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Originally published online as doi:10.1189/jlb.0204071 on April 23, 2004

Published online before print April 23, 2004
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(Journal of Leukocyte Biology. 2004;76:254-262.)
© 2004 by Society for Leukocyte Biology

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia

Gwenny M. Fuhler*, Karen A. Cadwallader{dagger}, Gerlinde J. Knol*, Edwin R. Chilvers{dagger}, A. Lyndsay Drayer{ddagger} and Edo Vellenga*,1

* Division of Hematology, Department of Medicine, University Hospital Groningen, The Netherlands;
{dagger} Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, United Kingdom; and
{ddagger} Sanquin Blood Bank North East Netherlands, Groningen, The Netherlands

1Correspondence: University Hospital Groningen, Department of Hematology Research, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: E.Vellenga{at}int.azg.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K–Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.

Key Words: MDS • signal transduction • cellular activation • phosphatidylinositol 3-kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of reactive oxygen species (ROS) by neutrophils is one of the most important defense mechanisms against infectious disease and can be stimulated by chemoattractants such as the bacterial peptide analog N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) [1 , 2 ]. Circulating neutrophils usually generate only limited amounts of oxygen radicals in response to such stimuli. However, proinflammatory agents released during infection, e.g., granulocyte macrophage-colony stimulating factor (GM-CSF), although not triggering ROS production directly, are capable of enhancing the neutrophil respiratory burst in response to other stimuli [3 ]. This process is known as "priming" and greatly augments the innate immune system. Incapacity of neutrophils to produce superoxide anions efficiently results in syndromes that are characterized by an increased occurrence of life-threatening bacterial and fungal infections, such as glycogen storage disease type b or chronic granulomatous disease [4 , 5 ].

The Myelodysplastic syndromes (MDS) are distinguished by a differentiation defect in the multipotent stem-cell compartment, resulting in disturbed proliferation and differentiation of the erythroid, myeloid, or megakaryocytic lineages [6 ]. The ensuing granulocytopenia and aberrant functioning of neutrophils make bacterial infections the most common cause of death in MDS patients [7 ]. We have recently shown that GM-CSF priming of the fMLP-triggered respiratory burst is severely impaired in neutrophils from MDS patients [8 ].

The molecular mechanisms underlying priming of ROS production are still unclear. Generation of ROS is mediated by the granulocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This enzyme consists of a membrane-bound component, flavocytochrome b558,and the cytosolic factors p47phox, p40phox, p67phox, and Rac [9 ]. Several reports have suggested that priming of the neutrophil respiratory burst results from enhanced translocation of flavocytochrome b558, which is stored in the membranes of specific granules and secundary vesicles, to the plasma membrane [10 , 11 ]. However, we have previously shown that whereas GM-CSF priming of ROS production is decreased in neutrophils from MDS patients, GM-CSF-induced increases in flavocytochrome b558 expression at the plasma membrane are normal in these cells [12 ]. This suggests that an additional mechanism involved in GM-CSF priming is affected in MDS patients.

Several signal-transduction molecules have been implicated in the priming of NADPH oxidase activity [13 ]. For example, the involvement of the lipid kinase phosphatidylinositol 3-kinase (PI-3K) in GM-CSF priming of fMLP-stimulated ROS production has been established by using specific inhibitors [8 , 14 ]. A number of class I isoforms of PI-3K have been identified; class IA catalytic subunits p110{alpha}, -ß, and -{delta} associate with the regulatory subunit p85, whereas the class IB catalytic subunit p110{gamma} associates with p101 [15 ]. Upon stimulation of neutrophils, these PI-3Ks produce the phosphoinositide PI(3,4,5)trisphosphate (PIP3), which acts as an important second messenger for superoxide anion generation [16 ]. Its immediate metabolic products, PI(3,4)P2 and PI(3)P1,have also been implicated in regulating ROS production through an interaction with p47phox and p40phox [17 , 18 ].

