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-1in human neutrophils Karen A.. However, the repertoire of PLD isoforms present in these primary cells, the precise me

Effect of priming on activation and localization of phospholipase D-1

in human neutrophils

Karen A Cadwallader1, Mohib Uddin1, Alison M Condliffe1, Andrew S Cowburn1, Jessica F White1, Jeremy

N Skepper2, Nicholas T Ktistakis3and Edwin R Chilvers1

1

Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, UK;2Department of Anatomy, University of Cambridge, UK;3Department 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, andthe impact of cell priming on

PLD activity and localization remain poorly defined

RT-PCR analysis showedthat both PLD1 andPLD2

iso-forms are expressedin human neutrophils, with PLD1

expressedat a higher level Endogenous PLD1 was detected

by immunoprecipitation andWestern blotting, andwas

predominantly membrane-associated under control and

primed/stimulated conditions Immunofluorescence showed

that PLD hada punctate distribution throughout the cell,

which was not alteredafter stimulation by soluble agonists

In contrast, PLD localizedto the phagolysosome membrane

after ingestion of nonopsonizedzymosan particles We

also demonstrate that tumour necrosis factor a greatly

potentiates agonist-stimulatedPLD activation,

myelo-peroxidase release, and superoxide anion generation, and that PLD activation occurs via a phosphatidylinositol 3-kinase-sensitive andbrefeldin-sensitive ADP-ribosylation factor GTPase-regulatedmechanism Moreover, propran-olol, which causes an increase in PLD-derived phosphatidic acidaccumulation, causeda selective increase in agonist-stimulated myeloperoxidase release Our results indicate that priming is a critical regulator of PLD activation, that the PLD-generatedlipidproducts exert divergent effects on neutrophil functional responses, that PLD1 is the major PLD isoform present in human neutrophils, andthat PLD1 actively translocates to the phagosomal wall after particle ingestion

Keywords: inflammation; lipidmediators; neutrophils; secondmessengers; 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 influencedby 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

priming agents promote a dramatic increase in the

functional responses evokedby 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-coupledreceptors leads to an increase in phospholipase D (PLD) activity andthe hydrolysis of Ptd Cho to Ptd OH and choline [2]; Ptd OH is then metabolizedto diacylglycerol (DAG) by the enzyme phosphatidate phosphohydrolase (PAP) Both PtdOH andDAG have been proposedto act as important second messengers linking cell stimulation to various effector functions including phagocytosis [3], degranulation [4], andrespiratory burst activity [5,6] It is now known that mammalian cells contain two major PLD isoforms, PLD1 andPLD2, 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 transformedcells 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: kc220@cam.ac.uk

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
 , superoxide anion; PtdCho,

phosphat-idylcholine; [ 3 H]PtdBut, [ 3 H]phosphatidylbutanol; CHAPS,

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GEF,

guanine nucleotide exchange factor.

(Received15 October 2003, revised5 May 2004,

accepted6 May 2004)

protein level in any primary cell Uncertainty also exists

over the nature of the PLD isoforms expressedin

neutrophils [9,10] andthe cosignals requiredfor PLD

activity, in particular the role of phosphatidylinositol

3-kinase (PI3-kinase), which we andothers have shown to

play a crucial role in the activation of NADPH oxidase

[11,12] Hence we have recently reportedthat the

metabolic product of PI3-kinase, phosphatidylinositol

3-phosphate, can activate the NADPH oxidase complex

by binding to the PX domain of the p40phox component

[13] Of note, the PX domain of p47phox has also been

shown to possess a binding site for PtdOH, although the

relevance of this to membrane localization andactivation

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-latedPLD enzymatic activity in neutrophils which parallels

the enhanced functional responses observed We identify

PLD1 as the major PLD isoform present in human

neutrophils andreveal 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 andbrefeldin-sensitive ARF

