Research Article The Subcellular Localization of the Receptor for … · 2019. 7. 31. ·...

Hindawi Publishing CorporationClinical and Developmental ImmunologyVolume 2013 Article ID 456407 11 pageshttpdxdoiorg1011552013456407

Research ArticleThe Subcellular Localization of the Receptor forPlatelet-Activating Factor in Neutrophils Affects Signaling andActivation Characteristics

Emil Andreacuteasson Karin Oumlnnheim and Huamei Forsman

Department of Rheumatology and Inflammation Research Goteborg University Guldhedsgatan 10A 413 46 Goteborg Sweden

Correspondence should be addressed to Huamei Forsman huameiforsmanrheumaguse

Received 26 April 2013 Accepted 22 July 2013

Academic Editor Richard L Gallo

Copyright copy 2013 Emil Andreasson et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The localization in neutrophils of the receptor for platelet-activating factor (PAFR) has been determined using subcellularfractionation and a receptor mobilization protocol We show that the PAFR is expressed primarily in the plasma membraneAlthough activation of neutrophils by PAF induces responses typical also of agonists that bind the formyl peptide receptors (FPR)known to be stored in mobilizable organelles some quantitative as well as qualitative differences were observed when neutrophilswere activated through these receptors PAF is equipotent to fMLF (high affinity agonist for FPR1) to cleave off L-selectin and toinduce granulevesicle secretion but is more potent than fMLF to induce a rise in intracellular Ca2+ Similar to fMLF PAF inducedalso a robust release of reactive oxygen species but with higher EC

50value and was less sensitive to a PI3K inhibitor compared to

the fMLF response Despite the lack of a granule localized storage pool of receptors the PAF-induced superoxide production couldbe primed receptor mobilization was thus not required for priming of the PAF response The desensitized PAFR could not bereactivated suggesting that distinct signaling pathways are utilized for termination of the responses triggered through FPR1 andPAFR

1 Introduction

Neutrophil granulocytes professional phagocytes of theinnate immune system are recognized and activated bychemoattractants soluble molecules serving as ldquodanger sig-nalsrdquo [1] Activation of the phagocytes is of great importancefor the outcome of the continuously ongoing combat withinvading microorganisms but accumulation of these cellsand their subsequent release of reactive oxygen species (ROS)and proteolytic enzymes are also responsible for the tissuedamage associated with a number of inflammatory diseaseconditions [2] Research on the structurefunctional rela-tionship of neutrophil chemoattractants and their receptorsas well as the downstream signaling pathways is thereforeof direct clinical importance and relevance and the list ofstructurally well-characterized receptor agonists as well asantagonistsinhibitors has steadily grown [3 4]

All chemoattractant receptors including the most exten-sively studied formyl peptide receptors (FPRs) exhibit some

sequence homologies and belong to the family of pertus-sis toxin sensitive G protein-coupled family of receptors(GPCRs) The neutrophil GPCRs specifically recognize dif-ferent agonists that most commonly are naturally occurringpeptidesproteins with a defined structure There are how-ever also some receptors that display high affinity binding forlipid metabolites such as lipoxin A4 (LXA4) leukotriene B4(LTB4) and platelet-activating factor (PAF) [5ndash7] The latterwas the first bioactive phospholipid identified and it has adefined and characteristic structure (an alkyl ether linkage atthe sn-1-position of the glycerol backbone) and is named afterits effect on platelets activation and aggregation [8 9] Thislipid agonist exerts all its basic functions through bindingto and activation of the G-protein-coupled PAF receptor(PAFR) [3 10] Research during the last years has demon-strated important roles for PAF in many pathophysiologicalconditions including asthma psoriasis and endotoxic shock[11] Additionally PAFR is expressed in neutrophils and PAFactivated neutrophils also serve a prominent source for the

2 Clinical and Developmental Immunology

generation of PAF and other lipid inflammatory mediatorssuch as LTB4 suggesting a role for this receptorligand pairnot only in host defence but also inmodulating inflammatoryresponses Similar to most neutrophil GPCRs occupationof the PAFR promotes secretion of granule constituents andmobilization of cell surface receptors cellular migration [12]and priming of the cells to generate increasing amounts ofROS [13] PAF has been shown by others to directly activatethe electron transporting system (the NADPH oxidase) thatgenerates ROS in eosinophils and in our hands PAF alsodirectly activates this system in neutrophils [14]

Some chemoattractants are rather poor ROS inducers inneutrophils but the response induced can be dramaticallyincreased (primed) by nonactivating agents such as tumornecrosis factor alpha (TNF-120572) and lipopolysaccharides (LPS)derived from Gram-negative bacteria [15 16] The prim-ing phenomenon has been extensively investigated and itsbiological significance in many disease processes has beenwell documented With respect to priming of neutrophils inresponse to endogenous lectins of the galectin family andreceptor specific agonists for FPR1 and FPR2 we and othershave suggested granule mobilization resulting in surfacereceptor upregulation as a key event [17 18] Accordinglyin resting neutrophils the mobilizabled FPRs and receptorsfor galectins are found primarily in secretory organelles thatfuse with the plasmamembrane in response to different typesof secretagogues [19ndash22] The precise subcellular localizationof PAFRs in human neutrophils has however not yet beenexamined

In the present study we determined the subcellularlocalization of the PAFR in resting neutrophils and ourstudies demonstrate that PAFRs are localized in a lightmembrane fraction containing plasmamembranes and secre-tory vesicles Mobilization data show that the cells lack aneasily mobilizable PAFR pool suggesting that the receptoris present primarily in the plasma membrane Still the PAF-induced ROS production in neutrophils could be enhancedby TNF-120572 and cytoskeleton disrupting agents suggesting thatour proposed mechanism for priming does not apply forthe PAFPAFR receptor-ligand pair The PAFR shared manysignalling properties and basic functional characteristics withthe FPRs but there were also many quantitative as well asqualitative differences possibly linked to the difference insubcellular localization between the two receptors

2 Materials and Methods

21 Chemicals The hexapeptide WKYMVM was synthe-sized and HPLC purified by KJ Ross-Petersen (Holte Den-mark) The formylated tripeptide formyl-methionyl-leucyl-phenylalanine (fMLF) isoluminol cytochalasin B (CytB)pertussis toxin (PTX) TNF120572 latrunculin A and phorbolmyristate acetate (PMA) were obtained from Sigma (SigmaChemical Co St Louis MO USA) Peptides were dissolvedin DMSO and stored at minus70∘C until use Subsequent dilutionsof all peptides were made in Krebs-Ringer phosphate buffer(KRG pH 73 120mM NaCl 5mM KCl 17mM KH

2PO4

83mM NaH2PO4 and 10mM glucose) supplemented with

Ca2+ (1mM) and Mg2+ (15mM) The PAFR antagonistWEB2086 was from Tocris bioscience (Bristol UK) andPAF was from Avanti Polar Lipids (Avanti Polar Lipids IncAlabama) Ficoll-Paque was obtained from Amersham Phar-macia Biotech AB (Uppsala Sweden) Horseradish peroxi-dase (HRP) was obtained from Boehringer Mannheim (Ger-many) Kinase inhibitors were obrained from Calbiochem(Darmstadt Germany) FURA-2 was fromMolecular Probes(Eugene OR USA) The MMP9 ELISA was from RampDsystems the ELISA-kit for NGAL was from Bioporto Diag-nostics Antibodies against CR3 and L-selectinwere fromBDBiosciences (Franklin Lakes NJ USA)

22 Isolation of Human Neutrophils Human peripheralbloodneutrophils were isolated frombuffy coats fromhealthyblood donors using dextran sedimentation and Ficoll-Paquegradient centrifugation as described [23] The remainingerythrocytes were hypotonic lysed and the neutrophils werewashed twice and resuspended inKRG and stored onmeltingice until useThis isolation process permits cells to be purifiedwith minimal granule mobilization

