Tumor Necrosis Factor- et Modulates Monocyte/Macrophage Apoprotein E Gene Expression

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1 Tumor Necrosis Fctor- et Modultes Monocyte/Mcrophge Apoprotein E Gene Expression Hongwei Dun, Zhigo Li, nd Theodore Mzzone Deprtments of Medicine nd Biochemistry, Rush Medicl College, Chicgo, Illinois Abstrct po E hs been shown to modulte cholesterol blnce in rteril wll cells. Production of po E by mcrophges in therosclerotic plques could thereby influence the development of the plque lesion. Cytokines, including TNF, hve been identified in humn lesions, therefore, we undertook series of studies to evlute the effect of TNF on monocyte/ mcrophge po E production. The ddition of TNF to freshly isolted humn monocytes led to four- to fivefold increse of po E mrna bundnce. The ddition of TNF to fully differentited mcrophges either hd no effect or modestly inhibited po E mrna expression. THP1 humn monocytic cells lso responded to TNF in phenotypespecific mnner. Tretment of these cells with TNFx produced dose- nd time-dependent increse in po E mrna. This increse ws reflected in po E synthesis nd ws ssocited with inhibition of DNA synthesis, nd with induction of c-fos nd ICAM-1 gene expression. Cell-permnent nlogues of cermide did not reproduce TNF effect on po E, but ntgonists of protein kinse C did inhibit its effect. TNF induction of po E mrna bundnce ws ssocited with stimultion of po E promoter-dependent gene trnscription. In summry, TNF stimultes po E gene trnscription, mrna bundnce, nd protein synthesis in the monocyte/mcrophge in phenotype-specific mnner. Such regultion could significntly modify the mount of po E present in vessel wll lesions. (J. Clin. Invest : ) Key words: cytokines * therosclerosis * mcrophge ctivtion * protein kinse C * cermide Introduction po E is n importnt structurl nd functionl constituent of plsm lipoproteins, including VLDL, HDL, nd chylomicron remnnts (1). Although po E is primrily synthesized by the liver, significnt mounts re lso produced by vriety of peripherl tissues nd cells (2), including mcrophges, where its synthesis is regulted by the cholesterol content of the cells (3, 4). Histochemicl studies hve indicted tht mcrophges Address correspondence to Theodore Mzzone, M.D., Rush Medicl College, 1653 W. Congress Prkwy, Chicgo, IL Phone: ; FAX: Received for publiction 24 October 1994 nd ccepted in revised form 19 April J. Clin. Invest. () The Americn Society for Clinicl Investigtion, Inc /95/08/0915/08 $2.00 Volume 96, August 1995, re the mjor source of loclly derived po E in developing vessel wll theromtous lesions (5). Locl production of poe in the vessel wll could impct on proximte cellulr cholesterol flux nd modify the locl vilbility of bioctive molecules such s growth fctors (1). Furthermore, endogenous production of po E hs been shown to influence mcrophge cholesterol flux by regulting the rte of cellulr cholesterol clernce to HDL3 (6). Thus, modultion of mcrophge po E secretion in the vessel wll could significntly impct therom progression or regression. Regultion of mcrophge po E expression is complex: it is regulted not only by cholesterol but lso by ctivtors of protein kinse C nd by endotoxin (7-9). TGF,8 hs been demonstrted to induce mcrophge po E expression (10). On the other hnd, the monocyte colony stimulting fctor (MSCF)' hs been shown, in seprte reports, both to inhibit nd ugment mcrophge po E expression ( 10, 11). A potentil regultory role for other cytokines, for exmple, TNF, hs been even less cler (10, 12). Locl production of TNF is n importnt component of the vessel wll response to injury. TNF positivity hs been found in the cytoplsm of mcrophges, nd in the cytoplsm nd ttched to cell membrnes of smooth muscle cells nd endothelil cells of the humn therom ( 13, 14). These