|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PHAGOCYTES
From the Department of Haematology, The Royal Free and
University College, London Medical Schools, London, England.
Arachidonic acid (AA) generated by phospholipase A2
(PLA2) is thought to be an essential cofactor for phagocyte
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.
Both enzymes are simultaneously primed by cytokines such as
granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor
necrosis factor- The production of the oxygen radical superoxide and
the unsaturated fatty acid arachidonic acid (AA) are both essential for phagocyte function, the former mediating microbicidal
activity,1,2 and the latter being the rate-limiting step
for eicosanoid synthesis.3,4 The activity of the enzymes
that produce superoxide and arachidonate, namely nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase and phospholipase A2
(PLA2), respectively, are tightly regulated, and the
activity of both enzymes can be enhanced rapidly by many-fold if
phagocytes are exposed to cytokines, growth factors, and inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF),5-7 tumor necrosis factor- There is evidence that the activation of the NADPH oxidase may require
AA. Early work showed that exogenous arachidonate added to neutrophils
was a potent activator of superoxide production.14,15 Arachidonate may act by directly modifying the molecular components of
the NADPH oxidase and is reported to increase the affinity of NADPH for
its binding site on the oxidase16 and regulate a proton
channel17 situated on the gp91phox
protein.18 Arachidonate may also facilitate the formation
of the NADPH oxidase complex on the membrane by enabling the
translocation of cytosolic proteins,19 increasing the
affinity of guanosine 5'-triphosphate (GTP) binding sites in the plasma
membrane,20 and enhancing the dissociation of the rac G
protein from its regulatory molecule, GDI.21 Additionally,
arachidonate may act as a signal transduction molecule by activating
protein kinase cascades, such as protein kinase
C,22,23 mitogen-activated protein kinase (MAPK),
p42ERK2,23,24 and stress-activated protein
kinase, p38SAPK,23 or by modulating
intracellular calcium levels25,26 upstream from the NADPH
oxidase. AA that is required for NADPH oxidase activity may derive from
several lipid sources through the activity of PLA2. Several
classes of PLA2, namely Group II,9 Group V sPLA2,27 Group IV complementary
(c)PLA2,9,28 and Group VI iPLA2,29 can release arachidonate from human
neutrophils. cPLA2 was recently demonstrated to be
essential for NADPH oxidase activity in human myeloid PLB-985 cells via
activation of a proton channel.30,31
Arachidonate may also mediate the priming of the NADPH
oxidase.6 Although not directly activating superoxide
production, 1 µM arachidonate caused enhancement of NADPH
oxidase activity when the cells were subsequently stimulated by the
chemotactic peptide FMLP.7 In addition, inhibition of
arachidonate release by mepacrine inhibited the priming of NADPH
oxidase by GM-CSF without inhibiting superoxide production by unprimed
cells.7 At present it is unclear which PLA2
enzymes are activated during cytokine-mediated priming of arachidonate
release, although it was recently reported that both Group IV
cPLA2 and Group II sPLA2 are activated by TNF
priming of neutrophils.9 Furthermore, different
PLA2 may be involved in arachidonate release for
eicosanoid production rather than for superoxide
generation.32
The mechanisms by which cytokines regulate the different
PLA2 have not been fully elucidated. Serine phosphorylation
is important for regulating cPLA2.33,34
Several kinases are reported to phosphorylate cPLA2
including protein kinase C,35 and
12-O-tetradecanoylphorbol 13-acetate (TPA) (the potent
agonist of protein kinase C) in combination with calcium ionophore
induces massive arachidonate release.36 The kinases
p42ERK2,35,37 and
p38SAPK,34 can also induce phosphorylation of
cPLA2. In platelets stimulated with collagen or thrombin,
both serine (S)505 and S727 residues of cPLA2 are
phosphorylated, and inhibition of p38SAPK was shown to
partially inhibit the phosphorylation of PLA2 on both S505
and S727.34 As only the S505 residue lies within a MAPK
consensus sequence, the involvement of another kinase downstream of
p38SAPK is suggested. Both GM-CSF6,28 and
TNF- The aim of this study was to investigate the role of
p42ERK2 and p38SAPK as well as AA production in
the activation and priming of the neutrophil respiratory burst. We show
that NADPH oxidase and PLA2 have a differential dependence
on p42ERK2 and p38SAPK activity and that
arachidonate release is not obligatory for eliciting the priming of
FMLP- or C5a-stimulated superoxide production.
Materials
Cytokines.
Stock solutions of recombinant human (rh)GM-CSF (expressed in
Escherichia coli) (Hoechst, Hounslow, England) and
rhTNF- Agonists.
FMLP, C5a, TPA, and calcium ionophore (A23187) (all from Sigma
Chemical, Poole, England) were used. A stock solution of A23187, prepared in 5 mg/mL dimethyl sulphoxide (DMSO) and stored at
Inhibitors.