23 Neutrophil NADPH-Oxidase Activity The NADPH-oxidase activity was determined using isoluminol-enhancedchemiluminescence (CL) [24 25] The CL activity was mea-sured in a six-channel Biolumat LB 9505 (BertholdCoWild-bad Germany) using disposable 4mL polypropylene tubeswith a 900-microliter reaction mixture containing 105 cellsisoluminol (2 times 10minus5M) and HRP (2U) Intracellular ROSwas measured by luminol (cell permeable) in the presenceof superoxide dismutase and catalase [25] The tubes wereequilibrated in the Biolumat for 5min at 37∘C after whichthe stimulus (100120583L) was added and the light emission wasrecorded continuously By a direct comparison of the super-oxide dismutase-inhibitable reduction of cytochrome c andsuperoxide dismutase-inhibitable CL 72 times 107 counts werefound to correspond to the production of 1 nmol superoxide(using a millimolar extinction coefficient for cytochrome cof 211) When experiments were performed with primingand antagonistsinhibitors these substances were included inthe CL reaction mixture for different time period (controlcells received no treatment but were incubated at the samecondition) before stimulation with agonists

24 Subcellular Fractionation and Marker Analysis Subcel-lular fractionation was performed as described [26] Brieflyneutrophils were treated with the serine protease inhibitordiisopropyl fluorophosphates (DFP 8 120583M) disintegrated bynitrogen cavitation (Parr Instruments Co Moline IL) andthe postnuclear supernatant was fractionated on two- (105and 112 gL) or three-layer (112 109 and 105 gL) Percollgradients and centrifuged at 15000 g for 45minutes in a fixed-angle JA-20Beckman rotor Fractions of 15mLwere collectedby aspiration from the bottom of the centrifuge tube Thelocalization of subcellular granules was determined by gran-ule marker analysis Alkaline phosphatase (marker for secre-tory vesiclesplasma membrane) was measured by hydrolysisof p-nitrophenyl phosphate at pH 105 in a sodium barbital

Clinical and Developmental Immunology 3

buffer Myeloperoxidase (a marker for azurophil granules)and gelatinase and neutrophil gelatinase-associated lipocalin(NGAL) were measured by Western blotting and ELISArespectively

25 SDS-PAGE and Western Blotting Percoll gradient frac-tions prepared as describe above were diluted in nonreduc-ing sample buffer boiled for 5min and applied to 12 SDS-polyacrylamide gels The separated proteins were transferredto nitrocellulose membranes followed by blocking the mem-brane at room temperature for 1 hr in 1 BSA After blockingthe blots were incubated overnight at 4∘C with primaryantibody against PAFR (Cat number SC-8742 Santa Cruzbiotechnology an affinity purified polyclonal goat antibodydirected against a peptide at the C-terminus of the humanreceptor) Nonbound antibodies were removed by washingwith PBS Tween An HRP-conjugated secondary antibodywas used for visualization of the receptors

26 Calcium Mobilization Cells at the density of 1ndash3 times 106per mL were washed with Ca2+-free KRG and centrifugedat 220timesg The cell pellets were resuspended at a density of2times 10

7 cellsmL in KRG that contained 01 BSA and loadedwith 2120583M Fura 2AM for 30 minutes at room temperatureThe cells were then diluted to twice the original volume withRPMI 1640 culture medium without phenol red (PAA Labo-ratories GmbH Pasching Austria) and centrifuged Finallythe cells were washed once with KRG and resuspended in thesame buffer at a density of 2 times 107 cellsmL Calcium mea-surements were carried out in a Perkin Elmer fluorescencespectrophotometer (LC50) with excitation wavelengths of340 nm and 380 nm an emission wavelength of 509 nmand slit widths of 5 nm and 10 nm respectively The tran-sient rise in intracellular calcium is presented as the ratioof fluorescence intensities (340 nm 380 nm) detected Themeasuring cuvette contained catalase (2000U) to counteractinactivation of the chemoattractants by the MPO-H

2O2-

system [27]The concentration of EGTA required to achieve a

calcium-free environment was determined by titration ofthe ionomycin-triggered production of oxidants by neu-trophils as described [27] A small volume (10 120583L) of EGTA-containing buffer was added to the measuring vial and 20seconds later the cells were activated by the addition ofionomycin (5 times 10minus7M final concentration) The lowest con-centration of EGTA that inhibited the ionomycin-inducedresponse maximally (around 90) was used in subsequentstudies to ensure that no Ca2+ entered the cells across theplasma membrane

27 Granule Secretion and Assessment of Surface Moleculesby FACS and ELISA Human neutrophils (2 times 106 cells)were stimulated with PAF fMLF (100 10 or 2 nM) or bufferas control and incubated at 37∘C for 10min Samples wereplaced on ice and centrifuged at 335timesg for 10min at 4∘CSupernatants were removed and centrifugated once more at1500timesg for 5min then stored at minus70∘C and used for markeranalysis The cells were resuspended in ice-cold PBS and 5 times

105 cellssample were labeled with antibodies against CR3 orL-selctin for 30min at 4∘C in the dark The cells were thenwashed twice before resuspended in PBS and analysed byFACS

28 Statistic-Analysis One-way Anova with Dunnettrsquos multi-ple comparison test was used for statistical analysis 119875 lt 005was considered statistically significant

3 Results

31 The PAFR Is Localized Primarily in the Neutrophil PlasmaMembrane Only smaller fractions of earlier characterizedneutrophil chemoattractant receptors such as the FPRsbelonging to the family of GPCRs are localized in theplasmamembrane whereasmost of these receptors are storedin the secretory organelles that is secretory vesicles andspecific granules [18 28 29] We have now determined thesubcellular localization of the neutrophil receptor for PAFusing a subcellular fractionation technique and a receptormobilization protocol combined with FACS analysis Two- orthree-layer Percoll gradients were used and the localizationof PAFR was determined by immunoblotting with a receptorspecific antibodyWhen analyzing the localization in a three-layer Percoll gradient which can separate not only the mainorganelles azurophil granules and specific granules fromthe light membrane fraction but also the gelatinase gran-ules from the somewhat denser specific granules (Figure 1upper panel) it is clear that the PAFR cannot be found inthe granules (Figure 1 lower panel) The PAFR was foundonly in the light membrane fraction enriched in plasmamembranes and secretory vesicles (Figure 1 lower panel)This distribution was further confirmed using a two-layerPercoll gradient in which all the specificgelatinase granulesare concentrated in the same fractions (see supplemen-tary Figure 1 in Supplementary Material available online athttpdxdoiorg1011552013456407)

The secretory vesicles are very easy mobilized membranestorage organelles and upon secretion the receptors present inthe vesicles are exposed on the cell surface [22 30] To deter-mine the role of the easily mobilizable secretory organellesas a storage pool for PAFRs neutrophils were incubatedat 37∘C for 20min in the presence or absence of TNF-120572The cells gradually increased their surface expression of themarker control CR3 a cell surface receptor localized not onlyin the plasma membrane but also in the secretory vesiclesand in the secretory granules (Figure 2(b)) Mobilization ofCR3 was not associated with any increase in the surfaceexposure of PAFRs indicating the lack of a mobilizable poolof PAFR in neutrophils (Figure 2(a)) Taken together thesedata show that the PAFR is present primarily (or maybe evenexclusively) in the plasma membrane

32 PAF Triggers Granule Secretion and a Release of ROS fromHuman Neutrophils Interaction of neutrophils with PAF hasbeen shown to induce many cellular responses includingsecretion of granule constituents Accordingly we show thatthe addition of PAF to human neutrophils activated the cells


