Effect of priming on activation and localization of phospholipase D-1
in human neutrophils
Karen A. Cadwallader
1
, Mohib Uddin
1
, Alison M. Condliffe
1
, Andrew S. Cowburn
1
, Jessica F. White
1
, Jeremy
N. Skepper
2
, Nicholas T. Ktistakis
3
and Edwin R. Chilvers
1
1
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and
Papworth Hospitals, Cambridge, UK;
2
Department of Anatomy, University of Cambridge, UK;
3
Department of Signalling, Babraham
Institute, Cambridge, UK
Phospholipase D (PLD) plays a major role in the activation
of the neutrophil respiratory burst. However, the repertoire
of PLD isoforms present in these primary cells, the precise
mechanism of activation, and the impact of cell priming on

mechanism of a wide range of inflammatory diseases. The
extent of neutrophil activation is influenced by the prior
exposure of these cells to agents such as tumour necrosis
factor a (TNFa), granulocyte macrophage colony stimu-
lating factor or platelet activating factor (PAF). These
Ôpriming agentsÕ promote a dramatic increase in the
functional responses evoked by subsequent exposure to
secretagogue agonists such as fMLP or interleukin-8, and
this excessive activation is thought to be one of the key
events underlying neutrophil-mediated tissue damage
in vivo [1]. Despite the recognition that priming is such
an important regulator of neutrophil physiology, compar-
atively little is known of the signalling mechanisms
underlying this process.
Neutrophil activation induced by an array of
G-protein-coupled receptors leads to an increase in
phospholipase D (PLD) activity and the hydrolysis of
PtdChotoPtdOHandcholine[2];PtdOHisthen
metabolized to diacylglycerol (DAG) by the enzyme
phosphatidate phosphohydrolase (PAP). Both PtdOH
and DAG have been proposed to act as important second
messengers linking cell stimulation to various effector
functions including phagocytosis [3], degranulation [4],
and respiratory burst activity [5,6]. It is now known that
mammalian cells contain two major PLD isoforms, PLD1
and PLD2, as well as additional splice variants. Both
isoforms have an absolute requirement for the lipid
phosphatidylinositol 4,5-bisphosphate, but activation of
PLD1 also requires interaction with ADP-ribosylation
factor (ARF), RhoA or protein kinase Ca [7], whereas

neutrophils [9,10] and the cosignals required for PLD
activity, in particular the role of phosphatidylinositol
3-kinase (PI3-kinase), which we and others have shown to
play a crucial role in the activation of NADPH oxidase
[11,12]. Hence we have recently reported that the
metabolic product of PI3-kinase, phosphatidylinositol
3-phosphate, can activate the NADPH oxidase complex
by binding to the PX domain of the p40
phox
component
[13]. Of note, the PX domain of p47
phox
has also been
shown to possess a binding site for PtdOH, although the
relevance of this to membrane localization and activation
of the oxidase complex has yet to be determined [14].
In this study, we show for the first time that priming with
TNFa causes a substantial up-regulation of agonist-stimu-
lated PLD enzymatic activity in neutrophils which parallels
the enhanced functional responses observed. We identify
PLD1 as the major PLD isoform present in human
neutrophils and reveal that PLD localizes to the phago-
somal membrane after particle ingestion but not to the
plasma membrane after stimulation with soluble agonists.
Furthermore, we demonstrate that PLD activation occurs
via a PI3-kinase-sensitive and brefeldin-sensitive ARF
GTPase-regulated mechanism and provide evidence that
the lipid products formed after PLD activation have an
unexpected and differential effect in supporting degranula-
tion and O

zymosan was supplied by Molecular Probes (Eugene, OR,
USA).
Isolation of human neutrophils
Human neutrophils were isolated from venous blood of
normal healthy volunteers using dextran sedimentation
followed by centrifugation on plasma-Percoll gradients as
previously detailed [16]. The viability of cells, as assessed
by trypan blue exclusion, was >97% and the purity of
neutrophil preparations was routinely >96% with <0.1%
mononuclear cell contamination.
Measurement of degranulation
Agonist-stimulated myeloperoxidase (MPO) release was
determined by the 3,3-dimethoxybenzidine method as
described previously [17] with the following minor modifi-
cations. Human neutrophils (10
6
) were suspended in NaCl/
P
i
with Ca
2+
and Mg
2+
(80 lL) and incubated with TNFa
(200 UÆmL
)1
)orNaCl/P
i
at 37 °C for 30 min followed by
fMLP (100 n

