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* Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, and
Inositide Laboratory, Babraham Institute, Babraham, Cambridge, United Kingdom; and
Medical Research Council Center for Inflammation Research, Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh, Teviot Place, Edinburgh, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and GM-CSF causes a
dramatic increase in the response of these cells to secretagogue
agonists and affects the capacity of neutrophils to induce tissue
injury. In view of the central role of phosphatidylinositol 3-kinase
(PI3-kinase) in regulating NADPH oxidase activity we examined the
influence of priming agents on agonist-stimulated phosphatidylinositol
3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human
neutrophils. Pretreatment of neutrophils with TNF- or GM-CSF, while
not influencing fMLP-stimulated PtdIns(3,4,5)P3
accumulation at 5 s, caused a major increase in
PtdIns(3,4,5)P3 at later times (10–60 s), which paralleled
the augmented superoxide anion (O2-) response.
The intimate relationship between PtdIns(3,4,5)P3
accumulation and O2- release was confirmed
using platelet-activating factor, which caused full but transient
priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor,
and genistein, a tyrosine kinase inhibitor, caused parallel inhibition
of O2- generation and
PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which
inhibits receptor-mediated activation of p85 PI3-kinase, had no effect
on either response. Despite major increases in PI3-kinase activity
observed in p85 and anti-phosphotyrosine immunoprecipitates in
growth factor-stimulated smooth muscle cells, no such increase was
observed in primed/stimulated neutrophils. In contrast, both fMLP and
TNF- alone caused a 3-fold increase in PI3-kinase activity in
p110
PI3-kinase immunoprecipitates. p21ras
activation (an upstream regulator of PI3-kinase) was unaffected by
priming. These data demonstrate that timing and magnitude of
PtdIns(3,4,5)P3 accumulation in neutrophils correlate
closely with O2- generation, that
PI3-kinase- is responsible for the enhanced
PtdIns(3,4,5)P3 production seen in primed cells, and that
factors other than activation of p21ras
underlie this response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, or GM-CSF) greatly enhances
subsequent agonist-induced respiratory burst activity and degranulation
responses (1). While this event improves the ability of
neutrophils to locate and kill invading microorganisms, priming is also
implicated in neutrophil-mediated tissue injury both in vitro and in
vivo (2, 3). Hence, the recruitment of large numbers of
such hyperresponsive cells at an inflammatory focus may predispose to
tissue injury and contribute to a variety of neutrophil-mediated
diseases, including acute respiratory distress syndrome, rheumatoid
arthritis, and ischemia-reperfusion injury (4). Priming
also has a direct effect on neutrophil shape, deformability,
integrin/selectin expression, and longevity (1, 5), and,
as a consequence, has a profound effect on the rheological, adhesive,
and survival properties of these cells in vivo (6). More
recently we have shown that priming is a reversible process and that
neutrophils can cycle among unprimed, primed, deprimed, and reprimed
states (7), underscoring the dynamic nature of this
process. Despite the important role of neutrophil priming in a range of
serious and often treatment-resistant conditions, the signaling
mechanisms underlying this process remain poorly characterized.
Previous reports have indicated a pivotal role for
phosphatidylinositol 3-kinase
(PI3-kinase)4 in
neutrophil activation (8, 9). In mammalian cells the type
Ia (PI3-kinase-
) and type Ib (PI3-kinase-) PI3-kinases
catalyze the conversion of phosphatidylinositol 4,5-bisphosphate
(PtdIns(4, 5)P2) to phosphatidylinositol
3,4,5-trisphosphate (PtdIns(3, 4, 5)P3)
(10), and this molecule has been shown to be involved in
the recruitment and activation of a variety of regulatory proteins via
an interaction with their PH domains (11). In the
neutrophil, agonist activation or the addition of GTP to permeabilized
cells results in a rapid and robust increase in
PtdIns(3, 4, 5)P3 accumulation (10, 12), and inhibitors of PI3-kinase abolish agonist-stimulated
superoxide anion (O2-)
generation (8). Moreover, we have recently demonstrated
that a metabolic product of PtdIns(3, 4, 5)P3,
PtdIns(3)P, is generated in phagocytic membranes (13) and
directly regulates the neutrophil oxidase complex by binding to the PX
domain of p40phox (14).
While priming is an obligate step required for full activation of the
NADPH oxidase, the impact of this process on agonist-stimulated
PtdIns(3, 4, 5)P3 accumulation and the precise
PI3-kinase isoforms responsible for this response have not been
defined. Our preliminary studies using
[32P]Pi-labeled
neutrophils (1) and those of Kodama et al.
