High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar macrophages

Biochimica Elsevier

103

et Biophysrca Acta 922 (1987) 103-110

BBA 52678

High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar maerophages Natsuko Mori a, Nobuhiro Yamada a, Shun Ishibashi a, Masanobu Kawakami b, Keiichi Takahashi a, Hitoshi Shimano ‘, Michio Fujisawa a, Fumimaro Takaku a and Toshio Murase a * The Third Deparrment of Internal Medicine. Fact&y oi Medicine, University of Tokyo and ’ Division of Pathophysioiogy,

The Cfinicul Research Institute, (Received

Key words:

Lipoprotein

lipase; VLDL;

National Medical Center, Tokyo (Japan)

12 June 1987)

Cholesterol;

Lipoprotein;

(Rat alveolar

macrophage)

We have previously shown that cultured rat alveolar macrophages synthesize and secrete lipoprotein lipase into the medium. The purpose of the present experiments is to examine whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion and the lipid accum~a~on in macrophages. Macrophages incubated with the VLDL obtained from rats fed a normal diet secreted 2-fold higher amounts of li~protein lipase than those without li~proteins. Intermediate-, low- and very-low-density lipoproteins from rats fed a high-cholesterol diet also enhanced the lipoprotein lipase secretion. Normal high- and low-density lipoprotiens, and high-density lipoproteins from hypercholesterolemic animals did not cause any increase in the lipoprotein lipase secretion. The lipoproteins which stimulated the lipoprotein lipase secretion caused intracellular accumulation of both triacylglycerol and cholesterol. It is speculated that macrophages residing in the environment rich in lipoproteins, especially hy~rcholesterolemic lipoproteins, take them up and accumulate lipids in~acellularly, and that this process links with the lipoprotein lipase secretion. Tbe secreted lipoprotein lipase could facilitate, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins. Probably such a series of events is of importance in the foam cell formation of macrophages.

Introduction Macrophages have been implicated as precursors of arterial wall foam cells. A number of recent studies emphasize the importance of the

Abbreviations: VLDL, very-low-density lipoprotein; IDL., intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Correspondence: nal Medicine, Hongo, Tokyo

T. Murase, The Third Department of InterFaculty of Medicine, University of Tokyo, 113, Japan.

0001%2760/87/$03.50

0 1987 Elsevier Science Publishers

macrophages in the development of atherosclerosis [1,2]. Macrophages take up a variety of lipoproteins via specific receptors [2] and both cholesterol ester [2] and triacylglycerol [3] accumulate in the cytoplasma. When excessive amounts of lipids accumulate intracellularly, macrophages give a foamy appearance [2]. Recently, Khoo et al. [4] found that macrophages synthesize and secrete lipoprotein lipase, and the report has attracted attention in connection with potential atherogenesity of t~acylglycerol-rich lipoproteins 13.41. Lindqvist et al. [5] focused their attention on the uptake of triacylglycerol-rich lipoproteins by mac-

B.V. (Biomedical

Division)

104

rophages and suggested that macrophage lipoprotein lipase enhances, by generating remnants of triacylglycerol-rich lipoproteins, the uptake of chylomicrons and VLDLs. Although previous studies demonstrated the secretion of lipoprotein lipase by cultured macrophages [4,6-81 and the possible function of this enzyme [S], regulatory factors of the enzyme activity have not yet been assessed. In the present study, we first examined the effects of normal lipoproteins on the lipoprotein lipase secretion by rat alveolar macrophages. Furthermore, we also examined whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion, because the predominant lipid found in the atheromatous lesion is cholesterol ester and much attention has been focussed previously on the uptake of cholesterol-rich lipoproteins by macrophages [2,9-131. The data demonstrate that normal VLDL increases lipoprotein lipase secretion in rat alveolar macrophages in culture. In adcholesterol-enriched lipoproteins were dition, shown to cause stimulation of the secretion of lipoprotein lipase, with concomitant increases of cellular lipids. Materials and Methods Materi& Sprague-Dawley male rats, weighing 300-350 g, were used. RPM1 1640 medium was purchased from Gibco Laboratories. Plastic petri dishes were obtained from Falcon, Division of becton, Dickinson and Co. Fetal bovine serum was obtained from Gibco Laboratories. Insulin and heparin were purchased from Novo Industries. Preparation and culture of rat aheolar macrophages. The methods of isolation and preparation of rat alveolar macrophages have been published previously 181. Under anesthesia with sodium pentobarbital, 40 mg per kg of body weight, rats were bled from abdominal aorta and the lungs were taken out. To collect alveolar macrophages, the lungs were perfused through the inserted cannula with 0.9% saline solution (three times, with 10 ml per lavage), and the fluid from the lavage was used to isolate macrophages for studies. The lavages were centrifuged at 100 X g for 10 min and the cell pellets thus obtained were then suspended at 5.10’ cells per dish in RPM1 1640