Evidence of priming at a molecular level comes from studies showing increased fMLP-stimulated PI-3K activation in GM-CSF-primed neutrophils compared with unprimed cells [19 , 20 ]. Furthermore, phosphorylation of the PI-3K downstream target protein kinase B (PKB/Akt) can be enhanced by GM-CSF pretreatment of neutrophils. Previously, we have shown that PKB/Akt activation in neutrophils from MDS patients is defective, suggesting disturbed PI-3K signaling in these cells [8 ].

Rac, a member of the Rho family of small GTPases, plays an important role in ROS production by regulating the assembly of the NADPH oxidase complex [21 ]. In resting cells, Rac is bound to guanosine 5'-diphosphate (GDP) and hence, is inactive. Guanine nucleotide exchange factors (GEFs), stimulated upon neutrophil stimulation, activate Rac by allowing the exchange of GDP for guanosine 5'-triphosphate (reviewed in ref. [22 ]). Inactivation occurs as a result of intrinsic GTPase activity, which can be enhanced by GTPase-activating proteins (GAPs). Different GEFs for Rac have been described to possess a PI-3K-binding domain, resulting in PI-3K-dependent activation of Rac (reviewed by Welch et al. [23 ]). In return, Rac can activate PI-3K itself, although the mechanism for this is still unclear. Another GTPase capable of triggering PI-3K activity is Ras, a member of the well-studied Ras–Raf–mitogen-activated protein kinase kinase–extracellular-regulated kinase (ERK) signaling pathway [24 ]. Ras can also activate the GTPase Ral through PI-3K-dependent or independent pathways [25 ]. However, Ras-independent mechanisms of Ral activation have also been identified [26 ]. The cellular function of Ral is still unclear, although recent evidence suggests that a downstream target of Ral, Ral-binding protein 1 (RLIP1), may play a regulatory role in Rac activation [27 ].

In this study, we addressed whether the disturbed GM-CSF priming of fMLP-induced ROS production in MDS neutrophils might reflect a defect in the activation of the GTPases Rac, Ras, and Ral and the production of PIP3. Our results demonstrate that in contrast to healthy donor neutrophils, Rac activation and PIP3 mass accumulation in response to fMLP stimulation could not be enhanced by GM-CSF in neutrophils from MDS patients. These impaired responses did not appear to be a result of a general GM-CSF or fMLP receptor-signaling defect, as the fMLP-triggered activation of Ral and GM-CSF priming of Ras activity were normal in MDS neutrophils. Taken together, our data suggest that in neutrophils from MDS patients, an impaired priming of the PI-3K–Rac signaling pathway results in a decreased GM-CSF priming of ROS production and that this defect is located at the level of PI-3K.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
fMLP was obtained from Sigma Chemical Co. (St. Louis, MO). Recombinant (r) human GM-CSF was supplied by Sandoz (Uden, The Netherlands). The PI-3K inhibitor LY294002 was purchased from Alexis (Läufelfingen, Switzerland). The glutathione S-transferase (GST)–p21-activated kinase 1 (PAK)–cdc42 and Rac-interactive binding (CRIB), GST–Raf–Ras-binding domain (RBD), and GST–RLIP–RBD constructs were a generous gift from Dr. Paul J. Coffer (Department of Pulmonary Diseases, University Medical Center, Utrecht, The Netherlands). Anti-Rac, anti-Ras, and anti-Ral antibodies were supplied by Transduction Laboratories (Lexington, KY). [Inositol-1-[3H](N)]-1,3,4,5-tetrakisphosphate {[3H]Ins(1,3,4,5)P4} was obtained from NEN Life Sciences (Boston, MA), and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was purchased from Cell Signals (Lexington, KY).

Patients
Eleven patients with MDS were studied. MDS was classified as refractory anemia (RA) or RA with ringed sideroblasts, according to French-American-British Cooperative Group criteria [28 ]. Informed consent was obtained from all patients. The Human Subject Review Board of the University Hospital Groningen (The Netherlands) approved the protocol.