GTPase-regulated mechanism and provide evidence that

the lipidproducts formedafter PLD activation have an

unexpectedanddifferential effect in supporting

degranula-tion andO2
responses

Materials and methods

Materials

Cytochrome c, superoxide dismutase, fMLP,

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

(CHAPS) andphosphate-bufferedsaline (NaCl/Piwith or

without CaCl2 andMgCl2) were purchasedfrom Sigma

Chemical Company (Poole, Dorset, UK) Percoll, dextran

andthe ECL detection kit were obtainedfrom Amersham

Biosciences (Amersham, Bucks., UK) Human recombinant

TNFa was suppliedby R & D Systems (Abingdon, Oxon,

UK) Wortmannin, brefeldin A and propranolol were

obtainedfrom Calbiochem (Nottingham, UK) Fetal

bovine serum,L-glutamine andRPMI 1640 medium were

from Gibco (Flow Laboratories, Rickmansworth, Herts.,

UK) The pan-PLD1/2 antibody was generated in rabbits

using the C-terminal thirdof hPLD1 (amino acids 770–

1075) as antigen The PLD1-specific antibody was generated

as described previously [15] Secondary antibodies were

obtainedfrom Dako (Ely, Cambs., UK) Nonopsonized

zymosan was suppliedby Molecular Probes (Eugene, OR,

USA)

Isolation of human neutrophils

Human neutrophils were isolatedfrom venous bloodof

normal healthy volunteers using dextran sedimentation

followedby centrifugation on plasma-Percoll gradients as

previously detailed [16] The viability of cells, as assessed

by trypan blue exclusion, was >97% andthe purity of

neutrophil preparations was routinely >96% with <0.1%

mononuclear cell contamination

Measurement of degranulation Agonist-stimulatedmyeloperoxidase (MPO) release was determined by the 3,3-dimethoxybenzidine method as described previously [17] with the following minor modifi-cations Human neutrophils (106) were suspended in NaCl/

Piwith Ca2+andMg2+(80 lL) andincubatedwith TNFa (200 UÆmL)1) or NaCl/Piat 37C for 30 min followedby fMLP (100 nM) or NaCl/Pi for 10 min For inhibitor studies, cells were preincubatedwith compoundor vehicle for 10 min before agonist stimulation The reactions were terminatedby using ice-coldNaCl/Pi Supernatants were incubatedwith phosphate buffer (pH 6.2), containing 3,3-dimethoxybenzidine (0.125 mgÆmL)1) and H2O2 (0.001%, v/v), for 20 min, at 37C NaN3 (0.1%, w/v) was added, and the amount of MPO released was measured spectrophotometrically (460 nm) andexpressedas a per-centage of the total MPO activity present in 0.2% (v/v) Triton X-100-lysedcells

Measurement of O2
 generation Respiratory burst activity was assessedby measuring the generation of O2
using superoxide dismutase-inhibitable reduction of cytochrome c as described previously [18] Determination of PLD activity

PLD activity was assayedin [3H]lyso-PAF (1-O-[3 H]oct-adecyl-sn-glycero-3-phosphocholine)-labelledneutrophils

by measuring the formation of [3H]phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol as described previously [19] Human neutrophils (5· 106per 0.24 mL) prelabelledwith [3H]lyso-PAF were suspended in NaCl/Piwith Ca2+andMg2+in the presence of 0.3% (v/v) butan-1-ol Cells were incubatedfor 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 [3H]PtdBut formation was measured by liquid-scintillation counting after TLC separation as detailed previously [20]

RT-PCR procedures Total RNA was isolatedusing RNeasy mini spin columns (Qiagen, Crawley, West Sussex, UK) RNA (2 lg) was transcribedinto cDNA using oligo(dT) primers (Invitrogen Life Technologies, Paisley, UK) and50 U reverse transcrip-tase (Promega, Southampton, UK) PCR amplification was performedusing primer sets specific for PLD1 (sense: 5¢-ATGAGACACCCGGATCATGT; antisense: 5¢-ACT

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

2 U ampliTaq DNA polymerase (Bioline, London, UK) PCR products were analyzed by 1% agarose gel electro-phoresis and imaged with ethidium bromide under UV light

