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

Leukocyte lipid body formation and generation: Cyclooxygenase-independent inhibition by (nonsteroidal antiinflammatory agents/cyclooxygenase knockout// endoperoxide H synthase/) 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 , Beth Israel Hospital, Harvard Medical School, Boston, MA 02215-5491; tDepartment of Pathology, University of North Carolina, Chapel Hill, NC 27599-7525; and *Laboratory of Experimental Carcinogenesis and Mutagenesis, 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 review April 8, 1996)

ABSTRACT Lipid bodies, cytoplasmic inclusions that de- inducible structures. Lipid bodies are prominent in leukocytes velop in cells associated with , are inducible at sites of natural and experimental inflammation, including structures that might participate in generating inflammatory leukocytes from joints of patients with inflammatory arthritis . Cis-unsaturated fatty acids (arachidonic and (5-7), the airways of patients with acute respiratory distress oleic acids) rapidly induced lipid body formation in leuko- syndrome (8), and casein- or lipopolysaccharide-elicited cytes, and this lipid body induction was inhibited by aspirin guinea pig peritoneal exudates (9). The formation of lipid and nonsteroidal antiinflammatory drugs (NSAIDs). Several bodies is a regulated process that involves activation of intra- findings indicated that the inhibitory effect of aspirin and cellular signaling pathways, including kinase C and NSAIDs on lipid body formation was independent of cycloox- (10, 11). Inhibitors of mRNA and protein ygenase (COX) inhibition. First, the non-COX inhibitor, synthesis inhibit lipid body formation, indicating that induction , was as potent as aspirin in inhibiting lipid of lipid body formation depends on the activation of specific body formation elicited by cis-fatty acids. Second, cis-fatty expression and new protein synthesis (11). The possibility acid-induced lipid body formation was not impaired in mac- that lipid bodies have a role in the formation of eicosanoid rophages from COX-1 or COX-2 genetically deficient mice. mediators of inflammation is suggested by their components. Finally, NSAIDs inhibited -induced lipid Lipid bodies in several cell types contain COX and 5-lipoxy- body formation likewise in macrophages from wild-type and genase (refs. 12-14 and unpublished observations), both of COX-1- and COX-2-deficient mice. An enhanced capacity to which use arachidonic acid (AA) to initiate synthesis of generate eicosanoids developed after 1 hr concordantly with inflammatory eicosanoids. In addition, stores ofAA have been cis-fatty acid-induced lipid body formation. Arachidonic and localized to lipid bodies in leukocytes, including polymorpho- oleic acid-induced lipid body numbers correlated with the nuclear leukocytes (PMNs) and eosinophils (15, 16). More- enhanced levels of leukotrienes B4 and C4 and produced after submaximal calcium ionophore stimula- over, Bozzaetal. (11) have demonstrated, in -activating tion. Aspirin and NSAIDs inhibited both induced lipid body factor (PAF)-stimulated PMNs, a correlation between en- formation and the enhanced capacity for forming leukotrienes hanced capacity for eicosanoid formation and levels of lipid as well as . Our studies indicate that lipid body body induction. formation is an inducible early response in leukocytes that Aspirin and other nonsteroidal antiinflammatory agents correlates with enhanced eicosanoid synthesis. Aspirin and (NSAIDs), widely used in treating inflammatory processes, NSAIDs, independent of COX inhibition, inhibit cis-fatty including arthritis (17, 18), are believed to act principally by acid-induced lipid body formation in leukocytes and in con- inhibition of COX thus blocking the initial step in prostaglan- cert inhibit the enhanced synthesis of leukotrienes and pros- din synthesis (19). This mechanism may not, however, fully taglandins. account for the antiinflammatory