases might also play a role in inhibiting neutrophil
apoptosis. To investigate the role of Src kinase and PI3K in apoptosis
of neutrophils, we incubated freshly isolated neutrophils for 20 min
with various concentrations of PP1 or 20 µM LY294002 before addition
of GM-CSF. To measure apoptosis, we used Annexin-V, which binds to
phosphatidylserine (PS), present on the outer leaflet of the plasma
membrane only in those cells that have initiated an apoptotic program.
In Figure 6A
dot plots are shown in which the lower left corner represents living
cells, and the lower right corner represents early apoptotic cells. Few
apoptotic cells were observed in freshly isolated neutrophils, whereas
after 12 h, approximately 50% of the cells were apoptotic.
Stimulating cells with GM-CSF results in a decrease of the number of
apoptotic cells from 50% to 20%, and PP1 completely blocks this
GM-CSF rescue (Fig. 6A)
. In Figure 6B
, we analyzed different time
points of apoptosis using several concentrations of PP1. Figure 6B
shows that neutrophils initiate the apoptotic program after 4–8 h of
isolation and that this process is not blocked but delayed by GM-CSF
for approximately 4 h. This delay is completely blocked with PP1
at a concentration of 20 µM, as well as with 20 µM LY294002.
Neutrophils treated with PP1 alone give similar kinetics for apoptosis
as neutrophils without treatment (unpublished results).
 View larger version (35K): [in a new window] |
Figure 6. Src kinases and PI3K are involved in GM-CSF-mediated survival.
Neutrophils were preincubated for 20 min with DMSO or increasing
concentrations of PP1 before adding GM-CSF. Apoptosis of the cells was
measured using Annexin-V in combination with propidium iodide followed
by FACS analysis. Cells positive for Annexin-V represent cells in
apoptosis. FACS results are presented in dot plots (A), or cells
positive for Annexin-V were plotted in a graph (B). Results of the
graph are expressed as percentage apoptosis (n=3).
|
 View larger version (17K): [in a new window] |
Figure 7. Kinetics of PP1 inhibition of F-actin polymerization and oxygen
consumption in neutrophils. (A) Human neutrophils were pretreated with
DMSO or PP1 (20 µM) for 20 min before stimulation with fMLP (1 µM).
At the indicated time points, cells were fixed, and F-actin staining
was performed followed by FACS analysis. The results are expressed as
the relative F-actin content ± SE (n=3).
(B) Respiratory burst was measured as oxygen consumption by neutrophils
as described in Materials and Methods. Cells were stimulated with
GM-CSF (10-10 M) for 30 min before the
addition of DMSO, 50 µM PP1, or 20 µM LY294002 for 5 min.
Subsequently, fMLP (1 µM) was added to activate the respiratory
burst, and oxygen consumption was continually measured for several min.
^, Addition of fMLP.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Recent studies show that PP1 can inhibit several members of the Src-kinase family including Fyn, Lck, Lyn, slightly Fgr, but not Hck in vitro [28 , 33 ]. In our study, we found that Lyn kinase was slightly more sensitive to PP1 than Hck kinase (Fig. 1A and 1B) . The finding that Hck is also inhibited is in contrast with a previous study [33 ]. However, in this study, only 2 µM PP1 was used, whereas we found inhibition of Hck-kinase activity between 2.5 and 10 µM PP1. Previous studies have shown that neutrophils stimulated with GM-CSF have enhanced phosphorylation of STAT1, STAT3, and STAT5 [65 , 66 ]. Whereas Src kinases have recently been implicated in cytokine-induced STAT activation in various cell types [34 35 36 , 67 ], little is known in neutrophils. In this study, we have demonstrated that Src kinases are apparently not involved in GM-CSF-induced STAT5 phosphorylation in human neutrophils (Fig. 1C) . This also demonstrates that PP1 is not a general tyrosine-kinase inhibitor because cytokine-induced tyrosine phosphorylation of STAT is not perturbed. Importantly, responses such as fMLP-induced migration and also PMA-activated respiratory burst are not sensitive for the inhibitor PP1, indicating that PP1 is not simply toxic to these cells.