Gelatinase

120574

0 5 10 15 20 25 30

3

0

1

2

3

10 11 12 1413 15 16 17 18 19 20

3 10 11 12 14 15 20 21 22 29

120572 1205731 1205731

120574120572 1205731

1205732

48kDaPAFR

Fraction number

Am

ount

of m

arke

r (au

)97kDa

Figure 1 Subcellular localization of the PAFR in resting neutrophilsNeutrophil subcellular organelles from disintegrated cells werefractionated on a three-layer Percoll gradient Upper panel Thedistribution of gelatinase (marker for the 120573-fractions) visualizedthrough immunoblotting Middle panel localization of azurophilgranules (120572-fraction marker MPO) specific granules (1205731-fractionmarkers NGAL) and plasma membranesecretory vesicles (120574-fraction ALP) The markers (MPO = 998771 NGAL = ∙ ALP = ◼) areexpressed in arbitrary units Lower panel proteins from selectedfractionswere separated by SDS-PAGE and the localization of PAFRwas determined by immunoblotting with a specific antibody againstPAFR

and induced granule secretion as reflected by a shedding ofthe adhesion molecule L-selectin (CD62L a protein highlyexpressed on the surface of resting cells) an increasedcell surface expression of CR3 (a marker for the secretorygranules) and secretion of the granule localized proteinsNGAL (a marker for specific granules) and gelatinase (amarker for gelatinase granules) (supplementary Figure 2)PAF was found to be as potent as fMLF (high affinity agonistfor FPR1) and WKYMVM (high affinity agonist for FPR2)to cleave off L-selectin and to induce fusion between thegranulesvesicles and the plasmamembrane (shown for fMLFand PAF in supplementary Figure 2)

PAF is generally considered as a nonactivating or verypoorweak ROS inducer [13] contrasting the activity inducedby many other chemoattractants such as fMLF which isregarded as a strong inducer In our hands PAF at aconcentration of 100 nM promoted a robust release of ROSand the magnitude of the response was comparable to thatinduced by fMLF andWKYMVM (shown for PAF and fMLFin Figure 3) The level of ROS productionrelease induced byPAF increased dose dependently with an EC

50of asymp 800 nM

(Figure 3(b)) which should be compared to an EC50

value

of 50 nM for fMLF (Figure 3(a)) The time courses differedbetween the PAF- and the fMLF-induced cellular responsesin that the peak of the response was reached at an earlier timepoint and the response declined to baseline somewhat morerapidly with PAF compared to the FPR1-mediated or FPR2-mediated responses (shown for PAF and fMLF in Figure 4)No ROS production was detected intracellularly (data notshown) suggesting that PAF triggers exclusively an extracellu-lar release of ROS in neutrophils As expected a specific PAFRantagonist (WEB2086) completely and selectively abolishedthe release of ROS upon PAF stimulation demonstratingthat PAFR is the responsible receptor in mediating PAF-induced ROS production (Figure 4) Further pertussis toxinabolished the PAF response showing that a pertussis-toxinsensitive G-protein is involved in signaling downstream ofthe PAFR (supplementary Figure 3) As a control the cellstreatedwith pertussis toxinwere nonresponding also to fMLFbut fully responsive to PMA (a ROS inducer that signalsindependent of the PTX-sensitive G-protein) (supplemen-tary Figure 3)

Taken together these data clearly show that PAF is notonly a potent secretagogue but also a potent ROS activatorand the production is mainly due to an assembly of theoxidase in the plasma membrane and a release of the ROSThe PAF response reaches a peak value as well as returnsto the baseline at an earlier time point than the responsesmediated by the FPRs suggesting that different signalingpathways are utilized downstream of the PAFR and the FPRs

33 The PAFR Allows Priming but Not Reactivation Non-activating concentrations of PAF primed neutrophils toenhanced ROS production with fMLF as the second agonist[31 32] which is a well known phenomenon and it was to beexpected since PAF is a potent secretagogue (supplementaryFigure 2 [33]) Basically priming is defined as a hyperreactivestate of neutrophils induced through an exposure of thecells to a nonactivating priming agent such as TNF-120572 Tofurther elucidate the function of the PAFR we determinedwhether PAF-mediated ROS production could be primedby well-known and earlier characterized priming agentsTNF-120572 CytB and latrunculin A TNF-120572 treated neutrophilsproduced higher amount of ROS upon subsequent stim-ulation with PAF compared to nontreated control cells(Figure 5) showing that also the PAF response could beprimed Additionally we found that pretreatment of cellswith the cytoskeleton disrupting agents CytB or latrunculinA significantly increased the amount of ROS release also inresponse to PAF (Figure 5)

The signalling of an occupied GPCR rapidly ceases asthe receptor is transferred to desensitized (nonrespond-ingsignaling) state achieved in neutrophils through a bind-ing of the ligand occupied receptor to the actin cytoskeleton[34ndash36] Accordingly when binding of the chemoattractantfMLF to FPR1 takes place at low temperature (le15∘C) theactivating signalling state of the receptor is bypassed andit is directly deactivateddesensitized but the receptor canbe reactivatedresensitized by cytoskeleton-disrupting drugs


101 102 103 104 105 106 107

PAFR

Num

ber o

f cel

ls

Ice

+TNF120572

MFI

0

2000

4000

37∘C

Ice +TNF12057237∘C

(a)

101 102 103 104 105 106 107

CR3

Num

ber o

f cel

ls

MFI

0

500

1000

Ice

+TNF12057237∘C

Ice +TNF12057237∘C

(b)

Figure 2 TNF-120572 induced receptor mobilization does not involve the PAFR Human neutrophils (5 times 105) were incubated on ice (solid lines)or at 37∘C without additive (dotted lines) or with TNF-120572 (10 ngmL dashed lines) for 20min The cells were then fixed with 2 ice-coldparaformaldehyde and labeled with specific antibodies against PAFR (A) or complement receptor 3 (CR3 B) Surface receptor expressionwas determined by flow cytometry and a representative histogram is shown Abscissa fluorescence intensity Ordinate number of cells Theinsets show the mean fluorescence intensity (MFI) plusmn SEM (119899 = 5)

0

50

100

150

001 01 1

001 01

O2minus

prod

uctio

n (M

cpm

)

1

fMLF (120583M)

00

05

10

15

Nor

mal

ized

resp

onse

EC50 sim 20nM

fMLF (120583M)

(a)

0

20

40

60

80

01 1 10

O2minus

prod

uctio

n (M

cpm

)

00

05

10

15

Nor

mal

ized

resp

onse

01 1 10

EC50 sim 870nM

PAF (120583M)

PAF (120583M)

(b)

Figure 3 PAF and fMLF dose dependently trigger the release of superoxide anions from human neutrophils Human neutrophils (105 cells)were incubated at 37∘C for 5min in measuring vials containing isoluminol and HRP Various concentrations of fMLF (A) or PAF (B) wereadded and the release of superoxide anions was recorded continuously Data are expressed as mean peak value plusmn SEM (119899 = 5) Abscissaagonist concentration ordinate superoxide production expressed as counts per minute times 106 (Mcpm) The insets show one representativenormalized dose-response experiment with respective EC

50values calculated for fMLF (A) and PAF (B) respectively

(Figure 6) Such cytoskeleton-dependent receptor reactiva-tion occurs not only with desensitized FPR1 but also withFPR2 and C5aR (data not shown) Interestingly preincubat-ing cells with PAF at low temperature (le15∘C) deactivatedthe PAFR but when transferred to 37∘C these cellsreceptorscould not be reactivated by cytoskeleton disrupting agents(Figure 6) Taken together our data show that PAF-inducedROS production can be enhanced by the priming agent

TNF-120572 as well as through disruption of the cytoskeletonbut in contrast to FPR1 the desensitized PAFR could notbe reactivated through a disruption of the cytoskeletonsuggesting that distinct signaling pathways are utilized fordesensitizationtermination of FPRs and PAFR

34 Basic Signaling Downstream of the PAFR Include aRelease of Calcium from Intracellular Stores Agonist binding


0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)

PAF sim 10fMLF sim 40

Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)

Figure 4 PAF triggers a rapid and WEB2086 sensitive release ofsuperoxide anions from human neutrophils Human neutrophils(105 cells) were incubated at 37∘C for 5min in measuring vialscontaining isoluminol and HRP in the absence (control solid linein the main figure) or presence of the PAFR antagonist WEB2086(dashed line in the main figure) The cells were then activatedwith PAF (100 nM added at arrow) and the release of superoxideanions was recorded continuously Inset the kinetics of superoxideproduction induced by PAF (100 nM solid line) was comparedto that induced by fMLF (100 nM dashed line) and the timesrequired to reach the respective peak of the response are givenData are derived from one representative experiment out of at leastfive Abscissa time of study (min) ordinate superoxide productionexpressed as counts per minute times 106 (Mcpm main figure) ornormalized to compare the time courses (inset)

to GPCRs initiates a chain of events starting with dissocia-tion of the receptor-associated G-protein and subsequentlyactivation of a number of downstream signaling pathways[37 38] One such early pathway is the release of Ca2+ fromintracellular stores and an increase in the cytosolic concentra-tion of free calcium ions [Ca2+]i resulting from the bindingof the PIP

