β-VLDL and acetylated-LDL binding to pigeon monocyte macrophages

Atherosclerosis, 78 (1989) 47-60 Elsevier Scientific Publishers Ireland,

47 Ltd.

ATH 04341

P-VLDL and acetylated-LDL binding to pigeon monocyte macrophages Dru Anne Henson Departmeni

*, Richard

of ParhologV and the Arteriosclerosis

W. St. Clair and Jon C. Lewis

Research Center, Wake Fores1 University, Winston-Salem, NC 27103 (U.S.A.)

The Bowman Gray School of Medicine,

(Received 2 January, 1989) (Revised received 27 February, 1989) (Accepted 1 March, 1989)

Summary

Blood-derived monocytes are an important source of foam cells in atherosclerotic lesions of White Carneau pigeons. Based upon studies with cultured blood monocytes (monocyte macrophages) and peritoneal macrophages from a variety of mammalian species, it has been proposed that these cells become loaded with cholesteryl esters through the uptake of lipoproteins including /&migrating very low density lipoproteins (P-VLDL) and low density lipoproteins that have been chemically modified in a manner analogous to experimental acetylation (AC-LDL). The purpose of this study was to determine whether similar mechanisms functioned in pigeon monocyte macrophages. Radioiodinated pigeon /3-VLDL and AC-LDL were incubated with White Carneau pigeon monocyte macrophages that had been maintained in culture for 7 days. Scatchard analysis of the specific binding data revealed the presence of specific and saturable receptors for both P-VLDL and AC-LDL. P-VLDL receptors had both low and high affinity binding components, whereas AC-LDL receptors displayed a single class of high affinity binding sites. /3-VLDL binding remained relatively constant from 3 to 10 days in culture while AC-LDL binding increased with time in culture. Competition studies demonstrated a high degree of binding specificity for lz51-AC-LDL, but less for ‘251-/?-VLDL. Binding of ‘251-/3-VLDL was not competed for by AC-LDL, but was by P-VLDL and by low density lipoproteins from both normal and hypercholesterolemic pigeons. Following binding of /S-VLDL and AC-LDL, the lipoproteins were rapidly internalized and degraded. Although the majority of degradation was secondary to internalization by the monocyte macrophages, approx. 5% of the degradation resulted from enzymatic activity in the culture medium, presumably due to secretion of proteolytic enzymes by the cells. As measured by esterification of [ l4 Cloleate to cholesterol, it was shown that the cholesterol liberated from the degradation of both /3-VLDL and AC-LDL stimulated cholesteryl ester synthesis in pigeon monocyte macrophages. These studies confirm the existence of specific P-VLDL and AC-LDL receptors on the surface of pigeon monocyte macrophages which facilitate both internalization of the lipoproteins and subsequent stimulation of cholesteryl ester synthesis. This is the first demonstration of ,8-VLDL and AC-LDL receptors on monocyte macrophages from an avian species, and

* Present address: Biology Department, Appalachian State University, Boone, NC 28608, U.S.A. Correspondence to: Dr. Jon C. Lewis, Department of Patho-

0021-9150/89/$03.50

0 1989 Elsevier Scientific

Publishers

Ireland,

logy, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103, U.S.A. Tel. (919) 748-2675.

Ltd.

48 the findings support the potential role for the receptor-mediated uptake of a variety of abnormal lipoproteins in the formation of monocyte-derived foam cells in the arterial wall of White Cameau pigeons during the development of atherosclerosis.

Key words: Atherosclerosis;

Lipoproteins;

Monocytes;

Introduction Atherosclerosis is a complex disease involving focal changes in the arterial intima that include proliferation of vascular smooth muscle cells, accumulation of macrophages, infiltration of blood and blood constituents, accumulation of connective tissue components, and accumulation ‘of lipids [1,2]. The lipids can be found within the extracellular space of the arterial intima or within cholesteryl ester-rich foam cells. For many years, foam cells were thought to arise principally from arterial smooth muscle cells [1,2]; however, more recent ultrastructural and biochemical studies have revealed that many foam cells represent monocyte-derived macrophages which have migrated from the blood into the intima and become engorged with lipid [2,3]. The White Carneau pigeon is a unique animal model for atherosclerosis. Naturally occurring aortic atherosclerosis in this breed of pigeon develops in all animals by 4 years of age, and the lesions resemble those in human beings, both histologically and biochemically [4]. In addition, the extent of aortic atherosclerosis and the rate of lesion development can be significantly accelerated by feeding the animals a cholesterol-supplemented diet [5]; and within a few months of cholesterol consumption, lesions predictably occur at a precise location near the celiac bifurcation [4]. As with many human atherosclerotic lesions, both naturally occurring and cholesterol-aggravated atherosclerotic lesions in the White Cameau pigeon are characterized ,by the presence of large, lipid-filled foam cells in the arterial intima. Many of these cells appear to originate from blood monocytes [6,7]. The mechanism by which monocyte-derived foam cells accumulate lipid after entering the artery wall is now beginning to be understood.