Inhibitors included the 5-lipoxygenase activating protein inhibitor
MK88642 (gift from Merck-Frosst, Kirkland, Quebec,
Canada). A 100 µM stock solution in DMSO was prepared immediately
prior to use. A stock solution of N-ethyl-maleimide
(NEM) (Sigma) in 100 mM PBS was prepared daily. Stock solutions of 30 mM PD98059 (Calbiochem-Novabiochem, La Jolla, CA) and 30 mM SB203580
(Alexis, Nottingham, England) in DMSO were stored at Purification of neutrophils
Measurement of p38 MAP kinase activity
The lysates were centrifuged at 12 000g for 10 minutes at
4°C, and the supernatants were incubated on ice for 30 minutes with 2 µg/mL of a polyclonal anti-p38 MAPK antibody (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) and then with an equal volume of protein
G Sepharose beads (Pharmacia Biotech) for 30 minutes at 4°C with
rotation. The immune complexes were washed twice with wash
buffer We added 2 µL magnesium/ATP (adenosine 5'-triphosphate) cocktail
(prepared according to the manufacturer's instructions) and 0.185 MBq (5 µCi) Detection of p38 phosphorylation by immunoblotting Neutrophils (1 × 106 cells per mL) were stimulated with either diluent (0.01% FCS), 1 µM FMLP, 500 U/mL TNF- , or 10 ng/mL GM-CSF at 37°C. At timed intervals 100-µL
samples were taken, and the reaction was terminated by addition of 2 mM
NEM followed by rapid centrifugation at 12 000g for 30 seconds. The pellet was resuspended in 50 µL ice-cold lysis buffer
(composition as given above, but without Pefabloc). We added 50 µL of
2 times Laemmli sample buffer, and the samples were heated at 95°C
for 10 minutes. Proteins equivalent to 1-2 × 105 cells
per lane were analyzed by 15% SDS-PAGE (acrylamide:bis percentage,
30:0.8) at 150 V for 3.5 hours. The resolved proteins were transferred
to PVDF membranes (Immobilon; Millipore, Watford, England). The
nonspecific binding sites were blocked in Tris-buffered saline/Tween-20
(TBS-T) 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20supplemented with 5% (wt/vol) nonfat dried milk for 2 hours at
room temperature. The blots were incubated overnight at 4°C with
either a goat polyclonal primary p38 MAPK or phosphospecific p38 MAPK
rabbit antibody (New England Biolabs, Beverly, MA) (1:1000 dilution,
specifically against tyrosine 182 and threonine 180). The membranes
were washed 3 times with TBS-T and then incubated for 1 hour at room
temperature with a 1:2000 dilution of either secondary horseradish
peroxidase-conjugated antirabbit or antigoat immunoglobulin G (IgG)
antibodies (Dako, High Wycombe, England). After 2 washes with TBS-T and
one wash with TBS, the phosphorylated p38 and total p38 MAP kinase
bands were detected by enhanced chemiluminescence (ECL)
(Amersham Pharmacia).
Measurement of superoxide production Superoxide generation was measured at 37°C by the superoxide dismutase-inhibitable reduction of ferri-cytochrome c in a dual-beam spectrophotometer as previously described.43 Purified human neutrophils (1 × 106 cells per mL) were incubated in 1-mL cuvettes with or without inhibitors (PD98059 for 10 minutes or SB203580 for 30 minutes) prior to addition of either 500 U/mL TNF-, 10 ng/mL GM-CSF, or 0.01% FCS diluent for 30 minutes. The samples were stimulated with 1 µM FMLP or 100 ng/mL C5a
for 10 minutes, and the reactions were stopped with NEM at a final
concentration of 2 mM.
Release of hydrogen-3-AA Release of hydrogen-3 (3H)-AA from purified human neutrophils was measured as previously described and confirmed using thin-layer chromatography.7 We incubated 5 × 106 cells per mL in PBS, 5 mM glucose, and 0.01% vol/vol FCS with 0.38 MBq (0.5 µCi) 5,6,8,9,11,12,14,15-3H-AA (specific activity, 7.33 TBq/mM (202 Ci/mM) (Amersham Pharmacia) for 2 hours at room temperature. The radio-labeled cells were then centrifuged at 300g for 7 minutes to remove unincorporated radioactivity and then washed 3 times in PBS. The pellet was resuspended to 2 × 106 cells per mL in PBSG. Neutrophil samples (0.5 mL in duplicate) were incubated with 200 nM MK886 (to inhibit metabolism of AA by the 5-lipoxygenase pathway) for 5 minutes at 37°C. Then cells were incubated with either 0.01% FCS as diluent control, 500 U/mL TNF-, or 10 ng/mL GM-CSF for 20 minutes followed by stimulation with 1 µM calcium ionophore A23187, 1 µM FMLP, or 100 ng/mL C5a for
20 minutes at 37°C. The reaction was stopped by placing the samples
on ice and then centrifuged at 12 000g for 4 minutes; 0.4-mL aliquots of the supernatants were assayed for radioactivity by
scintillation spectroscopy.
