Induction of Interleukin 18 Expression from Human Peripheral Blood Monocyte-derived Macrophages by 9-Hydroxyoctadecadienoic Acid*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Amerian Soiety for Biohemistry and Moleular Biology, In. Vol. 267, No. 20, Issue of July 15, pp ,1992 Printed in U. S.A. Indution of Interleukin 18 Expression from Human Peripheral Blood Monoyte-derived Marophages by 9-Hydroxyotadeadienoi Aid* (Reeived for publiation, Otober 21, 1991) George KuS, Craig E. Thomas, Ann L. Akeson, and Rihard L. Jakson From the Marion Merrell Dow Researh Institute, Cininnati, Ohio Oxidatively modified low density lipoproteins (LDL) haps by marophages or endothelial ells, plays a major role have reently been proposed to play a role in athero- in the pathogenesis of atheroslerosis (1). With regard to genesis by promoting foam ell formation and endothe- foam ell formation, these modified LDL are reognized and lial ell toxiity. The purpose of the present study was internalized by a savenger reeptor on marophages, resultto determine whether modified LDL ould also indue ing in lipid aumulation. Sine this reeptor is not downmarophage release of interleukin 18 (IL-lB), a yto- regulated by ytoplasmi holesterol (2), internalization of kine whih enhanes vasular smooth musle ell pro- lipoprotein holesterol results in massive aumulation of liferation, another feature of the atherosleroti pro- holesteryl esters within the marophages leading to foam ell ess. LDL were oxidatively modified by inubation with formation (3). either Cu2+ (Cu2+-LDL) or human peripheral blood The proliferation of smooth musle ells in the atheroslemonoyte-derived marophages (M-LDL). Inubation of these modified LDL with marophages (6 X 10 ells/ roti lesion is promoted by a number of growth fators (4). ulture) resulted in a dose-dependent indution of IL- One of these growth fators is interleukin-i@ (IL-16) (5-8), a 18 release. At 300 pg protein/ml, Cu2+-LDL and M- LDL indued 422 and 333 pg of IL-l@/ulture, respetively. Saponified Cu2+-LDL and M-LDL were shown to ontain 9- and 13-hydroxyotadeadienoi aid (HODE), lipid oxidation produts of linoleate. When tested for ativity in marophage ulture (3 X lo6 ells/ ulture), it was found that 9-HODE and 13-HODE (final onentration 33 p ~ indued ) the release of 122 lipid hydroperoxides (lo), we hypothesized that lipid oxidaand 43 pg of IL-l@/ulture, respetively, whereas untreated ells released only 4 pg of IL- lb/ulture. Inubation of marophages with holesteryl-9-hode also indued IL-18 release; however, the degree of indution of IL- 18 release by 9-HODE or its holesteryl ester relative to modified LDL suggests that other omponents in oxidized LDL may also ontribute to IL-18 indution. 9-HODE was rapidly taken up by maro- phages, and the kinetis were similar IL- to 18 release. A 1.6- to 6-fold inrease in the level of IL-18 mrna was deteted as little as 3-h post-9-hode treatment. The indution of IL- 18 release from human monoytederived marophages by 9-HODE and holesteryl-9- HODE suggests a role for modified LDL, and its assoiated linoleate oxidation produts, in vasular smooth musle ell proliferation. Reent evidene suggests that the ell-mediated oxidative modifiation of plasma low density lipoproteins (LDL), per- * The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked advertisement in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. $ To whom orrespondene should be addressed Marion Merrell Dow Researh Inst., 2110 E. Galbraith Rd., Cininnati, OH Tel.: ; Fax: The abbreviations used are: LDL, low density lipoproteins; ALDL, aetylated LDL; Cu2+-LDL, opper-oxidized LDL; M-LDL, marophage-oxidized LDL; ELISA, enzyme-linked immunosorbent assay; HODE, hydroxyotadeadienoi aid; HPODE, hydroperoxyotadeadienoi aid; IL-16, interleukin 18; LPS, lipopolysaharide; TBARS, thiobarbituri aid-reative substanes; TNFa, tumor nerosis fator a; HPLC, high performane liquid hromatography ytokine known to be released by ativated