Proc. Natl. Acad. Sci. USAVol. 93, pp. 11091-11096, October 1996Medical Sciences

Leukocyte lipid body formation and eicosanoid generation:Cyclooxygenase-independent inhibition by aspirin

(nonsteroidal antiinflammatory agents/cyclooxygenase knockout/leukotrienes/prostaglandin endoperoxide H synthase/lipoxygenase)

PATRICIA T. BOZZA*, JENNIFER L. PAYNE*, ScoTr G. MORHAMt, ROBERT LANGENBACHt, OLIVER SMITHIESt,AND PETER F. WELLER*§*Harvard Thorndike Laboratory and Charles A. Dana Research Institute, Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, MA02215-5491; tDepartment of Pathology, University of North Carolina, Chapel Hill, NC 27599-7525; and *Laboratory of Experimental Carcinogenesis andMutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

Communicated by Seymour J. Klenbanoff, University of Washington School of Medicine, Seattle, WA, July 31, 1996 (received for reviewApril 8, 1996)

ABSTRACT Lipid bodies, cytoplasmic inclusions that de-velop in cells associated with inflammation, are induciblestructures that might participate in generating inflammatoryeicosanoids. Cis-unsaturated fatty acids (arachidonic andoleic acids) rapidly induced lipid body formation in leuko-cytes, and this lipid body induction was inhibited by aspirinand nonsteroidal antiinflammatory drugs (NSAIDs). Severalfindings indicated that the inhibitory effect of aspirin andNSAIDs on lipid body formation was independent of cycloox-ygenase (COX) inhibition. First, the non-COX inhibitor,sodium salicylate, was as potent as aspirin in inhibiting lipidbody formation elicited by cis-fatty acids. Second, cis-fattyacid-induced lipid body formation was not impaired in mac-rophages from COX-1 or COX-2 genetically deficient mice.Finally, NSAIDs inhibited arachidonic acid-induced lipidbody formation likewise in macrophages from wild-type andCOX-1- and COX-2-deficient mice. An enhanced capacity togenerate eicosanoids developed after 1 hr concordantly withcis-fatty acid-induced lipid body formation. Arachidonic andoleic acid-induced lipid body numbers correlated with theenhanced levels of leukotrienes B4 and C4 and prostaglandinE2 produced after submaximal calcium ionophore stimula-tion. Aspirin and NSAIDs inhibited both induced lipid bodyformation and the enhanced capacity for forming leukotrienesas well as prostaglandins. Our studies indicate that lipid bodyformation is an inducible early response in leukocytes thatcorrelates with enhanced eicosanoid synthesis. Aspirin andNSAIDs, independent of COX inhibition, inhibit cis-fattyacid-induced lipid body formation in leukocytes and in con-cert inhibit the enhanced synthesis of leukotrienes and pros-taglandins.

The capacity of cells to generate eicosanoid mediators ofinflammation may be enhanced by inducible mechanisms. Onesuch mechanism is the induction of expression of a specificisoform of prostaglandin endoperoxide H synthase [cycloox-ygenase (COX)], COX-2. Various cytokines and pro-inflammatory stimuli elicit the de novo synthesis of COX-2over two or more hours that leads to increased formation ofCOX pathway-derived eicosanoids (1-4). Inducible mecha-nisms that enhance eicosanoid production are attractive aspotential targets for selective antiinflammatory treatments.Here we discuss a new mechanism for enhanced eicosanoidsynthesis based on the induction of lipid body in leukocytes.

Lipid bodies are lipid-rich cytoplasmic inclusions that char-acteristically develop in vivo in cells associated with inflam-mation; although neither the mechanisms of formation nor thefunction of lipid bodies are fully understood, lipid bodies are

inducible structures. Lipid bodies are prominent in leukocytesat sites of natural and experimental inflammation, includingleukocytes from joints of patients with inflammatory arthritis(5-7), the airways of patients with acute respiratory distresssyndrome (8), and casein- or lipopolysaccharide-elicitedguinea pig peritoneal exudates (9). The formation of lipidbodies is a regulated process that involves activation of intra-cellular signaling pathways, including protein kinase C andphospholipase C (10, 11). Inhibitors of mRNA and proteinsynthesis inhibit lipid body formation, indicating that inductionof lipid body formation depends on the activation of specificgene expression and new protein synthesis (11). The possibilitythat lipid bodies have a role in the formation of eicosanoidmediators of inflammation is suggested by their components.Lipid bodies in several cell types contain COX and 5-lipoxy-genase (refs. 12-14 and unpublished observations), both ofwhich use arachidonic acid (AA) to initiate synthesis ofinflammatory eicosanoids. In addition, stores ofAA have beenlocalized to lipid bodies in leukocytes, including polymorpho-nuclear leukocytes (PMNs) and eosinophils (15, 16). More-over, Bozzaetal. (11) have demonstrated, in platelet-activatingfactor (PAF)-stimulated PMNs, a correlation between en-hanced capacity for eicosanoid formation and levels of lipidbody induction.