observtions suggest potentil for TNF to modulte the evolution of the therosclerotic plque. Further, such modultion would be likely to occur vi effects on monocyte/mcrophge cells becuse these re primry trgets of TNF ction. In the present study, we investigted the effect of TNF on poe expression in the humn monocyte/mcrophge. We exmined phenotypedependent effects of TNF on po E gene expression nd compred these with its effects on the expression of other monocyte/ mcrophge genes (c-fos nd intercellulr dhesion molecule [ICAM] -1 ) nd cell cycle trverse. We lso investigted potentil roles for cermide second messengers nd protein kinse C (pkc) ctivtion for trnsducing the observed effects of TNF on po E. Methods Mterils. [35S]Methionine (10 Ci/mmol), [32P]dCTP (800 Ci mmol), nd [3H]thymidine (5 Ci/mmol) were purchsed from Amershm Corp. (Arlington Heights, IL). Humn recombinnt TNF ws purchsed from Genzyme, Corp. (Boston, MA). C2-cermide nd H7 were purchsed from BIOMOL Reserch Lbortories, Inc. (Plymouth Meeting, PA). Endotoxin-free BSA cme from Clbiochem Corp. (L 1. Abbrevitions used in this pper: ICAM, intercellulr dhesion molecule; MSCF, monocyte colony stimulting fctor; NFKB, nuclese fctor-kb; pkc, protein kinse C. TNF Modultes po E Gene Expression 915

2 Joll, CA). All other mterils were obtined from previously identified sources (6, 9, 15, 16). Cell culture. Freshly isolted humn monocytes were purified by elutrition. The cell popultion used for experiments ws > 95% monocytic, s determined by differentil counts of Wright stined smers. These cells were incubted using conditions described in the figure legends. For experiments using fully differentited humn monocytederived mcrophges, cells were llowed to differentite in serum-contining medium for 7 d before the strt of the incubtion with TNF. The THPl humn monocytic cell line ws obtined from Americn Type Culture Collection (ATCC, Rockville, MD), nd cells were grown in culture flsks (Flcon Plstics, Cockeysville, MD) in RPMI-1640 medium contining 10% het-inctivted FBS s described (15). For experiments, ll cells were wshed twice with serum-free RPMI-1640 nd were plced in RPMI-1640 medium supplemented with 0.2% endotoxin-free BSA unless otherwise indicted. Immunoprecipittion of po E. THP1 cells were pulse-lbeled with L- [35S ] methionine, s described in detil previously (16). Briefly, cells were plted t density of 5 x 106 cells in 60-mm dish in triplicte. 5 ng/ml of TNF ws dded, nd the cells were incubted for 72 h t 37TC. After incubtion, dherent cells nd cells in suspension were wshed twice with methionine-free RPMI-1640 nd then incubted for 2 h in 1 ml of methionine-free RPMI-1640 medium supplemented with 100,uCi/ml of [35S]methionine supplemented with 10 um unlbeled L-methionine. After incubtion, the medium ws collected fter centrifugtion to remove cells. Adherent nd cells in suspension were wshed twice in ice-cold phosphte-buffered sline nd lysed in 2.0% SDS t 950C for 3 min. The lyste ws then diluted with lysis buffer (10 mm N2HPO4, 15 mm NCl, 10 mm L-methionine, 1% Triton X-100, 1% deoxycholte, ph 7.4) to finl concentrtion of 0.2% SDS. Cell extrcts nd culture medi were nlyzed by immunoprecipittion followed by 10% SDS-PAGE nd fluorogrphy s previously described (16). 