Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0504306 on November 23, 2004

Published online before print November 23, 2004
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(Journal of Leukocyte Biology. 2005;77:257-266.)
© 2005 by Society for Leukocyte Biology

Impaired interleukin-8- and GRO{alpha}-induced phosphorylation of extracellular signal-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia

Gwenny M. Fuhler*, Gerlinde J. Knol*, A. Lyndsay Drayer{dagger} and Edo Vellenga*,1

* Division of Hematology, Department of Medicine, University Hospital Groningen, The Netherlands; and
{dagger} Sanquin Blood Bank North East Netherlands, Groningen

1 Correspondence: Division of Hematology, Department of Medicine, University Hospital Groningen, 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
 
Patients with myelodysplasia suffer from recurrent bacterial infections as a result of differentiation defects of the myeloid lineage and a disturbed functioning of neutrophilic granulocytes. Important physiological activators of neutrophils are the cytokines interleukin-8/CXC chemokine ligand 8 (IL-8/CXCL8), which activates CXC chemokine receptor 1 and 2 (CXCR1 and CXCR2), and growth-related oncogene (GRO{alpha})/CXCL1, which stimulates only CXCR2. In this study, we show that migration toward IL-8/GRO{alpha} gradients is decreased in myelodysplastic syndrome (MDS) neutrophils compared with healthy donors. We investigated the signal transduction pathways involved in IL-8/GRO{alpha}-induced migration and showed that specific inhibitors for extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol-3 kinase (PI-3K) abrogated neutrophil migration toward IL-8/GRO{alpha}. In accordance with these results, we subsequently showed that IL-8/GRO{alpha}-stimulated activation of ERK1/2 was substantially diminished in MDS neutrophils. Activation of the PI-3K downstream target protein kinase B/Akt was disturbed in MDS neutrophils when cells were activated with IL-8 but normal upon GRO{alpha} stimulation. IL-8 stimulation resulted in higher migratory behavior and ERK1/2 activation than GRO{alpha} stimulation, suggesting a greater importance of CXCR1. We then investigated IL-8-induced activation of the small GTPase Rac implicated in ERK1/2-dependent migration and found that it was less efficient in neutrophils from MDS patients compared with healthy donors. In contrast, IL-8 triggered a normal activation of the GTPases Ras and Ral, indicating that the observed defects were not a result of a general disturbance in CXCR1/2 signaling. In conclusion, our results demonstrate a disturbed CXCR1- and CXCR2-induced neutrophil chemotaxis in MDS patients, which might be the consequence of decreased Rac-ERK1/2 and PI-3K activation within these cells.

Key Words: MDS • granulocytes • signal transduction • ERK1/2 • Rac • migration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders that are characterized by a disturbed proliferation and differentiation of the erythroid, myeloid, and megakaryocytic cell lineages [1 ]. The observation that the high mortality rate in MDS patients is generally a direct result of recurrent bacterial infections underscores the importance of granulocytes in the pathology of the disease [2 ]. The responses of neutrophils leading to the eradication of invading bacteria can be divided into a number of steps: adhesion to and rolling along endothelial lining of blood vessels, migration toward the site of inflammation, diapedesis through the endothelial layer, degranulation, and finally, phagocytosis of bacteria in conjunction with production of bactericidal reactive oxygen species (ROS). In MDS, the differentiation defect in the multipotent stem cell compartment not only leads to neutropenia but also results in aberrant neutrophil functioning. For instance, the cell-membrane expression of the CD11b/CD18 complex, which regulates neutrophil adherence, migration, and diapedesis, is decreased on neutrophils from MDS patients [3 ]. Furthermore, reduced activity of granule enzymes and defects in granule-membranes have been reported in neutrophils from MDS patients [4 5 6 ]. Other studies have shown that in MDS neutrophils, phagocytosis of bacteria and the production of ROS are defective [7 8 9 10 11 12 ]. Lastly, a deficit in neutrophil chemotaxis has been reported in MDS cases [13 , 14 ].

Granulocytes are attracted to sites of infection by inflammatory mediators that are being produced at these sites. Of particular importance in the recruitment of neutrophils is the 72-amino acid polypeptide interleukin-8 or CXC chemokine ligand 8 (IL-8 or CXCL8) [15 , 16 ]. This chemokine is produced by monocytes and endothelial cells and allows granulocytes to arrive in the general vicinity of the infection [17 , 18 ]. Neutrophils subsequently migrate toward a more localized gradient of "end-target" chemoattractants such as the bacterial product N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) [19 , 20 ].