Western blot analysis

After the above treatments, human neutrophils (10· 106

cellsÆmL)1) were washedtwice in NaCl/Piwithout Ca2+and

Mg2+, lysedin 1 mL detergent lysis buffer [50 mM Tris/

HCl, pH 7.5, 150 mM NaCl, 5 mM 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 AppliedScience, Lewes, East Sussex, UK

(Complete tablets)] andleft on ice for 30 min Cells were

homogenizedor briefly sonicatedandthen spun (5 min,

15 000 g) to remove insoluble material The supernatant

was collectedandimmunoprecipitatedwith protein A–

Sepharose andthe pan-PLD1/2 antibody for 2 h After

being washedandboiledin sample buffer, samples were

analyzedby SDS/PAGE (10% gel)

For fractionation experiments, cells were lysedin 1 mL

hypotonic lysis buffer [10 mM Tris/HCl, pH 7.5, 5 mM

MgCl2, 1 mMdithiothreitol, 1 mMphenylmethanesulfonyl

fluoride plus a broad-spectrum proteinase inhibitor tablet

(see above)] andleft on ice for 30 min Cells were

homogenizedor briefly sonicatedandthen spun to remove

insoluble material (5 min, 1500 g) The supernatants were

collectedandrespun (100 000 g, 30 min), andthe pellet

(membrane fraction) was resuspended in membrane lysis

buffer (50 mM Tris/HCl, pH 7.5, 50 mM NaCl, 5 mM

MgCl2, 1 mMdithiothreitol, 1 mMphenylmethanesulfonyl

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 andmembrane fracconcentra-tions were

immunoprecipitatedandWestern blottedas 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 andCCL39) or PLD2 (Rat1) only Differences in the

molecular mass of the bandcorresponding to PLD were

observedaccording to the presence of PLD1 or PLD2 in

these cell lines (data not shown) CHO cells transfected with

human PLD1 were also usedas a positive control [15]

Immunofluorescent staining for PLD

Nonopsonizedzymosan was sonicatedandaddedto

neutrophils (25· 106 cellsÆmL)1 in NaCl/Pi containing

CaCl2andMgCl2) in a 5 : 1 particle to cell ratio at 37C

After 10 min, cells were diluted 10-fold in autologous serum

andimmediately cytospun (28 g, 5 min) The cytospins were

fixed [4% (v/v) paraformaldehyde, 10 min] and

permeabi-lized[0.1% (v/v) Triton X-100, 10 min] before blocking with

NaCl/Pi/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 usedat a

dilution of 1 : 300 The same methodwas usedto examine

PLD distribution under primed/stimulated conditions (see

above)

Electron microscopy

Neutrophils were fixed in 4% (v/v) formaldehyde in 0.1M

Pipes buffer for 1 h, cryoprotectedin 25% (v/v)

polypro-pylene glycol, frozen in propane, andfreeze-substitutedin

methanol containing 0.01% (w/v) uranyl acetate at)90 C

They were subsequently embedded in Lowicryl HM20 at )50 C [21] Thin sections mountedon nickel film were incubated in primary antibodies to PLD diluted 1 : 100 in NaCl/Picontaining 2% BSA, 0.1% Triton X-100 and0.1% Tween 20 (w/v/v/v) for 16 h Primary antibody-binding sites were visualizedby incubation with colloidal goldparticles (10 nm) conjugated to species-specific secondary antibodies The sections were stainedwith uranyl acetate andlead citrate andviewedin a Philips CM 100

Statistical analysis All values are expressedas mean ± SEM from n separate experiments Where appropriate, results were analyzedby analysis of variance followedby Student–Newman–Keuls post-test with the statistical program PRISM (GraphPad Software, San Diego, CA, USA) Differences were consid-eredsignificant when P < 0.05

