Báo cáo khoa học: Effect of priming on activation and localization of phospholipase D-1 in human neutrophils potx

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

PLD activity and localization remain poorly defined.
RT-PCR analysis showed that both PLD1 and PLD2 iso-
forms are expressed in human neutrophils, with PLD1
expressed at a higher level. Endogenous PLD1 was detected
by immunoprecipitation and Western blotting, and was
predominantly membrane-associated under control and
primed/stimulated conditions. Immunofluorescence showed
that PLD had a punctate distribution throughout the cell,
which was not altered after stimulation by soluble agonists.
In contrast, PLD localized to the phagolysosome membrane
after ingestion of nonopsonized zymosan particles. We
also demonstrate that tumour necrosis factor a greatly
potentiates agonist-stimulated PLD activation, myelo-
peroxidase release, and superoxide anion generation, and
that PLD activation occurs via a phosphatidylinositol 3-
kinase-sensitive and brefeldin-sensitive ADP-ribosylation
factor GTPase-regulated mechanism. Moreover, propran-
olol, which causes an increase in PLD-derived phosphatidic
acid accumulation, caused a selective increase in agonist-
stimulated myeloperoxidase release. Our results indicate that
priming is a critical regulator of PLD activation, that the
PLD-generated lipid products exert divergent effects on
neutrophil functional responses, that PLD1 is the major
PLD isoform present in human neutrophils, and that PLD1
actively translocates to the phagosomal wall after particle
ingestion.
Keywords: inflammation; lipid mediators; neutrophils;
second messengers; signal transduction.
Neutrophils play a critical role in host defence against
invading pathogens, but have also been implicated in the

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

PLD2 has no such requirement.
Although the mechanisms of PLD activation have been
studied extensively in many cells including neutrophils
[2,6,8], much of this work has been conducted in
transformed cells using overexpression systems. Hence to
date, PLD expression has yet to be demonstrated at a
Correspondence to K. Cadwallader, Respiratory Medicine Division,
Department of Medicine, University of Cambridge School of Clinical
Medicine, Level 5, Box 157, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 2QQ, UK. Fax: + 44 1223 762007,
Tel.: + 44 1223 762007, E-mail:
Abbreviations:TNFa, tumour necrosis factor a; PAF, platelet acti-
vating factor; PLD, phospholipase D; DAG, diacylglycerol; ARF,
ADP-ribosylation factor; PI3-kinase, phosphatidylinositol 3-kinase;
MPO, myeloperoxidase; O
2
À
, superoxide anion; PtdCho, phosphat-
idylcholine; [
3
H]PtdBut, [
3
H]phosphatidylbutanol; CHAPS, 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate; GEF,
guanine nucleotide exchange factor.
(Received 15 October 2003, revised 5 May 2004,
accepted 6 May 2004)
Eur. J. Biochem. 271, 2755–2764 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04204.x
protein level in any primary cell. Uncertainty also exists
over the nature of the PLD isoforms expressed in

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

À
2
responses.
Materials and methods
Materials
Cytochrome c, superoxide dismutase, fMLP, 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS) and phosphate-buffered saline (NaCl/P
i
with or
without CaCl
2
and MgCl
2
) were purchased from Sigma
Chemical Company (Poole, Dorset, UK). Percoll, dextran
and the ECL detection kit were obtained from Amersham
Biosciences (Amersham, Bucks., UK). Human recombinant
TNFa was supplied by R & D Systems (Abingdon, Oxon,
UK). Wortmannin, brefeldin A and propranolol were
obtained from Calbiochem (Nottingham, UK). Fetal
bovine serum,
L
-glutamine and RPMI 1640 medium were
from Gibco (Flow Laboratories, Rickmansworth, Herts.,
UK). The pan-PLD1/2 antibody was generated in rabbits
using the C-terminal third of hPLD1 (amino acids 770–
1075) as antigen. The PLD1-specific antibody was generated
as described previously [15]. Secondary antibodies were
obtained from Dako (Ely, Cambs., UK). Nonopsonized

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

M
)orNaCl/P
i
for 10 min. For inhibitor
studies, cells were preincubated with compound or vehicle
for 10 min before agonist stimulation. The reactions were
terminated by using ice-cold NaCl/P
i
. Supernatants were
incubated with phosphate buffer (pH 6.2), containing
3,3-dimethoxybenzidine (0.125 mgÆmL
)1
)andH
2
O
2
(0.001%, v/v), for 20 min, at 37 °C. NaN
3
(0.1%, w/v)
was added, and the amount of MPO released was measured
spectrophotometrically (460 nm) and expressed as a per-
centage of the total MPO activity present in 0.2% (v/v)
Triton X-100-lysed cells.
Measurement of O
À
2
generation
Respiratory burst activity was assessed by measuring the
generation of O
À

