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NeutrophilsArachidonic Acid Release from Humanin2and Secretory Phospholipase A

2Involvement of Cytosolic Phospholipase A

Barry RubinDowney David A Ford Peihong Zhu Paul Walker andJohn Marshall Eric Krump Thomas Lindsay Gregory

httpwwwjimmunolorgcontent16442084doi 104049jimmunol16442084

2000 1642084-2091 J Immunol

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Involvement of Cytosolic Phospholipase A2 and Secretory

Phospholipase A2 in Arachidonic Acid Release from Human

Neutrophils1

John Marshall Eric Krump2Dagger Thomas Lindsay Gregory Downeydagger David A Fordsect

Peihong Zhu Paul Walker and Barry Rubin3

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2) calcium-independent PLA2 and cytosolic

PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils While fMLP induced the release of

extracellular sPLA2 activity and AA 70 of sPLA2 activity remained associated with the cell Treatment with the cell-imper-

meable sPLA2 inhibitors DTT or LY311-727 or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity but had

minimal effect on neutrophil AA mass release In contrast coincubation of streptolysin-O toxin-permeabilized neutrophils with

DTT LY311-727 or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled fMLP-stimulated cells Exposure to fMLP resulted

in a decrease in the electrophoretic mobility of cPLA2 a finding consistent with cPLA2 phosphorylation and stimulated the

translocation of cPLA2 from cytosolic to microsomal and nuclear compartments The role of cPLA2 was further evaluated with

the cPLA2 inhibitor methyl arachidonyl fluorophosphonate which attenuated cPLA2 activity in vitro and decreased fMLP-

stimulated AA mass release by intact neutrophils but had no effect on neutrophil sPLA2 activity Inhibition of calcium-indepen-

dent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release These results

indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human

neutrophils The Journal of Immunology 2000 164 2084ndash2091

Phospholipase A2 (PLA2)4 hydrolyzes arachidonic acid

(AA) from the sn-2 position of glycerophospholipids (1)

The hydrolysis of AA by PLA2 is a critical regulatory step

in the development of an inflammatory response Neutrophils

(polymorphonuclear leukocytes (PMN)) release the proinflamma-

tory mediator AA via the function of PLA2 when activated by

bacterial peptides phorbol esters calcium ionophores and other

agonists Released AA may then be converted to biologically ac-tive metabolites such as prostaglandins leukotrienes lipoxins and

thromboxanes which are thought to directly modulate the inflam-

matory response (2)

Multiple PLA2 isoforms have been described in human neutro-

phils An 85-kDa cytosolic PLA2 (cPLA2) that selectively hydro-

lyzes glycerophospholipids with AA in the sn-2 position and trans-

locates to the nucleus in a Ca2-dependent manner in response to

agonists (3 4) has been identified in PMN (5) Neutrophils also

have Ca2-independent PLA2 (iPLA2) activity (6) iPLA2 hasbeen identified in murine macrophages and is thought to function

in membrane remodeling in these cells (7 8) In addition neutro-

phils contain low mw secretory PLA2 (sPLA2) that are stored in

granules (9ndash11) sPLA2 isoforms have six to eight disulfide

bridges require micromolar to millimolar levels of Ca2 for cat-

alytic activity (12) and do not exhibit specificity for phospholipids

with AA in the sn-2 position (13ndash15) The catalytic activity of the

low mw sPLA2 isoforms in contrast to cPLA2 and iPLA2 is

inhibited by the reducing agent DTT (15) and by the substituted

indole LY311-727 (16 17)

The enzyme(s) that mediates AA release from activated PMN

has not been completely characterized Evidence that cPLA2 di-

rectly mediates AA release includes the demonstration that cPLA2

is activated in response to bacterial agonists (18) that pharmaco-

logical inhibitors of cPLA2 attenuate PMN AA release (19) and

that macrophages from mice in which the gene for cPLA2 was

disrupted exhibited decreased AA release in response to PMA and

calcium ionophores (20) Evidence that sPLA2 directly mediates

AA release includes the observation that exogenous addition of

group V sPLA2 to PMN (21) or recombinant synovial PLA2 to

murine macrophages (21 22) directly results in AA release In

addition treatment with the cell-permeable sPLA2 inhibitors

SB203347 and Scalaradial prevented Ca2 ionophore-stimulated

AA mass release (23 24) While these studies support a role for

sPLA2 in PMN AA release (23 24) they are complicated by the

Divisions of Vascular Surgery and daggerRespiratory Diseases Max Bell Research Cen-ter Toronto General Hospital University Health Network Toronto Ontario CanadaDaggerDivision of Cell Biology Sick Childrenrsquos Hospital Toronto Ontario Canada andsectDepartment of Biochemistry and Molecular Biology St Louis University HealthSciences Center St Louis MO 63104

Received for publication August 2 1999 Accepted for publication December 6 1999

The costs of publication of this article were defrayed in part by the payment of pagecharges This article must therefore be hereby marked advertisement in accordancewith 18 USC Section 1734 solely to indicate this fact

1 This work was supported by The Heart and Stroke Foundation of Canada (Grant

NA-3497 BR) The Medical Research Council of Canada (GD) and by the Amer-ican Heart Association Grant-in-Aid 9750096N and National Heart Lung and BloodInstitute Grants R01-HL-42665-11 and K04-HL-03316-05 (to DAF) BR is therecipient of the Wylie Scholar Award in Academic Vascular Surgery from The PacificVascular Research Foundation San Francisco and a Bickel Foundation Award AMedical Research Council of Canada Fellowship supported EK

2 Deceased

3 Address correspondence and reprint requests to Dr Barry Rubin EC5-302a To-ronto General Hospital Toronto Ontario Canada M5G-2C4 E-mail addressbarryrubinuhnonca

4 Abbreviations used in this paper PLA2 phospholipase A2 AA arachidonic acidcPLA2 Ca2-dependent cytosolic PLA2 DFP diisopropyl fluorophosphate HELSShaloenol lactone suicide substrate or ( E )-6-(bromomethylene)tetrahydro-3-(1-naph-thalenyl)-2 H -pyran-2-1 HSD highly significant difference iPLA2 Ca2-indepen-dent cytosolic PLA2 KRPD Krebs-Ringer-phosphate dextrose MAFP methylarachidonyl fluorophosphonate PMN neutrophil SLO streptolysin O sPLA2 se-cretory PLA2

Copyright copy 2000 by The American Association of Immunologists 0022-176700$0200