2cleavage product IP

3to its receptor located in

storage organelles Emptying of the storage organelles leadsto the entry of extracellular Ca2+ through store-operatedcalcium channels in the plasma membrane thereby prolong-ing the increase in [Ca2+]i It has been suggested that someGPCRs for example FPR2 can directly open the channelin the plasma membrane without any involvement of theintracellular storage organelles but we have earlier shownthat signaling through FPR1 as well as through FPR2 involvesprimarily an emptying of the stores [39] A rise in [Ca2+]idepending on an emptying of the stores is by definition notinhibited through addition of a Ca2+ chelator (ie EGTA)([40] shown for fMLF in supplementary Figure 4)The PAF-induced increase in [Ca2+]i was largely insensitive to EGTAsuggesting that it primarily reflects a release of ions fromintracellular stores (supplementary Figure 3) It should alsobe noticed that PAF is more potent than fMLF in inducinga rise in [Ca2+]i with an EC

50value of 5 times 10minus10M for PAF

compared to 5 times 10minus9M for fMLF (Figure 7)

8

TNF-120572CytBLatrunculin A

6

4

2

0fMLF PAF

Fold

incr

ease

Figure 5 The PAF-induced neutrophil response is primed byTNF-120572 and inhibitors of actin polymerization Human neutrophilswere incubated at 37∘C for 20min with the priming agents TNF-120572 (10 ngmL black bars) or 5min with either CytB (5120583gmL greybars) or latrunculin A (50 ngmL white bars) Control cells wereincubated at the same conditions but in the absence of any primingagents The cells were then activated with fMLF (100 nM) or PAF(100 nM) and the release of superoxide was recorded continuouslyData are expressed as fold increase of the peak values of the responsefrom primed cells compared to nonprimed control (mean plusmn SEM119899 = 3)The dashed line in the graph denotes the value expected witha nonactive priming agent

35 Distinct Utilization of Phosphoinositide 3-Kinase by fMLFand PAF We have previously shown that both FPR1 (thatbinds fMLF) and CXCR2 (that binds IL-8) signal throughthe p38MAPK as well as the PI3 K pathways [21] Todetermine whether there is a signaling difference betweenfMLF and PAF in activation the NADPH-oxidase we usedwell-characterized pharmacological kinase inhibitors Theinhibitory effects of SB203580 (p38MAPK kinase inhibitor)Staurosporin and RO318220 (protein kinase C inhibitors)and genistein (tyrosine kinase inhibitor) were the same forthe fMLF and the PAF-induced responses (data not shown)The fMLF response was however found to be more sensitivethan the PAF response to the PI3 K inhibitor wortmannin(Figure 8) suggesting that the PI3 K pathway is preferentiallyutilized by fMLF

4 Discussion

Activation of neutrophils by the lipid chemoattractantPAF induces neutrophil responses typical of many otherGPCRs agonists including a transient rise in intracellularcalcium [Ca2+]i accomplished by a mobilization of granuleconstituents to the cell surface secretion of granule proteinsand ROS Our interest to compare the similarities anddifferences between the PAFR and the FPRs arouses when wedisclosed a fundamental difference in subcellular localizationbetween the two receptors in human neutrophils The two


0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)

Figure 6The desensitized PAFR is not reactivated by CytB Neutrophil was desensitized with fMLF (100 nM FPR1 agonist solid line) or PAF(100 nM PAFR agonist dashed line) at 15∘C for 10minThe cells were then transferred to 37∘C and the incubation was continued for another10minThe cells were then challenged with CytB (5 120583gmL added at the time point indicated by arrow) and reactivation of the receptor wasdetermined through the release of superoxide One representative experiment out of three is shown Abscissa time of study (min) ordinatesuperoxide production expessed as counts per minute times 106 (Mcpm)

FPRs (FPR1 and FPR2) have earlier been shown to belocalized to a minor part in the plasma membrane of restingcells whereas the majority of the receptors are stored inmobilizable organelles that is the secretory vesicles thegelatinase granules and the specific granules [18 29] Incontrast we show that the PAFR is localized primarilyin the plasma membrane and the functional differencesbetween the neutrophil responses induced by PAF and theFPR1 agonist fMLF will be discussed in light of this factThe difference in subcellular localization between the FPRsand the PAFR is most probably not directly related to thebiophysical properties of the respective specific agonists PAFbeing a lipid and the FPR agonists being peptidesproteins[4 41]This assumption is based on the fact that the receptorsfor the cytokine IL8 (CXCR1 and CXCR2) are also expressedsolely in the plasma membrane [21] and this suggeststhat the precise localization of the receptors in restingneutrophils is instead related to their specific functionsAgonistreceptor pairs can be categorized as so-called endtarget chemoattractantsreceptors or an intermediate groupof attractantsreceptors [1 42] In an inflammatory reactionmany chemoattractants are released from various locationsincluding the vascular endothelium the complement systemand possibly microbial intruders Facing an environmentwith complex chemoattractants neutrophils have tosenserespond and to make moving decision towards one orthe other of multiple gradients of different chemoattractantsBy definition the group of intermediate attractants isgenerated at an early time point (possibly at the surface ofendothelial cells) of the response and these attractant alsomediate their functions in the initial phase of the responsewhereas the group of end target chemoattractants (possibly ofmicrobial origin) is of importance for guiding the cells to the

source of their generation in the tissue [21 42] It is reasonableto believe that the end-type chemoattractant receptors (FPR1FPR2 and C5aR) needed to take action at the late stages ofinflammatory process are primarily stored in the mobilizableorganelles whereas the intermediary ones (eg CXCR12BLT12) involved in an early inflammatory response arereadily expressed at the membrane surface Whether PAFRshould be categorized as ldquoendrdquo or ldquointermediaryrdquo type is notobvious as PAFR shares many similarities to CXCR12 butthere are fundamental differences between the two receptorswith respect to heterologous desensitization by FPR agonistsWe have very recently demonstrated that the PAF responseis not desensitized but actually primed by FPR1 agonistsand the primed PAF response involves a FPR-dependentsignaling [43]

We have previously demonstrated a strong correlationbetween increased ROS production and surface receptorupregulation and suggested that receptor mobilization is amajor mechanism for priming [17 18 20] This suggestionwas based on experimentsresults with the FPR family ofreceptors which in resting neutrophils primarily are localizedinmobilizable organelles Asmentioned the receptors for IL-8 similar to PAFR are localized at the plasma membrane andwe have earlier presented results on IL-8 that support thesuggested link between receptor localizationmobilizationand priming [21 44] Our data showing that the PAF responseterminates more rapidly than the fMLF response furthersupport this link It was however quite a surprise that thePAF-induced ROS response in contrast to the IL-8 inducedresponse could be significantly primed by TNF120572 as well as bycytoskeleton disrupting drugs (this study)These data suggestthat a novel mechanism is involved in priming of the PAFresponse Alternative mechanisms to receptor mobilization


0 20 40 60 80 100 120Time (min)

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1nM

100nM

minus2 minus1

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[Ca2+

] i(r

atio

chan

ge)

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Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

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2

[Ca2+

] i(r

atio

chan

ge)

0 1 2Log [fMLF] (nM)

Ca2+

resp

onse

indu

ced

by fM

LF

(b)

Figure 7 PAF induces a transient rise in intracellular Ca2+ in neutrophils Fura-2 loaded neutrophils (2 times 106mL) were triggered withdifferent concentrations of PAF (a) or fMLF (b) and the changes in fluorescence were followed using dual excitation at 340 nm and 380 nmrespectively and an emission wavelength of 510 nm A representative experiment out of at least three is shown and an increase in Ca2+ isvisualized as an increase in fluorescence when excited at 340 nm Tomake a direct comparison easy curves obtained with the different agonistconcentrations are shown on top of each other Abscissa time of study (sec) The time point for addition of an agonist is marked by arrowInset mean values of the transient rise in [Ca2+]i (arbitrary units plusmn SEM (119899 = 3))

have been proposed and these include a translocation tothe plasma membrane of signaling molecules such as proteinkinase C gp91phox and p47phoxp67phox (the membrane andcytosolic components resp of the NADPH-oxidase) oran elevation of intracellular calcium level per se [45ndash47]The latter mechanism could however be excluded since nochange in intracellular Ca2+ is induced by the priming agentsused and there is no direct link between oxidase activity andthe cytosolic concentration of Ca2+ [48]