4 x 106 TCA-precipitble counts/min were immunoprecipitted from ech cell lyste smple, nd 4 x 105 counts/min were immunoprecipitted from ech medium smple. Incorportion of lbeled methionine into po E protein in THP1 cells ws liner over the ssy period. For quntittion, po E bnds were excised from the gel nd digested in 30% H202 for scintilltion counting (16). Rdioctivity in the po E shown ws normlized for rte of totl protein synthesis nd secretion, which ws not significntly different between groups. RNA isoltion nd Northern hybridiztion. Cells were seeded t density of 10 x 106 cells in 100-mm dish. To isolte totl cellulr RNA, wshed cells were solubilized in gunidine isothiocynte s described, followed by sedimenttion of the extrct through CsCl (9). For some experiments, poly (A) + RNA ws purified fter pssing totl RNA through silnized oligo (dt) columns. For Northern nlysis, formldehyde-treted RNA smples were frctionted by electrophoresis in 1.0% grose, trnsferred to Nytrn (Schleicher & Schuell, Inc., Keene, NH), nd hybridized with lbeled po E cdna probe s previously described (9). Rdiolbeled po E cdna probe ws prepred by rndom-primed synthesis using phea4, which is 1.1-kb Atll- Hinf I frgment tht encompsses the entire protein coding region of the humn po E cdna subcloned into puc (9). Ech rection ws crried out using 50 ng of cdna with minimum of 1 x 107 cpm. To provide n internl control, Nytrn membrnes were stripped ccording to the mnufcturer's instructions nd reprobed with lbeled 0.7-kb PstI frgment of the cdna for bet-ctin. The c-fos probe, which ws 1.0-kb PstI subfrgment from thef os cdna nd the ICAM-1 probe, which ws 1.4-kb BgIII-SII subfrgment from the ICAM-1 cdna, ws lbeled using the sme method. Mesurement of DNA synthesis. Rtes of DNA synthesis were ssessed by determining [3H] thymidine incorportion into TCA precipitble mteril (17). After cultures were treted with the indicted mount of TNF for 24 h, cells were pulsed with 2.5 0Ci/ml [3H]thymidine for 2 h nd then wshed with RPMI-1640 five times. This ws followed by five wshes with ice-cold 5% TCA. Cells were then extrcted in 1 I p4. ; control TNF poe JP-ctin TNF :~ iwk W %O Figure 1. TNF enhnces po E mrna expression in humn monocyte/ mcrophges. Humn monocytes (isolted by elutrition, > 95% pure) were incubted in Teflon FEP culture bottles (Fischer, Pittsburgh, PA) in 0.2% endotoxin-free BSA plus or minus TNF t 10 ng/ml. After 24 h, cells were hrvested for nlysis by Northern hybridiztion. The intensity of the bnds for po E nd /l-ctin (s the internl control) ws determined by scnning densitometry. ml of 0.25 N NOH for scintilltion counting. Protein ws ssyed using Lowry's method (18). Cell trnsfection nd mesurement of luciferse ctivity. An po E- luciferse chimeric construct, -2300/ +24 po E pgl2, ws mde using stndrd recombinnt techniques (19). The Sm 1-Atll frgment of the po E gene ws cloned into the polyliker region of pgl-2 Bsic (Promeg Corp. Mdison, WI). THP1 cells were trnsfected using modified dextrn method (20). 1 x 107 cells were trnsfected with 2 pg of -2300/+24 po E pgl2 plsmid in 1 ml STBS buffer (25 mm Tris-Cl, 137 mm NCl, 5 mm KCl, 0.6 mm N2HPO4, 0.7 mm CCl2, 0.5 mm MgCl2) with 100 pg DEAE dextrn for 90 min nd shocked with 10% DMSO for 5 min. After trnsfection, the cells were incubted in RPMI medium with 10% FBS overnight. At tht time, ll trnsfected cells were mixed in single pool prior to being liquoted to experimentl tretments, to eliminte differentil trnsfection efficiency s n experimentl vrible. The medium ws then chnged to RPMI with 0.2% endotoxin-free BSA, nd 10 ng/ml of TNF- ws dded. Before hrvesting, the cells were left t room temperture for 20 min; the cells were then lysed, nd luciferse ctivity ws mesured using luciferse ssy kit nd luminometer (Enhnced Luciferse Assy Kit; Luminometer Monolight 2010; Anlyticl Luminescence Lbortory, Sn Diego, CA). Protein concentrtion ws mesured using DC protein ssy kit (Bio Rd Lbortories, Richmond, CA). Results Phenotype-specific effects oftnf on humn monocyte/mcrophge po E gene expression. Fig. 1 shows the effect of TNF t 10 ng/ml on po E gene expression in freshly isolted humn monocytes. A four- to fivefold increse of po E mrna bundnce resulted from tretment with TNF over 24-h period. Apo E gene expression in more fully differentited mcrophges. (fter 7 d in culture) showed little response to TNF t 5 ng/ 916 Dun et l.