Phosphorylation of p42ERK2 measured by gel retardation assay Samples of stimulated neutrophils were prepared for Western blot analysis as described above. Proteins equivalent to 1-2 × 105 cells per lane were separated by 15% SDS-PAGE (acrylamide:bis percentage, 15:0.075 [200:1]) for 15 hours at 120 V. After electrophoresis, proteins were electrophoretically transferred onto PVDF membranes (Millipore). Nonspecific binding sites on the membrane were blocked in TBS-T/5% (wt/vol) nonfat dried milk for 2 hours at room temperature. Membranes were incubated with 0.2 µg/mL of a rabbit primary anti-p42ERK2 antibody at a 1:1000 dilution in TBS-T/5% milk (C-14) for 1 hour and then with a secondary horseradish peroxidase-conjugated goat antirabbit IgG antibody at a 1:2000 dilution for 1 hour at room temperature. After 2 washes with TBS-T and one wash with TBS, phosphorylated p42ERK2 was detected by ECL as a band with retarded mobility.Immunoprecipitation of cPLA2 and detection of phosphorylation by gel retardation Purified neutrophils (2 × 107 cells per mL) were incubated at 37°C for 30 minutes with either PD98059 or DMSO as diluent control. Cells were then stimulated with either GM-CSF or FCS diluent for 5 minutes at 37°C. Immunodetection of phosphorylated cPLA2 was performed according to the method of Kramer et al.44 The reaction was terminated by addition of 1 mL ice-cold lysis buffer (final concentrations, 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM PMSF, 10 µg/mL leupeptin, 10 µM pepstatin A, 100 µg/mL aprotinin, 50 mM NaF, 200 µM Na3VO4, 10 mM Na4P2O7, and 1 µM microcystin) for 15 minutes. The cell lysates were subjected to centrifugation at 12 000g for 15 minutes at 4°C. The supernatants were incubated with a rabbit antihuman cPLA2 antibody (N-216, Santa Cruz Biotechnology) at a 1:500 dilution for 2 hours followed by 25 µL protein A Sepharose beads for 30 minutes at 4°C. The immunoprecipitates were washed 4 times with 1 mL wash buffer (0.5% Triton X-100 and 150 mM NaCl [pH 7.4]), then twice with wash buffer containing 750 mM NaCl, and finally twice with the initial wash buffer. The samples were resuspended in 2 times Laemmli sample buffer, incubated for 10 minutes at 60°C, and subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Hybond C extra, Amersham Pharmacia). The membranes were blocked overnight in 3% bovine serum albumin and then incubated with rabbit antihuman cPLA2 antibody at 1:500 dilution for 2 hours at room temperature. The membranes were washed 3 times with wash buffer and incubated with a secondary horseradish peroxidase-conjugated goat antirabbit IgG antibody at a dilution of 1:2000 for 1 hour. The membrane was washed 3 times with wash buffer, and immunoreactive cPLA2 bands were detected using ECL.Statistical analysis The data presented are the mean ± SE of the number of experiments given in the text. Analyses to determine the statistical significance employed the Student paired t test.
Activation of neutrophil p38SAPK by agonists and cytokines Purified neutrophils were stimulated with 1 µM chemotactic peptide FMLP; p38SAPK immunoprecipitates were prepared, and their kinase activity was measured by the phosphorylation of a MAPKAPK-2 substrate, as described in "Materials and methods." Figure 1A shows that p38SAPK was rapidly and transiently stimulated by FMLP. Activity was detected within 30 seconds, was maximal at one minute, but was no longer detectable at 5 minutes. Figure 1B-C shows that the cytokines TNF-
and GM-CSF also stimulate the activation of p38SAPK in
purified neutrophils, and phosphorylation of this molecule is
determined by immunoblotting of whole cell lysates with a
p38SAPK phosphospecific antibody. GM-CSF-induced
phosphorylation of p38SAPK was detectable within 2 minutes
and was sustained for 15 minutes (Figure 1C), whereas TNF--induced
phosphorylation was slightly slower in onset and more transient (Figure
1B). The kinetics of p38SAPK phosphorylation stimulated by
FMLP, when determined by immunoblotting with the phosphospecific
antibody, was similar to the activation of p38SAPK activity
(data not shown). The optimum inhibitory concentration of the
p38SAPK inhibitor SB20358045,46 was determined
using the p38 kinase assay. The data in Figure 1D show that SB203580
inhibited the phosphorylation of MAPKAPK-2 in a dose-dependent manner
with approximately 1 µM IC50 and complete inhibition at
30 µM.
Effect of inhibition of p38SAPK on neutrophil superoxide production and AA release Studies with SB203580 were performed to determine whether p38SAPK has a role in mediating the priming effects of GM-CSF and TNF- on either PLA2 or NADPH oxidase activity
stimulated with FMLP. Neutrophils were preincubated with SB203580, and
NADPH oxidase activity was measured by the superoxide
dismutase-inhibitable reduction of cytochrome c as
described in "Materials and methods." Figure
2A-B shows that SB203580 inhibited
unprimed as well as GM-CSF- and TNF-primed superoxide production
stimulated by FMLP. SB203580, at doses that inhibited
p38SAPK, did not inhibit NADPH oxidase activity stimulated
by the receptor-independent agonist TPA and thus did not inhibit the
assembly and activation of the oxidase stimulated via PKC
(Figure 2C).
PLA2 activity was determined as the extracellular release
of 3H-AA from prelabeled phospholipid stores in neutrophils
whose 5-lipoxygenase activity had been fully inhibited by the highly specific inhibitor MK886.42 This allowed maximal detection
of PLA2 rather than 5-lipoxygenase activity and allowed the
effect of the kinase inhibitors on PLA2 to be determined
without interference from any possible effect on the downstream
metabolism of AA. In 14 experiments the amount of AA released from
non-primed neutrophils stimulated with FMLP was not
significantly greater than background activity (2340 ± 239
cpm/106 unstimulated cells and 2474 ± 230
cpm/106 FMLP-stimulated cells), as we previously
reported,7 but AA release was significantly greater than
background in FMLP-stimulated cells primed with either TNF- The data presented in Table 1 show that
when neutrophils were preincubated with SB203580 under the conditions
that gave complete inhibition of p38SAPK and significant
inhibition of NADPH oxidase activity, there was no significant
inhibition of either GM-CSF- or TNF-primed FMLP-stimulated AA release.