marophages. Minimally modified LDL indue monoyte olony-stimulating fator and monoyte hemotati protein-1 expression in endothelial ells, and these fators are released during the atherogeni proess (9). Sine minimally modified LDL have been shown to ontain low levels of thiobarbituri aid-reative substanes (TBARS), whih are derived primarily from tion produts may play a role in ytokine indution. In this regard, a reent report has shown that probuol, a lipophili antioxidant, inhibits ex vivo lipopolysaharide (LPS)-indued IL-lP release from murine peritoneal marophages (11). Oxidized lipids have also been shown to be involved in LPS stimulation of marophages (12) and protein kinase C ativation (13). These various observations indiate that produts of lipid peroxidation may be involved in the signal transdution leading to IL-16 release. Sine oxidized derivatives of linolei aid and holesteryl linoleate, i.e. hydroxyotadeadienoi aids (HODEs) and holesteryl-hodes, are present in human atheroma (14-16), it was of interest to determine whether these lipid oxidation produts present in oxidatively modified LDL ould stimulate marophages to release IL-1P. The present studies demonstrate that 9-HODE or its orresponding holesteryl ester indues IL-16 release, impliating a pathologial onnetion between oxidized LDL and smooth musle ell proliferation in atherosleroti lesions. EXPERIMENTAL PROCEDURES Isolation of Mononulear Cells-Human peripheral blood was olleted in 10 mm sodium itrate from healthy volunteers. Erythroytes and neutrophils were removed by low speed entrifugation in Leuoprep tubes (Beton Dikinson, Oxnard, CA) aording to the manufaturer s suggested protool. The resulting mixture of platelets and mononulear ells was inubated in tissue ulture dishes (3 X lo6 ells/well of 24-well plate, Corning, Corning, NY) for 1 h at 37 C, after whih nonadherent platelets and other ells were removed. Fresh medium RPMI 1640 (GIBCO) was then added to the adherent ells. Flow ytometri analyses showed that 99.5% of the adherent ells were apable of internalizing a fluoresent aetylated LDL (ALDL) probe, suggesting a marophage-like phenotype bearing the savenger reeptor (data not shown). All experiments were performed with freshly adhered ells in the absene of serum. Preparation of Modified LDL-Human plasma was obtained from

2 HODE-indued IL-1B Release a loal blood enter. To eah pool of plasma (18-20 donors) was mm glass srew ap test tube. The mixture was dried to an oil under added 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 50 units/ ml aprotinin, and 0.01% sodium azide prior to LDL frationation. LDL were isolated by preparative ultraentrifugation in KBr (d = g/ml) as desribed previously (17). Five different bathes nitrogen and the tube flushed with oxygen and sealed. The oil was heated at 40 "C for 5 days, and the material was then dissolved in 5 ml of ethanol. Twenty-five mg of sodium borohydride were added and the mixture shaken gently at room temperature for 10 min. The ofldlwereused in this study. Prior to oxidation, EDTA was ethanol was evaporated, and 1 ml of water was added. The lipid was removed from the LDL by dialysis against phosphate-buffered saline extrated into heptane and subjeted to semi-preparative HPLC using (PBS), ph 7.4, at 4 "C in the dark. Copper-oxidized LDL (CuZ+-LDL) a Spherisorb 5 Si1 olumn (22.5 X 250mm, Phenomenex, Ranho were prepared by inubating LDL (2 mg LDL protein/ml) with 10 pm CuSO, for 16 h at 37 "C. Typially, Cu2+-LDL ontained 40 nmol of TBARS/mg of protein. Exess CuS04 was removed by dialysis. Palos Verdes, CA). The mobile phase onsisted of heptane:ethyl ether:isopropanol:aeti aid (1002:l:O.l); the flow rate was 8 ml/ min. Marophage-modified LDL (M-LDL) were prepared by inubating Two major peaks exhibiting signifiant diene onjugation, as mon- LDL (2 mg LDL protein/ml) with human peripheral blood monoytes itored by absorbane at 234 nm, were olleted. Following removal of (adherent ells) for 24 h at 