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potential inhibitory effects of SB203347 and Scalaradial on cPLA2

activity or Ca2 metabolism (25) sPLA2 and cPLA2 both appear

to function in AA release from PAF- and LPS-stimulated P388D1

macrophages and evidence has been presented in support of the

concept that activation of sPLA2 at the plasma membrane is de-

pendent on previous activation of cPLA2 (22 26) The role that

iPLA2 plays in AA release from neutrophils has not been com-

pletely defined

In this study we have systematically evaluated the roles of

cPLA2 sPLA2 and iPLA2 in fMLP-stimulated AA release from

human PMN We present some evidence that cPLA2 and an intra-

cellular or cell-associated sPLA2 are both involved in fMLP-stim-

ulated AA release by human PMN

Materials and Methods Materials

Butylated hydroxytoluene leupeptin pepstatin PMSF fMLP and[2D8]AA were from Sigma (St Louis MO) Reagents for Krebs-Ringer-phosphate dextrose buffer (KRPD) including NaCl KCl Na2HPO4K2HPO4 MgCl2 and CaCl2 were obtained from Mallinckrodt (Paris KY)[3H8]AA (sp act 180 Cimmol) was from New England Nuclear (Mis-sissauga ON Canada) Organic solvents were from Fisher (Toronto ONCanada) and were HPLC grade or better N -Methyl- N -(tert -butyldimeth-

ylsilyl) trifluoroacetamide was from Pierce (Rockford IL) Aminopropylcolumns were obtained from Burdick and Jackson (Muskegon MI) andSep-Pak silica columns were obtained from Waters (Toronto ON Cana-da) The DB-23 cyanopropyl gas chromatography column was purchasedfrom JampW Scientific (Folsom CA) The PLA2 inhibitors HELSS andMAFP were obtained from Calbiochem (San Diego CA) and the anti-cPLA2 Ab was from Santa Cruz Biotechnology (Santa Cruz CA) SLOtoxin was obtained from Scantibodies (South San Francisco CA) ThesPLA2 inhibitor 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane-phosphonic acid (LY311-727) was the kind gift of Dr E Mihelich (LillyResearch Laboratories Indianapolis IN) and the lysosomal-type Ca2-independent PLA2 inhibitor 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) (27) was kindly provided by Dr M K Jain(Department of Chemistry and Biochemistry University of DelawareNewark DE) The neutralizing anti-sPLA2 Ab 3F10 (28) and sPLA2 in-hibitor SB203347 (29) were kindly provided by Dr Lisa Marshall Smith

Kline Beecham Pharmaceuticals (King of Prussia PA) pldA

Escherichiacoli was the kind gift of Dr Peter Elsbach New York University (NewYork NY)

Isolation of neutrophils

Neutrophils from whole blood were isolated by dextran sedimentation anddiscontinuous plasma Percoll gradient centrifugation and resuspended inKRPD buffer without Ca2 at 2 107 cellsml (30 31) This procedureyielded 95 PMN with 98 viability as judged by trypan blue ex-clusion PMN were treated with 1 mM DFP for 10 min on ice beforeexperimental treatments radiolabeling cell lysis subcellular fractionationor other experimental procedures We found that DFP which preventsdegradation of PMN proteins (32 33) decreased nonspecific backgroundactivation and prevented cell clumping and markedly enhanced cell via-bility over extended treatment and experimental regimes

Experimental protocol

PMN were treated with 01 DMSO 10 M MAFP 16 M HELSS 10M SB203347 10 M Scalaradial 10 M LY311-727 or 1 mM DTT for05 h at 37degC as indicated in the figure legends PMN were then pelletedand resuspended at 2 107 cellsml in KRPD with 025 fatty acid-freehuman albumin and 1 mM CaCl2 Following readdition of 10 MSB203347 10 M Scalaradial 10 M LY311-727 or 1 mM DTT whichdo not bind covalently and could therefore be washed out when cells wereresuspended in KRPD with CaCl2 and albumin neutrophils were equili-brated for 5 min at 37degC Cells were subsequently treated with either 01DMSO or 5 M cytochalasin B for 2 min and 100 nM fMLP for 3 min at37degC Experiments were terminated by centrifugation for 10 s at 14000 g

Acid extraction of sPLA2

from neutrophils

PMN (2 107 ml in KRPD) were mixed with an equal volume of water

brought to pH 16 with concentrated H2SO4 and stirred overnight at 4degCFollowing centrifugation at 14000 g for 15 min the supernatant wasdialyzed against 500 vol of 10 mM sodium acetate buffer pH 45 for 24 h

The dialysate was collected and centrifuged at 14000 g for 15 min andthe supernatant was used as a source of sPLA2

Determination of extracellular and cell-associated sPLA2

activity collection of extracellular and cell-associated sPLA2

PMN (2 107) were stimulated with fMLP and the extracellular sPLA2

was collected in the supernatant by brief centrifugation at 14000 g Thesupernatant was concentrated over a Centricon 3000 NMWL ultrafilter anddiluted to a total of 270 l in assay buffer A (150 mM NaCl 10 mM CaCl225 mM HEPES pH 74 and 025 fatty acid-free human albumin) The

cell pellet was resuspended in 270 l sPLA2 assay buffer and disrupted onice water by three 15-s bursts of a 50-W probe sonicator set to 20 am-plitude (Sonics and Materials Danbury CT)

Radiolabeling of E coli membranes

The PLA2-deficient strain of E coli (pldA) was radiolabeled during thelog growth phase with [3H8]AA washed three times in KRPD with 025fatty acid-free BSA autoclaved and rewashed exactly as described (34)and then used as a substrate in the sPLA2 assay and cPLA2 assays

sPLA2

assay

A total of 60 l of assay buffer A was combined with 10 l of [3H8]AA-labeled E coli membranes (50000 dpmassay) and 5 l of DMSO orMAFP (10 M) HELSS (16 M) LY311-727 (10 M) or 3F10 (1 g ml) on ice sPLA2 reactions were initiated by the addition of 25 l of cell

lysates cell supernatants or sPLA2 from acid-extracted neutrophils After30 min at 37degC reactions were terminated by the addition of 900 l of tetrahydrofuran followed by centrifugation at 14000 g for 15 min at4degC The reaction was then applied to an aminopropyl column and thefatty acid fraction was selectively eluted with tetrahydrofuranacetic acid(491) followed by liquid scintillation counting (28) Results are expressedas the percentage of free fatty acid hydrolyzed (dpm generated nonspe-cific hydrolysis)total dpm added

[3 H 8

]AA release from [3 H 8

]AA-labeled SLO-permeabilized

neutrophils

Neutrophils were labeled with [3H8]AA for 2 h at 37degC washed three timesin KRPD with 025 BSA resuspended in KRPD with 1 mM CaCl 2 and025 BSA and incubated with 01 DMSO 10 M LY311-727 or 1 g3F10ml for 5 min at 37degC Cells were then treated with either SLO toxin(approximately 01 g per ml) or vehicle (KRPD) for 25 min in the pres-

ence of 5 M cytochalasin B before stimulation with fMLP The quantityof SLO added was optimized for each experiment Incubations werestopped after 10 min by centrifugation and the supernatant was collectedand counted for [3H8]AA release using a liquid scintillation counter (Beck-mann 6500) after the method of Mira et al (35)

AA mass release

Following cellular stimulation and centrifugation for 15 s at 14000 g15 ml of supernatant from 3 107 PMN was collected and immediatelytransferred to siliconized borosilicate test tubes with 6 ml of chloroform-methanol (21 vv) and 001 butylated hydroxytoluene A total of 8 pmolof the internal standard deuterated AA ([2D8]AA) was then added to eachsample After vortexing and addition of 15 ml NaCl samples were cen-trifuged at 1000 g for 5 min and the lower phase was collected Theupper phase was then reextracted twice with chloroform-methanol-058NaCl (86141) (36) Lipids from the pooled lower phase were then driedunder a stream of N2 reconstituted in hexane-methyl tert -butyl ether (2003) and applied to a silicic acid column Fatty acids were selectively elutedfrom the column with hexane-methyl tert -butyl ether-acetic acid (100202) (37) dried under N2 and derivatized with N -methyl- N -(tert -butyldi-methylsilyl)trifluoroacetamide (38 39) The tert -butyldimethylsilyl etherof AA was then separated from other fatty acids by gas chromatography(model 5890 Hewlett Packard Palo Alto CA) on a DB-23 02 mm 25 mcyanopropyl-methyl column Following electrospray ionization AA masswas quantified by selectively monitoring the intensities of AA (m z 361)and the internal standard [2D8]AA (mz 369) with a quadrupole mass spec-trometer (model 5971 Hewlett Packard)

