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Division of Respiratory Diseases, Toronto Hospital, and Departments of
Medicine,
Biochemistry, and
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Surgery, University of Toronto, Toronto, Ontario, Canada;
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Department of Signal Transduction, Parke Davis Research Division, Warner Lambert Co., Ann Arbor, MI 48105; and
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Division of Cell Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Activation of neutrophils is associated with a striking increase in the
phosphorylation of multiple polypeptides on serine, threonine, and, to
a lesser extent, tyrosine residues, which ostensibly regulates the
ensuing cellular responses (5). Recent studies have revealed a complex
network of signaling pathways triggered by membrane receptors, which
include a cascade of phosphorylation-dependent reactions (6). One of
these pathways, involving activation of members of the
mitogen-activated protein kinase
(MAPK)3 superfamily, is
highly conserved and is believed to represent an important signaling
pathway in many cell types, including neutrophils (7, 8). MAPKs can be
grouped into three families: the ERKs (extracellular signal regulated
kinases), c-Jun N-terminal kinase (also called SAPK), and p38 (9).
Several isoforms of ERK have been described, and at least two of them,
ERK-1 (p44MAPK) and ERK-2 (p42MAPK), are
expressed in neutrophils (10). Activation of ERKs can be initiated by
serpentine receptors (e.g., the FMLP and C5a receptors) that are linked
to heterotrimeric GTP binding proteins as well as by those that are
more closely linked to tyrosine kinase activity (e.g., GM-CSF and Fc
receptors) (11, 12, 13, 14). Receptor-mediated stimulation of ERK appears to
require sequential activation of the small m.w. GTPase
p21ras, Raf-1, and MEK (MAPK or ERK kinase) (8, 15, 16). MEKs are members of a family of dual specificity kinases that
are capable of phosphorylating MAPK on both tyrosine and threonine
residues (17). Studies using expression of either dominant negative or
constitutively activated forms of MEK suggest that these enzymes
constitute the principal route for ERK activation (18, 19, 20, 21). Three
isoforms of MEK, termed MEK-1 to -3 have been described (17, 22), and
both MEK-1 and MEK-2, which phosphorylate and activate ERK, have been
detected in neutrophils, where they are activated by bacterial
chemotactic peptides (23, 24).
Some of the functions attributed to MAPKs, such as cytoskeletal remodeling (25) and activation of phospholipase A2 (26), may be important to the microbicidal activity of neutrophils. Moreover, it has been suggested that MAPK may participate in the activation of the neutrophil oxidative burst. One of the cytosolic components of the NADPH oxidase, p47phox, contains two serine residues within a sequence that is recognized by proline-directed kinases such as ERK (27, 28), and phosphorylation of p47phox is associated with oxidase activation (2). However, a direct link between the ERK pathway and activation of neutrophil effector functions, including the oxidative burst and granule secretion, has not been established.
The purpose of the current study was to establish the physiologic significance of activation of the MEK-MAPK pathway in several aspects of the complex microbicidal response of neutrophils. To accomplish this goal, we employed the specific MEK inhibitor PD098059, developed previously in one of our laboratories (29). This compound blocks the activation (and phosphorylation) of MEK through an allosteric mechanism that does not involve inhibition of ATP binding (30). The potency and specificity of this inhibitor have been extensively documented in a variety of biologic systems, and, of potential importance for leukocytes, PD098059 does not block the activity of MKK3 and MKK4 (29, 30).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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FMLP, TPA, EGTA, HEPES, ATP, GM-CSF, zymosan, PMSF, aprotinin,
horseradish peroxidase, pepstatin A, leupeptin, ferricytochrome
c, superoxide dismutase, and RPMI 1640 were obtained from
Sigma Chemical Co. (St. Louis, MO). Albumin was obtained from
Calbiochem (La Jolla, CA). Protein A-Sepharose, dextran T-500, and
Ficoll-Paque were purchased from Pharmacia LKB Biotechnology, Inc.
