|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PHAGOCYTES
From the Division of Hematology, Department of
Medicine, University Hospital Groningen, The Netherlands,
and the Blood Bank Noord Nederland, Groningen, The
Netherlands.
Neutrophils from patients with myelodysplastic syndrome (MDS) show
a disturbed differentiation pattern and are generally dysfunctional. To
study these defects in more detail, we investigated reactive-oxygen species (ROS) production and F-actin polymerization in neutrophils from
MDS patients and healthy controls and the involvement of N-formyl-L-methionyl-L-lucyl-L-phenylaline (fMLP) and granulocyte macrophage-colony-stimulating factor (GM-CSF)-stimulated signal transduction pathways. Following fMLP stimulation, similar levels of respiratory burst, F-actin polymerization, and activation of the
small GTPase Rac2 were demonstrated in MDS and normal neutrophils. However, GM-CSF and G-CSF priming of ROS production were significantly decreased in MDS patients. We subsequently investigated the signal transduction pathways involved in ROS generation and demonstrated that
fMLP-stimulated ROS production was inhibited by the
phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the
MAPK/ERK kinase (MEK) inhibitor U0126. In contrast, ROS
production induced by fMLP stimulation of GM-CSF-primed cells was
inhibited by LY294002 and U0126. This coincides with enhanced protein
kinase B (PKB/Akt) phosphorylation that was PI3K dependent and enhanced
extracellular signal-regulated protein kinase 1 and 2 (ERK1/2)
phosphorylation that was PI3K independent. We demonstrated higher
protein levels of the PI3K subunit p110 in neutrophils from MDS
patients and found that though the fMLP-induced phosphorylation of
PKB/Akt and ERK1/2 could also be enhanced by pretreatment with GM-CSF in these patients, the degree and kinetics of PKB/Akt and ERK1/2 phosphorylation were significantly disturbed. These defects were observed despite a normal GM-CSF-induced signal transducer and activator of transcription 5 (STAT5) phosphorylation. Our results indicate that the reduced priming of neutrophil ROS production in MDS
patients might be caused by a disturbed convergence of the fMLP and
GM-CSF signaling routes.
(Blood. 2003;101:1172-1180) Myelodysplastic syndromes (MDS) are characterized
by a differentiation defect in the multipotent stem cell compartment,
resulting in a disturbed proliferation and differentiation of the
erythroid, myeloid, or megakaryocytic lineage.1 With
regard to the myeloid lineage, it has been shown that the intrinsic
defect leads to granulocytopenia and to aberrant functioning of
neutrophils. For instance, there have been reports indicating that
reorganization of the actin cytoskeleton2 and the
production of reactive-oxygen species (ROS)3,4 are affected
in neutrophils from MDS patients.
Bacterial infections are the most common cause of death in MDS
patients.5 The capacity of neutrophils to produce ROS such as O Respiratory burst and additional neutrophil effector functions such as
granule trafficking and phagocytosis are dependent on the organization
of the actin cytoskeleton.12-15 There are 2 forms of
actin, a monomeric form (G-actin) and a filamentous form (F-actin),
that exist in equilibrium in the cell. On the stimulation of
neutrophils with chemotactic factors, rapid conversion of G-actin to
F-actin occurs.16,17 In contrast to ROS production,
F-actin polymerization cannot be primed by proinflammatory factors.
Different signal transduction pathways have been identified that are
critical for the cellular functions of neutrophils. Rac2, a member of
the Rho family of small GTPases, acts as a molecular switch by cycling
between an inactive guanosine diphosphate (GDP)-bound state and an
active guanosine triphosphate (GTP)-bound state. Rac2 is involved in
the reorganization of the actin cytoskeleton18-20 and the
fMLP mediated respiratory burst, as demonstrated by experiments in
Rac In this study, we further characterized the disturbed neutrophil
responses in MDS and questioned which signal transduction pathways
might be involved in aberrant neutrophil functioning. We demonstrated
normal fMLP-mediated respiratory burst, F-actin polymerization, and
Rac2 activation in neutrophils from MDS patients but found a strongly
decreased priming effect of GM-CSF and G-CSF on ROS production.
Analysis of signal transduction pathways revealed higher protein
expression levels of the PI3K subunit p110 in neutrophils from MDS
patients. Further examination also indicated altered phosphorylation
levels of protein kinase B (PKB/Akt) and ERK1/2 in MDS patients.
