I(l~f)( I~;I I 173- lY-,..I , I~.~1I~l,~vk.'rScience Pubh.,hcr,.B.V. All right., rc~'r~t-d (HHL~-~7(dl/ql/'~413,.'~)

I~.~ hmu(a cl Ili~pla'w~u .4cm,

173

BBALIP 53778

Cholesterol efflux from macrophages mediated by high-dcnsity lipoprotein subfractions, which differ principally in apolipoprotein A-I and apolipoprotein A-II ratios Eberhard von Hodenberg ', Susanne Heinen i Kalhryn E. Howell -~, Claus Lulcy ~. Wolfgang Kiibler a and Heather M. Bond ~ ! I..'~z.'cr~ily Ih~vmul. Ih'l~urt~m'nt of('urdiohq,.y. Umtcrsil; o/llc.h'll,e~, lh'~d¢ll~'rg l(,'rm.el~ I. ' t "~mc~s~l~ ,,I ( ,d,,~,.I., Ah'dical Schtsd. IX'met. ( "0 (U.S...I.1. ~( "~'nlrul' I.atwrutor~'. UIm c~:~ilvIh~l~ml. I "mr cram ,d I%'/hu, c. t ~'tlu~, ¢( . ' r m . m l attd 4 UnJt cr$hy o f ~itpl¢,~. 2let/,Ih'dic~d .%hcsd, .V~qdc~ ¢llah l

(Received 2h Ap,I I~1 )

Key ~.'ord~.: I II)L: R¢,.,crsccholc.,lcroltran~,l~rl: ('h.lc~,lcroltran~.l~rl: Alxdil~ ~prolcinA-I: AlXdll'~pr~h.'m A-II. ( Mou',c macropha~c)

High.density lipoprotein (HDL) was fractionated by preparative isaeiectric forussin~ into six distinct subpopuluteams. The major difference between the subfrnctions was in the molar ratio of apolipoprotein A-I to apolipolm~tein A-li, ranging from 2.1 to 0.5. The least acidic particles had little apolipoprotein A-II, were larger and c~mlained the most lipid. The effiux capacity of the HDL subfcactions was tested with mouse peritoneal macn~hages aad a mouse mmcrophage cell line (P388D~), either fed with neetylated low-density lipopratein mr free cholesterol. All the HDL snbfractions were equally able to el/flux cholesteroL The emux was concentration dependant and linear for the first 6 h. The HDL sulWractions bound with high affinity (Ka = 6 . 7 - 7 . 9 / z g / m l ) at 4 ~C to the cell ~arface of P3881) u c~ls (211000-359000 sites/cell). IApnd blotting showed that all the HDL subfracthms bound to membrane pal~pep|ides at 60, I N , and 210 kDa. These HDL binding proteins ma) represent HDL rt~-plors. In ~ m m a r ) HDI. particles, which differed principally in ratio of apolipoprotein A-I to apolipoprotein A-II behaved in a similar manner for both eholesterol ¢ffiux and cell surface blndime.

Introduction Plasma levels of high-density lipoprolcin ( l t D I . ) inversely correlate with risk for coronary hcarl disease

[!.2]. HDL is coml~sed of al'~dil~prolcins principally al~)A-I and apoA-II with approximately 50'~ lipid. The plasma concentrations of apoA-I have hccn fi~und to

bc inversely correlated with lhc incidence ol o)ronar~ artery disca~ [3.4]. Whether the amount of apoA-II has any relationship to the risk of o ) r o n a r ~ after) di~ase [5] or not [6] is unclear. The protective aclion o f H D L is thought to bc duc to its ability to mcdialc

Abbreviali~)n~,:al~, al~lil~prolcin: I l l ) L high-dcn,,01~lipoprolcm: acclyl-LDL, acet).lalcd k~-dcn'.il~ lip~n~lcin, PBS. ~h~v,~phalcbuffi:red ,~alinc (1(| mM Na:IIP()~/NalI:PO~ Ipll 7.41. I~} mM Na('l): I)'1"]. dithi¢~lhr¢it~fl: 1( ,'5, b~mc b~'lal call ,,crum: B.NA,

I~winc ..c.rumalbumin. Corrc,.pondcncc: E. ".on thvJcnl~:rg. Mcdl,,ini'-,cl'k:Um*.cr..,lal~khml~. Berghcimcr %lr. ~i~.|)-hrANI I Icid¢lhcrg. Gcrman~.

chole,,lcred ¢tllux from peripheral cell,, m Ihc prqvcc~,~, of rc~,cr~c ch~flc,,tcrol Iran,,Ix~rl. fir.,! prof~v-,:d hy (ilom',c! 17]. Macrl~phagc h~am cell,, ~ith a high cholesterol o m t c n ! arc prc,,,~n! in earl~ alhcr, r~.'lcr,~tic le,,ion,, ;rod m~t~. t~¢ c,m,,idcrcd a,, a Inaj~r iflili;.|ting factor fl,r alhcr~gcnc,,i', [~]. t!1)i, ha', hcen ,,hl~n in cell cuhurc ,,y,,Icm,, to rcm,~vc cholc,,tcrol I m m ,,uch macrophagc,, 19.1(IL and therein, i,, comddcrcd to have a dJrccl anli-alhcrogcnic ctlccl. "Fhe l t D L fraction ( d I . | ~ 3 - 1.21 g / m l ) is comlx~,,cd o f h e t e r o g e n e o u s i)articic,,, and ha', l~cn furlt~.'r IracIf|mated into di,,tinct ,,uhpq~pulation,, ~ ~ , c r a l dilfcron[ mcth,vJ,,: including zonal ultaecnlrilugation [I IJ. ,,ingle vertical ?,pin ultraccnlrifugallon 1121. gradient gel electrophore,,b, ll3]. immun.affinity chromalography [ 14- ih]. i~x~:l¢clric tOcu~ing I 17- Vg]. chr(~maloli~.-u~,ing [211]. [rcc f h ~ i~tachophorcsi,, 121] and l tcparin-Scphar(~c alTinit~, chrl)mat(~g[aph.~ 122] The ,.uhiraclkm,, obtained differ in size. density, charge, alx)lilx~protcin content and lipid comlx~,ition. There arc maj~r diflcrencc', in Ihe ratio (1| alx~A-I to aix~A-II. I.~r~' amount,,