2
using superoxide dismutase-inhibitable
reduction of cytochrome c as described previously [18].
Determination of PLD activity
PLD activity was assayed in [
3
H]lyso-PAF (1-O-[
3
H]oct-
adecyl-sn-glycero-3-phosphocholine)-labelled neutrophils
by measuring the formation of [
3
H]phosphatidylbutanol
([
3
H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol as
described previously [19]. Human neutrophils (5 · 10
6
per
0.24 mL) prelabelled with [
3
H]lyso-PAF were suspended in
NaCl/P
i
with Ca
2+
and Mg
2+
in the presence of 0.3% (v/v)
butan-1-ol. Cells were incubated for the time periods

cellsÆmL
)1
) were washed twice in NaCl/P
i
without Ca
2+
and
Mg
2+
, lysed in 1 mL detergent lysis buffer [50 m
M
Tris/
HCl, pH 7.5, 150 m
M
NaCl, 5 m
M
EDTA, 1% (v/v)
Nonidet P40 and 0.5% (v/v) CHAPS supplemented with
1 tablet per 50 mL lysis solution of a broad-spectrum
proteinase (Roche Applied Science, Lewes, East Sussex, UK
(Complete tablets)] and left on ice for 30 min. Cells were
homogenized or briefly sonicated and then spun (5 min,
15 000 g) to remove insoluble material. The supernatant
was collected and immunoprecipitated with protein A–
Sepharose and the pan-PLD1/2 antibody for 2 h. After
being washed and boiled in sample buffer, samples were
analyzed by SDS/PAGE (10% gel).
For fractionation experiments, cells were lysed in 1 mL
hypotonic lysis buffer [10 m
M

tion of 0.5% (v/v). Cytosolic and membrane fractions were
immunoprecipitated and Western blotted as above. Anti-
body-bound proteins were detected by ECL. The specificity
of the pan-PLD1/2 antibody was confirmed using whole cell
lysates of several cell lines that express PLD1 (U937, NIH
3T3 and CCL39) or PLD2 (Rat1) only. Differences in the
molecular mass of the band corresponding to PLD were
observed according to the presence of PLD1 or PLD2 in
these cell lines (data not shown). CHO cells transfected with
human PLD1 were also used as a positive control [15].
Immunofluorescent staining for PLD
Nonopsonized zymosan was sonicated and added to
neutrophils (25 · 10
6
cellsÆmL
)1
in NaCl/P
i
containing
CaCl
2
and MgCl
2
) in a 5 : 1 particle to cell ratio at 37 °C.
After 10 min, cells were diluted 10-fold in autologous serum
and immediately cytospun (28 g,5min).Thecytospinswere
fixed [4% (v/v) paraformaldehyde, 10 min] and permeabi-
lized [0.1% (v/v) Triton X-100, 10 min] before blocking with
NaCl/P
i

Software, San Diego, CA, USA). Differences were consid-
ered significant when P<0.05.
Results
Effect of TNFa priming on O
À
2
generation, MPO release,
and PLD activation
Before investigating the effect of TNFa priming on PLD
activity, we wished to confirm that our experimental system
was optimal for demonstrating priming-mediated up-regu-
lation of neutrophil effector functions. Figure 1 shows that
both O
À
2
generation and myeloperoxidase (MPO) release
were minimal when either priming agent (TNFa)or
activating agent (fMLP) were added alone. However, if
cells were incubated with TNFa (200 UÆmL
)1
,30min)
before fMLP stimulation (100 n
M
,10 min),O
À
2
and MPO
responses are significantly increased. Detailed time-course
analysis of fMLP-stimulated O
À

H]PtdBut accumulating within the first
5 min of stimulation.
Effect of PI3-kinase and ARF inhibitors on O
À
2
and
PLD activation
Selective pharmacological inhibitors were used to assess
the role of PI3-kinase and ARF proteins on the activation
of PLD. As illustrated in Fig. 3A,B, the PI3-kinase
inhibitor wortmannin (100 n
M
) [11] markedly attenuated
both fMLP-stimulated O
À
2
generation and [
3
H]PtdBut
accumulation. Brefeldin A (100 lgÆmL
)1
), an inhibitor of
Ó FEBS 2004 PLD1 activation in neutrophil priming (Eur. J. Biochem. 271) 2757
ARF localization and the guanine nucleotide exchange
factors (GEFs) for ARF (BIG-1 and 2), was also found to
inhibit O
À
2
generation and PLD activity (Fig. 3C,D).
O