Reagents
Patients
Isolation of neutrophils Peripheral blood, anticoagulated with 0.32% sodium citrate, was obtained from healthy volunteers and patients with MDS. Neutrophils were isolated as described by Koenderman et al.32 In short, mononuclear cells were removed by centrifugation over Ficoll-Hypaque (Amersham, Uppsala, Sweden), and erythrocytes were lysed with ice-cold NH4Cl solution. Neutrophils were allowed to recover for 30 minutes 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 [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 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 more than 95% neutrophils as determined by May-Grünwald-Giemsa staining.F-actin polymerization assay F-actin polymerization was measured as described by Nijhuis et al.33 In short, neutrophils (2.5 × 106 cells/mL) were stimulated with 1 µM fMLP. At indicated time points, cells were fixed and permeabilized for 10 minutes at room temperature with ice-cold 3% formaldehyde in phosphate-buffered saline (PBS), containing 100 µg/mL lysophosphatidylcholine (Sigma, St Louis, MO). Polymerized F-actin was stained with 2.5 U/mL Oregon Green 514-phalloidin for 30 minutes at room temperature. Intracellular fluorescence was determined by flow cytometry (FACScalibur; Becton Dickinson Medical Systems, Sharon, MA).Respiratory burst measurement Production of H2O2 was measured as described by Mardiney et al,34 with some alterations. Briefly, neutrophils (2.5 × 106 cells/mL) were incubated with DHR123 for 15 minutes and were stimulated with 1 µM fMLP for 30 minutes. For priming experiments, cells were pretreated with 5 ng/mL GM-CSF, 2.5 ng/mL G-CSF, or both for 15 minutes before fMLP stimulation. When indicated, neutrophils were pretreated with signal transduction inhibitors. Stimulation of the neutrophils with fMLP was terminated by washing the cells with ice-cold PBS containing 1% HSA and placing them on ice. Oxidation of DHR123 to fluorescent rhodamine 123 was measured by fluorescence-activated cell sorter (FACS) analysis within 30 minutes.Alternatively, O Rac activation assay Activated Rac was precipitated using the CDC42 and Rac interactive binding (CRIB) domain of PAK (aa 56-272) as described previously.36 Briefly, neutrophils (1 × 106 cells/mL) were stimulated with 0.1 µM fMLP for the indicated time and were lysed for 10 minutes in lysis buffer (50 mM Tris [tris(hydroxymethyl)aminomethane] pH 7.4, 10% glycerol, 200 mM NaCl, 1% NP-40, 2 mM MgCl2, 2 mM sodium orthovanadate, and protease inhibitors [1 tablet Complete (Roche, Mannheim, Germany)/50 mL buffer]). Lysates were cleared by centrifugation, and GST-PAKcrib protein precoupled to glutathione-Sepharose beads (Pharmacia, Uppsala, Sweden) was added for 30 to 45 minutes at 4°C. Beads were washed 3 times with 1× lysis buffer and were boiled in Laemmli sample buffer. 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 was detected by probing the membrane with anti-Rac antibodies and rabbit antimouse peroxidase-conjugated antibodies (DAKO, Copenhagen, Denmark) followed by enhanced chemiluminescence.Western blotting The amount of ERK and PI3-kinase p110 protein and the phosphorylation of STAT5, ERK, and PKB/Akt were determined by Western blotting. Neutrophils (2 × 106 cells/mL) were preincubated with inhibitors and stimulated with 1 µM fMLP or 5 ng/mL GM-CSF, as indicated in the figures. Stimulation was terminated by placing the cells on ice, subjecting them to immediate centrifugation, and resuspending the cell pellets in 1× Laemmli sample buffer. After boiling for 10 minutes, 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 ERK1/2 (K23), PI3-kinase p110, phosphorylated STAT5A/B (Y694/Y699), phosphorylated ERK1/2, and phosphorylated PKB/Akt (Ser473) according to the manufacturer's protocols. Proteins were detected by enhanced chemiluminescence. The total amount of PKB/Akt present in the samples could not be quantified on membranes that were previously probed with antibodies against phosphorylated PKB/Akt because of high chemiluminescence of the phosphorylated protein. However, Western blot analysis of cell lysates from 3 MDS patients and 3 healthy volunteers run on one gel and probed with PKB/Akt antibody (Cell Signaling Technology, Beverly, MA) did not show differences in the amount of total PKB/Akt protein (data not shown). Quantification of phosphorylation levels was performed by densitometry of the films, using ImageMaster1D Elite (Pharmacia, Woerden, The Netherlands).Statistical analysis For respiratory burst measurements, differences in experimental values between healthy controls and MDS patients were calculated using the Mann-Whitney U test. Differences between paired samples were calculated using the Wilcoxon nonparametric test or the Student t test for samples treated with signal transduction inhibitors. For comparison of PI3K p110 levels in MDS patients and healthy donors, densitometry values of p110 protein were corrected for the amount of ERK protein present in the samples, and differences between the normalized values were calculated using the Student t test for unpaired samples. For quantification of phospho-PKB/Akt and phospho-ERK1/2 blots, differences in densitometry values of healthy controls as a percentage of that of MDS patients were calculated using the Student t test for paired samples. Data were expressed as mean ± SEM. P < .05 was considered significant.
fMLP-stimulated F-actin polymerization and Rac2 activation are not disturbed in neutrophils from MDS patients fMLP-induced F-actin polymerization was examined and compared in neutrophils from MDS patients and healthy controls (n = 8 for each group). On stimulation of neutrophils with 1 µM fMLP, a transient increase in the relative amount of F-actin was observed that was maximal within 30 seconds and declined after 2 to 5 minutes. The maximum relative increase of F-actin content in neutrophils from healthy subjects was 507% ± 22%. This was comparable to the maximum relative increase of F-actin in neutrophils from MDS patients (507 ± 52%; P = .963), though in MDS patients there was a wider variation in relative F-actin increase. The kinetics of F-actin polymerization in MDS neutrophils was similar to that of healthy volunteers (data not shown). Although patients and healthy controls showed no significant difference in the kinetics and relative amount of F-actin formed in response to fMLP, 3 MDS patients did have a subpopulation of neutrophils (21%, 48%, and 49% of the total population) that had virtually no detectable F-actin polymerization (Figure 1). These subpopulations could not be distinguished from the responsive neutrophil populations in dot-plot profiles because their scattering profiles were identical.