3 Tble L Effect of TNF on po E mrna Expression in Differentited Mcrophges Cell type po E/j3-ctin Fold chnge Tble H. Dose-dependent Effect of TNF on po E mrna Expression po E/f3-ctin Fold chnge THPl cells 24 h control h + TNF h control h + TNF Humn monocyte-derived mcrophges 48 h control 3.88± h + TNF 3.57± THPl cells in 10% serum were treted with 100 ng/ml of PMA for 3 d. At tht time the medium ws replced with 0.2% endotoxin-free BSA lone or with 5 ng/ml of TNF for the indicted time. Humn monocytes isolted by elutrition were llowed to differentite in serum contining medium for 7 d. At tht time, the medium ws replced with 0.2% endotoxin-free BSA lone or with 5 ng/ml TNF for 48 h. Cells were nlyzed by Northern blot by hybridiztion. The intensity of the bnds for po E nd /-ctin ws mesured by scnning densitometry. Results for THP1 cells re the verge of duplicte smples, which vried < 10%. Results from humn monocyte-derived mcrophge re men±sd of triplicte smples. ml fter 48-h incubtion period (Tble I). Addition of TNF to cells llowed to differentite over shorter periods of time produced 30-40% reduction of po E mrna levels (not shown). Similr phenotype-specific effects were observed in the humn monocytic THP1 cell line. In this cell line, we nd others hve shown tht tretment of THP1 monocytes with phorbol ester induced expression of the mcrophge phenotype in these cells s well s in po E gene expression (8, 9, 12). When TNF ws dded during the initil 18 h of differentition, po E mrna bundnce ws further incresed more thn twofold by the ddition of TNF (Fig. 2). When TNF ws dded to THP1 cells fter differentition hd progressed for 72 h, po PMA PMA+TNF 0 III PUA+TNF- * I I I po E/bet-ctin Figure 2. TNF enhnces po E mrna expression in erly differentiting mcrophges. THP1 cells in 0.2% endotoxin-free BSA were treted with 100 ng/ml of PMA lone or with 5 ng/ml of TNF for 18 h. Northern blots of poly (A) + RNA for po E nd /3-ctin were processed s described in Methods. The brs show the chnge of po E mrna bundnce fter correcting for the /3-ctin signl. Control TNF 5 ng/ml TNF 10 ng/ml TNF 15 ng/ml qg totl cellulr RNA, isolted from untreted cells or cells treted with the indicted concentrtion of TNF for 24 h, ws nlyzed by Northern hybridiztion. The intensity of the bnds corresponding to po E nd /3-ctin ws determined by scnning densitometry. The vlues shown re the verges of duplicte smples, which vried < 10%. E mrna bundnce ws reduced by 24 nd 36% fter n dditionl 24 nd 72 h, respectively, in TNF (Tble I). The results in both cell types, therefore, demonstrted reltionship between TNF effect on po E expression nd phenotypic expression of monocyte/mcrophge cells. Additionl studies were performed to investigte further TNF-medited induction of po E gene expression using the THP1 cell line. TNF induces po E gene expression in THPI cells in time- nd dose-dependent mnner. Tble II shows the results of representtive experiment to evlute the effect of TNF on po E gene expression over rnge of doses. In this experiment, po E messenger RNA bundnce incresed 3.6-fold in response to 5 ng/ml TNF nd 4.7-fold in response to 15 ng/ ml TNF. The 5 ng/ml dose ws chosen to evlute the time dependency of po E mrna response. As shown in Tble III, fter 24-h incubtion in TNF, slightly greter thn twofold increse in po E mrna bundnce ws detected. The gretest increse of po E mrna ws observed when the incubtion in TNF ws extended to 72 h, t which time 14-fold increse in po E messenger RNA bundnce ws mesured in treted s compred with control cells. Effect of TNF on po E synthesis nd secretion. Induction of messenger RNA levels for mcrophge trget genes of TNF re not lwys reflected in chnges in protein synthesis nd secretion (21, 22). We therefore directly evluted the effect of TNF on po E synthesis nd secretion by pulse lbeling cells Tble III. Time Course Effect of TNF on po E mrna Expression po E/,B-ctin Time Control TNF 18 h h h Mg of totl cellulr RNA isolted from untreted cells nd cells treted with 5 ng/ml of TNF for the indicted time ws nlyzed by Northern hybridiztion. The intensity of the bnds corresponding to po E nd to the internl control, f3-ctin, ws determined by scnning densitometry. The results shown re the verges of duplicte smples, which vried by < 10%. TNF Modultes po E Gene Expression 917