medium and incubated at 37” C in a humidified atmosphere of 5% CO, in air for 2 h. Then, the dishes were washed to remove non-adherent cells and incubated for 2 days in the medium supplemented with 10% fetal bovine serum. On day 3, the cells were washed twice with 1 ml phosphatebuffered saline (pH 7.4) and then culture was continued for the designated time periods. When lipoproteins were tested, the cells were incubated with the indicated lipoproteins. 2 days later, the medium was removed and replaced with fresh medium containing the lipoprrteiil +r be examined, and culture was co’ -nued for another 4 h. At termination, medium was collected to measure lipoprotein lipase activity. Lipoprotejns. Plasma collected from 5-7 male rats between 9 and 11 a.m. were pooled and used for the isolation of lipoproteins. Blood samples were withdrawn from the abdominal aorta into tubes containing EDTA (1 mg/ml blood), sodium azide (0.4 mg/ml) and benzamidine (0.3 mg/ml). Normal lipoprotein fractions were isolated from the plasma of non-fasting rats fed a laboratory chow. Hypercholesterole~c lipoproteins were obtained from the plasma of non-fasting rats fed a high cholesterol diet containing 1% cholesterol and 0.5% cholic acid for 3 weeks. Lipoprotein fractionation was carried out using a type 40-3 rotor (Beckman Instruments, Palo Alto, Ca. U.S.A.) [14]. The density was adjusted with KBr and KBr solution of known density. Before isolating VLDL, plasma was centrifuged for 1 h at 10000 rpm to minimize the contamination of chylomicrons. Then, VLDL (d < 1.006 g/ml), IDL (1.006-1.030), LDL (1.030-1.063) and HDL (1.063-1.210) were sequentially isolated by ultracentrifugation at 40000 rpm for 16, 18, 18 and 40 h, respectively. Each lipoprotein fraction was washed once by ultracent~fugatio~ at 40000 rpm for 20 h, for VLDL, IDL and LDL, and for 40 h for HDL, and then dialyzed for 2 days with several changes against 4 1 of phosphate-buffered saline (pH 7.4). This was sterilized by filtration through a 0.45~pm Milleux-HA filter (Millipore Corporation) prior to use. Both triacylglycerol [15] and cholesterol [16] concentrations in each lipoprotein fraction were measured enzymatically. Protein concentrations were determined by the method of Lowry et al. [17].

