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Composition and metabolism of lipid in macrophages from normally fed and cholesterol-fed rabbits
ESPERIhIENTAL
Ah’ll
hlOLECULAR
PATHOLOGY
Composition and Normally
Metabolism Fed and
28, 65-75
( 1978)
of Lipid in Macrophages Cholesterol-Fed Rabbits1
from
SUDIPTA KAR AND ALLAN J. DAY Department
of Physiology, Received
April
University 18, 1977,
and
of Melbourne,
Victoria,
Australia
in revised
August
8, 1977
form
3052
The composition and metabolism of lipid in peritoneal macrophages obtained from normally fed rabbits were compared with those of macrophages obtained from cholesterol-fed rabbits. Macrophages from cholesterol-fed rabbits had a higher cholesterol content and a markedly higher cholesterol ester content than normal macrophages. The increase in cholesterol ester content was most marked for cholesterol oleate and cholesterol linoleate, with the cholesterol ester fatty acid composition of the cholesterol-fed macrophages resembling that of foam cells derived from aortic lesions of similarly cholesterol-fed rabbits. Metabolic differences were also demonstrated between the cells obtained from normal and cholesterol-fed rabbits. In the latter, incorporation of “C-laheled acetate into cholesterol was almost completely suppressed whereas in macrophages from normally fed rabbits, W-labeled acetate was incorporated predominantly into cholesterol. Incubation in vitro of normal macrophages for periods up to 20 hr with hyperlipemic serum, however, was not associated with any appreciable suppression of cholesterol synthesis.
Human fatty streak lesions and cholesterol-induced atherosclerotic lesions in experimental animals are characterized by the intracellular accumulation of cholesterol and cholesterol esters in foam cells in the intima. Most of these cells originate as smooth muscle cells which are stimulated to proliferate and accumulate lipid in situ (Parker, 1960; Geer et al., 1961; Buck, 1962; Parker and Odland, 1966). Ultrastructural studies have demonstrated, however, that foam cells in arterial lesions have a varying morphology. While many of them have the characteristics of smooth muscle cells, namely, myofibrils, a limiting basement membrane, and pinocytotic vessels along the basement membrane, lipid-containing ceIIs with morphological and histochemical characteristics ‘of macrophages can also be seen in the developing atherosclerotic lesion (Balis et al., 1964; Geer, 1965a,b). Blood monocytes can be observed penetrating the endothelium (Poole and Florey, 1958), and lipid-containing macrophages circulate in the blood under various circumstances (Still and O’Neal, 1962; Simon et al., 1961; Suzuki and O’Neal 1967). Some of the foam cells in the atherosclerotic lesion may therefore be derived from tissue macrophages or blood monocytes. The synthesis and metabolism of lipid by macrophages obtained from the peritoneal cavity of normal rabbits have been extensively studied (Day, 1967). These cells contain 1 This Australia.
work
was
supported
by
a grant-in-aid
from
the
National
Heart
Foundation
of
65 0014-4800/78/0281-0065$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
KAR AN11 DAY
cholesterol-esterifying enzymes ( Day and Tume, 1969; Tumc~ and Day,. 1970)) and their uptake of cholesterol particles is associated with increased phospholipid synthesis (Day et al., 1966a). Recently Wurster and Zilversmit (1971) observed that peritoneal macrophages obtained from cholesterol-fed rabbits showed a marked increase in their cholesterol and cholesterol ester content compared with normal macrophages, whereas granulocytes collected from cholesterol-fed rabbits showed no such increase. In the present study, the composition and synthesis of lipid in peritoneal macrophages of cholesterol-fed rabbits have been further studied and compared with that of peritoneal macrophages obtained from normally fed rabbits. METHODS Collection of mucrophages. Male New Zealand White Rabbits approximately 6 months of age were used throughout the experiments. Normally fed rabbits received normal rabbit chow while cholesterol-fed rabbits were fed a diet containing 1% cholesterol and 3% peanut oil for periods of 3 to 4 months prior to collection of the macrophages. Five days after the intraperitoneal injection of sterile liquid paraffin (Nujol), macrophages were removed from the peritoneal cavity by saline washing as previously described (Day and Fidge, 1962). The cells were suspended in Media 199 and counted using a standard hemocytometer chamber. From 100 to 300 X lo6 cells were obtained from the washings in normal rabbits; in cholesterol-fed rabbits a somewhat smaller number of macrophages was obtained (from 50 to 100 x 10” cells). The cells were dispensed in appropriate volumes for extraction of lipid or for protein assay or were dispensed in culture flasks for metabolic study as set out below. Clzenzical Assam.For assay of protein, between 5 and 10 x 10F cells in duplicate were washed with normal saline and taken up in sodium hy,droxide. Total protein content was assayed by the method of Lowry et al. (1951). For lipid analysis, aliquots of the cells from either cholesterol-fed or normally fed rabbits were extracted with chloroform:methanol (2:1, v/v) and were washed by the method of Folch et al. ( 1957). Lipid phosphorus was determined in the lipid extract by the method of Bartlett (1959). Triglycerides were assayed by the method of Van Handel and Zilversmit (1957). Cholesterol and cholesterol ester were assayed by densitometry following separation of the lipid fractions, in aliquots of the lipid extracts by thin-layer chromatography on prewashed silica gel G plates using the solvent system, N-hexane:diethyl ether:acetic acid (112:38:3, v/v/v). After development the plates were sprayed with 5% phosphomolybdic acid in ethanol and then heated at 90°C for 15 min to develop color. The spots were scanned using a Schoeffel Model SD 3000 dual-beam spectrodensitometer at a wavelength of 560 nm. An integrated output was used to calculate mass. Assays of amounts less than 1 pg of cholesterol or cholesterol ester were possibly by this method. Gas-liquid chromatography. Phospholipid, triglyceride, and cholesterol ester in the lipid extracts were separated by thin-layer chromatography on silica gel G using the solvent system indicated above. Methyl esters of the fatty acids present in the three lipid fractions were then prepared using boron trifluoride (Morrison and Smith, 1964; Klopfenstein, 1971). The methyl esters were separated by gas-liquid chromatography using 6% diethyleneglycol succinate on
COMPOSITION
AND
METABOLISM
OF MACROPHAGES
67