Two receptors for IL-8 have been identified: CXC chemokine receptor 1 and 2 (CXCR1 and CXCR2). CXCR1 is activated exclusively by IL-8, whereas CXCR2 also binds to other chemokines that are produced at the site of inflammation, such as growth-related oncogene (GRO{alpha} or CXCL1) and neutrophil-activating peptide-2 (or CXCL7).

Despite the fact that the receptors for fMLP and IL-8 belong to the family of G protein-coupled seven-transmembrane-spanning domain receptors, the signals they transduce result in qualitatively different neutrophil effector functions [21 ]. For instance, in contrast to fMLP, IL-8 does not trigger ROS production directly in human neutrophils. However, IL-8 pretreatment of cells does result in an enhanced production of oxygen radicals in response to fMLP stimulation [22 ]. In this capacity, IL-8 resembles priming agents such as the proinflammatory cytokines granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor {alpha} [23 , 24 ]. Although fMLP and IL-8 trigger neutrophil migration, the mechanisms through which chemotaxis is achieved might differ. Recent studies have shown that fMLP-induced transmigration is dependent on CD18 expression, whereas IL-8-induced chemotaxis is not [25 ].

The signal transduction pathways involved in chemotaxis toward a fMLP or IL-8/GRO{alpha} gradient are as yet unclear. Several studies have indicated that although IL-8, GRO{alpha}, and fMLP stimulate the activation of extracellular-signal regulated kinase (ERK1/2), neutrophil chemotaxis induced by these agents was ERK1/2-independent [26 27 28 29 ]. In contrast, equally convincing studies did show involvement of ERK1/2 in IL-8- or fMLP-induced migration [30 31 32 ]. ERK1/2 is an important member of the Ras-Raf-mitogen-activated protein kinase (MAPK) kinase (MEK)-ERK1/2 signaling pathway. In addition, recent evidence suggests that ERK1/2 might also be activated by the small GTPase Rac in a Ras-independent manner [33 ]. Furthermore, activity of the lipid kinase phosphatidylinositol-3 kinase (PI-3K) has been implicated in neutrophil migration [26 , 27 ] and has been placed upstream of ERK1/2 activation in several cell systems [9 , 34 ].

So far, only fMLP-induced migration has been studied in neutrophils from MDS patients [13 , 14 ], and we have previously shown decreased activation of ERK1/2 and the PI-3K downstream product protein kinase B (PKB/Akt) in response to fMLP stimulation in MDS neutrophils [9 ]. However, differences in IL-8- and GRO{alpha}-induced responses between MDS and healthy neutrophils have as yet scarcely been investigated. In this study, we show that migration toward IL-8 and GRO{alpha} is impaired in neutrophils from MDS patients and that this migration is dependent on ERK1/2 and PI-3K activity. Furthermore, IL-8- and GRO{alpha}-induced phosphorylation of ERK1/2 was inefficient, as was the IL-8 stimulated activation of PKB/Akt and Rac. IL-8-induced activation of Ras and Ral was normal, demonstrating that the defects observed in MDS neutrophils were not a result of a general CXCR1 or CXCR2 defect. Our results indicate a disturbed IL-8/GRO{alpha}-induced neutrophil chemotaxis in MDS patients, which might be the consequence of decreased Rac, PI-3K, and ERK1/2 activation within these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Human recombinant IL-8 and GRO{alpha} were obtained from R&D Systems (Minneapolis, MN). New England Biolabs (Beverly, MA) supplied the MEK inhibitor U0126. The PI-3K inhibitor LY294002 was purchased from Alexis (Läufelfingen, Switzerland). Polyclonal antibody against ERK1/2 (K23) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies (mAb) against phosphorylated ERK1/2 (Thr202/Tyr204) and phosphorylated PKB/Akt (Ser473) were obtained from Cell Signaling Technology (Beverly, MA). mAb against Ras, Rac, and Ral were purchased from Transduction Laboratories (Lexington, KY).

Patients
Fourteen 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 [35 ]. 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 previously [36 ]. In short, mononuclear cells were removed by centrifugation over Fycoll-Paque (Amersham, Uppsala, 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.

Migration assay
The migration assay was performed using a microchamber transwell system with 5 µM pores (Corning Costar, Corning, NY). Neutrophils were resuspended in incubation buffer with or without inhibitors for 30 min. Subsequently, 1 x 105 neutrophils were applied to the upper compartment of the chamber. Migration was induced by 5, 10, or 50 ng/ml IL-8 or GRO{alpha} in incubation buffer, present in the lower compartment of the chamber. In experiments with signal transduction inhibitors, the inhibitors were also present in the lower compartment. Cells were allowed to migrate to the lower compartment for 4 h at 37°C. The upper well was removed, and transmigrated neutrophils were harvested from the lower chamber and counted under the microscope. The assay was done in duplicates. Results are expressed as percentage, which represents the ratio between transmigrated neutrophils and the total amount of neutrophils added to the upper well.