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

indicated, and reactions terminated with 0.94 mL ice-cold
chloroform/methanol (1 : 2, v/v). Total lipids were extrac-
ted, and [
3
H]PtdBut formation was measured by liquid-
scintillation counting after TLC separation as detailed
previously [20].
RT-PCR procedures
Total RNA was isolated using RNeasy mini spin columns
(Qiagen, Crawley, West Sussex, UK). RNA (2 lg) was
transcribed into cDNA using oligo(dT) primers (Invitrogen
Life Technologies, Paisley, UK) and 50 U reverse transcrip-
tase (Promega, Southampton, UK). PCR amplification was
performed using primer sets specific for PLD1 (sense:
5¢-ATGAGACACCCGGATCATGT; antisense: 5¢-ACT
CACTGGACGGGTGAAAG; 496 bp product) and
PLD2 (sense: 5¢-CTGCACCCCAACATAAAGGT; anti-
sense: 3¢-GTTCTCCAGAGTCCCTGCTG; 594 bp prod-
uct). For a control reaction, a specific primer set for b-actin
(sense: 5¢-GTGGGGCGCCCCAGGCACCA; antisense:
3¢-CTCCTTAATGTCACGCAGCACGATTTC; 548 bp
product) was used. PCR (35 cycles) was performed using
2UampliTaq DNA polymerase (Bioline, London, UK).
PCR products were analyzed by 1% agarose gel electro-
phoresis and imaged with ethidium bromide under UV
light.
2756 K. A. Cadwallader et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Western blot analysis
After the above treatments, human neutrophils (10 · 10
6

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

Tris/HCl, pH 7.5, 5 m
M
MgCl
2
,1m
M
dithiothreitol, 1 m
M
phenylmethanesulfonyl
fluoride plus a broad-spectrum proteinase inhibitor tablet
(see above)] and left on ice for 30 min. Cells were
homogenized or briefly sonicated and then spun to remove
insoluble material (5 min, 1500 g). The supernatants were
collected and respun (100 000 g, 30 min), and the pellet
(membrane fraction) was resuspended in membrane lysis
buffer (50 m
M
Tris/HCl, pH 7.5, 50 m
M
NaCl, 5 m
M
MgCl
2
,1m
M
dithiothreitol, 1 m
M
phenylmethanesulfonyl
fluoride plus a broad-spectrum proteinase inhibitor tablet).
CHAPS was added to both fractions to a final concentra-

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

/0.5% BSA/1% goat serum. The pan-PLD1/2 anti-
body was used at a 1 : 100 dilution, and the secondary
fluorescein isothiocyanate goat anti-rabbit Ig used at a
dilution of 1 : 300. The same method was used to examine
PLD distribution under primed/stimulated conditions (see
above).
Electron microscopy
Neutrophils were fixed in 4% (v/v) formaldehyde in 0.1
M
Pipes buffer for 1 h, cryoprotected in 25% (v/v) polypro-
pylene glycol, frozen in propane, and freeze-substituted in
methanol containing 0.01% (w/v) uranyl acetate at )90 °C.
They were subsequently embedded in Lowicryl HM20 at
)50 °C [21]. Thin sections mounted on nickel film were
incubated in primary antibodies to PLD diluted 1 : 100 in
NaCl/P
i
containing 2% BSA, 0.1% Triton X-100 and 0.1%
Tween 20 (w/v/v/v) for 16 h. Primary antibody-binding sites
were visualized by incubation with colloidal gold particles
(10 nm) conjugated to species-specific secondary antibodies.
The sections were stained with uranyl acetate and lead
citrate and viewed in a Philips CM 100.
Statistical analysis
All values are expressed as mean ± SEM from n separate
experiments. Where appropriate, results were analyzed by
analysis of variance followed by Student–Newman–Keuls
post-test with the statistical program
PRISM
(GraphPad

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

2
release showed that
maximal O
À
2
generation occurred by 1 min and that levels
were back to basal by 2 min. MPO release in response to
fMLP was likewise extremely rapid (Fig. 1B) with more
than 90% of the response occurring within 5 min.
PLD has a unique characteristic in that it can catalyse a
transphosphatidylation reaction such that, in the presence
of the primary alcohol butan-1-ol, PtdBut is formed rather
then PtdOH. PtdBut is a relatively stable product, and the
amount formed can be used as a measure of PLD activity.
Figure 2A shows that fMLP stimulated a concentration-
dependent accumulation of [
3
H]PtdBut in TNFa-primed
cells (EC
50
¼ 2.8 ± 0.08 n
M
) with a maximal response
achieved with 100 n
M
fMLP. TNFa priming significantly
enhanced fMLP-stimulated PLD activity compared with
the levels observed with TNFa or fMLP alone (Fig. 2B,C)
with > 90% of the [
3