effects of NSAIDs. Low doses of aspirin and other NSAIDs are required for COX The capacity of cells to generate eicosanoid mediators of inhibition in vitro and in vivo (19-21), whereas higher doses are inflammation may be enhanced by inducible mechanisms. One required for antirheumatic effects in vivo (17, 22). In addition, such mechanism is the induction of expression of a specific sodium salicylate has minimal activity as a COX inhibitor (19), isoform of prostaglandin endoperoxide H synthase [cycloox- but is as effective as aspirin for inhibiting inflammatory ygenase (COX)], COX-2. Various cytokines and pro- manifestations of arthritis in humans and experimental models inflammatory stimuli elicit the de novo synthesis of COX-2 (17, 22, 23). NSAIDs, by mechanisms other than COX inhi- over two or more hours that leads to increased formation of bition, also inhibit PMN activation, including the synthesis of COX pathway-derived eicosanoids (1-4). Inducible mecha- leukotrienes, eicosanoids formed by a lipoxygenase, and not a nisms that enhance eicosanoid production are attractive as COX, pathway (24-26). Thus, there is evidence to suggest that potential targets for selective antiinflammatory treatments. the antiinflammatory effects of aspirin-like drugs are not Here we discuss a new mechanism for enhanced eicosanoid solely attributable to COX inhibition. Correlation of lipid body synthesis based on the induction of lipid body in leukocytes. formation with inflammatory responses led us to investigate Lipid bodies are lipid-rich cytoplasmic inclusions that char- whether inhibition of lipid body formation could be a mech- acteristically develop in vivo in cells associated with inflam- mation; although neither the mechanisms of formation nor the Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; NSAID, function of lipid bodies are fully understood, lipid bodies are nonsteroidal antiinflammatory drug; OA, oleic acid; PAF, platelet- activating factor; PMN, polymorphonuclear leukocyte (neutrophil); DMSO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be sent at present address: Beth payment. This article must therefore be hereby marked "advertisement" in Israel Hospital, 330 Brookline Avenue, DA-617, Boston, MA 02215- accordance with 18 U.S.C. §1734 solely to indicate this fact. 5491. e-mail: [email protected]. 11091 Downloaded by guest on October 2, 2021 11092 Medical Sciences: Bozza et al. Proc. Natl. Acad. Sci. USA 93 (1996) anism for some of the antiinflammatory effects of aspirin and three times in Ca2+/Mg2+-free HBSS and analysis of incor- NSAIDs. porated 14C-AA in the cell pellet was performed by liquid scintillation counting. MATERIALS AND METHODS Statistical Analysis. Results, expressed as mean ± SEM, were analyzed by analysis of variance followed by the New- Materials. Aspirin, indomethacin, , man-Keuls Student's t test with the level of significance set at (sodium salt), oleic acid (OA), AA (Sigma), 14C-AA (1.5-2.2 P < 0.05. Correlation coefficients were determined by linear GBq/mmol; DuPont/NEN), A23187 (Calbiochem), and NS- regression, and correlation analysis was performed by Fisher's 398 (Biomol, Plymouth Meeting, PA) were obtained as noted. r to z transformation with the level of significance set at P < Human and Murine Leukocytes. Human PMNs were puri- 0.05. fied from blood obtained from healthy adult volunteers and collected into acidified citrate. After addition of 6% dextran 70 (McGaw, Irvine, CA), erythrocytes were allowed to sediment RESULTS for 1 hr at 25°C. The leukocyte-rich supernatant was overlaid Lipid Body Formation. Normal PMNs contain few lipid onto an equal volume of Ficoll/Paque (Pharmacia), and bodies (Fig. 1A); but PMNs at sites of inflammatory reactions, centrifuged at 400 x g for 20 min. PMNs (>95% pure, the such as septic arthritis (Fig. contain difference were eosinophils) were recovered from the pellet 1B), markedly increased and washed in Ca2+/Mg2+ free Hanks' balanced salt solution numbers of