(15) suggest that priming may enhance fMLP-stimulated
[32P]PtdIns(3, 4, 5)P3
accumulation, but the kinetics, magnitude, and mechanism of this effect
remain to be determined. Identifying the PI3-kinase(s) involved in this
response is complicated by the presence of an additional,
myeloid-restricted, G-regulated PI3-kinase (p101/PI3-kinase-)
in neutrophils and the lack of high-affinity p101 or PI3-kinase- Abs
or PI3-kinase isoform selective inhibitors. The potential importance of
PI3-kinase- in neutrophil activation is supported by data in p110
knockout mice where defects in respiratory burst and migratory
responses have been observed (9, 16). However, murine
neutrophils have a very attenuated respiratory burst response compared
with human neutrophils, and priming has not been clearly demonstrated
in this species.
In the current study we have measured
PtdIns(3, 4, 5)P3 accumulation using a recently
developed mass assay (17). We show that neutrophil priming
with TNF-, GM-CSF, cytochalasin B, or platelet-activating factor
(PAF) before fMLP stimulation leads to a substantially prolonged
PtdIns(3, 4, 5)P3 signal. Manipulation of the
O2- response using a number of
PI3-kinase and tyrosine kinase inhibitors, and also allowing
neutrophils to deprime after PAF exposure, resulted in parallel changes
in both PtdIns(3, 4, 5)P3 accumulation and
O2- release. Moreover,
significant PI3-kinase activity could be detected in p110, but not
p85 or phosphotyrosine immunoprecipitates, after TNF- priming or
fMLP stimulation. Finally, priming did not affect the extent of basal
or fMLP-stimulated p21ras activity, an upstream
regulator of PI3-kinase. Together, these results suggest that enhanced
PI3-kinase- activity underlies the augmented accumulation of
PtdIns(3, 4, 5)P3 and
O2- response seen in primed
neutrophils, and that a PI3-kinase regulatory protein other than
p21ras controls this activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Percoll, dextran, [-32P]ATP, and the
ECL detection kit were obtained from Amersham Pharmacia Biotech
(Piscataway, NJ). Cytochrome c, superoxide dismutase, fMLP,
LPS, and PBS (with or without CaCl2 and
MgCl2) were purchased from Sigma Chemical (Poole,
Dorset, U.K.). [Inositol-1-[3H]
(N)]-1,3,4,5-tetrakisphosphate was supplied by NEN Life
Science (Boston, MA) and inositol 1,3,4,5-tetrakisphosphate
(Ins(1, 3, 4, 5)P4) was supplied by Cell
Signals (Lexington, KY). Human rTNF- and GM-CSF were supplied
by R&D Systems (Abingdon, U.K.). The p110 Ab (LS1/127) was raised in
mice against a Sf9 cell-expressed His-tagged full-length protein
(porcine sequence); p85 and 4G10 Abs were from Upstate Biotechnology
(Lake Placid, NY). PAF-18
(1-O-stearyl-2-acetyl-sn-glycero-3-phosphocholine),
LY294002, pan-Ras Ab, staurosporine, genistein, cytochalasin B, and
radicicol were purchased from Calbiochem (La Jolla, CA). All other
reagents were obtained from commercial sources and were of the highest
purity available.
Preparation of human neutrophils
Human neutrophils were prepared from peripheral blood of healthy volunteers using dextran sedimentation and discontinuous plasma-Percoll gradients as previously described (18). This method routinely yielded cells that were unprimed and >95% pure with <0.1% mononuclear cell contamination.
O2- release assay
Purified neutrophils (106/90 µl PBS
containing CaCl2 and MgCl2)
were equilibrated at 37°C in a gently shaking water bath for 5 min.
Priming agents were added to give final concentrations of 200 U/ml
TNF-, 100 ng/ml GM-CSF, 5 µM cytochalasin B, or 1 µM PAF. Cells
were primed for the periods stated and then stimulated with fMLP (100
nM) or vehicle in the presence of prewarmed cytochrome c
(final concentration 1.2 mg/ml in PBS).
O2- release was calculated from
the superoxide dismutase-inhibitable reduction of cytochrome
c (1).
Ins(1, 3, 4, 5)P4 isotope dilution assay
Recombinant Ins(1, 3, 4, 5)P4 binding protein
(GAPIP4BP) was purified as previously described
(17, 19) and sample purity was analyzed by SDS-PAGE. Total
and nonspecific binding of
[3H]Ins(1, 3, 4, 5)P4 in
each preparation of GAPIP4BP was assessed by
serial dilution and inclusion of 0.1 nM InsP6,
respectively, and thereafter GAPIP4BP was used at a
concentration to achieve a maximum binding of 20% of the total
[3H]Ins(1, 3, 4, 5)P4 input.
Purified neutrophils (8 x 106) were primed
with TNF- (200 U/ml) or GM-CSF (100 ng/ml) for 30 min. Vehicle or
fMLP (100 nM) was added for the indicated times and the reactions were
stopped by the addition of methanol/chloroform (2:1, v/v). Lipid
extractions were performed as previously described (1).
