Intracellular platelet-activating factor regulateseicosanoid generation in guinea-pig resident peritonealmacrophages'Alastair G. Stewart & *Wayne A. Phillips
Department of Physiology, University of Melbourne, *Department of Medicine, Royal Melbourne Hospital,University of Melbourne, Parkville, Victoria 3052, Australia
1 The role of intracellular platelet-activating factor (Pat) in arachidonic -acid (AA) mobilizationfrom guinea-pig peritoneal macrophages has been investigated by use of the potent and selectivePaf receptor antagonists, WEB 2086 and CV 6209.2 Adherent macrophages contained cell-associated Paf which was increased by exposure toformyl-methionyl-leucyl-phenylalanine (fMLP), endotoxin and the ionophore, A23187. However,only endotoxin and A23187 caused release of detectable amounts of Paf into the extracellularmedium.3 Exogenous Paf and each of the above stimuli mobilized previously incorporated ["4C]-AA andincreased the generation of prostacyclin (PGI2) in resident macrophages.4 WEB 2086 (10-100pM) and CV 6209 (0.1-1OMm) reduced both basal and stimulated PGI2 gener-ation and WEB 2086 inhibited the mobilization of ['4C]-AA. In addition, WEB 2086 (10pM) inhib-ited fMLP- and Paf-induced superoxide anion generation. Responses to A23187 were not inhibitedby either antagonist.5 Activation of macrophages by fMLP caused a short burst of intracellular Paf generation butnone was detected in the supernatants. The time-course of PGI2 synthesis followed closely that ofPaf.6 These data suggest that intracellular Paf generation is important for subsequent AA mobi-lization and may have a wider role in signal transduction processes.
Introduction
Evidence for an important role for platelet-activatingfactor (Pat) in a number of models of allergy, shockand inflammation has accumulated rapidly followingthe availability of potent and specific receptorantagonists (see Braquet et al., 1987 for a review).These include endotoxin shock, pulmonary anaphy-laxis, cardiac anaphylaxis, thrombosis and allergicand non-allergic inflammation. The participation ofcells capable of synthesizing and releasing Paf iscommon to these disorders and includes macro-phages (Camussi et al., 1981; Roubin et al., 1986),polymorphonuclear leukocytes (PMNLs, Camussi etal., 1981; Mueller et al., 1983; Jouvin-Marche et al.,1984) and platelets (Chap et al., 1981). However,
'Author for correspondence.
macrophages and PMNLs also retain a high propor-tion of the synthesized Paf within the cell and thefunction of this intracellular Paf has not been estab-lished (Harris et al., 1985; Henson, 1987).We have recently shown that guinea-pig resident
peritoneal macrophages possess a Paf receptorlinked to prostacyclin generation which has lowerapparent affinity for the Paf antagonists BN 52021,L-652,731 and WEB 2086 (Stewart & Dusting, 1988)than do the Paf receptors mediating platelet andPMNL aggregation. It is likely that the Paf recep-tors on macrophages are a distinct subclass.However, it is also possible that the low apparentaffinities were due to decreased access of the antago-nists to a receptor located intracellularly. The possi-bility of an intracellular site of action is also
supported by our recent observation that Paf recep-tor antagonists inhibit bradykinin-induced prostacy-clin (PGI2) generation by cultured endothelial cells(Stewart et al., 1989).
In this study we have investigated the role of exo-genous and endogenous Paf in stimulating themetabolism of arachidonic acid in guinea-pig perito-neal macrophages using the potent, specific andstructurally distinct Paf antagonists, WEB 2086(Casals-Stenzel et al., 1987) and CV 6209 (Terashitaet al., 1987).