Results

Effect of TNFa priming on O


2 generation, MPO release, and PLD activation

Before investigating the effect of TNFa priming on PLD activity, we wishedto confirm that our experimental system was optimal for demonstrating priming-mediated up-regu-lation of neutrophil effector functions Figure 1 shows that both O2
generation andmyeloperoxidase (MPO) release were minimal when either priming agent (TNFa) or activating agent (fMLP) were added alone However, if cells were incubatedwith TNFa (200 UÆmL)1, 30 min) before fMLP stimulation (100 nM, 10 min), O2
andMPO responses are significantly increased Detailed time-course analysis of fMLP-stimulatedO2
 release showedthat maximal O2
generation occurredby 1 min andthat 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 formedcan be usedas a measure of PLD activity Figure 2A shows that fMLP stimulateda concentration-dependent accumulation of [3H]PtdBut in TNFa-primed cells (EC50¼ 2.8 ± 0.08 nM) with a maximal response achievedwith 100 nMfMLP TNFa priming significantly enhancedfMLP-stimulatedPLD activity comparedwith the levels observedwith TNFa or fMLP alone (Fig 2B,C) with > 90% of the [3H]PtdBut accumulating within the first

5 min of stimulation

Effect of PI3-kinase and ARF inhibitors on O


2 and PLD activation

Selective pharmacological inhibitors were usedto assess the role of PI3-kinase andARF proteins on the activation

of PLD As illustratedin Fig 3A,B, the PI3-kinase inhibitor wortmannin (100 nM) [11] markedly attenuated both fMLP-stimulatedO2
 generation and[3H]PtdBut accumulation Brefeldin A (100 lgÆmL)1), an inhibitor of

ARF localization andthe guanine nucleotide exchange

factors (GEFs) for ARF (BIG-1 and2), was also foundto

inhibit O


2 generation andPLD activity (Fig 3C,D)

O2
 generation andPLD activity were also measured

across a range of brefeldin A (0–300 lgÆmL)1) and

wortmannin (0–100 nM) concentrations (inserts in

Fig 3) These results indicate that PI3-kinase-sensitive

andbrefeldin 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

performedusing butan-1-ol, which diverts a proportion of

PtdCho hydrolysis into the formation of PtdBut rather than

PtdOH, and propranolol, which at 200 l , sequesters

Fig 1 Effect of TNFa priming on fMLP-stimulated respiratory burst

activity and degranulation Neutrophils were incubatedwith TNFa

(200 UÆmL)1) or NaCl/P i for 30 min at 37 C andthen stimulatedwith

fMLP (100 n M ) for 10 min or the times indicated O2
generation (A)

andMPO release (B) were determinedas describedin Materials and

methods The data in the inserts represent mean ± SEM from at least

three experiments performedin triplicate Data points for the time

courses in (A) and(B) show a representative experiment performedin

triplicate ***P < 0.001, significant increase in O2
generation and

MPO release over fMLP-stimulatedlevels.

Fig 2 Effect of TNFa priming on fMLP-stimulated PLD activity Cells were incubatedwith TNFa (200 UÆmL)1) or NaCl/P i for 30 min

at 37 C andthen stimulatedwith various concentrations of fMLP for

10 min (A) [3H]PtdBut accumulation was determined as described in Materials andMethods (B) Time course of [3H]PtdBut accumulation after treatment with TNFa (200 UÆmL)1) or NaCl/P i for 30 min at

37 C andthen fMLP (100 n M ) for the times indicated (C) [3H]PtdBut accumulation after TNFa (200 UÆmL)1) or NaCl/P i for 30 min at

37 C andthen fMLP (100 n M ) for 10 min The data represent mean ± SEM from at least three experiments performedin triplicate.

***P < 0.001, significant increase in [3H]PtdBut accumulation over fMLP-stimulatedlevels.

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 lM) suppressedfMLP-stimulatedO2


gen-eration in TNFa-primedcells by 30%, it markedly

potentiatedMPO release under both fMLP only and

TNFa-primed/fMLP-stimulated conditions (by 169 ±

37% and71 ± 9.2%, respectively) In contrast,

butan-1-ol (0.3%, v/v) causeda near complete inhibition of

TNFa-primed/fMLP-stimulated MPO release under conditions in

which O2
 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 regardto the lipidrepertoire requiredto support MPO

release andO2
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 isolatedneutrophils 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 couldonly be detectedin 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 O2
generation and[3H]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) andbrefeldin A (100 lgÆmL)1) (C, D) or vehicle were added 10 min before stimulation Data represent mean ± SEM from three experiments each performedin triplicate **P < 0.01, ***P < 0.001, significant inhibition of O2
generation and[ 3 H]PtdBut accumulation over untreatedcontrols Inserts show the concentration effects of brefeldin A or wortmannin on O2
generation andPLD activity Data represent a single experiment representative of three independent experiments performed in triplicate.