Cell fractionation

A total of 6 107 cells were lysed in 600 l 20 mM HEPES pH 74 150mM KCl 5 glycerol 1 mM EDTA 1 mM EGTA 1 mM NaF 1 mM

sodium orthovanadate 2 mM DFP 1 mM PMSF 1 mM benzamidinehydrochloride 50 M leupeptin 50 M pepstatin and 50 M chymostatinby brief pulses of probe sonication on ice water The insoluble cellular

2085The Journal of Immunology

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components including nuclei were removed by centrifugation at 14000 g for 5 min at 4degC The supernatant was then separated into microsomaland cytosolic fractions by centrifugation at 150000 g for 20 min at 4degC

Determination of PLA2

activity in PMN cytosol

cPLA 2

assay PLA2 activity in PMN cytosol was measured by combin-ing 75 l of assay buffer B (150 mM NaCl 2 mM 2-ME 25 mM HEPESpH 74 5 mM EDTA and 025 fatty acid-free human albumin) and 10 lof [3H8]AA-labeled E coli membranes to a final concentration 5 nmol lipidphosphorus (or 50000 dpmassay) and was initiated by the addition of

20 l of cytosol A total of 10 M MAFP 16 M HELSS or 10 MLY311-727 or their diluents (DMSO or NaCl) was added to the cytosol 5min before the initiation of the assay After a 30-min incubation at 37degCthe reaction was terminated by addition of tetrahydrofuran and centrifuga-tion at 14000 g for 15 min at 4degC Fatty acids were eluted from anaminopropyl solid-phase silica column as described for the sPLA2 assay(28) Results are expressed as the percentage of free fatty acid hydrolyzed(dpm generated nonspecific hydrolysis)total dpm added

SDS-PAGE

Cytosolic microsomal or nuclear fractions (200 l) were combined with50 l of 5 sample buffer (0312 M Tris pH 68 50 sucrose (wv) 25mM DTT 10 SDS and 05 bromophenol blue) to a total volume of 250l The samples were boiled for 5 min and 25-l aliquots were resolvedon 12 075-mm gels in a Bio-Rad (Richmond CA) mini protean II ver-tical electrophoresis apparatus at 100 V

Western blots

Gels were placed against nitrocellulose membranes in 25 mM Tris and 192mM glycine with 20 (vv) methanol and transferred at 100 V for 1 h Theblots were stained with 01 Ponceau Red to ensure equal protein loadingbefore destaining in water Blots were blocked for 2 h in TBST (20 mMTris pH 73 024 M NaCl 26 mM KCl and 005 Tween 20) containing5 (wv) skim milk powder and 1 (vv) goat normal serum The blot wasthen washed 2 5 min in 10 ml TBST and incubated with 11000 anti-cPLA2 overnight at 4degC before washing 1 5 min in 10 ml TBST andincubation with a 120000 dilution of goat anti-mouse secondary Ab con-

jugated to HRP for 1 h Blots were then washed 3 5 min in TBST beforedetection with 124 M 5-amino-23-dihydro-14-phthalazinedione (lumi-nol) 065 M 4-hydroxycinnamic acid ( p-coumaric acid) and 00001hydrogen peroxide against KODAK ECL blue film

Statistical analysis

Data were analyzed by ANOVA followed by comparison of all meanswith the Tukey-Kramer honestly significant difference (HSD) test usingSAS software (SAS Institute Carey NC) Results were considered to besignificantly different when p 005 was observed

Results A minor role for extracellular sPLA

2 in PMN AA release

We examined the kinetics of sPLA2 release by PMN in response to

fMLP and found that approximately 30 of the apparent cell-

associated sPLA2 activity was lost within the first 30 s after stim-

ulation (Fig 1 A) Concurrently the sPLA2 activity observed in the

supernatant rapidly increased in the first 30 s after treatment with

fMLP and then remained constant (Fig 1 B) Under these assayconditions cell-associated and extracellular PLA2 activity were

each inhibited more than 98 by coincubation with 10 M

LY311-727 (Fig 2 A) which selectively inhibits group IIa (16) and

group V sPLA2 activity (17) but has no effect on cPLA2 or iPLA2

activity (17) In a similar pattern to sPLA2 activity release the

greatest change in [3H8]AA release from neutrophils occurred

within 30 s after fMLP stimulation with a decline in the rate of

release over the next several minutes (Fig 1C ) Hence there was

a strong temporal correlation between the secretion of sPLA2 and

the release of [3H8]AA from fMLP-stimulated PMN

As the release of sPLA2 activity correlated with extracellular

[3H8]AA release we sought to determine whether extracellular

sPLA2 mediated AA mass release Stimulating PMN with fMLP in

the presence of either DTT or the cell-impermeable sPLA2 inhib-

itor LY311-727 (16) significantly inhibited extracellular sPLA2

activity (Fig 2 A) In contrast incubation with either DTT or

LY311-727 only decreased fMLP-stimulated AA mass release by

approximately 15 a difference that failed to reach statistical sig-

nificance (Fig 2 B) Therefore sPLA2-mediated hydrolysis of AA

from glycerophospholipids on the extracellular face of the plasma

membrane of PMN does not appear to be a major source of re-

leased AA under these conditions

A central role for cPLA2

in PMN AA release

cPLA2 has been implicated in AA release from human neutrophils

(40) Using immunoblot analysis we found that most of the cPLA2

in unstimulated neutrophils is present in the cytosolic fraction (Fig

3) The cPLA2 in the cytosolic fraction was the more rapidly mi-

grating form indicating that most of the cPLA2 in this fraction was

not phosphorylated (41) Comparatively little cPLA2 was detected

in the nuclear or microsomal fraction of unstimulated PMN In

fMLP-stimulated cells cPLA2 was detected in both nuclear and

microsomal fractions and a concomitant decrease in the amount

of cPLA2 in the cytosolic fraction was observed In addition the

majority of cPLA2 detected in fMLP-stimulated cells migrated

at a relatively higher mw demonstrating that most of the cPLA2

FIGURE 1 Effect of fMLP on cellular sPLA2 activity release of sPLA2

into the extracellular fluid and [3H8]AA release Neutrophils were incu-

bated for 5 min at 37degC stimulated with fMLP (time 0 min) and cen-

trifuged The sPLA2 activity in cell lysates ( A) or extracellular fluid ( B) was

then measured as described in Materials and Methods C Cells were met-

abolically labeled with [3H8]AA and the time course of [3H8]AA releasefollowing stimulation with fMLP was assessed Incubation with 01

DMSO did not result in any changes in cell-associated sPLA2 activity

extracellular sPLA2 activity or [3H8]AA release Results shown are the

mean SE of three determinations Treatment differences by ANOVA

were significant ( p 001)

2086 INTRACELLULAR PLA2 AND AA RELEASE

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that translocated to nuclear and microsomal fractions was

phosphorylated (41)