Methods
Macrophage cultures
Macrophages were obtained from healthy maleguinea-pigs (500-900 g) by peritoneal lavage (Stewart& Dusting, 1988). Following isolation by centrifu-gation (I000g, 40C, 10min), cells were resuspended inRPMI 1640 (20% foetal calf serum, FCS, 15mmHEPES, pH 7.40, penicillin 100uml-', streptomy-cin, 100lugml-') at a concentration of 1-2 x 106cells ml-', dispensed (0.5 ml) into Linbro 24 wellplastic culture plates (6 well plates were used for theexperiments described in Table 2) and allowed toadhere for 2h. In experiments involving the releaseof ["4C]-arachidonic acid (['4C]-AA), the macro-phage adherence was carried out in RPMI 1640(20% FCS) containing 0.2 pCi ml-1 [4C]-AA(58mCimmol-1) and indomethacin 2.8pM. At theend of the adherence period the non-adherent cellswere removed and the remaining cells (80-95%) werewashed once with RPMI 1640 (containing 0.25%bovine serum albumin, BSA in place of FCS) andpre-incubated for 15 min in RPMI 1640 (0.25% BSA)containing either the vehicle (culture medium), WEB2086 (10-100pM) or CV 6209 (0.1-1OpM). The cellswere exposed to a range of known stimuli for arachi-donic acid metabolism for 60 min periods(corresponding to the maximum release for allstimuli) or as indicated. At the conclusion of theincubation period, the supernatants were harvestedand cells were subjected to ethanol extraction asdescribed below.
Radio-immunoassay of6-oxo-PGFI.PGI2 generation by adherent macrophages wasassessed by radio-immunoassay (RIA) of itschemically-stable breakdown product, 6-oxo-PGFI.according to the method of Salmon (1978). None ofthe agents used in this study cross-reacted with the6-oxo-PGFm antiserum or altered the sensitivity ofthe assay which was carried out in RPMI 1640(0.25% BSA). One hundred microlitres of the 0.5ml
of the supernatant was used immediately in the RIAof 6-oxo-PGFl,, the remainder was stored at- 20°C for bioassay of Paf.
Paf bioassay
Paf was bioassayed on rabbit washed platelets(Vargas et al., 1982) suspended in Tyrode solutioncontaining indomethacin 2.8pM (Parente & Flower,1985). Criteria for identification included: ability toinduce an indomethacin-resistant platelet aggre-gation; inhibition of bioactivity by the selective Pafreceptor antagonist WEB 2086 (1 pM); co-elution ofpooled and re-extracted samples with authentic[3H]-Paf on thin layer chromatography (t.l.c.),mobile phase 65:35:6 chloroform: methanol: water.Supernatants (up to 50 pl) were assayed withoutprior extraction whereas cells were extracted into80% ethanol. Following removal of the precipitateby centrifugation, the extract was evaporated todryness under reduced pressure and reconstituted in250 p1 of Tyrode solution (0.25% BSA). The recoveryfrom this extraction was 94 + 3%, n = 53. The limitof detection of the assay was 6fmol. The maximumvolume used was 501. However, the effective detec-tion limit varied with the cell number used. Thus, inexperiments reported in Table 2, 6 well plates wereused (containing 3-4 x 106 cells per well) giving alimit of detection of 0.01 pmol per 106 cells. In time-course experiments, 24 well plates were used and thelimit of detection was 0.03 pmol per 106 cells.
Samples from different cultures were pooled, sub-jected to a Bligh-Dyer extraction and chromato-graphed on t.l.c. (Whatman LK SD) as describedabove. One centimetre segments of the scraped silicawere extracted into Tyrode solution (BSA 0.25%)and bioassayed on washed rabbit platelets.