(Baie d’Urle, Quebec, Canada). Prestained m.w. standards were
purchased from Bio-Rad (Hercules, CA). Immobilon filters were obtained
from Millipore (Bedford, MA). [-32P]ATP was purchased
from ICN (Costa Mesa, CA). Texas Red-labeled zymosan and Lucifer Yellow
were purchased from Molecular Probes (Eugene, OR). PD098059
(2-(2'-amino-3'-methoxyphenol)-axanapthalen-4-one) was synthesized as
previously described (29). Bicarbonate-free medium RPMI 1640 was
buffered to pH 7.3 with 25 mM Na-HEPES. The sodium-rich medium used for
incubation of intact cells contained 140 mM NaCl, 5 mM KCl, 10 mM
glucose, 1 mM MgCl2, 1 mM CaCl2, and 10 mM
HEPES (pH 7.4). Both media were adjusted to 290 ± 5 mOsm with the
major salt.
Abs and recombinant proteins
Polyclonal Abs recognizing MEK-1 were generated by immunizing rabbits with keyhole limpet hemocyanin coupled to the N-terminal domain of MEK-1 (PKKKPTPIQLNPNPEY) and were gifts from Dr. Gilles L’Allemain (Centre de Biochimie, CNRS, Université de Nice, France). A mAb (IgG2a) to an N-terminal 13.6-kDa fragment of MEK-2 was obtained from Transduction Laboratories (Lexington, KY). Polyclonal Abs to ERK-1 and 2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Soluble and agarose-coupled monoclonal anti-phosphotyrosine Abs (4G10) were obtained from Upstate Biochemicals, Inc. (Lake Placid, NY). Abs to the phosphophorylated form of p38 MAPK were purchased from New England Biolabs, Inc. (Beverly, MA).
The cDNA construct encoding a GST fusion of the catalytically inactive form of ERK-1, in which Lys63 is changed to Met (K63M), was provided by Dr. R. L. Erikson (Department of Cellular and Developmental Biology, Harvard University, Boston, MA). GST-ERK-1 (K63M) fusion proteins, referred to hereafter simply as GST-ERK, were bacterially produced and purified on glutathione-agarose beads as described by Crews et al. (31).
Neutrophil isolation
Neutrophils were isolated from fresh heparinized blood from healthy human volunteers. The majority of the RBC were removed by dextran sedimentation followed by Ficoll-Paque centrifugation. The remaining RBC were eliminated using hypotonic lysis. Neutrophils were counted using a model ZM Coulter counter (Coulter, Hialeah, FL), resuspended in HEPES-buffered RPMI 1640 at 107 cells/ml, and maintained in this medium at room temperature until use. In experiments involving immunoprecipitation, the cells were pretreated with 2.5 mM di-isopropylfluorophosphate for 30 min at room temperature. Cell viability was estimated for all experiments using trypan blue exclusion and was always >95%.
SDS-PAGE, immunoblotting, and ERK activity assay
Immunoblotting was performed essentially as previously described (23), after separation of the proteins by SDS-PAGE (32) and transfer onto Immobilon (Millipore). Phosphotyrosine was detected using a 1/5000 dilution of monoclonal anti-phosphotyrosine Abs, followed by a 1/5000 dilution of secondary, horseradish peroxidase-conjugated sheep anti-mouse Ab (Amersham, Arlington Heights, IL). The enhanced chemiluminescence (ECL) system from Amersham was used for detection. ERK activity was assayed after renaturation in gels polymerized in the presence of myelin basic protein as previously described (23, 24).
Immunoprecipitation and MEK kinase assay
Immunoprecipitation of phosphotyrosine-containing proteins was performed under denaturing conditions using monoclonal anti-phosphotyrosine Abs covalently bound to agarose. For immunoprecipitation, 1 x 107 cells were suspended in 1 ml of lysis buffer (10% glycerol, 150 mM NaCl, 50 mM HEPES, 1 mM EGTA, 10 mM sodium pyrophosphate, 10 mM NaF, 1 mM sodium vanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µM pepstatin, and 0.1% SDS, pH 7.2). The lysate was boiled for 5 min, then diluted with ice-cold lysis buffer (without SDS), sonicated, and sedimented in a microcentrifuge for 5 min. The resulting supernatant was incubated with agarose-conjugated anti-phosphotyrosine (50 µl of packed beads) for 2 h at 4°C, followed by washing of the beads (six times) with lysis buffer. The immunoprecipitated proteins were then solubilized using Laemmli sample buffer, separated on an SDS-PAGE gel, and used for immunoblotting.