Isolation of neutrophils
Peripheral blood, anticoagulated with 0.32% sodium citrate, was obtained from healthy volunteers and MDS patients. Neutrophils were isolated as described [8 , 29 ]. In short, mononuclear cells were removed by centrifugation over Fycoll-Paque (Amersham, Upsala, Sweden), and erythrocytes were lysed with ice-cold NH4Cl solution. Neutrophils were allowed to recover for 30 min at 37°C in RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 0.5% human serum albumin (HSA; CLB, Amsterdam, The Netherlands). Before stimulation, cells were resuspended in incubation buffer (20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 1 mM CaCl2, 0.5% HSA). In all cases, the cell population isolated consisted of >95% neutrophils as determined by May-Grünwald Giemsa staining.

Rac, Ras, and Ral activation assays
Activated Rac was precipitated using bacterial lysate containing GST–PAK–CRIB, activated Ras was pulled down using bacterial lysate containing GST–Raf–RBD, and activated Ral was precipitated using bacterial lysate containing GST–RLIP–RBD as described previously [8 , 26 ]. Briefly, neutrophils (1x106 cells/ml for Rac activation assay and 10x106 cells/ml for Ral and Ras activation assays) were stimulated with 1 µM fMLP for the indicated time and lysed for 10 min in lysis buffer [50 mM Tris, pH 7.4, 10% glycerol, 200 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2, 2 mM sodiumorthovanadate, and protease inhibitors; one tablet Complete (Roche, Mannheim, Germany) per 50 ml buffer]. Lysates were cleared by centrifugation, and GST–PAK–CRIB, GST–Raf–RBD, or GST–RLIP–RBD protein, precoupled to glutathione-sepharose beads (Pharmacia, Uppsala, Sweden), was added for 30–45 min at 4°C. Beads were washed three times with 1x lysis buffer and boiled in Laemmli sample buffer. The bound proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). Activated Rac, Ras, or Ral was detected by probing the membrane with anti-Rac, anti-Ras, or anti-Ral antibodies, respectively, and rabbit anti-mouse peroxidase-conjugated antibodies (Dako, Denmark) followed by enhanced chemiluminescence (ECL). Equal amounts of glutathione-sepharose beads were loaded in all samples as determined by Ponceau S staining of the membranes. Quantification of the amount of precipitated, active GTPase was performed by densitometry of the films, using ImageMaster1D Elite (Pharmacia, Woerden, The Netherlands). Results are presented as normalized densitometry values [arbitrary units (AU)].

PIP3 mass assay
r-Ins(1,3,4,5)P4-binding protein (GAPIP4BP) was purified as decribed previously [30 ], and sample purity was analyzed by SDS-PAGE. Total and nonspecific binding of [3H]Ins(1,3,4,5)P4 in each preparation of GAPIP4BP was assessed by serial dilution and inclusion of 0.1 nM InsP6, respectively. Thereafter the GAPIP4BP was used at a concentration to achieve a maximum binding of 20% of the total [3H]Ins(1,3,4,5)P4 input. Purified neutrophils (8x106) were stimulated with 1 µM fMLP or vehicle for the indicated times, with or without prior priming with GM-CSF (5 ng/ml) for 15 min. The reactions were stopped by the addition of methanol/chloroform (2:1, v/v), and lipid extractions were performed as described previously [20 , 31 ]. After drying, the samples were processed as previously detailed and stored at –20°C until analyzed. Immediately before the assay, samples were resuspended in dilute acetic acid to a final pH of 5. The Ins(1,3,4,5)P4 radioreceptor assay was performed as described using 0.005 µCi [3H]Ins(1,3,4,5)P4 per sample [16 ].