After drying, the samples were processed as detailed (17)
and stored at -20°C until analyzed. Immediately before the assay,
samples were resuspended in dilute acetic acid to a final pH of 5. The
Ins(1, 3, 4, 5)P4 radioreceptor assay was performed
as described (20) using 0.005 µCi of
[3H]Ins(1, 3, 4, 5)P4 per
sample.
[3H]Thymidine incorporation
Confluent and quiescent primary bovine tracheal smooth muscle (BTSM) cells were prepared as previously detailed (21) and washed twice in serum-free DMEM, and inhibitors and mitogens were added as described. Cells were incubated for a further 24 h, with [3H]thymidine (0.1 µCi/ml) included for the final 4 h of the incubation. Cells were washed twice in PBS, trichloroacetic acid (5% w/v), and ethanol before finally being solubilized with NaOH (0.3 M). [3H]Thymidine incorporation was determined by liquid scintillation counting.
PI3-kinase activity assays
Neutrophils (20 x 106 cells) were
primed and stimulated as described. After stimulation, 1 ml of ice-cold
lysis buffer (10 mM Tris-HCl (pH 7.8), 1.5 mM EDTA, 10 mM KCl, 0.1%
Nonidet P-40, 1 mM
Na3VO4, 0.1 mM
di-isopropylfluorophosphate, 0.1 mM
N--tosyl-L-lysine chloromethyl
ketone, 0.1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10
µg/ml leupeptin) was added and the samples were left on ice for 20
min with intermittent vortex mixing. Samples were sonicated (2 x
10 s at maximum setting) and then centrifuged. The supernatants
were collected and incubated with p85, 4G10, and p110 Ab overnight
and protein A Sepharose beads were added for a further 2 h. The
beads were collected and washed once with lysis buffer, once with 100
mM Tris/500 mM LiCl (pH 7.6), and twice with 200 mM HEPES, 40 mM
MgCl2, 600 mM NaCl (pH 7.4). The washed beads
were then incubated for 10 min at 30°C in buffer (200 mM HEPES, 40 mM
MgCl2, 600 mM NaCl ((pH 7.4)) together with
phosphatidylinositol/phosphatidylserine (3:1, 0.2 mg/ml) vesicles, 10
µCi [32P]ATP, and 50 µM unlabeled ATP.
Lipid extractions were performed as previously detailed
(22) and the lower organic phase was dried in an Eppendorf
concentrator, redissolved in chloroform, and spotted onto silica gel 60
TLC plates. The plates were run in a solvent system containing
chloroform/methanol/ammonia/water (20:14:3:5, v/v/v/v). The
32P-labeled phosphoinositide 3-phosphate band was
detected by autoradiography and [32P]
incorporation measured by liquid scintillation counting. In experiments
characterizing the mouse p110 mAb (LS1/127), PI3-kinase activity was
assayed as described by Stephens et al. (12) with the
lipid products deacylated before resolution by TLC.
Transfection of COS7 cells with EE-tagged p110
Rapidly growing COS7 cells were cultured to 40–60% confluence
in DMEM containing 10% FBS, washed twice in serum-free DMEM buffered
with 10 mM HEPES (pH 7.3), and incubated with 1 µg Glu-Glu
(EE-tagged) p110 DNA/0.5 ml serum-free DMEM together with 125 µg
DEAE-dextran (23). The cells were incubated for 90 min at
37°C with the DNA solution followed by 5 h with DMEM containing
100 µM chloroquine. Cells were used 24–48 h after transfection. Cell
lysis, protein immunoprecipitation (using LS1/127 Ab-coupled protein
G-Sepharose beads), SDS-PAGE, and Western blotting were performed as
previously detailed (24).
Measurement of activated p21ras using Raf1 Ras binding domain (RBD)
pGex-KG-RBD (encoding a fusion protein of glutathione
S-transferase (GST) and residues 1–149 of human cRaf1 RBD)
was used to measure activation of p21ras using
the method described by de Rooij and Bos (25). Neutrophils
(1.5 x 107/sample in 80 µl PBS with
divalent cations) were primed (200 U/ml TNF-, 100 ng/ml GM-CSF, or
appropriate diluent) for 30 min at 37°C and stimulated with 100 nM
fMLP or buffer for 10–300 s. Reactions were stopped with ice-cold
lysis buffer (100 mM NaCl, 50 mM HEPES (pH 7.5), 1% Triton X-100, 1 mM
EDTA (pH 8), 1 mM EGTA (pH 8), 5 mM NaF, 12 mM
MgCl2, 1 mM
Na3VO4, and 10 µg/ml
aprotinin, pepstatin, leupeptin, and antipain). Nuclei were removed by
centrifugation and precoupled GST-RBD beads added to the lysates before
incubation at 4°C for 30 min. Beads were washed three times with cold
PBS containing 5 mM MgCl2 and 0.1% Triton X-100,
and subsequently boiled in Laemmli sample buffer. Protein samples were
separated by SDS-PAGE and Western blotted with a pan-Ras Ab. Blots were
developed using ECL Plus detection reagents.