We then examined the effect of the cPLA2 inhibitor MAFP on

AA mass release from fMLP-stimulated cells We controlled these

pharmacological experiments with the putative sPLA2 inhibitors

SB203347 and Scalaradial which have previously been shown to

inhibit AA release (23 24) and with the iPLA2 inhibitor HELSS

We observed that MAFP decreased fMLP-induced AA mass re-

lease from intact PMN to levels similar to those observed with the

cell-permeable sPLA2 inhibitor SB203347 while preincubation

with Scalaradial resulted in a further decrease in AA mass release

(Fig 4) The iPLA2 inhibitor HELSS had little effect on AA re-

lease at a concentration that has previously been shown to decrease

cytosolic PMN iPLA2 activity by 80 (42) Importantly MAFP

had no effect on the catalytic activity of sPLA2 that had been acid

extracted (43) from neutrophils (Fig 5) As expected neutrophil

sPLA2 activity was significantly decreased by LY311-727 and theneutralizing anti-sPLA2 Ab 3F10 and was not affected by coin-

cubation with HELSS (Fig 5)

We then evaluated the effect of MAFP on cPLA2 activity in

intact cells by incubating cells with MAFP and evaluating PLA2

activity in the cytosolic fraction of these cells We found that the

FIGURE 2 Effect of LY311-727 or DTT on extracellular sPLA2 activ-

ity and fMLP-stimulated neutrophil AA mass release PMN were treated

with vehicle (DMSO) or fMLP in the presence of DMSO LY311-727 or

DTT The release of sPLA2 activity ( A) or AA mass ( B) into the superna-

tant was then measured as described in Materials and Methods Results

shown are the mean SE of four determinations Different lower caseletters indicate differences by the Tukey-Kramer HSD test p 005

FIGURE 3 Effect of fMLP on cPLA2 translocation and electrophoretic

mobility PMN were treated with DMSO or fMLP disrupted by sonication

and separated into cytosolic microsomal and nuclear fractions by centrif-

ugation After SDS-PAGE immunoblot analysis was performed with an

anti-cPLA2 Ab cPLA2 has a calculated molecular mass of 85 kDa but

migrates at a molecular mass of approximately 110 kDa on SDS-PAGE

(41) cPLA2 phosphorylation was detected by a decrease in electrophoretic

mobility Similar results were obtained in three separate experiments

FIGURE 4 Effect of cell-permeable PLA2 inhibitors on neutrophil AA

mass release Cells were pretreated with DMSO HELSS MAFP

SB203347 or Scalaradial before treatment with DMSO or fMLP AA massrelease was then assessed by gas chromatography-selective ion monitoring

mass spectrometry as described in Materials and Methods Results shown

are the mean SE of three determinations Different lower case letters

indicate differences by the Tukey-Kramer HSD test p 005

FIGURE 5 Effect of PLA2 inhibitors on acid-extracted neutrophil

sPLA2 activity sPLA2 was partially purified from neutrophils by incuba-

tion with H2SO4 The effect of LY311-727 3F10 HELSS or MAFP on

acid-extracted sPLA2 activity was then assessed as described in Materials

and Methods Results shown are the mean SE of four determinations

Different lower case letters indicate differences by the Tukey-Kramer HSD

test p 005


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PLA2 activity in PMN cytosol using [3H8]AA-labeled bacterial

membranes as a substrate was almost completely inhibited by

MAFP (Fig 6) To determine whether the PLA2 activity in PMN

cytosol was attributable to cPLA2 activity we controlled this assay

against the presence of iPLA2 activity using the iPLA2-specific

inhibitor HELSS and against sPLA2 activity using the sPLA2 in-

hibitor LY311-727 (16) We found that neither HELSS nor

LY311-727 had any effect on AA hydrolysis from bacterial mem-

branes under these conditions (Fig 6) Two other iPLA2 inhibitors(PACOCF3 and MJ33) also failed to inhibit the PLA2 activity in

PMN cytosol (data not shown) Hence we observed that MAFP

significantly blocked cPLA2 activity in the cytosol of human PMN

in vitro a finding consistent with the capacity of MAFP to inhibit

fMLP-induced AA release from intact cells

No role for iPLA2

in fMLP-induced PMN AA release

We used the suicide substrate HELSS (44) to study the role of

iPLA2 in PMN AA release HELSS has previously been shown to

selectively constrain the activity of iPLA2 and to be a poor inhib-

itor of cPLA2 activity (26) At a concentration that significantly

decreased PMN iPLA2 activity (42) and obliterated PMN super-

oxide production (45) HELSS failed to inhibit fMLP-induced AA

mass release (Fig 4) or to have any effect on neutrophil sPLA2

activity (Fig 5) These findings do not support a role for iPLA2 in

fMLP-stimulated neutrophil AA release

A potential role for an intracellular or cell-associated sPLA2

-

like activity in PMN AA release

We have provided evidence that extracellular sPLA2 and iPLA2

make little or no contribution to AA release (Figs 1 and 4) and

that the function of cPLA2 appears to be required for fMLP-in-

duced AA release (Figs 3 and 4) As a control for the AA mass

release experiments with the soluble cPLA2 inhibitor MAFP we

examined the effects of the putative sPLA2-specific inhibitors

SB203347 and Scalaradial (23 24) which like MAFP readily

penetrate intact cells We noted that pretreatment with SB203347

or Scalaradial inhibited AA mass release (Fig 4) findings consis-

tent with a role for an intracellular or cell-associated sPLA2 in this

process In this regard activity measurements showed that approx-

imately 70 of cellular sPLA2 remained within the cell following

stimulation with fMLP (Fig 1 A) and thus was inaccessible to

LY311-727 or DTT sPLA2 inhibitors that failed to affect fMLP-

induced AA mass release (Fig 2 B) Hence we considered whether

an intracellular or cell-associated sPLA2-like molecule could play

a role in PMN AA metabolism To evaluate this hypothesis wepermeabilized PMN with SLO to deliver LY311-727 or 3F10 di-

rectly to the putative intracellular or cell-associated sPLA2 As

noted above LY311-727 and 3F10 inhibit neutrophil sPLA2 ac-

tivity by approximately 98 and 80 respectively (Fig 5)

[3H8]AA release from permeabilized cells treated with DMSO or

DTT were used as controls After PMN were permeabilized with

SLO incubation with either LY311-727 or 3F10 resulted in a 50

inhibition of [3H8]AA release (Fig 7 SLO) while DTT de-

creased [3H8]AA release from permeabilized cells by approxi-

mately 40 (data not shown) When [3H8]AA-labeled cells were

not permeabilized with SLO the cell-impermeable sPLA2 inhibi-

tor LY311-727 and the neutralizing anti-sPLA2 Ab 3F10 failed to

prevent [3H8]AA release (Fig 7 SLO) Hence we were able to

provide some evidence that an intracellular or cell-associated

sPLA2-like molecule participates in fMLP-stimulated AA release

FIGURE 6 Effect of PLA2 inhibitors on neutrophil cPLA2 activity

Cells were disrupted by sonication and the cytosolic fraction was isolated

by centrifugation The effect of MAFP HELSS or LY311-727 on cPLA2

activity was then measured as described in Materials and Methods Re-sults shown are the mean SE of four determinations Different lower case

letters indicate differences by the Tukey-Kramer HSD test p 005

FIGURE 7 Effect of LY311-727 and 3F10 on [3H8]AA release from

permeabilized neutrophils Cells were metabolically labeled with [3H8]AA

washed and treated with SLO toxin (SLO) or vehicle (SLO) Neutro-

phils were then treated with vehicle or fMLP in the presence of DMSO

LY311-727 or 3F10 Following centrifugation the release of [3H8]AA was

measured as described in Materials and Methods Exposure of SLO-per-

meabilized cells (ie SLO) to fMLP induced a mean release of 2598cpm which was arbitrarily taken as 100 Results shown are the mean