Immunoprecipitation of MEK-1 and MEK-2 for in vitro kinase assays was
performed as previously described (24). Briefly, MEK-1 or MEK-2 were
immunoprecipitated under nondenaturing conditions and incubated with
[-32P]ATP and GST-ERK as substrate. The kinase
reaction was stopped with boiling Laemmli sample buffer, followed by
SDS-PAGE. Substrate phosphorylation and the autophosphorylation of
MEK-1 or MEK-2 were then quantified by phosphorimaging (see below).
Experiments using nonimmune rabbit serum or isotype-matched (IgG2a)
mAbs instead of the anti-MEK Abs and in the absence of primary Ab
demonstrated that there was no phosphorylation of the GST-ERK substrate
under these conditions (not shown).
Oxidative burst
NADPH oxidase activity was determined using the superoxide dismutase-inhibitable reduction of ferricytochrome c (33) as described previously (34). Treated cells (4 x 106 cells/ml) were incubated in 1 ml of Na buffer with 75 µM cytochrome c at 37°C in the presence or the absence of superoxide dismutase. The cell suspensions were then placed in a stirred, thermostatted cuvette, and the absorbance was measured at 550 nm in a dual beam spectrophotometer (Hitachi, Tokyo, Japan). Stimulation of the cells with opsonized zymosan was accomplished by adding zymosan to the cells and gently sedimenting the mixture in a microcentrifuge at room temperature, followed by resuspension of the cells in prewarmed assay buffer.
Phagocytosis
The phagocytic ability of polymorphonuclear leukocytes was assayed by incubating opsonized zymosan with cells in the presence of the impermeant fluid phase marker Lucifer Yellow. Cells (3 x 105) were allowed to settle and adhere to fibrinogen-coated coverslips for 10 min at room temperature. To synchronize phagocytosis, the serum-opsonized zymosan (6 x 105 particles) was added to the cells and allowed to bind for 10 min at 4°C. The temperature was then rapidly raised to 37°C, and incubation proceeded at this temperature for 3 min in the presence of Lucifer Yellow (2 mg/ml). The coverslips were immediately cooled using an ice-water bath. The number of phagosomes was counted using a fluorescence microscope (Nikon, Melville, NY). To ensure that the dye did not leave the phagosome, the cells were kept cool throughout the visualization period using a cooled microscope stage and precooled slides. It is important to note that PD098059 did not alter the binding of opsonized zymosan to neutrophils.
Flow cytometric analysis of CD63 and CD67 surface expression
For experiments measuring FMLP-induced degranulation, to maximize the response, cells were pretreated with 5 µM cytochalasin B for 2 min before addition of the activating stimulus. After stimulation, cells were sedimented and resuspended in 200 µl of PBS followed by the addition of 800 µl of 1.6% paraformaldehyde in PBS. The cells were left on ice for 30 min followed by an additional 30 min at room temperature. After fixation, the cells were washed and resuspended in PBS containing 1% BSA. For both CD63 and CD67, the primary Ab was added at a dilution of 1/400 for 2 h at room temperature in the presence of 1% BSA. The cells were again washed with 1% BSA in PBS, and the secondary, FITC-labeled, goat anti-mouse Ab was added at a 1/400 dilution. After 1 h at room temperature, the cells were washed and resuspended in 0.5 ml of PBS and analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA).
Actin polymerization
Actin polymerization was determined using the fluorescent marker
7-nitrobenz-2-oxa-1,3-diazole (NBD)phallacidin as described by Howard
and Meyer (35). Briefly, after stimulation, the cells were fixed and
permeabilized in PBS containing 3.7% formaldehyde and 1 mg/ml
L-
-monopalmitoyl lecithin (lyso-PC) for 5 min at
37°C. NBD-phallacidin was added to the cells at a final concentration
of 1.65 x 10-7 M for 10 min at 37°C. The intensity
of staining was analyzed by flow cytometry (FACScan, Becton Dickinson,
San Jose, CA).