Aspirin and other nonsteroidal antiinflammatory agents(NSAIDs), widely used in treating inflammatory processes,including arthritis (17, 18), are believed to act principally byinhibition of COX thus blocking the initial step in prostaglan-din synthesis (19). This mechanism may not, however, fullyaccount for the antiinflammatory effects of NSAIDs. Lowdoses of aspirin and other NSAIDs are required for COXinhibition in vitro and in vivo (19-21), whereas higher doses arerequired for antirheumatic effects in vivo (17, 22). In addition,sodium salicylate has minimal activity as a COX inhibitor (19),but is as effective as aspirin for inhibiting inflammatorymanifestations of arthritis in humans and experimental models(17, 22, 23). NSAIDs, by mechanisms other than COX inhi-bition, also inhibit PMN activation, including the synthesis ofleukotrienes, eicosanoids formed by a lipoxygenase, and not aCOX, pathway (24-26). Thus, there is evidence to suggest thatthe antiinflammatory effects of aspirin-like drugs are notsolely attributable to COX inhibition. Correlation of lipid bodyformation with inflammatory responses led us to investigatewhether inhibition of lipid body formation could be a mech-

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; NSAID,nonsteroidal antiinflammatory drug; OA, oleic acid; PAF, platelet-activating factor; PMN, polymorphonuclear leukocyte (neutrophil);DMSO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution.§To whom reprint requests should be sent at present address: BethIsrael Hospital, 330 Brookline Avenue, DA-617, Boston, MA 02215-5491. e-mail: pweller@bih.harvard.edu.

11091

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1

11092 Medical Sciences: Bozza et al.

anism for some of the antiinflammatory effects of aspirin andNSAIDs.

MATERIALS AND METHODSMaterials. Aspirin, indomethacin, piroxicam, salicylic acid

(sodium salt), oleic acid (OA), AA (Sigma), 14C-AA (1.5-2.2GBq/mmol; DuPont/NEN), A23187 (Calbiochem), and NS-398 (Biomol, Plymouth Meeting, PA) were obtained as noted.Human and Murine Leukocytes. Human PMNs were puri-

fied from blood obtained from healthy adult volunteers andcollected into acidified citrate. After addition of6% dextran 70(McGaw, Irvine, CA), erythrocytes were allowed to sedimentfor 1 hr at 25°C. The leukocyte-rich supernatant was overlaidonto an equal volume of Ficoll/Paque (Pharmacia), andcentrifuged at 400 x g for 20 min. PMNs (>95% pure, thedifference were eosinophils) were recovered from the pelletand washed in Ca2+/Mg2+ free Hanks' balanced salt solution(HBSS). Residual erythrocytes were lysed with hypotonicsaline. Eosinophils (>80% pure, the difference were neutro-phils) were obtained from a donor with idiopathic hypereosi-nophilic syndrome using the same procedure. Synovial fluidleukocytes were examined in samples of joint fluids submittedto the clinical hematology laboratory. COX-1 and COX-2genetically deficient mice were generated as described (27, 28).Experiments were carried out with 8- to 12-week-old mice.Macrophages from COX-1 and COX-2 genetically deficientmice and wild-type littermates were obtained by lavaging theperitoneal cavity with 5 ml of cold HBSS containing heparin(10 units/ml).

Lipid Body Induction and Enumeration. Leukocytes (106cells per ml) were incubated with concentrations of AA, OA,or vehicle (0.1% ethanol) at 37°C in a 5% C0295% 02atmosphere, and after 1 hr leukocytes (105 cells per slide) werecytocentrifuged onto glass slides. For inhibitor studies, leuko-cytes were pretreated for 1 hr with concentrations of inhibitorsor vehicle as indicated. Cell viability, determined by Trypanblue dye exclusion at the end of each experiment, was alwaysgreater than 90%. Stock solutions of A23187, indomethacin,aspirin, sodium salicylate, piroxicam, and NS-398, prepared indimethyl sulfoxide (DMSO) and stored at -20°C, were dilutedin Ca2+/Mg2+-free HBSS to the indicated concentration im-mediately before use. The final DMSO concentration wasalways lower than 0.1% and had no effect on lipid bodynumbers. Slides, while still moist, were fixed in 3.7% formal-dehyde in Ca2+/Mg2+-free HBSS (pH 7.4), rinsed in 0.1 Mcacodylate buffer (pH 7.4), stained in 1.5% OS04 (30 min),rinsed in H20, immersed in 1.0% thiocarbohydrazide (5 min),rinsed in 0.1 M cacodylate buffer, restained in 1.5% OS04 (3min), rinsed in H20, dried, and mounted. The morphology offixed cells was observed, and lipid bodies were enumeratedwith phase contrast microscopy and a 10OX objective lens in50 consecutively scanned leukocytes.