Statistical analysis
Differences between unstimulated and stimulated samples from the same donor and differences between samples treated with or without signal-transduction inhibitors were calculated using the Wilcoxon-nonparametric test for paired samples. Differences between healthy volunteers and MDS patients were calculated using the Mann-Whitney nonparamentric test for unpaired samples. Data were expressed as mean ± SEM. P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rac activation in response to fMLP can be primed by GM-CSF in neutrophils from healthy donors
As GM-CSF is capable of priming the fMLP-mediated respiratory burst in neutrophils from healthy volunteers, we investigated the effect of neutrophil priming on fMLP-triggered Rac activation. When neutrophils from healthy donors were stimulated with 1 µM fMLP, Rac was rapidly activated (Fig. 1a and 1b ). Pretreatment of neutrophils with GM-CSF, which in itself did not activate Rac, resulted in a significantly increased response to fMLP stimulation [t=10 s: 23±2 vs. 14±3 (AU), P=0.018; t=30 s: 25±2 vs. 14±2 (AU), P=0.03, n=7]. These results indicate that the fMLP-triggered Rac activation can indeed be primed by GM-CSF in neutrophils from healthy donors.



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  		Figure 1. GM-CSF priming of Rac activation is disturbed in neutrophils from MDS patients. Unprimed neutrophils and neutrophils that were primed with 5 ng/ml GM-CSF for 15 min were stimulated with 1 µM fMLP for the indicated times (s). Stimulation was stopped by adding 3x lysis buffer. Activated Rac was precipitated using GST–PAK–CRIB protein precoupled to glutathione-sepharose beads. Proteins were separated by 15% SDS-PAGE, and membranes were probed with Rac antibodies, followed by ECL. Rac activation was investigated in neutrophils from seven healthy controls and seven MDS patients. Two representative blots from healthy subjects (a) and three representative blots from MDS patients (c) are shown. Equal amounts of glutathione-sepharose beads were loaded in all samples, as determined by Ponceau S staining of the membranes (not shown). Protein levels of activated Rac were quantified by densitometry of the films. The means of the normalized values were calculated for the healthy donors (b) and the MDS patients (d). Significant differences between unprimed and GM-CSF-primed groups were calculated using the Wilcoxon-nonparametric test for paired samples and are indicated (*, P<0.05).

 
Decreased priming of Rac activation in neutrophils from MDS patients
We subsequently analyzed Rac activation in neutrophils from seven MDS patients. In accordance with our previous results [8 ], fMLP induced Rac activation in neutrophils from MDS patients in a similar manner to that in healthy neutrophils; a rapid and transient activation was observed in response to stimulation with 1 µM fMLP (Fig. 1c and 1d) . Rac activation in response to 10 s fMLP stimulation appeared to be higher in unprimed MDS neutrophils compared with neutrophils from healthy donors, although this dissimilarity was not statistically different. In contrast to the results obtained with healthy neutrophils, priming of MDS neutrophils with GM-CSF did not increase the amount of Rac activated in response to fMLP stimulation [Fig. 1c and 1d ; t=10 s: 19±3 vs. 22±3 (AU), P=0.3]. Stimulation of unprimed neutrophils for 30 s with fMLP resulted in an activation of Rac that was equal in neutrophils from MDS patients and healthy donors. Again, GM-CSF priming did not enhance this response in MDS neutrophils [t=30 s: 17±4 vs. 15±2 (AU), P=0.8], in contrast to neutrophils from healthy donors.

These results imply that in agreement with the decreased GM-CSF priming of fMLP-induced ROS production, priming of Rac activity by GM-CSF is also disturbed in neutrophils from MDS patients.

Rac activation is sensitive to the PI-3K inhibitor LY294002
To elucidate the sequence of activation of Rac and PI-3K upon fMLP stimulation, healthy neutrophils were preincubated for 15 min with the specific PI-3K inhibitor LY294002 before priming with GM-CSF for 15 min and/or stimulation with fMLP for 30 s. Figure 2a demonstrates that LY294002 treatment resulted in a significant attenuation of Rac activation in response to fMLP stimulation in unprimed or GM-CSF-primed cells. Densitometry of the blots revealed that inhibition of PI-3K activity reduced the fMLP-triggered Rac activation from 14 ± 2 to 8 ± 2 (AU, n=5, P=0.04) in unprimed cells. In GM-CSF-primed neutrophils, the fMLP-triggered Rac activation was reduced from 21 ± 1 to 7 ± 1 (AU, n=5, P=0.04) as a result of LY294002 treatment (Fig. 2b) . These results demonstrate that activation of the small GTPase Rac by fMLP could be a downstream event from PI-3K activation.