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

À
2
generation and PLD activity were also measured
across a range of brefeldin A (0–300 lgÆmL
)1
)and
wortmannin (0–100 n
M
) concentrations (inserts in
Fig. 3). These results indicate that PI3-kinase-sensitive
and brefeldin A-sensitive GEFs (ARF1 or ARF3) play an
important role in the activation of PLD.
Functional role of PLD-derived second messengers
To determine the extent to which the activation of
neutrophil functional responses was dependent on individ-
ual PtdCho-derived second messengers, experiments were
performed using butan-1-ol, which diverts a proportion of
PtdCho hydrolysis into the formation of PtdBut rather than
PtdOH, and propranolol, which at 200 l
M
, sequesters
Fig. 1. Effect of TNFa priming on fMLP-stimulated respiratory burst
activity and degranulation. Neutrophils were incubated with TNFa
(200 UÆmL
)1
)orNaCl/P
i
for 30 min at 37 °C and then stimulated with
fMLP (100 n
M

) 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

for 30 min at
37 °C and then fMLP (100 n
M
) for the times indicated. (C) [
3
H]PtdBut
accumulation after TNFa (200 UÆmL
)1
)orNaCl/P
i
for 30 min at
37 °C and then fMLP (100 n
M
) for 10 min. The data represent
mean ± SEM from at least three experiments performed in triplicate.
***P < 0.001, significant increase in [
3
H]PtdBut accumulation over
fMLP-stimulated levels.
2758 K. A. Cadwallader et al.(Eur. J. Biochem. 271) Ó FEBS 2004
PtdOH, preventing its conversion into DAG by PAP
activity and thus leads to PtdOH accumulation [5,22].
Figure 4 illustrates that, whereas preincubation with prop-
ranolol (200 l
M
) suppressed fMLP-stimulated O
À
2
gen-
eration in TNFa-primed cells by 30%, it markedly

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.

Identification of PLD in human neutrophils at a
protein level was notably more difficult. Hence PLD
could only be detected in immunoprecipitates prepared
from whole cell lysates in the presence of 1% Nonidet
Fig. 3. Effect of PI3-kinase and ARF inhibitors on fMLP-stimulated respiratory burst and PLD activity in TNFa-primed neutrophils. O
À
2
generation
and [
3
H]PtdBut accumulation were assessed after fMLP (100 n
M
, 10 min) stimulation of TNFa-primed (200 UÆmL
)1
, 30 min) cells. The inhibitors
wortmannin (100 n
M
) (A, B) and brefeldin A (100 lgÆmL
)1
) (C, D) or vehicle were added 10 min before stimulation. Data represent mean ± SEM
from three experiments each performed in triplicate. **P < 0.01, ***P < 0.001, significant inhibition of O
À
2
generation and [
3
H]PtdBut
accumulation over untreated controls. Inserts show the concentration effects of brefeldin A or wortmannin on O
À
2
generation and PLD activity.

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

M
)and
butan-1-ol (0.3%, v/v) or vehicle were added 10 min before stimula-
tion. O
À
2
generation (A) and MPO release (B) were then assessed
after fMLP (100 n
M
, 10 min) stimulation of TNFa-primed
(200 UÆmL
)1
, 30 min) cells. Data represent mean ± SEM from three
experiments performed in triplicate. ***P < 0.001, significant inhibi-
tion of MPO release; P<0.001, significant enhancement of MPO
release over untreated controls.
Fig. 5. PLD isoform expression and localization in human neutrophils.
(A) Total mRNA was extracted from freshly isolated human neu-
trophils using RNeasy spin columns (Qiagen), and RT-PCR was
carried out as described in Materials and methods. PLD1 was identi-
fied as the predominant isoform with only marginal expression of
PLD2 in two independent experiments. Equal amplification of b-actin
transcripts confirmed identical total RNA content of the samples.
(B) Human neutrophils (10 · 10
6
) were lysed in whole cell lysis buffer
and immunoprecipitated using either the pan-PLD1/2 antibody or a
PLD1-specific antibody as described. Samples were separated by SDS/
PAGE and analyzed by Western Blotting using the pan-PLD1/2
antibody. Control lysates from CHO cells transfected with human