Prostaglandin (PG)E2, Leukotriene (LT)B4, and LTC4 As-says. Leukocytes (106 cells per ml) were stimulated withconcentrations of AA, OA, or vehicle (0.1% ethanol) at 37°Cfor 1 hr to induce lipid body formation; after which a samplewas taken for lipid body enumeration, and the cells werewashed in Ca2+/Mg2+-free HBSS. Leukocytes, resuspended in1 ml of HBSS containing Ca2+/Mg2+, were stimulated with 0.5AM A23187 at 37°C for 15 min. Reactions were stopped on ice,and samples were centrifuged (800 x g for 10 min at 4°C). Ineach replicate supernatant or extracted cell pellet, PGE2,LTB4, and LTC4 were assayed in duplicate by ELISA (CaymanChemicals, Ann Arbor, MI).AA Uptake. Neutrophils (106 per ml) were labeled with

14C-AA (0.2 ,uM) together with either vehicle, aspirin (10,ug/ml), indomethacin (1 ,ug/ml), or sodium salicylate (10,ug/ml) for 1 hr. After the incubation period, lipid bodyformation was elicited by AA (15 ,uM). The cells were washed

three times in Ca2+/Mg2+-free HBSS and analysis of incor-porated 14C-AA in the cell pellet was performed by liquidscintillation counting.

Statistical Analysis. Results, expressed as mean ± SEM,were analyzed by analysis of variance followed by the New-man-Keuls Student's t test with the level of significance set atP < 0.05. Correlation coefficients were determined by linearregression, and correlation analysis was performed by Fisher'sr to z transformation with the level of significance set at P <0.05.

RESULTS

Lipid Body Formation. Normal PMNs contain few lipidbodies (Fig. 1A); but PMNs at sites of inflammatory reactions,such as septic arthritis (Fig. 1B), contain markedly increasednumbers of lipid bodies. Common alcohol-based hematologicstains cause dissolution of lipid bodies (Fig. 1D), which pre-cludes their recognition. However, with appropriate lipidfixation, increased lipid body numbers have been observed inmany types of leukocytes when these cells are involved in avariety of infectious, allergic, neoplastic, and other inflamma-tory diseases (15, 29). Normal PMNs can be stimulated withcis-unsaturated, but not saturated, fatty acids, such as AA andthe non-eicosanoid precursor OA, to rapidly form (within 1 hr)new lipid bodies (Fig. 1C) morphologically comparable to lipid

A.... ^.;

B

C D

A

F

FIG. 1. Lipid bodies in human PMNs. Lipid bodies are sparse innormal donor-derived blood PMNs (A) but very prominent in synovialfluid PMNs from a patient with septic arthritis (B) and in normaldonor blood-derived PMNs after stimulation in vitro with 5 IIMAA for1 hr at 37°C (C). Without prior lipid preservation, however, lipidbodies are solubilized by routine staining and become undetectable(D) in the same synovial fluid PMNs as in B. Murine peritonealmacrophages contain few lipid bodies in resting conditions (E); but canalso be stimulated in vitro with 15 ,uMAA for 1 hr at 37°C to form lipidbodies (F). Cytocentrifuged leukocytes were fixed in 3.7% formalde-hyde in HBSS and stained with osmium tetroxide, (A-C, E, and F) orwere fixed in methanol and stained with Diff-Quik (Baxter, Miami,FL) (D).

Proc. Natl. Acad. Sci. USA 93 (1996)

Jo40

dol.91

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1

Proc. Natl. Acad. Sci. USA 93 (1996) 11093

FIG. 2. (A and B) Aspirin (ASA) and (C) other NSAIDs inhibit AA- and OA-induced lipid body formation in human PMNs (A and C) andeosinophils (B). Lipid bodies/cell with vehicle alone (open bar), fatty acid stimulation (solid bar), fatty acids plus ASA pretreatment (striped bar)(A and B), and NSAID inhibition with indomethacin (dotted bar), sodium salicylate (vertically striped bar), and piroxicam (cross-hatched bar) (C)are indicated. Cells (106 per ml) were pretreated with aspirin (10 ,ug/ml), indomethacin (1 ,ug/ml), sodium salicylate (10 ,ug/ml), piroxicam (10jig/ml), or vehicle (0.1% DMSO) for 1 hr, and then stimulated with 15 ,uM AA, 10 ,tM OA, or vehicle (0.1% ethanol) for 1 hr at 37°C. Valuesare expressed as mean±SEM of 50 consecutively counted cells of one representative experiment from 3-5 independent assays. Statisticallysignificant differences (P < 0.05, Student's t test) between lipid bodies/cell with vehicle alone and fatty acid stimuli are noted by a triangle, andbetween fatty acid stimuli alone and pretreatment with ASA or NSAID by *.