To confirm these data, neutrophils treated with SB203580 was also
stimulated with 1 µM calcium ionophore A23187, and AA release was
measured. Table 1 shows that no inhibition of AA release from either
unprimed or primed cells was apparent; in fact, SB203580 significantly
enhanced AA release from ionophore-stimulated cells that had been
primed with TNF-
Activation of neutrophil p42ERK2 by agonists and cytokines Neutrophils were stimulated with FMLP, GM-CSF, and TNF-,
and analysis of p42ERK2 phosphorylation was by gel
retardation assay as described in "Materials and methods." Figure
3 shows that FMLP, GM-CSF, and TNF-
activate p42ERK2 in neutrophils in addition to activating
p38SAPK. FMLP stimulated the phosphorylation of
p42ERK2 within 30 seconds, and the activation was sustained
for at least 40 minutes (Figure 3A). TNF- stimulation of
p42ERK2 was only transient, with a weak band being detected
at 10 minutes after stimulation (Figure 3B), whereas GM-CSF induced
more sustained activation (Figure 3C). Activation of
p42ERK2 was inhibited by preincubation of neutrophils with
the noncompetitive MEK1 inhibitor PD98059,47 as shown in
Figure 3. Data from dose-response studies showed that complete
inhibition of p42ERK2 activation in neutrophils stimulated
by GM-CSF was achieved at 10 µM PD98059 (data not shown). Inhibition
of p42ERK2 kinase by PD98059 was achieved rapidly, a
preincubation of 5 minutes was sufficient to fully inhibit the enzyme,
and inhibition was sustained for at least 60 minutes after GM-CSF
stimulation (data not shown).
Effect of inhibition of p42ERK2 MAP kinase on neutrophil superoxide production and AA release Figure 2D-E shows that under the conditions where p42ERK2 activation was completely blocked, there was no observable concomitant inhibition of either unprimed (n = 4), GM-CSF-primed (n = 3), or TNF--primed (n = 3) NADPH oxidase
activity stimulated by FMLP. Neither did PD98059 at any dose inhibit
TPA-stimulated NADPH oxidase activity (n = 3) (Figure 2F). However,
the data given in Table 2 show that
PD98059 did partially inhibit both GM-CSF- and TNF--primed FMLP-stimulated PLA2 responses. To confirm the inhibitory
effect of PD98059 on arachidonate release, studies were performed using 1 µM calcium ionophore A23187 as stimulant. In 4 experiments, AA
release from unprimed neutrophils stimulated by A23187 was not
significantly inhibited by PD98059 at any concentration, whereas GM-CSF
and TNF- priming of AA release stimulated by A23187 was inhibited in
a dose-dependent fashion (Table 2).
Effect of the MAP kinase inhibitors on the phosphorylation of cPLA2 To investigate whether the target class of PLA2 that was inhibited by PD98059 was cPLA2, we investigated the effects of PD98059 on cPLA2 phosphorylation as determined by gel retardation. The data in Figure 4 show that phosphorylation of cPLA2 was stimulated by GM-CSF and that this was not inhibited by either 20 or 30 µM PD98059, doses that completely inhibit p42ERK2 activity (Figure 3A-C).
The effect of the PLA2 inhibitor MAFP on superoxide production and arachidonate release The data so far presented suggest that superoxide production and arachidonate release can be dissociated by selective inhibition of either p42ERK2 or p38SAPK. To further investigate whether superoxide production can occur independently from arachidonate production, the effect of PLA2 inhibitors on the respiratory burst and AA release was measured. Neutrophils were preincubated with the dual c and iPLA2 inhibitor MAFP48,49 before priming and stimulation with FMLP, and the effect on NADPH oxidase and arachidonate release was measured. Figure 5 shows that MAFP inhibited FMLP-stimulated AA release primed by GM-CSF or TNF- with
approximately 0.1 µM IC50, whereas this compound did not
inhibit either unprimed or primed FMLP-stimulated NADPH oxidase
activity unless used at a much higher concentration of 5 µM. In
control experiments measuring the activation of a different signal
transduction pathway, we showed that these high doses of MAFP did not
inhibit signal transducer and activator of transcription (STAT)5b
activation in neutrophils stimulated with GM-CSF (data not shown), thus
the inhibitory effects of MAFP were not due to generalized
cellular toxicity.
Studies with C5a Neutrophil superoxide production and AA release stimulated by C5a were also measured in samples that had been preincubated with either PD98059, SB203580, or MAFP. Although C5a was a weaker agonist than FMLP, similar results were found with regard to the sensitivity of neutrophils to the MAP kinase and phospholipase inhibitors. Figure 6A-B shows that superoxide production was inhibited by SB203580 in a dose-dependent manner, but not by PD98059, whereas these compounds had the reverse effect on arachidonate release (Figure 6C). In addition, 1 µM MAFP enhanced both unprimed and GM-CSF-primed C5a-stimulated superoxide production (Figure 6D), but inhibited to basal levels C5a-stimulated AA release (Figure 6E).