Macrophages

Tissue culture studies using a variety of mammalian monocyte-derived macrophage and peritoneal macrophage models have shown that normal plasma lipoproteins such as low density lipoproteins (LDL) are poorly taken up by cultural mammalian monocyte-derived macrophages. This is largely due to the limited expression of LDL receptors on the surface of these cells [8,9]. Certain abnormal forms of lipoproteins, however, are readily metabolized by mammalian monocyte-derived macrophages through receptor-mediated processes. These abnormal lipoproteins include LDL which have been made more electronegative by a variety of in vitro modifications [lO,ll], and the cholesteryl ester-rich, P-migrating very low density lipoproteins (/3-VLDL) present in the plasma of certain cholesterol-fed animals [12,13] and in human beings with type III hyperlipoproteinemia [14]. In vitro studies have shown that mouse peritoneal and human monocyte-derived macrophages [8,10-12,15,16] possess specific cellsurface receptors for /3-VLDL as well as “scavenger” receptors which recognize a variety of abnormal lipoproteins including acetylated LDL (AC-LDL). These receptors bind and internalize the lipoproteins and lead to a massive, unregulated, or poorly regulated, accumulation of intracellular cholesteryl ester. As a result, the cultured cells take on the morphological characteristics of foam cells. The previous studies3 have led to the suggestion that the receptor-mediated uptake of abnormal lipoproteins by monocyte-derived macrophages within the intima may play an important role in the formation of foam cells during the pathogenesis of atherosclerosis. Although this hypothesis is supported by the identification of specific receptors for P-VLDL and AC-LDL on the surface of cultured mammalian monocyte-derived macrophages, the presence of receptors for these poten-

49 tially atherogenic lipoproteins has not been demonstrated in avian models such as the pigeon. Therefore, the purpose of the present studies was to determine if monocyte-derived macrophages from White Cameau pigeons possess specific cell surface receptors for P-VLDL and AC-LDL. Methods Pigeon monocyte cultures Peripheral blood monocytes were obtained from randomly bred, male White Cameau pigeons housed at the pigeon facility of the Bowman Gray School of Medicine. All pigeons were at least 6 months of age and had been fed a cholesterol-free pelleted grain diet. At the time of blood collection, 27 ml of blood was obtained from each bird by cardiac puncture following a lethal dose of sodium pentobarbital (0.45 ml i.v. per bird). Blood was collected using a 16-gauge needle and a 30-ml polypropylene syringe containing 3 ml of 3.8% sodium citrate, pH 7.3. Blood samples from individual birds were treated and maintained separately for subsequent preparation of a monocyteenriched suspension by Isolymph density gradient centrifugation as described previously [17]. To determine the percentage of the various leukocyte types comprising both the original titrated blood and the monocyte-enriched suspension, Wrightstained cells were identified according to their staining characteristics as defined by Lucas [18]. The monocyte-enriched preparation was resuspended at a concentration of 2.5 X lo6 cells/ml in Eagle’s minimum essential medium (MEM) containing 10% (v/v) heat-inactivated (56 o C, 30 min) chicken serum (KC Biological) supplemented with Eagle’s vitamins, 200 mM L-glutamine, 10 mM Hepes, 1.5 mg dextrose/ml, 100 IU penicillin/ml, and 100 pg streptomycin/ml. Monocytes were isolated from this cell mixture by allowing them to adhere to tissue culture dishes for 2 h at 37” C. For this, 1 or 2 ml of the monocyte-enriched suspension were transferred to 22.6-mm tissue culture wells (Costar) or 35-mm tissue culture dishes (Corning), respectively. The non-adherent cells were removed and the adherent cells were washed 3 times with culture medium without serum. Fresh medium containing 10% heat-inactivated chicken serum was added to the adherent cells and the

cultures maintained for up to 7 days to allow in vitro differentiation into monocyte macrophages. As a result, in this paper we will refer to these cultured blood monocytes as monocyte macrophages. The culture medium was replaced after 24 and 48 h and every 2-3 days thereafter. All incubations were carried out in a humidified atmosphere of 95% air and 5% CO,. Adherent cells were identified as monocyte macrophages on the basis of morphological and functional criteria. Morphological assessment was based on the staining characteristics of Wrightstained cells. Functional criteria were based on measurement of phagocytic activity as determined by the ingestion of latex beads. For this, the adherent cells were incubated in culture medium with 0.05 ml of a 1% solution of fluorescent latex beads (1.0 pm diameter, Polysciences) per ml of culture medium. After incubation at 37OC for 1 h in the dark, the cells were washed and stained with Wright stain. The percentage of phagocytic cells was calculated from the number of total cells ingesting 2 or more latex beads as assessed by fluorescence microscopy. It should be noted that all cell cultures were established from individual birds. The cells from different birds were never mixed since preliminary studies with pooled cell suspensions resulted in a very low survival rate. As a result, the design of experiments with cultured pigeon monocyte macrophages was limited by the number of cells that could be isolated from one bird. Typically, a total of 2-5 X 10’ cells were obtained in the mononuclear cell-rich suspension from one bird. This yielded sufficient cells for 4-10 35-n-m dishes or 8-20 22.6~mm wells with approx. 1 x 10’ monocyte macrophages per 35-mm dish or 5 x lo6 cells per 22.6-mm well. Lipoproteins Pigeons were fasted overnight prior to collection of blood for lipoprotein isolation. Normocholesterolemic LDL (N-LDL) was isolated from the plasma of fasted, grain-fed White Carneau pigeons using the combined ultracentrifugation and agarose column chromatography procedure of Rude1 [19]. This procedure has been used extensively for the isolation of pigeon lipoproteins [13,20,21]. Lipoproteins from hypercholestero-