bodies formed in vivo (10, 15, 16). As with human cells, mousemacrophages contain only few lipid bodies under normalconditions (Fig. 1E) and can also be stimulated in vitro withcis-unsaturated fatty acids to form lipid bodies (Fig. 1F).

Aspirin and NSAID Inhibition of Lipid Body Induction.Since lipid body numbers increase during inflammatory pro-cesses and might be related to the formation of eicosanoidmediators of inflammation, we investigated aspirin's effects oncis-fatty acid-induced lipid body formation. Aspirin inhibitedlipid body formation induced by both AA and OA in humanPMNs and eosinophils (Fig. 2A and B), as did the structurallyunrelated NSAIDs, indomethacin and piroxicam (Fig. 2C).Moreover, sodium salicylate (which does not inhibit COX; seerefs. 19 and 25) was as potent as aspirin in inhibiting lipid bodyformation elicited by both cis-fatty acids (Fig. 2C), suggesting

8 12 0 4 8 12Fatty Acid, tiM Fatty Acid, gM

that NSAID inhibition of lipid body formation was indepen-dent of COX inhibition. Aspirin, indomethacin, and sodiumsalicylate did not inhibit 14C-AA uptake by human PMNsstimulated by AA (data not shown), indicating that theseagents were not acting by diminishing fatty acid uptake.

Aspirin Inhibition of Lipid Body Induction is COX Inde-pendent. To determine the role of the two COX isoforms incis-fatty acid-induced lipid body formation, macrophages fromwild-type and COX-1- or COX-2-deficient mice (27, 28) werestudied. Both AA and OA induced lipid body formation withno differences between wild type and either COX-1 or COX-2deficient macrophages (Fig. 3 A and B), indicating that lipidbody formation is not dependent on COX-1 or COX-2 activity.

Aspirin, sodium salicylate, and indomethacin inhibited AA-induced lipid body formation in macrophages from wild-type

wild-type (+/+) COX-1 (-/-) COX-2 (-/-)

FIG. 3. Aspirin and NSAIDs inhibit cis-fatty acid-induced-lipid body formation independent of COX. Peritoneal macrophages from wild type,and COX-1 (A) and COX-2 (B) deficient mice responded similarly to stimulation with AA and OA and pretreatment with NSAIDs (C). InA andB, AA and OA are represented by squares and circles, respectively; and solid and open symbols, respectively, represent wild-type and COX-deficientmice. Macrophages (106 per ml) were incubated with AA and OA at varying concentrations for 1 hr. Values (mean ± SEM) calculated from 50consecutive macrophages from four mice each counted blindly without knowledge of the genotypes of the mice. In C, for wild-type (+/+) andCOX-1- and COX-2-deficient (-/-) mice, pretreatment with aspirin (open bar), indomethacin (dotted bar), sodium salicylate (vertically stripedbar), or NS-398 (cross-hatched bar) each significantly suppressed lipid body induction compared with AA alone (P < 0.05, Student's t test).Macrophages (106 per ml) were pretreated with aspirin (10 ,ug/ml), indomethacin (1 ,ug/ml), sodium salicylate (10 ,ug/ml), NS-398 (10 mM), orvehicle (0.1% DMSO) for 1 hr, and then stimulated with 15 ,uM AA or vehicle (0.1% ethanol) for 1 hr. Values are expressed as the percent lipidbody numbers induced by 15 ,uM AA alone. (Values for unstimulated cells ranged from 2.8-4.7 lipid bodies/cell, whereas cells stimulated with15 ,uM AA ranged from 8.6-9.1 lipid bodies/cell in different experiments.)

Medical Sciences: Bozza et aL

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1

11094 Medical Sciences: Bozza et al.

AA, iM OA, ,M

0 3.77.5 15 0 2.5 5 10AA, jiM OA, jiM

'IN

;0)00

I40C

I-.

Q0

AA,jM OA, tM

-150 i

40

-100 x

Ur-50 g

*0.0

-800

-600 o0.r

40*400 ECZ

V.