To confirm the normal fMLP response of MDS neutrophils at the
molecular level, fMLP-stimulated Rac2 activation was studied by using a
GST-beads pull-down technique. Figure
2A demonstrates that maximal Rac2
activation in neutrophils from healthy subjects was achieved after 15 seconds of fMLP stimulation and declined to near baseline levels within
15 minutes. Similar results were obtained in neutrophils from MDS
patients (n = 8), though there was some variation in the extent and
time course of Rac2 activation among donors (Figure 2B). The mean
increase in Rac2 activation after 15 seconds of fMLP stimulation was
729% ± 202% in neutrophils from healthy donors (n = 8) versus
658 ± 179% in neutrophils from MDS patients, as determined by
densitometry of the films (P = .795; data not shown).
Western blot analysis of whole cell lysates of neutrophils from 5 MDS
patients and 3 healthy controls, run on one gel, demonstrated equal
amounts of total Rac protein present in all individuals (data not
shown).
Priming of the respiratory burst leads to an enhancement of superoxide radical production that is higher in healthy controls than in MDS patients Given that the production of ROS is an essential antibacterial defense mechanism of neutrophils, we studied the amount of superoxide produced in neutrophils in response to fMLP and the enhancement of this response by priming with GM-CSF, G-CSF, or both. H2O2 production was calculated by expressing the fluorescence of rhodamine 123 in stimulated cells as a percentage of the fluorescence of unstimulated cells. As shown in Figure 3A, fMLP stimulation induced the production of ROS to a similar level in neutrophils from healthy controls and MDS patients (196% ± 18% and 257% ± 48%, respectively; P = .753). In neutrophils from healthy donors, stimulation with GM-CSF or G-CSF alone did not lead to H2O2 production (data not shown). However, priming of the neutrophils with GM-CSF, G-CSF, or both, followed by fMLP stimulation, led to a significantly higher respiratory burst (656% ± 65%, 329% ± 44%, and 646% ± 60%, respectively) than when cells were stimulated with fMLP alone (P = .012 for all 3 groups). In neutrophils from MDS patients, the mean percentage increases in fluorescence intensity stimulated by 1 µM fMLP following GM-CSF and G-CSF priming were 323% ± 39% and 276% ± 50%, respectively, which was significantly higher than fMLP stimulation alone (P = .017 and P = .018, respectively). However, as shown in Figure 3B, the percentage increase in H2O2 production because of priming of neutrophils with GM-CSF, G-CSF, or both was significantly decreased in neutrophils from MDS patients than in healthy controls (P = .002, P = .012, and P = .003 respectively). These results indicate that whereas fMLP-triggered H2O2 production is normal in MDS patients, GM-CSF and G-CSF priming of the respiratory burst are considerably disturbed. These results were confirmed for O
PI3K is involved in fMLP- and GM-CSF-stimulated respiratory burst and PKB/Akt phosphorylation in neutrophils from healthy donors Our studies indicate that the priming effect of GM-CSF and G-CSF on the fMLP-induced respiratory burst is disturbed in MDS. To elucidate the signal transduction routes involved in the ROS production in healthy neutrophils, several chemical inhibitors of signal transduction pathways were used. First, we tested the effect of the specific PI3K inhibitor, LY294002, on the fMLP-stimulated respiratory burst in neutrophils, with or without GM-CSF pretreatment. Figure 5A demonstrates that the fMLP-induced respiratory burst could be significantly inhibited at a concentration of 1 µM LY294002 (32% ± 8%; P = .021). Higher concentrations of LY294002 led to an even more pronounced reduction in fMLP-stimulated ROS production (66% ± 5% and 85% ± 8% for 10 and 100 µM LY294002, respectively; P < .05). The GM-CSF-primed respiratory burst was also significantly attenuated by preincubation of neutrophils with all concentrations of LY294002 tested. At concentrations of 1 and 10 µM LY294002, the reduction in fMLP-stimulated ROS production was significantly higher in the GM-CSF-primed group (70% ± 4% and 89% ± 4%, respectively) than in the unprimed group (P < .05). Incubation of neutrophils with 100 µM LY294002 led to an inhibition of the GM-CSF-primed ROS production of 93% ± 3%, which was statistically similar to that in the unprimed group.