SE of four determinations Different lower case letters indicate differences

by the Tukey-Kramer HSD test p 005


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Discussion

We have systematically examined the roles of extracellular and

intracellular or cell-associated sPLA2-like enzymes cPLA2 and

iPLA2 in AA release using pharmacological and biochemical

techniques Our intention was to utilize an integrated approach in

an attempt to establish the location and identity of the PLA 2 iso-

forms that play a role in AA release from fMLP-stimulated

human PMN

No direct role for iPLA2

in fMLP-stimulated PMN AA release

The role of iPLA2 in AA metabolism appears to vary in different

cell lines In pancreatic cells smooth muscle cells and cardio-

myocytes treatment with the iPLA2 inhibitor HELSS decreased

agonist-stimulated AA release (46ndash48) HELSS has been shown

to be approximately 1000-fold more selective for inhibition of

iPLA2 compared with cPLA2 or sPLA2 under some conditions

and has no effect on other enzymes involved in AA metabolism

including arachidonyl-CoA synthetase lysophosphatidylcholine

arachidonyl acyl transferase or CoA-independent transacylase

(26 47 49 ndash51) Therefore the results obtained with HELSS were

interpreted to indicate a role for iPLA2 in AA release from pan-

creatic cells smooth muscle cells and cardiomyocytes In ad-dition human embryonic kidney 293 cells transfected with iPLA2

cDNA demonstrated increased basal release of [3H8]AA and in-

creased [3H8]AA release in response to serum compared with con-

trol cells (52) In contrast studies with P388D1 macrophages in

which HELSS or antisense iPLA2 oligonucleotides were used to

inhibit or deplete iPLA2 demonstrated that iPLA2 participates in

phospholipid remodeling and did not play a role in PAF-stimu-

lated AA release from these cells In the present study we found

that HELSS at a concentration that significantly inhibits neutro-

phil iPLA2 activity (42) had no significant effect on fMLP-stim-

ulated AA release We conclude that iPLA2 did not significantly

contribute to fMLP-stimulated PMN AA release under our assay

conditions

Central role for cPLA2

in PMN AA release

Multiple studies have implicated cPLA2 activation as a critical step

in PMN AA release (18 53ndash55) In agreement with these studies

immunoblot analysis of fMLP-stimulated cells demonstrated trans-

location of cPLA2 from cytosolic to microsomal and nuclear frac-

tions In addition exposure to fMLP resulted in a decrease in the

electrophoretic mobility of cPLA2 a finding consistent with

cPLA2 phosphorylation (41) which is known to increase the cat-

alytic activity of cPLA2 in vitro (55) Pretreatment of neutrophils

with MAFP which covalently binds to and inactivates cPLA2 (56)

inhibited fMLP-induced AA mass release In parallel studies con-

ducted in vitro MAFP obliterated the PLA2 activity in neutrophilcytosol While these results are consistent with a role for cPLA2 in

neutrophil AA release they do not rule out the possibility that

MAFP inhibited AA release through an effect on sPLA2 To eval-

uate this possibility sPLA2 was partially purified from PMN by

acid extraction with H2SO4 pH 16 (43) and the effect of MAFP

on neutrophil sPLA2 activity was assessed Coincubating acid-ex-

tracted sPLA2 with MAFP had no effect on sPLA2 activity (Fig 5)

indicating that MAFP did not decrease neutrophil AA release

through an effect on sPLA2 MAFP also inhibits iPLA2 in vitro

(56) but studies with HELSS indicated that iPLA2 does not par-

ticipate in neutrophil AA release Therefore the observation that

fMLP stimulated cPLA2 phosphorylation and translocation and

that MAFP inhibited fMLP-stimulated neutrophil AA mass release

and cPLA2 activity supports a central role for cPLA2 in governing

AA release from human PMN

Role for sPLA2

in PMN AA release

We showed that stimulation with fMLP for 30 s resulted in a

significant release of both sPLA2 activity and [3H8]AA from PMN

These findings are consistent with the notion that sPLA2 released

by PMN into the extracellular space can bind to the cell membrane

and hydrolyze membrane glycerophospholipids (26) In support of

this model addition of exogenous recombinant synovial PLA2 to

P388D1 macrophages or group V PLA2 to unstimulated human

PMN both led to a significant release of AA (21 22) In additionthe agonist-induced translocation of phosphatidylserine and phos-

phatidylethanolamine from the intracellular to the extracellular

face of the phospholipid membrane favors membrane hydrolysis

by an extracellular sPLA2 as sPLA2 binding to lipid bilayers is

promoted by negative charges (57 58) and group V sPLA2 pref-

erentially hydrolyzes phosphatidylethanolamine vesicles com-

pared with phosphatidylcholine vesicles (59) Our results did not

strongly support a role for an extracellular sPLA2 in PMN AA

release as inhibition of extracellular sPLA2 activity with LY311-

727 or DTT which constrains the catalytic activity of group IIa

and group V sPLA2 (16 17) only had a small inhibitory effect on

AA mass release from fMLP-stimulated cells This discrepancy

may be explained by the finding that brief exposure to exogenous

group IIa or group V PLA2 caused an initial release of fatty acids

from human white blood cells that was followed by resistance to

further hydrolysis of membrane phospholipids by either enzyme a

phenomenon that may be mediated by sPLA2 binding to a mem-

brane receptor (60) Alternatively it is possible that the affinity of

the surface sPLA2 receptors rapidly changed in response to agonist

stimulation and that stimulated cells did not effectively bind en-

dogenous extracellular sPLA2 In support of this we have previ-

ously shown that sPLA2 expression on the surface of PMN in-

creases 7-fold after 15 s of stimulation with fMLP but that surface

sPLA2 expression returned to baseline levels within 1 min of stim-

ulation (61) We conclude from our data that the sPLA2 that is

released from human PMN in response to fMLP did not directly

mediate a major portion of the AA released from these cells

Possible role for an intracellular or cell-associated sPLA2

-like

enzyme in PMN AA release

Previous studies have indicated that a sPLA2 might play a role in

AA release from within the cell (23 24) Three independent lines

of evidence were identified in this study that support this concept

First we observed that approximately 70 of cellular sPLA2 ac-

tivity remained associated with the cell following stimulation with

fMLP Second treatment with the cell-permeable sPLA2 inhibitors

SB203347 or Scalaradial both prevented AA mass release to an

extent similar to MAFP (Fig 4) However studies with SB203347

or Scalaradial must be interpreted cautiously (24) as both of theseinhibitors may constrain the activity of cPLA2 and Scalaradial