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  		Figure 2. Effect of LY294002 on Rac activation in unprimed and GM-CSF-primed, healthy neutrophils. Unprimed neutrophils and neutrophils that were primed with 5 ng/ml GM-CSF for 15 min were stimulated with 1 µM fMLP for 30 s. Where indicated, cells were pretreated with 10 µM LY294002 for 15 min. Activated Rac was precipitated as described in Figure 1 . Three representative blots are shown (a). Protein levels of activated Rac were quantified by densitometry of the films, and the means of the normalized values for five individual experiments are shown (b). Differences between LY294002 treated and untreated groups were calculated using the Wilcoxon-nonparametric test for paired samples, and significant reductions in Rac activation are indicated (*, P<0.05).

 
Disturbed GM-CSF priming of fMLP-triggered PIP3 mass accumulation in neutrophils from MDS patients
To establish whether a disturbed activity of PI-3K might be responsible for the decreased priming of Rac activity observed in MDS neutrophils, we investigated the accumulation of the PI-3K product PIP3 in neutrophils from five healthy donors and five MDS patients. As shown in Figure 3a in neutrophils from healthy donors and MDS patients, PIP3 levels increased after 10 s stimulation with 1 µM fMLP. After 45 s, a rapid decline in PIP3 levels to near baseline values was observed in healthy neutrophils. Although in neutrophils from MDS patients, the accumulation of PIP3 mass appeared to be prolonged slightly, this was not statistically different from the values in healthy neutrophils. Pretreatment of neutrophils from healthy donors with 5 ng/ml GM-CSF for 15 min resulted in a slight but significant increase in PIP3 accumulation (3.1±1 pmol/8x106 cells) compared with unstimulated neutrophils (1.4±1 pmol/8x106 cells, P=0.04). However, in accordance with previous results [20 ], subsequent stimulation of neutrophils with 1 µM fMLP led to a significantly increased and prolonged PIP3 signal compared with that with fMLP alone (t=45 s: 11.6±2 vs. 1.6 pmol/8x106 cells, P=0.04). In contrast, GM-CSF priming of neutrophils from MDS patients did not result in any enhancement of the fMLP-induced PIP3 accumulation (t=45 s: 4.4±2 vs. 5.4±2 pmol/8x106 cells, P=0.5).



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  		Figure 3. Impaired GM-CSF priming of PIP3 mass accumulation in neutrophils from MDS patients. (a) Neutrophils were stimulated with 1 µM fMLP for the indicated amount of time, with or without prior treatment with 5 ng/ml GM-CSF for 15 min. Reactions were stopped by the addition of MeOH/CHCl3 (2:1 v/v), lipids were extracted, and the Ins(1,3,4,5)P4 isotope dilution assay was performed as described in Materials and Methods. Results represent the mean PIP3 mass accumulation in samples from five healthy volunteers and five MDS patients. Statistical differences between unstimulated and fMLP-stimulated groups were calculated using the Wilcoxon-nonparametric test for paired samples, and significant differences are indicated (*, P<0.05). Significant differences between unprimed and GM-CSF-primed groups are indicated (**, P<0.05). (b) Neutrophils from healthy donors were primed with 5 ng/ml GM-CSF for 15 min before stimulation with 1 µM fMLP for 45 s. Where indicated, cells were preincubated with LY294002 (10 µM for 30 min). PIP3 measurement was performed in three independent experiments as described. PIP3 mass accumulation of a representative experiment is shown.

 
The involvement of a class I PI-3K in the accumulation of PIP3 in human neutrophils was confirmed by the inhibition of the fMLP-induced PIP3 signal after pretreatment of GM-CSF-primed, healthy neutrophils with the PI-3K inhibitor LY294002 (Fig. 3b) .