50 lemic animals were obtained from White Carneau pigeons that had consumed a cholesterol-supplemented diet for at least 12 weeks [13]. P-VLDL was isolated from hypercholesterolemic birds by ultracentrifugation overnight at d c 1.006 g/ml [21]. LDL from hypercholesterolemic birds (HLDL) was isolated by agarose column chromatography of the d > 1.006 g/ml fraction after removal of P-VLDL [21]. Acetylated LDL (AC-LDL) was prepared from H-LDL using the method of Fraenkel-Conrat [22] as modified by Basu [23]. Native and acetylated lipoproteins were labeled with 1251 using the iodine monochloride method [24]. Using this method, < 3% of the 1251 label was incorporated into the lipids of the lipoproteins and greater than 97% of the radioactive label was associated with intact proteins as determined by precipitation with 20% trichloroacetic acid (TCA). The specific activities of the iodinated lipoproteins ranged from 115 to 337 cpm/ng of protein. The electrophoretic mowas assessed bility of the ‘251-labeled lipoproteins by agarose electrophoresis [25]. Lipoproteins were sterilized through a 0.45-pm filter, stored at 4“ C, and used for tissue culture within 3 weeks. Determination of protease activity in monocyte macrophage-conditioned culture medium Pigeon monocytes were cultured at 37” C in. 35-mm dishes. Parallel dishes were cultured in culture medium without cells. After 7 days, the medium was removed from all the dishes, the cells were washed with phosphate-buffered saline (PBS), and fresh medium containing lipoprotein-deficient chicken serum (LPDS) was added. The chicken LPDS was prepared as described for calf serum [26]. Following incubation for 18 h at 37OC, 1 ml of the LPDS-containing medium was removed from the dishes with cells (cell conditioned medium) and from the dishes without cells (non-conditioned medium). The media were centrifuged to remove cellular debris and 0.5 ml of the media was added to 22.6-n-m tissue culture wells. ‘251-labeled P-VLDL or AC-LDL (20 pg/ml) were added to each well and the wells were incubated for 5 h at either 37 ’ C or 4 o C. Degradawas determined as the tion of ‘251-lipoproteins TCA-soluble, non-iodine 1251 present in the culture medium [27].

‘251-p-VLDL and lz51-AC-LDL binding and degradation Pigeon monocyte macrophages were cultured in 22.6~mm wells for 7 days after which time the culture medium was removed and the cells were washed twice with PBS. The cells were incubated with 1 ml of fresh culture medium containing 2.5 mg/ml of LPDS serum plus the indicated concentration of 125I-labeled lipoproteins, with or without a 20-fold excess of unlabeled homologous lipoprotein. Incubations were carried out at 4 o C for 4 h. Preliminary experiments indicated that binding had reached a steady state by this time. Following incubation, the cells were washed extensively [9] and the amount of cell-associated radioactivity was measured after digestion of the cells in 1 N NaOH [9]. An aliquot of the NaOH solution was also taken for determination of protein concentration using bovine serum albumin (BSA) as the standard [28]. In preliminary experiments, the proteolytic degradation and binding of ‘251-j3-VLDL and ‘251AC-LDL was determined on cells cultured for 3, 7 and 10 days. The cells were incubated at 37°C with 1 ml of fresh culture medium containing 2.5 mg/ml of LPDS serum plus the indicated conwith or centration of 125I-labeled lipop roteins, without a 20-fold excess of unlabeled homologous lipoprotein. The total cell-associated (bound plus was ‘25I-P-VLDL or ‘251-Ac-LDL internalized) determined after digestion of the cells with NaOH as described above. Proteolytic degradation of the ‘251-labeled lipoproteins was determined on 1 ml of post-incubation medium by measuring TCA1251 radioactivity [27]. Results soluble, non-iodine were corrected for residual TCA-soluble, noniodine ‘25I found in control dishes incubated without cells. Specific binding and degradation of 125I-P-VLDL and 125I-AC-LDL were determined by subtracting the radioactivity obtained in the presence of a 20-fold excess of unlabeled lipoprotein from that obtained in its absence [27]. Cholesterol esterification Pigeon monocytes cultured in 35-mm dishes for 5 days were incubated for 48 h in the presence of the indicated lipoproteins and [ I4 Cloleate ([ l-l4 C] oleic acid), 0.2 mM, specific activity 6832-11247 dpm/nmol) as described [29]. Following incuba-

51 TABLE

1

PIGEON LEUKOCYTE COUNTS IN PERIPHERAL BLOOD AND FOLLOWING ISOLYMPH SEPARATION Differential Peripheral blood a Monocytes Lymphocytes Heterophils Eosinophils Basophils

14.5 f 1.8 38.6rt2.1 43.4k2.8 1.4rto.3 2.1 rto.5

Following isolymph Monocytes Lymphocytes Thrombocytes

separation 38.3 k 1.6 43.8 f 2.3 17.9*1.4

counts (%)

a Peripheral blood cell counts did not include thrombocytes which represent approximately 65% of the total nonerythrocytic cells present in avian peripheral blood [18]. Values represented are mean f SEM; n = 22.