105

Lipoprotein lipase assay. Initial studies, including identification of the enzyme, were done with our earlier employed assay system, using a sonicated substrate [18]. Since the method enables us to measure only limited number of samples at once, we later adopted the method of Nilsson-Ehle and Schotz [19], using a stable, radioactive substrate emulsion. Briefly, 150 ~1 of enzyme were mixed with 50 ~1 of substrate containing 22.6 mM [ 3H]Triolein (1.4 pCi/lr,mol), phosphatidylcholine at 2.5 mg/ml, bovine serum albumin at 40 mg/ml, 33% (v/v) rat serum and 33% (v/v) glycerol in 0.27 M Tris-HCl (pH 8.1) and the mixture was incubated at 37” C for 30 min. The amounts of free oleic acid liberated were evaluated from the counts of 3H and the specific activity of [‘Hltriolein. The lipase activity was inhibited more than 90% by addition of 1 M NaCl and more than 80% by omission of serum, which is the source of apolipoprotein C-II needed for enzyme activity. These two methods demonstrated a good correlation between assays (r = 0.99, n = 8). When the lipoprotein lipase assay is carried out, it is possible that the lipoprotein added to culture medium may influence the assay system. This possibility has been checked by assaying the lipoprotein lipase-containing medium after addition of various lipoproteins. Normal lipoprotein fractions as well as hypercholesterolemic rat lipoproteins at concentrations of less than 50 pg protein did not influence the assay. Determination of intracellular lipids. After medium was collected to measure lipoprotein lipase activity, each monolayer was washed twice with 1 ml of phosphate-buffered saline (pH 7.4). 1 ml of hexane/isopropanol (3 : 2, v/v) was added to each dish and kept standing for 30 min at room temperature [20]. The organic phase was collected in a glass tube, and each monolayer was rinsed briefly with 0.5 ml of the same solvent. These solvent extracts were combined in the tube. The solvent was then evaporated to dryness under air and the lipids in each tube were resuspended in 50 ~1 of hexane/isopropanol solution. The solution was divided into two tubes for measuring triacylglycerol and cholesterol. After the lipids were extracted from the well, the cells in each monolayer were dissolved in 0.5 ml of 0.1 M NaOH and aliquots were used for protein determination by

the procedure of Lowry et al. [17]. Statistical analysis. Cultures were performed in quadruplicate, unless otherwise stated. Data are expressed as mean + SE. Statistical significance of the data was evaluated with Student’s t-test. Results Secretion of lipoprotein lipase from cultured macrophages Rat alveolar macrophages secreted a lipolytic enzyme into culture medium, as previously described [Xl. The enzyme had the properties identical with lipoprotein lipase, in that it was activated ll-fold by serum (apolipoprotein C-II) and was inhibited 78.4% by 3 mg/ml of protamine sulfate. Further, the enzyme bound to the heparin-sepharose affinity column and was eluted from the column with the solution containing 1.5 M NaCl [8,18]. The mode of lipoprotein lipase secretion by cultured macrophages was quite unique in that lipoprotein lipase can be released even in the absence of heparin, because in other tissues, such as adipose tissues and muscles, heparin is needed for the release of the enzyme from the tissues [21]. Initial studies conducted to observe the effect of heparin showed that heparin added to the medium at concentrations of 2 units/ml enhanced the lipoprotein lipase secretion of macrophages as well; however, the effect was quite variable (57 * 19% increase, n = 7). Lipoprotein lipase has been known to be a very labile enzyme. When the culture medium free from macrophages was kept stand at 37°C for 4 h, the enzyme activity decreased to 37 + 4% (n = 4) initial activity. When either normal or hypercholesterolemic VLDL was added to the lipoprotein lipase-containing culture medium, the enzyme activity decreased to either 44 f 4% (n = 4) with normal VLDL or 36 * 5% (n = 4) with hypercholesterolemic VLDL (no significant difference between normal and hypercholesterolemic VLDL). Thus, lipoproteins themselves do not seem to have any effect on the stability of lipoprotein lipase. Although lipoprotein lipase is a labile enzyme, the enzyme activity in the medium increased during the culture period of up to 8 h, at which it reached plateau. This can be explained by assuming that the rate of secretion is much higher than that of

106

degradation. The results of these time course studies were very similar to those reported on the macrophage cell line by Melmed et al. [22]. When the lipoprotein lipase secreted into culture medium was measured every 2 days it was detected 2 days after plating the cells and increased lineally for up to 8 days in culture. Based on those time-course studies, the experiments were performed under the conditions described in Materials and Methods. We have attempted to measure further cell-associated lipoprotein lipase and compared this with the enzyme secreted into culture medium. After the medium was removed, the cells were homogenized in 0.05 M NH,OH/NH,Cl buffer (pH 8.5), containing heparin (0.5 U/ml) for 1 h on ice. Lipoprotein lipase thus extracted from the cells was measured. In our initial studies in which the measurement of lipoprotein lipase was done with our earlier assay system [18], the lipoprotein lipase activity of culture medium was more than 6-times as high as that of cell-associated lipoprotein lipase (1621 2 89 vs. 287 f 16 nmol free fatty acid/h per lo6 cells). When enzyme activity was measured by the method of Nilsson-Ehle and Schotz [19], the cell-associated lipoprotein lipase activity was very low, even under the condition that the lipoprotein lipase secretion into culture medium was enhanced by the addition of cholesterol-enriched VLDL from cholesterol-fed rats. Table I shows the actural value in cpm in both culture medium and cell extract. The enzyme activity of cell extract was below the acceptable level of accuracy, as compared with the cpm of a blank (without the enzyme). Hence, in subsequent experiments, we