4 Tble IV. Effect of TNF on po E Synthesis nd Secretion Medium dpm Cell extrct Control 265±73 447±69 TNF 1163±49 822±178 5 x 106 THP1 cells in 60-mm dishes were incubted for 72 h in medium lone or with 5 ng/ml of TNF. The cells were then lbeled for 2 h with [35S]methionine to mesure the rte of po E synthesis nd secretion s described in Methods. The po E bnds were excised from the gels, nd rdioctivity in po E is shown in the lower pnel s SEM from triplicte smples. with [35S ]methionine nd quntittively immunoprecipipting newly synthesized po E from cell lystes nd medi. The results of representtive experiment re shown in Tble IV. In culture medi, po E rdioctivity ws incresed more thn fourfold in response to 72-h incubtion in TNF. In cell lystes, there ws 1.8-fold increse in po E rdioctivity in response to TNF. TNF induction of po E messenger RNA bundnce is ssocited with phenotypic modultion. Three mrkers for phenotypic modultion were ssessed to compre with chnges in po E mrna expression during our experimentl incubtions. TNF, dded t 5 ng/ml for 24 h, significntly depressed DNA synthesis (14.5±1.7 vs. 9.0±0.7 cpm X 103/mg protein, control versus TNF) in THP1 cells. In Tble V, expression of ICAM- 1 mrna is presented s function of TNF tretment durtion. This gene codes for n dhesion molecule tht is counterreceptor for LFA-1 expressed on other leukocytes; interction between these two proteins my enhnce leukocyte recruitment nd communiction (23). As shown, fter 6 h in TNF, ICAM- 1 messenger RNA species remined undetectble. However, fter 18 nd 24 h, there ws n increse in ICAM-1 messenger RNA bundnce produced by TNF tretment. TNF tretment is lso ssocited with rpid nd trnsient enhncement of c- fos gene expression (Tble V). Levels for c-fos messge were undetectble in untreted cells. However, these becme esily detectble fter 15 min in TNF nd remined elevted for t lest 30 min. Levels of the c-fos messge gin becme undetectble fter 60 min of TNF tretment. Trnsduction pthwys for TNF effect on po E gene expression. Mny of the effects of TNF hve been scribed to second messenger pthwy tht uses cermide s n importnt regultory molecule (24-26). These effects cn be reproduced by treting cells with cell permeble nlogues of cermide, nd we used this pproch to evlute the importnce of this second messenger for trnsducing the effect of TNF on po E gene expression. As shown in Fig. 3 (upper pnel), C2- cermide did not reproduce the effect of TNF on po E gene expression, nor did it potentite the effect of TNF on this gene. Similrly, C2-cermide did not reproduce the effect of TNF on ICAM-1 expression. In this cse, however, the TNF effect ws potentited by cermide (Fig. 3, middle pnel). Neither extending the incubtion in C2-cermide for up to 72 h, ddition of C6- cermide nlogues, nor tretment of cells with Tble V. Time Course Effect of TNF on c-fos nd ICAM-I mrna Expression c-fos//l-ctin Control N.D. TNF 15 min 0.13 TNF 30 min 0.15 TNF 60 min N.D. ICAM- l/,6i-ctin Control N.D. TNF 6 h N.D. TNF 18 h 0.41 TNF 24 h ug of totl cellulr RNA ws isolted from untreted cells or cells treted with 5 ng/ml of TNF for the indicted times nd nlyzed by Northern blot hybridiztion. The intensity of the bnds for ICAM-1 or c-fos long with,b-ctin ws determined by scnning densitometry. N.D. indictes tht there ws no c-fos or ICAM-1 signl detected for tht experimentl condition. Vlues shown re the verge of duplicte smples, which vried by < 10%. sphingomyelinse to generte endogenous cellulr cermide led to induction of po E mrna bundnce (dt not shown). TNF effects cn lso be medited by ctivtion of pkc in trnsduction pthwys seprte from those involving cermide (24-26). We therefore exmined whether pthwy inhibition of pkc ctivtion during TNF tretment would ttentute po E gene response. In the experiment shqwn in the bottom pnel of Fig. 3, tretment with TNF produced significnt increse in po E gene expression (P < 0.02). Concurrent tretment with the pkc inhibitor H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperzine, completely prevented this induction however. A similr inhibition of TNF stimultion ws produced by using stursporine to inhibit pkc ctivity (not shown). Effect of TNF on po E gene trnscription. We next considered the mechnism by which TNF increses po E mrna bundnce. For other mcrophge genes, TNF hs been shown to increse mrna bundnce by stbiliztion of mrna species without ny chnge in gene trnscription rte. To ssess the contribution of incresed po E gene trnscription, THP1 cells were trnsiently trnsfected with chimeric construct contining po E promoter sequences (from to +24 bp) fused to luciferse reporter. After trnsfection, single pool of cells ws creted to normlize trnsfection efficiency, nd this pool ws then liquoted for incubtions in TNF tretment for 24 nd 48 h. TNF induced the expression of this chimeric construct 2.2-fold t 24 h nd 2.1-fold t 48 h (Fig. 4). Discussion Our studies demonstrte tht tretment with TNF stimultes po E gene trnscription, mrna bundnce, nd po E synthesis nd secretion in monocyte/mcrophge cells in phenotypedependent mnner. Such concordnt response t ll mjor regultory loci is not demonstrted by ll mcrophge trgets of TNF modultion. For exmple, for FCy HIb, TNF hs been shown to decrese protein expression but increse mrna bun- 918 Dun et l.