lipid bodies. Common alcohol-based hematologic (HBSS). Residual erythrocytes were lysed with hypotonic stains cause dissolution of lipid bodies (Fig. 1D), which pre- saline. Eosinophils (>80% pure, the difference were neutro- cludes their recognition. However, with appropriate lipid phils) were obtained from a donor with idiopathic hypereosi- fixation, increased lipid body numbers have been observed in nophilic syndrome using the same procedure. Synovial fluid many types of leukocytes when these cells are involved in a leukocytes were examined in samples ofjoint fluids submitted variety of infectious, allergic, neoplastic, and other inflamma- to the clinical hematology laboratory. COX-1 and COX-2 tory diseases (15, 29). Normal PMNs can be stimulated with genetically deficient mice were generated as described (27, 28). cis-unsaturated, but not saturated, fatty acids, such as AA and Experiments were carried out with 8- to 12-week-old mice. the non-eicosanoid precursor OA, to rapidly form (within 1 hr) Macrophages from COX-1 and COX-2 genetically deficient new lipid bodies (Fig. 1C) morphologically comparable to lipid mice and wild-type littermates were obtained by lavaging the peritoneal cavity with 5 ml of cold HBSS containing heparin A B (10 units/ml). .... ^.; Lipid Body Induction and Enumeration. Leukocytes (106 cells per ml) were incubated with concentrations of AA, OA, or vehicle (0.1% ethanol) at 37°C in a 5% C0295% 02 atmosphere, and after 1 hr leukocytes (105 cells per slide) were cytocentrifuged onto glass slides. For inhibitor studies, leuko- cytes were pretreated for 1 hr with concentrations of inhibitors or vehicle as indicated. Cell viability, determined by Trypan blue dye exclusion at the end of each experiment, was always greater than 90%. Stock solutions of A23187, indomethacin, C D aspirin, sodium salicylate, piroxicam, and NS-398, prepared in dimethyl sulfoxide (DMSO) and stored at -20°C, were diluted Jo in Ca2+/Mg2+-free HBSS to the indicated concentration im- 40 mediately before use. The final DMSO concentration was dol. always lower than 0.1% and had no effect on lipid body A numbers. Slides, while still moist, were fixed in 3.7% formal- 91 dehyde in Ca2+/Mg2+-free HBSS (pH 7.4), rinsed in 0.1 M cacodylate 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 (3 F min), rinsed in H20, dried, and mounted. The morphology of fixed cells was observed, and lipid bodies were enumerated with phase contrast microscopy and a 10OX objective lens in 50 consecutively scanned leukocytes. Prostaglandin (PG)E2, (LT)B4, and LTC4 As- says. Leukocytes (106 cells per ml) were stimulated with concentrations of AA, OA, or vehicle (0.1% ethanol) at 37°C for 1 hr to induce lipid body formation; after which a sample was taken for lipid body enumeration, and the cells were washed in Ca2+/Mg2+-free HBSS. Leukocytes, resuspended in FIG. 1. Lipid bodies in human PMNs. Lipid bodies are sparse in 1 ml of HBSS containing Ca2+/Mg2+, were stimulated with 0.5 normal donor-derived blood PMNs (A) but very prominent in synovial AM A23187 at 37°C for 15 min. Reactions were stopped on ice, fluid PMNs from a patient with septic arthritis (B) and in normal and samples were centrifuged (800 x g for 10 min at 4°C). In donor blood-derived PMNs after stimulation in vitro with 5 IIM AA for each replicate supernatant or extracted cell pellet, PGE2, 1 hr at 37°C (C). Without prior lipid preservation, however, lipid LTB4, and LTC4 were assayed in duplicate by ELISA (Cayman bodies are solubilized by routine staining and become undetectable Chemicals, Ann Arbor, MI). (D) in the same synovial fluid PMNs as in B. Murine peritoneal AA macrophages contain few lipid bodies in resting conditions (E); but can Uptake. Neutrophils (106 per ml) were labeled with also be stimulated in vitro with 15 ,uM AA for 1 hr at 37°C to form lipid 14C-AA (0.2 ,uM) together with either vehicle, aspirin (10 bodies (F). Cytocentrifuged leukocytes were fixed in 3.7% formalde- ,ug/ml), indomethacin (1 ,ug/ml), or sodium salicylate (10 hyde in HBSS and stained with osmium tetroxide, (A-C, E, and F) or ,ug/ml) for 1 hr. After the incubation period, lipid body were fixed in methanol and stained with Diff-Quik (Baxter, Miami, formation was elicited by AA (15 ,uM). The cells were washed FL) (D). Downloaded by guest on October 2, 2021 Medical Sciences: Bozza et aL 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) and eosinophils (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 (10 jig/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. Values are expressed as mean±SEM of 50 consecutively counted cells of one representative experiment from 3-5 independent assays. Statistically significant differences (P < 0.05, Student's t test) between lipid bodies/cell with vehicle alone and fatty acid stimuli are noted by a triangle, and between fatty acid stimuli alone and pretreatment with ASA or NSAID by *. bodies formed in vivo (10, 15, 16). As with human cells, mouse that NSAID inhibition of lipid body formation was indepen- macrophages contain only few lipid bodies under normal dent of COX inhibition. Aspirin, indomethacin, and sodium conditions (Fig. 1E) and can also be stimulated in vitro with salicylate did not inhibit 14C-AA uptake by human PMNs cis-unsaturated fatty acids to form lipid bodies (Fig. 1F). stimulated by AA (data not shown), indicating that these Aspirin and NSAID Inhibition of Lipid Body Induction. agents were not acting by diminishing fatty acid uptake. Since lipid body numbers increase during inflammatory pro- Aspirin Inhibition of Lipid Body Induction is COX Inde- cesses and might be related to the formation of eicosanoid pendent. To determine the role of the two COX isoforms in mediators of inflammation, we investigated aspirin's effects on cis-fatty acid-induced lipid body formation, macrophages from cis-fatty acid-induced lipid body formation. Aspirin inhibited wild-type and COX-1- or COX-2-deficient mice (27, 28) were lipid body formation induced by both AA and OA in human studied. Both AA and OA induced lipid body formation with PMNs and eosinophils (Fig. 2A and B), as did the structurally no differences between wild type and either COX-1 or COX-2 unrelated NSAIDs, indomethacin and piroxicam (Fig. 2C). deficient macrophages (Fig. 3 A and B), indicating that lipid Moreover, sodium salicylate (which does not inhibit COX; see body formation is not dependent on COX-1 or COX-2 activity. refs. 19 and 25) was as potent as aspirin in inhibiting lipid body Aspirin, sodium salicylate, and indomethacin inhibited AA- formation elicited by both cis-fatty acids (Fig. 2C), suggesting induced lipid body formation in macrophages from wild-type

8 12 0 4 8 12 wild-type (+/+) COX-1 (-/-) COX-2 (-/-) Fatty Acid, tiM Fatty Acid, gM 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 and B, AA and OA are represented by squares and circles, respectively; and solid and open symbols, respectively, represent wild-type and COX-deficient mice. Macrophages (106 per ml) were incubated with AA and OA at varying concentrations for 1 hr. Values (mean ± SEM) calculated from 50 consecutive macrophages from four mice each counted blindly without knowledge of the genotypes of the mice. In C, for wild-type (+/+) and COX-1- and COX-2-deficient (-/-) mice, pretreatment with aspirin (open bar), indomethacin (dotted bar), sodium salicylate (vertically striped bar), 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), or vehicle (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 lipid body numbers induced by 15 ,uM AA alone. (Values for unstimulated cells ranged from 2.8-4.7 lipid bodies/cell, whereas cells stimulated with 15 ,uM AA ranged from 8.6-9.1 lipid bodies/cell in different experiments.) Downloaded by guest on October 2, 2021 11094 Medical Sciences: Bozza et al. Proc. Natl. Acad. Sci. USA 93 (1996)

-150 i

40 'IN -100 x Ur -50 g

*0 0;0) .0 0 I40C AA, iM OA, ,M AA,jM OA, tM -800

I-. -600 o 0.r

*400 40E Q0 CZ V.