174 of other apolipoprotcins are somctimcs present in specific subfractions ( a ~ t L apoA-IV, apoC-I, apoC-II. alr~K'-II I 1. The aim of our approach was to determine if any differences in the ability of individual specific HDL subfractions to remove cholestcn~l from cells might be critical for the anti-atherogenic action described for HDL. Specific roles for individual almdil~proteins have been suggested from liposome studies with purified al~li~proteins [Z'L24], reconstituted HDL particles 125-27], recombinant constructs [28]. and apolipoprotein specific antil'aglies [29.~1]. These studies indicate thai a~)A-! is im~)rlanl fi)r the interaction of HDL with cells. There is. however, al~) evidence that other ala~li~prolcins [24.31 ]. lipid coml~sition [32-34]. and particle size [35] may play additional roles. A clagsical HDL~ preparation is fractionated by preparative i.,a~electric fix:using. The six fractions obtained were analysed fi~r composition and size. The major differences fimnd were in the ratio of a ~ A - I to al~*A-II ranging from 2.1 to 11.5. The ability of the different HDL subfractions to induce cholesterol efflux was ass "ssed by the amount of cholesterol mass rcm~wed and by use of radioactive tracer cholesterol. Binding of the HDL subfractions to cell surface was determined. Materials and Methods

Lil~roteins l a ~ density lilra~protcin (I.DL) (d 1.1tlt~-1.1163 g/ml), total HDL (d I.II63-1.21 g/ml), and HDL 3 (d 1.125-1.21 g/ml)were prepared from human plasma of healthy young donors by sequential preparative ultracentrifugation [36]. The i.,a~lated lipoproteins were dialy~d againsl 10 mM Tris-HCI (pH 7.41. 11.3 mM EDTA (LDL) or 10 mM Tris-HCl lpH 7.4) (total HDL and HDL~)at 4°C.

Ch,'mh'al modificathm of LDL i.DL was acctylatcd according to the method of Basu el al. [37] with repeated additions of acetic anhydride. The protein content of LDL and acetyI-LDL was determined by a modification of the meth¢~l of lam, ry ct al. 1381.

iYelaaratum of liD~. subfra('tio,.s Fractionation of HDL.~ was performed by preparative isoclectric-fi~cusing (IEF) in 4c,; (w/vl dextran. Ultrodcx (LKB). The gel was prepared by hydrating 4 g Ultn~dex in 911 ml H,O at 25°C. subsequent addition of ampholytes (1.5 mi pH 3-10:3.5 ml pH 5-(~. Serva) and 5 ml HDL 3 (2.2 mg/ml). The gel was l'a~ured onto a glags tray 112 c m x 25.5 cm) and dried at na~m temperature until approximately 37G of the liquid had eval~rated. Focusing was carried out at I/¢,110V. 211

mA. 611 W at 4 - 7 ° C for 18 h. The lipoprotein band pattern was visualized by blotting the gel onto filter paper (Munktel. grade IF) and staining with Sudan black (5g Sudan black in 6110 ml ethanol + 4110 ml destillcd H,O). The filter paper was then placed below the gel tray and each HDL subfraction band excised. The corresl~mding HDL subfractions from two preparative gels were Ix~oled, elutcd by repeated washing in I11 mM Tris-HCI (pH 8.4), dialysed three times against 10 mM Tris-HCI (pH 8.4) and finally against PBS (pH 7.4). Each subfraction was concentrated with Amicon filters (PMI()) to a volume of 1.5 ml. Approximately 911c/~ of the applied lily,protein was recovered. Individual al'x~li~proteins were quantified by single radial immunodiffusion (Daiichi, Tokyo and Sigma). The sensitivity of the assay was > 17.8 mg/dl for apoA-I, > 4.9 mg/dl for apoA-ii, > 1.25 mg/dl for al~C-II, > 2.75 mg/dl for apoC-lll and > 1.2 mg/dl for al~E. The presence of aI~3A-IV was determined by immunoblotting. Cholesterol, triacylglycerol and phospholipids were measured enzymatically using the appropriate kits from Bochringer, Mannheim (Cat. No. 237574; 3111328), Bio Merieux (Cat. No. 61491), and Human, FRG, (Cat. No. H 522).

Cells and cell culture Mouse peritoneal macrophages were harvested in PBS at 4 o C. The cells from each mouse were checked for cont:;mination, pooled, and washed once with cold PBS. The pooled cells were resuspended in Dulbecco's m,~lified Eagle's medium (DMEM) [containing 20% FCS, penicillin (100 U / m i ) and streptomycin (100 p,g/ml)] and plated at I • 10' cells per 35 mm diameter tissue culture dish. After 24 h, at 37 ° C the cells were washed with DMEM to remove non-adherent cells, Average protein content of adherent cells was 30-45 jag protein/dish ((2-5)- I(I5 cells). P388D~ macrophage cell line was grown in alpha MEM medium containing I1)% FCS, 100 U / m l penicillin. 11111btg/ml streptomycin. 2 mM glutamine and 10 mM Hepes (pH 7.4).

Loading cells with choh,sterol Cholesteryl ester accumulation in mouse peritoneal macrophagcs or P388D~ was induced by incubating cells with medium containing 11111~,g acetyI-LDL protein/ml ( i . II1" cells/35 mm dish) for 24 h at 37°C. Alternatively cells were cholesterol loaded for 211 h by supplying a combination of [3H]cholesterol (0.2-2 taCi/ml) and unlabelled cholesterol (50 izg/ml)[39]. The t7(nb H]cholestcrol, 5 Ci/mmol (Amersham International pie.) was dried with N, to remove toluene, and a stock ~lution was prepared in 10 mg/ml unlabelled cholesterol solubilized in 100c~ ethanol. The cholesterol mixture was added to the DMEM media at 37 ° C (containing 2 mg/ml fatty acid free bovine serum

175 all" ,ran (BSA) and 10~ FCS) 30 min prior to labelling the :ells. The extent of cholesterol loading was assessed by staining for lipid droplets with oil- Red O.

were toward the center. Each fraction was lipid extracted, TLC performed and spots corresponding to free and esterified cholesterol were counted.