PAF has generally been considered as a poor ROS inducerand well recognized for its ability to attract neutrophilmigration and for its function as a potent priming agent forenhanced production and release of superoxide anions uponexposure to a second stimulus [31 33] Using a techniquewith high sensitive and temporal resolution to monitor ROSproduction in real time we show that PAF directly activatesneutrophils to produce superoxide (this study and [14]) Theldquointensityrdquo of the PAF-induced response is somewhat lowerthan the fMLF induced response and the duration is muchshorter characteristics that might explain the observationsmade by other investigators (using the much less sensitivecytochrome c reduction technique [49 50]) that PAF is a verypoor ROS inducer [13] In our hands PAF is indeed a potentROS inducer in human neutrophils and the PAF responsecould be further amplified by priming agentsThis knowledgeshould increase our understanding about the role of PAF inregulation of inflammatory responses

We show that FPRs- but not PAFR-desensitized cellscould be reactivated upon disruption of the actin cytoskele-ton It is clear that the PAFPAFR induced response ter-minates much more rapidly than the fMLFFPR1 response

0

50

100

PAFfMLF

lowastlowastlowast

10 100 10 100Wortmannin (nM)

Inhi

bitio

n (

cont

rol)

lowastlowastlowast

Figure 8 Effect of the PI3-kinase inhibitor wortmannin on thePAF and fMLF-induced release of superoxide anions Humanneutrophils (105 cells) were incubated without (control) or withwortmannin (10 nM or 100 nM) for 10min at 37∘C The cells werethen stimulated with PAF (100 nM) or fMLF (100 nM) and therelease of superoxidewas recorded continuously Effects of wort-mannin expressed in of superoxide production (peak valuescompared) obtained without any additive (mean plusmn SEM 119899 = 3)

and that the mechanism for desensitization differ betweenthe two receptors A physical linkageassociation to the


actin cytoskeleton of an agonist-occupied receptor is animportant mechanism for the termination of the superoxideanion production in neutrophils It has been suggested thatbinding to the cytoskeleton of the ligand-receptor complexlaterally separates the signaling receptor from the G-proteina physical separation that terminates the signaling fromthe occupied receptor [34] The fact that the fMLF as wellas the PAF-induced responses were not only augmentedbut also prolongated in the presence Cyt B suggests thatan association to the cytoskeleton constitutes an importantmechanism for termination of both the FPR1 and the PAFRtriggered responses The responses are however terminatedalso in the presence of drugs that disrupt the cytoskeletonsuggesting that there are also other (nondefined) ways toterminate signaling For FPR1 binding of the occupiedreceptors to the cytoskeleton is also an important mechanismfor desensitization of receptors and as a consequence adisruption of the actin cytoskeleton reactivates the desensi-tized FPR1 [48] We now show that desensitized PAFRs arenot reactivated when the cytoskeleton is disrupted meaningthat the desensitization process is differently regulated forPAFR and FPR1 A functional uncoupling from G proteinsand phosphorylation of PAFR induced by the activatedreceptor itself could be part of the termination processAlternatively rapid internalization anddownregulation of thetotal number of PAFRs in neutrophils by PAF stimulationmay form another part of the desensitization process [5152] An immediate consequence of FPR1 and PAFR acti-vation is the production of inositol 145-triphosphate anda subsequent transient elevation of intracellular Ca2+ Thelipid remodeling is mediated by phosphoinositide kinasesphospholipase (PL)D PLA

2 and a phosphoinositide-specific

PLC The Ca2+ transient is however as mentioned notlinked to the generation of an NADPH-oxidase activatingsignal [48] This notion is further supported by the factthat although PAF is as potent as fMLF in inducing Ca2+resposne it ismuchweaker than fMLF in triggeringNADPH-oxidase The ligation of FPR1 and PAFR activates multiplesignal cascades and signaling through receptors for endtarget chemoattractants such as fMLF has been suggestedto involve p38 MAPKs whereas the receptors recognizinginetermediary type chemoattractants such as IL-8 utilizethe PI3 KAkt pathway during neutrophil migration [42]Determining the inhibitory profiles of p38 MAPK and PI3Kinhibitors on neutrophil oxidative response we found thatactivation through end-type receptors as well as intermediatetype receptors involve both p38 MAPK and PI3K [21] Stim-ulation with PAF as well as with fMLF result in equivalentphosphorylation and activation of p38 MAPK however asignificant difference between FPR1fMLF and PAFRPAFhas been describedwith respect to p4244 (ERK)MAPK acti-vation [53] In this study we observed a signaling differencebetween FPR1 and PAFR with respect to the PI3 K pathwaythat is FPR1-mediated ROS production is more sensitiveto the PI3 K inhibitor wortmannin than the PAF-mediatedresponse In summary the data presented provide evidencethat PAF can modulate neutrophil functions and directlypromote the production of superoxide anion in addition to

the many other known effects described for PAF for exam-ple priming secretion and receptor mobilization Thesefindings not only point out a possibility that PAF-mediatedpathology may involve these yet unappreciated moleculesreleased by a direct PAF stimulation but they also stronglydemonstrate that unique signaling pathways are utilizeddownstream of PAFR leading to priming effect and agonistdriven desensitization In addition we show that there arefundamental differences between FPR1FPR2 and PAFR withrespect to their utilization of different mechanisms leading tooxidase activationdeactivation and the difference betweenthe receptors in their subcellular localization is possiblyreflected in the signaling differences between FPR1FPR2 andPAFR similar to that earlier described between FPR1FPR2and CXCRs The precise signaling pathways involved inpriming and desensitizationreactivation of the PAFR as wellas the missing direct link between signaling leading to a risein cytosolic Ca2+ mobilization of granules and activationof the oxidase in the fMLFPAF response have to be furtherinvestigated

Acknowledgments

H Forsman was supported by Swedish Medical ResearchCouncil This work was supported by the Swedish MedicalResearch Council the King Gustaf V 80-Year Foundationand the Swedish state under the ALF agreement The authorsthank for the suggestions given from the Phagocyte ResearchGroup

References

[1] B McDonald K Pittman G B Menezes et al ldquoIntravasculardanger signals guide neutrophils to sites of sterile inflamma-tionrdquo Science vol 330 no 6002 pp 362ndash366 2010

[2] G B Segel M W Halterman and M A Lichtman ldquoTheparadox of the neutrophilrsquos role in tissue injuryrdquo Journal ofLeukocyte Biology vol 89 no 3 pp 359ndash372 2011

[3] A FMiller and J J Falke ldquoChemotaxis receptors and signalingrdquoAdvances in Protein Chemistry vol 68 pp 393ndash444 2004

[4] R D Ye F Boulay M W Ji et al ldquoInternational union ofbasic and clinical pharmacology LXXIII Nomenclature forthe formyl peptide receptor (FPR) familyrdquo PharmacologicalReviews vol 61 no 2 pp 119ndash161 2009

[5] L Stephens LMilne and P Hawkins ldquoMoving towards a betterunderstanding of chemotaxisrdquo Current Biology vol 18 no 11pp R485ndashR494 2008

[6] W S Powell and J Rokach ldquoBiochemistry biology and chem-istry of the 5-lipoxygenase product 5-oxo-ETErdquo Progress inLipid Research vol 44 no 2-3 pp 154ndash183 2005

[7] M P Wymann S Sozzani F Altruda A Mantovani and EHirsch ldquoLipids on the move phosphoinositide 3-kinases inleukocyte functionrdquo Immunology Today vol 21 no 6 pp 260ndash264 2000