Reverse-phase h.p.l.c. of [1"C]-arachidonic acidmetabolites
At the end of the 60 min incubation period, super-natants from macrophages pre-labelled with [14C]-AA were harvested and immediately frozen andstored at - 20°C until chromatography. The super-natant pH was adjusted to 5.6, and brought to 65%methanol, centrifuged to remove the precipitate andfiltered twice (0.22pm millipore filter) before injec-tion onto the rp h.p.l.c. column. Prior to samplepreparation, approximately 4000 d.p.m. of [3H]-leukotrienes B4 and C4 and [3H]-5-hydroxy-eicosatetraenoic acid (5-HETE) were added toindicate the respective retention times. The h.p.l.c.system consisted of a radial compression modulewith a novapak C18 column and a mobile phase ofmethanol: H20:acetic acid, 65:35:0.1, pH 5.6,1 ml min' for 17 min followed by a 1 min linear gra-
INTRACELLULAR Paf AND EICOSANOID BIOSYNTHESIS 143
10-a)( 8Moa)
< E 48t3°' 6- n l100
00<0I 60
40
~OS 20
Basal Paf Endotoxin fMLP A23187
Figure 1 The effects of WEB 2086 (10-100M) on basalrelease of pre-incorporated ['4C]-arachidonic acid(['4C]-AA) and that stimulated by Paf (0.lpM) endo-toxin (lOOpgmIP'), fMLP (1pM) and A23187 (10pM).The upper panel indicates the absolute release of ["4C]-AA in non-pretreated macrophages during a 60 minincubation period. The lower panel represents therelease of ["C]-AA in the presence of WEB 2086expressed as a percentage of that in non-pretreated(control) macrophages (n = 11): WEB 2086 10pM opencolumns; 100pM closed columns. * P < 0.05, pairedStudent's t test, compared to untreated cells.
0
80o 10c ,
0 4
o~
x0 L
Basal Paf Endotoxin fMLP A23187
Figure 2 The effects of WEB 2086 (10-100pM) on thegeneration of 6-oxo-PGF,. in unstimulated (basal)macrophages and in those activated by Paf (0.1 gM),endotoxin (lOOpgml-'), fMLP (1 pM) and A23187(1OyM). The upper panel indicates the absolute gener-ation of 6-oxo-PGF1, in non-pretreated macrophagesduring a 60min period. The lower panel represents thegeneration of 6-oxo-PGF1. in WEB 2086-pretreatedmacrophages and the data are expressed as a percent-age of the generation by the corresponding non-
pretreated macrophages: WEB 2086 10pM opencolumns; 100pM closed columns. Data are presented as
the means with s.e.mean shown by vertical bar (n = 8).* P < 0.05, paired Student's t test.
dient to methanol:acetic acid, 100:0.1, for 20min.One ml fractions were collected, added to 4 mlEmulsifier-Safe Scintillant (Packard) and counted ina Packard 300C liquid scintillation spectrometerwith software for dual counting of 3H/14C.
Superoxide anion generation
Stimulus-induced generation of superoxide anion byadherent macrophages was assessed by cytochromeC reduction according to the method of Johnston etal. (1978). Briefly, macrophages were incubated in1 ml of Krebs-Ringer solution (composition, mM:NaCl 121, KCI 5, CaCl2 1.3, MgSO4 1.2, NaH2PO43.1, Na2HPO4 12.5, dextrose 11, pH 7.3) containingcytochrome C (80 gM) and were pretreated withWEB 2086 (10 gM) for 15 min. The cells were stimu-lated for a period of 90 min after which the super-natants were harvested and the absorbance read at550 nm. The amount of cytochrome C reduced wascalculated by the change in absorbance at 550 nmusing the differential molar extinction coefficient,21 x 103M- cm-'. The potential free radical scav-enging activity of WEB 208§ was examined witha hypoxanthine (100 gM)/xanthine oxidase(25muml-1) superoxide anion generating system inthe absence of cells.