Our data extend previous findings that
FMLP,50-52 GM-CSF,53-55 and
TNF- Parallel experiments measuring arachidonate release stimulated by FMLP and C5a showed little inhibition attributable to blocking p38SAPK, and in fact, arachidonate release was enhanced by 1-20 µM SB203580. The enhancing effect of SB203580 on arachidonate release was further confirmed in cells stimulated with calcium ionophore. These results show that (1) SB203580 was not a global inhibitor of either FMLP or C5a signaling upstream of the NADPH oxidase, and thus the site of action of p38SAPK is likely to be the oxidase itself; (2) priming and activation of neutrophil AA release is not dependent on p38SAPK; and (3) the signal transduction pathways important for activating the NADPH oxidase via FMLP or C5a receptors in either primed or unprimed cells are not the same as those for activating PLA2. Our observations are in agreement with those of Syrbu et al,38 who showed that SB203580 does not inhibit the phosphorylation of cPLA2 in FMLP-stimulated neutrophils, as determined by a gel-shift assay. However, Syrbu et al38 report that SB203580 did partially inhibit FMLP-stimulated AA release from intact cells. The differences between these findings and ours may be due to differences in techniques used, as Syrbu et al did not use an inhibitor of 5-lipoxygenase in these assays and therefore measured the additive effects of SB203580 on both PLA2 and 5-lipoxygenase. Indeed, we have shown that SB203580 does partially inhibit 5-lipoxygenase,59 but it fails to inhibit AA release in cells where 5-lipoxygenase is blocked with MK886. SB203580 similarly inhibits other enzymes, such as cyclo-oxygenase 2, which are downstream of PLA2.60 In contrast, inhibition of p42ERK2 had no effect on
superoxide production in unprimed or primed cells. Unprimed cells
stimulated with calcium ionophore, but not FMLP or C5a, released
arachidonate, but this release was not inhibited significantly
by the p42ERK2 pathway inhibitor. A dose-dependent, but
partial, inhibition of arachidonate release was only seen in
cytokine-primed cells. The data support other reports that activation
of the FMLP-stimulated respiratory burst is not dependent on
p42ERK2,61-63 but indicate that
primed PLA2 responses are dependent on this kinase. Our
data show that activation of NADPH oxidase and PLA2 in
GM-CSF- or TNF- To examine further the dissociation of the activation pathways from the
FMLP receptor to NADPH oxidase and arachidonate release, the effect of
the PLA2 inhibitor MAFP48,49 on these enzyme systems was studied. We previously showed that both GM-CSF-primed oxidase and PLA2 activity were inhibited by mepacrine, a
relatively nonspecific inhibitor of sPLA2, which suggests a
possible role for this enzyme in activating the primed
oxidase.7 Superoxide production by unprimed neutrophils
was not inhibited by mepacrine, suggesting that only the enhanced
superoxide production in response to priming was dependent on
concomitant arachidonate production. However, concentrations of MAFP
that inhibited FMLP- and C5a-stimulated arachidonate release primed by
GM-CSF and TNF- The ability to dissociate FMLP- and C5a-stimulated NADPH oxidase
activity from arachidonate release has important implications for the
role of arachidonate in respiratory burst activity. Arachidonate is
thought to interact with the NADPH oxidase in several
ways,15-21and specific binding sites for arachidonate on
the p67phox protein have been recently
elucidated.66 However, recent work with leukemic cells
lines induced to differentiate to mature cells have revealed
conflicting results. Lowenthal and Levy31 showed that
human myeloid PLB-985 cells transfected with antisense
cPLA2 lack respiratory burst activity following
differentiation with either retinoic acid or vitamin D3,
but that activity was restored by addition of exogenous arachidonate.
In contrast we previously showed that U937 cells differentiated with
interferon- Our data reveal a greater sensitivity of AA release stimulated by
calcium ionophore to PD98059 in primed versus unprimed neutrophils; thus it is possible that a different PLA2 enzyme may be
responsible for AA release in primed cells compared to unprimed cells.
The PD98059-sensitive PLA2 in primed cells may not be
cPLA2, as Syrbu et al38 have shown that
PD98059 inhibits neither FMLP- nor TNF- The aim of this work was to investigate the signaling pathway
responsible for priming PLA2 activity and to compare and
contrast it with those pathways responsible for the priming and
activation of the respiratory burst. Our results indicate that
superoxide production is dependent on p38SAPK, which is in
agreement with previous work.54,55,57,58 By contrast, when
we looked at PLA2, there was no dependence on the p38SAPK pathway, but there was a dependence on
p42ERK2 MAP kinase activity only in cytokine-primed cells.
Blocking arachidonate release with either a p42ERK2 pathway
inhibitor or PLA2 inhibitor did not concomitantly reduce NADPH oxidase activity. Thus the signaling pathways from the GM-CSF and
TNF-
The authors are grateful to Dr Asim Khwaja for help with determining p38 kinase activity and Dr Helen Wheadon for performing the STAT5b assay.
Submitted May 1, 2000; accepted December 13, 2000.
Supported by a grant from the Kay Kendall Leukaemia Fund, London, United Kingdom.
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: David C. Linch, Department of Haematology, The Royal Free and University College London Medical School, 98, Chenies Mews, London WC1E 6HX, England; e-mail: d.linch{at}ucl.ac.uk.
1. Segal AW, Abo A. The biochemical basis of the NADPH oxidase of phagocytes. Trends in Biochem Sci. 1993;18:43-47[CrossRef][Medline] [Order article via Infotrieve].