-200 e

0LE3

Priming AA: - + - + - +Wild-type COX-1 (-/-) COX-2 (-/-)

FIG. 4. Cis-fatty acids induce both lipid body formation and enhanced eicosanoid generation. AA- and OA-induced lipid body formation (solidbar) and priming (striped bar) for LTB4 (A), PGE2 (B) and LTC4 (C) production by human PMNs (A and B) and eosinophils (C). Cells (106 perml) were stimulated for 1 hr at 37°C with concentrations ofAA, OA, or vehicle (0.1% ethanol) alone. Data are mean ± SEM from 5-10 independentassays (A and B) or from quadruplicates of a representative experiment (C). With each fatty acid and each cell type, increasing numbers of lipidbodies correlated with increased production of each eicosanoid (r > 0.9, P < 0.05 for all, Fisher's r to z transformation). Asterisk indicatessignificantly greater than value without fatty acid stimulation (P < 0.05, Student's t test). (D) The effect of 15 ,uM AA or vehicle (0.1% ethanol)on lipid body formation (solid bar) and priming (striped bar) for PGE2 production was investigated in peritoneal macrophages from wild type andCOX-1 and COX-2 deficient mice. Data are mean ± SEM from 2-7 mice evaluated blindly without knowledge of the genotypes. Lipid bodies wereenumerated microscopically following osmium staining. LTB4, LTC4, and PGE2 in supernatants were measured by ELISA after incubation with0.5 ,uM A23187 for 15 min.

and COX-1- and COX-2-deficient mice (Fig. 3C). Thus,inhibition by these NSAIDs takes place even in the absence ofCOX-1 or COX-2. Furthermore, NS-398, recognized as aspecific COX-2 inhibitor (30, 31), also inhibited lipid bodyformation in wild-type, and COX-1- and even COX-2-deficientmacrophages (Fig. 3C). Overall, these results indicate thatNSAID inhibition of lipid body formation, including that withthe nominally specific COX-2 inhibitor, NS-398, are indepen-dent of COX inhibition.

Induction of Enhanced Eicosanoid Generation. Since lipidbodies are stores of the eicosanoid precursor AA (15, 16) andcontain eicosanoid-forming enzymes (12, 13, 14), we investi-gated whether increased numbers of lipid bodies correlatedwith enhanced generation of eicosanoids by PMNs and eosin-ophils. Unstimulated leukocytes respond to specific receptor-mediated agonists or submaximal concentrations of calciumionophore by generating low levels of eicosanoids. However, ina process known as "priming," prior stimulation with variousagents, including cis-fatty acids, protein kinase C activators,and PAF, renders cells capable of generating greater quantitiesof eicosanoids (11, 32-36). After PMNs and eosinophils wereincubated with concentrations of AA or OA for 1 hr, lipidbodies were enumerated and replicate leukocytes were stim-ulated with 0.5 ,tM calcium ionophore. AA as well as OA

dose-dependently induced concordant increases in both lipidbody numbers and priming for enhanced LTB4 (Fig. 4A4), PGE2(Fig. 4B), and LTC4 (Fig. 4C) production. Increased numbersof AA- and OA-induced lipid bodies in both cell typescorrelated with the increased quantities of each eicosanoidgenerated by the primed leukocytes. Notably, stimulation ofleukocytes with OA, not itself an eicosanoid precursor fattyacid, led to increments in the production of each eicosanoidthat quantitatively paralleled the incremental increases in lipidbody numbers (PGE2: r, 0.99, P < 0.01; LTB4: r, 0.99, P < 0.01;LTC4: r, 0.95, P < 0.05). Thus, the capacity of two differentleukocyte types to generate greater quantities of COX andlipoxygenase pathway-derived eicosanoids correlated with lev-els of lipid bodies in these cells.To determine which COX is responsible for enhanced

prostaglandin production, we evaluated the effects of COX-1or COX-2 deficiency on priming. Primed COX-2-deficientmacrophages were found to be indistinguishable from wild-type cells in both lipid body and PGE2 formation (Fig. 4D),indicating that COX-2 does not have a role in PGE2 productionby primed macrophages. However, primed COX-1-deficientmacrophages demonstrated severely diminished levels ofPGE2 (Fig. 4D), identifying COX-1 as the enzyme responsiblefor enhanced PGE2 production induced early in AA-treatedmacrophages.