Materials
All reagents and solvents used in this study were ofanalytical or higher grade. RPMI 1640 and FCSwere obtained from CSL (Australia) and FlowLaboratories, respectively. Chemicals were obtainedfrom the following sources: A23187; BSA, grade 5,essentially fatty acid-free; cytochrome C (horse hearttype III); dextran, clinical grade C; endotoxin E.Coli Serotype 011 B4; formyl-methiony-leucyl-phenylalanine (fMLP); hypoxanthine; indomethacin;superoxide dismutase (Sigma Chemical Co.); hexa-decyl platelet-activating factor (Paf, Bachem); pros-tacyclin (PGI2, Na salt, Wellcome); 6-oxo-PGF1,(Upjohn); 6-oxo-PGF1, antiserum (Dr J. Salmon,Wellcome Research Laboratories); 6-keto [5,8,9,11,12,14,15 (n)-3H] prostaglandin Fl., 170Cimmol-1;[1-'4C]-arachidonic acid, 58.3-59.6 rCi mmol-1(Amersham); [hexadecyl-1',2'-3H(N)]-2-acetyl-sn-glyceryl-3-phosphoryl-choline, 59.5 Ci mmol;-;[14,15-3H(N)]-leukotriene B4, 32 Ci mmol '; [14,15-3H(N)]-leukotriene C4, 39 Ci mmol-P'; [3H]-5-hydroxyeicosatetraenoic acid, 150 Ci mmolP;(New England Nuclear); HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid, (BDHchemicals); heparin (Fisons Pty. Ltd.), CV 6209 (2-[N-acetyl-N-(2-methoxy-3-octadecyl-carbamoyloxy-propoxycarbonyl)aminomethyl] - 1 - ethylpyridiniumchloride) (Takeda Chemical Industries Ltd.);WEB 2086 (3-(4-(2-chlorophenyl)-9-methyl-6H-
144 A.G. STEWART & W.A. PHILLIPS
1-=
OCD w T T
12OF
a_
CLL-
x
g 40CD-
L PafBasal Paf
Figure 3 The effects of CV 6209 (0.1-1OpM) on thegeneration of 6-oxo-PGF1, in unstimulated (basal)macrophages and in those activated by Paf (0.1 pM),endotoxin (lOO1gmlP1), fMLP (1 pM) and A23187(10pM). The upper panel indicates the absolute gener-
ation of 6-oxo-PGF1, in non-pretreated macrophagesduring a 60min period. The lower panel represents thegeneration of 6-oxo-PGF1. in CV 6209-pretreatedmacrophages and the data are expressed as a percent-age of the generation by the corresponding non-
pretreated macrophages: CV6209 0.1 pm open columns;1 yM hatched columns; 10pM closed columns. Data are
presented as the means with s.e.mean shown by verticalbars (n = 8). * P < 0.05, paired Student's t test.
thieno(3,2-f),(1,2,4)-triazolo,(4,3-a),(1,4)-diazepine-2-yl)-144-morpholinyl)-1-propanone), (BoehringerIngelheim, West Germany).
Results
[14C]-arachidonic acid release
Activation of adherent macrophages by all stimulicaused the release of pre-incorporated [14C]-AAinto the supernatant (Figure 1). However, there was
no detectable conversion of the released ["4C]-AA to5-lipoxygenase products (data not shown). The basalrelease of [14C]-AA and that induced by A23187were not affected by WEB 2086 pretreatment. Incontrast, WEB 2086 (10-100 pM) significantlyreduced (P < 0.05, paired t test) the release of ["C]-AA induced by Paf (0.1 pM), endotoxin (00 pgml- 1)and fMLP (1 M). The extent of the inhibition rangedfrom 80% for Paf to 30% for fMLP (Figure 1).
PGI2 generation
Each of the stimuli for ['4C]-AA release also elicitedan increase in the generation of PGI2 measured as
immunoreactive 6-oxo-PGF1, (Figures 2 and 3). Inmacrophages pretreated with WEB 2086 (10-100pM)
there was a similar pattern of inhibition for PGI2generation and for [14C]-AA release except that thebasal PGI2 generation, but not that of [14C]-AA,was markedly reduced by WEB 2086 (Figure 2). In aseparate series of experiments, the potent Paf recep-tor antagonist, CV 6209 (0.1-10pM) concentration-dependently inhibited basal and stimulated PGI2generation (Figure 3) except that induced by A23187.