) for 10 min or the times indicated. O
À
2
generation (A)
and MPO release (B) were determined as described in Materials and
methods. The data in the inserts represent mean ± SEM from at least
three experiments performed in triplicate. Data points for the time
courses in (A) and (B) show a representative experiment performed in
triplicate. ***P < 0.001, significant increase in O
À
2
generation and
MPO release over fMLP-stimulated levels.
Fig.2.EffectofTNFa priming on fMLP-stimulated PLD activity.
Cells were incubated with TNFa (200 UÆmL
)1
)orNaCl/P
i
for 30 min
at 37 °C and then stimulated with various concentrations of fMLP for
10 min (A). [
3
H]PtdBut accumulation was determined as described in
Materials and Methods. (B) Time course of [
3
H]PtdBut accumulation
after treatment with TNFa (200 UÆmL
)1
)orNaCl/P
i

potentiated MPO release under both fMLP only and
TNFa-primed/fMLP-stimulated conditions (by 169 ±
37% and 71 ± 9.2%, respectively). In contrast, butan-1-
ol (0.3%, v/v) caused a near complete inhibition of TNFa-
primed/fMLP-stimulated MPO release under conditions in
which O
À
2
generation was only marginally suppressed
(Fig 4). Butan-1-ol alone, up to a concentration of 3%, had
no direct inhibitory effect on the MPO assay (data not
shown). These data suggest that important differences exist
with regard to the lipid repertoire required to support MPO
release and O
À
2
generation, with the former response being
more dependent on PtdCho-derived PtdOH.
Identification and localization of PLD isoforms
in neutrophils
To identify the PLD isoform(s) present in neutrophils, PCR
primers were designed to unique regions of PLD1 or PLD2
as detected by sequence analysis (data not shown). With the
use of a semiquantitative RT-PCR technique, PLD1 was
identified as the predominant mRNA present in freshly
isolated neutrophils with far lower levels of expression of
PLD2 (Fig. 5A). Identical data were obtained using eosi-
nophil-depleted neutrophils, confirming that these signals
were not consequent on the 1–5% eosinophil contamination
present in our granulocyte preparations.

Data represent a single experiment representative of three independent experiments performed in triplicate.
Ó FEBS 2004 PLD1 activation in neutrophil priming (Eur. J. Biochem. 271) 2759
P40 and 0.5% CHAPS (Fig. 5B). Initial PLD immuno-
precipitates were generated with an antibody specific for
either pan-PLD1/2 or PLD1. Western blotting of these
immunoprecipitates with the pan-PLD1/2 antibody con-
firmed the presence of PLD1 in human neutrophils
(Fig. 5B) with an approximate size of 120 kDa. Lysates
of CHO cells transfected with human PLD1 were used as
positive controls.
The subcellular distribution of PLD and the conse-
quences of priming and activation were determined using
immunoprecipitation and Western blot analysis of neutro-
phil cytosol and membrane fractions using the pan-PLD1/2
antibody (Fig. 5C). PLD was found to be membrane
associated under all conditions, with no overt change in the
membrane/cytoplasm ratio after TNFa priming and/or
fMLP stimulation.
With the use of confocal microscopy and the PLD1-
specific antibody, PLD was found to exhibit a punctate
pattern of distribution (characteristic of granule membrane
staining) which did not alter after priming and/or stimula-
tion with soluble stimuli (Fig. 6A). However, intense PLD
immunostaining was apparent at the margin of the phag-
olysosome formed after ingestion of nonopsonized zymosan
particles (Fig. 6B). Identical results were observed using the
pan-PLD1/2 antibody and two other independent PLD1
Fig. 4. Effect of butan-1-ol and phosphatidate phosphohydrolase inhi-
bition on fMLP-stimulated respiratory burst and degranulation in
TNFa-primed neutrophils. The inhibitors propranolol (200 l

PLD1 or vector alone were also run. (C) Cells were incubated for
30mininthepresenceorabsenceofTNFa (200 UÆmL
)1
)beforesti-
mulation with fMLP (100 n
M
, 1 min). Cells were lysed and fraction-
ated into cytosolic and membrane fractions as described in Materials
and methods. Immunoprecipitations were performed with the pan-
PLD1/2 antibody. SDS/polyacrylamide gels were then blotted with the
pan-PLD1/2 antibody. s, Cytosolic fraction; p, membrane fraction.
2760 K. A. Cadwallader et al.(Eur. J. Biochem. 271) Ó FEBS 2004
antibodies (Cell Signaling, Beverly, MA, USA; data not
shown).
The distribution of immunogold label for PLD was
similar in all four treatments (Fig. 7). Gold label was seen
over both intact and degranulated vesicles and diffusely over
the cytoplasm of neutrophils. Label was absent over the
nuclei and plasma membrane. No difference in the distri-
bution was observed in any of the four treatment groups.
With the use of commercially available PLD2-specific
antibodies, PLD2 could not be detected by immunoprecip-
itation or immunofluorescence techniques under any treat-
ment condition.
Discussion
The recent cloning of the mammalian forms of PLD has led
to renewed interest in the regulation and downstream effects
of PLD. Although neutrophil priming has been previously
shown to result in a small up-regulation of agonist-
stimulated PLD activation [6,8,23], the underlying mecha-