Ras, Rac, and Ral activation assays
Activated Rac was precipitated using bacterial lysate containing glutathione S-transferase (GST)–cdc42/Rac-interactive binding (CRIB) domain of p21-activated kinse (PAK; GST-PAK-CRIB), activated Ras was pulled down using bacterial lysate containing GST–Ras binding domain of Raf (RBD; GST-Raf-RBD), and activated Ral was precipitated using bacterial lysate containing GST–Ral-binding domain of 76 kDa Ral-binding GTPase-activating protein (RLIP76; GST-RLIP-RBD) as described previously [37 , 38 ]. Briefly, neutrophils (10x106 cells/m) were stimulated with IL-8 for the indicated time and were 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 sodium orthovanadate, 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 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)].

Western blotting
The amount of phosphorylated ERK, phosphorylated PKB/Akt, and total ERK was determined by Western blotting. Neutrophils were stimulated with IL-8 or GRO{alpha} as indicated in the figures. Stimulation was terminated by placing the cells on ice, immediate centrifugation, and resuspending the cell pellets in 1x Laemmli sample buffer. After boiling for 10 min, the proteins were separated on 10% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Protran; Schleicher & Schuell, Dassel, Germany). Membranes were probed with antibodies against phosphorylated ERK1/2, phosphorylated PKB/Akt (Ser473), and ERK1/2 (K23) according to the manufacturer’s protocols. Proteins were detected by ECL. Quantification of phosphorylation levels was performed by densitometry of the films using ImageMaster1D Elite (Pharmacia, Woerden, The Netherlands).

Statistical analysis
Differences in experimental values between MDS patients and healthy donors in migration assays and the normalized densitometry values of Ral activation were calculated using the Mann-Whitney U nonparametric test for unpaired samples. Differences between migration of neutrophils and activation of Ras, Rac, and Ral with or without inhibitors were calculated using the Student’s t-test for paired samples. For quantification of phosphorylated ERK protein, densitometry values were divided by the densitometry values of total ERK1/2 protein present in the same samples, and differences between the normalized values of MDS patients and healthy donors on the same blot were calculated using the Wilcoxon Signed Ranks test for paired samples. Likewise, differences in Ras and Rac activation between healthy donors and MDS patients run on the same blot were analyzed using the Wilcoxon Signed Ranks test for paired samples. Data were expressed as mean ± SEM. P values ≤0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Decreased migration of neutrophils from MDS patients in response to IL-8 and GRO{alpha}
Migration of neutrophils from eight MDS patients and six healthy volunteers toward the chemotactic cytokine IL-8 was measured in a microchamber chemotaxis assay. Figure 1 shows that in healthy donors, increasing concentrations of IL-8 resulted in increasing percentages of migrating neutrophils (0 ng/ml, 2±1%; 5 ng/ml, 29±7%; 10 ng, 40±7%; 50 ng/ml, 80±8%). Neutrophils from MDS patients also showed increasing migratory behavior in response to higher IL-8 gradients (0 ng/ml, 2±1%; 5 ng/ml, 13±5%; 10 ng, 21±7%; 50 ng/ml, 40±9%). However, the IL-8-induced migration of neutrophils from MDS patients was significantly lower than that of their healthy counterparts (P=0.7, 0.05, 0.07, and 0.02, respectively). IL-8 stimulates CXCR1 and CXCR2. To establish whether one of these receptors was specifically responsible for the disturbed migration of MDS neutrophils, we applied the selective CXCR2 chemoattractant GRO{alpha}. The maximal migration of healthy neutrophils toward a GRO{alpha} gradient was reached between 10 and 50 ng/ml GRO{alpha}, as the percentage of cells that transmigrated at 10 ng/ml GRO{alpha} did not increase significantly when cells were allowed to migrate toward 50 ng/ml GRO{alpha} (14±2 vs. 19±4%, P=0.23, n=5). Of note, maximal GRO{alpha}-induced chemotaxis was much lower than the IL-8-induced migration (50 ng/ml GRO{alpha}, 19±4%; 50 ng/ml IL-8, 80±8%). When comparing the GRO{alpha}-stimulated chemotaxis between healthy donors (n=5) with MDS patients (n=4), it was apparent that chemotaxis was consistently lower in MDS neutrophils (10 ng/ml, 4±2 vs. 14±2%, P=0.05; 50 ng/ml, 9±3 vs. 19±4%, P=0.142).