Chemotaxis
Chemotaxis was measured using a micro-Boyden chamber (Neuroprobe, Cabin John, MD). The chamber consists of two wells separated from each other by filter paper. The chemoattractant (10-7 M FMLP in HEPES-buffered RPMI with 1% BSA at pH 7.4) was placed in the bottom well, and a 0.45-µm pore size trap filter was placed above, followed by a 3-µm pore size chemotaxis filter. The top chamber was secured in place, and the cells were added in HEPES-buffered RPMI with 1% BSA (3 x 105 cells/well). The chamber was incubated at 37°C for 2 h; the trap filter was removed, fixed, and stained with hematoxylin; and the number of cells present was counted.
Lipid extraction and measurement of arachidonate production
Extracellular release of arachidonic acid (AA) from polymorphonuclear leukocytes was used as an index of phospholipase A2 activity. Cells were incubated in Na+-rich medium containing 0.1% BSA to trap extracellular arachidonate. After agonist exposure, ice-cold KRPD was added to the cells followed by rapid centrifugation. After addition of the internal standard deuterated arachidonic acid, supernatants were extracted with 6 ml of chloroform/methanol (2/1, v/v), with 0.01% butyrated hydroxytoluene as an anti-oxidant. The lower phase was collected, and the upper phase was re-extracted twice with chloroform/methanol/0.9% NaCl (86/14/1, v/v/v). The pooled lower phase was dried under N2, reconstituted with hexane/methyl-tert-butyl ether (98/2, v/v), and applied to a Sep-Pak silicic acid column (Waters, Toronto, Ontario, Canada). After elution of individual lipid classes with solvent mixtures of increasing polarity, the fatty acid fraction was dried under N2 and derivatized with N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide. The t-butyldimethylsilyl ether of AA was then separated from other fatty acids on a 0.2-mm x 25-m DB-23 gas chromatography column and quantified by selective ion monitoring mass spectrometry, by monitoring the intensities of the M-57 ions at m/z 361 (AA) and 369 (deuterated arachidonic acid). The assay for AA was linear between 0.1 and 400 pM (r2 = 0.996; p < 0.001).
Apoptosis
Apoptosis was measured using propidium iodide staining according to the method of Nicoletti et al. (36). After preparation, the cells were suspended in HEPES-buffered RPMI containing 10% FCS at a concentration of 2.5 x 106 cells/ml and incubated in polypropylene tubes for approximately 24 h. Subsequently, the cells were sedimented and resuspended in 0.5 ml of a hypotonic solution (3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, and 0.1% Triton X-100) containing 50 µg/ml of propidium iodide. After a 30-min incubation at 4°C in the dark, nuclear fluorescence was quantified by flow cytometry (FACScan, Becton Dickinson).