Gaschrom P at 170°C in an F & M Model 5750 gas chromatograph. Column quantitation was checked using Applied Science fatty acid mixture standards. The measured composition agreed with the stated composition with a relative error of less than 570 for major components (greater than 10% of the total mixture) and less than 10% for minor components (less than 10% of the total mixture). Metabolic experiments. Two series of metabolic experiments were carried out. In the first, macrophages obtained from either normally fed or cholesterol-fed rabbits were incubated at 37°C in SO-ml conical flasks. From 10 to 20 x lo6 macrophages were dispensed into each flask and incubated with 4 ml of incubation medium containing Media 199:normal rabbit serum (l:l, v/v) to which had been added a known amount ( 15-20 $X/flask) of sodium [1J4C]acetate (Radiochemical Centre, Amersham, United Kingdom; specific activity, 58 mCi/ mmole). Under these circumstances the macrophages adhered to the surface of the flask as monolayers. Duplicate flasks were removed at 4, 8, and 20 hr, the incubation medium was removed, and the adherent cells were washed twice with warm 0.9% sodium chloride solution, Cells which separated during the period of the incubation or with the saline washings were centrifuged (1000 rpm for 5 min) and combined with the adherent cells prior to extraction with chloroform:methanol (2:1, v/v) as described by Folch et al. ( 1957). The lipid extracts were made up to 25 ml, and aliquots were taken for counting and thinlayer chromatography or for saponification and digitonin precipitation. Separation of the lipid extract into phospholipid, cholesterol, fatty acid, triglyceride, and cholesterol ester fractions was carried out by thin-layer chromatography on silica gel G as described above. Identification of the separated spots was made under uv light and the separated spots were scraped for counting using the dioxane scintillator described by Snyder ( 1964). In the second series of metabolic experiments the incorporation of [l-14C]acetate into digitonin precipitable sterol was investigated in normally fed rabbit macrophages incubated in vitro in the presence of medium containing either normal (cholesterol content approximately 40 mg/IOO ml) or hyperlipemic serum (cholesterol content 667-1080 mg/ 100 ml). Approximately 2 X 10” macrophages were dispensed into Leighton tubes in 1.5 ml of incubation medium and were incubated at 37°C for periods of 4, 8, and 20 hr. The incubation medium contained Media 199:normal rabbit serum (50:50, v/v; i.e., approximately 200 pg of cholesterol/ml of medium) or Media 199:normal serum: hyperlipemic rabbit serum (50:40:10, v/v/v; i.e., 830-1240 pg of cholesterol/ml of medium) together with a known amount (approximately 5 &i/tube) of [I-14C]acetate. Incubation was stopped after 4, 8, or 20 hr and the adherent cells were washed with warm 0.9% NaCl solution. Any cells present in the discarded media or washings were centrifuged, washed, and combined with the adherent cells prior to chloroform:methanol extraction. The cholesterol in aliquots of the chloroform:methanol extract was precipitated as the digitonide as described below. Preparation of digitonin-preoipitable sterol. Aliquots of the chloroform: methanol extracts were saponified (Abcll et al., 1952) and the free cholesterol was extracted with petroleum ether ,bp SO-SOOC). An aliquot was reserved for cholesterol assay by densitometry as described above and the cholesterol in the
6s
KAR
AND
DAY
remainder was precipitated as the digitonide by addition of 1.25% digitonin in 95% ethanol. The volume was kept to 1 ml, 1 mg of carrier cholesterol being added prior to precipitation. After standing overnight the digitonin precipitate was separated by centrifugation, washed twice with ether:acetone (2:1, v/v), and finally dried by heating (1 hr at 1lO’C). Free sterol was recovered from the digitonide by addition of pyridine and extraction with ether. The extracts were transferred to counting vials, evaporated, and counted using the method of Snyder (1964). RESULTS Table I shows the lipid composition of macrophages from normally fed and cholesterol-fed rabbits expressed as micrograms of lipid per milligram of protein. The cells from cholesterol-fed rabbits were characterized by significant increases in cholesterol ester, free cholesterol, and triglyceride when compared with those obtained from normally fed rabbits. There was no significant difference in the phospholipid content between the two groups of macrophages. The increase in cholesterol ester is particularly striking, increasing some 40 times compared with that in the macrophages from normally fed rabbits. The percentage of the cholesterol present as ester had risen accordingly in the cells from 7.30 k 1.13 to 61.2 +- 2.89%. The fatty acid composition of the triglyceride, the phospholipid, and the cholesterol ester of macrophages from both normally fed and cholesterol-fed rabbits is given in Table II. There is a lower proportion of palmitate and a higher proportion of linoleate in the triglyceride fraction from the macrophages from cholesterol-fed rabbits. The phospholipid from these cells contains a higher proportion of linoleate and a lower proportion of arachidonate than does that from the normal cells. The most obvious difference between the two types of cells however is in the cholesterol ester composition. Macrophages from cholesterol-fed rabbits have appreciably lower proportions of cholesterol palmitate and stearate and appreciably higher proportions of cholesterol oleate and linoleate than do those from the normally fed rabbits. The incorporation of W-labeled acetate into various lipid fractions by the normally fed and cholesterol-fed macrophages is shown in Table III for each TABLE Lipid
Composition
of blacrophnges
from
Fed
Lipid
composition
Normally Cholest,erol Cholesterol Triglyceride Lipid P a h 147 c
(free) (ester)
24.6 1.98 11.68 0.9s
I
Normally
fed + + + f
0.91 0.36 2.88 0.26
and Cholesterol-Fed (g/mg
PC
f 3.60 zt 20.10 f 3.7 f 0.79
Mean f REM of five experiments. Normally fed rabbit macrophages aontjained 135 + 6.7 g of protein,/106 3~ 16.0 rg of protein/106 cells for cholesterol-fed marrophagcs. By Students 1 teat.
b
of protein) fed
Cholesterol 15.7 79.8 33.40 7.75
l
cells compared
with
COMPOSITION
AND
METABOLISM TABLE
Percentage
Fatty
OF
MACROPHAGES
69
II
Acid Composition of Triglyceride Phoapholipid and Cholesterol of Normally Fed and Cholesterol-Fed Rabbits Fatty Normally
acid fed
composition
Ester
(YO)
Cholesterol
fed
Triglyceride0 Palmitic sb tearic Oleic Linoleic
(16:0) (18:O) (18: 1) (15:2)
35.4 11.3 32.5 16.3
l zt f zk
1.51 1.06 0.77 1.54
27.9 11.3 33.9 25.6
f + f xt
0.82 1.17 0.68 0.58
(16:0) (1S:O) (18: 1) (18:2) (20: 4)
25.2 16.2 21.9 21.4 11.6
f + + f f
0.66 0.42 0.64 1.26 0.57
24.7 15.0 2Y.O 28.3 8.5
+ f + i f
0.45 0.40 0.73 1.33 0.46
35.4 16.1 41.1 7.3
f f z!z z!z
0.93 2.26 1.14 1.14
15.1 7.0 57.1 20.0
f f f xk
1.08 1.04 2.00 1.07
Phospholipkk Palmitic Stearic Oleic Linoleic Arachidonic Cholesterol Palmitic Stearic Oleic Linoleic
estersh (16:0) (1Y:O) (18: 1) (15:2)
a Mean f SEM of seven experimenk. * Mean & SEM of four experiment,s cholesterol-fed macrophages.
