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(Journal of Leukocyte Biology. 2002;71:115-124.)
© 2002 by Society for Leukocyte Biology

Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning

Evert Nijhuis, Jan-Willem J Lammers, Leo Koenderman and Paul J. Coffer

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.

Key Words: signal transduction • protein kinase • cellular activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are critical effector cells in the killing and removal of microorganisms through the regulation of specific effector functions [1 ]. The recruitment of neutrophils from peripheral blood to the inflammatory locus is mediated by several processes consisting of rolling and later, firm adhesion to the vascular endothelium, followed by transmigration through the endothelium and migration to the specific site [2 ]. At the inflammatory locus, the killing of microorganisms is mediated by different cytotoxic-effector mechanisms including phagocytosis, release of cytotoxic proteins, and production of toxic oxygen metabolites initiated by a membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [3 ]. Furthermore, these leukocytes are also involved in maintaining inflammatory reactions by the release of cytokines and bioactive lipids.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
fMLP was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human GM-CSF (2.5x108 U/mg) was from Genzyme (Boston, MA). Human serum albumin (HSA) and human pasteurized plasma-protein solution (40 g/L) were obtained from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). Hepes-buffered RPMI 1640 medium with L-glutamine and Hyclone were purchased from Life Technologies (Breda, The Netherlands). Ficoll-Paque was from Pharmacia (Uppsala, Sweden). Polyclonal antiphospho-PKB (ser473), antiphospho-p44/42 MAPK (Thr202/Tyr204), antiphospho-p38 MAPK (Thr180/Thr182), and antiphospho-STAT5 (Tyr694) antibodies were from New England Biolabs (Westburg, Leusden, The Netherlands). ERK-1 (C-16), ERK-2 (C-14), Hck (N-30), and c-Fgr (N-47) polyclonal antisera were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-Lyn was purchased from Transduction Laboratories (Lexington, KY). Anti-PKB antisera has been described previously [29 ]. The inhibitors PP1, PD098059, and LY294002 were from Biomol (Plymouth, PA).

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 [{gamma}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 [{gamma}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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PP1 inhibits p53/p56-Lyn and Hck kinase activity but not STAT5 phosphorylation in neutrophils
To analyze the role of Src kinases in neutrophil signaling and effector functions, we used the recently identified inhibitor PP1 [28 ]. This compound has been described to specifically inhibit Src kinases including Fyn and Lck but not ZAP-70, JAK2, and epidermal growth factor receptor protein kinases. As mentioned previously, several members of the Src-kinase family including Lyn, Hck, and Fgr are expressed in neutrophils and have been shown to be activated by several cytokine and G-protein-coupled receptors [18 , 22 , 23 ]. Furthermore, a recent study found that the sensitivity of the various members of the Src-kinase family to PP1 was different [33 ]. To characterize the inhibition of Lyn, Hck, and Fgr by PP1, we stimulated neutrophils with fMLP, and an immunocomplex kinase assay was performed in the presence of different concentrations of PP1. Whereas Lyn and Hck demonstrated kinase activity, no Fgr kinase activity could be found in fMLP-stimulated neutrophils (unpublished results). As shown in Figure 1A and B, increasing concentrations of PP1 resulted in a concentration-dependent decrease in Lyn and Hck kinase activities.



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  		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.

 
Recently, it has been suggested that Src kinases are critically involved in the activation of STAT transcription factors in many cell types [34 ]. c-Src was found to be involved in interleukin-3-mediated activation of STAT3, and it has been shown that Lyn can enhance STAT5 activation [35 , 36 ]. To investigate whether Src kinases may be involved in STAT activation in neutrophils, cells were preincubated with increasing concentrations of PP1 and subsequently stimulated with GM-CSF. After stimulation, samples were analyzed by performing a Western blot using an activation-specific phospho-STAT5 antibody. Figure 1C shows no inhibition of STAT5 phosphorylation by PP1 at a concentration of 50 µM PP1, demonstrating that Src kinases are not involved in GM-CSF-induced STAT5 phosphorylation in neutrophils. This observation also establishes that PP1 does not aspecifically inhibit other tyrosine kinases.

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.



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  		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.

 
A role for Src kinases in GM-CSF- and fMLP-induced PKB activation
Recent studies have shown that cytokines and chemoattractants activate PI3K, which plays a critical role in regulating a variety of neutrophil-effector functions [7 , 12 , 13 , 42 43 44 45 ]. A role for tyrosine kinases in activation of the PI3K has also been proposed in neutrophils [17 , 18 , 46 ]. To investigate the role of Src kinases in PI3K activation in neutrophils, we used the phosphorylation status of PKB, a downstream target of PI3K [29 , 47 ], as a measurement of PI3K activation. First, we analyzed the ability of fMLP and GM-CSF to induce PKB activation in neutrophils using an activation-specific phospho-antibody against serine 473 of PKB. Figure 3 A shows a transient phosphorylation of PKB upon GM-CSF or fMLP stimulation. FMLP-induced PKB phosphorylation is much stronger and more rapid than by GM-CSF, similar to the kinetics of MAPK and PI3K activation by these two stimuli [7 , 18 ]. As shown in Figure 3B , increasing concentrations of PP1 resulted in a decrease in GM-CSF- and fMLP-induced PKB phosphorylation with a half-maximal inhibition of 1–5 µM PP1 and a complete block at 5–20 µM PP1, respectively. Addition of the specific PI3K inhibitor LY294002 [48 ] also completely blocks GM-CSF- and fMLP-induced PKB phosphorylation, indicating that PKB activation requires PI3K. Furthermore, we have found that PP1 does not directly inhibit PKB kinase activity (unpublished results). Taken together, our data demonstrate that Src kinases are critical in activation of the PI3K-PKB signaling.