2.
Babior BM.
NADPH oxidase: an update.
Blood.
1999;93:1464-1476
3.
Roshak A, Sathe G, Marshall LA.
Suppression of monocyte 85-kDa phospholipase A2 by antisense and effects on endotoxin induced prostaglandin biosynthesis.
J Biol Chem.
1994;269:25999-26005 4. Bauldry SA, Wooten RE. Leukotriene B4 and platelet activating factor production in permeabilized human neutrophils: role of cytosolic PLA2 in LTB4 and PAF generation. Biochim Biophys Acta. 1996;1303:63-73[Medline] [Order article via Infotrieve].
5.
Gasson JC.
Molecular physiology of granulocyte-macrophage colony-stimulating factor.
Blood.
1991;77:1131-1145
6.
Durstin M, Durstin S, Moleski TFP, Becker EL, Sha'afi RI.
Cytoplasmic phospholipase A2 translocates to the membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase.
Proc Natl Acad Sci U S A.
1994;91:3142-3146 7. Roberts PJ, Williams SL, Linch DC. The regulation of neutrophil phospholipase A2 by granulocyte-macrophage colony-stimulating factor and its role in priming superoxide production. Br J Haematol. 1996;92:804-814[CrossRef][Medline] [Order article via Infotrieve].
8.
Atkinson YH, Marasco WA, Lopez AF, Vadas MA.
Recombinant tumor necrosis factor- 9. Seeds MC, Jones DF, Chilton FH, Bass DA. Secretory and cytosolic phospholipases A2 are activated during TNF priming of human neutrophils. Biochim Biophys Acta. 1998;1389:273-284[Medline] [Order article via Infotrieve].
10.
Yuo A, Kitagawa S, Kasahara T, Matsushima K, Saito M, Takaku F.
Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors.
Blood.
1991;78:2708-2714 11. Forehand JR, Johnston RB, Bomalaski JS. Phospholipase A2 activity in human neutrophils: stimulation by lipopolysaccharide and possible involvement in priming for an enhanced respiratory burst. J Immunol. 1993;151:4918-4925[Abstract]. 12. Doerfler ME, Weiss J, Clark JD, Elsbach P. Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2. J Clin Invest. 1994;93:1583-1591. 13. Hallett MB, Lloyds D. Neutrophil priming: the cellular signals that say `amber' but not `green.' Immunol Today. 1995;16:264-268[CrossRef][Medline] [Order article via Infotrieve].
14.
Badwey JA, Curnutte JT, Karnovsky ML.
cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils.
J Biol Chem.
1981;256:12640-12643 15. Henderson LM, Moule SK, Chappell JB. The immediate activator of the NADPH oxidase is arachidonate not phosphorylation. Eur J Biochem. 1993;211:157-162[Medline] [Order article via Infotrieve]. 16. Rubinek T, Levy R. Arachidonic acid increases the activity of the assembled NADPH oxidase in cytoplasmic membranes and endosomes. Biochim Biophys Acta. 1993;1176:51-58[Medline] [Order article via Infotrieve]. 17. Henderson LM, Chappell JB. The NADPH oxidase-associated H+ channel is opened by arachidonate. Biochem J. 1992;283:171-175. 18. Henderson LM, Thomas S, Banting G, Chappell JB. The arachidonate-activatable NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91phox. Biochem J. 1997;325:701-705.
19.
Uhlinger DJ, Tyagi SR, Inge KL, Lambeth JD.
The respiratory burst oxidase of human neutrophils: guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.
J Biol Chem.
1993;268:8624-8631 20. Abramson SB, Lesczynska-Piziak J, Weissmann G. Arachidonic acid as a second messenger: interactions with a GTP-binding protein of human neutrophils. J Immunol. 1991;147:231-236[Abstract].
21.
Chuang T-H, Bohl BJ, Bokoch GM.
Biologically active lipids are regulators of Rac:GDI complexation.
J Biol Chem.
1993;268:26206-26211 22. Khan WA, Blobe GC, Hannun YA. Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C. Cell Signal. 1995;7:171-184[CrossRef][Medline] [Order article via Infotrieve].
23.
Hii CS, Huang ZH, Bilney A, et al.
Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL-60 promyelocytic leukemic cells, and human neutrophils: evidence for cell type-specific activation of mitogen-activated protein kinases.
J Biol Chem.
1998;273:19277-19282
24.
Rao GN, Baas AS, Glasgow WC, Eling TE, Runge MS, Alexander RW.
Activation of mitogen activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells.
J Biol Chem.
1994;269:32586-32592 25. Smith RJ, Sam LM, Justen JM, Leach KL, Epps DE. Human polymorphonuclear neutrophil activation with arachidonic acid. Br J Pharmacol. 1987;91:641-649[Medline] [Order article via Infotrieve]. 26. van der Zee L, Nelemans A, den Hertog A. Arachidonic acid is functioning as a second messenger in activating the Ca2+ entry process on H1-histaminoceptor stimulation in DDT1 MF-2 cells. Biochem J. 1995;305:859-864.
27.
Han SK, Kim KP, Koduri R, et al.
Roles of Trp31 in high membrane binding and proinflammatory activity of human group V phospholipase A2.
J Biol Chem.
