Immunology 1991 72 206-212

Involvement of leukotriene B4 and platelet-activating factor incytokine priming of human polymorphonuclear leucocytes

A. G. STEWART,T. HARRIS, M. DE NICHILO* & A. F. LOPEZ*Department of Physiology, University of Melbourne, Parkville, Victoria and *Division of Human Immunology,

Institute of Medical and Veterinary Science, Adelaide, Australia

Acceptedfor publication 24 September 1990

SUMMARY

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophagecolony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) forincreased superoxide anion (027) generation and for increased platelet-activating factor (PAF)biosynthesis and leukotriene B4 (LTB4) release. Both PAFand LTB4 are candidate mediators for theenhanced 02 generation in cytokine-primed PMN, since exogenous PAFor LTB4 primes PMN. Wemeasured the generation and release of these mediators and examined their potential roles in cytokinepriming using the PAF receptor antagonist, WEB2086, and the inhibitor of 5-lipo-oxygenase, CGS8515. rhTNF-a or rhGM-CSF, alone, increased PAFlevels in PMN, but did not cause PAFrelease or

LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detect-able and biologically active amounts of both LTB4 and PAFin primed, but not in non-primed PMN.However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the primingof 02 generation by rhTNF-a or rhGM-CSF. Simultaneous pretreatment of PMNwith WEB2086and CGS8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAFin the priming response, but indicate that the release of PAF and LTB4 do not mediate thisphenomenon. The ability of cytokines to amplify the production and release of lipids may represent a

mechanism to attract and localize the pro-inflammatory actions of stimulated PMNto regions wherecytokine levels are also elevated.

INTRODUCTION

Circulating polymorphonuclear leucocytes (PMN) undergo afinal maturation process during extravasation and migration tolocal sites of inflammation. This process is known as primingand may be induced in vitro by a number of cytokines, includingrecombinant human granulocyte-macrophage colony-stimu-lating factor (rhGM-CSF),' -6 recombinant human tumournecrosis factor-alpha (rhTNF-a)7'8 and recombinant humaninterleukin-1 alpha (rhIL-1a).9 Low levels of endotoxin alsoinduce priming which is associated with an increase in themagnitude of the respiratory burst,'0"' enhanced generation ofleukotriene B4 (LTB4)'2 and platelet-activating factor (PAF)." '3

The mechanism of priming has not been elucidated, despiteintensive investigation.6"4-2' Priming of PMNby rhGM-CSF is

Abbreviations: FMLP, N-formylmethionyl-leucyl-phenylalanine;GM-CSF, granulocyte-macrophage colony-stimulating factor; h,human; HEPES, N-2-hydroxyethylpiperazine-N-2-ethane sulphonicacid; LTB4, leukotriene B4; 02, superoxide anion; PAF, platelet-activating factor; PMN, polymorphonuclear leucocytes; r, recombi-nant; TNF-o, tumour necrosis factor-alpha.

Correspondence: Dr A. G. Stewart, Dept. of Physiology, Universityof Melbourne, Parkville, Victoria 3052, Australia.

not accompanied by activation of phosphatidylinositol phos-phate breakdown or activation of protein kinase C.'5 However,both rhGM-CSF and rhTNF-cx elicit the mobilization ofarachidonic acid and generation of PAF,4 9'22-24 suggestingactivation of phospholipase A2. Arachidonic acid may beconverted to leukotriene B4 (LTB4), which is reported toprime PMNfor an enhanced respiratory burst to the chemotac-tic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP).25Similarly, several studies indicate that PAF primes the respira-tory burst in PMN.'6'25 2" Thus, both PAF and LTB4 arecandidates for autocrine mediators of priming.

In the present study, we have examined the roles of PAFandLTB4 in the enhanced respiratory burst in cytokine-primedPMN. The role of PAF was evaluated using the specific andcompetitive PAF receptor antagonist, WEB2086.28 29 The roleof LTB4 was evaluated using the selective 5-lipo-oxygenaseinhibitor, CGS8515.30 In addition, we examined the generationand release of these phospholipid-derived mediators of inflam-mation from primed and stimulated PMN. The biosynthesis ofPAF and LTB4 was markedly increased in primed PMN, andsubsequent stimulation by FMLP led to the release of bio-logically relevant levels of PAF. However, our data do notsupport a role for released PAF or for LTB4 in the enhancedrespiratory burst in cytokine-primed PMN.