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  		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.

 
Src-kinase activity is required for activation of the respiratory burst in neutrophils
Stimulation of neutrophils with the chemotactic peptide fMLP induces the rapid formation of microbicidal oxidants, a process termed the respiratory burst, and is dependent on prior priming of cells with cytokines, chemoattractants, or lipopolysaccharide [1 , 4 , 5 , 7 ]. Previously, we have shown a role for PI3K but not ERK in this process [7 ]. This finding taken together with our result that PKB but not ERK activation is inhibited by PP1 suggests a possible role for Src kinases in fMLP-induced superoxide production. We analyzed the effect of preincubation of neutrophils with various concentrations of PP1 on the fMLP-induced respiratory burst using GM-CSF as a priming agent.

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.



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  		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.

 
Src kinases are not involved in fMLP-induced neutrophil migration
To reach the site of infection, chemoattractants guide neutrophils to the inflammatory locus by a process termed chemotaxis. To investigate the role of Src kinases in neutrophil migration, we used a modification of the method described by Boyden with a 48-well microchemotaxis chamber, where the stimuli was placed in the lower chamber and migratory activity was measured as described previously [7 ]. To induce migration, we used the chemoattractant fMLP, which is able to potently induce neutrophil migration (Fig. 5 A ).



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  		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).

 
To determine the role of Src kinases in neutrophil migration, we preincubated neutrophils with various concentrations of PP1 before performing Boyden chamber-migration assays. As is shown in Figure 5B , fMLP-induced migration is not blocked by PP1. Similar results were also obtained by incubating the cells with 20 µM LY294002 (Fig. 5B) .

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-{alpha}, 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).



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  		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).

 
PP1 inhibits fMLP-induced actin polymerization in neutrophils with the similar kinetics as for respiratory burst
Cytoskeletal rearrangement, which involves changes in levels of F-actin, is considered to be an essential event for several neutrophil-effector functions including the respiratory burst and migration. In neutrophils, chemoattractants such as fMLP and PAF induce a rapid increase in relative F-actin content [54 , 55 ]. To determine whether Src kinases may also be involved in actin polymerization in neutrophils, cells were preincubated with different concentrations of PP1 and subsequently stimulated with fMLP. Actin polymerization was determined by measuring F-actin content using NBD-phalloidin followed by FACS analysis as described in Materials and Methods. Cells pretreated with 20 µM PP1 demonstrate a similar rapid increase of F-actin as control cells, however the decline in F-actin, which normally occurs after several minutes of fMLP stimulation, occurs much more rapidly (Fig. 7 A ). This suggests a possible role for Src kinases in regulating the depolymerization reaction.



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  		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.

 
A link between actin polymerization and correct functioning of the NADPH oxidase has recently been proposed [56 ]. Furthermore, we also observe inhibition of F-actin polymerization and superoxide production by PP1 (Figs. 4 and 7A) . Using an oxygen probe, we analyzed the detailed kinetics of fMLP-induced oxygen consumption (Fig. 7B) . It is interesting that similar to actin polymerization, where the overall respiratory burst is reduced dramatically, the initial phase of activation appears to be unaffected. Although indirect, this supports a link between sustained actin polymerization and activation of the respiratory-burst complex.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During the last few years, increasing evidence has demonstrated that tyrosine kinases are involved in the coupling between cytokine and G-protein-coupled receptor activation of agonist-effector functions in granulocytes [57 58 59 ]. Among the tyrosine kinases, the family of Src kinases is activated upon stimulation of several cytokine and G-protein-coupled receptors in neutrophils [18 , 24 ] and therefore might play an important role in neutrophil signaling and effector functions. Data obtained using knock-out mice of different Src-family members demonstrate that these kinases are involved in degranulation [60 ] and migration [61 ] in human neutrophils, whereas other studies have shown a role of Lyn in superoxide production [33 ] and in regulation of neutrophil survival by cytokines [62 ]. Furthermore, a link between Src kinases/PI3K [18 , 63 ] and p38 MAPK [64 ] has been proposed in human neutrophils. Recently, a specific Src-family kinase inhibitor called PP1 has been developed [28 ]. By using this inhibitor, we have been able to determine the role of Src kinase in regulating human neutrophil signal-transduction and effector functions.

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{gamma} 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.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This work was supported by the Dutch Asthma Foundation (Grant 9768).

Received November 20, 2000; revised August 26, 2001; accepted August 27, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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