In conclusion, these results indicate that as for ROS production and Rac activation, priming of PI-3K activity by GM-CSF is severely disturbed in neutrophils from MDS patients.

Priming of Ras activation in neutrophils from healthy donors and MDS patients
Activation of the Ras GTPase has been placed upstream of PI-3K activity. To establish whether the observed disturbance in PIP3 accumulation in MDS neutrophils could be attributed to disturbed Ras signaling, we investigated the activation of this small GTPase in neutrophils from healthy donors compared with MDS patients. Stimulation of healthy neutrophils with 1 µM fMLP resulted in a transient activation of Ras, with a maximum activation after 30 s (Fig. 4a ). After 10 min of fMLP stimulation, levels of active Ras were reduced to baseline values (not shown). Preincubation of neutrophils with 5 ng/ml GM-CSF before fMLP stimulation resulted in a significantly increased and prolonged activation of Ras when compared with fMLP stimulation without GM-CSF pretreatment. Densitometry of the blots showed that this priming effect was significantly higher than the accumulative activation of Ras by fMLP and GM-CSF alone (Fig. 4b) . These data are consistent with the increased and prolonged fMLP-induced phosphorylation of the Ras downstream target ERK1/2, which we previously found in healthy GM-CSF-primed neutrophils [8 ].



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  		Figure 4. fMLP-induced Ras activation can be primed by GM-CSF in neutrophils from healthy donors and MDS patients. Unprimed neutrophils and neutrophils that were primed with 5 ng/ml GM-CSF for 15 min were stimulated with 1 µM fMLP for the indicated time (s). Activated Ras was precipitated using GST–Raf–RBD protein precoupled to glutathione-sepharose beads as described. Ras activation was investigated in neutrophils from five healthy controls and seven MDS patients. Two representative blots from healthy subjects (a) and three representative blots from MDS patients (c) are shown. Protein levels of activated Ras were quantified by densitometry of the films. The means of the normalized values were calculated for the healthy donors (b) and the MDS patients (d). Significant differences between unprimed and GM-CSF-primed groups were calculated using the Wilcoxon-nonparametric test for paired samples and are indicated (*, P<0.05).

 
We next tested the capacity of fMLP to activate Ras and the efficacy of GM-CSF in priming this activation in neutrophils from MDS patients. As shown in Figure 4c and 4d fMLP stimulated Ras activity in a similar manner in neutrophils from MDS patients (n=7) as in neutrophils from healthy volunteers (n=5), with a maximum after 30 s of stimulation. Furthermore, as in healthy neutrophils, GM-CSF was capable of priming the fMLP-triggered response.

We subsequently set out to investigate the position of Ras relative to PI-3K in signal transduction (Fig. 5a ). Incubation of neutrophils with 10 µM of the PI-3K inhibitor LY294002 before stimulation of the cells with fMLP did not reduce the amount of activated Ras present in the cells [14±4 vs. 12±4 (AU), P=0.7]. Likewise, priming of fMLP-stimulated Ras activation by GM-CSF pretreatment of neutrophils was also not affected by the PI-3K inhibitor [31±3 vs. 39±3 (AU), P=0.08; Fig. 5b ]. These results indicate that fMLP-induced Ras activation can be primed by GM-CSF in a PI-3K-independent manner and that this priming is normal in MDS neutrophils.



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  		Figure 5. Effect of LY294002 on Ras activation in unprimed and GM-CSF-primed, healthy neutrophils. Unprimed neutrophils and neutrophils that were primed with 5 ng/ml GM-CSF for 15 min were stimulated with 1 µM fMLP for 2 min. Where indicated, cells were pretreated with 10 µM LY294002 for 30 min. Activated Ras was precipitated as described previously. Two representative blots are shown (a). Protein levels of activated Ras were quantified by densitometry of the films, and the means of the normalized values for five individual experiments are shown (b).