Influence of culture medium
Approximately 80% of cells obtained by peritoneallavage adhered within the 2 h period. This adherencewas not dependent on the presence of foetal calfserum (FCS). The basal generation of PGI2 was con-siderably higher in cells which adhered in the pre-sence of FCS (Table 1). However, WEB 2086(100p#M) and CV 6209 (10pM) inhibited basal 6-oxo-PGF1i generation irrespective of the presence ofFCS in the culture medium.
Pafgeneration
Endotoxin and A23187 consistently stimulated thegeneration of Paf in macrophages, of which approx-imately 50% was released into the BSA-containingculture medium (Table 2). In unstimulated macro-phages and in those exposed to fMLP, detectablelevels of cell-associated Paf (>0.01 pmol per 106 cellsh-) were observed after 60min, but there was noconsistent release of Paf into the extracellularmedium. In pooled samples subjected to t.l.c., all theplatelet aggregating activity co-eluted with authenticPaf.
Temporal relationship between Pafand 6-oxo-PGFlggeneration
In fMLP (1 pM)-stimulated macrophages, cell-associated Paf was detectable after 6s (limit of detec-tion 0.03 pmol per 106 cells h- 1), reached amaximum at 2 min and returned towards controlwithin 20 min. An increase in 6-oxo-PGF1. wasobserved at 30s and levels peaked at 2-5 min afterfMLP (Figure 4). All the Paf synthesized by thefMLP-stimulated macrophages remained cell-associated and no Paf was detected in unstimulatedmacrophages during the 20min period.
Superoxide generation
Paf (0.1 pM) and fMLP (0.01 pM) stimulated gener-ation of superoxide anion during a 90min incu-bation (Table 3). WEB 2086 (10pM) completelyinhibited the response to Paf and reduced that tofMLP. However, this concentration of WEB 2086had no free radical scavenging activity since it didnot inhibit superoxide generation by the hypo-xanthine (100 pM)/xanthine oxidase (25 muml- 1)
OL.
INTRACELLULAR Paf AND EICOSANOID BIOSYNTHESIS 145
Table 1 The influence of the culture medium onthe inhibition of basal PGI2 generation by WEB2086 and CV 6209
Culture 6-oxo-PGF,,medium (pmol per 106 h- 1)
RPMI 1640'(20% FCS)RPMI 1640(no FCS)
WEB 2086 CV 6209100PM 1OpM(% inhibition)
2.74+0.89 96+2 94+2
0.81 +0.20 80+8 70+ 10
n = 4.1 Peritoneal cells from individual animals weredivided into 2 aliquots and adhered in the presenceor absence of FCS. After removal of the non-adherent cells by washing, incubations of eachgroup were carried out in RPMI 1640 (0.25%BSA).* P < 0.05, paired t test.
Table 2 Generation of Paf by macrophagesduring a 60 min incubation with endotoxin,formyl-methionyl-leucyl-phenylalanine (fMLP) andA23187
Stimulus
fMLP 1 MEndotoxin 100pgml-A23187 10pM
n = 8.ND = none detected.
Paf (pmol per 106 cells h-')Cell-associated Supernatant
Figure 4 Temporal relationship between Paf (opensymbols) and 6-oxo-PGF,. (closed symbols) generationin fMLP (1 Mm)-stimulated macrophages. The data are
presented as the means and s.e.mean are shown by ver-
tical bars where the latter exceed the size of the symbol(n = 4).
Table 3 Effects of WEB 2086 (10pM) on super-oxide anion generation by adherent macrophages
Superoxide anion WEB 2086 (10pM)Stimulus (nmol per 106 1.5 h - 1) (% inhibition)
Paf 0.1 M 2.43 + 0.77fMLP 0.01 gM 3.73 + 1.19
* P < 0.05, paired t test
system (control, 5.48; WEB 2086per ml h-').