The cytokine GM-CSF and G-protein-coupled receptor agonist fMLP are able to transiently activate ERK, p38, and PI3K-PKB signaling cascades in neutrophils [6 7 8 9 , 17 , 18 , 37 , 68 ]. Currently, little is known about the specific upstream signals by which receptors activate these cascades. In Figures 2 and 3B , we demonstrate that PP1 inhibits PKB phosphorylation but not ERK1/2 and p38 MAPK activities upon fMLP and GM-CSF stimulation. In the case of activation of p38, opposing data have been published showing that fMLP-stimulated neutrophils from Hck-/-Fg-/-Lyn-/- mice are unable to activate p38 MAPK [64 ]. This contradictory finding is difficult to reconcile with our data but may be a result of functional differences between murine- and human-derived neutrophils. The inability of Hck-/-Fgr-/-Lyn-/- mice to activate p38 could also be through a variety of alternative mechanisms not directly linked to receptor-mediated stimulation. Furthermore, the same study also demonstrates a partial inhibition of p38 MAPK phosphorylation with PP1 in human neutrophils. An alternate explanation for this opposing result might be a result of the different concentrations of fMLP used in these assays. We have also demonstrated that fMLP and GM-CSF require Src kinases to activate the PI3K-effector kinase PKB. It is likely that activation of PI3K is mediated by Src kinases because preincubation of cells with PP1 or the PI3K inhibitor LY294002 inhibited PKB activation. This is supported by data showing that a member of the Src-family kinases, Lyn, can bind to the GM-CSF receptor and to the p85 subunit of PI3K in neutrophils [18 , 69 ]. The observation that Src kinases are involved in fMLP-mediated activation of PKB is supported by data showing that in neutrophils stimulated with agonists of G-protein-coupled receptors, PI3K is activated through a genistein-sensitive target, presumably a protein tyrosine kinase. Again supporting a functional link between Src kinases and PI3K-PKB signaling, we demonstrated that PP1 and LY294002 inhibit GM-CSF-mediated survival (Fig. 6) . These data are supported by a study demonstrating a role of Lyn in GM-CSF-delayed apoptosis [62 ] and by a recent study showing that the PI3K inhibitor LY294002 blocked GM-CSF-dependent PKB and BAD (pro-apoptotic Bcl-2 family member) phosphorylation in neutrophils [70 ]. It has been shown in several cell types that PKB can phosphorylate BAD, which in turn prevents its association with anti-apoptotic Bcl-2 family members by association with 14-3-3 proteins, resulting in prolonged survival [71 , 72 ]. Thus, it is tempting to speculate that Src kinases act through PKB to inhibit pro-apoptotic Bcl-2 family members, resulting in a delay in neutrophil apoptosis.
The effector functions, migration, and superoxide production involve
cytoskeletal rearrangement requiring actin polymerization. Previous
studies have shown that fMLP induces actin polymerization in
neutrophils in suspension [54
, 73
]. Here,
we demonstrate that although the rapid induction of F-actin formation
is not inhibited by PP1, the decline in F-actin content is more rapid
in PP1-treated neutrophils (Fig. 7A)
. This suggests that Src kinases
may play a role in the stabilization and duration of actin
polymerization. This finding might be considered in contrast with our
data that Src kinases and PI3K do not affect fMLP-induced migration,
which also involves cytoskeletal rearrangement. However, the role of
PI3K in regulating migration is still controversial. Whereas some
studies, including use of PI3K knockout mice, show that PI3Ks play
an important role in neutrophil migration [12
,
13
, 45
, 74
], other studies
demonstrate that PI3K inhibitors do not inhibit fMLP-induced migration
[75
, 76
]. Furthermore, the small GTPase
p21Rac, which is an upstream regulator of actin polymerization,
superoxide production, and migration [77
,
78
], does not appear to require Src kinases or PI3Ks in
neutrophils [74
, 79
].