may affect AA release though inhibition of Ca2 mobilization

(25) As the reducing environment of the cytosol would inactivate

sPLA2 activity sPLA2 would likely have to be confined to intra-

cellular granules (9) or some other intracellular location to retain

catalytic activity To examine the potential role of an intracellular

or cell-associated sPLA2 in AA release under our conditions we

used the highly specific sPLA2 inhibitor LY311-727 (16) and the

neutralizing anti-sPLA2 mAb 3F10 (28) As neither LY311-727

nor 3F10 was likely to be able to enter cells effectively during the

time frame of these studies (minutes) we permeabilized PMN with

SLO so that the sPLA2 inhibitors could gain direct access to the

putative intracellular or cell-associated sPLA2 The nonspecific re-

ducing agent DTT which inhibits sPLA2 but not cPLA2 or iPLA2

activity was used as a control We found that both LY311-727 and


y

g

p j

g



3F10 inhibited the release of [3H8]AA from permeabilized PMN

(Fig 7) to an extent that was comparable with that of the nonspe-

cific reducing agent DTT thereby providing a third line of evi-

dence that an intracellular or cell-associated sPLA2 participates in

PMN AA release In contrast to our results Bauldry et al showed

that DTT had little effect on [3H8]AA release from [3H8]AA-la-

beled fMLP-stimulated SLO-permeabilized PMN (62) This dis-

crepancy may be explained by the fact that the cytosolic buffer

used for the permeabilization studies conducted by Bauldry et al

(62) did not support sPLA2 catalytic activity most likely because

a Ca2 concentration of 500 nM was used In the present study

cells were simultaneously exposed to fMLP and 1 mM Ca2 a

concentration of Ca2 that supports maximal sPLA2 activity

As SLO permitted a free diffusion of small molecules between

cytoplasm and medium (35) it was not clear whether the [3H8]AA

release inhibited by LY311-727 or 3F10 in the permeabilized cell

was in fact destined for extracellular release or if the [3H8]AA

leaked out of the cell from intracellular compartments such as

granules through the SLO-induced pores Since LY311-727 did

not inhibit cPLA2 activity (Fig 6) a finding consistent with a

recent report (17) we conclude that a low mw sPLA2 activity

remained within a noncytosolic compartment of the neutrophils

such as the granules (9) and participated in AA metabolism inresponse to the bacterial peptide fMLP

In summary our results demonstrate that cPLA2 plays a central

role in fMLP-stimulated AA release from human PMN We also

present some evidence in support of a role for an intracellular or

cell-associated sPLA2 in PMN AA release The possibility that

sPLA2 activity is regulated by the prior activation of cPLA2 (26)

in human PMN is currently being evaluated

Acknowledgments

We thank Dr Lisa Marshall Smith Kline Beecham Pharmaceuticals (no

relation to JGM) Dr Jim Clark and Dr Simon Jones Genetics Institute

(Cambridge MA) Dr Peter Elsbach New York University and Dr Ed

Mihelich Eli Lilly for helpful discussions and their invaluable contribu-tion of specialty reagents

References

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2 Serhan C N J Z Haeggstrom and C C Leslie 1996 Lipid mediator networksin cell signaling update and impact of cytokines FASEB J 101147

3 Clark J D A R Schievella E A Nalefski and L L Lin 1995 Cytosolicphospholipase A2 J Lipid Mediat Cell Signal 1283

4 Pouliot M P P McDonald E Krump J A Mancini S R McCollP K Weech and P Borgeat 1996 Co-localization of cytosolic phospholipaseA2 5-lipoxygenase and 5-lipoxygenase-activating protein at the nuclear mem-brane of A23187-stimulated human neutrophils Eur J Biochem 238250

5 Ramesha C S and D L Ives 1993 Detection of arachidonoyl-selective phos-pholipase A2 in human neutrophil cytosol Biochim Biophys Acta 116837

6 Smith D M and M Waite 1992 Phosphatidylinositol hydrolysis by phospho-lipase A2 and C activities in human peripheral blood neutrophils J Leukocyte

Biol 526707 Balboa M A J Balsinde S S Jones and E A Dennis 1997 Identity between

the Ca2-independent phospholipase A2 enzymes from P388D1 macrophages andChinese hamster ovary cells J Biol Chem 2728576

8 Tang J R W Kriz N Wolfman M Shaffer J Seehra and S S Jones 1997A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrinmotifs J Biol Chem 2728567

9 Rosenthal M D M N Gordon E S Buescher J H Slusser L K Harris andR C Franson 1995 Human neutrophils store type II 14-kDa phospholipase A2

in granules and secrete active enzyme in response to soluble stimuli Biochem Biophys Res Commun 208650

10 Cupillard L K Koumanov M G Mattei M Lazdunski and G Lambeau1997 Cloning chromosomal mapping and expression of a novel human secre-tory phospholipase A2 J Biol Chem 27215745

11 Han S K E T Yoon and W Cho 1998 Bacterial expression and character-ization of human secretory class V phospholipase A2 Biochem J 331353

12 Marshall L A and A McCarte-Roshak 1992 Demonstration of similar cal-cium dependencies by mammalian high and low molecular mass phospholipaseA2 Biochem Pharmacol 441849

13 Tischfield J A 1997 A reassessment of the low molecular weight phospholipaseA2 gene family in mammals J Biol Chem 27217247

14 Seilhamer J J W Pruzanski P Vadas S Plant J A Miller J Kloss andL K Johnson 1989 Cloning and recombinant expression of phospholipase A2

present in rheumatoid arthritic synovial fluid J Biol Chem 2645335

15 Vadas P E Stefanski and W Pruzanski 1985 Characterization of extracellularphospholipase A2 in rheumatoid synovial fluid Life Sci 36579

16 Schevitz R W N J Bach D G Carlson N Y Chirgadze D K ClawsonR D Dillard S E Draheim L W Hartley N D Jones E D Mihelich et al1995 Structure-based design of the first potent and selective inhibitor of humannon-pancreatic secretory phospholipase A2 Nat Struct Biol 2458

17 Yang H C M Mosior C A Johnson Y Chen and E A Dennis 1999Group-specific assays that distinguish between the four major types of mamma-lian phospholipase A2 Anal Biochem 269278

18 Durstin M S Durstin T F Molski E L Becker and R I Sharsquoafi 1994Cytoplasmic phospholipase A2 translocates to membrane fraction in human neu-trophils activated by stimuli that phosphorylate mitogen-activated protein kinaseProc Natl Acad Sci USA 913142

19 Surette M E N Dallaire N Jean S Picard and P Borgeat 1998 Mechanismsof the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4

in chemotactic peptide-stimulated human neutrophils FASEB J 121521

20 Bonventre J V Z Huang M R Taheri E OrsquoLeary E Li M A Moskowitzand A Sapirstein 1997 Reduced fertility and postischemic brain injury in micedeficient in cytosolic phospholipase A2 Nature 390622

21 Han S K K P Kim R S Koduri L Bittova N M Munoz A R LeffD C Wilton M H Gelb and W Cho 1999 Role of Trp31 in high membranebinding and proinflammatory activity of human group V phospholipase A2

J Biol Chem 27411881

22 Balsinde J M A Balboa and E A Dennis 1998 Functional coupling betweensecretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic

group IV phospholipase A2 Proc Natl Acad Sci USA 95795123 Marshall L A J D Winkler D E Griswold B Bolognese A Roshak

C M Sung E F Webb and R Jacobs 1994 Effects of Scalaradial a type IIphospholipase A2 inhibitor on human neutrophil arachidonic acid mobilizationand lipid mediator formation J Pharmacol Exp Ther 268709

24 Marshall L A R H Hall J D Winkler A Badger B Bolognese A RoshakP L Flamberg C M Sung M Chabot-Fletcher J L Adams et al 1995 SB203347 an inhibitor of 14 kDa phospholipase A2 alters human neutrophil ara-chidonic acid release and metabolism and prolongs survival in murine endotoxinshock J Pharmacol Exp Ther 2741254

25 Barnette M S J Rush L A Marshall J J Foley D B Schmidt andH M Sarau 1994 Effects of Scalaradial a novel inhibitor of 14 kDa phospho-lipase A2 on human neutrophil function Biochem Pharmacol 471661

26 Balsinde J and E A Dennis 1996 Distinct roles in signal transduction for eachof the phospholipase A2 enzymes present in P388D1 macrophages J Biol Chem