for
normally
fed macrophages
and of six experiments
for
of the time intervals studied. The data are expressed as counts per minute of [ 1-Wlacetate incorporated into phospholipid, triglyceride, cholesterol, or cholesterol ester per milligram of cell protein per lo6 cpm added to the incubation medium. In order to reduce the variation between experiments the incorporation of 14C-labeled acetate into the various lipid fractions for the cells from cholesterol-fed rabbits is expressed as a percentage of that in the normal cells and these data are also given in Table III. In normally fed rabbit macrophages most of the 14C-labeled acetate was incorporated into cholesterol throughout the time period studied. Saponification and fatty acid extraction of the separated cholesterol ester fraction indicated that only 12.7% of the acetate incorporated into cholesterol ester was present as fatty acid. In the cholesterol-fed rabbit macrophages incorporation into phospholipid and triglyceride was somewhat lower than in the corresponding normally fed macrophages, but the most marked difference between the cholesterol-fed and normally fed rabbit macrophages was the almost complete suppression of cholesterol synthesis in the former. The incorporation of 14C-labeled acetate into digitonin-precipitable sterol together with the specific activity of the digitonin preciptable cholesterol for both groups of cells is also shown in Table III. It can be seen that almost complete suppression of sterol synthesis has occurred in the cells from cholesterol-fed rabbits. Saponification and fatty acid extraction of the cholesterol ester fraction from the cholesterol-fed rabbit macrophagcs was not carried out in view of the few counts per minute in the fraction. However saponification and digitonin precip-
54
193 f
0 Data are expressed as counts per tonin-precipitable cholesterol. * Mean -or 8lM of three experimeuts.
minute
281
462 f
Incorporation LSpecific activity (cpm/pg of cholesterol)
86 48 194
130 73 ff 369 f
Cholest8erol ester Triglyceride Cholesterol Digit,onin-precipitable cholesterol
45b
123 f
4 hr
of [l-W]Acet,at,e
Phospholipid
Incorpnrat,ion
102
per milligram
324 f
741 Yk 3%
225 158 ff 123 78 580 + 263
276f119
fed
Variolls
8 hr
Normally
into
Flactions
of protein
313 f
809 f
200 322 f 686 f
371 f
20 hr
f
s
3 f
1.5
13.7 f
11 f3
medium,
0.7
4
28 49 ffll 13 2 11.7 f
14 23 10
zk38
fed
Normally
120
8 hr
Cholesterol
from
51
4 hr
incorporat,ion=
by blacrophages
III
per lo6 cpm in t,he incubation
9s
408
90 103 306
153
[IJ’C]Acetate
Lipid
TABLE
54
17
Specific
szk1.2
38 f
activity
data
4f2
99 30 5
63
4 hl
is also given
0.7
2 f -
22 12 0.8
5s 19 f s f
10
8 hr
for
incorporation fed/normally
Rabbits
48 f
Percentage (cholesterol
and Cholesterol-Fed
I33 43 ff 42 76 31f 9
IS2 f
20 hr
Fed
1
23 14 1
12
the digi-
-
.Tf
54 f 57 f 27 f 5+
20 hr
fed)
!s
G
5‘3
COMPOSITION
AND
METABOLISM
OF
MACROPHAGES
71
FIG. 1. Incorporation of [1-‘Clacetate into digitonin-precipitable sterol of normal macrophages incubated in medium supplemented with normal or hyperlipemic serum. Mean * SEM for four experiments. Differences between normal and hyperlipemic are not statistically significant at any time point.
itation of the extract from the cholesterol-fed rabbit cells (see Table III), in contrast to the macrophages from normally fed rabbits, showed that the free cholesterol fraction accounted for most of the digitonin-precipitable sterol. It can be assumed therefore that in the cholesterol fed rabbit cells most of the “C-labeled acetate incorporated into cholesterol ester is in the fatty acid fraction. The effect of the hyperlipemic serum added to the incubation medium on the incorporation of [l-14C]acetate into cholesterol in normally fed macrophages is shown in Fig. 1. In macrophages incubated with both normal and hyperlipemic sera appreciable incorporation into labeled cholesterol occurred with no significant difference between the two groups. Thus, despite the presence of a relatively large supplement of hyperlipemic serum there was no significant inhibition of cholesterol synthesis over the 20-hr time period observed. DISCUSSION The most obvious difference in lipid composition between the normal macrophages and the cholesterol-fed rabbit macrophages was the presence of a high concentration ,of cholesterol ester in the latter. While the total cholesterol of the cells was some four times higher in the cholesterol-fed than in the normally fed macrophages, the bulk of this increase was in the cholesterol ester fraction which rose from 7.3 to 61% of the cholesterol with cholesterol feeding. The total cholesterol content of the cells compared closely with the value reported by Wurster and Zilversmit (1971), although they note that in normal macrophages ester cholesterol accounted for as much as 3370. Werb and Cohn (1971) report cholesterol concentrations for normal mouse macrophages as 12 pg/mg of protein, i.e., less than half the value reported to us. They note that ester cholesterol was not detectable in the normal mouse macrophages.