TABLE

I

LIPOPROTEIN LIPASE ACTIVITY IN BOTH CULTURE MEDIUM AND CELL EXTRACT OF ALVEOLAR MACROPHAGES VLDL at concentrations of SO ng protein/ml were added to the culture medium. A blank (without the enzyme) was 2SO+ 21 cpm. Each value was obtained by subtracting the blank from actual cpm.

selectively measured the lipase secreted into culture

activity of lipoprotein medium.

Effects of normal lipoproteins on the secretion of lipoprotein lipase Plasma lipoproteins at concentrations of 50 pg/ml protein were added to the culture medium. As controls, 0.2% lactalbumin in place of lipoproteins was added. Constituents of each lipoprotein fraction are shown in Table II. VLDL from rats fed a standard laboratory chow was relatively rich in triacylglycerol, and both LDL and HDL were increased in cholesterol contents. In these rats, plasma IDL was very scanty and sufficient amounts of IDL fraction could not be obtained. As shown in Table II, normal VLDL caused a 2-fold increase in the secretion of lipoprotein lipase, while both LDL and HDL had no such an effect on it. In a separate set of experiment, cellular contents of lipids were measured. Incubation of macrophages with normal VLDL caused a marked cellular accumulation of triacylglycerol (0.595 rt 0.134 pg,/pg cell protein, as compared

TABLE

II

CHEMICAL FRACTIONS

COMPOSITION OF PLASMA ADDED TO THE CULTURE

Each value represents

the final concentration

in culture medium

(pg/ml). Normal diet-fed VLDL Triacylglycerol Cholesterol Protein

rats

High-cholesterol diet-fed rats

332 36 so

149 109 so

_

15 213 so

LDL Triacylglycerol Cholesterol Protein

13 31 so

8 139 so

HDL Triacylglycerot Cholesterol Protein

1 27 50

1 32 50

IDL Triacylglycerol Cholesterol Protein

Enzyme activity (cpm)

Hypercholesterolemic rat VLDL Controls (no addition)

medium

cell extract

726 i 80 341 i: 35

75f34 37+24

LIPOPROTEIN MEDIUM

107

with 0.088 + 0.018 pg/pg P < 0.05).

cell protein

for controls,

Effects of cholesterol-enriched lipoproteins from cholesterol-fed rats on the secretion of lipoprotein Iipase Rats fed a high-cholesterol diet showed hypercholesterolemia. Plasma VLDL, IDL and LDL from cholesterol-fed rats were all markedly rich in cholesterol compared with those from normal diet-fed rats (Table II). The properties of cholesterol-fed rat plasma lipoproteins analyzed by both agarose gel electrophoresis and SDS-polyacrylamide gel electrophoresis have been described previously [23]. The hypercholesterolemic rat VLDL fraction consists of a mixture of ,& VLDL and VLDL. and the LDL contains an TABLE

a-migrating

III

Macrophages were cultured with the indicated lipoproteins at concentrations of 50 pg protein/ml for 2 days. Thereafter, the medium was changed and the culture was continued in the presence of the identical lipoprotein. 4 h later, medium was collected and was assayed for its lipoprotein lipase activity. The results were expressed as percentage of each control, because the prepared lipoproteins were used for experiments as freshly as possible and all lipoprotein fractions were not always examined at the same time. Each value represents mean f S.E. (n = 4,5).