Other methods
Radioactivity incorporated into MEK or GST-ERK was quantified by phosphorimaging (Molecular Dynamics, Sunnyvale, CA), using ImageQuant software. For reproduction, the images were saved as 16-bit TIFF files, cropped using Adobe Photoshop, and labeled using Adobe Illustrator (Adobe Systems Inc., Mountain View, CA). For illustrations, immunoblots were scanned by a Hewlett-Packard Jet Scan II cx using Desk Scan II v2.1 software (Hewlett-Packard, Palo Alto, CA). For quantitation, immunoblots were scanned using a model DNA 35 high resolution flatbed scanner and analyzed using the PDI one-dimensional gel analysis software (Protein Databases Inc., Huntington Station, NY). Results are presented as typical radiograms or fluorescence traces or as the mean ± SE of the indicated number of replicates. Statistical analysis was performed using Student’s t test for paired or unpaired data or analysis of variance as indicated.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We have recently demonstrated that human neutrophils express MEK-1
and MEK-2, and that these enzymes are activated in response to diverse
soluble agonists such as FMLP and TPA (24). To determine whether
activation of MEKs could be inhibited by PD098059, human peripheral
blood neutrophils were incubated with the compound before exposure to
FMLP. Figure 1
illustrates that treatment
with 
50 µM PD098059 inhibited FMLP-induced activation of both MEK-1
and MEK-2 almost completely (>90%), as determined by an in vitro
immune complex kinase assay using recombinant, kinase-deficient ERK-1
as the substrate. These results confirm that this compound is both
membrane permeant and effective in neutrophils. Figure 1B illustrates
the dose-dependent inhibition of MEK1 by PD098059; the
IC50 for MEK1 was approximately 3.5 µM, comparable
to values (
4 µM) obtained in other cell types (29, 30). It should
be noted that PD098059 inhibited both the autophosphorylating activity
(solid arrowhead in Fig. 1A) as well as the ability
of MEK-1 and -2 to phosphorylate recombinant kinase-deficient ERK-1
(open arrowhead in Fig. 1A). To ensure the
specificity of PD098059, the effects of the compound on another member
of the MAPK superfamily, p38 MAPK, was assayed. Figure 1C
illustrates that 50 µM PD098059 had minimal effects (
30%) on
FMLP-induced phosphorylation of p38 MAPK, confirming the specificity of
the inhibitor.
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D illustrates that exposure of neutrophils to the phorbol
ester TPA (a direct activator of protein kinase C), opsonized zymosan
(a particulate stimulus acting via Fc and CR3 receptors), and GM-CSF
(acting via a tyrosine kinase-linked growth factor receptor) induced
MEK1 activation, and this was substantially (from 60–90%) inhibited
by PD098059. PD098059 inhibits tyrosine phosphorylation and activation of ERK-1 and -2
MEKs are dual specificity kinases that phosphorylate substrates on
both Tyr and Thr residues (17). The purported physiologic substrates of
MEK-1 and -2 are ERKs, members of the MAPK family (31). Human
neutrophils have been shown to express both ERK-1 and -2, and these
enzymes are activated in cells exposed to chemoattractants (10). To
determine the effects of MEK inhibition by PD098059 on the activation
of ERK-1 and -2 in intact cells, neutrophils were incubated with the
compound before chemoattractant exposure, as described above. The cells
were then lysed, and the phosphorylation and activity of ERK-1 and -2
were evaluated. PD098059 prevented the tyrosine phosphorylation of both
ERK-1 and -2 induced by FMLP (Fig. 2A). Moreover, at
concentrations of 50 µM, the compound almost completely (>90%)
abrogated the enhanced activity of ERK-1 and -2 as assayed by an in-gel
renaturation assay using myelin basic protein as the substrate (Fig. 2, B and C). A similar pattern of inhibition
of ERK-2 was noted using immunoprecipitation followed by an in vitro
kinase assay using myelin basic protein as the substrate (data not
shown). The IC50 for FMLP-induced ERK-1 and ERK-2
activation was about 6 µM, comparable to that for MEK inhibition
(cf., Fig. 1B). Together, these observations are
consistent with the idea that MEK-1 and/or MEK-2 are the predominant
upstream activators of ERK-1 and -2 in response to diverse
agonists.
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As discussed above, the MEK-ERK pathway has been tentatively
implicated in the activation of certain microbicidal functions of
neutrophils, such as the oxidative burst. Figure 3, A and B, shows
that treatment of neutrophils with 50 µM PD098059 substantially
(70%) inhibited the FMLP-induced oxidative burst. The inhibition
was statistically significant, using Student’s paired t
test (p < 0.05). Perusal of traces such as
that in Figure 3A revealed that the MEK inhibitor altered
mainly the rate of oxidant production, with little effect on the
duration of the burst. As the magnitude of the FMLP-induced oxidative
burst is relatively small, experiments such as that illustrated in
Figure 3A were performed in cells pretreated with
cytochalasin B, an actin-disrupting agent known to enhance the response
to the chemoattractant (8, 34). To ensure that the inhibitory effects
of PD098059 were exerted on FMLP-activated pathways (and not simply
blocking the enhancement due to cytochalasin B), the experiments were
repeated in the absence of cytochalasin B. Figure 3B
illustrates that although the magnitude was lower in the absence of
cytochalasin, PD098059 still exerted significant inhibitory effects on
the chemoattractant-induced oxidative burst.