5 0*0 *1 0 N0 so O." C.S0 4L.' - I - 01-Wd no_ 1I." I e*g. C" U 0 I l 4-0, L N.D. ND. nw-o UP0 control H7 TNF TNF+H7 Figure 3. Trnsduction pthwys for TNF ctivtion of po E gene expression. Cells cultured with RPMI-1640 contining 0.05% free-endotoxin BSA were treted with 5 ng/ml of TNF, 5 AM of C2-cermide, 10 jig/ml of H7, or combintion of these for 24 h. Northern blots for po E, ICAM-1, nd f3-ctin were processed s described previously. The upper pnel shows the effect of TNF nd C2-cermide on po E mrna bundnce fter correcting for,/-ctin signl. The middle pnel shows the TNF nd C2-cermide effect on ICAM-1 mrna bundnce fter the sme correction. N.D. indictes tht there ws no detected signl for tht experimentl condition. Vlues shown re the men of duplicte smples, which vried by < 10%. The lower pnel shows the effect of H7 inhibition on TNF stimultion of po E mrna bundnce fter correction for /l-ctin bundnce. Vlues shown re the men±sem from triplicte smples. dnce in mcrophges, nd this incresed mrna bundnce is due to chnge in mrna stbility (21). While our studies estblish tht incresed po E gene trnscription contributes to enhnced po E mrna bundnce fter TNF tretment, potentil role for po E mrna stbiliztion is not excluded, especilly for the lrge increse of po E mrna levels mesured fter prolonged incubtion in TNF (up to 13-fold increse fter 72 h). Alterntively, inclusion of dditionl po E TNF Modultes po E Gene Expression 919

6 "4) o 04 0 we1 24 h 48 h m I control TNF Figure 4. TNF promotes the expression of the po E gene promoter. Cells trnsfected with -2300/+24 po E pgl-2 were cultured with 10 ng/ml of TNF for the indicted times nd then hrvested for mesurement of luciferse ctivity. All cells were derived from single pool of trnsfected cells to normlize for trnsfection efficiency s described in Methods. The dt re shown s the men±sd of luciferse ctivity (reltive light units/mg protein X 10-6) from triplicte smples. gene elements my be necessry to mximize the trnscriptionl response of chimeric constructs to TNF. Others hve studied the effect of TNF on po E expression with inconsistent results. Menju et l. investigted the effect of TNF on THP1 cells fter these cells were treted for 4 d with PMA, 12-0-tetrdecrvolyphorbol-13-cette (12). Additionl tretment for 12 h in TNF produced no effect on po E mrna bundnce or protein synthesis. Zuckermn treted thioglycollte-elicited mouse peritonel mcrophges with TNF for longer period of time (48 h) nd found n 50% reduction of - po E protein secretion (10). Both of these reports re in ccord with our observtions regrding the phenotype-specific effect of TNF on po E gene expression. Monocyte/mcrophges re multipotentil cells tht cn respond to their humorl environment in divergent wys. Phenotype-dependent modultion of po E expression by TNF is consistent with the pleiotropic effects of TNF tht hve been reported for other mcrophge trgets (22, 27). Such phenotype-dependent response to cytokine stimultion my be relted to ctivtion of endogenous cytokine synthesis nd secretion by the fully differentited nd ctivted mcrophge. For exmple, PMA stimultion hs been shown to increse TNF mrna expression within 3 h nd TNF protein synthesis within 10 h in humn monocytes (28). However, if endogenous synthesis of TNF by differentited mcrophges ccounts for the lck of po E induction fter tretment with exogenous TNF, the suppression of po E expression observed by Zuckermn et l. (10) nd by us (Tble I) during prolonged incubtions in TNF would not be expected. Alterntively, phenotype-specific effects of TNF my relte to differences in cell-mtrix interctions between monocyte nd mcrophge phenotypes. Adhesion of cells to extrcellulr mtrix hs been shown to modify their response to TNF (29). Further, in the vessel wll, it is likely tht the presence of other cytokines will be involved in modulting monocyte/mcrophge response to TNF. Interferon-y, for exmple, hs been shown to be n importnt influence on the response of the IGF- 1 expression fter tretment of mcrophges with TNF (30). TNF