caused significant inhibition of both fMLP-stimulated PLD
activity and O
À
2
generation. Moreover, this inhibitor also
abolished the oxidative burst in response to nonopsonized
zymosan, whereas there was no effect on O
À
2
generation
with opsonized zymosan (data not shown). Together these
observations suggest a selective role for a brefeldin-sensitive
ARF–GEF complex in regulating granulocyte responses to
soluble stimuli [29,30].
Brefeldin-sensitive PLD activation and O
À
2
generation
have previously been described [31,32] and are thought to be
mediated by Class 1 ARFs such as ARF1 and ARF3. More
recently, ARF6 has been implicated in activating PLD and
functional responses in chromaffin cells [33], macrophages
[34] and epithelial cell lines [35], and in neutrophil-like PLB-
985 cells a specific role for ARF6 (controlled by brefeldin-
insensitive GEFs) has been implicated in the activation of
NADPH oxidase after fMLP stimulation [36]. We recog-
nize, however, that brefeldin A has also been reported to
block ARF binding to Golgi membranes and the trans-
location of proteins from the endoplasmic reticulum to the
Golgi and to cause disassembly of the Golgi complex [37]

staining was imaged using confocal microscopy. (B) For
immunofluorescence studies, neutrophils were incubated with non-
opsonized zymosan in a 5 : 1 particle to cell ratio, fixed, and perme-
abilized before incubation with the pan-PLD1/2 antibody.
Ó FEBS 2004 PLD1 activation in neutrophil priming (Eur. J. Biochem. 271) 2761
It should be noted, however, that the modest suppression of
fMLP-stimulated O
À
2
generation in TNFa-primed cells by
propranolol suggests that DAG is not the sole mediator of
the respiratory burst response. Our results therefore reveal
important differences in the lipid-dependency of the oxida-
tive and secretory responses in neutrophils. It is probable,
however, that DAG and PtdOH act together to activate
components of the NADPH oxidase. This theory is
supported by earlier work in intact neutrophils showing
that activation of O
À
2
generation occurs in parallel with
the elevation of both PtdOH and DAG levels [40] and in
experiments using cell-free systems where PtdOH and DAG
work collectively to activate NADPH oxidase components
[41]. It should also be noted that propranolol is a
b-adrenoceptor antagonist and has also been shown to
inhibit protein kinase C [42]. It is possible therefore that
some of the effect observed with propranolol reflected
protein kinase C inhibition.
Characterization of the PLD isoforms present in human

in RBL-2H3 cells localizes to secretory granules and
lysosomes of unstimulated cells and, on stimulation,
translocates to the plasma membrane [45]. Other investiga-
tors have reported that PLD1 is localized on the Golgi [46],
nucleus [47] and lysosomal/endosomal compartments [48].
It is possible therefore that the subcellular distribution of
PLD is cell specific and may depend on the nature of the
stimulus. Although previous studies in human neutrophils
have demonstrated translocation of ARF1-regulated PLD
activity from secretory granules to the plasma membrane
after fMLP stimulation [49], no shift in PLD protein was
apparent in our studies. This was despite the induction of
maximal degranulation (using an optimal priming/activa-
Fig. 7. Gold electron microscopy of PLD
localization in human neutrophils. Gold label is
present over intact vesicles (single arrowheads)
and degranulated vesicles (double arrow-
heads) and diffusely over the cytoplasm. Label
is not present over the nuclei (n). Scale bar is
100 nm. (A) Control; (B) TNFa treatment;
(C) fMLP treatment; (D) TNFa and fMLP.
2762 K. A. Cadwallader et al.(Eur. J. Biochem. 271) Ó FEBS 2004
tion strategy) and clear evidence of granule depletion on
electron microscopy (data not shown). Given that we found
no translocation of PLD from the cytosol to the crude
membrane fraction, we investigated the possibility that the
uplift in PLD activity reflected translocation of ARF rather
than PLD. Preliminary data (not shown) have indicated
that under basal and TNFa-primed conditions, ARF1 and
ARF6 are distributed equally between the cytosol and

Advanced Fellowship, and J.F.W. an MRC Clinical Training Fellow-
ship. K.A.C. is a British Lung Foundation Research Scientist. Electron
microscopy was carried out in the MIC which was established with
funding from the Wellcome Trust. We thank Dr Nancy Pryer, Onyx
Pharmaceuticals, USA for kindly donating the pan-PLD1/2 antibody
and Professor Michael Wakelam for helpful discussions.
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