to as HDL,

from rats fed a normal 100 204k29 * 83&10 96k 8

Lipoprotein fractions No addition VLDL IDL LDL HDL

from rats fed a high-cholesterol 100 269+36 ** 237k31 * 215510 ** 134*15

IV

EFFECTS OF VARIOUS LIPOPROTEIN FRACTIONS ON THE CELLULAR ACCUMULATION OF LIPIDS IN CULTURED MACROPHAGES Plasma tein/ml

lipoprotein fractions at concentrations were added to the culture medium. Cellular

VLDL Normal (4) High-cholesterol diet-induced (3) No addition (3) IDL Normal High-cholesterol diet-induced (4) No addition (5)

lipase activity

Lipoprotein fractions No addition VLDL LDL HDL

* Significantly different sponding values of the * * Significantly different sponding values of the

which may referred

As shown in Table III, hypercholesterolemic rat VLDL increased markedly the lipoprotein lipase activity in culture medium (2.34 k 0.32 nmol free fatty acid/h per pg cell protein vs. 0.87 + 0.18 for controls, P < 0.01). This cholesterol-enriched VLDL was slightly but not significantly stronger in its stimulating effect than normal VLDL. Both IDL and LDL fractions obtained from hypercholesterolemic rats also showed stimulating effects on the lipoprotein lipase secretion, while HDL did not. Hypercholesterolemic rat VLDL markedly increased the cellular content of both triacylglycerol and cholesterol (0.733 f 0.040 vs. 0.088 f 0.018 pg/pg cell protein for triacylglycerol, P < 0.01,

TABLE

EFFECTS OF PLASMA LIPOPROTEIN FRACTIONS FROM RATS FED EITHER A NORMAL DIET OR A HIGH-CHOLESTEROL DIET ON THE SECRETION OF LIPOPROTEIN LIPASE IN RAT ALVEOLAR MACROPHAGE IN CULTURE

Lipoprotein (percentage)

band,

v41.

diet

LDL Normal (4) High-cholesterol diet-induced (4) No addition (3)

diet

at least at P < 0.05 from controls (no addition). at least at P i 0.01 from controls (no addition).

HDL Normal (5) High-cholesterol diet-induced (5) No addition (4) correcorre-

of 50 ng pro-

lipids (p g/f.tg protein)

triacylglycerol

cholesterol

0.595 zbo.134 *

0.136 i 0.020

0.733 f 0.040 * * 0.088 f 0.018

0.301* 0.017 * * 0.084 * 0.008

_ 0.120*0.020 ** 0.041+ 0.003

0.128 +0.015 0.055 +0.012

*

0.103 * 0.015

0.113 kO.003

**

0.082 + 0.010 0.066 f 0.009

0.146+0.011 ** 0.094 * 0.002

0.086 f 0.009

0.101 kO.021

0.079 + 0.005 * 0.056 k 0.010

0.132kO.014 * 0.080 f 0.017

* Significantly different, at least at P < 0.05, sponding values of the controls (no addition). * * Significnatly different, at least at P < 0.01, sponding values of the controls (no addition).

from

corre-

from

corre-

108

HC-VLDL

ADDED (ug PROTEIN/ml

MEDIUM)

HC-IDL

ADDED (ug PROTEIN/ml

MEDIUM)

Fig. 1. Effects of varying amounts of plasma VLDL and IDL from hypercholesterolemic (HC) rats on the secretion of lipoprotein lipase and cellular accumulation of both triacylglycerol and cholesterol in rat alveolar macrophages in culture. Macrophages were cultured with the indicated amounts of lipoprotein from hypercholesterolemic rats for 2 days. Thereafter, the medium was changed and culture was continued in the presence of the same amount of lipoprotein. 4 h later, medium was collected and was assayed for its lipoprotein lipase activity. After washing twice with 1 ml of phosphate-buffered saline (pH 7.4) cellular lipids were extracted with 1 ml of hexane/isopropanol (3: 2, v/v) to measure triacylglycerol and cholesterol. After lipids were extracted, the cells were dissolved in 0.5 ml of 0.1 M NaOH and aliquots were used for protein determination. The values with bars indicate mean + S.E. of three samples and the values at 1 pg protein of IDL are the mean of two samples. FFA, free fatty acids.