tretment produced cell cycle rrest s indicted by mrkedly reduced rte of DNA synthesis. In ddition, TNF incresed the expression of c-fos within 15 min of its ddition. Rpid nd trnsient induction of c-fos expression serves s mrker for mcrophge differentition nd/or ctivtion (31). Also, TNcF enhnced the expression of the ICAM-1 gene. In rteril wll plques, ICAM-1 expression is found in rteril smooth muscle cells nd mcrophges (32), nd it hs previously been shown tht TNF induces the expression of ICAM-1 in rteril smooth muscle cells (33). The effect of TNF on po E gene expression cnnot be generlized to ll cytokines ssocited with monocyte/mcrophge ctivtion or differentition. Brnd et l. dded PMA nd interferon--y nd observed no effect on po E mrna bundnce in THP1 cells (34). THP1 cells hve been shown to express MCSF receptors (35); however, in our lbortory, the ddition of MCSF, with or without phorbol ester, to THP1 cells lso hd no effect on po E mrna bundnce. This grees with the report of Brnd et l., who mde similr observtion regrding the lck of MCSF effect on po E protein synthesis nd secretion in these cells (34). Treting cells with TNF hs reportedly produced sphingomyelin turnover s well s endogenous cellulr cermide (24-26). Investigtion of biologiclly importnt signl trnsduction pthwys for TNF hs focused on the role of cermide s second messenger s well s on potentil role for pkc ctivtion. There re exmples of pkc ctivtion nd cermide producing opposing effects in trget monocyte/mcrophge cells (26). In HL60 cells, TNF leds to incresed ctivity of mitogen-ctivted protein kinse nd the ccumultion of nuclese fctor-kb (NFkB) (36, 37). Both of these effects cn be reproduced by exogenously dded cermide nlogues. In Jurkt cells, TNF decreses DNA synthesis nd increses NFkB ccumultion (38). In these cells, cermide inhibits DNA synthesis but does not produce NFkB ccumultion, lthough it potentites the effect of TNF on NFkB ccumultion. These observtions support role for cermide s second messenger molecule mediting TNF effects. Recently, however, the importnce of cermide s TNF second messenger hs been 920 Dun et l.

7 questioned. For exmple, Betts et l. directly mesured cermide levels, nd they could find no difference produced by TNF tretment in HL60 cells or Jurkt cells, even though NFkB ctivtion ws mesurble in both cell types (39). Our dt demonstrte tht cermide is not involved in TNF modultion of the po E gene. Addition of cell-perment cermide nlogues did not increse po E messenger RNA bundnce nor did it potentite the effect of TNF on po E. We lso observed tht tretment of THP1 cells with sphingomyelinse, to generte endogenous cellulr cermide, did not ugment po E mrna bundnce (not shown). Inhibitors of pkc ctivtion, however, ttenuted the effect of TNF on po E gene expression, suggesting the importnce of this pthwy for mediting TNF effects on the po E gene. With respect to the mechnism for TNF induction of po E gene trnscription, it is noteworthy tht TNF tretment of THP1 cells induces expression of the c-fos gene. The protein product of this gene is component of the AP1 trnscription complex, which we hve previously shown to be importnt for enhnced po E gene trnscription in the monocyte/mcrophge (19). In summry, our results document significnt role for TNF in modulting monocyte/mcrophge po E gene expression. TNF stimultes po E gene trnscription, mrna bundnce, nd protein synthesis in the monocyte/mcrophge in phenotype-specific mnner. This effect is observed in monocyte cells or cells in the erly stge of monocyte/mcrophge differentition. In fully differentited/ctivted mcrophges, TNF hs no effect or reduces po E mrna bundnce. Mcrophges in vessel wll lesions re derived from the migrtion of bloodborne monocytes tht cross the endothelium nd trnsform, over time, into fully differentited mcrophges. Phenotypic monocytes s well s monocyte/mcrophges in more dvnced stges of differentition hve been identified in the vessel wll intim of hyperlipemic nimls (40, 41). 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