Cholesterol efflux

Iodination of HDL sub]ractions

Cholesterol loaded cells were washed 4 times with PBS, 0.5 mg/ml BSA at 37°C over a 45 rain period prior to the addition of the efflux media which contained different concentrations of the HDL subfractions or HDL~. At the end of the efflux period the medium was centrifuged. Adherent cells were washed three times in PBS and removed from the dishes by scraping with a rubber policeman. The cells were sonicared on ice for 30 s, the protein (Lowry) and lipid content (as described below) were analysed,

Proteins were iodinated using Iodogen reagent (Pierce Chemical Co.). The iodination reaction contained 100 p,g lodogen, 11.5 mg apolipoprotein in 200 p.I PBS and 1 mCi carrier-free NanZSl, 16.5 mCi/#g (Amersham International pie.). After 10 min at 4 ° C free ~'-Sl was .separated from the protein on a 10 ml Sephadex G50 column. The column was elutcd with PBS and the protein peak dialysed against PBS, 1 mM EDTA (pH 7.4) until less then 5% of the label was .soluble in 10% (w/v) trichloroacetic acid (TCA). Less than 5% of the label was associated with lipid, as determined after extraction with chloroform/methanol (2: !, v/v). The specific activity obtained was 2011-400 cpm/ng apolipoprotein.

Lipid extraction and analysis Lipid analysis was performed by thin-layer chromatography (TLC) [40], using cholesteryl formeate (Sigma) as an internal standard. Cell homogenates and medium were extracted twice with chloroformmethanol (2: 1, v/v), according to a modification of the Folch procedure [41]. Samples were dried under N, and dissolved in 20 p,I chloroform. External standards (0.5 /~1, containing free cholesterol, cholesteryl formeate, cholesteryl ester and triacylglyeerol, in concentrations of 0.1-0.6 ,ug/~l) were added to an equal volume of the extracted samples and applied to highperformance TLC (HPTLC) plates (10 x 20 em silica gel 60F 254/Merck) using a Camag Nanomat capillary dispenser. The separation of neutral lipids was performed in a linear developing chamber (Camag) with n-hexane/n-heptane/diethyl ether/acetic acid (63: 18.5 : 18.5 : I, v/v). The spots were detected using manganese chloride-sulphuric acid. Quantitation of cellular free cholesterol and cholesteryl esters was carried out by ~anning the HPTLC plates by fluore~ence (excitation at 366 nm, emission maxima at 4111 nm) using the Camag TLC scanner ll+ combined to a HewlettPackard-Basic integrator. The densitometry was carried out in the linear range for cholesterol and cholesterol esters between 11.1-11.6/xg//~l. The amount of 3H label in cholesterol or cholesteryl ester was measured by ~raping the TLC spots, solubilizing and counting in scintillation fluid.

Ligand blotting P388D n cells (2- 10~) wcrc washed twice in PBS and homogenized using a glass dounce in 10 mM Tris-HCI (pH 7.41, ! mM EDTA and a proteolytic inhibitor cocktail, containing I # g / m l pepstatin, I # g / m l leupeptin, I ,ug/mi aprotinin, I # g / m l antipain (proteinase inhibitor), and 500 #M benzamidine. A post nuclear supcrnatent was prepared by centrifugation at 20110 × g for 111rain and then centrifuged at 100000 × g for I h at 4°C. The total membrane pellet obtained was resuspended in 10 mM Tris-HCI (pH 7.41, (1.1 mM EDTA and was applied to SDS-PAGE (2011 ptg per lane of protein). The gel was blotted onto nitrocellulose (Schleicher and Schuell with a pore size of 0.45 /.tm) by electrotransfer at 70 mA tor 16 h at 4 ° C in 11.15 M glycine and 211 mM Tris-HCI, 20% methanol. The bands were visualized by Ponceau S (Serva) staining. The nitrocellulose strips were quenched for 3 h in 50 mM Tris-HCI (pH 7.4k 1511 mM NaCI, I mM EDTA, 5el (w/v) low fat milk powder (Ferma Reform). ~-'51-HDL subfractions at 2. 4 or 6 # g / m l were bound in quenching buffer at 37 °C for 4 h. Competition was with total HDL 15110 # g / m l ) which was prebound for 311 min at 37°C prior to binding the t2Sl-HDL. The strips were then washed four times with quenching buffer, dried and exposed to X-lay film for 24 h.

Cell [ractionation P388D I cells were fed with [3Hkholesterol for 411 h as described above, washed and were homogenized in 10 mM Hepes (pH 7.4), 0.25 M sucrose, 2 mM EDTA using a glass dounce. Post nuclear supernatants were prepared by centrifugation at 2000 × g for 10 rain. and were mixed with 20 ml 22~ Percoll in homogenization buffer. The gradients were centrifuged at 200110× g for 2 h at 4 o C. The lipid droplets were at the top of the gradient and the plasma membrane/endo,~mes

Binding of HDL subfractions Binding assays were performed on suspension grown P388D I cells. The cells were washed three times with Hank'., buffer (pH 7.4, I mg/ml BSA) at 4°C. For binding the cells were resuspended in 1 mi Hank's buffer (pH 7.4, 1 mg/ml BSA) and different concentrations of the n-"~I-HDLsubfractions were bound. Cells were then washed three times in Hank+s buffer (pH 7.4), and transferred to new tubes before gamma ra-

176 dioactivity was countcd. ('ompetition was performed by the addition of 500 #tg of unlabelled (total) HDL. Results

4.88) 5.04) 5.t2)

Preparatit'e isoelectri¢".focusing of HDL Preparative isoelectric focusing (IEF) of HDL 3 (d 1.125-1.21 g/roll was used to obtain sufficient amounts of HDL subfractions for comparison of their activities. The IEF gel resolved six major lipoprotein bands between pH 4.8 and 5.5. The bands (Fig.l) focusing at p l values of 5.50, 5.39, 5.24, 5.12, 5.04, and 4.88 were designated suhfractions I-6. The most abundant :,,bfractions were numhcrs 3 and 4 (Table !), with less material present in fractions numbers 1 and 6.