[8] G A Zimmerman T M McIntyre S M Prescott and D MStafforini ldquoThe platelet-activating factor signaling system andits regulators in syndromes of inflammation and thrombosisrdquoCritical Care Medicine vol 30 supplement 5 pp S294ndashS3012002


[9] S M Prescott G A Zimmerman D M Stafforini andT M McIntyre ldquoPlatelet-activating factor and related lipidmediatorsrdquoAnnual Review of Biochemistry vol 69 pp 419ndash4452000

[10] R D Ye and F Boulay ldquoStructure and function of leukocytechemoattractant receptorsrdquo Advances in Pharmacology C vol39 pp 221ndash289 1997

[11] D J Dupre C Thompson Z Chen et al ldquoInverse agonist-induced signaling and down-regulation of the platelet-activating factor receptorrdquo Cellular Signalling vol 19 no 10 pp2068ndash2079 2007

[12] L M McManus and R N Pinckard ldquoPAF a putative mediatorof oral inflammationrdquo Critical Reviews in Oral Biology andMedicine vol 11 no 2 pp 240ndash258 2000

[13] E J Welch R P Naikawadi Z Li et al ldquoOpposing effectsof platelet-activating factor and lyso-platelet-activating factoron neutrophil and platelet activationrdquoMolecular Pharmacologyvol 75 no 1 pp 227ndash234 2009

[14] T M Osborn C Dahlgren J H Hartwig and T P StosselldquoModifications of cellular responses to lysophosphatidic acidand platelet-activating factor by plasma gelsolinrdquo AmericanJournal of Physiology vol 292 no 4 pp C1323ndashC1330 2007

[15] F R Sheppard M R Kelher E E Moore N J D McLaughlinA Banerjee and C C Silliman ldquoStructural organization ofthe neutrophil NADPH oxidase phosphorylation and translo-cation during priming and activationrdquo Journal of LeukocyteBiology vol 78 no 5 pp 1025ndash1042 2005

[16] A M Condliffe E Kitchen and E R Chilvers ldquoNeutrophilpriming pathophysiological consequences and underlyingmechanismsrdquo Clinical Science vol 94 no 5 pp 461ndash471 1998

[17] J Almkvist J Faldt C Dahlgren H Leffler and A Karls-son ldquoLipopolysaccharide-induced gelatinase granule mobi-lization primes neutrophils for activation by galectin-3 andformylmethionyl-Leu-Pherdquo Infection and Immunity vol 69 no2 pp 832ndash837 2001

[18] J Bylund A Karlsson F Boulay and C DahlgrenldquoLipopolysaccharide-induced granule mobilization andpriming of the neutrophil response to Helicobacter pyloripeptide Hp(2-20) which activates formyl peptide receptor-like1rdquo Infection and Immunity vol 70 no 6 pp 2908ndash2914 2002

[19] J Almkvist CDahlgrenH Leffler andAKarlsson ldquoNewcastledisease virus neuraminidase primes neutrophils for stimula-tion by galectin-3 and formyl-Met-Leu-Pherdquo Experimental CellResearch vol 298 no 1 pp 74ndash82 2004

[20] A Karlsson P Follin H Leffler and C Dahlgren ldquoGalectin-3activates the NADPH-oxidase in exudated but not peripheralblood neutrophilsrdquo Blood vol 91 no 9 pp 3430ndash3438 1998

[21] H Fu J Bylund A Karlsson S Pellme and C Dahlgren ldquoThemechanism for activation of the neutrophil NADPH-oxidaseby the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8rdquo Immunology vol 112no 2 pp 201ndash210 2004

[22] M Faurschou and N Borregaard ldquoNeutrophil granules andsecretory vesicles in inflammationrdquoMicrobes and Infection vol5 no 14 pp 1317ndash1327 2003

[23] A Boyum D Lovhaug L Tresland and E M Nordlie ldquoSepa-ration of leucocytes improved cell purity by fine adjustments ofgradientmedium density and osmolalityrdquo Scandinavian Journalof Immunology vol 34 no 6 pp 697ndash712 1991

[24] H Lundqvist and C Dahlgren ldquoIsoluminol-enhanced chemi-luminescence a sensitive method to study the release of super-oxide anion from human neutrophilsrdquo Free Radical Biology andMedicine vol 20 no 6 pp 785ndash792 1996

[25] C Dahlgren and A Karlsson ldquoRespiratory burst in humanneutrophilsrdquo Journal of Immunological Methods vol 232 no 1-2 pp 3ndash14 1999

[26] L Kjeldsen H Sengeloslashv and N Borregaard ldquoSubcellular frac-tionation of human neutrophils on Percoll density gradientsrdquoJournal of Immunological Methods vol 232 no 1-2 pp 131ndash1431999

[27] J Karlsson J Bylund C Movitz L Bjorkman H Forsmanand C Dahlgren ldquoA methodological approach to studies ofdesensitization of the formyl peptide receptor role of the readout system reactive oxygen species and the specific agonist usedto trigger neutrophilsrdquo Journal of Immunological Methods vol352 no 1-2 pp 45ndash53 2010

[28] T Andersson C Dahigren P D Lew and O Stendahl ldquoCellsurface expression of fMet-Leu-Phe receptors on human neu-trophils Correlation to changes in the cytosolic free Ca2+ leveland action of phorbol myristate acetaterdquo Journal of ClinicalInvestigation vol 79 no 4 pp 1226ndash1233 1987

[29] H Sengelov F Boulay L Kjeldsen and N Borregaard ldquoSub-cellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophilsrdquoBiochemical Journal vol 299 no 2 pp 473ndash479 1994

[30] N Borregaard O E Soslashrensen and K Theilgaard-MonchldquoNeutrophil granules a library of innate immunity proteinsrdquoTrends in Immunology vol 28 no 8 pp 340ndash345 2007

[31] B Dewald and M Baggiolini ldquoActivation of NADPH oxidasein human neutrophils Synergism between fMLP and the neu-trophil products PAF and LTB4rdquo Biochemical and BiophysicalResearch Communications vol 128 no 1 pp 297ndash304 1985

[32] I Daniels M A Lindsay C I C Keany R P Burden JFletcher and A P Haynes ldquoRole of arachidonic acid andits metabolites in the priming of NADPH oxidase in humanpolymorphonuclear leukocytes by peritoneal dialysis effluentrdquoClinical andDiagnostic Laboratory Immunology vol 5 no 5 pp683ndash689 1998

[33] B Dewald and M Baggiolini ldquoPlatelet-activating factor as astimulus of exocytosis in human neutrophilsrdquo Biochimica etBiophysica Acta vol 888 no 1 pp 42ndash48 1986

[34] K N Klotz and A J Jesaitis ldquoNeutrophil chemoattractantreceptors and the membrane skeletonrdquo BioEssays vol 16 no 3pp 193ndash198 1994

[35] L Liu O Harbecke H Elwing P Follin A Karlsson andC Dahlgren ldquoDesensitization of formyl peptide receptors isabolished in calcium ionophore-primed neutrophils an asso-ciation of the ligand-receptor complex to the cytoskeleton isnot required for a rapid termination of the NADPH- oxidaseresponserdquo Journal of Immunology vol 160 no 5 pp 2463ndash24681998

[36] OHarbecke L LiuAKarlsson andCDahlgren ldquoDesensitiza-tion of the fMLP-induced NADPH-oxidase response in humanneutrophils is lacking in okadaic acid-treated cellsrdquo Journal ofLeukocyte Biology vol 61 no 6 pp 753ndash758 1997

[37] M J Berridge ldquoInositol trisphosphate and calcium signallingrdquoNature vol 361 no 6410 pp 315ndash325 1993

[38] G M Bokoch ldquoChemoattractant signaling and leukocyte acti-vationrdquo Blood vol 86 no 5 pp 1649ndash1660 1995

[39] J KarlssonA StenfeldtMRabiet J BylundH F Forsman andC Dahlgren ldquoThe FPR2-specific ligand MMK-1 activates the


neutrophil NADPH-oxidase but triggers no unique pathwayfor opening of plasma membrane calcium channelsrdquo CellCalcium vol 45 no 5 pp 431ndash438 2009