2716758

27 Kim T S C S Sundaresh S I Feinstein C Dodia W R Skach M K JainT Nagase N Seki K Ishikawa N Nomura and A B Fisher 1997 Identifi-

cation of a human cDNA clone for lysosomal type Ca2-independent phospho-lipase A2 and properties of the expressed protein J Biol Chem 2722542

28 McCord M M Chabot-Fletcher J Breton and L A Marshall 1994 Humankeratinocytes possess an sn-2 acylhydrolase that is biochemically similar to theU937-derived 85-kDa phospholipase A2 J Invest Dermatol 102980

29 Anderson K M A Roshak J D Winkler M McCord and L A Marshall1997 Cytosolic 85-kDa phospholipase A2-mediated release of arachidonic acid iscritical for proliferation of vascular smooth muscle cells J Biol Chem 27230504

30 Haslett C L A Guthrie M M Kopaniak R B Johnston Jr and P M Henson1985 Modulation of multiple neutrophil functions by preparative methods ortrace concentrations of bacterial lipopolysaccharide Am J Pathol 119101

31 Waddell T K L Fialkow C K Chan T K Kishimoto and G P Downey1994 Potentiation of the oxidative burst of human neutrophils a signaling rolefor L-selectin J Biol Chem 26918485

32 Amrein P C and T P Stossel 1980 Prevention of degradation of humanpolymorphonuclear leukocyte proteins by diisopropylfluorophosphate Blood 56

442

33 Oda T N Komatsu and T Muramatsu 1998 Diisopropylfluorophosphate(DFP) inhibits ricin-induced apoptosis of MDCK cells Biosci Biotechnol Bio-

chem 62325

34 Elsbach P and J Weiss 1991 Utilization of labeled Escherichia coli as phos-pholipase substrate Methods Enzymol 19724

35 Mira J P T Dubois J P Oudinet S Lukowski F Russo-Marie and B Geny1997 Inhibition of cytosolic phospholipase A2 by annexin V in differentiatedpermeabilized HL-60 cells evidence of crucial importance of domain I type IICa2-binding site in the mechanism of inhibition J Biol Chem 27210474

36 Shaikh N A 1994 Assessment of various techniques for the quantitative ex-traction of lysophospholipids from myocardial tissues Anal Biochem 216313

37 Hamilton J G and K Comai 1988 Rapid separation of neutral lipids free fattyacids and polar lipids using prepacked silica Sep-Pak columns Lipids 231146

38 Chilton F H and R C Murphy 1986 Remodeling of arachidonate-containingphosphoglycerides within the human neutrophil J Biol Chem 2617771

39 Wollard P M 1983 Selective silylation using tert -butyldimethylsilyl reagentstheir use in the quantification of fatty acids Biomed Mass Spectrom 10143

40 Doerfler M E J Weiss J D Clark and P Elsbach 1994 Bacterial lipopoly-

saccharide primes human neutrophils for enhanced release of arachidonic acidand causes phosphorylation of an 85-kD cytosolic phospholipase A2 J Clin

Invest 931583


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g

p j

g



41 Lin L L M Wartmann A Y Lin J L Knopf A Seth and R J Davis 1993cPLA2 is phosphorylated and activated by MAP kinase Cell 72269

42 Tithof P K M Peters-Golden and P E Ganey 1998 Distinct phospholipasesA2 regulate the release of arachidonic acid for eicosanoid production and super-oxide anion generation in neutrophils J Immunol 160953

43 Marki F and R Franson 1986 Endogenous suppression of neutral-active andcalcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Biochim Biophys Acta 87914944 Daniels S B E Cooney M J Sofia P K Chakravarty and

J A Katzenellenbogen 1983 Haloenol lactones potent enzyme-activated irre-versible inhibitors for -chymotrypsin J Biol Chem 25815046

45 Marshall J G E Krump P Zhu R Erickson G P Downey J T Curnutte

S Grinstein P M Walker and B B Rubin 1999 The haloenol lactone suicidesubstrate (HELSS) does not prevent the oxidative burst by inhibition of iPLA2

alone FASEB J 13A109646 Wolf M J and R W Gross 1996 The calcium-dependent association and

functional coupling of calmodulin with myocardial phospholipase A2 implica-tions for cardiac cycle-dependent alterations in phospholipolysis J Biol Chem

2712098947 Hazen S L L A Zupan R H Weiss D P Getman and R W Gross 1991

Suicide inhibition of canine myocardial cytosolic calcium-independent phospho-lipase A2 mechanism-based discrimination between calcium-dependent and -in-dependent phospholipases A 2 J Biol Chem 2667227

48 Ramanadham S M J Wolf B Li A Bohrer and J Turk 1997 Glucose-responsitivity and expression of an ATP-stimulatable Ca(2)-independent phos-pholipase A2 enzyme in clonal insulinoma cell lines Biochim Biophys Acta1344153

49 Hazen S L R J Stuppy and R W Gross 1990 Purification and character-ization of canine myocardial cytosolic phospholipase A2 a calcium-independentphospholipase with absolute f1-2 regiospecificity for diradyl glycerophospholip-

ids J Biol Chem 2651062250 Balsinde J I D Bianco E J Ackermann K Conde-Frieboes and E A Dennis

1995 Inhibition of calcium-independent phospholipase A2 prevents arachidonicacid incorporation and phospholipid remodeling in P388D1 macrophages Proc

Natl Acad Sci USA 928527

51 Ackermann E J and E A Dennis 1995 Mammalian calcium-independentphospholipase A2 Biochim Biophys Acta 1259125

52 Murakami M S Shimbara T Kambe H Kuwata M V WinsteadJ A Tischfield and I Kudo 1998 The function of five distinct mammalianphospholipase A2rsquos in regulation arachidonic acid release J Biol Chem 273

14411

53 Seeds M C D F Jones F H Chilton and D A Bass 1998 Secretory andcytosolic phospholipases A2 are activated during TNF priming of human neu-trophils Biochim Biophys Acta 1389273

54 Sharsquoafi R I and T F Molski 1988 Activation of the neutrophil Prog Allergy421

55 Leslie C C 1997 Properties and regulation of cytosolic phospholipase A2 J Biol Chem 27216709

56 Lio Y C L J Reynolds J Balsinde and E A Dennis 1996 Irreversibleinhibition of Ca(2)-independent phospholipase A2 by methyl arachidonyl fluo-rophosphonate Biochim Biophys Acta 130255

57 Burack W R M E Gadd and R L Biltonen 1995 Modulation of phospho-lipase A2 identification of an inactive membrane-bound state Biochemistry 3414819

58 Jain M K B Z Yu and A Kozubek 1989 Binding of phospholipase A2 tozwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles

Biochim Biophys Acta 98023

59 Chen Y and E A Dennis 1998 Expression and characterization of humangroup V phospholipase A2 Biochim Biophys Acta 139457

60 Wilson H A J B Waldrip K H Nielson A M Judd S K Han W ChoP J Sims and J D Bell 1999 Mechanism by which elevated intracellularcalcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2 J Biol Chem 26411494

61 Rubin B B J Marshall T F Lindsay D A Ford G P Downey S Shapira

S Liauw A D Romaschin and P M Walker 1997 Discovery of a novelphospholipase A2 isoform in human neutrophils Surg Forum XLVIII386

62 Bauldry S A R E Wooten and D A Bass 1996 Activation of cytosolicphospholipase A2 in permeabilized human neutrophils Biochim Biophys Acta