 
Ral activation in response to fMLP is normal in neutrophils from MDS patients
One of the described, downstream targets of Ras is the small GTPase Ral, which has been shown to play a regulatory role in the activity of Rac through its effector molecule RLIP1. To examine whether the priming of Ras activation in response to GM-CSF also resulted in priming of Ral activity, neutrophils from healthy volunteers (n=3) were pretreated with 5 ng/ml GM-CSF for 15 min before stimulation with 1 µM fMLP for the indicated time-points, and active Ral was precipitated using GST–RLIP–RBD fusion protein. As shown in Figure 6a and 6b fMLP stimulation of neutrophils resulted in a transient activation of the Ral GTPase, which returned to near baseline levels after 10 min (not shown). In contrast to Rac and Ras activation, fMLP-stimulated Ral activation could not be primed by GM-CSF treatment.



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  		Figure 6. fMLP-induced Ral activation cannot be primed by GM-CSF and is not inhibited by LY294002 in neutrophils from healthy donors. (a) Unprimed, healthy neutrophils and neutrophils that were primed with 5 ng/ml GM-CSF for 15 min were stimulated with 1 µM fMLP for the indicated time (s). Activated Ral was precipitated using GST–RLIP–RBD protein precoupled to glutathione-sepharose beads as described. Two representative blots are shown. (b) Protein levels of activated Ral were quantified by densitometry of the films. The means of the normalized values from three individual experiments were calculated. (c) Neutrophils were stimulated with 1 µM fMLP for 30 s, with or without prior incubation with 10 µM LY294002 for 30 min. Activated Ral was precipitated as described. Blots from two individual experiment are shown. (d) Active Ral precipitated in c was quantified by densitometry. The means of the normalized values from three individual experiments were calculated.

 
Neutrophils from healthy donors were subsequently preincubated with the PI-3K inhibitor LY294002 and stimulated with 1 µM fMLP for 30 s. No inhibition of Ral activation was observed as a result of LY294002 pretreatment (Fig. 6c and 6d) .

We next compared the fMLP-stimulated activation of Ral in neutrophils from healthy donors (n=7) and MDS patients (n=3). Figure 7a shows that as in healthy donors, Ral activation in response to fMLP was rapid and transient in neutrophils from MDS patients. Densitometry of the blots indicated that the degree of Ral activation was identical in MDS patients and healthy volunteers (Fig. 7b) .



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  		Figure 7. Ral activation by fMLP is normal in neutrophils from MDS patients. Neutrophils were stimulated with 1 µM fMLP for the indicated time (s), and activated Ral was precipitated using GST–RLIP–RBD protein precoupled to glutathione-sepharose beads as described. Two representative blots from healthy subjects (a) and three representative blots from MDS patients (b) are shown. Protein levels of activated Ral were quantified by densitometry of the films. The means of the normalized values were calculated for seven healthy donors and three MDS patients (c).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The production of ROS by neutrophils is extremely important in the defense against invading bacteria. In neutrophils from MDS patients, GM-CSF priming of ROS production is severely disturbed, whereas fMLP-triggered ROS production in unprimed cells is relatively normal [8 , 32 , 33 ]. The molecular mechanisms underlying priming of ROS production are still unclear. In the present study, we wanted to further elucidate the molecular effects of GM-CSF priming on human neutrophils. Our results demonstrate that pretreatment of neutrophils with GM-CSF leads to an increased activity of the small GTPase Ras in response to fMLP. Although the precise involvement of Ras in ROS production remains to be determined, the capacity of Ras to activate ERK1/2 and PI-3K is well established [34 , 35 ]. Studies with specific signal-transduction inhibitors have revealed the importance of these signal transducers in the GM-CSF-mediated priming of ROS production. Previously, it has been shown that fMLP-triggered activation of ERK1/2 and the PI-3K target PKB/Akt could be enhanced by GM-CSF pretreatment of neutrophils and that this activation was disturbed in neutrophils from MDS patients [8 ]. However, although Ras has frequently been reported to activate ERK1/2, the present study shows that activation of Ras by fMLP and GM-CSF priming thereof are not affected in neutrophils from MDS patients. These data suggest that the molecular defect leading to the disturbed ROS priming in neutrophils from MDS patients is located downstream of Ras activation. Furthermore, these results indicate that the disturbed priming of ROS production and activation of signal-transduction molecules in MDS granulocytes are not the result of a general defect in fMLP or GM-CSF receptor signaling. This is also confirmed by our results showing normal activation of the GTPase Ral in neutrophils from MDS patients.