tion, the cells were washed with PBS and scraped from the dishes. The cells were disrupted by sonication and aliquots taken for protein determination and for lipid extraction using the methods of Bligh and Dyer [30]. Cholesterol esterification was determined by measuring the incorporation of [‘4C]oleate into cholesteryl esters as previously described [29]. Results are expressed as nmol [ “C]oleate incorporated into cholesteryl oleate per mg cell protein per 48 h. Results The differential leukocyte counts obtained from Wright-stained pigeon peripheral blood smears and from preparations of a monocyte-enriched cell suspension obtained by Isolymph density gradient centrifugation are shown in Table 1. Prior to Isolymph separation, heterophils accounted for a majority (43.4%) of the peripheral blood leukocytes, while monocytes represented 14.5% of the total circulating white blood cells (WBC). When the WBC-rich plasma was separated from the peripheral blood of these same birds and fractionated with Isolymph density gradient centrifugation, a relatively enriched preparation of monocytes was obtained. Monocytes and lymphocytes accounted for a majority of the cells in this secondary isolate, with monocytes representing

38.3% and lymphocytes representing 43.8% of the total. Thrombocytes accounted for the remaining 17.9% of the isolated cells. This monocyte-rich suspension was used for the establishment of pigeon monocyte macrophage cultures which involved the additional purification by differential adhesion of the mononuclear cells to plastic tissue culture dishes. Although both monocytes and thrombocytes initially adhered to the culture dishes, there was a selective detachment of the thrombocytes throughout the first several days of culture such that after 3 days, most of the cells in the residual populations displayed the morphological characteristics of monocyte macrophages (Fig. 1). The macrophage-like characteristics of these cells at 3 days was verified by phagocytosis of fluorescent latex beads (Fig. 2). After 1 h > 85% of the cells ingested 2 or more of the latex beads. Studies with mammalian monocyte macrophages have shown that the expression of ,&VLDL and AC-LDL receptors changes depending on time in culture [11,16]. P-VLDL receptor activity is highest on newly cultured monocytes and decreases as these cells mature into macrophages with increased time in culture [16]. The opposite is true for AC-LDL receptors; expression of the AcLDL receptor is low initially, but increases with time in culture [ll]. To see if a similar temporal sequence of lipoprotein receptor expression occurred in pigeon monocyte macrophages, cells were maintained in culture for 3, 7 or 10 days then incubated for 5 h at 37 o C with ‘251-P-VLDL or 125I-AC-LDL. The amount of cell-associated (bound + internalized) and degraded 1251 was determined (Fig. 3). Although there were differences in the amount of ‘251-P-VLDL metabolized by cells which had been maintained in culture for up to 10 days (particularly cell-associated), there was not a consistent decrease in P-VLDL receptor expression with time in culture from 3 to 10 days. Van Lenten et al. [16] observed that the expression of mammalian monocyte macrophage P-VLDL receptor activity significantly decreased during the first 48 h of culture. We cannot eliminate the possibility that similar changes could have taken place in pigeon monocyte macrophages as well, since 3 days was the earliest time point studied. The reason for this was that prior to 3 days there

52

53 were still considerable numbers of thrombocytes in the culture dishes. Greater than 80% of the total ‘251-j3-VLDL metabolism at all time points was the result of specific processes. Also shown in Fig. 3 are similar studies done with ‘251-Ac-LDL. The absolute amount of uptake and degradation of ‘251-Ac-LDL was considerably higher than obin served for ‘251-P-VLDL (note the differences the ordinate scales) with most of the ‘251-Ac-LDL being metabolized by nonspecific processes. Specific uptake did occ’lr, however. The results for specific uptake were calculated for each of the 4--6 experiments, and in all cases some specific uptake was seen. The data in Fig. 3 include the mean and SEM for all experiments. Thus, the variability is much greater than observed in individual experiments. There was a tendency for specific uptake of ‘251-Ac-LDL to increase with maturation of the monocyte macrophages in culture. Therefore, to maintain consistency, all subsequent studies described in this paper were carried out on monocyte macrophages that had been maintained in culture for 7 days. Macrophages are capable of secreting a large number of proteolytic enzymes [31]. As a result, it was important to determine if the pigeon monocyte macrophages were secreting into the culture media proteases that were capable of degrading P-VLDL and/or AC-LDL extracellularly. To test this possibility, ‘251-labeled P-VLDL or Ac-LDL were added to 18-h monocyte macrophage-conditioned culture medium and allowed to incubate for an additional 5 h at either 37’C or 4” C. As shown in Table 2, incubations carried out at 37 o C in the cell-conditioned medium resulted in a 2-fold increase in the amount of ‘251-P-Vl,DL and 1251AC-LDL degradation relative to that observed with

medium not previously conditioned by the cells. When incubations were carried out at 4”C, no differences were observed between cell conditioned and non-conditioned medium, and the values were similar to those obtained with non-conditioned medium at 37°C. These results suggested that pigeon monocyte macrophages were secreting or releasing into the culture medium proteases that were capable of degrading /3-VLDL and AcLDL at 37” C. For this reason, in subsequent lipoprotein binding experiments, incubations were carried out at 4” C to measure surface binding and to minimize the actions of these putative proteolytic enzymes on the ‘251-lipoprotein ligands. When monocyte macrophages were incubated with increasing concentrations of 12’I-fi-VLDL at 4”C, saturable binding kinetics were observed (Fig. 4). At low concentrations, binding was predominantly specific with saturation occurring at concentrations greater than 20 pg of ‘251-j3-VI,DL protein/ml. When the specific binding data were analyzed according to the method of Scatchard [32] a biphasic binding curve resulted which suggested the presence of both low and high affinity binding sites for P-VLDL (Fig. 4). A similar biphasic binding curve for pigeon /3-VLDI. has been demonstrated in pigeon peritoneal macrophages [13]. The high-affinity site displayed an apparent dissociation constant (Kd) for binding of approx. 3.0 /lg of ‘251-/?-VLDL protein/mg and a maximum binding capacity (B,,) of approx. 80 ng of ‘251-j3-VLDL protein/mg cell protein (Fig. 4). The second site had a much lower binding affinity, but a higher capacity. The specificity of binding of ‘“‘I-/3-VLDL to pigeon monocyte macrophages at 4°C was characterized using different lipoproteins as competi-