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-

nisms and the relationship between this event and the
functional consequences of priming have not been explored.
Our data confirm that the extent of agonist-stimulated PLD
activity, O
À
2
generation, and MPO release in neutrophils is
exquisitely sensitive to whether these cells have been
previously primed. Moreover, we show that the onset and
maximum rate of PLD activity occurs concurrently with
O
À
2
generation and MPO release.
PI3-kinase has been shown to have an essential role in a
number of neutrophil functions including adhesion [24,25],
phagocytosis [26], chemotaxis [27], degranulation and
respiratory burst activity [11,12]. Here we show that
wortmannin, a PI3-kinase inhibitor, abolished both
fMLP-stimulated O
À
2
generation and [
3
H]PtdBut genera-
tion, providing further evidence that PI3-kinase is upstream
of both these responses. Yasui & Komiyama [28] reported
that NADPH oxidase is activated by PI3-kinase in the early
phase and by PLD in a later phase. Brefeldin A, an inhibitor
of ARF GTPases and therefore ARF activation, likewise

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]

and hence might be influencing processes other than
through a direct effect on ARF itself.
Having established a link between PLD activation and
the secretory and oxidative responses, we examined the
differential effects of the immediate PLD products, PtdOH
and DAG, on these processes. Butan-1-ol leads to the
preferential formation of PtdBut over PtdOH and
thereby reduces agonist-stimulated PtdOH accumulation.
Propranolol sequesters PtdOH away from the enzyme
phosphatidate phosphohydrolase and thereby augments the
accumulation of PtdOH. Hence the ability of propranolol to
enhance fMLP-stimulated MPO release in both primed and
unprimed cells suggests that PtdOH has a pivotal role in
supporting degranulation responses [4,38]. It is of particular
interest that propranolol increased the degranulation
response observed with fMLP alone to levels normally only
reached in primed/stimulated cells.
In contrast, our data would suggest that PLD-generated
DAG is more essential for supporting O
À
2
generation [39].
Fig. 6. PLD1 localizes to the phagosomal membrane on exposure to
particulate stimuli. (A) Neutrophils were treated with TNFa
(200 UÆmL
)1
, 30 min) or fMLP (100 n
M
, 1 min), fixed and permea-
bilized before incubation with the PLD1-specific antibody. Immuno-

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

neutrophils and indeed most other primary cells has been
a major challenge. PLD1 protein expression has previ-
ously been described in neutrophils, but the Western blots
were not shown [43], and PLD2 message has been shown
in peripheral blood leucocytes [44]. Previous investigators
have also described two additional human neutrophil
PLDs with varying molecular mass (92 kDa and 350–
400 kDa) [9,10]. Using RT-PCR, we have been able to
show the presence of both PLD1 and PLD2 isoforms in
human peripheral blood neutrophils, with PLD1 being the
more abundant isoform. At a protein level, the major
isoform was shown to be PLD1 as immunoprecipitation
with a PLD1-specific antibody and blotting with the pan-
PLD1/2 antibody gave a band of the same density as
immunoprecipitation and blotting with the pan-PLD1/2
antibody. Moreover, no staining was identified using
currently available commercial PLD2-specific antibodies
despite their ability to detect PLD2 in Rat1 cells (data not
shown). Using the pan-PLD1/2 antibody, we show that
PLD is associated with the membrane fraction under
control conditions and that this localization pattern did
not alter on priming or stimulation.
Unstimulated neutrophils exhibited a punctate pattern of
PLD immunostaining which we predict from our cell
fractionation data to represent localization to intracellular
granules. Soluble stimuli produced no change in PLD
localization. With particulate stimuli, however, PLD was
shown to translocate to the phagosomal membrane after
engulfment of nonopsonized zymosan particles. Others
have shown that an epitope-tagged form of PLD1 expressed

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

membrane fractions and that priming/stimulation increases
the amount of membrane-associated ARF1/6. Using im-
munogold electron microscopy, we again found no differ-
ence in PLD distribution between primed, stimulated or
primed/stimulated cells. Gold label was found over both
intact and degranulated vesicles and diffusely over the
cytoplasm of neutrophils in all treatment groups.
In summary, our data show that priming causes a major
up-regulation of agonist-stimulated PLD activity which
parallels the increase in O
À
2
generation and MPO release.
The products of PLD activation appear to have a differ-
ential effect on these responses, with PtdOH able to support
degranulation and DAG being more important for respir-
atory burst activity. PLD activation and O
À
2
generation
are both dependent on PI3-kinase and ARF1/3. We show
for the first time that the major PLD isoform present in
neutrophils is PLD1 and that only particulate stimuli cause
a major relocation of PLD protein within the cell. These
studies provide important information on the mechanisms
underlying granulocyte activation.
Acknowledgements
This work was funded by The Wellcome Trust, BBSRC, The Medical
Research Council and the British Lung Foundation. M.U. was
supported by an MRC studentship. A.M.C holds a Wellome Trust

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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