An important downstream target of PI3K is PKB/Akt.37,38 As shown in Figure 5B (upper panel), fMLP induced PKB/Akt serine phosphorylation in a time-dependent manner. Phosphorylation was maximal within 2 minutes and declined to near basal levels after 10 minutes of stimulation. GM-CSF alone stimulated PKB/Akt phosphorylation levels to a much lower extent (Figure 5B). Longer exposure demonstrated that GM-CF-stimulated phosphorylation occurred at a maximum at 2 minutes (Figure 5C). Interestingly, PKB/Akt phosphorylation by fMLP could be enhanced by pretreatment of the neutrophils with GM-CSF. Figure 5D demonstrates that the phosphorylation of PKB/Akt by stimulation of unprimed or GM-CSF-primed neutrophils with fMLP, or GM-CSF alone, could be partially inhibited by preincubation of the neutrophils with 1 µM LY294002 and completely inhibited with 10 µM. This indicates that the concentrations LY294002 used effectively inhibited PI3K activation. Our results demonstrate that fMLP stimulation of neutrophils leads to the production of reactive oxygen species at least partially by activating PI3K, and they imply that the priming effect of GM-CSF on the fMLP-induced neutrophil burst is completely dependent on the activation of PI3K. GM-CSF-mediated enhancement of fMLP-stimulated respiratory burst is inhibited by preincubation of healthy neutrophils with the ERK inhibitor, U0126 Next, we studied the role of the MEK/ERK pathway in the fMLP-induced respiratory burst in neutrophils by using the extremely potent, selective inhibitor of MEK1/2, U0126. Figure 6A shows that unprimed fMLP-stimulated ROS production was not significantly reduced by preincubation of the neutrophils with U0126 (39% ± 20%; P = .554). However, GM-CSF priming of the respiratory burst could be significantly inhibited by 10 µM U0126 for 65% ± 1% (P = .021). Similar results were obtained when neutrophils were preincubated with 50 µM PD98059, another chemical MEK1/2 inhibitor (data not shown).
We subsequently examined the activation of the MEK/ERK pathway at the protein level. Phosphorylation of Thr202/Tyr204 of ERK1/2 was observed within 30 seconds of fMLP stimulation (Figure 6B). GM-CSF stimulation alone led to ERK1/2 phosphorylation at a maximum at 2 minutes, though at a much lower level than fMLP-stimulated ERK1/2 phosphorylation. Priming of neutrophils with GM-CSF, followed by fMLP stimulation, resulted in enhanced ERK1/2 phosphorylation compared with the effects of either fMLP or GM-CSF alone. The MEK1/2 inhibitor U0126, when added at a concentration of 10 µM, was capable of completely inhibiting ERK1/2 phosphorylation in unprimed and GM-CSF-primed neutrophils stimulated with 1 µM fMLP and in neutrophils stimulated with 5 ng/mL GM-CSF alone (Figure 6C). Together these data suggest that the MEK-ERK route is not involved in the unprimed ROS production but that it does play a role in the GM-CSF-priming effect of the respiratory burst. To establish the place of ERK in the signal transduction cascade,
neutrophils were preincubated with LY294002 before fMLP or GM-CSF
stimulation. As shown in Figure 7, ERK1/2
activation by fMLP, with or without GM-CSF priming, was not inhibited
by preincubation of the cells with increasing concentrations of
LY294002. In contrast, the ERK1/2 activation induced by GM-CSF
stimulation alone was completely inhibited at 10 µM LY294002. These
results indicate that whereas GM-CSF-mediated ERK1/2 phosphorylation
is a downstream event of PI3K activation, the fMLP-stimulated ERK1/2 phosphorylation in GM-CSF-primed cells is PI3K independent.
Altered PI3K p110 protein levels and decreased PKB/Akt phosphorylation in neutrophils from MDS patients Because our results indicate involvement of PI3K in the priming of the respiratory burst and disturbed priming of this burst in neutrophils from MDS patients, we investigated the protein levels of the PI3K subunit p110 in neutrophils from healthy donors (n = 5) and MDS patients (n = 7). Figure 8 shows that though the p110 levels differed between individual donors (Figure 8A), the amount of p110 present in the neutrophil lysates of MDS patients was significantly higher than in healthy volunteers after correction for ERK levels (Figure 8B, P < .05). These results indicate increased protein expression levels of the catalytically active p110 subunit in MDS neutrophils, possibly resulting in a disturbed regulation of the PI3K signaling pathway.
We subsequently examined the activation of PKB/Akt in neutrophils from
MDS patients (n = 5). Isolated neutrophils were stimulated with fMLP
with or without GM-CSF pretreatment or GM-CSF alone, and proteins were
separated by SDS-PAGE. In each experiment, lysates of neutrophils from
healthy controls, stimulated under the same experimental conditions,
were run as controls. Time-curve experiments in neutrophils from MDS
patients demonstrated maximal PKB/Akt phosphorylation after 30 seconds
of fMLP stimulation and after 2 minutes of GM-CSF stimulation in most
patients (Figure 9A-C). As in healthy
donors, GM-CSF priming of the neutrophils from the MDS patients
resulted in enhanced phosphorylation of PKB/Akt compared with the
effects of fMLP or GM-CSF alone. Interestingly, in all cases studied,
the total amount of phosphorylated PKB/Akt present after stimulation
with fMLP with or without priming appeared lower in MDS neutrophils
than in healthy controls (compare lanes 3 and 9 and lanes 6 and 10 in
Figure 9A and lanes 3 and 11 and lanes 6 and 12 in Figure 9B-C).
Quantification of the phosphorylation levels by densitometry indicated
that the amount of phosphorylated PKB/Akt present in fMLP-stimulated
neutrophil lysates was significantly higher in healthy donors than in
MDS patients (P < .05). In GM-CSF-primed cells, fMLP
stimulation also led to a higher phosphorylation of PKB/Akt in healthy
donors than in MDS patients, though this was not significant, because
of the high SEM (P = .073) (Figure 9D). Furthermore, a
difference in the kinetics of phosphorylation was noted between MDS
neutrophils and healthy controls. In 4 of 5 patients studied, the
amount of fMLP-induced phosphorylation of PKB/Akt following GM-CSF
priming was identical at 30 seconds and 2 minutes of stimulation,
whereas in most healthy controls the phosphorylation level was
significantly higher at 2 minutes than at 30 seconds (Figure 5B and
data not shown). Protein loading was equal in all samples, as assessed
by Western blotting for ERK1/2. These data indicate that in neutrophils
from MDS patients, the kinetics and the maximal levels of PKB/Akt
phosphorylation induced by fMLP stimulation and GM-CSF priming are
affected.