5.24)

5.39) 5.50)

Analysis of HDL suhfractions To compare the HDL subfractions the amount of the different components was calculated relative to a constant apoA-! concentration. Significant differences were found for the subfractions for the amount of apoA-I relative to apoA-Ii and lipid/protein ratios. The polypeptide composition was visualized by SDSPAGE, 10/.¢g of apoA-! were loaded of each subfrac-

Fig. I. Isoelectric fi~cusing of t i D E HDL 3 (d 1.125-1.21 g/roll was fractionated by oreparalive IEF in a 4~7¢ UItrtnlex gel with a pH gradient of 4-6 as described in Materials and Methods. The subfraction bands were visualized by Sudan black staining of a blot taken from the UItrodex IEF gel.

TABLE I

Anall:ds of Itl)L suh#a(tums Apoprotein concentrations were assayed by radial immunodiffusion and lipids were enzymatically measured. To calculate the molar ratios of apoA-I to apoA-II, subunits 28 kDa and 8.5 kDa wcrc used. CE, cholesterol esters: FC. free cholesterol; PL phospholipid; TG triacylglycerides. IlI)L subfraetion I pl of I IDL subfractions pl 5.511

2

3 5.30

4 5.24

Al~protein (apoA-I plus apoA-IlJyield from 12 mg IIDL3 mg O,gt + 0.2 1.24 + 0.2 2.24 ~: 0.33 Apoprotein ¢oml'~sition relative to apoA-I ( IlXl m g / d l ) al~)A-I IIl(J I(K) IIM) al'a)A-II 15. I + 0.0 23,4 + 4.7 35. I ± 4.0 Apo('-II < 1.25 < 1.25 < 1.25 apo('-III < 2.75 < 2.75 ~ 2.75 apoF: < 1.2 < 1.2 < 1.2 Lipid cornlm)sition relative to alm~A-!(mg/dl) ('E 43.fi5 3.3 33.6± 9.0 FC 19.15 0.7 18.15 6.t~ PL 82.3_+ 4.7 80.5± 7.b TG 52.8± 14.3 24.0± 11.2 Molar ratio ara~A-I/apoA-II 2.1

33.0± 12.1± 76,25 15.65

6.4 2.7 I1.¢~ 5.6

5

6

ttDL

5.12

5.114

4.88

3.31 + 0.01

1.57 +_11.33

1.24 511.24

I0{1 48.5 + 2 ] < 1.25 < 2.75 < 1.2 35.6±5.1 11.11_+2.8 74.2±7.7 12.65 2.0

IIN) 611.1 5 7.ll < 1.25 < 2.75 < 1.2 311.9± 6.2 14.9± 6.11 64.75 8.8 18.4+ 10.8

I(N) 61,7 5 3.7 5.65 1.8 3.05 0.8 < 1.2 31.1± 18.8+ fi6.1 5 19.45

5,5 0,1 6,6 12.7

I(MI 46.7 5 1.11 1.750.1 2,8±0.8 < 1.2 38.3± 8.6 15.6± 2.1 71.7_+ 18.11 25.1 5 I.I

1.37

0.30

II.f~6

0.52

0.51

0.69

Ratio of al~)AI/alm~AII (by weight) (~.92 4.51

3.116

2.17

1.71

1.68

2.14

Lipid/protein ratio (by weight ) 1.73

1.114

11.91

I).81

11.85

11.943

1.23

)77

HDL ~ubfractions 2 3 4 5

1

200

6

926645-

"•

31-

./s

100 I I IIIIIII III

.......

21-

~,//

¢0

14-

~

~

~"

_1111 [I III

"""

I I1~.

-

~)

.

L

1

2

.

3 4 5 HDL subfractions

.

6

HDL 3 c

Fig. J. Ability of the dff6:rcnt ItDL subfracli~ms ',o deplete mou~c Fig. 2. SDS-PAGE analysis of the HDL subt'ractkms I0 /zg of apoA-I of each subfraction (lanes l-h) were mixed with sample buffer (without DTI" reductkm) and heated It2 95°C for 5 min. The samples were applied to a 5-15% gradient SD,?,-p~dyacrylamide gel. subjected to ¢lectrophoresis and stained wire Coomassi¢ blue. ApoA-I (28 kDa) and apoA-|l (|7 kDa directs) were identified by Western blotting with specific p~)lyc[,u*a!antibodies.

tion (Fig. 2). The two major apolipoproteins of HDL were separated, apoA-I (28 kDa) and the dimeric apoA-ll (17 kDa). When the samples were treated with disulphide reducing agent (DTT) monomers of apoA-ll were seen (not shown). The apolipoprotein content was quantitated by radial immunodiffusion (Table i). Fraction I with a p l of 5.5 had a higher ratio of apoA-I HOL subfractlon I

J

pcriloneal macrophagcs of cladcstcryl esters. Mouse peritoneal

macrophages t 1" It)" celk/35 mm dish) wcrc loaded with twt~batches of I[X; p,g/m{ acety(-LOL over a 24 h periled. One set or'dishes was harvested and free/cstcrified cholcslcrol ma~;s measured (loaded cells, L). The ~sthcr dishes were incubitted vdth medium containing Illl) p.g/ml of apoA.I of each of the diffi:rent H D L suhfri:cthms ( i - h ) or in the absence of lilx~prolein acccptor (cemtroL C). After 2~) h of incubation the ceils we=,: cxtcnsk'e|y washed with PBS and analysed h)r cellular lyre and c*,terified cholcslerol mass. The results arc expressed as means of duplicate measurements from t~o s,.:parate experiments.

with little apoA-II (molar ratio of 2.1, al~)A-I/apoA-ID whereas the more acidic fractions (5 and ~) contained relatively higher amounts of apoA-II (molar ratio

HOL subfracllon 2

HDL subfracllon 3

50 ¸

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40-

40' 30'

20' 10

10'

10

0 •

•It

¢~

.~

.

~,'?

.

¢~

.