and 0.301 * 0.017 vs. 0.084 + 0.008 pg/pg cell protein for cholesterol, P < 0.01). Also, the accumulation of cellular cholesterol was stimulated by hypercholesterolemic rat IDL, LDL and HDL (Table IV), although the increases were minimal. We further examined the effects of increasing amounts of cholesterol-enriched VLDL and IDL added to the culture medium on changes of both lipoprotein lipase secretion and cellular accumulations of lipids. The lipoprotein lipase secretion increased markedly when VLDL at concentrations of as low as 1 pg protein was added to the medium; however, since the added VLDL contained only limited amounts of lipids, this did not cause increases of cellular lipids (Fig. 1, left). We also made an observation in a separate series of experiments that VLDL at concentrations of 0.5 pg protein showed a significant stimulating effect on the lipoprotein lipase secretion (data not shown). When 50 pg protein of VLDL were added, cellular triacylglycerol and cholesterol contents were increased 8- and 3-fold, respectively. In

this experiment, hypercholesterolemic rat IDL was examined in parallel with VLDL. Hypercholesterolemic rat IDL also caused a significant increase of lipoprotein lipase secretion, with concomitant increases of cellular lipids; however, such stimulating effects of IDL seemed to be less than those of VLDL (Fig. 1, right). Discussion

The present study demonstrates that hypercholesterolemic rat VLDL, IDL and LDL as well as normal VLDL stimulate the secretion of lipoprotein lipase in cultured rat alveolar macrophages. Macrophages incubated with such lipoproteins accumulated lipids intracellularly. It is speculated that high-cholesterol diet-induced lipoproteins containing P-VLDL stimulate the secretion of lipoprotein lipase, which degrades lipoproteins into triacylglycerol-depleted particles, and that the lipoprotein lipase-modified lipoprotein

109

particles could be more easily taken up by macrophages. Although the roles for lipoprotein lipase in both tissues and lipoprotein metabolism have been well documented previously [25,26] and factors regulating tissue lipoprotein lipase activities have been studied extensively [27-291, only limited information is available so far on the macrophage lipoprotein lipase. Melmed et al. [22] indicated that cyclic AMP modified the secretion of lipoprotein lipase in macrophages. Recently, Goldberg and Khoo [30] reported that thioglycollate-elicited mouse peritoneal macrophages showed an increased synthesis and secretion of lipoprotein lipase. As far as a role for lipoprotein lipase in the metabolism of VLDL is concerned, Stalenhoef et al. [31] suggested that the removal of VLDL is dependent upon the initial action of lipoprotein lipase from the study of lipoprotein lipase-deficient subject, and lipoprotein lipase is a key enzyme for VLDL metabolism. Lindqvist et al. [5] showed a similar function of lipoprotein lipase in macrophage, suggesting that VLDL is modified by the action of macrophage lipoprotein lipase and resulting partially hydrolized VLDL can be taken up by macrophages, causing intracellular accumulation of lipids. Our present study shows that normal VLDL stimulates the lipoprotein lipase secretion, whereas both normal LDL and HDL have no effects on it. We have demonstrated further that lipoproteins fractionated as VLDL, IDL and LDL from rats fed a high-cholesterol diet also enhance the lipoprotein lipase secretion. Macrophages cutured with the indicated lipoproteins accumulated both triacylglycerol and cholesterol intracellularly and, in support of this, the cells had plenty of lipid droplets histochemically, with concomitant increases in their cell size (data not shown). The mode of cellular uptake of lipoprotein lipase-modified lipoproteins is thought to be mediated by specific receptors [32]. A number of recent works have shown that macrophages possess receptors for both native and chemically modified forms of lipoproteins on their cell membrane, and take up a variety of lipoproteins through the receptors [11,13,33-361. Recent studies showed that polyvalent binding of lipoproteins containing several apolipoproteins E to the LDL receptor