[40] T Andersson C Dahlgren and T Pozzan ldquoCharacterization offMet-Leu-Phe receptor-mediated Ca2+ influx across the plasmamembrane of human neutrophilsrdquo Molecular Pharmacologyvol 30 no 5 pp 437ndash443 1986

[41] H Fu J Karlsson J Bylund C Movitz A Karlsson and CDahlgren ldquoLigand recognition and activation of formyl peptidereceptors in neutrophilsrdquo Journal of Leukocyte Biology vol 79no 2 pp 247ndash256 2006

[42] B Heit S Tavener E Raharjo and P Kubes ldquoAn intracellularsignaling hierarchy determines direction ofmigration in oppos-ing chemotactic gradientsrdquo Journal of Cell Biology vol 159 no1 pp 91ndash102 2002

[43] H Forsman K Onnheim E Andreasson et al ldquoReactivationof desensitized formyl peptide receptors by platelet activatingfactor a novel receptor cross talk mechanism regulating neu-trophil superoxide anion productionrdquo PLoS One vol 8 ArticleID e60169 2013

[44] K Onnheim J Bylund F Boulay CDahlgren andH ForsmanldquoTumour necrosis factor (TNF)-120572 primes murine neutrophilswhen triggered via formyl peptide receptor-related sequence2 the murine orthologue of human formyl peptide receptor-like 1 through a process involving the type I TNF receptor andsubcellular granule mobilizationrdquo Immunology vol 125 no 4pp 591ndash600 2008

[45] C Elbim S Bailly S Chollet-Martin J Hakim and M AGougerot-Pocidalo ldquoDifferential priming effects of proinflam-matory cytokines on human neutrophil oxidative burst inresponse to bacterial N-formyl peptidesrdquo Infection and Immu-nity vol 62 no 6 pp 2195ndash2201 1994

[46] J El-Benna P M Dang and M Gougerot-Pocidalo ldquoPrimingof the neutrophil NADPH oxidase activation role of p47phoxphosphorylation and NOX2 mobilization to the plasma mem-branerdquo Seminars in Immunopathology vol 30 no 3 pp 279ndash289 2008

[47] N J McLaughlin A Banerjee S Y Khan et al ldquoPlatelet-activating factor-mediated endosome formation causes mem-brane translocation of p67phox and p40phox that requiresrecruitment and activation of p38 MAPK Rab5a and phos-phatidylinositol 3-kinase in human neutrophilsrdquo Journal ofImmunology vol 180 no 12 pp 8192ndash8203 2008

[48] J Bylund A Bjorstad DGranfeldt A Karlsson CWoschnaggand C Dahlgren ldquoReactivation of formyl peptide receptorstriggers the neutrophil NADPH-oxidase but not a transient risein intracellular calciumrdquo Journal of Biological Chemistry vol278 no 33 pp 30578ndash30586 2003

[49] J Bylund K L Brown C Movitz C Dahlgren and A Karls-son ldquoIntracellular generation of superoxide by the phagocyteNADPH oxidase how where and what forrdquo Free RadicalBiology and Medicine vol 49 no 12 pp 1834ndash1845 2010

[50] C Dahlgren A Karlsson and J Bylund ldquoMeasurement of res-piratory burst products generated by professional phagocytesrdquoMethods in Molecular Biology vol 412 pp 349ndash363 2007

[51] W Zhou and M S Olson ldquoA mechanism for phorbol ester-mediated regulation of the PAF receptor in human neutrophilsrdquoArchives of Biochemistry and Biophysics vol 313 no 1 pp 179ndash183 1994

[52] D J Dupre Z Chen C Le Gouill et al ldquoTrafficking ubiqui-tination and down-regulation of the human platelet-activating

factor receptorrdquo Journal of Biological Chemistry vol 278 no 48pp 48228ndash48235 2003

[53] J A Nick N J Avdi S K Young et al ldquoCommon and distinctintracellular signaling pathways in human neutrophils utilizedby platelet activating factor and FMLPrdquo Journal of ClinicalInvestigation vol 99 no 5 pp 975ndash986 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


generation of PAF and other lipid inflammatory mediatorssuch as LTB4 suggesting a role for this receptorligand pairnot only in host defence but also inmodulating inflammatoryresponses Similar to most neutrophil GPCRs occupationof the PAFR promotes secretion of granule constituents andmobilization of cell surface receptors cellular migration [12]and priming of the cells to generate increasing amounts ofROS [13] PAF has been shown by others to directly activatethe electron transporting system (the NADPH oxidase) thatgenerates ROS in eosinophils and in our hands PAF alsodirectly activates this system in neutrophils [14]

Some chemoattractants are rather poor ROS inducers inneutrophils but the response induced can be dramaticallyincreased (primed) by nonactivating agents such as tumornecrosis factor alpha (TNF-120572) and lipopolysaccharides (LPS)derived from Gram-negative bacteria [15 16] The prim-ing phenomenon has been extensively investigated and itsbiological significance in many disease processes has beenwell documented With respect to priming of neutrophils inresponse to endogenous lectins of the galectin family andreceptor specific agonists for FPR1 and FPR2 we and othershave suggested granule mobilization resulting in surfacereceptor upregulation as a key event [17 18] Accordinglyin resting neutrophils the mobilizabled FPRs and receptorsfor galectins are found primarily in secretory organelles thatfuse with the plasmamembrane in response to different typesof secretagogues [19ndash22] The precise subcellular localizationof PAFRs in human neutrophils has however not yet beenexamined

In the present study we determined the subcellularlocalization of the PAFR in resting neutrophils and ourstudies demonstrate that PAFRs are localized in a lightmembrane fraction containing plasmamembranes and secre-tory vesicles Mobilization data show that the cells lack aneasily mobilizable PAFR pool suggesting that the receptoris present primarily in the plasma membrane Still the PAF-induced ROS production in neutrophils could be enhancedby TNF-120572 and cytoskeleton disrupting agents suggesting thatour proposed mechanism for priming does not apply forthe PAFPAFR receptor-ligand pair The PAFR shared manysignalling properties and basic functional characteristics withthe FPRs but there were also many quantitative as well asqualitative differences possibly linked to the difference insubcellular localization between the two receptors

2 Materials and Methods

21 Chemicals The hexapeptide WKYMVM was synthe-sized and HPLC purified by KJ Ross-Petersen (Holte Den-mark) The formylated tripeptide formyl-methionyl-leucyl-phenylalanine (fMLF) isoluminol cytochalasin B (CytB)pertussis toxin (PTX) TNF120572 latrunculin A and phorbolmyristate acetate (PMA) were obtained from Sigma (SigmaChemical Co St Louis MO USA) Peptides were dissolvedin DMSO and stored at minus70∘C until use Subsequent dilutionsof all peptides were made in Krebs-Ringer phosphate buffer(KRG pH 73 120mM NaCl 5mM KCl 17mM KH

2PO4

83mM NaH2PO4 and 10mM glucose) supplemented with

Ca2+ (1mM) and Mg2+ (15mM) The PAFR antagonistWEB2086 was from Tocris bioscience (Bristol UK) andPAF was from Avanti Polar Lipids (Avanti Polar Lipids IncAlabama) Ficoll-Paque was obtained from Amersham Phar-macia Biotech AB (Uppsala Sweden) Horseradish peroxi-dase (HRP) was obtained from Boehringer Mannheim (Ger-many) Kinase inhibitors were obrained from Calbiochem(Darmstadt Germany) FURA-2 was fromMolecular Probes(Eugene OR USA) The MMP9 ELISA was from RampDsystems the ELISA-kit for NGAL was from Bioporto Diag-nostics Antibodies against CR3 and L-selectinwere fromBDBiosciences (Franklin Lakes NJ USA)

22 Isolation of Human Neutrophils Human peripheralbloodneutrophils were isolated frombuffy coats fromhealthyblood donors using dextran sedimentation and Ficoll-Paquegradient centrifugation as described [23] The remainingerythrocytes were hypotonic lysed and the neutrophils werewashed twice and resuspended inKRG and stored onmeltingice until useThis isolation process permits cells to be purifiedwith minimal granule mobilization