106+7*28 + 5*
1OpM, 5.78 nmol
Discussion
It is well established that much of the Paf generatedby pro-inflammatory cells remains intracellular(Henson, 1987), but it is not known whether thisintracellular Paf has any functional consequences.Our observations suggest a role for Paf in signaltransduction. Peritoneal macrophages from healthyguinea-pigs mobilized arachidonic acid (AA) inresponse to several stimuli, by a mechanism whichwas susceptible to inhibition by two structurally dis-similar, potent and specific Paf receptor antagonists,WEB 2086 (Casals-Stenzel et al., 1987) and CV 6209(Terashita et al., 1987). The same stimuli causedintracellular Paf formation. These data suggest thatintracellular Paf releases AA from its phosphatidestores, leading to eicosanoid generation. We haverecently proposed that intracellular Paf has a role inAA mobilization in cultured endothelial cells(Stewart et al., 1989). fMLP-stimulated superoxideanion generation was also reduced by WEB 2086indicating that Paf generation may have a wider rolein macrophages.
Guinea-pig resident peritoneal macrophagesincorporated ["4C]-AA which was released on sub-sequent stimulation with optimal concentrations ofPaf, fMLP, endotoxin and A23187. WEB 2086 inhib-ited the release of ["4C]-AA by all stimuli exceptA23187. However, the inhibition was most markedfor endotoxin-stimulated macrophages. This effectcannot be ascribed to inhibition of cyclo-oxygenasesince indomethacin was present throughout theseexperiments. Inhibition of lipoxygenase is unlikely toexplain the reduction in ['4C]-AA release for therewas no detectable conversion of released ['4C]-AAto ["4C]-5-lipoxygenase products. It may be con-cluded that the inhibition takes place at the level ofthe generation of non-esterified AA. The stimuli usedin this study have been reported to elicit significantleukotriene formation in other macrophage popu-lations (Rouzer et al., 1982; Rhodes et al., 1985; Wil-liams et al., 1986; Schade et al., 1987; Kouzan et al.,1988). The present findings may be explained by ouruse of ["4C]-AA whereas other workers have mainly
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146 A.G. STEWART & W.A. PHILLIPS
used the higher specific activity [3H]-AA (Rouzer etal., 1982; Kouzan et al., 1988) or radioimmunoassay(Rhodes et al., 1985; Williams et al., 1986) whichmay provide greater sensitivity. Alternatively, the 5-lipoxygenase pathway may not be expressed in theguinea-pig resident peritoneal macrophage popu-lation.Data from the metabolism of pre-incorporated
['4C]-AA predicted that stimulated release of cyclo-oxygenase products from macrophages should alsobe susceptible to inhibition by WEB 2086 since it iswell established that the formation of cyclo-oxygenase products is limited by the availability ofnon-esterified arachidonic acid. WEB 2086 inhibitedPGI2 generation and ['4C]-AA release by stimu-lated macrophages to similar extents. Although themaximum inhibition of PGI2 generation dependedon the stimulus used, with each stimulus the inhibi-tion by WEB 2086 and CV 6209 was similar. Theseobservations support the proposed role of intracellu-lar Paf in regulating non-esterified AA levels.
