Aggregated Low Density Lipoprotein Induces and Enters Surface-connected Compartments of Human Monocyte-Macrophages

Aggregation of low density lipoprotein (LDL) stimulates its uptake by macrophages. We have now shown by electron microscopic and chemical experiments that aggregated LDL (produced by vortexing (VxLDL) or treatment with phospholipase C) induced and became sequestered in large amounts within surface-connected compartments (SCC) of human monocyte-derived macrophages. This occurred through a process different from phagocytosis. Formation of SCC and accumulation of aggregated LDL in SCC are cell-mediated processes that were temperature-dependent (10 × greater cell association at 37 °C than at 4 °C) and blocked by cytochalasin D but not by nocodazole. Because of the surface connections of SCC, trypsin could release aggregated LDL from SCC. Degradation of125I-VxLDL through the SCC pathway showed delayed and a lower rate of degradation (10–55%) compared with nonaggregated125I-acetylated LDL that did not enter SCC. However, similar to 125I-acetylated LDL degradation,125I-VxLDL degradation occurred through a chloroquine-sensitive pathway. Uptake of VxLDL into SCC was not mediated by the LDL receptor. Methylation of LDL prevents its binding to the LDL receptor. However, methylated LDL still entered SCC after it was aggregated by vortexing. On the other hand, degradation of125I-VxLDL was substantially decreased by methylation of LDL and by cholesterol enrichment of macrophages, which decreases macrophage LDL receptor expression. The results suggest that whereas uptake of aggregated LDL into SCC occurs independently of the LDL receptor, movement of aggregated LDL from SCC to lysosomes may depend in part on LDL receptor function. Sequestration into SCC is a novel endocytosis pathway for uptake of aggregated LDL that allows the macrophage to store large amounts of this lipoprotein before it is further processed.

Previously, we reported that human monocyte-macrophages display an unusual mode of endocytosis in which they sequester cholesterol crystals into an extensive labyrinth of cytoplasmic compartments (1). These compartments remain connected to the surface of the macrophage and to the extracellular space. Thus, this endocytotic process is different from phagocytosis, where particles are taken into the macrophage within vacuoles that form from pinched-off regions of the plasma membrane and do not maintain any connection to the extracellular space (2).
Besides cholesterol crystals, we observed that acetylated low density lipoprotein (AcLDL) 1 could induce and enter SCC of human monocyte-macrophages (1). However, we subsequently discovered that this occurred only for some lots of AcLDL and not with other lots. AcLDL that entered these compartments was aggregated in linear strands. We suspected that some aggregation of AcLDL occurred during its storage and that this aggregated AcLDL could have been responsible for the formation of the macrophage SCC.
Other investigators have shown that aggregation of lipoproteins enhances their uptake by macrophages. Oxidation and thiolation of LDL or treatment of LDL with sialidase, phospholipase C, or sphingomyelinase plus lipoprotein lipase (in the presence of cultured smooth muscle cells) and other treatments cause LDL to aggregate and to show enhanced degradation by macrophages (3)(4)(5)(6)(7)(8)(9)(10). Khoo et al. (11) showed that aggregation of normal LDL by vortexing converted LDL to a form that was readily taken up by mouse peritoneal macrophages through an LDL receptor-dependent mechanism. The endocytotic pathway for uptake of the vortexed low density lipoprotein (VxLDL) was not described, but it was suggested that this LDL entered the macrophages by phagocytosis.
In the present study, we examined whether aggregated LDL has the capacity to induce and accumulate within monocytemacrophage SCC. Not only did aggregated LDL induce and accumulate within SCC, this occurred by a mechanism that did not depend on the classical LDL receptor.

MATERIALS AND METHODS
Preparation of Lipoproteins-Human LDL was prepared as described previously (12) and was obtained from PerImmune (Rockville, MD). AcLDL was prepared as described by Basu et al. (13). Methylation of LDL was performed according to Weisgraber et al. (14). 125 I-Labeled LDL and AcLDL with specific activities that ranged 70 -200 Ci/mg of protein were obtained from Biomedical Technologies (Stoughton, MA). Lipoproteins that were not to be aggregated by vortexing were centrifuged at 14,000 ϫ g for 10 min to remove any spontaneously formed aggregates from these lipoprotein preparations. To prepare aggregated lipoproteins, 40 l of LDL or methylated LDL (5 mg/ml for unlabeled and 1-3 mg/ml for labeled lipoproteins) was placed into a 0.5-ml silicone-coated polypropylene tube and vortexed (VWR vortex mixer) at the maximal speed for 1 min. LDL aggregated with phospholipase C treatment was prepared as described previously (7,8).