37 "C; M-LDL ontained nmol of the mobile phase under vauum, the residues were dissolved in TBARS/mg of protein. M-LDL were re-isolated from the ulture heptane. Fast atom bombardment-mass spetrometry indiated both medium by preparative ultraentrifugation in KBr (d = g/ml). ALDL were prepared aording to the method of Fraenkel- Conrat (18). To ontrol for possible endotoxin ontamination during dialysis and re-isolation, modified LDL were routinely passed over a prepaked endotoxin affinity olumn (Detoxigel, Piere Chemial produts to have a moleular weight idential to the hydroxylated derivative of holesteryl linoleate. The identity of regio-speifiity of the produts was onfirmed by saponifiation and analytial HPLC analysis of the free fatty aids as desribed above. One produt ontained a fatty aid whih o-migrated with authenti 13-HODE Co.). As shown by the Limulus polyphemus ameboyte lysate test and the other with 9-HODE. The amount of eah produt was (Sigma; sensitivity 1 ng of endotoxin), this Detoxigel olumn has the determined by omparison of the areas of the free fatty aids to a apaity of removing 2 mg of endotoxin. Furthermore, the affinity of standard urve prepared from standards of 9- and 13-HODE. Heptane endotoxin for the olumn is unaffeted by the presene of LDL (data wasremoved from eah isolated fration under nitrogen and the not shown). Sine endotoxin is equally potent in induing IL-10 and holesteryl ester produts reonstituted in ethanol at a final onentumor nerosis fator a (TNFa), and sine oxidized LDL only stim- tration of 1 mg/ml (3.3 mm with respet to HODE ontent). ulate IL-lP but not TNFa release (see below), a TNFa enzyme-linked Determination of ZL-10 mrna-totalrnawas isolated from immunosorbent assay (ELISA, sensitivity 20 pg) was used routinely human peripheral blood monoytes using guanidine thioyanate and for oxidized LDL-stimulated ulture supernatants to monitor for phenol/hloroform extrations by a modifiation of the method of potential endotoxin ontamination. In every experiment in whih Chomzynski and Sahi (22). Briefly, ells were washed twie with Cu'+-LDL, M-LDL, and ALDLwere ompared with native LDL, old PBS and released from the plate with a ell sraper into 1 ml of LDL were from the same lipoprotein preparation. Human IL-10 and PBS ontaining 40 units of RNasin (Stratagene, La Jolla, CA). The TNFa ELISA kits were from Cistron (Pinebrook, NJ), and both were ells were pelleted and resuspended in 1 ml of GTC extration buffer used aording to the manufaturer's suggested protool. Latate (4 M guanidine thioyanate, 25 mm sodium itrate, ph 7.0, 40 mm 2- dehydrogenase levels were routinely measured by a linial analyzer meraptoethanol, and 0.5% sodium lauryl sarosine). The ellular (Coulter, Hialeah, FL). lysate was sheared by passing three times through an 18-gauge needle Determination of Lipid Peronidation Produts-Lipid peroxidation and four to five times through a 25-gauge needle. The lysate was kept produts were determined by HPLC analysis of saponified lipids. For on ie and extrated by addition of 0.2 ml of 1 M sodium aetate, 1.0 LDL and modified LDL, the lipids were extrated by dropwise addi- ml of water-saturated phenol, and 0.2 ml of h1oroform:isopropanol tion of the sample (0.4 mg of protein) into 2 ml of ether:ethanol (3:l) (49:l) with mixing on a vortex after eah addition and for 30 s at the with mixing. The protein was pelleted by entrifugation and the end. The tubes were immediately entrifuged for 20 min at 4 "C and supernatant fration deanted and dried under nitrogen. The dried 10,000 rpm. The aqueous phase was transferred to a new tube and lipids were reonstituted in 0.5 ml of ethanol to whih was added 0.05 the RNA preipitated, following addition of an equal volume of old ml of 10 N NaOH, followed by heating at 60 "C for 20 min to generate isopropanol at -80 "C for 10 min. The pellet was redissolved in 200 the free aids. The solution was neutralized by the addition of 0.03 pl of Tris-EDTA buffer (10 