Proc. Natl. Acad. Sci. USAVol. 79, pp. 922-926, February 1982Medical Sciences

Lymphocyte-conditioned medium protects human monocyte-macrophages from cholesteryl ester accumulation

(atherosclerosis/lipoprotein receptors/lympholdnes)

ALAN M. FOGELMAN*, JANET SEAGER*, MARGARET E. HABERLAND*, MARTHA HOKOM*,RICHARD TANAKA*, AND PETER A. EDWARDS*t*Division of Cardiology, Department of Medicine, and tDepartment of Biological Chemistry, School of Medicine, University of California, Los Angeles,California 90024

Communicated by William N. Valentine, October 16, 1981

ABSTRACT Exposure ofhuman monocyte-macrophages to aslittle as 50 IAI of culture medium from lymphocytes stimulated byconcanavalin A (Con A) resulted in a dramatic decrease in the ac-tivities of the low density lipoprotein (LDL) receptor pathway, theLDL-dextran sulfate pathway, and the scavenger receptor path-way. This effect was not seen when the monocyte-macrophageswere exposed to culture medium from lymphocytes cultured with-out Con A or with Con A together with a-methyl mannoside orcontrol medium without lymphocytes. The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase also decreased in. mono-cyte-macrophages exposed to culture medium from stimulatedlymphocytes. Acyl-CoA:cholesterol O-acyltransferase activity,protein synthesis, protein content, phagocytosis of heat-killedyeast, and non-receptor-mediated endocytosis were not inhibited.Monocyte-macrophages exposed to malondialdehyde altered-LDLin the presence of stimulated lymphocyte culture medium accu-mulated substantially less cholesteryl esters than did cells in con-trol medium. We propose that substances produced by stimulatedlymphocytes may be useful in protecting macrophages from cho-lesteryl ester accumulation.

There now is substantial evidence that many of the foam cellsin atherosclerotic lesions are macrophages derived from bloodmonocytes (1-5). On the basis of studies from this laboratory(6-8) we concluded that normal low density lipoprotein (LDL)does not cause cholesteryl ester accumulation in macrophagesderived from human monocytes. However, cholesteryl esteraccumulation of the degree found in foam cells was inducedwhen LDL was modified by malondialdehyde. This malondi-aldehyde-modified LDL (MDA-LDL) entered the cells througha receptor for negatively charged proteins, the scavenger re-ceptor (6-10). Dextran sulfate complexed with LDL (11) entersmacrophages by a different pathway and also causes cholesterylester accumulation. Because it has been reported that the cho-lesterol content of activated macrophages from lower animalsis higher than that of nonstimulated macrophages (12, 13), wereasoned that activation of human macrophages by stimulatedlymphocytes might promote the uptake of exogenous choles-terol. Therefore we tested the effect of supernatants from hu-man lymphocytes exposed to concanavalin A (Con A) on thecholesterol metabolism ofmonocyte-macrophages isolated fromthe same person. Unexpectedly, we found that both the influxof exogenous cholesterol and the activity of the rate-limitingenzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglu-taryl-coenzyme A (HMG-CoA) reductase, were markedly re-duced. On the basis ofthese studies we propose that substancesproduced by stimulated lymphocytes may be useful in protect-ing macrophages from cholesteryl ester accumulation.

METHODS

Lymphocytes and Macrophages Derived from Monocytes.Pure human lymphocytes and monocytes were prepared fromthe blood of normal individuals by using counterflow centrif-ugation as described (method BB in ref. 8) except that the lym-phocyte fractions were collected at 8.8, 10.2, 11.5, and 12.5 ml/min, respectively. The monocytes (106 cells) were cultured in35 X 10 mm plastic tissue culture dishes in 1 ml of 30% au-tologous serum in Dulbecco's modified Eagle's medium (GIBCO,no. 430-1600) supplemented with 24 mM NaHCO3, 10 mMHepes, insulin (8 ,pg/ml), glucose (2 mg/ml), and antibiotic-antimycotic (GIBCO, no. 600-5245) (1:100) [hereafter referredto as medium B in order to be consistent with our previouspublications (6-8)]. Viability was determined by trypan blueexclusion and ability to ingest heat-killed Candida albicans (14,15). In most experiments the lymphocytes (3 x 106 cells per ml)were cultured in 25-cm2 tissue culture flasks (Coming, no.25100) in 30% autologous serum in medium B supplementedwith 0.350 nM MnCl2. Unless otherwise stated, lymphocyte-conditioned medium (LCM) was prepared by incubating thelymphocytes for 2 days with Con A (Sigma, no. C-2010) at 25tug/ml. Parallel flasks with Con A but without lymphocytesprovided the control medium (CM). At the conclusion of theincubation the lymphocytes were removed by centrifugation,methyl a-D-mannoside (Sigma, no. M-6882) was added to pre-vent further action of the Con A, the supernatant was filteredthrough a 0.45-,um-pore-diameter filter, and aliquots werecombined with fresh medium (30% autologous serum in me-dium B) prior to addition to the monocyte-macrophages.