Superoxide anion generation by macrophages pro-vided an AA-independent and functionally impor-tant response to examine our hypothesis that Paf hasa second messenger role in this cell type. Superoxideanion generation induced by fMLP was reduced byWEB 2086 which suggests that the involvement ofPaf in signal transduction processes extends beyondregulation of eicosanoid generation. Incomplete inhi-bition of the response to fMLP suggests that thereare other mechanisms involved in the intracellularpathway for superoxide anion generation. Intracellu-lar Paf may be responsible for endotoxin-inducedpriming of enhanced fMLP-induced superoxideanion generation in human PMNLs (Worthen et al.,1988). Thus, Paf may act to upregulate other trans-duction pathways. Superoxide anion production byguinea-pig alveolar macrophages in response tofMLP is inhibited by another Paf receptor antago-nist, Ro 19-3704 (Lagente et al., 1988). These authorssuggested that this effect was due to an ability toscavenge free radicals but did not directly test thishypothesis. WEB 2086 does not have free radicalscavenging activity since it had no effect on super-oxide anion generation by hypoxanthine/xanthineoxidase nor does it inhibit A23187-induced super-oxide anion generation in rabbit PMNLs (Stewart,unpublished observations).An important consideration in the present study is
the specificity of the antagonists. WEB 2086 is a tri-azolodiazepine which is reported to lack thesedative/hypnotic properties of the other compoundsin this class (Casals-Stenzel & Weber, 1987; Casals-Stenzel et al., 1987). CV 6209 is a recently developedphospholipid analogue of Paf which has structuralsimilarity to an earlier compound, CV 3988(Terashita et al., 1983). Phospholipid analogue
antagonists of Paf have the potential for non-specificactions due to physicochemical interactions withlipid membranes. However, at the concentrationsused in the present study CV 6209 is devoid of suchactions (Terashita et al., 1987). Furthermore, there isindirect evidence that neither WEB 2086 nor CV6209 are inhibitors of phospholipase, cyclo-oxygenase or PGI2 synthetase since they failed toinhibit PGI2 generation or mobilization of pre-incorporated [14C]-AA induced by a maximallyeffective concentration of A23187. In addition, noneof BN 52021 (Nunez et al., 1986), L-652,731 (Hwanget al., 1985), WEB 2086 (Casals-Stenzel et al., 1987)or CV 6209 (Terashita et al., 1987) has significantinhibitory actions on cyclo-oxygenase activity atconcentrations which inhibit basal PGI2 generation(Stewart & Dusting, 1988, this study). Further evi-dence against a non-specific action of WEB 2086 orCV 6209 is apparent from the ability of these com-pounds to inhibit differentially a range of stimuliwhich produce a similar extent of mobilization ofAA.The failure of WEB 2086 to inhibit the basal
release of pre-incorporated ["4C]-AA may beexplained by a non-uniform labelling of phospho-lipid pools by ['4C]-AA. There is evidence for theexistence of multiple pools of arachidonic acid inmacrophages (Humes et al., 1982; Tanaka &Shibata, 1985; Schade et al., 1987) consistent withthe possibility that, under the conditions of thisstudy, ["4C]-AA may not label the pool of AA whichcontributes to basal eicosanoid production.The inhibition of basal PGI2 generation by Paf
receptor antagonists implied that Paf was formedunder the conditions of adherence. It was also con-sidered possible that the FCS used in the culturemedium could contain Paf or stimuli for its gener-ation. Nevertheless, cells adhered in the absence ofFCS were equally susceptible to the inhibitoryactions of the Paf receptor antagonists, even thoughthey produced less PGI2. Cell-associated, but notextracellular Paf was observed in basal adherentmacrophages giving support to the suggestion thatmacrophages become partly activated during adher-ence (Kouzan et al., 1987) and providing an explana-tion of the inhibition of 'basal' PGI2 generation bythe Paf antagonists.Even though the ionophore A23187 was the most
effective stimulus for Paf generation, neither antago-nist inhibited A23187-induced AA mobilization. Thisobservation appears to conflict with the suggestionthat intracellular Paf generation is a key event in AAmobilization. However, unlike physiological stimuli,A23187 causes persistent and large increases in intra-cellular Ca2+ which would be expected to activatedirectly the Ca2'-dependent enzyme, phospholipaseA2 irrespective of any action of intracellular Paf. It is