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Role of LTB4 and PAFin cytokine priming of PMN

MATERIALS ANDMETHODS

MaterialsAll reagents and solvents were of analytical or higher grade.Chemicals were obtained from the following sources: bovineserum albumin (BSA; grade 5, essentially fatty acid free),cytochrome C (horse heart type III), dextran, clinical grade Cformyl-methionylleucyl-phenylalanine (FMLP), HEPES(N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid), indometh-acin, superoxide dismutase (Sigma Chemical Co., St Louis,MO); rh granulocyte-macrophage colony-stimulating factor(rhGM-CSF; batch number 84; Scherring Plough Corporation);rh tumour necrosis factor-alpha (rhTNF-a; lot no. N9017AX;specific activity 4 x 107 U/mg protein; generous gift fromGenentech, South San Francisco, CA); rh interleukin- 1 alpha(rhIL-loc; specific activity exceeds 108 thymocyte mitogenesisU/mg protein; generous gift from Dr S. Gillis, ImmunexCorporation, Seattle, WA); hexadecyl platelet-activating factor(PAF; Novachem, Bukendorf, Switzerland); (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 (New Eng-land Nuclear, Boston, MA); leukotriene B4 antiserum (Dr J.Salmon, Wellcome Research Laboratories, Beckenham, Kent,U.K.); leukotriene B4 (Dr J. Rokach, Merck Frosst Laborator-ies, Montreal, Quebec, Canada); WEB2086 (3-(4-(2-chlorophe-nyl)-9-methyl-6H-thieno (3,2,-f), (1,2,4)-triazolo, (4,3-a), (1,4)-diazepine-2-yl)- I -(4-morpholinyl)-propanone) (BoehringerIngelheim, Ingelheim, Germany).

Preparation of human polymorphonuclear leucocytesPMNwere obtained from buffy coats supplied by the Red CrossSociety, South Melbourne, Australia. The interval between theinitial venepuncture and the experiment was approximately 5hr. Similar results were obtained with PMNobtained fromvolunteers for which the time between venepuncture and thecommencement of the experiment was 2 5-3 hr. PMNwereisolated by dextran (T500; Pharmacia, Uppsala, Sweden)sedimentation of red blood cells and density gradient centrifu-gation through Lymphoprep (endotoxin-tested) using pre-viously described methods.293' Contaminating red blood cellswere lysed with NH4Cl (0-15 M containing 10 mmHEPES, pH7-0). The PMNwere then washed twice in Tyrode buffercontaining 0 25% BSA [composition (mM): 5, HEPES; 137,NaCl; 1 1, D( + )-glucose; 11 9, NaHCO3; 2-7, KCl; 0-26 MgCl2;0-4, NaH2PO4; 1-8 CaCl2] and resuspended in Tyrode at 6 x 106/ml. Cell purity, assessed by morphology, exceeded 98% andviability, assessed by exclusion of trypan blue, exceeded 99%.None of the drugs/vehicles used in this study had any significanteffect on cell viability. Buffer reagents were tested for endotoxincontamination by the supplier (Sigma Chemical Co.) andreported to contain less than 0-01 ng/ml. This level is consider-ably less than that known to prime PMN(10 ng/ml)."' 3' Bufferswere prepared using water for injection (BP).

Isolation of plateletsRabbits (2-4 kg), C.S.L., Parkville, Australia were anaesthe-tized by intravenous infusion of propanidid and exsanguinatedvia a sterile cannula inserted in the carotid artery. The blood wasimmediately mixed with trisodium citrate (0-38% w/v, finalconcentration) and platelet-rich plasma (PRP) was prepared bycentrifugation at room temperature for 20 min at 100 g. Washedplatelets were prepared as previously described in detail.32 The

platelet concentration was adjusted to 2 x 108/ml and plateletswere stored in Tyrode solution overnight at 4° before use on thefollowing day.