-200 e E30L

0 3.77.5 15 0 2.5 5 10 Priming AA: - + - + - + AA, jiM OA, jiM 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 (solid bar) and priming (striped bar) for LTB4 (A), PGE2 (B) and LTC4 (C) production by human PMNs (A and B) and eosinophils (C). Cells (106 per ml) 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 independent assays (A and B) or from quadruplicates of a representative experiment (C). With each fatty acid and each cell type, increasing numbers of lipid bodies correlated with increased production of each eicosanoid (r > 0.9, P < 0.05 for all, Fisher's r to z transformation). Asterisk indicates significantly 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 and COX-1 and COX-2 deficient mice. Data are mean ± SEM from 2-7 mice evaluated blindly without knowledge of the genotypes. Lipid bodies were enumerated microscopically following osmium staining. LTB4, LTC4, and PGE2 in supernatants were measured by ELISA after incubation with 0.5 ,uM A23187 for 15 min. and COX-1- and COX-2-deficient mice (Fig. 3C). Thus, dose-dependently induced concordant increases in both lipid inhibition by these NSAIDs takes place even in the absence of body numbers and priming for enhanced LTB4 (Fig. 4A4), PGE2 COX-1 or COX-2. Furthermore, NS-398, recognized as a (Fig. 4B), and LTC4 (Fig. 4C) production. Increased numbers specific COX-2 inhibitor (30, 31), also inhibited lipid body of AA- and OA-induced lipid bodies in both cell types formation in wild-type, and COX-1- and even COX-2-deficient correlated with the increased quantities of each eicosanoid macrophages (Fig. 3C). Overall, these results indicate that generated by the primed leukocytes. Notably, stimulation of NSAID inhibition of lipid body formation, including that with leukocytes with OA, not itself an eicosanoid precursor fatty the nominally specific COX-2 inhibitor, NS-398, are indepen- acid, led to increments in the production of each eicosanoid dent of COX inhibition. that quantitatively paralleled the incremental increases in lipid Induction of Enhanced Eicosanoid Generation. Since lipid body numbers (PGE2: r, 0.99, P < 0.01; LTB4: r, 0.99, P < 0.01; bodies are stores of the eicosanoid precursor AA and LTC4: r, 0.95, P < 0.05). Thus, the capacity of two different (15, 16) leukocyte types to generate greater quantities of COX and contain eicosanoid-forming (12, 13, 14), we investi- lipoxygenase pathway-derived eicosanoids correlated with lev- gated whether increased numbers of lipid bodies correlated els of lipid bodies in these cells. with enhanced generation of eicosanoids by PMNs and eosin- To determine which COX is responsible for enhanced ophils. Unstimulated leukocytes respond to specific - prostaglandin production, we evaluated the effects of COX-1 mediated agonists or submaximal concentrations of calcium or COX-2 deficiency on priming. Primed COX-2-deficient ionophore by generating low levels of eicosanoids. However, in macrophages were found to be indistinguishable from wild- a process known as "priming," prior stimulation with various type cells in both lipid body and PGE2 formation (Fig. 4D), agents, including cis-fatty acids, protein kinase C activators, indicating that COX-2 does not have a role in PGE2 production and PAF, renders cells capable of generating greater quantities by primed macrophages. However, primed COX-1-deficient of eicosanoids (11, 32-36). After PMNs and eosinophils were macrophages demonstrated severely diminished levels of incubated with concentrations of AA or OA for 1 hr, lipid PGE2 (Fig. 4D), identifying COX-1 as the responsible bodies were enumerated and replicate leukocytes were stim- for enhanced PGE2 production induced early in AA-treated ulated with 0.5 ,tM calcium ionophore. AA as well as OA macrophages. Downloaded by guest