Assay of Cell Association and Degradation of 125 I-Lipoproteins-The cell association and degradation of lipoproteins by human monocytederived macrophages was performed according to the methods of Goldstein et al. (15) using 125 I-VxLDL and 125 I-AcLDL. Human monocytederived macrophages were cultured as described previously except that * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Lipoprotein degradation was quantified by measurement of trichloroacetic acid-soluble organic iodide radioactivity in supernatants of media samples that were centrifuged at 15,000 ϫ g for 10 min. Values obtained in the absence of cells were Ͻ5% those values obtained with cells. These control values were subtracted. Cell-associated 125 I-lipoproteins were determined by rinsing macrophages 5 times with Dulbecco's phosphate-buffered saline (DPBS) containing Ca 2ϩ , Mg 2ϩ , and 0.35% bovine serum albumin (3 quick rinses and two 10-min incubations on ice). After a final rinse with DPBS plus Ca 2ϩ and Mg 2ϩ , macrophages were dissolved overnight in 0.1 N NaOH. Aliquots of NaOH-solubilized cell samples were assayed for 125 I radioactivity. Values of 125 I radioactivity determined for wells incubated with 125 I-lipoproteins but without macrophages were subtracted. These values were Ͻ1% those of the cell-associated 125 I-lipoproteins. Macrophage protein content was determined by the method of Lowry et al. (16) using bovine serum albumin as a standard. Protein contents of cultures generally ranged between 0.2 and 0.3 mg/well. For Experiment I in Table II, passaged normal human skin fibroblasts (GM09503, National Institute of General Medical Sciences Cell Repository, NIH, Camden, NJ) were seeded into 6-well (35-mm diameter) Plastek C culture plates at a density of 10 5 cells/well. These cultures were maintained 3 days with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and then for another 2 days with Dulbecco's modified Eagle's medium containing 10% human lipoprotein-deficient serum (PerImmune) before incubations with 125 Ilipoproteins were carried out.
Trypsin Release of 125 I-Lipoproteins from SCC-After incubation with the indicated amount of 125 I-lipoprotein, monocyte-macrophages were rinsed 3 times with RPMI 1640 and incubated for 30 min in 1 ml of RPMI 1640 with 25 l of DPBS plus Ca 2ϩ and Mg 2ϩ or that solution containing 50 g of trypsin (2,740 units/mg of protein; Life Technologies Inc.) at 37°C. Then cells were rinsed and assayed for cell-associated 125 I-lipoprotein and cell protein. Organic 125 I-radioactivities in soluble and trichloroacetic acid-insoluble fractions of media were also measured.

Effect of Cytochalasin D and Chloroquine on 125 I-Lipoprotein Cell
Association and Degradation, Respectively-Monocyte-macrophages were rinsed and incubated with 1 ml of RPMI 1640 medium and 125 I-labeled lipoproteins containing 4 g/ml cytochalasin D (Calbiochem) to demonstrate the actin dependence of cell association. Chloroquine and cytochalasin D sensitivity of degradation of cell-associated 125 I-VxLDL was determined with pulse-chase experiments. Monocytemacrophages were incubated with 125 I-VxLDL in 1 ml of RPMI 1640 for 5 h, rinsed, and then incubated in RPMI 1640 without or with 100 M chloroquine (Sigma) or 4 g/ml cytochalasin D. For cytochalasin D controls, macrophages were incubated with 1 ml of RPMI 1640 containing 2 l of Me 2 SO, the solvent for cytochalasin D.
Assay of Cholesterol Content of Monocyte-Macrophages-Unesterified and esterified cholesterol contents of macrophages were determined enzymatically according to the fluorometric method of Gamble et al. (17). For these assays, macrophages were harvested by scraping into distilled water and processed as described previously (12).
Electron Microscopy-Extracellular and intracellular membranes were differentiated with ruthenium red according to the method of Luft (18). Monocyte-macrophage cultures were ruthenium red-stained and prepared for electron microscopy as described previously (1).