Extraction and bioassay of PAFPAF was bioassayed on washed rabbit platelets as describedpreviously.33 PMNsupernatants were assayed without furtherextraction using a maximum volume of 50 ,l and the amountof PAF-like bioactivity was quantified by reference to theaggregation induced by the hexadecyl PAFstandard. The PMNcell pellets were extracted into ice-cold 80% ethanol, the proteinprecipitate was removed by centrifugation (1000 g, 4°, 5 min)and the extract was evaporated to dryness under reducedpressure before reconstitution in the assay buffer (containing0 25% BSA to facilitate the resuspension of PAF). The criteriafor the identification of the bioactivity as PAF includedsusceptibilty to antagonism by a selective concentration of thePAF receptor antagonist, WEB2086 (1 ,UM), and resistance toinhibition by indomethacin (2-8 ,M). In addition, samplescontaining PAF-like bioactivity were pooled, subjected to aBligh-Dyer extraction and chromatographed on silica gelTLC (chloroform: methanol: water: acetic acid, 65:35:6:0-1)together with authentic [3H]Paf (approximately 4000 d.p.m.).The co-migration of the bioactivity of pooled and re-extractedsamples with authentic PAF further indicated that the activity inthe non-extracted samples was due to PAF.

Superoxide anion assayThe generation of 07 was measured by the superoxide dis-mutase (SOD)-inhibitable reduction of cytochrome C.34.35Following 30 min pretreatment with cytokines or the vehicle,0-25% BSA in Tyrode buffer, PMNwere resuspended at1-2 x 106/ml in Tyrode buffer containing 80 pM cytochrome C.The response to FMLPwas complete within 5 min, at whichtime the samples were centrifuged (1000 g, 4', 5 min). Theabsorbance (550 nm) of the supernatants was determined in aHitachi U2000 spectrophotometer. The generation of 02 wascalculated by the superoxide dismutase (30 U/ml)-inhibitableincrease in absorbance at 550 nm using the differential molarextinction coefficient, 21 x 103/M/cm.

Radioimmunoassay of LTB4LTB4 was measured in the supernatants of human PMNprimedwith cytokines then stimulated with FMLP for 5 min. Non-extracted supernatants were subject to radioimmunoassay usingthe LTB4-specific antisera and the methods of Salmon et al.36The limit of detection of the assay was 0-5 pmol/106 PMN.

RESULTS

Generation of PAFand release of LTB4 in cytokine-primed PMN

In freshly isolated PMNincubated for 30 min in the cytokinevehicle (Tyrode, 0-25% BSA) a low level of cell-associated PAFwas detected (Fig. 1), but there was no release of PAF into thesupernatants (data not shown). Upon stimulation with FMLP(100 nM) cell-associated PAF levels increased significantly(P < 0-05; Fig. 1) but no PAFwas detectable in the supernatants(Fig. 2). Similarly, no LTB4 release was detected in non-primedcells stimulated with FMLP(Table 1).

PMNwere exposed to concentrations of cytokines known tobe near maximally active in other assay systems. Quantitative

207

A. G. Stewart et al.

0 r

riAQ

i C n±IControl GM-CSF TNF-a IL-la

za_V-

04_06

OLU-

a1_M ,L'J.- r

0

Figure 1. Cell-associated PAF levels in control and cytokine-primedPMN(open histograms) and in those subsequently stimulated withFMLP (filled histograms). Data are presented as the means andstandard error of the mean of 3-13 observations. *P < 0 05 WilcoxonRank sum test, compared with corresponding control value.

IOr

0

0

I'0

00

0

0 0

0

0

*Ap At &x

Table 1. Release of LTB4 from non-primed and rh GM-CSF-primedPMN

LTB4(pmol/106 PMN)

Priming agent Stimulation Control CGS8515 3 yM

None FMLP100 nm 0 0GM-CSF 1 nm None 0 0GM-CSF 1 nM FMLPl0OnM 5 57+3-41* 1-79+1-68t

0=less than 0 50 pmol/106 PMN(limit of detection of the assay).Data are presented as the means and standard error of the means of fiveobservations.

* P< 0-05, Wilcoxon rank sum test, compared with level in non-primed PMN.

t P< 0 05, Wilcoxon rank sum test, compared with the correspond-ing level in control PMN.

P< 005). PAF release ranged from 0% up to 60% for GM-CSF-primed, FMLP-treated PMNand from 2%up to 78% forthose primed with TNF-cx.