.

.

.

.

.

•

.

.

.

.

.

.

.

.

~

.

.

.

~

0¢=

nm HOL s u b f r a ~ l o n 4

HOL ,~Jbffa,~lon 6

50'

50-

40.

40"

40"

30-

|

20" 10" ~ t m m ~

0

nm

Fig. 3. Size of H D L subfractions. H D L subfraclions were visualized by negative stain and c|ectron microcopy. Ilislograms sh,w. the measured diameters of [(M)particles of c~ch subfraction.

178 T A B L E II

A,m6"sis of size distrih,;ion of ttDI, .s~thJ?acthms The analysis is T h e number of standard error from the mean

calculated from the si;,_e distribution shown in Fig. 3. particles measured for each subfraction was I|~). The (S.E.I and standard devialion (S.D.I are calculated values.

Sub fraction

I

2

3

Mode

I 1.6

I 1.6

11.6

9.33

9.33

Median

11.6

I 1.67

I 1.67

9.33

I 1.67

Mean S.E. S.D.

13.(~9 0.369 4.02

11.77 0.252 2.6fi

1 3 . 1 ( 1 1{).68 12.111 0 , 3 7 0 11.199 0.438 4.049 2.224 J,.992

4

5

6 7.0 9.33 111.47 0,4(14 4.255

apoA-l/apoA-ll of 0.5). This distribution is consistant with known p i values of p l = 5.6-5.7 for apoA-! and p l = 4.9-5.0 for apoA-1l [42]. Low amounts of apoC-ll and apoC-lll were present in fraction 6. ApoE or apoA-IV were not detected by either radial immuno.E

6

®

50-

~

40-

0

-

~

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I 2 3 4 • 5 "---O"-- 6

30--

20-

0

g

50

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i

100 150 apoA-I(i,Lg/rnl)

Cholesterol efflux by HDL subfractions Cholesterol efflux experiments were performed with two different cell systems. Mouse peritoneal macro-

i

200 500000"

60

~

50

~ o

40 ~

30

,~

20

__~

1 2

E

HDLsubftaclions (100pg/mlapoA-I)

400000 300000 "

200000

4 5

•

2

A

3

~

4

•

5 6

3

¢D =~

diffusion or by Western blotting with specific antibodies (data not shown). The lipid composition of the different subfractions is shown in Table 1. The total lipid/apolipoprotein ratio decreases from 1.73 to 0.85 in fraction 1 to 6. Therc were relatively higher amounts of cholcsteryl esters (43.6 mg/dl), phospholipids (82.3 mg/dl), and triacylglyceroi (52.8 mg/dl) in fraction 1, than in fractions 2-6 (cholesteryl esters 30.9-35.6 mg/dl, phospholipids 64.7-80.5 mg/dl, and triacylglycerol 12.6-24.0 mg/dl). The subfractions were analysed for size by negative staining and visualized by electron microscopy. The frequency of particles at different sizes is shown in Fig. 3 and the statistical analysis of this data in Table II. The mean size of particles in subfraction 1 was 13.09 nm and in subfraction 6, 10.47 nm. The difference between the subfractions is statistically significant. There was a gradient of size, with the largest particles in subfraction 1 and smaller particles in subfraction 6. This gradient in size of the subfractions was confirmed by non denaturating electrophoresis (not shown). The particles which contained most lipid also had increased apoA-! to apoA-II ratios and larger sizes. Whereas the more acidic particles containing less lipids and relatively more apoA-ll, were smaller (fraction 5 and 6).

& •

100000

HDL3 None

6

tI

o 0

50

100

150

200

apoA-I(ggtrnl) Fig. 5. Ability of H D L subfractions to deplete P388D I cells of cholesteryl esters and free cholesterol. P388D t cells (I - I11¢' cells/35 m m dish) were loaded with two batches of ItXJ p . g / m l acetyI-LDL over a 24 h period. ('ells were either incubated with 25. 511, 100 or 2iX) p.g apoA-I of each subfraction (1-61. A f t e r l0 h of incubation the cells were extensively washed with PBS and analysed for cholesteryl esters and free cholesterol mass. The results are an average of duplicates and are representative of three seperate experiments.

time(h) Fig. 6. T i m e course of e m u x from mouse peritoneal macrophages.

Mouse peritoneal macrophages were loaded with free l~HJcholesterol 12(1 # C i / l . 11)6 cells per 35 mm dish) in the presence of 2 mg/ml BSA and 10% FCS for 20 h. After washing with PBS 11.2% BSA the cells contained 1.6" 106 dpm (20% cholesteryl esters and 80% free cholesterol). The efflux media (1.2 ml/dish) contained 100 # g / m l of apoA-I of the different subfractions (I-6), H D L ~ or no acceptor. A t time points up to 24 h the media was removed and counted for radioactivity. T L C analysis showed that all the d p m effluxed was as free cholesterol. T h e results are expressed as m e a n s of duplicate determinations.

179 phages and the macrophage cell line P388D~ were fed with acetyl-LDL for a 24 h period. These macrophages accumulated 120-144) #g cholesteryl esters and 20-30 p g free cholesterol/mg ceil protein. Whereas P338D~ ceils supplied with the same acetyI-LDL accumulated 50-60 # g cholesteryl esters and 15-20 # g free cholesterol. Prior to loading, in both cases the amount of free cholesterol and cholesteryl ester was less than 5 p g / m g cell protein. Cholesterol feeding it~lf did not alter the protein content of the cells. After feeding for 24 h the cells were washed to remove excess acetyI-LDL or

N

N

N

R

R

R

1

3

6

1

3

6

92 -

66-

46-

31. - - - a u l l m ~ l l l 4 1 b

- ~poA-,

21-

A Time course of efflux 14-

400000 • -o

HDL subfractaons (50~gtrnl apoA-I)

300000 " 200000'

~

"1o 100000 '

~

--

0

4

6

Nor'e

•

time (h)

20

B. Concentration dependence (4 h) 200000HDL subfract~ons 150000 S

¢

2 3

1000OO

4

• -

'

5 ~

6

50000

' 20

. . . 40 . . . apoA-I(pg~ml)

60

"

Fig. 8. Autoradiograph of iodinated t I D L suhfractions. Suhfraclions I. 3 and f) were labelled with n~'~l using kvJogen. I - l 0 s dpm of each subfraction were applied to a 5 -lSc; SDS-polyacrylamide g e l either after reduction w'ith DTY (R) or non-reduced iN).