1299223


y

g

p j

g



Involvement of Cytosolic Phospholipase A2 and Secretory

Phospholipase A2 in Arachidonic Acid Release from Human

Neutrophils1

John Marshall Eric Krump2Dagger Thomas Lindsay Gregory Downeydagger David A Fordsect

Peihong Zhu Paul Walker and Barry Rubin3

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2) calcium-independent PLA2 and cytosolic

PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils While fMLP induced the release of

extracellular sPLA2 activity and AA 70 of sPLA2 activity remained associated with the cell Treatment with the cell-imper-

meable sPLA2 inhibitors DTT or LY311-727 or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity but had

minimal effect on neutrophil AA mass release In contrast coincubation of streptolysin-O toxin-permeabilized neutrophils with

DTT LY311-727 or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled fMLP-stimulated cells Exposure to fMLP resulted

in a decrease in the electrophoretic mobility of cPLA2 a finding consistent with cPLA2 phosphorylation and stimulated the

translocation of cPLA2 from cytosolic to microsomal and nuclear compartments The role of cPLA2 was further evaluated with

the cPLA2 inhibitor methyl arachidonyl fluorophosphonate which attenuated cPLA2 activity in vitro and decreased fMLP-

stimulated AA mass release by intact neutrophils but had no effect on neutrophil sPLA2 activity Inhibition of calcium-indepen-

dent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release These results

indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human

neutrophils The Journal of Immunology 2000 164 2084ndash2091

Phospholipase A2 (PLA2)4 hydrolyzes arachidonic acid

(AA) from the sn-2 position of glycerophospholipids (1)

The hydrolysis of AA by PLA2 is a critical regulatory step

in the development of an inflammatory response Neutrophils

(polymorphonuclear leukocytes (PMN)) release the proinflamma-

tory mediator AA via the function of PLA2 when activated by

bacterial peptides phorbol esters calcium ionophores and other

agonists Released AA may then be converted to biologically ac-tive metabolites such as prostaglandins leukotrienes lipoxins and

thromboxanes which are thought to directly modulate the inflam-

matory response (2)

Multiple PLA2 isoforms have been described in human neutro-

phils An 85-kDa cytosolic PLA2 (cPLA2) that selectively hydro-

lyzes glycerophospholipids with AA in the sn-2 position and trans-

locates to the nucleus in a Ca2-dependent manner in response to

agonists (3 4) has been identified in PMN (5) Neutrophils also

have Ca2-independent PLA2 (iPLA2) activity (6) iPLA2 hasbeen identified in murine macrophages and is thought to function

in membrane remodeling in these cells (7 8) In addition neutro-

phils contain low mw secretory PLA2 (sPLA2) that are stored in

granules (9ndash11) sPLA2 isoforms have six to eight disulfide

bridges require micromolar to millimolar levels of Ca2 for cat-

alytic activity (12) and do not exhibit specificity for phospholipids

with AA in the sn-2 position (13ndash15) The catalytic activity of the

low mw sPLA2 isoforms in contrast to cPLA2 and iPLA2 is

inhibited by the reducing agent DTT (15) and by the substituted

indole LY311-727 (16 17)

The enzyme(s) that mediates AA release from activated PMN

has not been completely characterized Evidence that cPLA2 di-

rectly mediates AA release includes the demonstration that cPLA2

is activated in response to bacterial agonists (18) that pharmaco-

logical inhibitors of cPLA2 attenuate PMN AA release (19) and

that macrophages from mice in which the gene for cPLA2 was

disrupted exhibited decreased AA release in response to PMA and

calcium ionophores (20) Evidence that sPLA2 directly mediates

AA release includes the observation that exogenous addition of

group V sPLA2 to PMN (21) or recombinant synovial PLA2 to

murine macrophages (21 22) directly results in AA release In

addition treatment with the cell-permeable sPLA2 inhibitors

SB203347 and Scalaradial prevented Ca2 ionophore-stimulated

AA mass release (23 24) While these studies support a role for

sPLA2 in PMN AA release (23 24) they are complicated by the

Divisions of Vascular Surgery and daggerRespiratory Diseases Max Bell Research Cen-ter Toronto General Hospital University Health Network Toronto Ontario CanadaDaggerDivision of Cell Biology Sick Childrenrsquos Hospital Toronto Ontario Canada andsectDepartment of Biochemistry and Molecular Biology St Louis University HealthSciences Center St Louis MO 63104

Received for publication August 2 1999 Accepted for publication December 6 1999

The costs of publication of this article were defrayed in part by the payment of pagecharges This article must therefore be hereby marked advertisement in accordancewith 18 USC Section 1734 solely to indicate this fact

1 This work was supported by The Heart and Stroke Foundation of Canada (Grant

NA-3497 BR) The Medical Research Council of Canada (GD) and by the Amer-ican Heart Association Grant-in-Aid 9750096N and National Heart Lung and BloodInstitute Grants R01-HL-42665-11 and K04-HL-03316-05 (to DAF) BR is therecipient of the Wylie Scholar Award in Academic Vascular Surgery from The PacificVascular Research Foundation San Francisco and a Bickel Foundation Award AMedical Research Council of Canada Fellowship supported EK

2 Deceased

3 Address correspondence and reprint requests to Dr Barry Rubin EC5-302a To-ronto General Hospital Toronto Ontario Canada M5G-2C4 E-mail addressbarryrubinuhnonca

4 Abbreviations used in this paper PLA2 phospholipase A2 AA arachidonic acidcPLA2 Ca2-dependent cytosolic PLA2 DFP diisopropyl fluorophosphate HELSShaloenol lactone suicide substrate or ( E )-6-(bromomethylene)tetrahydro-3-(1-naph-thalenyl)-2 H -pyran-2-1 HSD highly significant difference iPLA2 Ca2-indepen-dent cytosolic PLA2 KRPD Krebs-Ringer-phosphate dextrose MAFP methylarachidonyl fluorophosphonate PMN neutrophil SLO streptolysin O sPLA2 se-cretory PLA2

Copyright copy 2000 by The American Association of Immunologists 0022-176700$0200

y

g

p j

g










pletely defined
















from neutrophils











sPLA2

assay



[3 H 8



neutrophils



AA mass release


Cell fractionation




y

g

p j

g






cPLA 2



SDS-PAGE


Western blots






2 in PMN AA release





























in PMN AA release
























y

g

p j

g




phosphorylated (41)














































test p 005


y

g

p j

g
















No role for iPLA2












-
















































y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g










pletely defined
















from neutrophils











sPLA2

assay



[3 H 8



neutrophils



AA mass release


Cell fractionation




y

g

p j

g






cPLA 2



SDS-PAGE


Western blots






2 in PMN AA release





























in PMN AA release
























y

g

p j

g




phosphorylated (41)














































test p 005


y

g

p j

g
















No role for iPLA2












-
















































y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g






cPLA 2



SDS-PAGE


Western blots






2 in PMN AA release





























in PMN AA release
























y

g

p j

g




phosphorylated (41)














































test p 005


y

g

p j

g
















No role for iPLA2












-
















































y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g




phosphorylated (41)














































test p 005


y

g

p j

g
















No role for iPLA2












-
















































y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g
















No role for iPLA2












-
















































y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g



Discussion







human PMN


























conditions


in PMN AA release


























Role for sPLA2

in PMN AA release




































-like


























y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g
































Acknowledgments





References

































2716758








442


chem 62325









Invest 931583


y

g

p j

g





















14411













1299223


y

g

p j

g





















14411













1299223


y

g

p j

g

izolovanje pla2

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