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1

Proc. Natl. Acad. Sci. USA 93 (1996) 11095

FIG. 5. Aspirin and sodium salicylate inhibit cis-fatty acid-induced priming for enhanced leukotriene production. (A) LTB4 production by humanPMNs treated with vehicle control (open bars), cis-fatty acid (solid bars), or cis-fatty acid plus aspirin (striped bars), or sodium salicylate (verticallystriped bars). PMNs (106 per ml), pretreated with aspirin (10 jig/ml), sodium salicylate (10 ,ug/ml), or vehicle (0.1% DMSO) for 1 hr at 37°C, werethen stimulated with 5 ,uM AA, 10 ,uM OA, or vehicle (0.1% ethanol) for 1 hr. Data are mean ± SEM from four to eight independent assaysperformed'in duplicate. (B) LTC4 production by human eosinophils treated with vehicle control (open bars), cis-fatty acid (solid bars), or cis-fattyacid plus aspirin (striped bars). Eosinophils (106 per ml), pretreated with aspirin (10 ,ug/ml) or vehicle for 1 hr at 37°C, were then stimulated with15 ,uM AA, 10 t.M OA or vehicle (0.1% ethanol) for 1 hr. Data are mean ± SEM from quadruplicates of a representative experiment. Afterincubation with 0.5 ALM A23187 for 15 min, LTB4 and LTC4 in supernatants were measured by ELISA. Statistically significant differences (P <0.05, Student's t test) between vehicle alone and fatty acids are represented by a triangle, and between fatty acids alone and with aspirin or sodiumsalicylate pretreatment are indicated by *.

Aspirin Inhibition ofEnhanced Eicosanoid Generation. Weevaluated whether aspirin inhibition of lipid body formationwould affect cis-fatty acid-induced priming for enhanced pro-duction of both COX- and lipoxygenase-derived eicosanoids.Aspirin pretreatment inhibited PGE2 production by humanPMNs and eosinophils in either vehicle- or cis-fatty acid-stimulated leukocytes (data not shown), but this inhibitionmight be due solely to aspirin inhibition of COX. In contrast,aspirin is devoid of direct inhibitory effects upon lipoxygenasesin leukocytes and platelets (37-39). We found that aspirinsignificantly inhibited AA- and OA-induced priming for en-hanced production of 5-lipoxygenase-derived LTB4 by PMNs(Fig. SA) and LTC4 by eosinophils (Fig. 5B) and that sodiumsalicylate likewise inhibited cis-fatty acid-induced priming forenhanced LTB4 production by PMNs (Fig. 5A). Aspirin wasnot acting to block pathways of AA mobilization or metabo-lism activated by calcium ionophore, since aspirin failed toinhibit calcium ionophore-induced LTB4 and LTC4 productionin cells not prestimulated with cis-fatty acids (Fig. 5), and wasnot inhibiting eicosanoid export (no residual intracellulareicosanoids were detectable). Aspirin's inhibition of primingfor enhanced eicosanoid production correlated with its abilityto inhibit cis-fatty acid-induced lipid body formation.

DISCUSSIONOur findings indicate that lipid bodies have roles in theenhanced formation of eicosanoid mediators of inflammation.Lipid bodies are specialized intracellular domains whose for-mation in leukocytes is rapidly inducible within 1 hr bycis-unsaturated fatty acids or by PAF (10, 11). The inductionof lipid body formation correlated with development of anenhanced capacity for eicosanoid formation, a process termed"priming." Interestingly, priming agents, including cis-fattyacids, protein kinase C activators, and PAF, in the concentra-tions and times previously recognized to effectively primePMNs for enhanced leukotriene production (11, 32, 33, 35, 36),are identical with stimulatory agents and conditions necessaryto induce lipid body formation (10, 11). With bothAA and OAstimulation, numbers of lipid bodies correlated with enhancedlevels of LTB4, LTC4, and PGE2 formed by human PMNs andeosinophils (Fig. 3). Likewise, with PAF stimulation, numbers

of lipid bodies induced in PMNs correlated with enhancedlevels of lipoxygenase (LTB4)- and COX (PGE2)-derivedeicosanoids formed in response to submaximal calcium iono-phore (11). Additional support for a relationship between lipidbody formation and heightened eicosanoid formation comesfrom studies with inhibitors. In this study, aspirin and variousNSAIDs both inhibited cis-fatty acid-induced lipid body for-mation and inhibited the enhanced formation of lipoxygenase(LTB4, LTC4)- and COX (PGE2)-derived eicosanoids (Figs. 2,3C, and 5). Inhibition of PAF-induced lipid body formation inPMNs with the mRNA and protein synthesis inhibitors, acti-nomycin D and cycloheximide, respectively, also inhibitedenhanced LTB4 and PGE2 formation (11). Thus, lipid bodyformation may provide a cellular basis for "priming" thataugments eicosanoid generation.