H

~

"

)

L subfrachons

150000

~

o

-

~ 100000

2

4 •

5

6 50000

0

20 40 60 80 apoA-I plus apoA41(tig/mf)

cholesterol. The cells were then incubated with the HDL subfractions to measure efflux for peri(~ls up to 20 h. After the efflux period free and esterified cholesterol mass were measured, in the absence of HDL (control) there was only a small decrease in either cholcstcryl esters and frcc cholesterol. This represents background of efflux in the absence of acccptor and cellular metabolism of cholesterol and cholesteryl esters in the absence of serum. All thc HDL subfractions resulted in the cfflux of significant amounts of cholesterol such that the cholesteryl ester content of the cells compared to the control cells in thc absence of HDL

" 8'0

C. Concentration dependence (4 h) 200000

lo

apoA-II (2n)

!

10

0

t~

-

- apoA-II (ln)

3

---0---

m

100

Fig. 7. Ability of ItDL subfraclions Io elllux chole~,+crol from P388D I cells. P3~[)= were loaded with 8 # ( i [;tlJcht~leslcrol/4 ml per e,. i(+~ cells per f~l mm dish as described in the Materials and MelhiKls. At 4ll h the cells contained 6.h- IIY" dpm of v,.-hich ~Ic~ was cholestetyl ester and 40r; free cholesterol. After 40 h the ceils were v,ashed and incubated with efflux media containing each of the dift:erent suhfractions. At the indicaled time points of efflux. 100 ,ul of the media was counted for radioactivity. TLC analysis showed thai all of the dpm effluxed was as free choleslerol. The results shown are expressed as means of duplicates from one experiment, which was represeniive of three different experiments. (A). Time course of cfflux with 50 p.g/ml apoA-I of the different subfractions ( I -h). The background for the efflux was in the presence of 50 pg/ml BSA. (B). Concenlrali~m dependance of efflux at a 4 h time point. Increasing amounts (I(). 30 or 60 pg al'~A-l/ml) of the t|DL suhfractions (1-6) were incubated with the cholesterol-loaded P388DI cells for 4 h. (('). Concentration dependance of efflux, data of Fig. 7B expressed on a basis of al~)A-I + apoA-It.

180 A. HDL subfraction 1

h a d dccrcase:l by 35-45r/c (Figs. 4 a n d 5). T h e differe n c e s b e t w e e n t h e s u b f r a e t i o n s were minimal, m i n o r d i f f e r e n c e s b e t w e e n s u b f r a c t i o n s were not r e p r o d u c e d for any specific s u b f r a c t i o n in o t h e r e x p e r i m e n t s . Neit h e r the a p o A - I I n o r lipid c o n t e n t h a d a n y significant effect on the capacity o f the individual H D L s u b f r a c tion to efflux cholesterol. T o analyse in m o r e detail t h e relative activity o f t h e different H D L s u b f r a c t i o n s m o u s e p e r i t o n e a l m a c r o p h a g e s a n d P388D~ cells were labelled with free [3H]-

lO

x

.B

-

0

A. Total and non-specific binding at 4~C loo ]

w -

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w

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-

20 30 40 ng bound (specific)

50

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B. HDL subfraction 3

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JD

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0

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B.S )ecific binding 80-

40' 20"

i

60

S

1

o

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i 120

C. Specific binding

2

80'

0

O .

0

60" o

F i g . IlL S c a t c h a r d

10

20 30 40 ng bound (specific)

analysis of IIDL

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50

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P38~1)= cells. The specific binding shown in Fig. 9B is used to calculate the amount bound/free fl~r Scatchard analysis and the binding is expressed as ng I:SI-HDL subfraction bound per I-l06 cells. Lines were drawn using a best fit.

40'

20' 0

-

50

C. HDL subfraction 6

60'

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20 30 40 ng bound (specific)

v

2o

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,o 60 80 , o o , 2 o ap0A-I (pg/ml) Fig. 9. Binding of ltDL subfractions to P3X~,i)~ cells at 4°C. (A). Each binding incubation at 40( ` l~r 2 h contained I.II)" P388D I cells in suspension and increasing amounts (#g of apoA-! + apoA-II) of ~-~I-ItDL subfractions I(n). 3( ,~ )or 6(~). Non-specific binding was estimated in the presence of 54X)p.g of unlahelled HDL (total fraction), for each ItDL suhfraction I(11). 3(A) and 6(e). (B). Specific binding is calculated as total binding minus non-specific binding for suhfractions I( El L 3( ~ ) and 6(.~,). (C). Specific binding: data t~f Fig. 9B ~xpressed on a basis of apt~A-! ahme.

cholesterol. T h e free c h o l e s t e r o l w a s u s e d in t h e p r e s e n c e o f B S A (essentially fatty acid free) w h i c h b i n d s u n e s t e r i f i e d c h o l e s t e r o l t h u s stabilizing t h e free c h o l e s terol in solution. A f t e r l o a d i n g with free [3H]cholesterol t h e cells c o n t a i n e d 4 0 - 5 0 /zg cholesteryl e s t e r s a n d 2 0 - 3 0 / , t g free chole,,/.erol p e r m g cell protein. A f t e r a 24 h labelling p e r i o d m o u s e p e r i t o n e a . macrophages contained 1.6. l06 dpm/dish of [-~H]cholesterol label (20% esterified c h o l e s t e r o l a n d

181

subftaction 2 p.g 2 4 6

subfraction 4 4 6

subfraction 6 2 4 6

+ unlabelled 2 4 6 4 4 4

200-

%

11692-

66-

46-

312114-

i0i

i

It

Fig. I I. Ligand blotting of I:~i-IIDL subfractions.A membrane fraction from P388DI cells (2(k)/Jg/lanc) "~,asapplied to 5-15'; SI)S-PA(;t(non-reducing} and transl~crred to nitnK'ellulose.After quenching 2. 4 or h .ug/ml {apoA-I plus ap~}A-II)o[ each t2~l-Ill)l. ,mbfraclitm~'er¢ bound fi)r 6 h at 37°( ". Competition was in the presence of 5{Xl #g/ml of Iotal Ill)l,. The nitroccllulo,,e ~,lrips were s~ashcd and autoradiographed.