Fig. 1. Morphology of pigeon monocytes after 7 days in culture as observed and photographed by Nomarski differential interference contrast (DIC) optics. Cell cultures were established as described in Methods. After 7 days, the cells were washed, fixed, stained with Wright stain, and examined by Nomarski DIC optics. The cells can be seen to exhibit a characteristic spread morphology, a variability in shape from oval to elongated, and numerous plasma membrane ruffles and veils. Nuclei and intracellular inclusions can be seen as raised bumps on the surface of the cells. Magnification, x 900. Fig. 2. Phagocytosis of fluorescent-labeled latex beads by pigeon monocyte macrophages. Cell cultures were established as described in Methods. After 3 days, the cells were washed and fresh culture medium containing fluorescent-labeled latex beads (1.0 pm diameter) was added to the cells. Incuations were carried out for 1 h after which time the cells were washed, stained with Wright stain and examined by fluorescent microscopy. Phagocytized latex beads can be seen as bright particles within the cells. Magnification, x 600.

54 /3 -VLDL

AC-LDL Cell-Associated

0

T

d

NowSpecIfic

OL 3 Days

7 Days

10 Days

T

.II

1

3 Days

Days in Culture

lays

10 Days

Days in Culture

Degraded

0 3 Days

7 Days

10 Days

Days in Culture

0 3 Days

7 Days Days in Culture

1 10 Days

Fig. 3. Effect of time in culture on cell-associated and degraded lZ51-j3-VLDL and 1251-Ac-LDL at 37 ’ C by pigeon monocyte macrophages. Cells were cultured as described in Methods. After the period of time shown on the abscissa, the cells were washed and 1 ml of LPDS culture medium containing 10 pg of ‘251-b-VLDL or ‘251-Ac-LDL (in the presence or absence of a lo-fold excess of unlabeled p-VLDL or AC-LDL, respectively) was added to the cells. After 5 h at 37 o C, the amount of degraded 1251-/3-VLDL or ‘251-A~-LDL was determined by measuring TCA-soluble, non-iodide radioactivity in the culture medium (lower panel). The cells were washed extensively, digested for 1 h with 1 N NaOH, and aliquots were taken for measurement of cell-associated ‘251-/3-VLDL Values for specific cell-associated and degraded “‘1-P-VLDL and or 1251-Ac-LDL (upper panel) and protein concentration. 1251-Ac-LDL were calculated as described in Methods. Degradation values have been corrected for material degraded in wells without cells. Results are the meanf SEM of 4-6 different pigeon monocyte macrophage cultures studied in duplicate.

tors (Fig. 5). A 20-fold excess of unlabeled pVLDL effectively inhibited 81% of the binding of ‘251-P-VLDL. This is similar to the degree of competition seen with P-VLDL at 37 o C (Fig. 3). Likewise, LDL from normal and hypercholesterolemic pigeons were effective competitors for 12’1/3-VLDL binding. They were, however, less effective than fl-VLDL when added at equivalent protein concentrations. AC-LDL did not compete for demonstrating that the binding of 125I-P-VLDL, P-VLDL was binding to a site distinct from the scavenger receptor.

Cells incubated at 4“ C with increasing concentrations of ‘251-Ac-LDL also displayed saturable binding kinetics (Fig. 6). In these experiments done at 4O C, specific binding accounted for a greater percentage of total binding than was seen at 37 o C (Fig. 3). In contrast to P-VLDL binding, Scatchard analysis revealed the presence of a single, high-affinity binding site for AC-LDL with a Kd of approx. 12.0 pg of AC-LDL protein/ml and a B,,,,of approx. 170 ng of AC-LDL protein/mg cell protein (Fig. 6). As shown in Fig. 7, this binding site was specific for AC-LDL since

a 20-fold excess of AC-LDL effectively inhibited 70% of the binding of t2’I-AC-LDL, while /3-VLDL and LDL from normal and hypercholesterolemic pigeons failed to compete. These observations were consistent with the presence of a receptor on pigeon monocyte macrophages similar to the scavenger receptor described for mammalian macrophages [lo]. The experiments described thus far revealed that pigeon monocyte macrophages possess specific cell surface receptors that bind P-VLDL and AC-LDL. These binding sites presumably facilitate the internalization and delivery of the cholesteryl ester-rich lipoproteins to intracellular sites of degradation. In order to determine if the cholesterol liberated from /3-VLDL and AC-LDL degradation stimulated cholesteryl ester synthesis in the pigeon monocyte macrophages, the rate of cholesteryl esterification was measured in the cells incubated with P-VLDL or AC-LDL for 2 days (Table 3). The rate of cholesterol esterification was approximately twice as high in cells incubated with P-VLDL or AC-LDL as compared to control cells incubated without added lipoproteins. As a