Statistics
Data are presented as the mean ± SEM of n separate experiments. Data were analyzed using the Student t test or ANOVA followed by the Student-Kewan-Keuls post-test. Results were considered to be significant with p < 0.05. The Kd and maximal binding capacity of [3H]Ins(1, 3, 4, 5)P4 were determined by Scatchard analysis of [3H]Ins(1, 3, 4, 5)P4 displacement with unlabeled D-Ins(1, 3, 4, 5)P4 after correction for isotopic dilution and nonspecific binding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PtdIns(3, 4, 5)P3 has been proposed to be an important second messenger in O2- generation in phagocytic cells (8, 26). Traditional methods of measuring PtdIns(3, 4, 5)P3 accumulation involve metabolic labeling of cells with [32P]PO4 followed by HPLC analysis of individual sample extracts. Moreover, the incubation times and conditions required to achieve true isotopic equilibrium labeling of the agonist-sensitive PtdIns(4, 5)P2 pool in neutrophils invariably result in significant basal priming. These factors have precluded a detailed analysis of the effects of cytokine-mediated priming on agonist-stimulated PtdIns(3, 4, 5)P3 accumulation in neutrophils.
To address these problems we have used a recently described
PtdIns(3, 4, 5)P3 mass assay (17)
based on the ability of boiling KOH to convert, in a quantitative
manner, cell-extracted PtdIns(3, 4, 5)P3 to its
water-soluble counterpart, Ins(1, 3, 4, 5)P4. This
product can be measured by competitive radioreceptor assay using a
bacterially expressed GST-tagged
GAP-Ins(1, 3, 4, 5)P4 binding protein
(GAPIP4BP). A typical calibration curve for the
displacement of
[3H]Ins(1, 3, 4, 5)P4 from
the GAPIP4BP by unlabeled
Ins(1, 3, 4, 5)P4 is shown in Fig. 1
a
(Kd 3.9 ± 0.38 nM;
n = 5). The specificity of the assay for
Ins(1, 3, 4, 5)P4 was confirmed in this system using
authentic Ins(1, 4, 5)P3. As shown in Fig. 1a, no significant displacement of
[3H]Ins(1, 3, 4, 5)P4
binding was observed with up to 120 pmol of Ins(1, 4, 5)P3. The recovery of
Ins(1, 3, 4, 5)P4 during the extraction process
(determined using spiked
[3H]Ins(1, 3, 4, 5)P4) was
93% (data not shown). Assay values for each sample were corrected for
these losses and the efficiency of
PtdIns(3, 4, 5)P3 to
Ins(1, 3, 4, 5)P4 conversion (62%) and expressed as
pmol PtdIns(3, 4, 5)P3/8 x
106 neutrophils.
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As previously demonstrated, quiescent neutrophils or those treated
with TNF- (200 U/ml) or fMLP (100 nM) alone released minimal
O2-, whereas cells primed with
TNF- and subsequently stimulated with fMLP showed greatly enhanced
O2- release (Fig. 1
b).
A detailed time course of fMLP-stimulated
PtdIns(3, 4, 5)P3 mass accumulation was undertaken
in TNF--primed or unprimed neutrophils. Cells exposed to TNF- or
vehicle alone showed minimal PtdIns(3, 4, 5)P3
accumulation compared with values obtained in freshly isolated cells
(Fig. 1c). fMLP stimulation of unprimed cells resulted in a
rapid but transient increase in PtdIns(3, 4, 5)P3
accumulation with peak values between 5 and 15 s (Fig. 1c). In contrast, cells primed with TNF- and then
stimulated with fMLP showed a greater increase in
PtdIns(3, 4, 5)P3 accumulation at early times and a
sustained elevation in PtdIns(3, 4, 5)P3 levels
lasting for at least 60 s (Fig. 1c). Similar data were
obtained with the alternative priming agent GM-CSF (Fig. 1, d and e), suggesting that the ability of TNF-
to enhance fMLP-stimulated PtdIns(3, 4, 5)P3
accumulation is a "class effect" and is shared by other priming
agents.
To test this hypothesis further, the effects of an alternative
nonreceptor-acting priming agent, cytochalasin B, were also examined.
Table I shows that cytochalasin B
significantly increased both
O2- generation and
PtdIns(3, 4, 5)P3 accumulation in a similar manner
to TNF- and GM-CSF, suggesting that a broad array of priming agents
activate these responses through the same signaling pathways.
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We have demonstrated previously that neutrophils primed with PAF
spontaneously "deprime" over a period of 120 min even if left
continuously exposed to PAF (7). Fig. 2a confirms that the ability
of PAF to enhance fMLP-stimulated
O2- generation in neutrophils
is transient, with the priming effect completely lost by 120 min.