8()~ free cholesterol). A time course experiment (Fig. 6) was carried out to determine whether there were any differences in efficiency at early time points of efflux. Efflux could be detected with all the subfractions as early as 30 min. The steady-state rate of efflux at 3 7 ° C was linear for the first 6 h fi)r all thc subfractions and continued fi)r at least 20 h. Although. even fit 20 h a true plateau had not been reached. The background in the absence of an acceptor was I()r~,: of the amount released with HDL. After 20 h all the HDL subfractions and HDL~ had resulted in a similar net cfflux of cholesterol, which represented 19-25C,: of the cellular [~H]cholesterol label. The amount of cholesterol released measured by label compared to the mass measurements is likely to be an underestimate due to an incomplete distribution of the label into the differcnt subcellular pt~ls. All the [~H]cholcsterol label released into the medium by the cells was free cholesterol. The HDL preparations contained no detectable lecithin:cholesterol acyltransfera~ (LCAT) activity, measured with [~H]cholesterol labelled apoA-I lipo-

somes (not shown). In the absence of LCAT no reesterifieation of the effluxed cholesterol would be expected. The cell line. P388D L fed with [~H]cholesterol was also used to test the activity of the different subfractions. After an incubation period of 4(1 h the intracellular label was found to be ()()r/~ in cholesteryl esters and 40F,: in free c h o l e s t e r o l . To analyse the intra-

TA FILl': III Bindm,¢. o/t:~l-IIDI. ~zd,lmctio.~., 1~3,v,?IDtccll~

The binding alfinitic~, are derived from the' Scatchard plots q,, .vn in Fig. t~. "[hc M, (avt:rage apparent molecular weight) o[ the sublractlons v.ere c;.,limalcd from an noll-denaturing gel IIDI ,,uhfraction

.U,

K,j (pg/ml)

# max (rig/I - lip cells)

Itigh-affinity ~,it¢~, per cell

I 3 6

151 (XMI 123(XNI t)j 2tilt

h.74 7.(,~5 7,64

53 47 54

211tHH) 232(KN! 3591NX)

182 cellular distribution of t.he labelled cholesterol the cells were homogenized to seperate lipid droplets and plasma/endosomal membranes on Percoll density gradients. [~H]cholesterol label was found as cholesteryl esters in the lipid droplet fraction (5(}%) and as free cholesterol (30%) associated with the plasma and cndommal membranes. When this fractionation was performed after an efflux period of 20 h with HDL, there was 58% !oss of esterified cholesterol from the lipid droplets, in addition to a 3(1% loss of free cholesterol from the membrane fraction. A time course and concentration dependance of efflux are shown in Fig. 7. The six different subfractions were all equally able to efflux cholesterol from the cells, at both early and longer time points whether compared on a basis of apoA-! content or total apolipoprotein content (apoA-I + apoA-il). As with the mouse macrophages there was a linear rate of efflux for the first 6 h which continued until 2() h (Fig. 7A). The maximum amount at 21) h represented an efflux of 311% of the [3H]cholesterol initially cell associated. A 4 h time point was used to compare the effectiveness of different concentrations of the subfractions (Figs. 7B and C). The experiment was performed with equal amounts of al~A-I from each subfraction thus the subfractions differed principally in the amount of apoA-li. The efflux measured was concentration dependant for all the subfractions in the range of 10-611 p,g/ml. The results were also calculated and plotted on an apoA-I + a p o A - l l basis, which in this case shows only minor non-significant differences between the subfractions (Fig. 7C),

Binding of HDL subfractions The differcnt HDL subfractions were tested for their ability to bind to P388D t macrophages at 4 ° C . To measure binding, the subfractions were labelled with ~251. Fig. 8 shows an autoradiograph of an 5-15% SDS-polyacrylamide gel of the subfractions, either after treatment with dithiothreitol (D'IT) or non reduced. Both apoA-I and apoA-II polypeptides, as well as apo C-ll and al~)C-IIl were labelled with L'51. When the samples were reduced the M-'51-apoA-ll polypeptides migrated as monomers (8.5 kDa) and when non-reduced, dimers were seen (17 kDa). There was a clear difference between the relatively low amount of L~51-apoA-il in subfraction ! compared to the increased amounts of apoA-I! in subfraction 6. Even with a longer exposure no other high molecular weight polypeptides were visible. Binding was performed for 2 h at 4 ° C by which time the binding of HDL to the cell surface has reached a plateau. As for the efflux data, binding was compared on a basis of either apoA-I + apoA-ll (Fig. q A / B ) or apoA-I (Fig. 9C). The Scatchard plots of this data on a

total protein basis (Fig. 10) show a single high-affinity binding site for each HDL-subfraction. The binding affinities (Table !!1) were similal for the different subfractions ( K a = 6 . 7 - 7 . 9 p g / m I ) with 211000359000 sites per P388D~ macrophage cell. The J251-HDL subfractions were also tested for binding to membrane proteins prepared from P388D~ macrophages by ligand blotting. All the subfractions were found to specifically bind to three polypeptides at 60, 101L and 210 kDa (Fig. 1 i). The amount of binding increased with higher amounts of each ~2Sl-HDL subfraction and was competed by :mlabelled HDL 3. These binding polypeptides may represent potential cell surface 'receptors" for HDL. Discussion