Statistical Analysis-All data are presented as means Ϯ S.E. The means were determined from 3 culture wells for each data point. No standard error is shown when it was smaller than the height of the graphic symbol.

RESULTS
Aggregated LDL Accumulated within SCC of Human Monocyte-Macrophages-Ruthenium red is an electron dense membrane stain. Because ruthenium red does not penetrate membranes of aldehyde-fixed cells, it stains only membranes in continuity with the extracellular space. Electron microscopic examination of macrophages incubated with VxLDL showed accumulation of lipoprotein particles within ruthenium redoutlined SCC not within phagocytic vacuoles (Fig. 1). As we reported previously (1), SCC were tortuous interconnecting membranous compartments. VxLDL also accumulated in some ruthenium red negative membranous compartments that had the same appearance as SCC. These ruthenium red negative FIG. 1. Accumulation of aggregated LDL in SCC of monocyte-macrophages. Two-week-old monocyte-macrophage cultures were incubated with 100 g/ml VxLDL (a and b) or 100 g/ml phospholipase C-treated LDL (c) for 3 h. Then macrophage cultures were fixed and stained with ruthenium red and processed for electron microscopy as described previously (1). Thin sections were prepared and examined unstained. The contrast in a was increased photographically to enhance ruthenium red staining. The arrowheads in a and c delineate the region of SCC. The arrow in a indicates microvillus folds at the macrophage cell surface. The arrows in b show the ruthenium red-stained delimiting membranes of macrophage SCC that contain VxLDL particles. The bars in a, b, and c represent 1, 0.5, and 1 m, respectively. membranous compartments most likely are SCC that were not open to the extracellular space at the time of fixation of the macrophages. The lipoprotein particles in all SCC were often larger than native LDL (which is about 22 nm) and often resembled beads on a string. The increased size of LDL particles can be explained by fusion of LDL particles induced by vortexing (19). LDL that aggregated by treatment with phospholipase C also induced and accumulated within SCC (Fig.  1c). The aggregates produced by phospholipase C were more rounded rather than linear as was VxLDL. No macrophage SCC were induced by LDL that had not been vortexed or by AcLDL centrifuged to remove any spontaneously formed aggregates.
Comparison of Metabolism of 125 I-VxLDL, 125 I-AcLDL, and 125 I-LDL-Because VxLDL entered SCC and centrifuged AcLDL did not enter these compartments, it was possible to compare the metabolism of AcLDL and VxLDL as they were processed by two different endocytosis pathways. The time courses of the metabolism of 125 I-VxLDL and 125 I-AcLDL were compared. Cell association of 125 I-VxLDL and 125 I-AcLDL both began to reach a plateau by 2 h. However, the cell association of 125 I-VxLDL was greater than 5-times that of 125 I-AcLDL by this time (Fig. 2a). Cell association of nonvortexed LDL was small compared with that of 125 I-AcLDL and 125 I-VxLDL.
Though cell association of 125 I-VxLDL was high compared with 125 I-AcLDL, degradation of 125 I-VxLDL was less (about one-half at 4 h) than the degradation of AcLDL (Fig. 2b). Degradation of 125 I-VxLDL compared with 125 I-AcLDL could even be lower than this. In another experiment not shown, although 125 I-VxLDL cell association was 4 times that of 125 I-AcLDL by 3 h, degradation of the 125 I-VxLDL was only about one-fifth that of 125 I-AcLDL and was not significantly different from the degradation of nonvortexed 125 I-LDL.
Part of the difference in the amounts of degradation of 125 I-VxLDL and 125 I-AcLDL could be explained by differences in their initial rates of degradation. 125 I-AcLDL showed a linear increase in its degradation during incubation with the macrophages. In contrast, there was a delay in degradation of 125 I-VxLDL. Its maximal rate of degradation was not reached until 1 h after its addition to macrophage cultures. However, even when comparing their maximal rates of degradation (rather than their absolute amounts of degradation), 125 I-VxLDL was degraded at Ͻ55% the rate of degradation of 125 I-AcLDL. Cell Association of Aggregated LDL Was an Active Cell-mediated Process-Uptake into SCC was an active cell-mediated process that was dependent on temperature and actin microfilament function. Cell association of 125 I-VxLDL was 10 times greater at 37°C compared with 4°C and 5 times greater at 37°C compared with 20°C (Fig. 3). Cytochalasin D, an inhibitor of actin microfilament function (20), inhibited cell association of 125 I-VxLDL by 72 Ϯ 3.5% (n ϭ 3 experiments) during 5 h of incubation with 50 g/ml 125 I-VxLDL (see Fig. 4 for results of one experiment). Nocodazole, an inhibitor of microtubule function, did not inhibit cell association (data not shown). Electron microscopy showed that cytochalasin D blocked formation of SCC induced by VxLDL similar to what we have observed for compartments induced by cholesterol crystals (1). The temperature dependence and cytochalasin D sensitivity of 125 I-VxLDL cell association showed that most cell association occurred through an active cell-mediated process and was not due to nonspecific sticking of lipoprotein aggregates to the culture dish.