Sera and Lipoproteins. Autologous serum, LDL, and MDA-LDL were prepared and the lipoproteins and human serumalbumin (hereafter referred to as albumin) (Sigma, no. A-8763)were radioiodinated as described (7).

Assays. The proteolytic degradation of "2I-labeled native-LDL ('25I-native-LDL) and "2I-labeled MDA-LDL (125I-MDA-LDL) was used as a measure of receptor-mediated endocytosis(LDL receptor pathway, LDL-dextran sulfate pathway, andscavenger receptor pathway) and the degradation of "2I-labeledalbumin ("2I-albumin) was used as a measure of non-receptor-mediated endocytosis. The proteolytic degradation of the 125I-labeled proteins was measured by assaying the amount of 5I-labeled trichloroacetic acid-soluble (noniodide) material formedby the cells and excreted into the culture medium as previouslydescribed (6, 7, 16). Free and esterified cellular cholesterolcontents were determined using gas/liquid chromatography

Abbreviations: LDL, low density lipoprotein; MDA-LDL, LDL mod-ified by malondialdehyde; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; Con A, concanavalin A; LCM, lymphocyte-conditionedmedium; CM, control medium prepared without lymphocytes.

922

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 79 (1982) 923

.0*2.

-e _O_______=--_-_______

1.6 B

1 .2 1.

0 CM No LCM ConA All CM No LCM ConA AllConA + fresh Con A -> fresh

aMeMan caMeMan

FIG. 1. Effect of LCM on LDL and scavenger receptor activities. Monocytes were cultured in 1 ml of 30% autologous serum in medium B. After2 days the medium was removed and replaced with 0.5 ml of fresh medium of the same composition together with 0.5 ml of LCM (heavily stippledbars) or 0.5 ml of CM. Other controls included 0.5 ml of the supernatant from lymphocytes incubated without Con A (No Con A), lymphocytes in-cubated with Con A together with a-methyl mannoside from the start of the incubation (lightly stippled bars, Con A + aMeMan), all fresh me-dium-i.e., 30% autologous serum in medium B (All fresh). Three days later the medium was removed, the cells were washed, and 20 pug of 1251-native-LDL (411 cpm/ng of protein) with or without a 50-fold excess of nonradioactive-LDL (A) or 10 ,ug of 1251-MDA-LDL (427 cpm/ng protein)with or without a 50-fold excess of nonradioactive MDA-LDL (B) was added. After 4 hr at 370C the amount of 1251-labeled acid-soluble materialin the medium was determined. The values shown are the mean ± 1 SD of quadruplicate dishes incubated without excess nonradioactive lipoprotein.Subtracting the values obtained in the presence of excess nonradioactive lipoprotein from the values obtained in the absence of nonradioactivelipoprotein revealed that 97 ± 1% and 95 ± 1% (CM, A and B, respectively) and 96 ± 1% and 94 ± 1% (LCM, A and B, respectively) of the releaseof radioactivity were due to degradation by high-affinity receptors.*Pf < 0.001 compared to CM.