TABLE

TABLE 3 STIMULATION OF CHOLESTERYL ESTER FORMATION IN PIGEON MONOCYTE MACROPHAGES INCUBATED WITH B-VLDL OR AC-LDL Addition medium

to

Incorporation of [“C]oleate into cholesteryl [14C]oleate (nmol/mg protein)

________

P-VLDL None

104 42

AC-LDL None

75 40

-

Pigeon monocytes were isolated and established in culture as described in Methods. After 5 days, the cells were washed and 1 ml of LPDS culture medium containing 50 pg of /I-VLDL or AC-LDL and 0.2 mM [‘4C]01eate was added to the cells. Control cells received LPDS culture medium and the [‘4C]01eate substrate only. The cells were incubated for an additional 2 days after which time the cholesteryl [i4C]01eate content of the cells was determined as described m Methods. Results are the mean of 2 different pigeon monocyte cultures studied in duplicate.

result, internalization and degradation of /?-VLDL and AC-LDL promoted the delivery of cholesterol to pigeon monocyte macrophages as seen from the stimulation of cholesterol esterification.

2

EFFECT OF CULTURE MEDIUM CONDITIONED BY PIGEON MONOCYTE MACROPHAGES ON 1251-fi-VLDL AND ‘25 I-AC-LDL DEGRADATION Degradation lipoproteins 37OC /I-VLDL Non-conditioned Cell conditioned

medium

AC-LDL Non-conditioned Cell conditioned

medium medium

437f 50 997 + 139

3151130 1040+169

of “‘I-labeled (cpm/dish) 4OC

462 & 161 459& 91

413+ 521*

33 19

Cell conditioned and non-conditioned media were prepared as described in Methods by incubation in the presence or absence of cells for 18 h at 37 o C. 0.5 ml of each medium was added to 22.6~mm tissue culture wells along with 10 pg of ‘251-/3-VLDL Or ‘251-Ac-LDL. After 5 h incubation at either 37 o C or 4” C, degradation products in the medium were determined as TCA-soluble, non-iodide ‘251. For P-VLDL, results are the mean f SEM of 5 different pigeon monocyte macrophage cultures carried out in triplicate for each experiment. For AC-LDL, results are the mean+ SEM of 3 different pigeon monocyte macrophage cultures studied in triplicate.

Discussion The present studies have demonstrated for the first time that monocyte macrophages from an avian species, the White Carneau pigeon, express surface receptors for /3-VLDL and AC-LDL, and that these receptors can mediate the uptake and degradation of these lipoproteins and promote cholesterol esterification. Kinetic analysis of AC-LDL binding revealed that pigeon monocyte macrophages bind AC-LDL by a saturable and specific process. The binding of the AC-LDL displayed many characteristics of the scavenger receptor previously reported to exist on the surface of White Carneau pigeon peritoneal macrophages [13] and on peritoneal [9,10,15] and monocyte-derived macrophages [ 1 l] from mammalian sources. As shown by Scatchard analysis, AC-LDL bound to a single class of high affinity receptors with a K, of approx. 12 pg/ml. This compares favorably to previous studies with pigeon (13 pg/ml) [13] and mammalian (5 pg/ml)

Total + AC-LDL + /3-VLDL + H-LDL + N-LDL

26 6.26

12.5

25

W&VLDL

50

Concentration

14

(uglml)

J

0 t-Competing

.0172 .0164 .0136 I? % 5 li3

.0116 .OlcQ .a362 .3064.0046.0026 .OOlO

\

’ 20

Bound

.*....

h 100

60

....

140

(nglmg)

Fig. 4. Effect of ‘*‘I-P-VLDL concentration on cell-associated ‘251-j3-VLDL at 4OC by pigeon monocyte macrophages in culture. Pigeon monocyte macrophages were cultured as described in Methods. After 7 days, the cells were washed and 1 ml of LPDS culture medium containing the indicated concentrations of ‘251-b-VLDL with (A) or without (0) a 20-fold excess of unlabeled /3-VLDL were added to the cells. The cells were incubated for 4 h at 4“ C after which time the amount of cell-associated ‘251-/3-VLDL (panel A) was determined. Values for specific cell-associated *251-p-VLDL (m) were calculated as described in Methods. Results are the mean f SEM of 4 different pigeon monocyte macrophage cultures studied in duplicate. Panel B displays the Scatchard plot obtained from the specific cell-associated data in the above experiment. Bound/free represents the lipoprotein bound (ng protein/well) divided by the lipoprotein free in the medium (ng/ml).

[15] peritoneal macrophages. Binding to the scavenger receptor was specific for AC-LDL as evidenced by the failure of pigeon LDL or /?-VLDL to compete for binding while AC-LDL competed effectively. The expression of the scavenger receptors in human monocyte macrophages is coupled to the maturation of the macrophages in culture [ll]. Scavenger receptor activity is low soon after adding human blood monocytes to tissue culture

Lipoprotein

Fig. 5. Competition studies illustrating the ability of AC-LDL, @-VLDL, N-LDL, and H-LDL to inhibit the cell-association of ‘251-~-VLDL at 4°C with pigeon monocyte macrophages in culture. Pigeon monocyte macrophages were cultured as described in Methods. After 7 days, the cells were washed and 1 ml of LPDS culture medium containing 20 pg of ‘251-/3-VLDL (with or without a 20-fold excess of the unlabeled lipoprotein indicated) was added to the cells. After incubation for 4 h at 4O C, the cells were washed extensively and the cell-associated 1251-j3-VLDL determined as described in Methods. Results are the mean f SEM of 3 different pigeon monocyte macrophage cultures studied in duplicate. * Indicates difference from total (P -e 0.05) using single factor analysis of variance.