Priming of the respiratory burst

In parallel with changes in PAFand LTB4 release, both rhTNF-aand rhGM-CSF primed PMNfor an increased generation of 02in response to FMLP (100 nM) (Table 2). rhIL-la, whichinduced a smaller increase in the generation of PAF, did notconsistently increase FMLP-induced 02 generation (Table 2).

0 5 10 20 30Total PAF (pmol / 107 cel Is)

Figure 2. Release of PAF (supernatant) plotted against the total PAFformation (cell-associated+supernatant). Data are presented as indi-vidual observations and indicate PAF levels in non-primed or primedPMNsubsequently stimulated with FMLP. In rhGM-CSF (squares)and TNF-a (filled circles) pretreated PMN10/13 and 5/5 donors showedPAF release, respectively, whereas PAF levels were below the limit ofdetection (0 05-0-10 pmol/107 PMN) in IL-la (triangles)-pretreatedPMNfrom four donors. In non-primed PMN(control, stars), 12/13donors failed to release detectable PAF levels upon stimulation withFMLP.

and qualitative changes in mediator generation and releasetook place in PMNprimed with rhGM-CSF (I nM), rhTNF-a(0-3 nM) or rhIL-a(60 pM). The basal generation of cell-associated PAF increased with each of these priming agents, as

did the levels following FMLP stimulation (Fig. 1). Impor-tantly, rhGM-CSF and rhTNF-a, which induced a quanti-tatively greater degree of priming than rhIL-la, also inducedPAF release in PMNstimulated with FMLP(Fig. 2), whereaslevels remained undetectable in the supernatants of rhIL-la-pretreated, FMLP-stimulated PMN. Furthermore, in rhGM-CSF-primed PMN, FMLP induced the release of detectableamounts of LTB4 (Table 1).

The release of PAFshowed considerable variability betweendonors (Fig. 2), but there were significant correlations betweenthe amount released and the total amount formed for both GM-CSF (r2=072, n= 14, P<0-05) and TNF-a (r2=0-35, n=5,

Involvement of PAF in the enhanced respiratory burst

It is established that exogenous PAF acts as a priming agent,inducing an increase in the 02 generation in response to FMLP.Since we observed both PAF release and an enhanced 02generation from rhTNF-oa- and rhGM-CSF-primed andFMLP-stimulated PMN, the possibility that released PAFcontributed to the priming was examined using the PAFreceptor antagonist, WEB2086. Pretreatment of PMNwithPAF(0 1-1000 nM) for 5 min caused a concentration-dependentincrease in FMLP-stimulated 02 generation, which was in-hibited significantly (P < 0-05) by preincubation of PMNwith1 gMWEB2086 (Fig. 3). In contrast, preincubation with WEB2086 had no effect (P> 005) on priming induced by eitherrhGM-CSF or by rhTNF-x (Table 2). It is noteworthy that thisconcentration of WEB2086 prevented completely the en-

hancing actions of PAF used at levels in the range detectedin supernatants of primed and FMLP-stimulated PMN(0 1-1 nM).

Involvement of LTB4 in the enhanced respiratory burst

The possible contribution of LTB4 to priming was examinedusing the selective 5-lipo-oxygenase inhibitor, CGS8515 (3 pM).The generation and release of LTB4 in rhGM-CSF-primed,FMLP-stimulated PMNwas prevented by pretreatment withCGS8515 (Table 2). However, CGS8515, either alone or incombination with WEB2086, had no effect (P>0 05) on theenhancement of 02 generation in primed PMN. Nonetheless,CGS8515 did reduce the respiratory burst in non-primed PMN.

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Role of LTB4 and PAF in cytokine priming of PMN

Table 2. Effects of pretreatment with WEB2086, CGS8515 and the combination onthe respiratory burst induced by FMLPin cytokine-primed PMN

02 (% FMLP)*

WEB2086 CGS8515 WEB2086 andPriming agent Control 1 JIM 3 yM CGS8515

None 100 102+4 (6) 75±+7 (8) 83±+6 (7)10-54+ 1-69t

GM-CSF MnM 174 + 20§ (14) 184 + 23§ (6) 150 + 26§ (8) 121 + 10§ (7)1531 + 1-59t

TNF-c 0-3 nM 181 +22§ (14) 185+28§ (6) 137+ 18§ (8) 123+ 13§ (7)15 70+ 1-65t

IL-la 60 pM 108+6 (10) 118+9 (6) 85+5 (4) 75+4 (3)9-34+ 1-24t

* Data are presented as means and standard errors of means of (n) observations.Data have been expressed as a percentage of the response to FMLPin non-primed cellsnot treated with inhibitor (control).

t Absolute values of 02 generation (nmol/106 PMN).t P< 0-05, Wilcoxon rank sum test, compared with value in control PMN.§ P<-005, Wilcoxon rank sum test, compared with value in

non-primed PMN.