7”
KAR AND DAY
Comparison of the data for macrophages with that published for foam cells (Day et al., 196613)indicates that rabbit aortic foam cells contain approximately the same concentration of lipid P (1.15 pg of lipid P/lo6 cells), but about fourfold as much cholesterol as the cholesterol-fed rabbit macrophages (72.0 rf~ 17.9 pg of cholesterol/lOF cells for the foam cells). The content of cholesterol in foam cells isolated from aortic lesions depencls however on the severity of the lesion (Peters and De Duve, 1974). The similarity of macrophages from cholesterol-fed rabbits to foam cells is apparent when the fatty acid compositions of the accumulating cholesterol esters are compared. The cholesterol esters in the cholesterol-fed rabbit macrophages are markedly different from those present in the normal macrophages but, resemble closely those for foam cells isolated from aortic lesions of cholesterol-fed rabbits (Peterson et al., 1971). It is possible that this composition simply relates to the serum cholesterol ester fatty acid composition which is high in cholesterol oleate and linoleate in the cholesterol-fed rabbit. However, it has been observed previousl) that the foam cell cholesterol esters are significantly higher in oleic acid and lower in linoleic acid than the corresponding serum (Peterson et al., 1971). Macrophages do not in any case readily take up lipoprotein cholesterol (CasleySmith and Day, 1966; Wurster and Zilversmit, 1971), so that one must assume that at least some of the cholesterol ester, comes from synthesis in the respective cells. Both normal and cholesterol-fed cells contain some paraffin oil droplets as a result of the method of collection and it is possible that such droplets may physically trap some of the lipid in the cholesterol-fed rabbit cells. However, the difference in composition of the cholesterol ester of the cells from that of the serum would preclude this possibility. In any case, as indicated above, lipoprotein is not readily taken up by similarly prepared cells, nor does exposure of the normal cells, with their contained paraffin droplets, to hyperlipemic serum result in interiorization of the cholesterol and suppression of cholesterol synthesis (data presented in Fig. 1.). The difference in fatty acid composition of the triglyceride and phospholipid between the two types of cells may in part be due to the composition of the diet which is high in both triolein and trilinolein. Since the normal cells contain a relatively low proportion of linoleic acid this fraction increases most obviously in the cholesterol-fed cells. The reduction in arachidonic acid in the phospholipids from the cholesterol-fed cells is of interest. Since the total phospholipid content does not increase, this change represents a real and not just a relative reduction in arachidonate content. This may be due to the suppression of the conversion of linoleic acid to arachidonic acid in the cholesterol-loaded cells. Preliminary data from our laboratory (Shamgar, unpublished) indicate that such a suppression occurs in aortas of cholesterol-fed rabbits when compared with normal aortas, Considerable work has been carried out in our laboratory on the synthesis and metabolism of lipid by normal macrophages (reviewed by Day, 1967). It has previously been demonstrated that most of the ‘%-labeled acetate taken up and converted to lipid in these cells is incorporated into the cholesterol fraction, This observation is in contrast to the report of Werb and Cohn ( 1971). Their model of cholc,stcrol handling by macrophagcs is b~serl on observations with mouse macrophages and it has already been pointed out that these cells contain
COMPOSITION
AND
METABOLISM
OF MACROPHAGES
73
only about half the cholesterol content of the normal rabbit macrophages. Metabolic differences between macrophages from different sources can be observed and may occur between species. The preferential incorporation of acetate into cholesterol in normal rabbit macrophages previously reported (Day and Fidge, 1964) has been confirmed in the present report where over 60% of the acetate incorporated into lipid is present in the cholesterol fraction, either as free cholesterol or combined as cholesterol ester. Labeling of the latter fraction also indicates the action of cholesterol-esterifying enzymes in rabbit peritoneal macrophages. In contrast to the present work and that reported in other studies (Day and Tume, 1969; Tume and Day, 1970), Werb and Cohn (1971) were not able to demonstrate cholesterol-esterifying activity in preparations of mouse macrophages. Clearly there are significant differences in lipid metabolism among macrophages from different species. The incorporation of [ l-Y3]acetate into cholesterol in the cholesterol-fed rabbit macrophages is less than 5% of that in the normal macrophages. It can be reasonably assumed that the syntheses of phospholipid, triglyceride, and cholesterol draw from a common pool of acetyl-CoA. Reduced incorporation into phospholipid and triglyceride is not marked for the cholesterol-fed compared with the normal cells, so that possible differences in the size of the acetate pool between the two groups of cells can be excluded. Recent studies have demonstrated that cholesterol synthesis is normally controlled by HMG CoA reductase (Siperstein and Fagan, 1966). In fibroblasts (Goldstein and Brown, 1974), aortic smooth muscle cells in culture (Weinstein et al., 1976), and in leukocytes (Fogelman et al., 1975), exp osure to cholesterol contained in serum lipoprotein results in inhibition of cholesterol synthesis. It is suggested that such inhibition is due to the uptake of LDL by receptors at the cell surface, although the data for leukocytes (Fogelman et al., 1975) suggest that the cholesterol level in the cells and its consequent action on cellular HMG CoA reductase is due to varying loss of cholesterol into the medium. Chemical and ultrastructural studies with macrophages indicate that ‘these cells do not take up appreciable quantities of lipoprotein ( Casley-Smith and Day, 1966). The effect of addition of hyperlipemic serum to the incubation medium on the incorporation of [1-lC]acetate into lipid by normal macrophages was therefore investigated. As indicated in Fig. 1, the addition of 10% hyperlipemic serum did not result in any appreciable inhibition of cholesterol synthesis by the cells. Experiments carried out using rabbit aortic smooth muscle cells in tissue culture under similar conditions resulted in ahnost complete inhibition of the incorporation of [l-lC]acetate into cholesterol when hyperlipemic serum was added (Day et al., 1977). There is little doubt that the high cholesterol content of the cholesterol-fed rabbit macrophages has “turned ofI” cholesterol synthesis in these cells. Exposure of normal macrophages to high levels of lipoprotein in the incubation medium however fails to do so. It seems therefore that macrophages, unlike fibroblasts and aortic smooth muscle cells, do not possessthe lipoprotein receptors which are necessary for the uptake and interiorization of lipoprotein cholesterol synthesis until lipid synthesized and accumulated in situ influences endogenous cholesterol synthesis. A ready source of cholesterol for esterification is therefore assured and the increased cholesterol-esterifying activity of the cholesterol-fed
74
KAR
AND
DAY
macrophages promotes the formation and packaging of cholesterol droplets in the cell and the formation of lipid laden cells.
ester
as
ACKNOWLEDGMENTS We are grateful Mrs. Jill Duda for
to Mr. technical
Dennis Vickery, Mr. assistance throughout
D. Falkiner, bliss these studies.
Margaret
Ackland,
and
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COMPOSITION
PARKER, F. (1960).