Aggregated LDL That Had Accumulated in SCC Could Be Released by Trypsin-After cell association of 20 g/ml 125 I-VxLDL with macrophages for 5 h, then up to 90% could be released from the macrophages with subsequent trypsin treatment (Fig. 5). Incubation of macrophages for 5 h with 50 g/ml 125 I-VxLDL showed an average (of three experiments) cell association of 19.9 Ϯ 1.3 g 125 I-VxLDL/mg of cell protein, 67 Ϯ 2% of which could be released by trypsin. There was a progressive decrease with time in the percentage of cell-associated 125 I-VxLDL that could be released by trypsin (Fig. 4). 86, 70, and 64% cell-associated 125 I-VxLDL could be released after 1-, 3-, and 5 h-incubations with 50 g/ml 125 I-VxLDL, respectively. Between 5 and 24 h, there was no further decrease in the percent of cell-associated 125 I-VxLDL that could be released by trypsin. Cell association of 125 I-LDL that had been aggregated with phospholipase C was less than that of 125 I-VxLDL. However, similar to 125 I-VxLDL, most cell association of phospho- lipase C-treated 125 I-LDL was inhibited by cytochalasin D and could be released by trypsin (Fig. 6).
About one-third of cell-associated 125 I-VxLDL could not be released with trypsin. However, this trypsin-resistant cell-as-sociated 125 I-VxLDL did accumulate within macrophages by an actin-dependent process. This was shown by incubating macrophages for 5 h with 50 g/ml 125 I-VxLDL in the presence of cytochalasin D and then exposing the macrophages to trypsin. In this case, release of cell-associated 125 I-VxLDL was almost complete, being 4 Ϯ 0% that of the amount found with macrophages not treated with cytochalasin D or trypsin. The occurrence of some trypsin-resistant cell-associated 125 I-VxLDL could be explained by the electron microscopic findings of some VxLDL having accumulated within ruthenium red negative SCC that lacked connections to the cell surface (see electron microscopy results above), incomplete release by trypsin of VxLDL from open SCC, and some uptake of VxLDL into non-SCC endocytic compartments. However, at a minimum, onethird VxLDL was taken up into SCC (i.e. one-third of cellassociated 125 I-VxLDL was both cytochalasin D-inhibitable and trypsin-releasable).
Two-thirds of the degradation of cell-associated 125 I-VxLDL could be accounted for by the degradation of the pool of 125 I-VxLDL that was trypsin-releasable (i.e. was within SCC or associated with macrophage cell surfaces). This was shown by incubating monocyte-macrophages with 50 g/ml 125 I-VxLDL for 5 h to produce a large pool of cell-associated 125 I-VxLDL. Then, the macrophages were treated without or with trypsin for 30 min as described in Fig. 4 to remove 125 I-VxLDL from SCC. The amount of 125 I-VxLDL degraded during a subsequent 24-h incubation in RPMI 1640 medium was determined for those macrophages with 125 I-VxLDL accumulated in SCC and for those macrophages that had their 125 I-VxLDL removed by trypsin treatment. Trypsin treatment removed 66 Ϯ 1% cellassociated 125 I-VxLDL, and this decreased subsequent 125 I-VxLDL degradation during the chase period by a similar amount at 64 Ϯ 1%.
Degradation of 125 I-VxLDL occurred in lysosomes. When macrophages were first incubated 5 h with 125 I-VxLDL and then chased 1 day in RPMI 1640 medium with the lysosome inhibitor chloroquine, degradation of cell-associated 125 I-Vx-

FIG. 4. Inhibition of cell association of 125 I-VxLDL by cytochalasin D and release of cell-associated 125 I-VxLDL by trypsin.