300 r

250

CLJ

_OU-

0

200 F

150 F1001-

50L

12

10

28a-w

06 'E

4.-0

2

0

10 9 8 7 6 Cz - O-Log 1PAF] 1_

Figure 3. The enhancing effect of PAF (0- 1 - 1000 nm, open circles) on the02 generation in response to FMLP(100 nM). Inhibition of the PAF-induced enhancement by pretreatment with WEB2086 (I pM, filledcircles) is also shown. Data are presented as the means and standarderror of the mean of five observations (in duplicate). Data are expressedas percentages of the response to FMLP(100 nM) in the absence of PAFpretreatment. The basal and FMLP(100 nM)-induced 02 generation(nmol /106 PMN) are presented as histograms on the right hand side ofthe figure.

DISCUSSION

Our findings indicate that cytokine-priming of human PMNresults in an increased generation of PAFwhich may be furtherincreased by stimulation with the chemotactic peptide, FMLP.More importantly, primed PMNrelease detectable amounts ofPAFupon stimulation. Weexamined the role of PAFand LTB4in the priming of PMNfor increased 02 generation. Althoughreleased in increased amounts, neither of these lipid mediatorsaccounts for the priming of 02 generation.

Previous studies have established that rhGM-CSF-primedPMN release increased amounts of LTB4 in response to

stimulation by a variety of neutrophil agonists, including FMLPand serum-treated zymosan.4.5'24 37 Furthermore, LTB4 isreported to prime human PMNfor increased FMLP-induced02 generation.25 The selective inhibitor of 5-lipo-oxygenase,CGS851530 reduced LTB4 levels in rhGM-CSF-primed andFMLP-stimulated PMNsupernatants by 70%, but failed toinhibit the priming of 02 generation. Thus, it seems unlikelythat released LTB4 plays a role in the priming phenomenon.Cellular retention of LTB4 has been suggested38 and severalstudies have provided evidence for second messenger functionsof 5-lipo-oxygenase products in PMN.39A2 Retention of LTB4 isalso unlikely to explain the priming phenomenon since thesynthesis inhibitor would also have reduced these levels of LTB4.Inhibition by CGS8515 of the respiratory burst in non-primedPMNis consistent with previous suggestions, based on theinhibitory effects of relatively non-selective agents such as

nordihydroguiaretic acid, that 5-lipo-oxygenase products act as

second messengers in human PMN. However, the apparentcontribution of products of the 5-lipo-oxygenase pathway to thetransduction processes was not increased by priming, since therewas no increase in the percentage inhibition of 02 generation byCGS8515 in primed PMN.

Several studies report that FMLPis an ineffective stimulusfor LTB4 generation unless exogenous arachidonic acid is alsoadded,4543 indicating that phospholipase activity is rate limitingfor PMNleukotriene generation. The present study, and otherson the influence of cytokines on PMNeicosanoid generation,45suggest that rhGM-CSF overcomes the requirement for exo-

genous arachidonic acid. Indeed, one of the biochemicalresponses to rhTNF-a and rhGM-CSF is the mobilization ofarachidonic acid.22 Activation of phospholipase A2 is also likelyto explain the enormous increase in PAF levels in cytokine-primed PMN, since this enzyme is the initial step in theremodelling pathway of PAF biosynthesis.44 The failure ofGM-CSF to stimulate LTB4 release may be related to its

209

210 A. G. Stewart et al.

inability to stimulate phospholipase C and thereby increaseintracellular Ca2+, which is necessary for the activation of5-lipo-oxygenase.