mm Tris, 1 mm EDTA, ph 7.5) with 4 ml of glaial aeti aid and dried under nitrogen. To the lipid was units RNasin. The RNA was further purified by extrating one time added 1 ml of water and then 1 ml of heptane. Following mixing and with an equal volume of h1oroform:isoamyl aloho1:phenol (49:1:50) entrifugation at 1,800 rpm for 5 min, 0.8 ml of the heptane layer and one time with an equal volume of h1oroform:isoamyl alohol was removed, dried under nitrogen, and re-solubilized into 0.15 ml of (24:l). The RNA was preipitated at -80 "C for 10 min or -20 "C for heptane. Fatty aid oxidation produts were separatedusing a normal- 1 h or longer following addition of 0.1 volume of 3 M sodium aetate phase silia olumn (Zorbax Sil, 25 m X 4.6 mm, 5 pm) aording to and 2 volumes of old ethanol. After entrifugation, the RNA was Teng and Smith (19) with minor modifiation. The mobile phase was resuspended in 25 p1 of the Tris-EDTA buffer and was quantitated a quaternary mixture of heptane:diethyl ether:isopropanol:aeti aid spetrophotometrially. The relative ellular levels of IL-10 were (100:10:0.9:0.1) run at a flow rate of 2 ml/min; onjugated dienes were determined by Northern blot analysis of 3 or 10 pg of total RNA. The monitored at 234 nm. Standards of linoleate hydroperoxides, i.e. 9(S)- RNA was eletrophoresed in a 1% agarose/formaldehyde gel (23) and and 13(S)-HPODE, and linoleate hydroxides, i.e. 9(S)- and 13(S)- blot-transferred to Nytran (Shleiher and Shuell). An EoRIIXbaI HODE (is and trans isomers, Cayman Chemials, Ann Arbor, MI) 480-base pair portion of the mature IL-10 DNA (Bekman, Fullerin ethanol were dried under nitrogen, re-solubilized in heptane, and ton, CA) and a PstIIXbaI 516-base pair portion of human skeletal subjeted to hromatography as desribed for the saponified lipid musle atin DNA (24) were used as hybridization probes and were frations. radiolabeled by random priming with [32P]dCTP(25). Following For determination of onjugated dienes in ells, the lipids were autoradiography, band intensities were determined with a densitomextrated as desribed by Shade et al. (20) with slight modifiations. eter (Moleular Dynamis, Sunnyvale, CA). Cells (25 X IO6) were lysed with 0.75 ml of 0.25% deoxyholate and then the lysate was transferred to 13 X 100-mm polypropylene tubes. The plates were washed with 0.75 ml of water and the wash ombined RESULTS with the deoxyholate lysate. This mixture was then extrated with Inubation of human peripheral blood monoytes with 1.8 mlof h1oroform:methanol:aeti aid (2OO:lOOl) followed by Cu2+-LDL or M-LDL for 24 h indued a dose-dependent entrifugation at 1,500 rpm for 5 min. The hloroform layer (0.9 ml) inrease of IL-1@ release into the medium (Fig. 1). At the was removed and dried under nitrogen. The lipids were saponified as desribed above and subjeted to hromatography. Quantitation was highest onentration tested (300 pg protein/ml), Cu2'-LDL ahieved by omparison to a standard urve (0-1 pg) generated from and M-LDL indued an average of 422 f 8 and 333 k 7 pg of the ommerially available standards of 9(S)- and 13(S)-HPODE IL-lp/ulture (6 X lo6 ells/ulture), respetively. Control and 9(S)- and 13(S)-HODE. LDL whih were subjeted to inubation at 37 "C for 24 h but Preparation of Cholesteryl-9-HODE-Cholesteryl-9-HODE was in the absene of Cu2+ or ells did not indue IL-lP release, prepared by a modifiation of the method desribed by Peers and Coxon (21) for 2-linoleoyl-1,3-dipalmitoylglyerol. Briefly, 50 mg of as ompared with untreated ells (65 pg of IL-lplulture at holesteryl linoleate (Nu-hek Prep, Elysian, MN) were dissolved in 300 pg of LDL protein). ALDL differs from Cu2'-LDL and heptane and mixed with 5 mg of a-toopherol (Sigma) in a 16 X 125- M-LDL in that it is modified by aetylation of lysine residues