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  		Figure 1. Decreased IL-8- and GRO{alpha}-induced migration of neutrophils from MDS patients. Neutrophils from healthy donors and MDS patients were applied to the upper compartment of a microchamber transwell system with 5 µM pores. Migration was induced by 5, 10, or 50 ng/ml IL-8 and 10 or 50 ng/ml GRO{alpha} in incubation buffer present in the lower compartment of the chamber. Cells were allowed to migrate for 4 h at 37°C after which the transmigrated neutrophils were harvested from the lower chamber and counted under the microscope. The assay was done in duplicates. Results are expressed as the ratio between transmigrated neutrophils and the total amount of neutrophils added to the upper well. The means of eight MDS patients and six healthy donors for IL-8-induced migration and four MDS patients and four healthy volunteers for GRO{alpha}-induced migration are shown.

 
Taken together, these results show that in terms of cellular function, MDS neutrophils respond less adequately to IL-8 and GRO{alpha} stimulation than neutrophils from healthy donors. Furthermore, the disturbed GRO{alpha}-induced response in MDS neutrophils implies a CXCR2 signaling defect. However, the percentage reduction of migration of MDS neutrophils in response to IL-8 is greater than maximal GRO{alpha}-induced chemotaxis. Therefore, it cannot be explained solely by the disturbed CXCR2 signaling and indicates that impaired CXCR1 signaling is also involved.

Migration of healthy neutrophils toward IL-8 or GRO{alpha} is dependent on ERK1/2 and PI-3K activation
To establish whether ERK1/2 and PI-3K are involved in IL-8- and GRO{alpha}-induced neutrophil migration in our system, we applied their specific inhibitors U0126 and LY294002, respectively, in the transwell assay. As is shown in Figure 2a , pretreatment of healthy neutrophils with 10 µM U0126 for 30 min resulted in ~50% attenuation of migration toward 50 ng/ml IL-8 (92±12 vs. 40±8%, P=0.06, n=3). Furthermore, the PI-3K inhibitor LY294002 (92±12 vs. 64±12%, P=0.014, n=3) also significantly inhibited IL-8-induced migration. Likewise, Figure 2b demonstrates that pretreatment of healthy neutrophils with the ERK1/2 inhibitor or the PI-3K inhibitor also attenuated migration toward GRO{alpha} (U0126, 22±3 vs. 11±5%, P=0.03; LY294002, 22±3 vs. 9±4%, P=0.06, n=3).



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  		Figure 2. Effect of the ERK1/2 inhibitor U0126 and the PI-3K inhibitor LY294002 on IL-8- and GRO{alpha}-induced migration of healthy neutrophils, which from three healthy donors, were resuspended in incubation buffer with or without U0126 (10 µM for 30 min) or LY294002 (10 µM for 30 min). Subsequently, 1 x 105 neutrophils were applied to the upper compartment of a microchamber transwell system with 5 µM pores, and migration was induced by 50 ng/ml IL-8 (a) or 50 ng/ml GRO{alpha} (c) in incubation buffer containing U0126 or LY294002, present in the lower compartment of the chamber. Cells were allowed to migrate for 4 h at 37°C, after which the transmigrated neutrophils were harvested from the lower chamber and counted under the microscope. The assay was done in duplicates. Results are expressed as the ratio between transmigrated neutrophils and the total amount of neutrophils added to the upper well. (b and d) Neutrophils were pretreated with 10 µM U0126 or 10 µM LY294002 for 30 min where indicated and subsequently stimulated with IL-8 (b) or GRO{alpha} (d) for the indicated times. Stimulation of the neutrophils was stopped by boiling the samples in 1x Laemmli buffer. ERK1/2 activation was detected by Western blotting, using antibodies against phosphorylated ERK1/2 (p-ERK; top panels). PKB activation was detected using antibodies against phosphorylated PKB (p-PKB; middle panels). The blots were reprobed with total ERK1/2 antibody to control for equal loading (bottom panels). Three independent experiments were performed, and one representative experiment is shown.

 
The concentration U0126 used completely abolished IL-8- and GRO{alpha}-stimulated activation of ERK1/2 in healthy neutrophils (n=3; Fig. 2c ). Toxicity of the inhibitor was ruled out, as phosphorylation of PKB/Akt was not inhibited by pretreatment of cells with U0126. The concentration LY294002 used inhibited IL-8- and GRO{alpha}-triggered activation of PKB/Akt, whereas ERK phosphorylation was hardly affected. (Out of four independent experiments, no inhibition was shown in two experiments, and slight inhibition was observed in two others.)

Together, these data indicate that IL-8- and GRO{alpha}-induced migration is at least partially dependent on the activation of ERK1/2 and PI-3K.