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C illustrates that
PD098059 significantly inhibited the respiratory burst induced by the
phagocytic stimulus, opsonized zymosan. These findings imply that
although soluble and particulate agonists use different signal
transduction pathways (37), they may share some of the downstream
events. In contrast to the formyl peptide and opsonized zymosan, the
respiratory burst in response to TPA was minimally inhibited by
PD098059 (Fig. 3D). These results underscore two
important points. First, the MEK inhibitor does not inhibit the NADPH
oxidase directly. Second, although the phorbol ester activates both MEK
and ERK, activation of the oxidative burst in response to this potent
agonist may proceed by alternate (perhaps parallel) pathways. PD098059 partially inhibits phagocytosis of opsonized zymosan
Another key component of the microbicidal function of neutrophils
is their ability to ingest foreign organisms by phagocytosis. To
determine whether the MEK-MAPK pathway is involved in regulation of
this important function, the effects of PD098059 on the phagocytosis of
opsonized zymosan were analyzed. Figure 4
illustrates the experimental protocol as well as the results of such
determinations. Phagocytosis was quantified by a combination of
phase-contrast and fluorescence microscopy. Successful phagosome
formation was scored by assessing the number of Texas Red-labeled yeast
particles that were colocalized with the trapped fluid phase marker
Lucifer Yellow. This procedure allowed us to clearly discern
extracellular zymosan (arrows in Fig. 4, B–D) from
those trapped within the cells (arrowheads). As illustrated in Figure 4A, compound PD098059 induced a partial (25%), yet
significant, inhibition of phagocytosis. Importantly, PD098059 did not
diminish binding of the opsonized zymosan to neutrophils.
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Another important facet of the bactericidal function of
neutrophils is the regulated exocytosis of the contents of the various
secretory granules (3). To ascertain the importance of the MEK-MAPK
pathway in the secretion of granular contents, we used
immunofluorescence and flow cytometry to quantify the surface
expression of granule membrane markers (38). CD63 was used as a marker
of primary granule secretion, while CD67 (CD66a) was used as a marker
for secondary granules. The summarized data from three such experiments
of each kind are presented in Figure 5,
A and B. Treatment of neutrophils with either
FMLP or opsonized zymosan resulted in increased plasma membrane
expression of markers for both primary (CD63) and secondary (CD67)
granules, in good agreement with earlier findings (39). This increased
surface expression of granule markers detected by immunofluorescence
has been shown to correlate closely with conventional assays of
granular content release (38, 39). The effect of PD098059 was tested
next. At concentrations that virtually eliminated the activity of MEK,
the inhibitor (50 µM) had no significant effect on the exocytosis of
primary (Fig. 5A) or secondary (Fig. 5B) granules in response to either soluble (FMLP) or
particulate (zymosan) stimuli.
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Neutrophils must be able to emigrate from the vascular space,
across the endothelium, and through interstitial tissues to a site of
infection. This requires coordinated cell movement along a
gradient of attractant molecules that is termed chemotaxis (40). The
coordinated assembly, disassembly, and redistribution of the actin
cytoskeleton is crucial for this motile response (reviewed in 40 .
To determine the involvement of MEK and MAPK in these responses, we
studied the effects of PD098059 on actin assembly, measured by binding
of NBD-phallacidin staining and flow cytometry. In Figure 6, representative flow cytometric
histograms illustrate the increase in F-actin induced by exposure of
neutrophils to either FMLP or opsonized zymosan. Figure 6 summarizes
data from three similar experiments in which the effect of PD098059 was
evaluated. The assembly of F-actin induced by either the soluble or
particulate agonists was essentially unaffected by PD098059.
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, the strong chemoattractant effect
exerted by FMLP was not significantly affected by PD098059 under
conditions shown previously to inhibit activation of both MEK-1 and -2
as well as ERK.
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Activation of phospholipase A2 is an early and
purportedly important response of stimulated neutrophils (41).