AND
METABOLISM
OF
MACROPHACES
7.5
An electron microscopic study of experimental atherosclrrosis. Amer. 1. 36, 19-53. PARKER, F., and ODLAND, G. F. ( 1966). A correlative, histochemical, biochemical and electron microscopic study of experimental atherosclerosis in the rabbit aorta with special reference to the myointimal cell. Amer. J. Pathol. 48, 197-239. PETERS, T. J., and DE DUVE, C. (1974). Lysosomes of the arterial wall: II. Subcellular fractionation of aortic cells from rabbits with experimental atheroma. Exp. Mol. Pothot. 20, 228-256. PETERSON, M., DAY, A. J., Tuarn, R. K., and EISENBERG, E. (1971). Ultrastructure, fatty acid content, and metabolic activity of foam cells and other fractions separated from rabbit atherosclerotic lesions. Exp. MOE. Puthol. 15, 157-169. POOLE, J. C. F., and FLOREY, H. W. ( 1958). Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits. j. Pathol. Bacterial. 75, 245-252. SIMON, R. C., STILL, W. J., and O'NEAL, R. M. (1961). The circulating lipophage and experimental atherosclerosis. J. Atheroscler. Res. 1, 395-400. SIPERSTEIN, M. D., and FAGAN, V. M. (1966). Feedb ac k control of mevalonate synthesis by dietary cholesterol. J. Biol. Chem. 241, 602-609. SNYDER, F. ( 1964). Radioassy of thin-layer chromatograms: A high-resolution zonal scraper for quantitative Cl4 and H3 scanning of thin-layer chromatograms. Anal. &o&em. 9, 183-196. STILL, W. J. S., and O’NEAL, R. M. (1962). Electron microscopic study of experimental atherosclerosis in the rat. Amer. J. Pathol. 40, 21-35. SUZUKI, M., and O’NEAL, R. M. (1967). Circulating lipophages, serum lipids and atherosclerosis in rats. Arch. Pathol. 83, 169-174. TUME, R. K., and DAY, A. J. ( 1970). Cholesterol esterifying activity of rabbit alveolar macrophages. J. Reticuloendothel. Sot. 7, 338-354. VAN HANDEL, E., and ZILvERshPIT, D. B. (1957). M’ rcromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50, 152-157. WEINSTEIN, D. B., CAREW, T. W., and STEINBERG, D. (1976). Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis. Biochim. Biophys. Acta 424, 404-421. WERB, Z., and COHN, Z. A. (1971). Cholesterol metabolism in the macrophage: I. The regulation of cholesterol exchange. J. Exp. Med. 134, 1545-1569. WURSTER, N. B., and ZILVERSMIT, D. B. ( 1971). Th e role of phagocytosis in the development of atherosclerotic lesions in the rabbit. Atherosclerosis 14, 309322. Puthol.
Ah’ll
hlOLECULAR
PATHOLOGY
Composition and Normally
Metabolism Fed and
28, 65-75
( 1978)
of Lipid in Macrophages Cholesterol-Fed Rabbits1
from
SUDIPTA KAR AND ALLAN J. DAY Department
of Physiology, Received
April
University 18, 1977,
and
of Melbourne,
Victoria,
Australia
in revised
August
8, 1977
form
3052
The composition and metabolism of lipid in peritoneal macrophages obtained from normally fed rabbits were compared with those of macrophages obtained from cholesterol-fed rabbits. Macrophages from cholesterol-fed rabbits had a higher cholesterol content and a markedly higher cholesterol ester content than normal macrophages. The increase in cholesterol ester content was most marked for cholesterol oleate and cholesterol linoleate, with the cholesterol ester fatty acid composition of the cholesterol-fed macrophages resembling that of foam cells derived from aortic lesions of similarly cholesterol-fed rabbits. Metabolic differences were also demonstrated between the cells obtained from normal and cholesterol-fed rabbits. In the latter, incorporation of “C-laheled acetate into cholesterol was almost completely suppressed whereas in macrophages from normally fed rabbits, W-labeled acetate was incorporated predominantly into cholesterol. Incubation in vitro of normal macrophages for periods up to 20 hr with hyperlipemic serum, however, was not associated with any appreciable suppression of cholesterol synthesis.
Human fatty streak lesions and cholesterol-induced atherosclerotic lesions in experimental animals are characterized by the intracellular accumulation of cholesterol and cholesterol esters in foam cells in the intima. Most of these cells originate as smooth muscle cells which are stimulated to proliferate and accumulate lipid in situ (Parker, 1960; Geer et al., 1961; Buck, 1962; Parker and Odland, 1966). Ultrastructural studies have demonstrated, however, that foam cells in arterial lesions have a varying morphology. While many of them have the characteristics of smooth muscle cells, namely, myofibrils, a limiting basement membrane, and pinocytotic vessels along the basement membrane, lipid-containing ceIIs with morphological and histochemical characteristics ‘of macrophages can also be seen in the developing atherosclerotic lesion (Balis et al., 1964; Geer, 1965a,b). Blood monocytes can be observed penetrating the endothelium (Poole and Florey, 1958), and lipid-containing macrophages circulate in the blood under various circumstances (Still and O’Neal, 1962; Simon et al., 1961; Suzuki and O’Neal 1967). Some of the foam cells in the atherosclerotic lesion may therefore be derived from tissue macrophages or blood monocytes. The synthesis and metabolism of lipid by macrophages obtained from the peritoneal cavity of normal rabbits have been extensively studied (Day, 1967). These cells contain 1 This Australia.
work
was
supported
by
a grant-in-aid
from
the
National
Heart
Foundation
of
65 0014-4800/78/0281-0065$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
KAR AN11 DAY
cholesterol-esterifying enzymes ( Day and Tume, 1969; Tumc~ and Day,. 1970)) and their uptake of cholesterol particles is associated with increased phospholipid synthesis (Day et al., 1966a). Recently Wurster and Zilversmit (1971) observed that peritoneal macrophages obtained from cholesterol-fed rabbits showed a marked increase in their cholesterol and cholesterol ester content compared with normal macrophages, whereas granulocytes collected from cholesterol-fed rabbits showed no such increase. In the present study, the composition and synthesis of lipid in peritoneal macrophages of cholesterol-fed rabbits have been further studied and compared with that of peritoneal macrophages obtained from normally fed rabbits. METHODS Collection of mucrophages. Male New Zealand White Rabbits approximately 6 months of age were used throughout the experiments. Normally fed rabbits received normal rabbit chow while cholesterol-fed rabbits were fed a diet containing 1% cholesterol and 3% peanut oil for periods of 3 to 4 months prior to collection of the macrophages. Five days after the intraperitoneal injection of sterile liquid paraffin (Nujol), macrophages were removed from the peritoneal cavity by saline washing as previously described (Day and Fidge, 1962). The cells were suspended in Media 199 and counted using a standard hemocytometer chamber. From 100 to 300 X lo6 cells were obtained from the washings in normal rabbits; in cholesterol-fed rabbits a somewhat smaller number of macrophages was obtained (from 50 to 100 x 10” cells). The cells were dispensed in appropriate volumes for extraction of lipid or for protein assay or were dispensed in culture flasks for metabolic study as set out below. Clzenzical Assam.For assay of protein, between 5 and 10 x 10F cells in duplicate were washed with normal saline and taken up in sodium hy,droxide. Total protein content was assayed by the method of Lowry et al. (1951). For lipid analysis, aliquots of the cells from either cholesterol-fed or normally fed rabbits were extracted with chloroform:methanol (2:1, v/v) and were washed by the method of Folch et al. ( 1957). Lipid phosphorus was