IL-8-induced ERK phosphorylation is higher than GRO{alpha}-stimulated ERK1/2 activation
Migration in response to IL-8 was much higher than the GRO{alpha}-induced chemotaxis of healthy and MDS neutrophils. To investigate whether these differences between IL-8 and GRO{alpha} were reflected by differences in activation of signal transduction pathways, we next stimulated neutrophils from healthy donors with increasing concentrations of IL-8 and GRO{alpha} and compared the activation of ERK1/2 and PKB/Akt. As shown in Figure 3 , maximal ERK1/2 activation was reached at 50 ng/ml GRO{alpha}, whereas ERK1/2 phosphorylation in response to IL-8 reached a maximum at 100 ng/ml IL-8. Furthermore, as for migration, the level of IL-8-induced ERK1/2 phosphorylation was higher than the GRO{alpha}-stimulated ERK1/2 activation. PKB/Akt activation also increased with increasing amounts of IL-8, although this was less apparent for GRO{alpha} stimulation. Furthermore, the differences in height of PKB/Akt activation by IL-8 or GRO{alpha} were less pronounced than observed for ERK1/2 phosphorylation.



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  		Figure 3. Differences in IL-8- and GRO{alpha}-induced ERK1/2 and PKB phosphorylation in healthy neutrophils, which were stimulated with an increasing concentration of IL-8 or GRO{alpha} for the indicated times. Stimulation of the neutrophils was stopped by boiling the samples in 1x Laemmli buffer. ERK1/2 activation was detected by Western blotting, using antibodies against phosphorylated ERK1/2 (p-ERK; top panel). PKB activation was detected using antibodies against phosphorylated PKB (p-PKB; middle panel) The blots were reprobed with total ERK1/2 antibody to control for equal loading (bottom panel). Three independent experiments were performed, and one representative experiment is shown.

 
Reduced IL-8-stimulated ERK1/2 and PKB/Akt phosphorylation in neutrophils from MDS patients
As we found that neutrophil migration in response to an IL-8 gradient was dependent on the activation of ERK1/2, we wondered whether the disturbed migration of MDS neutrophils might be a result of ineffective ERK1/2 activation in these cells. We therefore examined the phosphorylation of this signal transducer in neutrophils from MDS patients (n=5) and healthy volunteers (n=5). Figure 4 shows that in neutrophils from MDS patients and healthy donors, IL-8 stimulation resulted in a rapid and transient activation of ERK1/2. In healthy neutrophils, increasing concentrations of IL-8 (10 ng/ml vs. 50 ng/ml) induced a significantly increased phosphorylation of ERK1/2 (23±4 vs. 36±3 AU at t=30 s, P=0.04; 2±1 vs. 7±2 AU at t=2 min, P=0.04; Fig. 4b ). In neutrophils from MDS patients, a similar concentration-dependent increase in ERK1/2 activation was observed (10±1 vs. 20±43 AU at t=30 s, P=0.04; 0 vs. 1±1 AU at t=2 min, P=0.3). When the amount of phosphorylated ERK1/2 present in neutrophil lysates from MDS patients was expressed as a percentage of that present in lysates from healthy neutrophils run on the same Western blot, it was apparent that IL-8 induced a much weaker response in MDS neutrophils (Fig. 4c) . Furthermore, in four out of five patients, phosphorylation of PKB/Akt was also decreased when compared with neutrophil lysates of healthy donors run on the same Western blot (Fig. 4a , middle panels). These results demonstrate that in MDS patients, IL-8-induced ERK1/2 and PKB/Akt phosphorylation is impaired, likely resulting in the disturbed migration toward an IL-8 gradient.



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  		Figure 4. Disturbed ERK1/2 activation by IL-8 in neutrophils from MDS patients. (a) Neutrophils from five healthy volunteers and five MDS patients were stimulated with 10 or 50 ng/ml IL-8 for the indicated times. Western blotting was performed using an antibody against phosphorylated ERK1/2 (p-ERK; top panels) or phosphorylated PKB (p-PKB; middle panels). Equal loading was demonstrated by reprobing the blots with an antibody against ERK1/2 (bottom panels). Two independent experiments are shown. (b) For quantification of phosphorylated ERK protein, densitometry values were divided by the densitometry values of total ERK1/2 protein present in the same samples. Means of five independent experiments are shown. *, Significant differences between groups stimulated with 10 or 50 ng/ml IL-8 are indicated (P<0.05). **, Significant differences between healthy donors and MDS patients are indicated (P<0.05). (c) The level of phosphorylation of ERK1/2 in neutrophil lysates from MDS patients was expressed as a percentage of the phosphorylation observed in cell lysates from healthy donors run on the same gel. *, Significant differences are indicated (P<0.05).