dishes. As the monocytes mature to more macrophage-like cells in culture, there is an increase in scavenger receptor activity [ll] and a decrease in expression of /3-VLDL receptors [16]. These changes are maximum for AC-LDL by about 12 days in culture for human monocyte macrophages. In the present studies we consistently observed a significant number of thrombocytes in the cell cultures throughout the first 3 days of culture. As a result, it was not possible to measure AC-LDL receptor activity prior to day 3. Given the low levels of AC-LDL receptor activity at 3 days in culture, it is unlikely that AC-LDL receptor activity was higher prior to day 3. Nevertheless, a similar pattern of expression of scavenger receptor activity with time in culture was seen. Specific uptake of AC-LDL was low at 3 days and increased 3-fold by day 7 and 13-fold by day 10. A somewhat surprising finding was the large amount of nonspecific binding of AC-LDL at all times in culture when incubated at 37 o C. This is particularly evident in the studies shown in Fig. 3. Whether this reflects a difference in avian versus

57

m

Total

3 [7

+ AC-LDL

H

+&VLDL

m

+ H-LDL

m

+ N-LDL

b

6.25

25

12.5 ts51-Ac-LDL

50

Concentration

(ug/ml) Competing

0160 B

’

Bound

(nglmg)

Fig 6. Effect of ‘251-A~-LDL concentration on cell-associated ‘251-Ac-LDL at 4O C by pigeon monocyte macrophages in culture. Pigeon monocyte macrophages were cultured as described in Methods. After 7 days, the cells were washed and 1 ml of LPDS culture medium containing the indicated concentrations of 1251-Ac-LDL with (A) or without (0) a 20-fold excess of unlabeled AC-LDL was added to the cells. The cells were incubated for 4 h at 4 o C after which time the amount of cell-associated ‘251-Ac-LDL (panel A) was determined. Values for specific cell-associated 12’1-AC-LDL (w) were calculated as described in Methods. Results are the mean f SEM of 3 different pigeon monocyte macrophage cultures studied in duplicate. Panel B displays the Scatchard plot obtained from the specific cell-associated data in the above experiment. Bound/free represents the lipoprotein bound (ng protein/well) divided by the lipoprotein free in the medium (ng/ml).

mammalian macrophages relative to the pathogenesis of atherosclerosis is unclear. The higher nonspecific binding with AC-LDL was not seen at 4OC. Pigeon monocyte macrophages also displayed saturable binding kinetics for P-VLDL. In contrast to the AC-LDL receptor, however, Scatchard analysis suggested that the binding was mediated by at least 2 classes of binding sites which exhibited high and low affinities for P-VLDL. Al-

Lipoprotetn

Fig. 7. Competition studies illustrating the ability of AC-LDL, /3-VLDL, N-LDL, and H-LDL to inhibit the cell-association of ‘251-Ac-LDL at 4O C by pigeon monocyte macrophages in culture. Pigeon monocyte macrophages were cultured as described in Methods. After 7 days, the cells were washed and 1 ml of LPDS culture medium containing 20 pg of ‘251-Ac-LDL (with or without a 20-fold excess of the unlabeled lipoprotein indicated) was added to the cells. After incubation for 4 h at 4’ C, the cells were washed extensively and the cell-associated ‘251-Ac-LDL determined as described in Methods. Results are the meanf SEM of 3 different pigeon monocyte macrophage cultures studied in duplicate. * Indicates difference from total (P < 0.05) using single factor analysis of variance.

though previous investigators working with mammalian macrophages [15] have reported binding of P-VLDL exclusively to 1 class of high affinity receptors sites, the present findings are consistent with those of Adelman and St. Clair [13] who showed the presence of both high and low affinity sites for pigeon P-VLDL on elicited peritoneal macrophages from White Cameau and Show Racer pigeons. The high affinity binding component for pigeon P-VLDL on pigeon monocyte macrophages in the present study had a K, of approx. 3.0 pg/rnl, which was in the same range as that previously reported for pigeon (8 pg/ml) [13] as well as mouse (1 pg/rnl) [15] peritoneal macrophages. The significance of the lower affinity binding site for pigeon P-VLDL is unclear. It could represent a general ‘lipoprotein binding site’ similar to that previously demonstrated in a number of tissues including rabbit liver membranes [33] and pigeon smooth muscle cells [34]. The binding sites for P-VLDL on the pigeon monocyte macrophages appear to be distinct from