Phosphorylation of ERK1/2 is decreased in neutrophils from MDS patients Because we demonstrated that the MEK/ERK pathway is also involved in the GM-CSF-mediated priming of the respiratory burst, we compared the phosphorylation pattern of ERK1/2 after stimulation in neutrophils from healthy volunteers and MDS patients. FMLP-induced phosphorylation in MDS neutrophils could be enhanced by GM-CSF pretreatment in a manner similar to that in neutrophils from healthy controls (Figure 9A-C, middle panels). In all cases it was observed that the total amount of phosphorylated ERK1/2 present after 2 minutes of fMLP stimulation was slightly, but significantly, higher in GM-CSF-primed and unprimed neutrophils from healthy donors than in those from MDS patients (P = .013 and P = .010, respectively), though this was less pronounced than for phosphorylated PKB/Akt (Figure 9D).Finally, to exclude the possibility that the GM-CSF receptor in MDS is
defective in general, we examined the capability of GM-CSF to induce
tyrosine phosphorylation of STAT5. Figure
10 shows that GM-CSF-induced STAT5
phosphorylation, though varying slightly between persons, did not
differ significantly between MDS patients and healthy subjects. These
data suggest that GM-CSF receptors on neutrophils from MDS
patients are functional.
The present study demonstrates that the responses of neutrophils from MDS patients are significantly disturbed with regard to GM-CSF and G-CSF priming of ROS production, whereas the fMLP-mediated cellular effects are not affected. In contrast to our findings, Carulli et al2 demonstrated a defect in the F-actin polymerization in neutrophils from MDS patients, but that applied especially to patients with refractory anemia (RA) with excess blasts (RAEB), RAEB in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML).2 In the present study, with patients with RA and RA with ringed sideroblasts (RARS), no significant decrease in actin polymerization was observed, though in 3 of 8 patients a neutrophil subpopulation was observed that did not polymerize F-actin in response to fMLP. However, respiratory burst measurements in these patients did not reveal separate neutrophil subpopulations; all neutrophils were capable of producing H2O2 following fMLP stimulation, indicating that the lack of F-actin polymerization was not attributed to an fMLP receptor defect. These results were further supported by our finding that Rac2 activation after fMLP stimulation was normal in neutrophils from MDS patients. In contrast, the priming effects of GM-CSF and G-CSF on ROS production
were severely disturbed in neutrophils from MDS patients. Previous
studies by Zabernigg et al3 and Ohsaka et al4
also demonstrated disturbed GM-CSF priming of the respiratory burst in
neutrophils from most, but not all, MDS patients. In contrast to our
study, Ohsaka et al4 found increased fMLP-induced
O The respiratory burst in neutrophils is produced by the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase complex, which in its inactive state consists of flavocytochrome b558, stored in the membrane of specific granules, and the cytosolic components p47phox, p67phox, p40phox, and Rac2. On stimulation of the neutrophils, the expression of the flavocytochrome on the plasma membrane is up-regulated, and the cytosolic components are transported to the membrane.39,40 DeLeo et al41 have shown that lipopolysaccharide (LPS) priming of the neutrophil respiratory burst is accompanied by enhanced recruitment of flavocytochrome b558 and association of p47phox, p67phox, and Rac2 with the plasma membrane. Recently, it has been described that PI3K binds and activates the NADPH complex, including p40phox and p47phox, suggesting that it plays a major role in the respiratory burst.28,29 This is confirmed by our finding that the PI3K inhibitor LY294002 is capable of inhibiting fMLP-stimulated ROS production in unprimed and GM-CSF-primed neutrophils. To what extent the PI3K downstream target PKB/Akt is involved in the activation of the NADPH complex is unknown. Its involvement is often assumed as a result of the corresponding kinetics between PKB/Akt activation and respiratory burst, as measured by cytochrome c reduction in response to chemoattractants.42 Furthermore, as for ROS production, we show that the response of neutrophils to fMLP with regard to PKB/Akt phosphorylation can be primed by GM-CSF. fMLP-induced p47phox phosphorylation has been shown to be mediated by ERK1/2.43 However, the role of ERK1/2 in the chemoattractant-stimulated respiratory burst is disputed. Some studies indicate that MAPK is at least partially responsible for fMLP-induced ROS formation,30,43-45 whereas in additional studies, no effect of ERK inhibitors on the neutrophil respiratory burst were observed.26,46 The present study demonstrates that fMLP-mediated ROS production is not significantly attenuated by the MEK inhibitor U0126. Moreover, it appeared that LY294002 at 1 and 10 µM significantly inhibited the fMLP-triggered respiratory burst without an effect on the fMLP-stimulated ERK1/2 phosphorylation in unprimed or GM-CSF-primed neutrophils, indicating a PI3K-independent activation of ERK. These data corroborate findings from Kodama et al,46 who demonstrated that GM-CSF priming of ERK activity could not be inhibited by LY294002. However, ROS formation in response to fMLP in GM-CSF primed neutrophils was significantly inhibited by the MEK inhibitor U0126. These findings underscore the role of ERK in ROS production of primed granulocytes