determined in the lipid extract by the method of Bartlett (1959). Triglycerides were assayed by the method of Van Handel and Zilversmit (1957). Cholesterol and cholesterol ester were assayed by densitometry following separation of the lipid fractions, in aliquots of the lipid extracts by thin-layer chromatography on prewashed silica gel G plates using the solvent system, N-hexane:diethyl ether:acetic acid (112:38:3, v/v/v). After development the plates were sprayed with 5% phosphomolybdic acid in ethanol and then heated at 90°C for 15 min to develop color. The spots were scanned using a Schoeffel Model SD 3000 dual-beam spectrodensitometer at a wavelength of 560 nm. An integrated output was used to calculate mass. Assays of amounts less than 1 pg of cholesterol or cholesterol ester were possibly by this method. Gas-liquid chromatography. Phospholipid, triglyceride, and cholesterol ester in the lipid extracts were separated by thin-layer chromatography on silica gel G using the solvent system indicated above. Methyl esters of the fatty acids present in the three lipid fractions were then prepared using boron trifluoride (Morrison and Smith, 1964; Klopfenstein, 1971). The methyl esters were separated by gas-liquid chromatography using 6% diethyleneglycol succinate on
COMPOSITION
AND
METABOLISM
OF MACROPHAGES
67
Gaschrom P at 170°C in an F & M Model 5750 gas chromatograph. Column quantitation was checked using Applied Science fatty acid mixture standards. The measured composition agreed with the stated composition with a relative error of less than 570 for major components (greater than 10% of the total mixture) and less than 10% for minor components (less than 10% of the total mixture). Metabolic experiments. Two series of metabolic experiments were carried out. In the first, macrophages obtained from either normally fed or cholesterol-fed rabbits were incubated at 37°C in SO-ml conical flasks. From 10 to 20 x lo6 macrophages were dispensed into each flask and incubated with 4 ml of incubation medium containing Media 199:normal rabbit serum (l:l, v/v) to which had been added a known amount ( 15-20 $X/flask) of sodium [1J4C]acetate (Radiochemical Centre, Amersham, United Kingdom; specific activity, 58 mCi/ mmole). Under these circumstances the macrophages adhered to the surface of the flask as monolayers. Duplicate flasks were removed at 4, 8, and 20 hr, the incubation medium was removed, and the adherent cells were washed twice with warm 0.9% sodium chloride solution, Cells which separated during the period of the incubation or with the saline washings were centrifuged (1000 rpm for 5 min) and combined with the adherent cells prior to extraction with chloroform:methanol (2:1, v/v) as described by Folch et al. ( 1957). The lipid extracts were made up to 25 ml, and aliquots were taken for counting and thinlayer chromatography or for saponification and digitonin precipitation. Separation of the lipid extract into phospholipid, cholesterol, fatty acid, triglyceride, and cholesterol ester fractions was carried out by thin-layer chromatography on silica gel G as described above. Identification of the separated spots was made under uv light and the separated spots were scraped for counting using the dioxane scintillator described by Snyder ( 1964). In the second series of metabolic experiments the incorporation of [l-14C]acetate into digitonin precipitable sterol was investigated in normally fed rabbit macrophages incubated in vitro in the presence of medium containing either normal (cholesterol content approximately 40 mg/IOO ml) or hyperlipemic serum (cholesterol content 667-1080 mg/ 100 ml). Approximately 2 X 10” macrophages were dispensed into Leighton tubes in 1.5 ml of incubation medium and were incubated at 37°C for periods of 4, 8, and 20 hr. The incubation medium contained Media 199:normal rabbit serum (50:50, v/v; i.e., approximately 200 pg of cholesterol/ml of medium) or Media 199:normal serum: hyperlipemic rabbit serum (50:40:10, v/v/v; i.e., 830-1240 pg of cholesterol/ml of medium) together with a known amount (approximately 5 &i/tube) of [I-14C]acetate. Incubation was stopped after 4, 8, or 20 hr and the adherent cells were washed with warm 0.9% NaCl solution. Any cells present in the discarded media or washings were centrifuged, washed, and combined with the adherent cells prior to chloroform:methanol extraction. The cholesterol in aliquots of the chloroform:methanol extract was precipitated as the digitonide as described below. Preparation of digitonin-preoipitable sterol. Aliquots of the chloroform: methanol extracts were saponified (Abcll et al., 1952) and the free cholesterol was extracted with petroleum ether ,bp SO-SOOC). An aliquot was reserved for cholesterol assay by densitometry as described above and the cholesterol in the
6s
KAR
AND
DAY
remainder was precipitated as the digitonide by addition of 1.25% digitonin in 95% ethanol. The volume was kept to 1 ml, 1 mg of carrier cholesterol being added prior to precipitation. After standing overnight the digitonin precipitate was separated by centrifugation, washed twice with ether:acetone (2:1, v/v), and finally dried by heating (1 hr at 1lO’C). Free sterol was recovered from the digitonide by addition of pyridine and extraction with ether. The extracts were transferred to counting vials, evaporated, and counted using the method of Snyder (1964). RESULTS Table I shows the lipid composition of macrophages from normally fed and cholesterol-fed rabbits expressed as micrograms of lipid per milligram of protein. The cells from cholesterol-fed rabbits were characterized by significant increases in cholesterol ester, free cholesterol, and triglyceride when compared with those obtained from normally fed rabbits. There was no significant difference in the phospholipid content between the two groups of macrophages. The increase in cholesterol ester is particularly striking, increasing some 40 times compared with that in the macrophages from normally fed rabbits. The percentage of the cholesterol present as ester had risen accordingly in the cells from 7.30 k 1.13 to 61.2 +- 2.89%. The fatty acid composition of the triglyceride, the phospholipid, and the cholesterol ester of macrophages from both normally fed and cholesterol-fed rabbits is given in Table II. There is a lower proportion of palmitate and a higher proportion of linoleate in the triglyceride fraction from the macrophages from cholesterol-fed rabbits. The phospholipid from these cells contains a higher proportion of linoleate and a lower proportion of arachidonate than does that from the normal cells. The most obvious difference between the two types of cells however is in the cholesterol ester composition. Macrophages from cholesterol-fed rabbits have appreciably lower proportions of cholesterol palmitate and stearate and appreciably higher proportions of cholesterol oleate and linoleate than do those from the normally fed rabbits. The incorporation of W-labeled acetate into various lipid fractions by the normally fed and cholesterol-fed macrophages is shown in Table III for each TABLE Lipid
Composition
of blacrophnges
from
Fed
Lipid
composition
Normally Cholest,erol Cholesterol Triglyceride Lipid P a h 147 c
(free) (ester)
24.6 1.98 11.68 0.9s
I
Normally
fed + + + f
0.91 0.36 2.88 0.26
and Cholesterol-Fed (g/mg
PC
f 3.60 zt 20.10 f 3.7 f 0.79
Mean f REM of five experiments. Normally fed rabbit macrophages aontjained 135 + 6.7 g of protein,/106 3~ 16.0 rg of protein/106 cells for cholesterol-fed marrophagcs. By Students 1 teat.