- =o

-0

b.D

-~4 S

LCM Con A CM LCM Con A CM LCM Con A CM

aMeMan aMeMan aMeMan

FIG. 2. Effect of LCM on scavenger receptor activity. Monocytes were cultured in 1 ml of 30% autologous serum in medium B. After 2 days themedium was removed and replaced with 0.5 ml of fresh medium of the same composition together with 0.5 ml of LCM (heavily stippledbars) preparedwith Con A at 5 Ag/ml (A) or 25 Ag/ml (B and C), or with the same concentrations of Con A together with a-methyl mannoside (aMeMan; 50mM in A and B, 250 mM in C) from the beginning of the incubation (lightly stippled bars) or with 0.5 ml of CM from parallel incubations withoutlymphocytes (open bars). Three days later the medium was removed, the cells were washed, and 10 'ug of 125I-MDA-LDL (213 cpm/ng of protein)was added. After 4 hr at 370C the content of 125I-labeled acid-soluble material in the medium was determined. The values shown are the mean+ SD of quadruplicate dishes.*P < 0.001 compared to CM.

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924 Medical Sciences: Fogelman et at

(6, 7), and HMG-CoA reductase activity was determined inKyro EOB (Procter and Gamble) extracts (17, 18). Acyl-CoA:cholesterol O-acyltransferase activity in whole cells wasdetermined by measuring the incorporation of label from[14C]oleate albumin into cholesteryl ['4C]oleate as described byBrown et aL (19). Protein was determined by the method ofLowry et al (20).

RESULTSThe degradation of both 125I-native-LDL and '25I-MDA-LDLby high-affinity receptors dramatically decreased (Fig. 1) whenmonocyte-macrophages were exposed to medium from lym-phocytes cultured with Con A (LCM). The lymphocyte super-natant had no significant effect when Con A was omitted (Fig.1). When 50 mM a-methyl mannoside was added together withthe Con A from the beginning ofthe lymphocyte incubation theresulting effect upon the receptor activities of the monocyte-macrophages was substantially blunted (Fig. 1). The experi-ments shown in Fig. 2 demonstrate that at higher ratios of a-methyl mannoside to Con A the inhibitory effect of the lym-phocyte supernatant upon the receptor activities of the mono-cyte-macrophages was completely blocked. Taken together(Figs. 1 and 2), these data constitute strong evidence that lym-phocytes stimulated by Con A produce substance(s) that result

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LCM, ml

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FIG. 3. Effect of LCM on the degradation of '2rI-native-LDL, 1251_MDA-LDL, and 1251-albumin. Monocytes were cultured in 1 ml of 30%autologous serum in medium B. After 2 days the medium was removedand replaced with a volume of fresh medium of the same compositiontogether with the volume of LCM shown on the abscissa such that thetotal volume was 1 ml. Three days later the medium was removed, thecells were washed, and 20 lg of 125I-native-LDL (e, 201 cpm/ng ofprotein), 10 pg of 1251-MDA-LDL (o, 187 cpm/ng of protein), or 25 mgof 125I-albumin (A, 143 cpm/ng of protein) was added. After4 hr at37Cthe content of 125I-labeled acid-soluble material in the medium wasdetermined. The values shown are the mean ± SD of quadruplicatedishes.

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FIG. 4. Effect of LCM on receptor activities, HMG-CoA reductaseactivity, cholesterol acyltransferase activity, protein synthesis, andprotein content of monocyte-macrophages. Monocytes were culturedin 1 ml of 30% autologous serum in medium B. After 2 days the mediumwas removed and replaced with 0.65 ml of fresh medium of the samecomposition together with 0.35 ml ofLCM or CM. Three days later themedium was removed, the cells were washed, and 20 ,ug of 25I-native-LDL (267 cpm/ng of protein) (lightly stippled bar, CM, and open bar,LCM, in A) or 10 pug of '25I-MDA-LDL (247 cpm/ng of protein) (dottedbar, CM, and heavily stippled bar, LCM, in A) was added. After 4 hrat 370C the content of 125I-labeled acid-soluble material in the mediumwas determined. (B) HMG-CoA reductase activity was determined incells that hadbeen incubated inCM (stippled bar) or inLCM (open bar).(C) Incorporation of label from ["4Cjoleate albumin (0.13 mM, 13,203cpm/nmol) into cholesteryl ['4Cloleate was determined in cells thathad been incubated in CM (stippled bar) or in LCM (open bar). (D) In-corporation of L-[4,5-3H]leucine (83 Ci/mmol) into trichloroacetic acid-precipitable protein was determined in cells that had been incubatedin CM (stippled bar) or in LCM (open bar). (E) Protein content of cellsthat had been incubated in CM (stippled bar) or in LCM (open bar) wasalso determined. The values shown in C are duplicate determinationsand their averages from 8 dishes were pooled from each condition. Allother values are the mean + SD of quadruplicate dishes.*P < 0.001 compared to CM.