3 (26), whereas Cu2+-LDL and M-LDL are modified by oxidative proesses. However, ALDL (300 pg protein/ml), whih ontained less than 0.5 nmol of TBARS/mg of protein and undetetable levels of onjugated dienes, did not indue IL- 16 release. TNFa was not deteted (sensitivity of assay 20 pg) in any ulture supernatants (data not shown). In a total of five separate experiments, Cu2+-LDL indued a 3-15-fold inrease in IL-lP release when ompared with medium ontrol, whereas M-LDL indued a 4-8-fold inrease (Table I). HPLC analysis of the saponified lipid fration from Cu2+- LDL and M-LDL showed the presene of 9- and 13-HODE and 9- and 13-HPODE, produts of linoleate oxidation (Fig. 2). In Fig. 2A, peaks 1-4 represent standards of 13-HPODE, g-hpode, 13-HODE, and 9-HODE, respetively. Cu2'-LDL (Fig. 2B) ontained eight major peaks representing isomers of the four produts, whereas M-LDL ontained primarily 9- HODE and 13-HODE (3.06 and 5.90 pg/mg LDL protein, respetively) (Fig. 2C). Prior to 9-HODE-indued IL-10 Release oxidation, freshly isolated LDL ontained less than 10% of these lipid dienes relative to M-LDL (Fig. 20). To assess the ontribution of these oxidized lipids to the release of IL-lP by modified LDL, marophages (3 x lo6 ells/ ulture) were inubated with authenti 9- and 13-HODE. As shown in Fig. 3, at 33 PM the HODEs signifiantly inreased IL-lP release relative to medium ontrol. Interestingly, 9- HODE indued a muh greater release of IL-lp than 13- HODE (122 uersus 43 pg of IL-l@/ulture, respetively), suggesting a speifiity in the signal transdution pathway leading to IL-lP indution. The absolute amounts of IL-1p in- 500 OJ Protein ilg;ml FIG. 1. Stimulation of IL-lfl release by Cu2+-LDL and M- LDL. 6 X lo6 human peripheral blood monoyte-derived marophages were inubated in 24-multiwell plates with medium RPMI 1640 alone (O), LDL (0), ALDL (A), Cu*+-LDL (O), or M-LDL (W) for 24 h. Sereted IL-10 was measured with an IL-10 ELISA. Data are mean k S.E. of quadrupliate ultures. Experiment Donor LPS" ALDL M-LDL Vehile CuZ'-LDL TABLE I Indution of IL-16, bv " oxidized LDL LDL dued by 9-HODE varied among donors. However, IL-1P levels were onsistently higher than that of ontrol ultures, but were 2-8-fold lower than that of LPS-stimulated ells (Table 11). Although free 9-HODE an be produed and released by endothelial ells from linolei aid via a ylooxygenasedependent mehanism, in the ontext of atheroslerosis (27), the majority of linoleate-derived 9-HODE present in oxidized LDL ours as esters of holesterol or phospholipid. Therefore, holesteryl-9-hode was synthesized and its effets on IL-lP indution determined. Both holesteryl-9-hode and 9-HODE indued a similar dose-dependent inrease in IL-lP release (Fig. 4). The plateau observed with holesteryl-9- HODE may reflet its relative insolubility in the ulture medium. To determine the kinetis of IL-1P indution, marophages were inubated with 9-HODE for 30, 60, 120, and 300 min, after whih ulture supernatants were olleted for IL-1P determination. Analysis of the ulture supernatants showed that extraellular IL-lP was detetable as early as 5 h after the addition of 9-HODE (Fig. 5A). To determine whether the kinetis of indution orrelated with the uptake of 9-HODE, the hydroxy fatty aid was quantitated both in the ell and the medium. HPLC analysis of ells at these various times showed a time-dependent assoiation of 9-HODE with the ells (Fig. 5B). The sum of ell-assoiated 9-HODE and 9- HODE in the medium was similar to the amount of 9-HODE reovered from ell-free medium at eah time point, suggesting that 9-HODE was not signifiantly metabolized by the ells during this time period. We next determined whether 9-HODE affeted the expression of IL-1P by marophages. By Northern blot analysis of total RNA, the levels of IL-10 mrna in unativated peripheral monoytes were undetable. However, adherene to plasti indued detetable levels of IL-lP mrna in mediumtreated ontrol ells (Fig. 6), onsistent with earlier reports (28, 29). In the experiment shown in Fig. 6, after a 3-h exposure to 33 PM 9-HODE, ells from a single donor had a 2-fold inrease of IL-lp mrna level ompared with the untreated ells from the same donor. For nine different marophage preparations tested (Fig. 7), IL-lP mrnas inreased from 1.5- to 6-fold with a mean of 3.67? 