on October 2, 2021 Medical Sciences: Bozza et aL 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 human PMNs treated with vehicle control (open bars), cis-fatty acid (solid bars), or cis-fatty acid plus aspirin (striped bars), or sodium salicylate (vertically striped 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, were then 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 assays performed'in duplicate. (B) LTC4 production by human eosinophils treated with vehicle control (open bars), cis-fatty acid (solid bars), or cis-fatty acid 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 with 15 ,uM AA, 10 t.M OA or vehicle (0.1% ethanol) for 1 hr. Data are mean ± SEM from quadruplicates of a representative experiment. After incubation 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 sodium salicylate pretreatment are indicated by *. Aspirin Inhibition ofEnhanced Eicosanoid Generation. We of lipid bodies induced in PMNs correlated with enhanced evaluated whether aspirin inhibition of lipid body formation levels of lipoxygenase (LTB4)- and COX (PGE2)-derived would affect cis-fatty acid-induced priming for enhanced pro- eicosanoids formed in response to submaximal calcium iono- duction of both COX- and lipoxygenase-derived eicosanoids. phore (11). Additional support for a relationship between lipid Aspirin pretreatment inhibited PGE2 production by human body formation and heightened eicosanoid formation comes PMNs and eosinophils in either vehicle- or cis-fatty acid- from studies with inhibitors. In this study, aspirin and various stimulated leukocytes (data not shown), but this inhibition NSAIDs both inhibited cis-fatty acid-induced lipid body for- might be due solely to aspirin inhibition of COX. In contrast, mation and inhibited the enhanced formation of lipoxygenase aspirin is devoid of direct inhibitory effects upon (LTB4, LTC4)- and COX (PGE2)-derived eicosanoids (Figs. 2, in leukocytes and (37-39). We found that aspirin 3C, and 5). Inhibition of PAF-induced lipid body formation in significantly inhibited AA- and OA-induced priming for en- PMNs with the mRNA and protein synthesis inhibitors, acti- hanced production of 5-lipoxygenase-derived LTB4 by PMNs nomycin D and cycloheximide, respectively, also inhibited (Fig. SA) and LTC4 by eosinophils (Fig. 5B) and that sodium enhanced LTB4 and PGE2 formation (11). Thus, lipid body salicylate likewise inhibited cis-fatty acid-induced priming for formation may provide a cellular basis for "priming" that enhanced LTB4 production by PMNs (Fig. 5A). Aspirin was augments eicosanoid generation. not acting to block pathways of AA mobilization or metabo- Aspirin and NSAIDs inhibited cis-fatty acid-induced lipid lism activated by calcium ionophore, since aspirin failed to body formation. This inhibition was independent of inhibition inhibit calcium ionophore-induced LTB4 and LTC4 production of either COX-1 or COX-2. AA- and OA-induced lipid body in cells not prestimulated with cis-fatty acids (Fig. 5), and was induction in macrophages from COX-1- and COX-2-deficient not inhibiting eicosanoid export (no residual intracellular mice was not impaired; and NSAIDs, including aspirin, sodium eicosanoids were detectable). Aspirin's inhibition of priming salicylate, indomethacin, and NS-398, inhibited AA-induced for enhanced eicosanoid production correlated with its ability lipid body formation equally in macrophages from wild-type to inhibit cis-fatty acid-induced lipid body formation. and COX-1- or COX-2-deficient mice (Fig. 3). Other evidence also suggests COX-2 does not participate in lipid body forma- DISCUSSION tion. COX-2 induction takes 2-48 hr (1-4), whereas lipid body formation occurs within 1 hr. Furthermore, dexamethasone, a Our findings indicate that lipid bodies have roles in the