INTRACELLULAR Paf AND EICOSANOID BIOSYNTHESIS 147
also possible that the lack of inhibition was due tothe use of a maximally effective concentration ofA23187. The amount of intracellular Paf generatedby A23187, which greatly exceeded that produced bythe more physiological stimuli, may have sur-mounted the antagonism by WEB 2086 and CV6209. Variations in the extents of inhibition of theother stimuli may be related to differing mechanismsof activation of macrophages.Henson and co-workers have suggested that Paf
accumulates in cells in concentrations (- 10- M) suf-ficient to have direct physicochemical actions onmembranes (Harris et al., 1985; Henson, 1987). Inthe present study, the stimuli which were susceptibleto inhibition by the Paf antagonists elicited synthesisof cell-associated Paf (- -0.2 pmol per 106) whichwould lead to intracellular concentrations of theorder of 10-8_10-7M, well in excess of exogenousconcentrations required to activate macrophage AAmetabolism (Stewart & Dusting, 1988). In contrast,the extracellular Paf concentrations (< 10 -M) werebelow the EC50 for activation of AA metabolism inmacrophages stimulated with endotoxin and extra-cellular Paf could not be detected in basal or fMLP-stimulated macrophages. The low or undetectableconcentrations of Paf in these supernatants may be aresult of its rapid metabolism by macrophages(Roubin et al., 1986). These observations on the dis-tribution of Paf are not consistent with an extra-cellular site of action.The time-course of fMLP-stimulated Paf and
PGI2 generation provides evidence which is consis-tent with an intracellular action since Paf was detect-able within 6s of exposure to fMLP whereas PGI2was not detected in the extracellular medium until30s. However, the interpretation of this temporalrelationship as evidence for a causal link betweenthese lipids may be influenced by the relative detec-tion limits of the assays. It could be suggested thatsince both Paf and PGI2 synthesis are dependent onphospholipase A2 activity these lipids should be syn-thesized simultaneously. However, phospholipase A2
activity is clearly not essential for Paf synthesis in ratperitoneal macrophages even though inhibitors ofphospholipase A2 reduce the release of the imme-diate precursor, lyso-Paf (Parente & Flower, 1985).It would appear that resting lyso-Paf levels are ade-quate to support Paf synthesis, at least over shortperiods. In addition, it is possible that the alterna-tive, physiological pathway involving choline phos-photransferase (Braquet et al., 1987) may subservethe generation of intracellular Paf. In this pathway,the requirement for phospholipase A2 activity isfurther removed from the ultimate synthetic stepthan it is for the acetyltransferase pathway. Theapparent rapid metabolism of intracellular Paf byguinea-pig macrophages would confer greatercontrol on Paf as an intracellular messenger and is acommon' feature of other second messengers(Berridge, 1984).
In conclusion, we have shown that a number ofcompounds which stimulate macrophages alsostimulate the generation of Paf within the cells andthis is an important prelude to AA mobilization andeicosanoid generation. These data raise the possi-bility that Paf has the role of a second messenger oralters the gain of other signal transduction processeswithin these cells.
This study was supported by the National Health andMedical Research Council of Australia. A.G.S. was sup-ported by the C.R. Roper bequest to the Faculty of Medi-cine, University of Melbourne and W.A.P. is supported bya D.W. Kier Fellowship from the Royal Melbourne Hospi-tal. We thank; Dr J. Salmon, Wellcome Research Labor-atories (U.K.) for a gift of 6-oxo-PGF1, antiserum; theWellcome Foundation Ltd. for prostacyclin; Dr J. Rokach,Merck Frosst, Canada for synthetic leukotrienes B4, C4and D4; Dr N. Latt, Boehringer Ingelheim (Australia) forWEB 2086; Dr Oka, Takeda Chemical Industries, Japanfor CV 6209. We are grateful to T. Harris and D. Strode forexpert technical assistance and to M. Fenwick for secre-tarial assistance in the preparation of this manuscript. Wealso thank Dr G.J. Dusting for helpful comments on thismanuscript.
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(Received January 30, 1989Revised March 20, 1989Accepted April 6, 1989)