1.6 in response to 9-HODE. In Fig.7, the relative levels of IL-1p mrna in vehile- and 9-HODE-treated ells were determined by nor- malizing to the level of atin mrna in eah sample as deteted on the same blot. DISCUSSION In the present study, we demonstrate that oxidized LDL, but neither ALDL nor native LDL, were apable of induing pg/ulture* 1 R. S. 33 f ' 228 f f 22' 2 T. D. I f 2 4 f Id 55 k 2d 3 T. M. 84 & f 25' 228 k 30' 369 f 39' 72 f 23' 4 J. M. 100 f k 27' 124 k 10' 5 M. T. 33 f 3 37 f 6B 486 f 2@ 32 f 11" 6 M. K. 74 k 9 82 f Sh 355 k 2Oh 7 B. G. 95 f 4 83 f 28' 384 f 4' 8 A. M. 22 k 3 20 f 6' 166 k 7' The LPS onentration was 1 pg/ml/ulture. Data are mean f S.E. of tripliate ultures. ' The lipoprotein onentrations were 250,' 300: 100,' 200,' 250: IL-lP 100," 200,' and 400' pg of protein/ml/ulture, respetively. 904 f 9

4 HODE-indued IL-lp Release A a" = F r t 2 [I [I 5 1 I I I TIME (min) FIG. 2. HPLC analysis of lipid peroxidation produts. A, 13(S)-HPODE (l), 9(S)-HPODE (2), 13(S)-HODE (3), and 9(S)- HODE (4) standards were separated using a normal phase silia olumn. B, saponified lipid profile from Cu2+-LDL. All four, 9- and 13-HODE and 9- and 13-HPODE, were deteted. C, saponified lipid profile from M-LDL. The two major lipid peroxidation produts found in M-LDL were 9- and 13-HODE. D, saponified lipid profile of native LDL. IL-1p release from human peripheral blood monoyte-derived marophages. The lak of effet with ALDL suggests that binding to the savenger reeptor alone is insuffiient to indue IL-la release and that other omponents of oxidized LDL are involved in the stimulation of IL-lB release. Sine oxidized LDL did not indue TNFa release, and sine endotoxin indues both IL-1p and TNFa release, it is unlikely that IL-1/3 release indued by oxidized LDL was due to endotoxin ontamination. In an effort to identify omponent(s) of oxidized LDL to whih this biologial ativity ould be attributed, the lipid fration was separated and haraterized by HPLC. Analysis of the saponified lipid fration from Cu2'-LDL showed the presene of both Z,E- and E,E-isomers of 9- and 13-hydroxy and 9- and 13-hydroperoxy derivatives of linoleate, as expeted from free radial-mediated linoleate oxidation (19). A reent report has haraterized the lipid fration of Cu2+-LDL by HPLC and gas hromatography-mass spetrometry analyses (30). In agreement with our results, it was found that 0 FIG. 3. HODE-indued stimulation of IL-10 release by human peripheral blood monoyte-derived marophages. Cultures of marophages (3 X lo6) in 24-multiwell plates were inubated with 33 PM of 9-HODE (uertial bar) or 13-HODE (horizontal bar). Eight lots of 9- and 13-HODE have been tested. 10 pl of 95% ethanol (vehile) to I-ml ulture (final 1% ethanol) did not affet the ph of the medium, ell viability, nor the ellular IL-la response to LPS. Conentrations of 9- and 13-HODE below 50 pm were not ytotoxi, as indiated by the release of latate dehydrogenase. Data are mean f S. E. of quadrupliate ultures of a representative experiment. Experiment TABLE I1 Indution of IL-1B bv 9-HODE Donor IL-10 Vehile 9-HODE" LPSb pgfulture' 1 R.S f3 2 T. M. 28 & D.M. 12&2 247t f54 4 C.B E. M f44 6 E. H. 21 f f 38 7 M.R f 16 8 D.D. lo f 15 R. 9 D pm. * 200 ng/mi ulture. e Data are mean + S.E. of tripliate ultures. 350/ 3ooJ Conentrailon UM FIG. 4. Dose-dependent indution of IL-la seretion by 9- HODE and holesteryl-9-hode. Cultures of marophages in 24- multiwell plates were inubated with 11,22,33, or 44 PM of 9-HODE (0) or holesteryl-9-hode (0). Data are mean -t S.E. of tripliate ultures of a representative experiment. 67% of the oxidized linoleate produts were present as 9- and 13-derivatives. Additionally, onjugated dienes derived from arahidonate are also generated by oxidation, although at approximately 10% the level of linoleate oxidation produts.