independent of the PI3K cascade and are in line with the recent findings of Hedin et al.47 They demonstrate that ERK activation by Gi protein is regulated in part by a Ras- and a PI3K-independent pathway that might be modulated by tyrosine kinase. One of signaling cascades that might be involved in this process is the GTPase Rap, which can activate ERK independently of Ras.48 Limited studies have been focused on the signaling events in hematopoietic cells of MDS patients. STAT5 DNA binding in response to erythropoietin (EPO) was shown to be impaired in cells of the erythroid lineage.49,50 EPO exerts its STAT5-activating effect through the phosphorylation of Janus kinase 2 (JAK2),51,52 suggesting a central role for JAK2 in the disturbed signaling in MDS. GM-CSF stimulation of neutrophils from MDS patients led to STAT5 tyrosine phosphorylation that was comparable to that in healthy controls, implying that GM-CSF receptor and JAK2 activation are normal in MDS. In contrast, a significant defect was observed with regard to PKB/Akt and ERK1/2 phosphorylation. fMLP-stimulated PKB/Akt phosphorylation in unprimed and GM-CSF-primed neutrophils was drastically lowered in MDS patients. Moreover, the kinetics of PKB/Akt phosphorylation were different than they were in healthy controls. These findings might be related to the altered p110 protein levels found in MDS neutrophils. The abundance of the catalytically active PI3K subunit p110 might result in a disturbed activation of the PI3K pathway. A recent study suggests that the overexpression of myr-p110 in murine fibroblasts, resulting in constitutive PI3K activation, does not lead to constitutive PKB/Akt activation but rather to a reduced ability of stimuli to induce the phosphorylation of PKB.53 Differences similar to those for PKB/Akt activation were observed for ERK1/2 phosphorylation, albeit to a lesser extent. Because the fMLP-mediated cellular responses were normal in MDS patients and GM-CSF could properly stimulate STAT5 activation, it is tempting to speculate that the convergence of fMLP and GM-CSF signaling is disturbed in MDS patients. It is conceivable that altered PI3K-PKB/Akt and ERK1/2 activation patterns found in MDS patients might lead to decreased priming of the respiratory burst by preventing proper assembly of the NADPH enzyme complex. In conclusion, our studies indicate that PI3-kinase p110 protein levels are increased in neutrophils from MDS patients and that the phosphorylation of PKB/Akt and, to a lesser extent, ERK1/2 is disturbed in neutrophils from these patients. Further research will have to elucidate to what extent these alterations in signaling events are responsible for the disturbed neutrophil effector functions in MDS.
We thank all the patients and volunteers who participated in the study. We also thank Drs Paul J. Coffer and Evert Nijhuis for their assistance regarding technical procedures and for their helpful suggestions.
Submitted October 4, 2001; accepted September 30, 2002.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: E. Vellenga, Department of Medicine, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands; e-mail: e.vellenga{at}int.azg.nl.
1. Kouides PA, Bennett JM. Morphology and classification of the myelodysplastic syndromes and their pathologic variants. Semin Hematol. 1996;33:95-100[Medline] [Order article via Infotrieve]. 2. Carulli G, Sbrana S, Minnucci S, et al. Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes: a flow cytometric study. Leuk Res. 1997;21:513-518[CrossRef][Medline] [Order article via Infotrieve]. 3. Zabernigg A, Hilbe W, Eisterer W, Greil R, Ludescher C, Thaler J. Cytokine priming of the granulocyte respiratory burst in myelodysplastic syndromes. Leuk Lymph. 1997;27:137-143[Medline] [Order article via Infotrieve]. 4. Ohsaka A, Kitagawa S, Yuo A, et al. Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on respiratory burst activity of neutrophils in patients with myelodysplastic syndromes. Clin Exp Immunol. 1993;91:308-313[Medline] [Order article via Infotrieve].
5.
Kouides PA, Bennett JM.
Understanding the myelodysplastic syndromes.
Oncologist.
1997;2:389-401
6.
Babior BM.
Oxidants from phagocytes: agents of defense and destruction.
Blood.
1984;64:959-966 7. McPhail LC, Henson PM, Johnston RB Jr. Respiratory burst enzyme in human neutrophils: evidence for multiple mechanisms of activation. J Clin Invest. 1981;67:710-716[Medline] [Order article via Infotrieve].
8.
Weisbart RH, Kwan L, Golde DW, Gasson JC.
Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants.
Blood.
1987;69:18-21 9. Kitagawa S, Yuo A, Souza LM, Saito M, Miura Y, Takaku F. Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide. Biochem Biophys Res Commun. 1987;144:1143-1146[CrossRef][Medline] [Order article via Infotrieve]. 10. Gomez-Cambronero J, Veatch C. Emerging paradigms in granulocyte-macrophage colony-stimulating factor signaling. Life Sci. 1996;59:2099-2111[CrossRef][Medline] [Order article via Infotrieve]. 11. Clark SC. Biological activities of human granulocyte-macrophage colony-stimulating factor. Int J Cell Cloning. 1988;6:365-377[Abstract]. 12. Grogan A, Reeves E, Keep N, et al. Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J Cell Sci. 1997;110:3071-3081[Abstract]. 13. Mukherjee G, Quinn MT, Linner JG, Jesaitis AJ. Remodeling of the plasma membrane after stimulation of neutrophils with f-Met-Leu-Phe and dihydrocytochalasin B: identification of membrane subdomains containing NADPH oxidase activity. J Leukoc Biol. 1994;55:685-694[Abstract].