Proc. Nad Acad. Sci. USA 79 (1982)

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Proc. Natl. Acad. Sci. USA 79 (1982) 925

1.2

~0.-0.4

CM LCM CM LCM CM LCM

FIG. 5. Effect of LCM on the degradation of 125I-native-LDL, 125I_LDL-dextran sulfate, and I-MDA-LDL. Normal monocytes werecultured in 1 ml of 30% autologous serum in medium B. After 2 daysthe medium was removed and replaced with 0.65 ml of fresh mediumof the same composition together with 0.35 ml of LCM or CM. Threedays later the medium was removed, the cells were washed, and 20 ,gof i25I-native-LDL (268 cpm/ng of protein) (open bars), 20 ,g of 125I_native-LDL (268 cpm/ng of protein) together with dextran sulfate (Mr500,000) at 5 ug/ml (stippled bars), or 10 ,ug of 125I-MDA-LDL (270cpm/ng protein) (dotted bars) was added. After 4 hr at 370C the contentof 1 5I-labeled acid-soluble material in the medium was determined.The values shown are the mean ± SD of quadruplicate dishes.*P < 0.001 compared to CM.

in decreased LDL and scavenger receptor activities ofthe mon-ocyte-macrophages. Using the formula

,ug degraded in CM - gg degraded in LCMjig degraded in CM

x 100%

to analyze the data from 14 different donors, we found that theactivity of the LDL receptor pathway decreased 40 ± 14%(range 19-63%) and the activity of the scavenger receptor path-way decreased 67 ± 16% (range 25-90%) in the LCM as com-pared to the CM.

The potency and specificity of the LCM is shown in Fig. 3.As little as 50 A1 of LCM in a 1-ml incubation mixture caused

60 - A

g50 -

20 -

30-0

20-

CM LCM CM LCM+ +

MDA- MDA-LDL LDL

a substantial reduction in LDL and scavenger receptor activi-ties. The uptake and degradation of "2I-albumin by the mon-ocyte-macrophages was linearly related to the concentration ofprotein and therefore consistent with non-receptor-mediatedendocytosis, as has been reported to be the case for human fi-broblasts (9). In contrast to the effect ofthe LCM upon the LDLand scavenger receptor activities of the monocyte-macro-phages, as much as 500 A.l of the LCM had no significant effecton the degradation of "2I-albumin, indicating that non-recep-tor-mediated endocytosis was not affected. In other experi-ments it was found that the LCM also had no effect on thephagocytosis of heat-killed yeast. Greater than 99% of mono-cyte-macrophages excluded trypan blue and ingested the yeastwhether they were cultured in LCM or CM. Cells cultured inLCM or CM ingested 42 ± 13 and 43 ± 11 yeast cells per mon-ocyte-macrophage, respectively.

It might have been anticipated that a decrease in LDL re-ceptor activity in cells maintained in a medium rich in choles-terol acceptors (30% autologous serum) would result in a net lossof cholesterol from the cells and a compensatory increase inHMG-CoA reductase (21, 22). However, as shown by the ex-periments in Fig. 4, HMG-CoA reductase activity in fact de-creased (Fig. 4B) and cholesterol acyltransferase activity in-creased (Fig. 4C). Neither the incorporation of leucine intoproteins nor the protein content of the cells was significantlyaffected (Fig. 4 D and E).

Basu et aL (11) have demonstrated that LDL forms complexeswith high molecular weight dextran sulfate, and these com-plexes are taken up and degraded by macrophages via a high-affinity pathway that is distinctly different from the LDL re-ceptor pathway or the scavenger receptor pathway. As shownin Fig. 5, the activity of the LDL-dextran sulfate pathway wasalso markedly reduced in cells exposed to LCM.We previously demonstrated that MDA-LDL causes cho-

lesteryl ester accumulation in monocyte-macrophages of thedegree found in arterial foam cells (6, 7). The protective effectof the LCM is clearly demonstrated in Fig. 6. Monocyte-mac-rophages incubated in LCM accumulated substantially less cho-

60 - B

50

40-

340-

20-

10*

CM LCM+ +

MDA- MDA-LDL LDL

FIG. 6. Effect of LCM on cholesteryl ester accumulation. Monocytes were cultured in 1 ml of 30% autologous serum in medium B. After 2 daysthe medium was removed and replaced with 0.65 ml of 10% autologous serum in medium B together with 0.35 ml of LCM (open bars) orCM (stippledbars) containing 10% autologous serum. The next day 500 ,g of MDA-LDL was added to some of the dishes (+ MDA-LDL). Six days later the cellswere washed and the free (A) and esterified (B) cholesterol contents were determined. The values shown are the mean ± SD of quadruplicatedeterminations.*P < 0.001 compared to control.