Several studies have shown that the GTPase Ras can activate PI-3K [24 ]. However, we now show that although priming of PIP3 accumulation, a result of PI-3K activity, was absent in neutrophils from MDS patients, Ras actvation was normal. This suggests that another signal transducer capable of activating PI-3K might be affected in MDS neutrophils. Alternatively, the defect is located at the level of PI-3K itself, a suggestion corroborated by our finding that protein levels of PI-3K subunits are disturbed in MDS neutrophils [8 ]. Other studies have revealed that the level of PIP3 within cells is carefully balanced by different lipid kinases and phosphatases [36 ]. It can therefore not be formally excluded that one or more of these enzymes are responsible for the disturbed PIP3 accumulation in neutrophils from MDS patients.

Neutrophils from PI-3K{gamma}–/– mice, which do not produce PIP3 and ROS in response to fMLP, show normal activation of the small GTPase Rac after fMLP stimulation [37 ]. This would indicate a PI-3K-independent Rac activation, which is not involved in the neutrophil respiratory burst. In contrast, studies describing a patient with an inhibitory Rac2 mutation have clearly established the importance of this GTPase in ROS production [38 ]. Furthermore, other studies indicate that Rac activation in response to fMLP is PI-3K-dependent in unprimed and GM-CSF-primed neutrophils [39 ]. In the present study, attenuation of Rac activation, although significant, was not complete. This suggests that Rac activation might occur through multiple signal-transduction routes, including a PI-3K-independent pathway.

Early reports suggested that the diversity in PI-3Ks (PI-3K{alpha}, -ß, -{delta}, and -{gamma}) contributes to the distinct PI-3K-dependent, cellular functions activated upon stimulation of cells with different stimuli (reviewed in ref. [15 ]). For example, G protein-coupled receptors (GPCRs), e.g., the fMLP receptor, were described to exclusively activate p110{gamma}, whereas p110{alpha}, -ß, and -{delta} activation was attributed to receptor tyrosine kinases and cytokine receptors such as the GM-CSF receptor [40 41 42 ]. Recent studies, however, have shown that GPCRs can activate class IA PI-3Ks, in addition to PI-3K{gamma} [43 44 45 ]. Another recent study demonstrated that in p85{alpha}–/– mice, GM-CSF priming of fMLP-stimulated ROS production is severely decreased, indicating the involvement of class IA PI-3Ks in the GM-CSF-priming response [46 ]. However, equally convincing studies have shown that p110{gamma} is the primary subunit responsible for the enhanced PIP3 production seen in primed neutrophils [20 ]. It is conceivable that full activation of class IA and class IB PI-3Ks is neccessary in GM-CSF-primed neutrophils to get a full fMLP-induced response, as was recently described for stromal cell-derived factor-1-induced migration of leukemic T cells [47 ]. To what extent activation of the different PI-3K classes is affected in MDS neutrophils remains to be elucidated.

In conclusion, we show a disturbed GM-CSF priming of fMLP-induced PIP3 accumulation and Rac activation in neutrophils from MDS patients. Activation of Ras could be primed normally by GM-CSF in MDS neutrophils, and fMLP-triggered activity of the Ras target Ral was likewise undisturbed in these cells. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K–Rac signaling pathway results in a decreased GM-CSF priming of ROS production and that this defect is located at the level of PI-3K activity.


   ACKNOWLEDGEMENTS
 
The European Molecular Biology Organization (EMBO) and the Dutch Cancer Society (RUG 2003-2920) supported this work. The Wellcome Trust and British Lung Foundation supported K. A. C. and E. R. C. We thank all patients and healthy volunteers who participated in this study.

Received February 5, 2004; revised March 12, 2004; accepted March 13, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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