14.
Serrander L, Skarman P, Rasmussen B, et al.
Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils.
J Immunol.
2000;165:2451-2457
15.
Rizoli SB, Rotstein OD, Parodo J, Phillips MJ, Kapus A.
Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling.
Am J Physiol Cell Physiol.
2000;279:C619-C633
16.
Howard TH, Meyer WH.
Chemotactic peptide modulation of actin assembly and locomotion in neutrophils.
J Cell Biol.
1984;98:1265-1271
17.
Watts RG, Howard TH.
Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes.
Blood.
1993;81:2750-2757
18.
Hall A.
Rho-GTPases and the actin cytoskeleton.
Science.
1998;279:509-514
19.
Ma AD, Metjian A, Bagrodia S, Taylor S, Abrams CS.
Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine exchange factor, and Rac.
Mol Cell Biol.
1998;18:4744-4751
20.
Chung CY, Lee S, Briscoe C, Ellsworth C, Firtel RA.
Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells.
Proc Natl Acad Sci U S A.
2000;97:5225-5230
21.
Kim C, Dinauer MC.
Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways.
J Immunol.
2001;166:1223-1232
22.
Ambruso DR, Knall C, Abell AN, et al.
Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.
Proc Natl Acad Sci U S A.
2000;97:4654-4659
23.
Williams DA, Tao W, Yang F, et al.
Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.
Blood.
2000;96:1646-1654
24.
Dorseuil O, Reibel L, Bokoch GM, Camonis J, Gacon G.
The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.
J Biol Chem.
1996;271:83-88
25.
Ding J, Vlahos CJ, Liu R, Brown RF, Badwey JA.
Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils.
J Biol Chem.
1995;270:11684-11691 26. Coffer PJ, Geijsen N, M'rabet L, et al. Comparison of the roles of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function. Biochem J. 1998;329:121-130[Medline] [Order article via Infotrieve].
27.
McLeish KR, Knall C, Ward RA, et al.
Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF- 28. Kanai F, Liu H, Field SJ, et al. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol. 2001;3:675-678[CrossRef][Medline] [Order article via Infotrieve]. 29. Ellson CD, Gobert-Gosse S, Anderson KE, et al. PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox. Nat Cell Biol. 2001;3:679-682[CrossRef][Medline] [Order article via Infotrieve]. 30. Djerdjouri B, Lenoir M, Giroud JP, Perianin A. Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils. Biochem Biophys Res Commun. 1999;264:371-375[CrossRef][Medline] [Order article via Infotrieve]. 31. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol. 1982;51:189-199[Medline] [Order article via Infotrieve]. 32. Koenderman L, Kok PT, Hamelink ML, Verhoeven AJ, Bruijnzeel PL. An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals. J Leukoc Biol. 1988;44:79-86[Abstract].
33.
Nijhuis E, Lammers JW, Koenderman L, Coffer PJ.
Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning.
J Leukoc Biol.
2002;71:115-124
34.
Mardiney M III, Jackson SH, Spratt SK, Li F, Holland SM, Malech HL.
Enhanced host defense after gene transfer in the murine p47phox-deficient model of chronic granulomatous disease.
Blood.
1997;89:2268-2275
35.
Franssen CF, Huitema MG, Muller Kobold AC, et al.
In vitro neutrophil activation by antibodies to proteinase 3 and myeloperoxidase from patients with crescentic glomerulonephritis.
J Am Soc Nephrol.
1999;10:1506-1515
36.
Sander EE, van Delft S, ten Klooster JP, et al.
Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.
J Cell Biol.
1998;143:1385-1398 37. Coffer PJ, Jin J, Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J. 1998;335:1-13[Medline] [Order article via Infotrieve]. 38. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J. 2000;346:561-576[CrossRef][Medline] [Order article via Infotrieve].
39.
Ward RA, Nakamura M, McLeish KR.
Priming of the neutrophil respiratory burst involves p38 mitogen-activated protein kinase-dependent exocytosis of flavocytochrome b558-containing granules.
J Biol Chem.
2000;275:36713-36719
40.
Babior BM.
NADPH oxidase: an update.
Blood.
1999;93:1464-1476 41. DeLeo FR, Renee J, McCormick S, et al. Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J Clin Invest. 1998;101:455-463[Medline] [Order article via Infotrieve].
42.
Tilton B, Andjelkovic M, Didichenko SA, Hemmings BA, Thelen M.
G-protein-coupled receptors and Fc
43.
Dewas C, Fay M, Gougerot-Pocidalo MA, El Benna J.
The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils.
J Immunol.
2000;165:5238-5244
44.
Downey GP, Butler JR, Tapper H, et al.
Importance of MEK in neutrophil microbicidal responsiveness.
J Immunol.