Activation of this enzyme yields arachidonate, which has been
implicated in cytoskeletal alterations (42) and in triggering the
oxidative burst (43). It has been demonstrated that ERKs are able to
phosphorylate and activate phospholipase A2 (26). If ERKs
are the prevailing upstream activators of phospholipase A2
in neutrophils, then inhibition of MEK (and therefore ERK) would be
predicted to diminish or abrogate arachidonate production. This
possibility was analyzed by a combination of gas chromatography and
mass spectrometry. In accordance with earlier findings, both FMLP and
the phorbol ester TPA triggered the release of arachidonate from human
neutrophils (Fig. 8). Treatment of
unstimulated cells with PD098059 resulted in a significant elevation in
arachidonate release. More importantly, treatment with PD098059 did not
alter the net amount of arachidonate released by either FMLP or TPA
(Fig. 8). These observations suggest that ERK-1 and -2 are not the main
activators of phospholipase A2 in stimulated
neutrophils.
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Apoptosis is a process of programmed cell death that results in
degradation and fragmentation of chromosomal DNA. Apoptosis is also
accompanied by alterations in the cell membrane that allow recognition
and clearance of effete inflammatory cells by macrophages, a process of
major importance in the resolution of inflammation (44). Peripheral
blood neutrophils, in their unactivated state, are relatively short
lived (t1/2 = 6–8 h) and undergo spontaneous
apoptosis when maintained in culture (44). However, exposure to
cytokines such as GM-CSF has been shown to delay apoptosis (45),
thereby prolonging the half-life of these cells, potentially
contributing to host defense or, under other circumstances, tissue
injury (46). It is noteworthy that GM-CSF exposure has also been
reported to activate ERKs (47) and that recent evidence points to a
role of these kinases in preventing or delaying apoptosis (48, 49).
Figure 9A confirms the former
findings and additionally illustrates that PD098059 effectively
inhibits the tyrosine phosphorylation of both ERK-1 and -2 induced by
GM-CSF. To determine whether ERK activation was involved in the delay
of apoptosis, nucleic acid fragmentation was estimated by staining with
propidium iodide and flow cytometry. The cells were incubated with
PD098059 before and during exposure to GM-CSF, and apoptosis was
measured 24 h later. As detailed in Figure 9B, the
cytokine substantially reduced the fraction of apoptotic cells. More
importantly, PD098059 increased the number of apoptotic cells observed
after a 24-h incubation in the presence of GM-CSF. The MEK inhibitor
did not completely reverse the effect of GM-CSF, but the increase noted
was statistically significant (p < 0.05).
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Reports from many groups have documented a prominent activation of the MEK/MAPK pathway after exposure of neutrophils to diverse inflammatory mediators (8, 10, 23, 24, 50, 51). Although the activation of these kinases was found to precede or parallel several physiologic responses, no definitive proof of a causal relationship between these events was provided. In principle, the role of specific kinases in defined responses can be studied by transfection or microinjection of constitutively activated or dominant negative (interfering) mutants. These approaches, however, are not applicable to neutrophils. These are terminally differentiated cells with a remarkably short biologic half-life, precluding transfection. Myeloid cell lines are notoriously difficult to transfect and have phenotypes that do not appropriately reflect the function of mature neutrophils. Microinjection is similarly intractable; neutrophils are small, pliable, and react to contact with glass.