5 ~. ~ ~."~ 9-HODE-indued IL-IP Release oTA 1.8 / E 1.4._ a I i:\ IL-la rnrna Alm rnrna FIG HODE indution of IL-Ij3 mrna expression. Human peripheral hlood monoyte-derived marophages were ultured in 60-mm ulture dishes. Medium RPMI 1640 ( opn hnrs) or9-hode (3.7 pm) (losed bars) was added to ultures for 3 h, after whih total RNAs were isolated and were eletrophoresed in a 1'; agaroseformaldehyde gel and blotted onto Nytran. II.-lP mrna and atin mrna levels were determined hy Northern hlot analysis with I 2 P - labeled DNA probes. The relative hand intensities were quantitated hy densitometri sanning of the autoradiograph. Data are from a representative experiment with ells from a single donor. 1., o- "1)- i, 0!\r:::" ~..~ ~~ ~~ " ~~ fio Mlnules FIG. 5. Kinetis of 9-HODE-indued IL-IS release. Human peripheral hlood monoyte-derived marophages (20 X 10") were ultured in 60-mm tissue ulture dishes. 9-HODE (10 pl of a stok solution of 3.3 mm in 9.5% ethanol, final onentration was 33 pm) was added to ultures at 0 h. A, time-dependent seretion of IL-10 inubated with 9-HODE (0) or medium RPMI 1640 (0). R, timedependent hanges of 9-HODE levels in ells (0). ulture supernatant (A), or ell-free medium ('>). The sum of 9-HODE in ells and ulture supernatants is represented hy open squares. Data are mean 2 S.E. of tripliate ultures of a representative experiment. It must also be onsidered that yli endoperoxides and aldehydi ompounds whih are not detetable as onjugated dienes are generated during LDL oxidation. The potential ontribution of these additional produts to IL-1P release by oxidized LDL is suggested by the demonstration that mg of M-LDL, ontaining pg of 9-HODE, indued IL- lp release omparable with 9.5 pg (33 p ~ of ) free 9-HODE. This may be explained, in part, by the limited uptake of 9- HODE by marophages whih ahieved a level of pg/ 10" ells after a 5-h inubation (Fig. 5B). Uptake of M-LDL or Cu2+-LDL via the savenger reeptor may be more effiient at delivering 9-HODE to the ells, or alternatively, other omponents present in modified LDL may also ontribute to IL-la release. In ontrast to Cu"-LDL, saponified M-LDL ontained mainly 9- and 13-HODE in agreement with preliminary results reported by Rankin et al. (31). This differene is onsistent with the ability of ells to redue 9- and 13-HPODE to their respetive HODE. We were unable to aurately plaques revealed the presene of oxidized holesterol esters (16). It annot be determined from these studies whether LDL determine the ability of HPODEs to indue IL-lP, as they are lipid oxidation by marophages is an oxygen radial-mediated unstable in medium RPMI 1640 and are degraded to uniden- proess or is lipoxygenase atalyzed (32), sine both mehatified produts (data not shown). They exhibited very limited nisms result in the formation of oxidized linoleate produts. ability to indue IL-ls (data not shown) whih refleted the Support for free radial-mediated lipid oxidation is provided net result of ontinual "deterioration" of HPODEs and ell- by demonstration that the oxidized lipids exist as a raemi mediated onversion of HPODEs to HODEs. In agreement, no HPODE and only a small level of HODE was found in W sz :E E r n. ; o m - b E U t j E >r.- I - mg a U Donor FIG. 7. Inrease in IL-ID mrna levels by 9-HODE in ells from multiple donors. Monoyte-derived marophages from nine donors were treated with 33 pm 9-HODE for 3 h and total RSA isolated as desrihed in the legend of Fig. 6 and under "Experimental Proedures." The Northern hlots were first probed for II,-ld mrsa levels and then for atin mrna levels. Following autoradiography. hand intensities were determined and levels of IL-Id mhsa in ontrol (open bars) and 9-HODE-treated samples (losed hats) normalized to the levels of atin mrna in the same samples. For eah donor, the ontrol sample was assigned 8 relative ILld mrna/atin MRNA level of 1. For the samples from the nine donors tested. the mean level of IL-10 mrna indution hv 9-HODE WRS 3.67 k 1.6 (mean k S.D.). ells inubated with 9- or 13-HPODE. As few onjugated dienes were observable in modified LDL without saponifiation, it is likely that oxidation involves holesteryl esters or phospholipids. Strutural identifiation of oxygenated fatty aid derivatives in human atherosleroti mixture of Z,E- and E,E-isomers of 9- and 13-HODE (34). It annot be ruled out, however, that the initial lipid oxidation