58 the scavenger receptor. This was evidenced by the finding that AC-LDL only competed for about 20% of the binding of ‘251-P-VLDL, while at the same time &VLDL competed to greater than 80%. The small amount of competition by AC-LDL could simply reflect nonspecific binding of 1251-j3VLDL or competition of AC-LDL for the low affinity ‘251-fi-VLDL binding site. The P-VLDL binding sites however, were not specific for pVLDL alone, since LDL from normal and hypercholesterolemic pigeons also competed for 1251-/3VLDL binding. The observation that LDL is recognized by P-VLDL receptors is consistent with previous reports using pigeon peritoneal macrophages [13]. /3-VLDL receptors on human monocyte-derived [16] and mouse peritoneal [9,35] macrophages also have been shown to recognize LDL. In the mammalian system however, LDL is a significantly less effective competitor than P-VLDL, since 5- [16] to lOO-fold [9,35] more LDL than P-VLDL is re‘ 251-P-VLDL binding by 50%. In quired to inhibit the present studies, we also observed that at the same protein concentration LDL was 14-23% less efficient at competing for ‘251-/3-VLDL binding than was P-VLDL (Fig. 5). However, since the competing lipoproteins were added at equivalent protein concentrations, and not at equivalent particle numbers, it is possible that this effect was due to differences in the number of P-VLDL and LDL particles used as competitors. /3-VLDL from White Cameau pigeons is composed of approx. 10% protein, while the protein compositions of LDL from normal and hypercholesterolemic pigeons are approx. 14% and 20%, respectively [20]. Thus, at equivalent protein concentrations, as was the case in the present experiment, up to twice as many /3-VLDL as LDL particles were available to compete for the binding of ‘251-P-VLDL. This probably accounts for the differences in 1251-pVLDL displacement observed for P-VLDL and LDL, and suggests that unlike binding of mammalian /3-VLDL and LDL to mammalian macrophage receptors, pigeon /3-VLDL and LDL probably bind with similar affinities. This conclusion is supported by the fact that pigeons lack apoprotein E [20] which is the protein that confers the higher binding affinity of /3-VLDL for the mammalian P-VLDL receptor [36]. When this is coupled with

the fact that pigeon LDL and P-VLDL have remarkably similar apoprotein compositions [20], it is not surprising that these lipoproteins might be recognized similarly by macrophage receptors. Recent studies have suggested that the mammalian P-VLDL receptor is not a unique receptor for fi-VLDL, but instead represents an altered form of the apo B,E (LDL) receptor [35]. Previous studies by Adelman and St. Clair [13] working with pigeon peritoneal macrophages have shown that P-VLDL binding in the pigeon system also displays several characteristics of the classical LDL receptor pathway [37]. These include a requirement for the divalent cation Ca2+, sensitivity to pronase treatment, and the ability to be downregulated by cholesterol loading. Due to limitations in the number of cells available for study, it was not possible to evaluate these characteristics in pigeon monocyte macrophages. Other characteristics appear identical however, between the monocyte macrophage and peritoneal macrophage P-VLDL receptor. If indeed this conclusion is correct, and if the /3-VLDL receptor in avian macrophages is a modified LDL receptor [35], then it is intriguing that this receptor is expressed in monocyte macrophages and peritoneal macrophages in culture and not in pigeon smooth muscle cells or skin fibroblasts in culture [34,38]. In addition to the ability of pigeon monocyte macrophages to take up and degrade lipoproteins by receptor-mediated processes, these cells also secreted ‘protease-like’ activity into the culture medium that was able to promote the degradation of P-VLDL and AC-LDL into products that were not precipitable by TCA. The amount of this activity appears to be low relative to receptor mediated degradation of /3-VLDL and AC-LDL. For example, given the data shown in Table 2, a maximum of about 10 ng of lipoprotein was degraded per dish in 5 h when incubated with cell conditioned medium. The cell conditioned medium was produced by incubation with monocyte macrophages for 18 h. If secretion of proteolytic activity is linear over time, then only about 25% of this activity would have been secreted in 5 h. Thus, in a 5 h incubation at 37 o C with cells, about 2-5 pg of lipoprotein would be degraded (2.5 ng/mg cell protein) by proteolytic enzymes in the culture medium. This compares with a minimum of about

59 500 pg/mg of cell protein of total degradation in 5 h in the presence of cells (Fig. 3) or about 5% of the total. As a result, degradation by proteolytic enzymes secreted into the culture medium does not significantly alter the conclusion that the levels of degradation of /3-VLDL and AC-LDL by pigeon monocyte macrophages in culture is the result of receptor-mediated endocytosis and lysosomal degradation. However, it does leave open the possibility, that in situ the secretion of proteolytic enzymes from macrophages in the atherosclerotic lesions could play an important role in degradation of lipoproteins within the the extracellular matrix. Such degradation could result in particles that would be more readily recognized by scavenger lipoprotein receptors on macrophages or alternatively be taken up by phagocytosis. This and previous studies from our laboratory have shown that pigeons, like all the mammalian species that have been studied, have a variety of mechanisms available for the recognition and internalization of lipoproteins, Whether differences in the quantitative or qualitative nature of any of these pathways are responsible for differences in atherosclerosis susceptibility is unclear. The pigeon is a potentially useful model to investigate these possibilities given the well known differences in atherosclerosis susceptibility among breeds [39]. Acknowledgements The authors wish to thank Mrs. Bobbie Lindsay for assistance in the preparation of this manuscript. We are also grateful for the excellent technical assistance provided by Ms. Molly Leight, Dr. Richard Taylor, Dr. W. Gray Jerome, Mr. Kenneth Grant and Mrs. Susie Hester. This work was supported by the Specialized Center of Research (SCOR) in Atherosclerosis Grant HL-14164 from the National Heart, Lung, and Blood Institute. Dr. Henson was supported by an Institutional National Research Service Award Grant HI.-07115 and by a Special Fellowship from R.J. Reynolds Industries. References 1 Haust, M.D., The morphogenesis early atherosclerotic 1

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