Previous studies have shown that cells exposed to PAF in this way
remain viable throughout this incubation period, revert to a fully
depolarized state with normal CD11b expression, and indeed are capable
of being "reprimed" by a second priming agent such as TNF-
(7).
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, c
and e). In agreement with the previous data with TNF- and
GM-CSF, cells treated with PAF for 10 min and then stimulated with fMLP
showed a large increase in O2-
generation and PtdIns(3, 4, 5)P3 accumulation
compared with unprimed, fMLP-stimulated cells (Fig. 2). In contrast,
cells treated with PAF for 120 min displayed
O2- and
PtdIns(3, 4, 5)P3 responses identical to unprimed
cells (Fig. 2). Time-matched controls were included where cells were
incubated for 2 h before the addition of PAF, showing that the
O2- and
PtdIns(3, 4, 5)P3 responses of these cells were
identical to those seen in freshly isolated cells. These results
demonstrate that the enhanced fMLP-stimulated
PtdIns(3, 4, 5)P3 accumulation seen in acutely
primed cells is lost when the cells are deprimed and that this effect
parallels O2- responsiveness
and does not reflect changes in cell integrity or metabolic status. Effects of PI3-kinase and protein kinase inhibitors on O2- and PtdIns(3, 4, 5)P3 generation
Consistent with previous results, LY294002, a well-characterized
and highly selective inhibitor of PI3-kinase activity, inhibited
fMLP-stimulated O2- generation
and PtdIns(3, 4, 5)P3 accumulation in both primed
and unprimed cells (Fig. 3, a
and e). Identical data were obtained with the alternative
PI3-kinase inhibitor wortmannin (data not shown). These results confirm
the involvement of a type I or III PI3-kinase in
O2- generation and
PtdIns(3, 4, 5)P3 accumulation in human
neutrophils.
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b) and a matched inhibition of
PtdIns(3, 4, 5)P3 accumulation (Fig. 3f). Genistein was effective only at concentrations >10
µM, suggesting that its effect may be due to inhibition of
alternative (non-tyrosine) kinases.
Staurosporine is a broad-spectrum serine/threonine protein kinase and
protein kinase C (PKC) inhibitor that has the paradoxical ability to
augment rather than inhibit agonist-stimulated respiratory burst
activity in neutrophils (27). Fig. 3, c and
g, demonstrates the ability of staurosporine to increase
both fMLP-stimulated O2-
generation and PtdIns(3, 4, 5)P3 accumulation in
primed and unprimed cells. This again demonstrates the close
association between these two events and the potential involvement of a
PKC-dependent pathway in inhibiting
PtdIns(3, 4, 5)P3 accumulation.
The most discriminating inhibitor used in this set of
experiments was radicicol, which is an antifungal antibiotic with
selective anti-tyrosine kinase properties that has been reported
to block Lyn-dependent activation of p85 PI3-kinase and yet has
no direct inhibitory effect on the G-regulated p101/p110
(28). Importantly, radicicol was found to have no effect
on fMLP-stimulated O2-
generation or PtdIns(3, 4, 5)P3 accumulation in
primed or unprimed cells (Fig. 3, d and h),
despite causing a concentration-dependent and complete inhibition of
platelet-derived growth factor (PDGF)-stimulated
[3H]thymidine incorporation in BTSM (Fig. 3i); we have previously shown this latter response to
reflect activation of a tyrosine kinase-dependent p85 PI3-kinase
pathway (21). Radicicol was also found to inhibit
thrombin-stimulated DNA synthesis in BTSM cells (data not shown).
These results in primed, deprimed, and kinase inhibitor-manipulated
cells underscore the close correlation between
PtdIns(3, 4, 5)P3 accumulation and
O2- generation in neutrophils.
The radicicol data in particular also suggest the involvement of the
G-regulated p101/p110 PI3-kinase in the augmented
PtdIns(3, 4, 5)P3 and
O2- responses seen under
TNF--primed conditions.
Determination of which PI3-kinase isoforms are responsible for enhanced PtdIns(3, 4, 5)P3 accumulation under primed/stimulated conditions
To determine more precisely which PI3-kinase isoforms are
responsible for the enhanced PtdIns(3, 4, 5)P3
production observed in primed neutrophils, selective
immunoprecipitation-based PI3-kinase assays were performed. Positive
control data for the experiments involving 4G10 and p85
immunoprecipitations were obtained using primary BTSM cells stimulated
with PDGF (20 ng/ml for 10 min). Fig. 4a demonstrates the
substantial increase in PI3-kinase activity associated with 4G10
anti-phosphotyrosine immunoprecipitates in BTSM cells after
stimulation with PDGF. In contrast, 4G10 (Fig. 4b) and p85
(Fig. 4d) immunoprecipitates prepared from primed and
fMLP-stimulated (45 s) neutrophils showed no increase in PI3-kinase
activity over control levels. Of note, wortmannin was able to fully
inhibit p85-associated PI3-kinase activity (Fig. 4d),
confirming the specificity of this assay. These results concur with the
data obtained using radicicol and imply that the increases observed in
PtdIns(3, 4, 5)P3 accumulation under
primed/stimulated conditions do not reflect activation of a tyrosine
kinase-linked p85/p110, , or PI3-kinase.