The release of PAFfrom primed and stimulated PMNand itsestablished capacity to rapidly prime PMN(present study)"' 25-27raised the possibility that PAF mediates the priming pheno-menon, at least for 02 generation. This possibility was furthersupported by the failure of rhIL- 1 a to prime for PAF release orconsistently increase FMLP-induced 02 generation. rhIL-ladid increase cell-associated PAF levels, as reported for humanendothelial cells,45 but to a lesser extent than either rhGM-CSFor rhTNF-ox. In addition, a recent study indicated that PAFrelease during phagocytosis could increase intracellular calciumby an autocrine mechanism.46 Unequivocal evidence thatreleased PAF does not account for rhTNF-a- or rhGM-CSF-induced priming was provided by experiments using the com-petitive PAF receptor antagonist WEB2086.28 This compoundcompletely prevented priming of PMNinduced by 01-1 nMPAF, and reduced responses to higher concentrations of PAF(10-1000 nM), but had no effect on cytokine priming. In thestudy of Wirthmueller et al.27 the potential role of PAF as amediator of priming was proposed on the basis that total lipidextracts of primed and stimulated PMNcould prime PMNfor02 generation. However, these studies took no account of theamount of PAF released and therefore available to cell-surfacereceptors. Our results provide clear evidence against an auto-crine priming action of PAF released from cytokine-treatedPMN, but it is not possible to exclude a role for cell-associatedPAF in this phenomenon.

Wehave suggested that cell-associated PAF functions as asecond messenger in phagocytes and endothelial cells.33'35'47 Athigher concentrations than that used in the present study, PAFreceptor antagonists inhibit partially the non-primed respira-tory burst in rabbit PMN35and in human PMNstimulated withFMLP (A. G. Stewart and T. Harris, unpublished observa-tions)26 or with rhTNF-x.48 Further examination of the roleof cell-associated PAF in the priming response awaits theavailability of an effective and selective inhibitor of PAFbiosynthesis.

PAF release was consistently observed in rhTNF-ot- andrhGM-CSF-primed, FMLP-stimulated PMN, but not in non-primed PMNor in those exposed to rhIL-loc. Considerablevariation in the values for PAF release from FMLP-stimulatedPMNreported in the literature may be explained by the presenceof trace quantities of endotoxin in the reagents.31 Endotoxininduces priming of human PMN, and also by itself, as reportedhere for GM-CSFand TNF-o, markedly increases PAF levels. "The conditions of PMNincubation (cell number/extracellularpH; constant renewal of extracellular medium) may be critical indetermining the proportion of PAF released by PMN.49.50 Therelationship, if any, of these findings to the ability of cytokinesto increase PAF release is not clear. A recent study reported thatFMLPstimulated PAF release from human PMNand that therelease could be increased by pretreatment with GM-CSF, butcell-associated PAF levels were not measured.5' The use ofcytochalasin B, which disrupts the normal physiology of PMNby interfering with microtubule function, limits the conclusionsthat can be drawn from latter study. Moreover, these workersused a radiochemical method to determine PAF release whichmay not produce results indicative of changes in endogenouslevels.

An important consideration is whether the release of PAFbycytokine-primed PMNis simply a result of over-loading thecellular storage capacity or whether a specific transport mecha-nism is involved. The latter is suggested by the observation thatthe ionophore, A23187, stimulates the generation of signifi-cantly more PAF than does FMLPbut results in the release of asmaller proportion of the total amount of PAF.52

In conclusion, release of the phospholipid-derived media-tors, LTB4 and PAF, either singly or in combination, does notcontribute to the rhTNF-a or rhGM-CSF priming of PMNforenhanced 02 generation. However, the release of these media-tors by primed PMNis likely to be of importance for thepathogenesis of inflammatory conditions4448 involving PMNinfiltration and in immune regulation.53

ACKNOWLEDGMENTS

WeThank Dr H. Heuer (Boehringer Ingelheim, Ingelheim, Germany),Dr J. Turk (Ciba-Geigy, Summit, NJ), Dr S. Gillis (Immunex Corpo-ration, Seattle, WA), Dr J. Rokach (Merck Frosst Laboratories,Montreal, Quebec, Canada) and Dr J. Salmon (Wellcome ResearchLaboratories, Beckenham, Kent, U.K.) for gifts of WEB2086, CGS8515, rhIL-lot, LTB4 and LTB4 antiserum, respectively. Wealso thankAnna Christov for excellent technical assistance. This study wassupported by grants from the NH& MRC(Australia).

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