b
of protein) fed
Cholesterol 15.7 79.8 33.40 7.75
l
cells compared
with
COMPOSITION
AND
METABOLISM TABLE
Percentage
Fatty
OF
MACROPHAGES
69
II
Acid Composition of Triglyceride Phoapholipid and Cholesterol of Normally Fed and Cholesterol-Fed Rabbits Fatty Normally
acid fed
composition
Ester
(YO)
Cholesterol
fed
Triglyceride0 Palmitic sb tearic Oleic Linoleic
(16:0) (18:O) (18: 1) (15:2)
35.4 11.3 32.5 16.3
l zt f zk
1.51 1.06 0.77 1.54
27.9 11.3 33.9 25.6
f + f xt
0.82 1.17 0.68 0.58
(16:0) (1S:O) (18: 1) (18:2) (20: 4)
25.2 16.2 21.9 21.4 11.6
f + + f f
0.66 0.42 0.64 1.26 0.57
24.7 15.0 2Y.O 28.3 8.5
+ f + i f
0.45 0.40 0.73 1.33 0.46
35.4 16.1 41.1 7.3
f f z!z z!z
0.93 2.26 1.14 1.14
15.1 7.0 57.1 20.0
f f f xk
1.08 1.04 2.00 1.07
Phospholipkk Palmitic Stearic Oleic Linoleic Arachidonic Cholesterol Palmitic Stearic Oleic Linoleic
estersh (16:0) (1Y:O) (18: 1) (15:2)
a Mean f SEM of seven experimenk. * Mean & SEM of four experiment,s cholesterol-fed macrophages.
for
normally
fed macrophages
and of six experiments
for
of the time intervals studied. The data are expressed as counts per minute of [ 1-Wlacetate incorporated into phospholipid, triglyceride, cholesterol, or cholesterol ester per milligram of cell protein per lo6 cpm added to the incubation medium. In order to reduce the variation between experiments the incorporation of 14C-labeled acetate into the various lipid fractions for the cells from cholesterol-fed rabbits is expressed as a percentage of that in the normal cells and these data are also given in Table III. In normally fed rabbit macrophages most of the 14C-labeled acetate was incorporated into cholesterol throughout the time period studied. Saponification and fatty acid extraction of the separated cholesterol ester fraction indicated that only 12.7% of the acetate incorporated into cholesterol ester was present as fatty acid. In the cholesterol-fed rabbit macrophages incorporation into phospholipid and triglyceride was somewhat lower than in the corresponding normally fed macrophages, but the most marked difference between the cholesterol-fed and normally fed rabbit macrophages was the almost complete suppression of cholesterol synthesis in the former. The incorporation of 14C-labeled acetate into digitonin-precipitable sterol together with the specific activity of the digitonin preciptable cholesterol for both groups of cells is also shown in Table III. It can be seen that almost complete suppression of sterol synthesis has occurred in the cells from cholesterol-fed rabbits. Saponification and fatty acid extraction of the cholesterol ester fraction from the cholesterol-fed rabbit macrophagcs was not carried out in view of the few counts per minute in the fraction. However saponification and digitonin precip-
54
193 f
0 Data are expressed as counts per tonin-precipitable cholesterol. * Mean -or 8lM of three experimeuts.
minute
281
462 f
Incorporation LSpecific activity (cpm/pg of cholesterol)
86 48 194
130 73 ff 369 f
Cholest8erol ester Triglyceride Cholesterol Digit,onin-precipitable cholesterol
45b
123 f
4 hr
of [l-W]Acet,at,e
Phospholipid
Incorpnrat,ion
102
per milligram
324 f
741 Yk 3%
225 158 ff 123 78 580 + 263
276f119
fed
Variolls
8 hr
Normally
into
Flactions
of protein
313 f
809 f
200 322 f 686 f
371 f
20 hr
f
s
3 f
1.5
13.7 f
11 f3
medium,
0.7
4
28 49 ffll 13 2 11.7 f
14 23 10
zk38
fed
Normally
120
8 hr
Cholesterol
from
51
4 hr
incorporat,ion=
by blacrophages
III
per lo6 cpm in t,he incubation
9s
408
90 103 306
153
[IJ’C]Acetate
Lipid
TABLE
54
17
Specific
szk1.2
38 f
activity
data
4f2
99 30 5
63
4 hl
is also given
0.7
2 f -
22 12 0.8
5s 19 f s f
10
8 hr
for
incorporation fed/normally
Rabbits
48 f
Percentage (cholesterol
and Cholesterol-Fed
I33 43 ff 42 76 31f 9
IS2 f
20 hr
Fed
1
23 14 1
12
the digi-
-
.Tf
54 f 57 f 27 f 5+
20 hr
fed)
!s
G
5‘3
COMPOSITION
AND
METABOLISM
OF
MACROPHAGES
71
FIG. 1. Incorporation of [1-‘Clacetate into digitonin-precipitable sterol of normal macrophages incubated in medium supplemented with normal or hyperlipemic serum. Mean * SEM for four experiments. Differences between normal and hyperlipemic are not statistically significant at any time point.
itation of the extract from the cholesterol-fed rabbit cells (see Table III), in contrast to the macrophages from normally fed rabbits, showed that the free cholesterol fraction accounted for most of the digitonin-precipitable sterol. It can be assumed therefore that in the cholesterol fed rabbit cells most of the “C-labeled acetate incorporated into cholesterol ester is in the fatty acid fraction. The effect of the hyperlipemic serum added to the incubation medium on the incorporation of [l-14C]acetate into cholesterol in normally fed macrophages is shown in Fig. 1. In macrophages incubated with both normal and hyperlipemic sera appreciable incorporation into labeled cholesterol occurred with no significant difference between the two groups. Thus, despite the presence of a relatively large supplement of hyperlipemic serum there was no significant inhibition of cholesterol synthesis over the 20-hr time period observed. DISCUSSION The most obvious difference in lipid composition between the normal macrophages and the cholesterol-fed rabbit macrophages was the presence of a high concentration ,of cholesterol ester in the latter. While the total cholesterol of the cells was some four times higher in the cholesterol-fed than in the normally fed macrophages, the bulk of this increase was in the cholesterol ester fraction which rose from 7.3 to 61% of the cholesterol with cholesterol feeding. The total cholesterol content of the cells compared closely with the value reported by Wurster and Zilversmit (1971), although they note that in normal macrophages ester cholesterol accounted for as much as 3370. Werb and Cohn (1971) report cholesterol concentrations for normal mouse macrophages as 12 pg/mg of protein, i.e., less than half the value reported to us. They note that ester cholesterol was not detectable in the normal mouse macrophages.