Aspirin and NSAIDs inhibited cis-fatty acid-induced lipidbody formation. This inhibition was independent of inhibitionof either COX-1 or COX-2. AA- and OA-induced lipid bodyinduction in macrophages from COX-1- and COX-2-deficientmice was not impaired; and NSAIDs, including aspirin, sodiumsalicylate, indomethacin, and NS-398, inhibited AA-inducedlipid body formation equally in macrophages from wild-typeand COX-1- or COX-2-deficient mice (Fig. 3). Other evidencealso suggests COX-2 does not participate in lipid body forma-tion. COX-2 induction takes 2-48 hr (1-4), whereas lipid bodyformation occurs within 1 hr. Furthermore, dexamethasone, apotent inhibitor of COX-2 expression (40), did not block lipidbody formation (data not shown). Thus, COX-2 is unlikely tohave a role in the induction of lipid bodies, and since thisinduction can still occur in COX-1-deficient macrophagesneither COX is essential in this process.Whereas stimulation of COX-2 formation is one inducible

mechanism to heighten eicosanoid synthesis, the induction oflipid body formation constitutes another mechanism for en-hancing the synthesis of eicosanoid mediators of inflamma-tion. Lipid body induction develops more rapidly than COX-2induction (1-4). Moreover, whereas COX-2 induction leads tothe enhanced generation of only COX-derived eicosanoids,lipid body induction is associated with the enhanced formationof both lipoxygenase- and COX-derived eicosanoids. Theenhanced COX-derived eicosanoids associated with lipid bodyinduction are likely COX-1 derived since enhanced PGE2

Medical Sciences: Bozza et aL

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1

11096 Medical Sciences: Bozza et al.

generation was deficient in COX-1-, but not COX-2-, deficientmacrophages (Fig. 4D). These findings indicate that COX-1,and not solely induced COX-2, may have a significant role ininflammatory processes, a possibility supported by previousdata demonstrating diminished AA-induced inflammation inCOX-1-deficient mice (27,28). Finally, of special pertinence tounderstanding the antiinflammatory activities of aspirin, as-pirin and NSAIDs, by a COX-independent mechanism, inhibitthe early induction by cis-fatty acids of both lipid bodyformation and priming for enhanced eicosanoid formation inleukocytes. This action of aspirin and related NSAIDs extendstheir antiinflammatory capacity to include suppression ofmore than COX pathway-derived eicosanoids and indicatesthat inhibition of induction of lipid body formation mayconstitute a novel target for antiinflammatory therapies.

This work was supported by National Institutes of Health Grant Al22571 (to P.F.W). P.T.B. is a Pew Fellow in Biomedical Sciences anda recipient of a postdoctoral fellowship from Conselho Nacional dePesquisas (Brazil). S.G.M is a fellow from American Cancer Society(PF #3973). We thank Dr. Craig Gerard, Dr. Anne Nicholson-Weller,Dr. Rodolfo Acuna-Soto, and Dr. Wengui Yu for comments on thework and manuscript, and Lila Tulin for providing synovial fluidsamples.

1. Reddy, S. T. & Herschman, H. R. (1994) J. Biol. Chem. 269,15473-15480.

2. Smith, W. L., Meade, E. A. & DeWitt, D. L. (1994) Ann. N.YAcad. Sci. 714, 136-142.

3. O'Sullivan, M. G., Chilton, F. H., Huggins, E. M., Jr., & McCall,C. E. (1992) J. Biol. Chem. 267, 14547-14550.

4. Lee, S. H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W.,Zhong, H., Liou, S., Simmons, D. & Hwang, D. (1992) J. Biol.Chem. 267, 25934-25938.

5. Coimbra, A. & Lopes-Vaz, A. (1971) J. Histochem. Cytochem. 19,551-557.

6. Schlesinger, P. A., Stillman, M. T. & Peterson, L. (1982)ArthritisRheum. 25, 1365-1368.

7. Weinstein, J. (1980) Arch. Intern. Med. 140, 560-561.8. Triggiani, M., Oriente, A., Seeds, M. C., Bass, D. A., Marone, G.

& Chilton, F. H. (1995) J. Exp. Med. 182, 1181-1190.9. Robinson, J. M., Karnovsky, M. L. & Karnovsky, M. J. (1982) J.

Cell Bio. 95, 933-942.10. Weller, P. F., Ryeom, S. W., Picard, S. T., Ackerman, S. J. &

Dvorak, A. M. (1991) J. Cell Biol. 113, 137-146.11. Bozza, P. T., Payne, J. L., Goulet, J. L. & Weller, P. F. (1996) J.

Exp. Med. 183, 1515-1525.12. Dvorak, A. M., Morgan, E., Schleimer, R. P., Ryeom, S. W.,

Lichtenstein, L. M. & Weller, P. F. (1992) J. Histochem. Cyto-chem. 40, 759-769.