P40 and0.5% CHAPS (Fig 5B) Initial PLD

immuno-precipitates were generatedwith an antibody specific for

either pan-PLD1/2 or PLD1 Western blotting of these

immunoprecipitates with the pan-PLD1/2 antibody

con-firmedthe presence of PLD1 in human neutrophils

(Fig 5B) with an approximate size of 120 kDa Lysates

of CHO cells transfectedwith human PLD1 were usedas

positive controls

The subcellular distribution of PLD and the

conse-quences of priming andactivation were determinedusing

immunoprecipitation andWestern blot analysis of

neutro-phil cytosol andmembrane 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 andthe 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 formedafter ingestion of nonopsonizedzymosan 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 O2
generation (A) andMPO 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 performedin triplicate ***P < 0.001, significant

inhibi-tion of MPO release; P < 0.001, significant enhancement of MPO

release over untreatedcontrols.

Fig 5 PLD isoform expression and localization in human neutrophils (A) Total mRNA was extractedfrom freshly isolatedhuman neu-trophils using RNeasy spin columns (Qiagen), andRT-PCR was carriedout as describedin Materials andmethods PLD1 was identi-fiedas the pred ominant isoform with only marginal expression of PLD2 in two independent experiments Equal amplification of b-actin transcripts confirmedid entical total RNA content of the samples (B) Human neutrophils (10 · 10 6 ) were lysedin whole cell lysis buffer andimmunoprecipitatedusing either the pan-PLD1/2 antibody or a PLD1-specific antibody as described Samples were separated by SDS/ PAGE andanalyzedby 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 incubatedfor

30 min in the presence or absence of TNFa (200 UÆmL)1) before sti-mulation with fMLP (100 n M , 1 min) Cells were lysedandfraction-atedinto cytosolic andmembrane fractions as describedin Materials andmethods Immunoprecipitations were performedwith the pan-PLD1/2 antibody SDS/polyacrylamide gels were then blotted with the pan-PLD1/2 antibody s, Cytosolic fraction; p, membrane fraction.

antibodies (Cell Signaling, Beverly, MA, USA; data not

shown)

The distribution of immunogold label for PLD was

similar in all four treatments (Fig 7) Goldlabel was seen

over both intact anddegranulatedvesicles anddiffusely over

the cytoplasm of neutrophils Label was absent over the

nuclei andplasma membrane No difference in the

distri-bution was observedin 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 renewedinterest in the regulation anddownstream effects

of PLD Although neutrophil priming has been previously shown to result in a small up-regulation of agonist-stimulatedPLD activation [6,8,23], the underlying mecha-nisms andthe relationship between this event andthe functional consequences of priming have not been explored Our data confirm that the extent of agonist-stimulated PLD activity, O2
generation, andMPO 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

O2
generation andMPO 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, abolishedboth fMLP-stimulatedO2
generation and[3H]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 andby PLD in a later phase Brefeldin A, an inhibitor

of ARF GTPases andtherefore ARF activation, likewise causedsignificant inhibition of both fMLP-stimulatedPLD activity andO2
generation Moreover, this inhibitor also abolishedthe oxidative burst in response to nonopsonized zymosan, whereas there was no effect on O2
generation with opsonizedzymosan (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 O2
generation have previously been described[31,32] andare thought to be mediated by Class 1 ARFs such as ARF1 and ARF3 More recently, ARF6 has been implicatedin activating PLD and functional responses in chromaffin cells [33], macrophages [34] andepithelial cell lines [35], andin neutrophil-like

PLB-985 cells a specific role for ARF6 (controlledby brefeldin-insensitive GEFs) has been implicatedin 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 andto cause disassembly of the Golgi complex [37] andhence might be influencing processes other than through a direct effect on ARF itself