An alternative strategy is to use specific, cell-permeant, pharmacologic inhibitors with adequate specificity toward a defined enzyme. The present study used this approach, taking advantage of the well-documented selectivity of PD098059, an antagonist of MEK activation. The resulting findings suggest that the MEK-MAPK pathway provides substantial input into some important effector responses of neutrophils, including the oxidative burst and phagocytosis. These conclusions are in agreement with a recent report by Avdi et al. (52), which appeared while this work was in progress, that treatment of neutrophils with PD098059 partially inhibited the FMLP-induced respiratory burst. It should be noted that a correlation between ERK activation and activation of the NADPH oxidase has not been observed by all investigators. For example, Yu et al. (53) recently reported that increased intracellular cAMP resulted in inhibition of the FMLP-stimulated oxidative burst, whereas MAPK activity remained under these conditions. However, it is possible that only partial activity of MAPK (perhaps of a minor fraction or isoform) suffices for activation of the oxidative burst. Alternatively, multiple pathways, each capable of independent input, may contribute to initiation of the oxidative burst. Indeed, our observations that PD098059 did not completely inhibit superoxide generation or phagocytosis, while virtually obliterating MEK and ERK activity, suggests that multiple, possibly redundant, signals can initiate these responses. That the respiratory burst can be triggered by a variety of seemingly unrelated second messengers, including calcium, agonists of protein kinase C, arachidonate and other lipid metabolites, and agents that favor phosphotyrosine accumulation (54, 55, 56, 57) provides additional support for this idea. Recent studies have indicated that another MAPK family member, p38 MAPK, may provide input into the pathways leading to activation of the oxidative burst (58, 59), adherence, and chemotaxis (58). As there are multiple phosphorylation sites on p47phox (2), it is conceivable that phosphorylation of this cytosolic component by both ERKs and p38 MAPK is required for maximal activity of the oxidase.
The use of a pharmacologic inhibitor in the current study engenders
certain limitations pertaining primarily to issues of specificity. For
example, PD098059 had a small, but consistent, inhibitory effect on p38
MAPK phosphorylation (Fig. 1C). Nonetheless, it is
unlikely that this small effect of the MEK inhibitor on p38 MAPK could
have accounted for the inhibition of the oxidative burst or
phagocytosis, since complete inhibition of p38 MAPK by a more potent
and specific inhibitor (SB203580) had no effect on these microbicidal
functions (60).
Our findings that PD098059 attenuated the GM-CSF-induced delay in apoptosis has important implications for the regulation of the inflammatory process. In an inflammatory response initiated by bacterial infection, prolongation of leukocyte survival might be expected to facilitate the killing of invading microbes by these phagocytic cells. On the other hand, in circumstances of inflammation-mediated tissue damage such as the systemic inflammatory response syndrome (53), the persistence of tissue neutrophilia might be undesirable. Clearly, the fate of inflammatory cells must be carefully regulated (44). Our results suggest that the delay of apoptosis induced by cytokines such as GM-CSF is mediated in part by activation of the MEK-MAPK pathway. This idea is in good agreement with recent observations in cells of neural origin, in which apoptosis induced by withdrawal of nerve growth factor was found to be mediated by activation of Jun kinase and p38 MAPK in concert with inhibition of p42ERK and p44ERK (48). It follows from the above considerations that by accelerating the apoptosis of tissue neutrophils, judicious use of MEK-ERK inhibitors might afford protection against inflammatory tissue injury (44). Conversely, the use of these inhibitors is not indicated when the microbicidal function of the leukocytes is more important.
In summary, the inhibitor PD098059 largely prevented chemoattractant-induced activation of MEK-1 and -2 and of ERK-1 and -2, and substantially inhibited the oxidative burst and phagocytosis. Other neutrophil functional responses, including exocytosis, actin polymerization, and motility, as well as activation of phospholipase A2 were relatively unaffected by the compound. Importantly, PD098059 prevented the delay of apoptosis promoted by the growth factor GM-CSF. We conclude that activation of MEK and ERK provides substantial input into the signaling pathways regulating the oxidative burst, phagocytosis, and apoptosis. Other pathways appear to be largely responsible for secretion, actin assembly, chemotaxis, and release of arachidonate by neutrophils. These results demonstrate that distinct intracellular signaling pathways lead to activation of the various responses and highlight the ability of neutrophils to differentially regulate these important microbicidal functions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregory Downey, Room 6264, Medical Sciences Building, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail address:
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase; MEK, mitogen-activated protein kinase or extracellular signal-regulated kinase kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; TPA, tetradecanoyl phorbol myristate acetate; GST, glutathione-S-transferase; NBD, 7-nitrobenz-2-oxa-1,3-diazole; AA, arachidonic acid.
Received for publication April 3, 1997. Accepted for publication September 18, 1997.
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