1999;274:11881-11888 28. Nahas N, Waterman WH, Sha'afi RI. Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils. Biochem J. 1996;313:503-508. 29. Larsson Forsell PKA, Runarsson G, Ibrahim M, Bjorkholm M, Claesson H-E. On the expression of cytosolic calcium independent phospholipase A2 (88 kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes. FEBS Letters. 1998;434:295-299[CrossRef][Medline] [Order article via Infotrieve].
30.
Dana R, Leto TL, Malech ML, Levy R.
Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase.
J Biol Chem.
1998;273:441-445
31.
Lowenthal A, Levy R.
Essential requirement of cytosoloic phospholipase A2 for activation of the H+ channel in phagocyte-like cells.
J Biol Chem.
1999;274:21603-21608
32.
Tithof PK, Peters-Golden M, Ganey PE.
Distinct phospholipases A2 regulate the release of arachidonate acid for eicosanoid production and superoxide generation in neutrophils.
J Immunol.
1998;160:953-960
33.
de Carvalho MG, McCormack AL, Olson E, et al.
Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes.
J Biol Chem.
1996;271:6987-6997
34.
Borsch-Haubold AG, Bartoli F, Asselin J, et al.
Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells.
J Biol Chem.
1998;273:4449-4458
35.
Nemenoff RA, Winitz S, Qian N-X, Putten VV, Johnson GL, Heasley LE.
Phosphorylation and activation of a high molecular weight form of phospholipase A2 by microtubule-associated protein 2 kinase and protein kinase C.
J Biol Chem.
1993;268:1960-1964
36.
Lin L-L, Lin AY, Knopf JL.
Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.
Proc Natl Acad Sci U S A.
1992;89:6147-6151 37. Lin L-L, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993;72:269-278[CrossRef][Medline] [Order article via Infotrieve].
38.
Syrbu SI, Waterman WR, Molski TFP, Nagarkatti D, Hajjar J-J, Sha'afi RI.
Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils.
J Immunol.
1999;162:2334-2340
39.
Okuda K, Sanghera J, Pelech SL, et al.
Granulocyte-macrophage colony-stimulating factor, interleukin-3, and steel factor induce rapid tyrosine phosphorylation of 242 and p44 MAP kinase.
Blood.
1992;79:2880-2889
40.
Waterman WH, Sha'afi RI.
Effects of granulocyte-macrophage colony stimulating factor and tumour necrosis factor- 41. Corey S, Eguinoa A, Puyana-Theall K, et al. Granulocyte-macrophage colony-stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells. EMBO J. 1993;12:2681-2690[Medline] [Order article via Infotrieve]. 42. Ford-Hutchinson AW. FLAP: a novel drug target for inhibiting the synthesis of leukotrienes. Trends Pharmacol Sci. 1991;12:68-70[CrossRef][Medline] [Order article via Infotrieve].
43.
Roberts PJ, Devereux S, Pilkington GR, Linch DC.
Fc
44.
Kramer RM, Roberts EF, Manetta JV, Hyslop PA, Jakubowski JA.
Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets.
J Biol Chem.
1993;268:26796-26804 45. Cuenda A, Rouse J, Doza YN, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Letters. 1995;364:229-233[CrossRef][Medline] [Order article via Infotrieve]. 46. Cohen P. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 1997;7:353-361.
47.
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem.
1995;270:27489-27494 48. Huang Z, Payette P, Abdullah K, Cromlish WA, Kennedy BP. Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2. Biochemistry. 1996;35:3712-3720[CrossRef][Medline] [Order article via Infotrieve]. 49. Lio Y-C, Reynolds LJ, Balsinde J, Dennis EA. Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochim Biophys Acta. 1996;1302:55-60[Medline] [Order article via Infotrieve]. 50. Nick JA, Avdi NJ, Young SK, et al. Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP. J Clin Invest. 1997;99:975-986[Medline] [Order article via Infotrieve].
51.
Krump E, Sanghera JS, Pelech SL, Furuya W, Grinstein S.
Chemotactic peptide N-formyl-Met-Leu-Phe activation of p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils.
J Biol Chem.
1997;272:937-944
52.
Zu Y-L, Qi J, Gilchrist A, et al.
p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF- 53. Molski TF, Huang CK, Fernandez GA, Sha'afi RI. Divergent cytosolic phospholipase A2 (cPLA2) phosphorylation pathways in human neutrophils [abstract]. FASEB J. 1996;10:416a.
54.
McLeish KR, Knall C, Ward RA, et al.
Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF
55.
Suzuki K, Hino M, Hato F, Tatsumi N, Kitagawa S.
Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor- 56. Waterman WH, Molski TF, Huang CK, Adams JL, Sha'afi RI. Tumour necrosis factor-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils. Biochem J. 1996;319:17-20. 57. Mollapour E, Khwaja A, Linch DC, Roberts PJ. Activation and priming of neutrophil NADPH oxidase and phospholipase A2 are dissociated by inhibition of ERK2 and p38 kinases [abstract]. Blood. 1998;92:533. 58. Lal AS, Clifton AD, Rouse J, Segal AW, Cohen P. Activation of the neutrophil NADPH oxidase is inhibited by SB 203580, a specific inhibitor of SAPK2/p38. Biochem Biophys Res Commun. 1999;259:465-470[CrossRef][Medline] [Order article via Infotrieve]. 59. Mollapour E. The role of arachidonic acid mobilisation in myeloid cells [PhD thesis]. London, England: University of London; 1999.