13. Dvorak, A. M., Schleimer, R. P., Dvorak, A. M., Lichtenstein,L. M. & Weller, P. F. (1992) Int. Arch. AllergyAppl. Immunol. 99,208-217.

14. Dvorak, A. M., Morgan, E., Tzizik, D. M. & Weller, P. F. (1994)Int. Arch. Allergy Appl. Immunol. 105, 245-250.

15. Weller, P. F., Ackerman, S. J., Nicholson-Weller, A. & Dvorak,A. M. (1989) Am. J. Pathol. 135, 947-959.

16. Weller, P. F., Monahan-Earley, R. A., Dvorak, H. F. & Dvorak,A. M. (1991) Am. J. Pathol. 138, 141-148.

17. Weissmann, G. (1991) Sci. Am. 264, 84-90.18. Vane, J. (1994) Nature (London) 367, 215-216.19. Vane, J. R. (1971) Nat. New Biol. 231, 232-235.20. Roth, G. J., Machuga, E. T. & Ozols, J. (1983) Biochemistry 22,

4672-4675.21. Patrignani, P., Filabozzi, P. & Patrono, C. (1982) J. Clin. Invest.

69, 1366-1372.22. Roth, G. J. & Calverley, D. C. (1994) Blood 83, 885-898.23. Zurier, R. B. & Quagliata, F. (1971) Nature (London) 234,

304-305.24. Abramson, S. B., Leszczynska-Piziak, J., Haines, K. & Reibman,

J. (1991) Biochem. Pharmacol. 41, 1567-1573.25. Abramson, S., Korchak, H., Ludewig, R., Edelson, H., Haines, K.,

Levin, R. I., Herman, R., Rider, L., Kimmel, S. & Weissmann, G.(1985) Proc. Natl. Acad. Sci. USA 82, 7227-7231.

26. Villanueva, M., Heckenberger, R., Strobach, H., Palmer, M. &Schror, K. (1993) Br. J. Clin. Pharmacol. 35, 235-242.

27. Morham, S. G., Lagenbach, R., Loftin, C. D., Tiano, H. F.,Vouloumanos, N., Jennette, J. C., Mahler, J. F., Kluckman,K. D., Ledford, A., Lee, C. A. & Smithies, 0. (1995) Cell 83,473-482.

28. Langenbach, R., Morham, S. G., Tiano, H. F., Loftin, C. D.,Ghanayem, B. I., Chulada, P. C., Mahler, J. F., Lee, C. A., Gould-ing, E. H., Kluckman, K. D., Kim, H. S. & Smithies, 0. (1995)Cell 83, 483-492.

29. Galli, S. J., Dvorak, A. M., Peters, S. P., Schulman, E. S., Mac-Glashan, D. W., Jr., Isomura, T., Pyne, K., Harvey, V. S., Ham-mel, I., Lichtenstein, L. M. & Dvorak, H. F. (1985) Prostaglan-dins, Leukotrienes, and Lipoxins, ed. Bailey, J. M. (Plenum, NewYork), pp. 221-239.

30. Copeland, R. A., Williams, J. M., Giannaras, J., Nurnberg, S.,Covington, M., Pinto, D., Pick, S. & Trzaskos, J. M. (1994) Proc.Natl. Acad. Sci. USA 91, 11202-11206.

31. Futaki, N., Takahashi, S., Yokoyama, M., Arai, I., Higuchi, S. &Otomo, S. (1994) Prostaglandins 47, 55-59.

32. Bauldry, S. A., Wykle, R. L. & Bass, D. A. (1988) J. Biol. Chem.263, 16787-16795.

33. Raulf, M. & Konig, W. (1988) Immunology 64, 51-59.34. Haines, K. A., Giedd, K. N., Rich, A. M., Korchak, H. M. &

Weissmann, G. (1987) Biochem. J. 241, 55-62.35. Glaser, K. B., Asmis, R. & Dennis, E. A. (1990) J. Biol. Chem.

265, 8658-8664.36. Liles, W. C., Meier, K. E. & Henderson, W. R. (1987) J. Immu-

nol. 138, 3396-3402.37. Cerletti, C., Livio, M., Doni, M. G. & De Gaetano, G. (1983)

Biochim. Biophys. Acta 759, 125-127.38. Cerletti, C. (1985) Int. J. Tissue React. 7, 309-312.39. Punnonen, K. & Uotila, P. (1984) Prostaglandins Leukotrienes.

Med. 15, 177-185.40. Kujubu, D. A. & Herschman, H. R. (1992) J. Biol. Chem. 267,

7991-7994.

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 10

, 202

1