Cholesterol enriched macrophages (foam cells) are predominant in the early atherosclerotic lesion, therefore HDL-mediated cholesterol efflux from these cells could play a major anti-atherogenic role. By using HDL subfraetions which contained a constant amount of apoA-I but differed in the content of apoA-II, the effect of apoA-11 on the H D L binding to macrophages and its induction of cholesterol efflux from cholesterol loaded macrophages could be determined. Mouse peritoneal macrophages and a macrophage cell line (P388D~ cells) were cholesterol loaded by incubation with acetyI-LDL or with free cholesterol ,so that cholesteryl esters accumulated in lipid droplets, it was important to use both methods as it could not be certain that the type of pools of cholesterol accumulated were equivalent to those of in vivo foam cells. However, the results for both systems were essentially the same. All the H D L subfractions caused a depletion of cellular cholesteryl esters and efflux of free cholesterol. The apoA-ll content did not significantly affect either the rate or amount of efflux. There were differences for the lipid/apolipoprotein ratio for the subfractions, in particular, fractions 1 and 2 had higher levels of free cholesterol. Since our data show that all the subfractions were equally efficient for cholesterol efflux, the differences in free cholesterol content were not substantially effecting the process. A net loss of cholesteryl ester mass was measured after efflux, however, there may also be ,some cholesterol exchange between cells and HDL. This exchange would be a component of the [3H]cholesterol effluxed by HDL. It is unlikely that the acetyI-LDL used to load the cells is contributing directly to the loss of esterified cholesterol as after feeding, the cells were washed extensively over a period of 45 min, during which the surface acetyI-LDL will have been removed or metabolized by the cells. Any release of acetyI-LDL ,;hould be independant of HDL in the media, thus will be a

183 component of the background levels in the absence of acceptor. There are several different models proposed for the mechanism of cholesterol efflux. Data has been reported [10,43,44] which indicates that HDL is endocytosed by macrophages and subsequently r e c y c l e d having gained cholesterol. Alternatively kinetic studies suggest that the cholesterol acquisition by HDL occurs at the cell surface [45,46] or by aqueous diffusion directly from the plasma membrane [33,47]. There is also evidence that H D L can promote the tran~u)cation of cholesterol from the intracellular membranes to the cell surface for efflux [46]. Our results with the differcnt subfractions indicate that if a receptor is required for efflux the apoA-ll content is not critical. However, the efflux measured could be by either of these pathways. Adipocytes can also be induced to accumulate a large amount of free cholesterol, which subsequently can be accepted by HDL [23,48]. In this case, liposomes or HDL particles containing high amounts of apoA-! compared to apoA-ll were more effective in inducing cholesterol efflux than those with a higher apoA-ll content, suggesting an antagonistic effect of apoA-ll [23]. However in the same adipocyte system it has recently been shown that apoA-i, apoA-I! and apoA-IV all bind to the same polypeptides (receptors) at 80 and 92 kDa by ligand blotting [49]. It is possible that binding and efflux are mediated by different HDL apolipoproteins. Our results showed that all the different HDL subfractions were able to bind with the ~ m e affinity to the cell surface. The binding affinity and number of binding sites per cell obtained for the different HDL subfractions were similar to that previously described fi)r H D L [28]. it is also shown that the HDL subfractions bind to the same membrane polypeptides at 60, l(M), and 210 kDa on a ligand blot. From disulphide bond reduction experiments it appears that at least some of the 100 kDa band exists as directs of 210 kDa and that the 60 kDa band is unrelated [54]. Both apoA-I in a recombinant construct with protein A from S t a p h y l o c o c c u s a u r e u s as apoA-l-protein A [28], and purified apoA-li [50] bind to polypeptides of this size. The I(~P kDa H D L binding polypeptide may well be similar to the 110 kDa HDL binding I~)lypeptide detected in macrophages by Graham and Oram [51], the I(M) kDa (HB2) H D L binding protein purified from liver by Tozuka and Fidge [52], the 92 kDa HDL binding protein purified from adipose cells by Barbaras et al. [49] or to the 120 kDa protein in human placenta dc~ribed by Keso et al. [53]. it is however possible that more than one receptor exists in the various cell types which process H D L in different ways. When .sequence information for those HDL binding proteins is avail-

able, it should be possible to evaluate their physiological roles in the different cell types and tissues. in conclusion HDL subfractions with different apoA-I and apoA-il composition and lipid/protein ratio were cqually effective to efflux cholesterol from macrophages and to bind with high affinity to cell surface binding sites (receptors) on macrophages. The content of apoA-ll of the HDL particle therefore did not significantly modify these activities.

Acknowledgments Part of this work was suprmrted by a grant f r o m Bundesmini:;tcrium ffir Forsehung und Technologic, BMFT, No. 0706332/5 and a grant from ICI Co., Germany. H.M.B. was supported by a fclk)wships from Boehringer Mannheim and the EEC. Support by the American Heart As~)ciation Grant-in-Aid 98-(k653 to K.E.H, is acknowledged. We thank Steven Fuller and Marck Cryklaff for the electron microscopic analysis of the HDL subfractions, Hans AIois Dresel for helpful discussion and critical reading of the manuscript, Armin Stcinmetz for supplying an antibody specific for apoAIV, and Sabina I ~ p p and Marion Forstcr fiw excellent technical assistance.

References I Wilson. P.W.F.. A h , tl. R.D. and ('ast,i'.i.W.P. (19Y-~) Arlerio~.'lero~b, ~. 737-741. 2 Miller. (LJ. and Miller. N.E. (1975) I ante! 4. 16-19. 3 Avogaro. P.. Bitmlo P~m. (i.. ('asmlalo. (;.. C)uinei. (LB. and Belw,si. F. (1978) Artery 4. 3~(5-394. 4 Maci¢iko. J.J.. lhdmu?.. D.R.. Konke. B.A.. Zinsm¢isler. A.R.. l)inh. I).M. and Map. SJ.T. (I'~83) N. [':ngl. J. M e d . . ~ . ~,~5-389. 5 Fager. G.. Wiklund. O.. Ohff~mn. S.O.. Wilhelmseu. I.. aml l~mdjvrs. (L (1981) Arlcrio~.clerosi~ I. 273-27(L 6 l)uchoi~. P.. Kandou~,~i. A.. Fievcl. P.. Fourricr. J.l... Bertrand. M.. Korcn. I'. and Drucharl. J.C. (19S7} Alheroselero~.i~ 6N. 7 (Hom~cl. J.A. I 1 9 ~ ) J. Lipid Re~,.