60.
Borsch-Haubold AG, Pasquet S, Watson SP.
Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059.
J Biol Chem.
1998;273:28766-28772
61.
Yu H, Suchard SJ, Nairn R, Jove R.
Dissociation of mitogen-activated protein kinase activation from the oxidative burst on differentiated HL-60 cells and human neutrophils.
J Biol Chem.
1995;270:15719-15724 62. Kuroki M, O'Flaherty JT. Differential effects of a mitogen-activated protein kinase kinase inhibitor on human neutrophil responses to chemotactic factors. Biochem Biophys Res Commun. 1997;232:474-477[CrossRef][Medline] [Order article via Infotrieve]. 63. Coffer PJ, Geijsen N, M'Rabet L, et al. Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function. Biochem J. 1998;329:121-130.
64.
Balsinde J, Dennis EA.
Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages.
J Biol Chem.
1996;271:6758-6765
65.
Hazen SL, Zupan LA, Weiss RH, Getman DP, Gross RW.
Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2: mechanism-based discrimination between calcium-dependent and -independent phospholipases A2.
J Biol Chem.
1991;266:7227-7236 66. Marshall J, Han C-H, Thatcher B, et al. Discovery of the arachidonic acid binding sites on p67phox. Surg Forum. 1999;XL:363-366.
67.
Roberts PJ, Devalia V, Faint R, et al.
Differentiation-linked activation of the respiratory burst in a monocytic cell line (U937) via Fc 68. Burke JR, Davern LB, Gregor KR, Owczarczak LM. Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation. Biochem Biophys Res Comm. 1999;260:232-239[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. Ayilavarapu, A. Kantarci, G. Fredman, O. Turkoglu, K. Omori, H. Liu, T. Iwata, M. Yagi, H. Hasturk, and T. E. Van Dyke Diabetes-Induced Oxidative Stress Is Mediated by Ca2+-Independent Phospholipase A2 in Neutrophils J. Immunol., February 1, 2010; 184(3): 1507 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Aponte, W. Jiang, M. Lakkis, M.-J. Li, D. Edwards, L. Albitar, A. Vitonis, S. C. Mok, D. W. Cramer, and B. Ye Activation of Platelet-Activating Factor Receptor and Pleiotropic Effects on Tyrosine Phospho-EGFR/Src/FAK/Paxillin in Ovarian Cancer Cancer Res., July 15, 2008; 68(14): 5839 - 5848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Morgan, V. V. Cherny, A. Finnegan, J. Bollinger, M. H. Gelb, and T. E. DeCoursey Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2{alpha} activity J. Physiol., March 1, 2007; 579(2): 327 - 344. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Kilpatrick, S. Sun, D. Mackie, F. Baik, H. Li, and H. M. Korchak Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of {delta}-PKC and ERK1/2 J. Leukoc. Biol., December 1, 2006; 80(6): 1512 - 1521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sakamoto, F. Kuribayashi, M. Nakamura, and K. Takeshige Involvement of p38 MAP Kinase in Not Only Activation of the Phagocyte NADPH Oxidase Induced by Formyl-methionyl-leucyl-phenylalanine but Also Determination of the Extent of the Activity J. Biochem., November 1, 2006; 140(5): 739 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Sheppard, M. R. Kelher, E. E. Moore, N. J. D. McLaughlin, A. Banerjee, and C. C. Silliman Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation J. Leukoc. Biol., November 1, 2005; 78(5): 1025 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Jo, H.-Y. Lee, Y.-N. Lee, J. I. Kim, H.-K. Kang, D.-W. Park, S.-H. Baek, J.-Y. Kwak, and Y.-S. Bae Group IB Secretory Phospholipase A2 Stimulates CXC Chemokine Ligand 8 Production via ERK and NF-{kappa}B in Human Neutrophils J. Immunol., November 15, 2004; 173(10): 6433 - 6439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Harfi, S. D'Hondt, F. Corazza, and E. Sariban Regulation of Human Polymorphonuclear Leukocytes Functions by the Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide after Activation of MAPKs J. Immunol., September 15, 2004; 173(6): 4154 - 4163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Widder, T. Behr, D. Fraccarollo, K. Hu, P. Galuppo, P. Tas, C. E Angermann, G. Ertl, and J. Bauersachs Vascular endothelial dysfunction and superoxide anion production in heart failure are p38 MAP kinase-dependent Cardiovasc Res, July 1, 2004; 63(1): 161 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bylund, A. Bjorstad, D. Granfeldt, A. Karlsson, C. Woschnagg, and C. Dahlgren Reactivation of Formyl Peptide Receptors Triggers the Neutrophil NADPH-oxidase but Not a Transient Rise in Intracellular Calcium J. Biol. Chem., August 15, 2003; 278(33): 30578 - 30586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhou, J. Zhao, T. Wiedmer, and P. J. Sims Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1 Blood, May 13, 2002; 99(11): 4030 - 4038. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
20°C.
glycerophosphate
-phosphorous-32
(
) or (B, D) 500 U/mL TNF-
) and stimulated with either 1 µM FMLP (
). (A, B) Arachidonate release was measured as described
in "Materials and methods." Stimulation with FMLP was for 30 minutes. (C, D) Superoxide production was measured by the reduction of
cytochrome c in a dual-beam spectrophotometer. Stimulation
with FMLP was for 10 minutes.