23 Neutrophil NADPH-Oxidase Activity The NADPH-oxidase activity was determined using isoluminol-enhancedchemiluminescence (CL) [24 25] The CL activity was mea-sured in a six-channel Biolumat LB 9505 (BertholdCoWild-bad Germany) using disposable 4mL polypropylene tubeswith a 900-microliter reaction mixture containing 105 cellsisoluminol (2 times 10minus5M) and HRP (2U) Intracellular ROSwas measured by luminol (cell permeable) in the presenceof superoxide dismutase and catalase [25] The tubes wereequilibrated in the Biolumat for 5min at 37∘C after whichthe stimulus (100120583L) was added and the light emission wasrecorded continuously By a direct comparison of the super-oxide dismutase-inhibitable reduction of cytochrome c andsuperoxide dismutase-inhibitable CL 72 times 107 counts werefound to correspond to the production of 1 nmol superoxide(using a millimolar extinction coefficient for cytochrome cof 211) When experiments were performed with primingand antagonistsinhibitors these substances were included inthe CL reaction mixture for different time period (controlcells received no treatment but were incubated at the samecondition) before stimulation with agonists

24 Subcellular Fractionation and Marker Analysis Subcel-lular fractionation was performed as described [26] Brieflyneutrophils were treated with the serine protease inhibitordiisopropyl fluorophosphates (DFP 8 120583M) disintegrated bynitrogen cavitation (Parr Instruments Co Moline IL) andthe postnuclear supernatant was fractionated on two- (105and 112 gL) or three-layer (112 109 and 105 gL) Percollgradients and centrifuged at 15000 g for 45minutes in a fixed-angle JA-20Beckman rotor Fractions of 15mLwere collectedby aspiration from the bottom of the centrifuge tube Thelocalization of subcellular granules was determined by gran-ule marker analysis Alkaline phosphatase (marker for secre-tory vesiclesplasma membrane) was measured by hydrolysisof p-nitrophenyl phosphate at pH 105 in a sodium barbital






2O2-






3 Results





Gelatinase

120574

0 5 10 15 20 25 30

3

0

1

2

3

10 11 12 1413 15 16 17 18 19 20

3 10 11 12 14 15 20 21 22 29

120572 1205731 1205731

120574120572 1205731

1205732

48kDaPAFR

Fraction number

Am

ount

of m

arke

r (au

)97kDa




50of asymp 800 nM


value






101 102 103 104 105 106 107

PAFR

Num

ber o

f cel

ls

Ice

+TNF120572

MFI

0

2000

4000

37∘C

Ice +TNF12057237∘C

(a)

101 102 103 104 105 106 107

CR3

Num

ber o

f cel

ls

MFI

0

500

1000

Ice

+TNF12057237∘C

Ice +TNF12057237∘C

(b)


0

50

100

150

001 01 1

001 01

O2minus

prod

uctio

n (M

cpm

)

1

fMLF (120583M)

00

05

10

15

Nor

mal

ized

resp

onse

EC50 sim 20nM

fMLF (120583M)

(a)

0

20

40

60

80

01 1 10

O2minus

prod

uctio

n (M

cpm

)

00

05

10

15

Nor

mal

ized

resp

onse

01 1 10

EC50 sim 870nM

PAF (120583M)

PAF (120583M)

(b)







0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)


Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)








8


6

4

2

0fMLF PAF

Fold

incr

ease



4 Discussion



0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2O2-






3 Results





Gelatinase

120574

0 5 10 15 20 25 30

3

0

1

2

3

10 11 12 1413 15 16 17 18 19 20

3 10 11 12 14 15 20 21 22 29

120572 1205731 1205731

120574120572 1205731

1205732

48kDaPAFR

Fraction number

Am

ount

of m

arke

r (au

)97kDa




50of asymp 800 nM


value






101 102 103 104 105 106 107

PAFR

Num

ber o

f cel

ls

Ice

+TNF120572

MFI

0

2000

4000

37∘C

Ice +TNF12057237∘C

(a)

101 102 103 104 105 106 107

CR3

Num

ber o

f cel

ls

MFI

0

500

1000

Ice

+TNF12057237∘C

Ice +TNF12057237∘C

(b)


0

50

100

150

001 01 1

001 01

O2minus

prod

uctio

n (M

cpm

)

1

fMLF (120583M)

00

05

10

15

Nor

mal

ized

resp

onse

EC50 sim 20nM

fMLF (120583M)

(a)

0

20

40

60

80

01 1 10

O2minus

prod

uctio

n (M

cpm

)

00

05

10

15

Nor

mal

ized

resp

onse

01 1 10

EC50 sim 870nM

PAF (120583M)

PAF (120583M)

(b)







0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)


Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)








8


6

4

2

0fMLF PAF

Fold

incr

ease



4 Discussion



0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Gelatinase

120574

0 5 10 15 20 25 30

3

0

1

2

3

10 11 12 1413 15 16 17 18 19 20

3 10 11 12 14 15 20 21 22 29

120572 1205731 1205731

120574120572 1205731

1205732

48kDaPAFR

Fraction number

Am

ount

of m

arke

r (au

)97kDa




50of asymp 800 nM


value






101 102 103 104 105 106 107

PAFR

Num

ber o

f cel

ls

Ice

+TNF120572

MFI

0

2000

4000

37∘C

Ice +TNF12057237∘C

(a)

101 102 103 104 105 106 107

CR3

Num

ber o

f cel

ls

MFI

0

500

1000

Ice

+TNF12057237∘C

Ice +TNF12057237∘C

(b)


0

50

100

150

001 01 1

001 01

O2minus

prod

uctio

n (M

cpm

)

1

fMLF (120583M)

00

05

10

15

Nor

mal

ized

resp

onse

EC50 sim 20nM

fMLF (120583M)

(a)

0

20

40

60

80

01 1 10

O2minus

prod

uctio

n (M

cpm

)

00

05

10

15

Nor

mal

ized

resp

onse

01 1 10

EC50 sim 870nM

PAF (120583M)

PAF (120583M)

(b)







0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)


Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)








8


6

4

2

0fMLF PAF

Fold

incr

ease



4 Discussion



0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















101 102 103 104 105 106 107

PAFR

Num

ber o

f cel

ls

Ice

+TNF120572

MFI

0

2000

4000

37∘C

Ice +TNF12057237∘C

(a)

101 102 103 104 105 106 107

CR3

Num

ber o

f cel

ls

MFI

0

500

1000

Ice

+TNF12057237∘C

Ice +TNF12057237∘C

(b)


0

50

100

150

001 01 1

001 01

O2minus

prod

uctio

n (M

cpm

)

1

fMLF (120583M)

00

05

10

15

Nor

mal

ized

resp

onse

EC50 sim 20nM

fMLF (120583M)

(a)

0

20

40

60

80

01 1 10

O2minus

prod

uctio

n (M

cpm

)

00

05

10

15

Nor

mal

ized

resp

onse

01 1 10

EC50 sim 870nM

PAF (120583M)

PAF (120583M)

(b)







0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)


Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)








8


6

4

2

0fMLF PAF

Fold

incr

ease



4 Discussion



0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 300

05

10

Time (min)

Nor

mal

ized

resp

onse

0 1 2 30

5

10

15

Time (min)

Time to peak (sec)


Control+ WEB 2086

O2minus

prod

uctio

n (M

cpm

)








8


6

4

2

0fMLF PAF

Fold

incr

ease



4 Discussion



0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

0 2 4Time (min)

fMLFPAF

CytB

O2minus

prod

uctio

n (M

cpm

)






0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus2 minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)

0 1 2

Ca2+

resp

onse

indu

ced

by P

AF

Log [PAF] (nM)

(a)

0 20 40 60 80 100 120Time (min)

01nM

1nM

100nM

minus1

0

1

2

[Ca2+

] i(r

atio

chan

ge)


Ca2+

resp

onse

indu

ced

by fM

LF

(b)





0

50

100

PAFfMLF

lowastlowastlowast


Inhi

bitio

n (

cont

rol)

lowastlowastlowast








Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Subcellular Localization of the Receptor for … · 2019. 7. 31. ·...

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Transcript of Research Article The Subcellular Localization of the Receptor for … · 2019. 7. 31. ·...