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926 Medical Sciences: Fogelman et al.

lesteryl ester when exposed to MDA-LDL than did cells in-cubated in CM.

DISCUSSIONMacrophages possess a number of recognition sites for choles-terol-rich particles (6-11, 23-25). The activities ofthree ofthesepathways (LDL receptor pathway, LDL-dextran sulfate path-way, and scavenger receptor pathway) were markedly de-creased in cells exposed to as little as 50 A.l of medium fromlymphocytes stimulated by Con A. This effect was not seenwhen the monocyte-macrophages were exposed to mediumfrom lymphocytes not exposed to Con A, or lymphocytes ex-posed to Con A together with a-methyl mannoside or controlmedium not containing lymphocytes. Taken together, this evi-dence strongly suggests that lymphocytes stimulated by Con Aproduced potent substance(s) that inhibited these three path-ways. Depression of the LDL-dextran sulfate pathway and thescavenger receptor pathway indicates that these substances areprobably not specific regulators of cellular cholesterol homeo-stasis but may be acting upon receptor-mediated endocytosisin general. However, there was no change in non-receptor-mediated endocytosis or in phagocytosis of heat-killed yeast.Under these conditions cholesterol acyltransferase activity in-creased slightly while protein synthesis and protein content didnot change, providing further evidence that the effect of theLCM upon the monocyte-macrophages was not a generaldepression of cellular functions. In preliminary experiments,partially purified lymphokines from an established lymphocyteline kindly provided by David W. Golde and Jerome E. Groop-man markedly decreased the activities of the LDL and scav-enger receptor pathways, suggesting that the substances re-leased from the Con A-stimulated lymphocytes may belymphokines.The greater decrease in scavenger receptor activity (67 +

16%) as compared to LDL receptor activity (40 ± 14%) suggestseither that there are differences in the regulation of the tworeceptors or that there are different substances (lymphokines)affecting the receptor activities. The LCM also inhibited theactivity of the rate-controlling enzyme in cholesterol biosyn-thesis, HMG-CoA reductase. Despite the decrease in the up-take of exogenous cholesterol and the presumed decrease inendogeneous cholesterol synthesis, the total cholesterol contentof cells incubated in the absence of added lipoproteins did notdecline appreciably. One explanation for these observationswould be a decrease in cholesterol efflux that partially com-pensated for reduced cholesterol influx and synthesis. How-ever, at present there are no data to substantiate this hypothesisand the observation remains unexplained.

Addition of MDA-LDL to cells in CM produced cholesterylester accumulation. However, addition of MDA-LDL to cellsexposed to LCM resulted in substantially less cholesteryl esteraccumulation. Thus, the net result of exposure to the LCM wasthat the macrophages were protected from cholesteryl esteraccumulation without apparent adverse effects. The isolationof the substance(s) responsible for these metabolic changesshould allow new insights into the mechanisms ofreceptor-me-

diated endocytosis and the relationship between these pro-cesses and cellular cholesterol homeostasis. Moreoever, thesesubstance(s) may have therapeutic potential in preventingatherosclerosis.

We thank Ms. Mercedes Limon and Mr. Daniel Flores for experttechnical assistance. A.M.F. is the recipient of a U.S. Public HealthService Research Career Development Award (HL-00426). P.A.E. wasan Established Investigator of the American Heart Association duringthe course of this work. This work was supported in part by U. S. PublicHealth Service Grants HL 25590, HL 19063, 1T32 HL 07412, and RR865; a grant from the American Heart Association, Greater Los AngelesAffiliate (649-P5); the Laubisch Fund; and a gift from Mr. and Mrs.Louis DaSilvacuriel and family.

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