Having establisheda link between PLD activation and the secretory andoxidative responses, we examinedthe differential effects of the immediate PLD products, PtdOH andDAG, 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-stimulatedMPO release in both primedand unprimedcells suggests that PtdOH has a pivotal role in supporting degranulation responses [4,38] It is of particular interest that propranolol increasedthe degranulation response observedwith fMLP alone to levels normally only reachedin primed/stimulatedcells

In contrast, our data would suggest that PLD-generated DAG is more essential for supporting O
generation [39]

Fig 6 PLD1 localizes to the phagosomal membrane on exposure to

particulate stimuli (A) Neutrophils were treatedwith TNFa

(200 UÆmL)1, 30 min) or fMLP (100 n M , 1 min),

fixedandpermea-bilizedbefore incubation with the PLD1-specific antibody

Immuno-staining was imagedusing confocal microscopy (B) For

immunofluorescence studies, neutrophils were incubated with

non-opsonizedzymosan in a 5 : 1 particle to cell ratio, fixed,

andperme-abilizedbefore incubation with the pan-PLD1/2 antibody.

It should be noted, however, that the modest suppression of

fMLP-stimulatedO2
generation in TNFa-primedcells 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 andsecretory responses in neutrophils It is probable,

however, that DAG andPtdOH act together to activate

components of the NADPH oxidase This theory is

supportedby earlier work in intact neutrophils showing

that activation of O2
generation occurs in parallel with

the elevation of both PtdOH andDAG levels [40] andin

experiments using cell-free systems where PtdOH and DAG

work collectively to activate NADPH oxidase components

[41] It shouldalso be notedthat 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 observedwith propranolol reflected

protein kinase C inhibition

Characterization of the PLD isoforms present in human

neutrophils andindeedmost 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], andPLD2 message has been shown

in peripheral bloodleucocytes [44] Previous investigators

have also described two additional human neutrophil

PLDs with varying molecular mass (92 kDa and350–

400 kDa) [9,10] Using RT-PCR, we have been able to

show the presence of both PLD1 andPLD2 isoforms in

human peripheral bloodneutrophils, 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 andblotting 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 associatedwith the membrane fraction under control conditions and that this localization pattern did not alter on priming or stimulation

Unstimulatedneutrophils exhibiteda 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 nonopsonizedzymosan particles Others have shown that an epitope-taggedform of PLD1 expressed

in RBL-2H3 cells localizes to secretory granules and lysosomes of unstimulatedcells and, on stimulation, translocates to the plasma membrane [45] Other investiga-tors have reportedthat PLD1 is localizedon the Golgi [46], nucleus [47] andlysosomal/endosomal compartments [48]

It is possible therefore that the subcellular distribution of PLD is cell specific andmay dependon the nature of the stimulus Although previous studies in human neutrophils have demonstratedtranslocation of ARF1-regulatedPLD 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 Goldlabel is present over intact vesicles (single arrowheads) anddegranulatedvesicles (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 andfMLP.

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 investigatedthe possibility that the

uplift in PLD activity reflectedtranslocation of ARF rather

than PLD Preliminary data (not shown) have indicated

that under basal and TNFa-primedconditions, ARF1 and

ARF6 are distributed equally between the cytosol and

membrane fractions andthat priming/stimulation increases

the amount of membrane-associatedARF1/6 Using

im-munogoldelectron microscopy, we again foundno

differ-ence in PLD distribution between primed, stimulated or

primed/stimulatedcells Goldlabel was foundover both

intact anddegranulatedvesicles anddiffusely over the

cytoplasm of neutrophils in all treatment groups

In summary, our data show that priming causes a major

up-regulation of agonist-stimulatedPLD activity which

parallels the increase in O2
generation andMPO 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 andO2
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 andthat 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 andthe British Lung Found ation 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 carriedout in the MIC which was establishedwith

funding from the Wellcome Trust We thank Dr Nancy Pryer, Onyx

Pharmaceuticals, USA for kindly donating the pan-PLD1/2 antibody

andProfessor Michael Wakelam for helpful discussions.

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