The difference in sensitivity of the respiratory burst and GM-CSF-delayed apoptosis and also between fMLP and GM-CSF activation of PKB might suggest that different Src kinases are involved in these processes. Therefore, we tested the sensitivity of Lyn and Hck kinases to PP1. However, the difference between the sensitivity of Lyn and Hck to PP1 is too small to draw any definitive conclusions. Furthermore, the sensitivity of Lyn and Hck to PP1 is probably different whether PP1 is added to an in vitro kinase assay compared with adding PP1 to cells, as has been suggested by others [28 ]. This difference may be attributed in part to permeability of PP1 and its distribution within the cell.
The finding that Src kinases are involved in PKB phosphorylation but not in MAPK activation together with the comparison of data of the Src-kinase inhibitor PP1 with those of PI3K inhibitor LY294002 suggests that Src kinases and PI3K-PKB act in the same pathway that is responsible for GM-CSF-induced survival, prolonging the fMLP-induced respiratory burst and regulating F-actin polymerization. Thus, the regulation of Src-kinase family members by cytokine and chemoattractant receptors provides a critical upstream control point modulating human neutrophil function.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Received November 20, 2000; revised August 26, 2001; accepted August 27, 2001.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  H. Jin, N. J. Lanning, and C. Carter-Su JAK2, But Not Src Family Kinases, Is Required for STAT, ERK, and Akt Signaling in Response to Growth Hormone in Preadipocytes and Hepatoma Cells Mol. Endocrinol., August 1, 2008; 22(8): 1825 - 1841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Fuhler, A. L. Drayer, S. G. M. Olthof, J. J. Schuringa, P. J. Coffer, and E. Vellenga Reduced activation of protein kinase B, Rac, and F-actin polymerization contributes to an impairment of stromal cell derived factor-1 induced migration of CD34+ cells from patients with myelodysplasia Blood, January 1, 2008; 111(1): 359 - 368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Sandoval, J. P. Riquelme, M. D. Carretta, J. L. Hancke, M. A. Hidalgo, and R. A. Burgos Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils J. Leukoc. Biol., November 1, 2007; 82(5): 1266 - 1277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Fumagalli, H. Zhang, A. Baruzzi, C. A. Lowell, and G. Berton The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine J. Immunol., March 15, 2007; 178(6): 3874 - 3885. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Lehman, M. Di Fulvio, N. McCray, I. Campos, F. Tabatabaian, and J. Gomez-Cambronero Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2 Blood, November 15, 2006; 108(10): 3564 - 3572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sato, K. Iwabuchi, I. Nagaoka, Y. Adachi, N. Ohno, H. Tamura, K. Seyama, Y. Fukuchi, H. Nakayama, F. Yoshizaki, et al. Induction of human neutrophil chemotaxis by Candida albicans-derived {beta}-1,6-long glycoside side-chain-branched {beta}-glucan J. Leukoc. Biol., July 1, 2006; 80(1): 204 - 211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rosas, P. F. Dijkers, C. L. Lindemans, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer IL-5-mediated eosinophil survival requires inhibition of GSK-3 and correlates with {beta}-catenin relocalization J. Leukoc. Biol., July 1, 2006; 80(1): 186 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M.-C. Dang, C. Elbim, J.-C. Marie, M. Chiandotto, M.-A. Gougerot-Pocidalo, and J. El-Benna Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity FASEB J, July 1, 2006; 20(9): 1504 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Daoud, E. Rassart, A. Masse, and J. Lafond Src family kinases play multiple roles in differentiation of trophoblasts from human term placenta J. Physiol., March 15, 2006; 571(3): 537 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhang, H. Cao, and G. N. Rao Fibroblast Growth Factor-2 Is a Downstream Mediator of Phosphatidylinositol 3-Kinase-Akt Signaling in 14,15-Epoxyeicosatrienoic Acid-induced Angiogenesis J. Biol. Chem., January 13, 2006; 281(2): 905 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Condliffe, K. Davidson, K. E. Anderson, C. D. Ellson, T. Crabbe, K. Okkenhaug, B. Vanhaesebroeck, M. Turner, L. Webb, M. P. Wymann, et al. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils Blood, August 15, 2005; 106(4): 1432 - 1440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Verploegen, L. Ulfman, H. W. M. van Deutekom, C. van Aalst, H. Honing, J.-W. J. Lammers, L. Koenderman, and P. J. Coffer Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function Blood, August 1, 2005; 106(3): 1076 - 1083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gaudreault, C. Thompson, J. Stankova, and M. Rola-Pleszczynski Involvement of BLT1 Endocytosis and Yes Kinase Activation in Leukotriene B4-Induced Neutrophil Degranulation J. Immunol., March 15, 2005; 174(6): 3617 - 3625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-S. Suh, M.-O. Kim, and S. C. Lee Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Signaling and Microglial Proliferation by Anti-CD45RO: Role of Hck Tyrosine Kinase and Phosphatidylinositol 3-Kinase/Akt J. Immunol., March 1, 2005; 174(5): 2712 - 2719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. van Rossum, A. A. Rarok, M. G. Huitema, G. Fassina, P. C. Limburg, and C. G. M. Kallenberg Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies J. Leukoc. Biol., December 1, 2004; 76(6): 1162 - 1170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Yan, D. M. Byers, and R. Bortolussi Role of Protein Tyrosine Kinase p53/56lyn in Diminished Lipopolysaccharide Priming of Formylmethionylleucyl- phenylalanine-Induced Superoxide Production in Human Newborn Neutrophils Infect. Immun., November 1, 2004; 72(11): 6455 - 6462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Maruyama, Y. Yamamoto, A. Shimizu, H. Masuda, N. Sakai, R. Sakurai, H. Asada, and Y. Yoshimura Pyrazolo Pyrimidine-Type Inhibitors of Src Family Tyrosine Kinases Promote Ovarian Steroid-Induced Differentiation of Human Endometrial Stromal Cells In Vitro Biol Reprod, January 1, 2004; 70(1): 214 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gomez-Cambronero, J. Horn, C. C. Paul, and M. A. Baumann Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway J. Immunol., December 15, 2003; 171(12): 6846 - 6855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Kim, C. C. Marchal, J. Penninger, and M. C. Dinauer The Hemopoietic Rho/Rac Guanine Nucleotide Exchange Factor Vav1 Regulates N-Formyl-Methionyl-Leucyl-Phenylalanine-Activated Neutrophil Functions J. Immunol., October 15, 2003; 171(8): 4425 - 4430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. G. Shenoy, G. J. Gleich, and L. L. Thomas Eosinophil Major Basic Protein Stimulates Neutrophil Superoxide Production by a Class IA Phosphoinositide 3-Kinase and Protein Kinase C-{zeta}-Dependent Pathway J. Immunol., October 1, 2003; 171(7): 3734 - 3741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-Y. Yang, J. J. Arcaroli, and E. Abraham Early Alterations in Neutrophil Activation Are Associated with Outcome in Acute Lung Injury Am. J. Respir. Crit. Care Med., June 1, 2003; 167(11): 1567 - 1574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Gilbert, S. Levasseur, P. Desaulniers, A.-A. Dusseault, N. Thibault, S. G. Bourgoin, and P. H. Naccache Chemotactic Factor-Induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils. II. Effects of LFM-A13, a Specific Btk Inhibitor J. Immunol., May 15, 2003; 170(10): 5235 - 5243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuhler, A. L. Drayer, and E. Vellenga Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia Blood, February 1, 2003; 101(3): 1172 - 1180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Li, A. Yamauchi, C. C. Marchal, J. K. Molitoris, L. A. Quilliam, and M. C. Dinauer Chemoattractant-Stimulated Rac Activation in Wild-Type and Rac2-Deficient Murine Neutrophils: Preferential Activation of Rac2 and Rac2 Gene Dosage Effect on Neutrophil Functions J. Immunol., November 1, 2002; 169(9): 5043 - 5051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Parameswaran, G. Cox, K. Radford, L. J. Janssen, R. Sehmi, and P. M. O'Byrne Cysteinyl Leukotrienes Promote Human Airway Smooth Muscle Migration Am. J. Respir. Crit. Care Med., September 1, 2002; 166(5): 738 - 742. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