yields a high affinity for LDL receptor, followed by the receptor-mediated uptake of lipoproteins [37,38]. We speculate that lipoproteins including normal VLDL, hypercholesterolemic-VLDL, -1DL and -LDL used in this study can be taken up by either LDL receptor or P-VLDL receptor (which is possibly LDL receptor [39,40]), since these lipoproteins have more apolipoprotein E on them than normal LDL and hypercholesterolemic VLDL contain P-VLDL, as we reported previously [23]. Another possible mechanism for the accumulation of triacylglycerol in macrophages is that triacylglycerol in lipid droplets can also be derived from free fatty acid released after the hydrolysis of triacylglycerol-rich lipoproteins by macrophage lipoprotein lipase. Our results indicate that the lipoproteins which stimulate the lipoprotein lipase secretion also cause cellular accumulation of lipids. In a separate series of experiments, we have observed that hypercholesterolemic rat VLDL at concentrations of as low as 0.5 pg protein showed a stimulating effect on the lipoprotein lipase secretion. It would be interesting to know how lipoproteins stimulate the lipoprotein lipase secretion in macrophages, and study is now under way. Although the precise mechanism remains to be assessed, we tentatively propose the following process. When macrophages reside in an environment rich in the indicated lipoproteins, they take them up and store lipids intracellularly. The process probably links with the secretion of lipoprotein lipase. The secreted lipoprotein lipase further facilitates, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins by macrophages. Thus, macrophages pursue their scavenger function. The enhancement of lipoprotein lipase secretion by hypercholesterolemic rat lipoproteins is of particular interest from the standpoint of foam cell formation of macrophages, because high-cholesterol diet-induced lipoproteins such as ,&VLDL are known to be highly atherogenic [2]. In summary, our present study shows the secretion of lipoprotein lipase in cultured rat alveolar macrophages. Both normal VLDL and hypercholesterolemic rat lipoproteins stimulated significantly the lipoprotein lipase secretion, with the increase of cellular lipids in cultured rat alveolar macrophages.

110

Acknowledgements We wish to thank Misses Ishikawa, Miyake, Izawa and Ohta for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 60570287) and Life Science Foundation of Japan. References 1 Gerrity, R.G. (1981) Am. J. Pathol. 103, 181-200 2 Brown, MS. and Goldstein, J.L. (1983) Annu. Rev. Biothem. 52, 223-261 3 Gianturco, S.H., Bradley, W.A., Gotto, A.M. Jr., Morrisett, J.D. and Peavy, D.L. (1982) J. Clin. Invest. 70, 168-178 4 Khoo, J.C., Mahoney, E.M. and Witztum, J.L. (1981) J. Biol. Chem. 256, 7105-7108 5 Lindqvist, P., Ostlund-Lindqvist, A.M., Witztum, J.L., Steinberg, D. and Little, J.A. (1983) J. Biol. Chem. 258, 9086-9092 6 Chait, A., Iverius, P.H. and Brunzell, J.D. (1982) J. Clin. Invest. 69, 490-493 P., Ungar, A., Bliumis, J., Bukberg, P.R., I Wang-Iverson, Gibson, J.C. and Brown, W.V. (1982) Biochem. Res. Commun. 104, 923-928 8 Okabe, T., Yorifuji, H., Murase, T. and Takaku, F. (1984) Biochem. Biophys. Res. Commun. 125, 273-278 9 Brown, M.S., Ho, Y.K. and Goldstein, J.L. (1980) J. Biol. Chem. 255, 9344-9352 10 Mahley, R.W., Innerarity, T.L., Brown, MS., Ho, Y.K. and Goldstein, J.L. (1980) J. Lipid Res. 21, 970-980 11 Fogelman, A.M., Shechter, I., Seager, J., Hokom, M., Child, J.S. and Edwards, P.A. (1980) Proc. Natl. Acad. Sci. USA 77, 2214-2218 T.L., Pitas, R.E. and Mahley, R.W. (1982) 12 Innerarity, Arteriosclerosis 2, 114-124 13 Via, D.P., Plant, A.L., Craig, IF., Gotto, A.M. Jr. and Smith, L.C. (1985) Biochim. Biophys. Acta 833, 417-428 14 Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353 15 Bucolo, G. and David, H. (1973) Clin. Chem. 19, 476-482 W. and 16 Allain, CC., Poon, L.S. Chan, C.S.G., Richmond, Fu, P.C. (1974) Clin. Chem. 20, 470-475

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