7”
KAR AND DAY
Comparison of the data for macrophages with that published for foam cells (Day et al., 196613)indicates that rabbit aortic foam cells contain approximately the same concentration of lipid P (1.15 pg of lipid P/lo6 cells), but about fourfold as much cholesterol as the cholesterol-fed rabbit macrophages (72.0 rf~ 17.9 pg of cholesterol/lOF cells for the foam cells). The content of cholesterol in foam cells isolated from aortic lesions depencls however on the severity of the lesion (Peters and De Duve, 1974). The similarity of macrophages from cholesterol-fed rabbits to foam cells is apparent when the fatty acid compositions of the accumulating cholesterol esters are compared. The cholesterol esters in the cholesterol-fed rabbit macrophages are markedly different from those present in the normal macrophages but, resemble closely those for foam cells isolated from aortic lesions of cholesterol-fed rabbits (Peterson et al., 1971). It is possible that this composition simply relates to the serum cholesterol ester fatty acid composition which is high in cholesterol oleate and linoleate in the cholesterol-fed rabbit. However, it has been observed previousl) that the foam cell cholesterol esters are significantly higher in oleic acid and lower in linoleic acid than the corresponding serum (Peterson et al., 1971). Macrophages do not in any case readily take up lipoprotein cholesterol (CasleySmith and Day, 1966; Wurster and Zilversmit, 1971), so that one must assume that at least some of the cholesterol ester, comes from synthesis in the respective cells. Both normal and cholesterol-fed cells contain some paraffin oil droplets as a result of the method of collection and it is possible that such droplets may physically trap some of the lipid in the cholesterol-fed rabbit cells. However, the difference in composition of the cholesterol ester of the cells from that of the serum would preclude this possibility. In any case, as indicated above, lipoprotein is not readily taken up by similarly prepared cells, nor does exposure of the normal cells, with their contained paraffin droplets, to hyperlipemic serum result in interiorization of the cholesterol and suppression of cholesterol synthesis (data presented in Fig. 1.). The difference in fatty acid composition of the triglyceride and phospholipid between the two types of cells may in part be due to the composition of the diet which is high in both triolein and trilinolein. Since the normal cells contain a relatively low proportion of linoleic acid this fraction increases most obviously in the cholesterol-fed cells. The reduction in arachidonic acid in the phospholipids from the cholesterol-fed cells is of interest. Since the total phospholipid content does not increase, this change represents a real and not just a relative reduction in arachidonate content. This may be due to the suppression of the conversion of linoleic acid to arachidonic acid in the cholesterol-loaded cells. Preliminary data from our laboratory (Shamgar, unpublished) indicate that such a suppression occurs in aortas of cholesterol-fed rabbits when compared with normal aortas, Considerable work has been carried out in our laboratory on the synthesis and metabolism of lipid by normal macrophages (reviewed by Day, 1967). It has previously been demonstrated that most of the ‘%-labeled acetate taken up and converted to lipid in these cells is incorporated into the cholesterol fraction, This observation is in contrast to the report of Werb and Cohn ( 1971). Their model of cholc,stcrol handling by macrophagcs is b~serl on observations with mouse macrophages and it has already been pointed out that these cells contain
COMPOSITION
AND
METABOLISM
OF MACROPHAGES
73
only about half the cholesterol content of the normal rabbit macrophages. Metabolic differences between macrophages from different sources can be observed and may occur between species. The preferential incorporation of acetate into cholesterol in normal rabbit macrophages previously reported (Day and Fidge, 1964) has been confirmed in the present report where over 60% of the acetate incorporated into lipid is present in the cholesterol fraction, either as free cholesterol or combined as cholesterol ester. Labeling of the latter fraction also indicates the action of cholesterol-esterifying enzymes in rabbit peritoneal macrophages. In contrast to the present work and that reported in other studies (Day and Tume, 1969; Tume and Day, 1970), Werb and Cohn (1971) were not able to demonstrate cholesterol-esterifying activity in preparations of mouse macrophages. Clearly there are significant differences in lipid metabolism among macrophages from different species. The incorporation of [ l-Y3]acetate into cholesterol in the cholesterol-fed rabbit macrophages is less than 5% of that in the normal macrophages. It can be reasonably assumed that the syntheses of phospholipid, triglyceride, and cholesterol draw from a common pool of acetyl-CoA. Reduced incorporation into phospholipid and triglyceride is not marked for the cholesterol-fed compared with the normal cells, so that possible differences in the size of the acetate pool between the two groups of cells can be excluded. Recent studies have demonstrated that cholesterol synthesis is normally controlled by HMG CoA reductase (Siperstein and Fagan, 1966). In fibroblasts (Goldstein and Brown, 1974), aortic smooth muscle cells in culture (Weinstein et al., 1976), and in leukocytes (Fogelman et al., 1975), exp osure to cholesterol contained in serum lipoprotein results in inhibition of cholesterol synthesis. It is suggested that such inhibition is due to the uptake of LDL by receptors at the cell surface, although the data for leukocytes (Fogelman et al., 1975) suggest that the cholesterol level in the cells and its consequent action on cellular HMG CoA reductase is due to varying loss of cholesterol into the medium. Chemical and ultrastructural studies with macrophages indicate that ‘these cells do not take up appreciable quantities of lipoprotein ( Casley-Smith and Day, 1966). The effect of addition of hyperlipemic serum to the incubation medium on the incorporation of [1-lC]acetate into lipid by normal macrophages was therefore investigated. As indicated in Fig. 1, the addition of 10% hyperlipemic serum did not result in any appreciable inhibition of cholesterol synthesis by the cells. Experiments carried out using rabbit aortic smooth muscle cells in tissue culture under similar conditions resulted in ahnost complete inhibition of the incorporation of [l-lC]acetate into cholesterol when hyperlipemic serum was added (Day et al., 1977). There is little doubt that the high cholesterol content of the cholesterol-fed rabbit macrophages has “turned ofI” cholesterol synthesis in these cells. Exposure of normal macrophages to high levels of lipoprotein in the incubation medium however fails to do so. It seems therefore that macrophages, unlike fibroblasts and aortic smooth muscle cells, do not possessthe lipoprotein receptors which are necessary for the uptake and interiorization of lipoprotein cholesterol synthesis until lipid synthesized and accumulated in situ influences endogenous cholesterol synthesis. A ready source of cholesterol for esterification is therefore assured and the increased cholesterol-esterifying activity of the cholesterol-fed
74
KAR
AND
DAY
macrophages promotes the formation and packaging of cholesterol droplets in the cell and the formation of lipid laden cells.
ester
as
ACKNOWLEDGMENTS We are grateful Mrs. Jill Duda for
to Mr. technical
Dennis Vickery, Mr. assistance throughout
D. Falkiner, bliss these studies.
Margaret
Ackland,
and
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B. B.,
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BHODIE,
cholesterol
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