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-regulated isoform of PI3-kinase
(p101/p110) activity under primed/activated conditions, a mouse mAb
(LS1/127, IgM isotype-µ chain) was raised against His-tagged p110
expressed in Sf9 insect cells (29). The LS1/127 Ab was
able to immunoprecipitate EE-tagged p110 from transfected COS7 cells
with a similar efficiency to EE beads (Fig. 5a) and identify specific
cytoplasmic staining in Myc-p110 transfected COS7 cells (data not
shown). LS1/127 was also able to immunoprecipitate p110 activity
from porcine neutrophils (Fig. 5b) (but no other porcine
tissues; data not shown) and from EE-p110 transfected COS7 cells
(Fig. 5b).
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immunoprecipitates prepared from cells treated with
TNF- (200 U/ml for 30 min) or fMLP (100 nM for 45 s) alone and
under primed/stimulated conditions compared with control levels (Fig. 5c). Of note, Western blot analysis confirmed that an
identical amount of p110 was immunoprecipitated from neutrophil
lysates irrespective of the treatment conditions (data not shown). This
result further supports the suggestion that priming-induced increases
in fMLP-stimulated PtdIns(3, 4, 5)P3 accumulation
are mediated via enhanced activation of PI3-kinase-. Effect of priming on fMLP-stimulated p21ras activation
In view of these data and the previous demonstration that priming
up-regulates mitogen-activated protein kinase activity
(15), we investigated the effect of priming on
p21ras activation, an upstream regulator of both
PI3-kinase and MEK1/2 responses. Human neutrophils primed with GM-CSF
or TNF- were stimulated with fMLP, and p21ras
activation was determined. While no GTP-bound
p21ras was detected in either control cells or
cells treated with TNF- alone (Fig. 6, upper panel), GM-CSF induced a slight increase in
detectable GTP-Ras (Fig. 6, lower panel). Stimulation with
fMLP alone induced dramatic activation of
p21ras, maximal at 30–60 s and diminishing to
near baseline by 5 min (Fig. 6). Priming with TNF- did not augment
this response. The minor increase in the fMLP-stimulated
p21ras signal seen in GM-CSF-pretreated cells
was purely additive when quantified by densitometry (data not shown).
Thus, increased availability of GTP-bound p21ras
does not appear to underlie the enhancement of
PtdIns(3, 4, 5)P3 generation seen in
primed/stimulated neutrophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -, while specialized leukocyte
functions are the province of the predominantly leukocyte-restricted
PI3-kinase- and - isoforms. While defects in chemotaxis and
oxygen radical formation seen in neutrophils derived from animals
lacking PI3-kinase- support this premise, these results cannot be
extrapolated to human neutrophils, which undergo a far more intense and
primeable O2- response compared
with their murine counterparts. While Naccache et al. (30)
have also demonstrated PI3-kinase activity in p110 but not p85
immunoprecipitates in fMLP-stimulated neutrophils, the impact of
priming, which is an essential regulator of
O2- generation, was not
studied.
The main aims of this research were to extend the observation
that PtdIns(3, 4, 5)P3 accumulation
correlates with, and may signal for,
O2- generation, and to
determine which PI3-kinase isoforms are responsible for the increases
in fMLP-stimulated PtdIns(3, 4, 5)P3 mass observed
in primed cells. Because there is a lack of reagents, both Abs and
inhibitors specific for the p110/p101 PI3-kinase- isoform, several
different experimental strategies were used to address this issue.
A radioligand displacement assay was used to measure total
PtdIns(3, 4, 5)P3 mass accumulation in primed and
unprimed neutrophils after fMLP stimulation. This assay relies on the
quantitative conversion of PtdIns(3, 4, 5)P3 to
Ins(1, 3, 4, 5)P4 under strong alkaline hydrolysis
conditions and assay of the Ins(1, 3, 4, 5)P4
generated using a specific radioreceptor assay incorporating the
GAPIP4BP. As previously reported (30, 31), fMLP alone led to a rapid but transient increase in
PtdIns(3, 4, 5)P3 accumulation maximal at 5–15 s.