 
Reduced GRO{alpha}-stimulated ERK1/2 phosphorylation in neutrophils from MDS patients
As GRO{alpha}-induced migration was also disturbed in neutrophils from MDS patients, we next investigated the GRO{alpha}-stimulated ERK1/2 and PKB/Akt phosphorylation in neutrophils from five MDS patients and five healthy donors (Fig. 5a ). As for IL-8 stimulation, ERK phosphorylation in response to 10 ng/ml GRO{alpha} was much lower in MDS neutrophils compared with their healthy counterparts (6±1 vs. 25±2 AU at t=10 s, P=0.04; 12±3 vs. 50±5 AU at t=30 s, P=0.04; 1±1 vs. 6±4 AU at t=2 min, P=0.3; Fig. 5b ). In contrast, PKB/Akt phosphorylation was not decreased in neutrophils from MDS patients (Fig. 5a , middle panels). These data imply that although ERK and PI-3K are involved in CXCR2-induced migration, PKB/Akt is not responsible for the decreased chemotactic response to GRO{alpha} observed in MDS neutrophils. More likely, impaired signaling through the ERK1/2 pathway results in the disturbed GRO{alpha}-induced migration. Furthermore, the normal phosphorylation of PKB/Akt indicates that the inefficient migration and ERK phosphorylation seen in neutrophils from MDS patients are not a result of a general CXCR2 defect.



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  		Figure 5. Disturbed ERK1/2 phosphorylation by GRO{alpha} in neutrophils from MDS patients. (a) Neutrophils from five healthy volunteers and five MDS patients were stimulated with 10 ng/ml GRO{alpha} for the indicated times. Western blotting was performed using an antibody against phosphorylated ERK1/2 (p-ERK; top panels) or phosphorylated PKB (p-PKB; middle panels). Equal loading was demonstrated by reprobing the blots with an antibody against ERK1/2 (bottom panels). Two independent experiments are shown. (b and c) Quantification was performed as described in Figure 4b and 4c . *, Significant differences are indicated (P<0.05).

 
IL-8-stimulated activation of Ras is normal in neutrophils from MDS patients
As activation of ERK1/2 appeared to be the common denominator in IL-8- and GRO{alpha}-induced, disturbed migration of MDS neutrophils, we wished to investigate whether this defect was located at the level of ERK1/2 itself or whether it was caused by disturbed activation of an upstream signaling molecule. As GRO{alpha}-induced responses were marginal compared with those triggered by IL-8 [compare migration (Figs. 1 and 2) and ERK phosphorylation (Fig. 3) ], we chose to focus our study on IL-8.

As ERK1/2 is known to be an important member of the well-described Ras-Raf-MEK-ERK1/2 signaling pathway, we next investigated the activation of the small GTPase Ras by using a pull-down assay in which only active GTPase is precipitated. We first excluded the possibility of Ras being activated by PI-3K by showing normal Ras activation in response to 10 ng/ml IL-8 in the presence of the PI-3K inhibitor LY294002 (Fig. 6a ). We next tested the activation of Ras in neutrophils from MDS patients (n=6) and healthy donors (n=6). Figure 6b shows that stimulation of cells with IL-8 resulted in a rapid and transient activation of Ras that was equal in neutrophils from healthy donors and MDS patients (14±3 vs. 15±2 AU at t=10 s, P= 0.8; 30±5 vs. 34±6 AU at t=30 s, P=0.9).



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  		Figure 6. Ras activation in neutrophils from healthy donors and MDS patients. (a) Neutrophils were stimulated with 10 ng/ml IL-8 for the indicated times with or without pretreatment with 10 µM LY294002 for 30 min. Activated Ras was precipitated using GST-Raf-RBD protein and visualized by Western blotting with Ras antibodies. Four independent experiments were performed, of which two are shown. (b) Neutrophils from six healthy controls and six MDS patients were stimulated with 10 ng/ml IL-8 for the indicated times, and activated Ras was precipitated. Three representative experiments are shown. Protein levels of activated Ras were quantified by densitometry of the films. The means of the normalized values were calculated for the MDS patients and healthy donors.