potent inhibitor of COX-2 expression (40), did not block lipid enhanced formation of eicosanoid mediators of inflammation. body formation (data not shown). Thus, COX-2 is unlikely to Lipid bodies are specialized intracellular domains whose for- have a role in the induction of lipid bodies, and since this mation in leukocytes is rapidly inducible within 1 hr by induction can still occur in COX-1-deficient macrophages cis-unsaturated fatty acids or by PAF (10, 11). The induction neither COX is essential in this process. of lipid body formation correlated with development of an Whereas stimulation of COX-2 formation is one inducible enhanced capacity for eicosanoid formation, a process termed mechanism to heighten eicosanoid synthesis, the induction of "priming." Interestingly, priming agents, including cis-fatty lipid body formation constitutes another mechanism for en- acids, protein kinase C activators, and PAF, in the concentra- hancing the synthesis of eicosanoid mediators of inflamma- tions and times previously recognized to effectively prime tion. Lipid body induction develops more rapidly than COX-2 PMNs for enhanced leukotriene production (11, 32, 33, 35, 36), induction (1-4). Moreover, whereas COX-2 induction leads to are identical with stimulatory agents and conditions necessary the enhanced generation of only COX-derived eicosanoids, to induce lipid body formation (10, 11). With both AA and OA lipid body induction is associated with the enhanced formation stimulation, numbers of lipid bodies correlated with enhanced of both lipoxygenase- and COX-derived eicosanoids. The levels of LTB4, LTC4, and PGE2 formed by human PMNs and enhanced COX-derived eicosanoids associated with lipid body eosinophils (Fig. 3). Likewise, with PAF stimulation, numbers induction are likely COX-1 derived since enhanced PGE2 Downloaded by guest on October 2, 2021 11096 Medical Sciences: Bozza et al. Proc. Natl. Acad. Sci. USA 93 (1996) generation was deficient in COX-1-, but not COX-2-, deficient 15. Weller, P. F., Ackerman, S. J., Nicholson-Weller, A. & Dvorak, macrophages (Fig. 4D). These findings indicate that COX-1, A. M. (1989) Am. J. Pathol. 135, 947-959. and not solely induced COX-2, may have a significant role in 16. Weller, P. F., Monahan-Earley, R. A., Dvorak, H. F. & Dvorak, inflammatory processes, a possibility supported by previous A. M. (1991) Am. J. Pathol. 138, 141-148. data demonstrating diminished AA-induced inflammation in 17. Weissmann, G. (1991) Sci. Am. 264, 84-90. COX-1-deficient mice (27,28). Finally, of special pertinence to 18. Vane, J. (1994) Nature (London) 367, 215-216. understanding the antiinflammatory activities of aspirin, as- 19. Vane, J. R. (1971) Nat. 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Abramson, S., Korchak, H., Ludewig, R., Edelson, H., Haines, K., This work was supported by National Institutes of Health Grant Al Levin, R. I., Herman, R., Rider, L., Kimmel, S. & Weissmann, G. 22571 (to P.F.W). P.T.B. is a Pew Fellow in Biomedical Sciences and (1985) Proc. Natl. Acad. Sci. USA 82, 7227-7231. a recipient of a postdoctoral fellowship from Conselho Nacional de 26. Villanueva, M., Heckenberger, R., Strobach, H., Palmer, M. & Pesquisas (Brazil). S.G.M is a fellow from American Cancer Society Schror, K. (1993) Br. J. Clin. Pharmacol. 35, 235-242. (PF #3973). We thank Dr. Craig Gerard, Dr. Anne Nicholson-Weller, 27. Morham, S. G., Lagenbach, R., Loftin, C. D., Tiano, H. F., Dr. Rodolfo Acuna-Soto, and Dr. Wengui Yu for comments on the Vouloumanos, N., Jennette, J. C., Mahler, J. F., Kluckman, work and manuscript, and Lila Tulin for providing synovial fluid K. D., Ledford, A., Lee, C. A. & Smithies, 0. (1995) Cell 83, samples. 473-482. 28. Langenbach, R., Morham, S. 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