However, after priming with either TNF- or GM-CSF, fMLP resulted in
a more robust and prolonged increase in
PtdIns(3, 4, 5)P3 accumulation, with
PtdIns(3, 4, 5)P3 levels remaining elevated for up
to 2 min. Our earlier studies in metabolically labeled cells failed to
detect any defect in the metabolism of
PtdIns(3, 4, 5)P3 to
PtdIns(3, 4)P2 in these cells (1),
suggesting that the augmented accumulation of
PtdIns(3, 4, 5)P3 reflects enhanced production
rather than inhibition of metabolism. Moreover, the reported ability of
the NADPH oxidase to activate the
PtdIns(3, 4, 5)P3-5 phosphatase Src homology 2
domain-containing inositol phosphatase-1 via a Lyn-dependent
mechanism would predict that the metabolism of
PtdIns(3, 4, 5)P3 would be enhanced rather than
inhibited after fMLP stimulation (32). These data provide
the first evidence of a difference in
PtdIns(3, 4, 5)P3 mass accumulation between
fMLP-stimulated and primed/stimulated neutrophils. We also found that,
while TNF- treatment alone had no effect on
PtdIns(3, 4, 5)P3 levels at 30 min, GM-CSF
incubation over the same period resulted in a small but
consistent increase in PtdIns(3, 4, 5)P3
accumulation. This agrees with previous work using metabolically
labeled cells and estimates of
[32P]PtdIns(3, 4, 5)P3
accumulation (30, 33). Of note, the priming status of the
cells used in these experiments was confirmed by performing
O2- release assays alongside
each PtdIns(3, 4, 5)P3 mass and immunoprecipitation
assay to ensure that no biochemical analysis was undertaken in cells
that had been inadvertently primed during cell isolation. Hence, any
cells showing evidence of basal priming (i.e., having a ratio between
TNF-/fMLP and fMLP O2-
generation of <4) were discarded.
Other priming agents were also examined to determine whether all such
agents caused similar increases in
O2- and
PtdIns(3, 4, 5)P3 responses. Cytochalasin B was
found to prime both O2-
generation and PtdIns(3, 4, 5)P3 accumulation after
45 s of fMLP stimulation in a similar manner to that observed with
TNF-. Cytochalasin B is a non-G protein receptor-mediated agonist
that has been widely used to induce neutrophil priming; however, its
exact mode of action is not known and may involve other pathways
besides PI3-kinase. The effects of LPS were also examined and, while
both O2- and
PtdIns(3, 4, 5)P3 responses were increased, in the
absence of serum the effects were much less dramatic than those
observed with the other priming agents used (data not shown).
The ability of neutrophils to spontaneously "deprime" after
exposure to PAF for 120 min was also investigated. Previous work from
our group has shown that full priming (equivalent to that seen with
TNF- or GM-CSF at 30 min) is observed 10 min after PAF addition;
thereafter the cells regain their nonpolarized rounded morphology, and
CD11b/18 expression and function and
O2- responses return to normal
(7). Our results confirm normalization of the
O2- responses after a 120-min
incubation with PAF and show a parallel decline in fMLP-stimulated
PtdIns(3, 4, 5)P3 accumulation at a time point (45
s) that coincides with maximal respiratory burst activity
(1).
Using a variety of PI3-kinase and other kinase inhibitors we have been
able to show a remarkably similar response profile with respect to
O2- generation and
PtdIns(3, 4, 5)P3 accumulation. Both
PtdIns(3, 4, 5)P3 and
O2- generation was severely
inhibited by LY294002, confirming the involvement of a PI3-kinase in
both of these responses. The tyrosine kinase inhibitor genistein was
also found to inhibit both PtdIns(3, 4, 5)P3 and
O2- generation, albeit at
relatively high concentrations. Staurosporine, a serine/threonine
protein kinase and PKC inhibitor, was found to increase both
fMLP-stimulated O2- generation
and PtdIns(3, 4, 5)P3 accumulation to levels equal
to or higher than those observed with TNF- priming. This rather
surprising "priming" effect of staurosporine has also been observed
by other workers (27); however, the mechanism remains to
be defined. More importantly, the lack of inhibition observed with
radicicol, a selective tyrosine kinase inhibitor that lacks activity
against the G-regulated PI3-kinase (28) but
abolished p85-regulated PI3-kinase activity in BTSM cells (Fig. 3i and Ref. 21), suggested that the enhanced
O2- and
PtdIns(3, 4, 5)P3 signal observed under priming
conditions occurs via the p101/p110 PI3-kinase rather than through a
tyrosine kinase-regulated process. However, it is possible that a
component of the fMLP-stimulated PtdIns(3, 4, 5)P3
response observed in unprimed cells originates from an alternative
PI3-kinase; the data of Ptasznik et al. (28), who have
shown that radicicol can inhibit fMLP-stimulated
PtdIns(3, 4, 5)P3 production in unprimed cells,
support this conclusion.
Using more traditional lipid kinase assays to measure PI3-kinase
activity, we found that fMLP was unable to stimulate PI3-kinase
activity in either p85 or 4G10 immunoprecipitates, irrespective of the
priming status of these cells. These data were obtained in the face of
clearly positive signals in PDGF-stimulated BTSM cells and again point
to the lack of involvement of a tyrosine kinase-regulated form of
PI3-kinase in the PtdIns(3, 4, 5)P3 responses
observed at 45 s post-agonist addition. While such data agrees
with that obtained by Naccache et al. (30), Stephens et
al. (10), and Vlahos et al. (34), other
groups have reported enhanced PI3-kinase activity in p85
immunoprecipitates from neutrophils (35, 36). However, in
the latter study (36), lysates were prepared from adherent
cells treated with TNF- and it is possible that this, as well as the
use of different primary Abs, may underlie such differences.