 
Inefficient IL-8-induced activation of Rac in neutrophils from MDS patients
Another GTPase implicated in ERK1/2-dependent and ERK1/2-independent migration is Rac. Stimulation of Rac activation by IL-8 was not inhibited by pretreatment of neutrophils with the PI-3K inhibitor LY294002 (Fig. 7a ; n=5). Figure 7b shows a high basal activity of Rac present in unstimulated neutrophils. Stimulation of neutrophils from healthy donors (n=5) with 10 ng/ml IL-8 resulted in a rapid and transient activation of Rac with a maximum at 10 s of stimulation (9±1 at t=0 vs. 15±2 at t=10 s, P=0.04; 9±1 at t=0 vs. 11±1 at t=30 s, P=0.08). Although there was no significant difference in the amount of activation of Rac in MDS patients in direct comparison with healthy donors (12±1 vs. 15±2 at t=10 s, P=0.35), Rac activation in MDS neutrophils did not reach statistical significance at 10 s of IL-8 stimulation (8±1 at t=0 vs. 12±1 at t=10 s, P=0.14; 8±1 at t=0 vs. 12±1 at t=30 s, P=0.08). These results show that Rac activation is less efficient in MDS patients, possibly resulting in the disturbed ERK activation observed in MDS cases.



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  		Figure 7. Rac activation in neutrophils from healthy donors and MDS patients. (a) Neutrophils were stimulated with 10 ng/ml IL-8 for the indicated times with or without pretreatment with 10 µM LY294002 for 30 min. Activated Rac was precipitated using GST-PAK-CRIB and visualized by Western blotting with Rac antibodies. Five independent experiments were performed, of which two are shown. (b) Neutrophils from five healthy controls and five MDS patients were stimulated with 10 ng/ml IL-8 for the indicated times, and activated Rac was precipitated. Three representative experiments are shown. Protein levels of activated Rac were quantified by densitometry of the films. The means of the normalized values were calculated for the MDS patients and healthy donors. *, Significant differences between unstimulated and IL-8-stimulated groups are indicated (P<0.05).

 
Normal Ral activation in neutrophils from MDS patients
Another signal transduction molecule that has been implicated in cell migration and cytoskeletal reorganization is the small GTPase Ral [39 , 40 ]. We first ruled out that activation of Ral is dependent on PI-3K or ERK1/2 activity. Figure 8a shows that IL-8-induced Ral activity was not attenuated by the PI-3K inhibitor LY294002 or the ERK1/2 inhibitor U0126. Next, we studied the activation of Ral in neutrophils from healthy donors (n=4) and MDS patients (n=4). As shown in Figure 8b and 8c , Ral activation in response to 10 ng/ml IL-8 was rapid and transient in neutrophils from healthy donors and similar to that in neutrophils from MDS patients (67±9 vs. 60±5 AU for t=30 s, P=0.7; 15±4 vs. 17±2 AU for t=2 min, P=0.7).



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  		Figure 8. Ral activation in neutrophils from healthy donors and MDS patients. (a) Neutrophils were stimulated with 10 ng/ml IL-8 for the indicated times with or without pretreatment with 10 µM LY294002 or 10 µM U0126 for 30 min. Activated Ral was precipitated using GST-RLIP-RBD protein and visualized by Western blotting with Ral antibodies. Four independent experiments were performed, of which two are shown. (b) Neutrophils from six healthy controls and four MDS patients were stimulated with 10 ng/ml IL-8 for the indicated times, and activated Ral was precipitated. Two representative experiments are shown. Protein levels of activated Ral were quantified by densitometry of the films. The means of the normalized values were calculated for the MDS patients and healthy donors.

 
Taken together, these results demonstrate that IL-8-triggered Ras and Ral activation is normal in MDS neutrophils, thereby eliminating the possibility that the decreased migration and ERK phosphorylation observed in neutrophils from MDS patients are a result of a general IL-8 receptor defect.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neutrophils from patients suffering from myelodysplasia generally show impaired neutrophil functioning. Several processes involved in the attraction of neutrophils toward sites of inflammation and the killing of invading bacteria have been shown to be affected. For example, chemotaxis of neutrophils from patients with MDS toward a fMLP gradient were shown to be impaired [13 , 41 ]. We now show that neutrophil migration toward IL-8 and GRO{alpha} chemotactic gradients are also disturbed in MDS patients.

Motility of neutrophils is preceded by polymerization of cytoplasmic F-actin and pseudopod extension. A recent study demonstrated that whereas activation of PI-3K was essential for fMLP-induced pseudopod extension, IL-8-stimulated pseudopod extension was much less dependent on PI-3K [42 ]. Furthermore, IL-8- but not fMLP-induced F-actin polymerization, pseudopod extension, and migration have been shown to be enhanced by pretreatment of cells with GM-CSF or insulin [30 , 42 , 43 ]. Moreover, this GM-CSF priming was PI-3K-dependent, whereas priming by insulin was PI-3K-independent. These data clearly indicate the existence of multiple pathways leading to migration in response to different stimuli. This would seem logical; neutrophils encountering an IL-8 gradient in the human body are recruited to the general vicinity of a source of infection, where intracellular signaling hierarchy dictates that cells subsequently migrate toward end-target chemoattractants such as fMLP by overriding IL-8-induced signaling [19 , 20 ].