1998;160:434-443 45. Rane MJ, Carrithers SL, Arthur JM, Klein JB, McLeish KR. Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol. 1997;159:5070-5078[Abstract]. 46. Kodama T, Hazeki K, Hazeki O, Okada T, Ui M. Enhancement of chemotactic peptide-induced activation of phosphoinositide 3-kinase by granulocyte-macrophage colony-stimulating factor and its relation to the cytokine-mediated priming of neutrophil superoxide-anion production. Biochem J. 1999;337:201-209[CrossRef][Medline] [Order article via Infotrieve].
47.
Hedin KE, Bell MP, Huntoon CJ, Karnitz LM, McKean DJ.
Gi proteins use a novel beta gamma- and Ras-independent pathway to activate extracellular signal-related kinase and mobilize AP-1 transcription factors in Jurkat T lymphocytes.
J Biol Chem.
1999;274:19992-20001
48.
Garcia J, de Gunzburg J, Eychene A, Gisselbrecht S, Porteu F.
Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways.
Mol Cell Biol.
2001;21:2659-2670
49.
Hoefsloot LH, Amelsfoort vMP, Broeders LCAM, et al.
Erythropoietin-induced activation of STAT5 is impaired in the myelodysplastic syndrome.
Blood.
1997;89:1690-1700 50. Fontenay-Roupie M, Bouscary D, Guesnu M, et al. Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction. Br J Haematol. 1999;106:464-473[CrossRef][Medline] [Order article via Infotrieve]. 51. Wakao H, Harada N, Kitamura T, Mui AL, Miyajima A. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways. EMBO J. 1995;14:2527-2535[Medline] [Order article via Infotrieve].
52.
Pallard C, Gouilleux F, Charon M, Groner B, Gisselbrecht S, Dusanter-Fourt I.
Interleukin-3, erythropoietin, and prolactin activate a STAT5-like factor in lymphoid cells.
J Biol Chem.
1995;270:15942-15945 53. Auger KR, Wang J, Narsimhan RP, Holcombe T, Roberts TM. Constitutive cellular expression of PI 3-kinase is distinct from transient expression. Biochem Biophys Res Commun. 2000;272:822-829[CrossRef][Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  G. M. Fuhler, A. L. Drayer, S. G. M. Olthof, J. J. Schuringa, P. J. Coffer, and E. Vellenga Reduced activation of protein kinase B, Rac, and F-actin polymerization contributes to an impairment of stromal cell derived factor-1 induced migration of CD34+ cells from patients with myelodysplasia Blood, January 1, 2008; 111(1): 359 - 368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Sandoval, J. P. Riquelme, M. D. Carretta, J. L. Hancke, M. A. Hidalgo, and R. A. Burgos Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils J. Leukoc. Biol., November 1, 2007; 82(5): 1266 - 1277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Kasper, E. Brandt, S. Brandau, and F. Petersen Platelet Factor 4 (CXC Chemokine Ligand 4) Differentially Regulates Respiratory Burst, Survival, and Cytokine Expression of Human Monocytes by Using Distinct Signaling Pathways J. Immunol., August 15, 2007; 179(4): 2584 - 2591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. H. Heijink, P. Marcel Kies, A. J. M. van Oosterhout, D. S. Postma, H. F. Kauffman, and E. Vellenga Der p, IL-4, and TGF-beta Cooperatively Induce EGFR-Dependent TARC Expression in Airway Epithelium Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 351 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuhler, N. R. Blom, P. J. Coffer, A. L. Drayer, and E. Vellenga The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation J. Leukoc. Biol., February 1, 2007; 81(2): 449 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shikama, T. Shichishima, I. Matsuoka, P. T. Jubinsky, C. A. Sieff, and Y. Maruyama Accumulation of an intron-retained mRNA for granulocyte macrophage-colony stimulating factor receptor common {beta} chain in neutrophils of myelodysplastic syndromes J. Leukoc. Biol., May 1, 2005; 77(5): 811 - 819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuhler, G. J. Knol, A. L. Drayer, and E. Vellenga Impaired interleukin-8- and GRO{alpha}-induced phosphorylation of extracellular signal-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia J. Leukoc. Biol., February 1, 2005; 77(2): 257 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Drayer, A.-K. Boer, E. L. Los, M. T. Esselink, and E. Vellenga Stem Cell Factor Synergistically Enhances Thrombopoietin-Induced STAT5 Signaling in Megakaryocyte Progenitors through JAK2 and Src Kinase Stem Cells, February 1, 2005; 23(2): 240 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fuhler, K. A. Cadwallader, G. J. Knol, E. R. Chilvers, A. L. Drayer, and E. Vellenga 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 J. Leukoc. Biol., July 1, 2004; 76(1): 254 - 262. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
/
, p110 
, and p110 
associate with the
regulatory subunit p85, whereas the class 1B catalytic
subunit p110
associates with p101. Evidence for PI3K involvement in
neutrophil respiratory burst comes from studies with specific PI3K
inhibitors.
OD 550) between the
ferricytochrome c reduction test in the absence and in the presence of
SOD. Each test was performed in quadruplicate.
) and neutrophils that
were stimulated for 10 minutes with 5 ng/mL GM-CSF (+) were analyzed
for 3 MDS patients and 3 healthy volunteers. Western blot analysis was
performed, and proteins were detected using an antibody against
phosphorylated STAT5 (upper panel). Equal loading was confirmed by
reprobing the blot with antibodies against ERK1/2.