In agreement with Naccache et al. (30), we were able to
show significant involvement of the p110 subunit in neutrophil
PI3-kinase activity after fMLP treatment. However, the increased
PI3-kinase activity observed in p110 immunoprecipitates prepared
from cells treated with TNF- alone was surprising, because there was
no accompanying increase in PtdIns(3, 4, 5)P3
accumulation. While it is possible that TNF- enhances
PtdIns(3, 4, 5)P3 degradation (and this would
concur with the small increases in
[32P]PtdIns3P accumulation in TNF--treated
neutrophils previously reported (1)), effects of priming
agents on Src homology 2 domain-containing inositol phosphatase-1 and
the PtdIns(3, 4)P2-4 phosphatases are not
described. It is also possible that the enzyme has limited access to
PtdIns(4, 5)P2 or, more likely, that the absence
of activated G subunits precludes PI3-kinase- activation in
the intact cell. Similar explanations could pertain to the lack of
difference in PI3-kinase- activity observed between unprimed and
primed cells treated with fMLP. New insights into how 3-phosphorylated
lipids might control the assembly and hence activity of the neutrophil
NADPH oxidase complex have come from recent work demonstrating that
components of the enzyme complex, namely p47phox
and p40phox, bind
PtdIns(3, 4)P2 and PtdIns3P (both breakdown
products of PtdIns(3, 4, 5)P3), respectively, via
PX domains (14). Therefore, it seems likely that
PtdIns(3, 4, 5)P3 degrading enzymes may be
intimately involved in the control of the oxidase response.
The p85 and G-regulated forms of PI3-kinase have both been shown
to bind p21ras, and indeed ras is
thought to be an important upstream regulator of PI3-kinase. Additional
data suggest that p21ras can also be a target of
PI3-kinase (37). Ras also regulates the activity of the
MEK1/2 pathway, which is also known to be up-regulated after priming
(15). Despite this, we demonstrated that TNF- had no
effect on the amount of active, GTP-bound p21ras
present in neutrophils and failed to influence the strength of the
fMLP-stimulated signal. Likewise, fMLP alone caused robust activation
of p21ras, with GM-CSF having an intermediate
effect. While we have not excluded the possibility that priming agents
alter the activity of p21ras within the
detergent-insoluble neutrophil fraction, our data concur with those
reported by Coffer et al. (35) and suggest a dissociation
between p21ras activity and both
O2- release and
PtdIns(3, 4, 5)P3 accumulation. Thus, these data
indicate that priming agents enhance PI3-kinase- activity by factors
other than p21ras. Because previous studies have
shown that priming-mediated effects on receptor expression are
temporally dissociated from the up-regulation of secretory responses
and that fMLP-stimulated Ins(1, 4, 5)P3
accumulation is unaffected by priming (1), other
mechanisms, including the regulation of Gi
subunit expression (38) or factors affecting PI3-kinase
localization or substrate access, need to be considered.
In conclusion, priming represents a major checkpoint controlling the
functional responsiveness of neutrophils to secretagogue agonists and,
as such, their capacity to induce tissue damage in vivo. We have
demonstrated the close relationship between fMLP-stimulated
PtdIns(3, 4, 5)P3 accumulation and
O2- generation under conditions
of (TNF-, GM-CSF, cytochalasin B, and PAF-induced) priming,
depriming, and kinase inhibition and present evidence to suggest that
the G-regulated PI3-kinase (P101/p110) is the dominant
PI3-kinase involved in this response.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TJ, U.K.
3 Address correspondence and reprint requests to Dr. Edwin R. Chilvers, Respiratory Medicine Division, Department of Medicine, Box 157, Level 5, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K. E-mail address: erc24{at}cam.ac.uk
4 Abbreviations used in this paper: PI3-kinase, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; O2-, superoxide anion; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; GST, glutathione S-transferase; PAF, platelet-activating factor; BTSM, bovine tracheal smooth muscle; RBD, Ras binding domain; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PDGF, platelet-derived growth factor, EE, Glu-Glu.
Received for publication April 5, 2002. Accepted for publication July 15, 2002.
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) and inositol 3,4,5-trisphosphate (
)
at the indicated concentrations was measured in a final assay
volume of 0.4 ml after a 30-min incubation on ice with recombinant
GAPIP4BP. Each point represents a determination in
triplicate ± SEM of three independent experiments.
b and d, O2-
generation in human neutrophils. Cells were primed with 200 U/ml
TNF-
, Control; 
, fMLP; 
,
TNF-