IL-8 stimulates two receptors, CXCR1 and CXCR2, whereas GRO{alpha} specifically activates CXCR2. The disturbed migration toward a GRO{alpha} gradient in MDS neutrophils indicates that CXCR2 signaling is affected. It is therefore likely that at least part of the reduced migration of MDS neutrophils toward IL-8 is a result of impaired CXCR2 signaling. However, the maximal achievable migration induced by GRO{alpha} was much lower than that induced by IL-8, indicating that CXCR1 plays the major part in IL-8-stimulated migration. Furthermore, the reduction of IL-8-induced migration in MDS neutrophils was too large to be explained solely by disturbed CXCR2 signaling, indicating that CXCR1 signaling is also affected.

In the present study, we show that IL-8- and GRO{alpha}-induced migration was dependent on the activation of the MAPK ERK1/2. However, despite complete abrogation of ERK1/2 phosphorylation by U0126, migration was not completely abolished, implicating the involvement of other signal transduction pathways. Indeed, we also demonstrated that the lipid kinase PI-3K was involved in IL-8- and GRO{alpha}-induced migration. As ERK1/2 phosphorylation was not significantly affected by the PI-3K inhibitor LY294002, it can be concluded that activation of CXCR1 and CXCR2 leads to migration through multiple, independent pathways. Indeed, a recent study has shown that yet another MAPK, p38, might also regulate directional granulocyte migration toward IL-8 [44 ].

The dependency of neutrophil migration on ERK1/2 and PI-3K activation prompted us to investigate the activation of these signal transduction pathways in MDS neutrophils. We showed that the activation of ERK1/2 was severely impaired in neutrophils from MDS patients. Not merely the height of activation but also the duration of activation of ERK1/2 were disturbed in MDS neutrophils in response to IL-8. Although the GRO{alpha}-stimulated chemotaxis was shown to be attenuated by the ERK1/2 and PI-3K inhibitors, only ERK1/2 activation was affected in MDS patients. This indicates that CXCR2-activated PI-3K activation possibly results in migration through a PKB/Akt-independent pathway. However, studies using cell lines transfected with dominant-negative or constitutive-active PKB/Akt constructs have clearly demonstrated its involvement in migration of these cell lines [45 , 46 ].

Several signal transducers have been shown to be able to activate ERK1/2 in vitro. The best described of these is the small GTPase Ras. Our studies indicate that Ras activation in response to IL-8 stimulation is normal in MDS neutrophils. The GTPase Rac has also been described to activate ERK1/2 [47 , 48 ] via PAK [33 ]. Our studies show that IL-8-triggered activation of Rac is less effective in neutrophils from MDS patients, possibly resulting in the disturbed ERK1/2 activation and subsequently disturbed migration. In addition, Rac and ERK activation have been described to lead to migration through two separate mechanisms [49 ]. The disturbed IL-8-induced migration of MDS neutrophils could therefore also be a result of disturbed activation by Rac and ERK1/2 of two independent pathways leading to migration.

Upon stimulation of the neutrophils with IL-8, its receptors become desensitized and are less susceptible to subsequent restimulation with IL-8. Desensitization results in a decreased migration and phosphorylation of IL-8 receptor targets [50 ]. A recent study has suggested that in MDS patients, serum levels of IL-8 are elevated [51 ]. It would therefore be conceivable that in these patients, in vivo desensitization of CXCR1 and CXCR2 might result in a decreased in vitro stimulation of IL-8 receptor functions. However, the normal IL-8-stimulated Ras and Ral activation that was observed in MDS neutrophils makes it unlikely that desensitization plays an important role in this study.

In conclusion, we showed that ERK1/2-dependent migration toward IL-8 is impaired in neutrophils from MDS patients and that IL-8-stimulated activation of Rac and phosphorylation of ERK1/2 and PKB/Akt are disturbed in MDS neutrophils, despite a normal activation of Ras and Ral. Furthermore, impaired GRO{alpha}-stimulated migration and ERK1/2 activation indicate that disturbed CXCR2 signaling, besides a defective CXCR1 signaling, plays a role in this. Our results suggests a defective IL-8-induced Rac-ERK and PI-3K signaling in MDS patients, which might result in an increased susceptibility to bacterial infections by affecting neutrophil chemotaxis.


   ACKNOWLEDGEMENTS
 
This work was supported by the Dutch Cancer Society (RUG 2003-2920). The authors thank all patients and healthy volunteers who participated in this study.

Received May 27, 2004; revised September 29, 2004; accepted October 21, 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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