6 HODE-indued IL-lP Release produts are stereo- andlor regio-speifi hydroperoxides whih are onverted to raemi mixtures via subsequent transition metal-atalyzed reations. The 9-HODE present in modified LDL would exist predominantly as holesteryl-9-hode whih we have shown an also indue IL-1p release (Fig. 4). Sine holesteryl-9-hode ould oneivably be de-esterified by marophage lysosomal esterases to 9-HODE, 9-HODE ould be an intermediate metabolite in holesteryl-9-hode stimulation of IL-lp release. Experiments are in progress to determine whether de-esterifiation of holesteryl-9-hode by the marophage is required for indution of IL-16 release. It also remains to be asertained whether 9-HODE serves as a preursor or substrate for the ultimate ativating agent. Kineti experiments showed that within 5 h of stimulation, the amount of 9-HODE reovered from the medium and ells was similar to that added at zero time, indiating that 9-HODE was not signifiantly metabolized and that 9-HODE ould be the intraellular signal. However, radiolabeled 9-HODE is needed to determine whether the ell-assoiated 9-HODE is derived from the medium or is a produt of oxidation of endogenous ellular linolei aid. Preedene for a diret role of 9-HODE in ativation has been provided by the finding that HODE ativates rat brain protein kinase C (11) by fulfilling the requirement for phospholipid. A similar role in indution of IL-lP expression may be envisioned. Northern blot analyses of aorti lesions obtained from holesterol-fed non-human primates show a signifiant inrease of IL-lp mrna levels ompared with aortas from ontrol animals (33). In addition, a 640-fold inrease of IL-lp mrna levels was found in human atherosleroti lesions (34). These observations support a potential atherogeni role of IL- 10 and is onsistent with reports that IL-10 stimulates vasular smooth musle ell proliferation (5, 6). Oxidized LDL have been shown previously to promote holesteryl ester aumulation in marophages (4) and to be ytotoxi toward endothelial ells, vasular smooth musle ells, and fibroblasts (35,36). The present demonstration that oxidized LDL indue the expression and release of IL-1p further supports a ritial role of oxidized LDL in atherogenesis. Aordingly, inhibition of LDL oxidation may be a plausible therapeuti approah for prevention of atheroslerosis. Aknouledgrnents-We thank David Ohlweiler for assistane in the HPLC analyses and Rosanne Dennin for typing of the manusript. We appreiate the suggestion of Dr. Alan R. Brash (Vanderbilt University) onerning the methodology for the preparation of holesteryl-9-hode. REFERENCES 1. Steinberg, D., Parthasarath, S, Carew, T. E., Khoo, J. C., and Witztum, J. L. (1989) N. E8l. J. dd. 320, Brown, M. S., and oldstem, J. L. (1983) Annu. Reu. Eiohem. 62, 223-3fil 3. Go%tein. J. L.. Ho. Y. K.. Basu. S. K.. and Brown. M. S. (1979) Pro. Natl. Aad. Si. U. S. A. 76, Ross, R. (1986) N. Engl. J. Med. 314, Libby, P., Warner, S. J. C., and Friedman, G. B. (1988) J. Clin. Inuest. 81, Fous 10,l Gunnin, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L. (19837 Mol. Cell. Biol. 3, Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biohem. 132, Brown, M. S., Basu, S. K., Falk, J. R., Ho, Y. K., and Goldstein, J. L. (1980) J. Supramol. Strut. 13, Kadue, T. L., Figard, P. H., Leihr, R., and Spetor, A. A. (1989) J. Biol. Chem. 264, Haskill, S., Johnson, C., Eierman, D., Beker, S., and Warren, K. (1988) J. Immunol. 140, Unanue E. R., and Allen P. M. (1987) Siene 236, Lenz, M. L., Hu hes, H., Mithell,, J. R., Via, D. P., Guyton, J. R., Taylor, A. A,, Gotto, 1. M., Jr., and Smlth, C. V. (1990) J. L~pd Res. 31, nm 31. RaG& S. M., Parthasarathy, S., and Steinberg. D. (1990) Arterioslerosis 10,828a 32. MNally, A. K., Chisolm, G. M., 111, Morel, D. W., and Cathart, M. K. (1990) J. Immunol. 145, Ross R Masuda J. Raines, E. W., Gown, A. M., Katsuda, S., Sasahara, M., Malden, L. T., Masuko, H., and Sato, H. (1990) Siene 248, Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Pro. Natl. Aad. Si. U. S. A. 86, Evenorn, S. A., Galdal, K. S., and Nilsen, E. (1983) Atheroslerosis 49, Morel, D. W., Hessler, J. R., and Chisolm, G. M. (1983) J. Lipid Res. 24,