First, 2-week-old monocyte-macrophage cultures were incubated 1 (a), 3 (b), or 5 h (c) with 50 g/ml 125 I-VxLDL in the absence and presence of 4 g/ml cytochalasin D. These cultures were rinsed, harvested, and analyzed for their content of protein and cell-associated 125 I-VxLDL. Other duplicate sets of cultures incubated with 50 g/ml 125 I-VxLDL were rinsed three times with RPMI 1640 and incubated for 30 min at 37°C in 1 ml RPMI 1640 containing either 25 l of DPBS plus Ca 2ϩ and Mg 2ϩ or DPBS plus Ca 2ϩ , Mg 2ϩ , and 50 g/ml trypsin. Then, these cultures were rinsed, and their contents of cell-associated 125 I-VxLDL were determined. Note that the scales of the y axes for a, b, and c are different. cyto. D, cytochalasin D.

FIG. 5. Effect of trypsin concentration on release of cell-associated 125 I-VxLDL.
Two-week-old monocyte-macrophage cultures were incubated for 5 h with 20 g/ml 125 I-VxLDL. Then cultures were rinsed 3 times with RPMI 1640 and incubated at 37°C for an additional 30 min with varying amounts of trypsin added to 1 ml of RPMI 1640. After this incubation, cultures were rinsed, harvested, and analyzed for their content of protein and cell-associated 125 I-VxLDL. Total organic 125 I contents of media were measured to determine how much 125 I-VxLDL was released into the media by trypsin. Trypsin did not cause the detachment of macrophages from the culture surface because the protein contents of cultures incubated without or with trypsin were similar.
FIG. 6. Release of cell-associated phospholipase C-treated 125 I-LDL by trypsin. Two-week-old monocyte-macrophage cultures were incubated for 3 h with 50 g/ml 125 I-LDL that had been aggregated with phospholipase C (PlLDL) (7,8). Then these cultures were processed as described in Fig. 4. cyto. D, cytochalasin D.
LDL was inhibited Ͼ90% (Table I). Chloroquine inhibition of 125 I-VxLDL degradation resulted in a 2-fold increase in loss of nondegraded (i.e. trichloroacetic acid-insoluble) 125 I-VxLDL from the macrophages during the chase period. However, most (Х80%) of the 125 I-VxLDL that chloroquine blocked from being degraded remained associated with the macrophages. This was shown by the observation that cell-associated 125 I-VxLDL was increased for macrophages incubated with chloroquine compared with cell-associated 125 I-VxLDL for macrophages incubated without chloroquine (Table I).
Above, it was shown that cytochalasin D blocked uptake into SCC. We also determined whether cytochalasin D blocked subsequent degradation of 125 I-VxLDL that was already accumulated in SCC. Macrophages were first incubated 5 h with 50 g/ml 125 I-VxLDL and then chased 1 day in RPMI 1640 medium without or with cytochalasin D. During the chase period 5.5 Ϯ 0.2 g/mg of cell protein of 125 I-VxLDL was degraded without cytochalasin D, but only 0.9 Ϯ 0.1 g/mg of cell protein of 125 I-VxLDL was degraded with cytochalasin D. Thus, transport of 125 I-VxLDL from SCC to lysosomes was also an actindependent process.
Uptake of Aggregated LDL into SCC Was Not Mediated by the LDL Receptor-Cell-association of 125 I-VxLDL was not mediated through the classical LDL receptor, but some of its degradation may have been. Reductive methylation of LDL is known to prevent its binding to the macrophage LDL receptor (21). However, methylated 125 I-VxLDL became cell-associated with monocyte-macrophages through a cytochalasin D-inhibitable process that could not have been mediated by the LDL receptor (Experiment I in Table II). Also, a one-day incubation of monocyte-macrophages with RPMI 1640 medium containing 200 g/ml methylated VxLDL caused the total cholesterol content of the macrophages to increase from 83 Ϯ 2 to 260 Ϯ 9 nmol/mg of cell protein. Eighty-eight percent of the increase in cholesterol content could be inhibited by cytochalasin D. Moreover, electron microscopy showed that methylated VxLDL induced and entered SCC similar to that shown for VxLDL in Fig.  1.
On the other hand, although cell association of methylated 125 I-VxLDL was even greater than that of 125 I-VxLDL, degradation of methylated 125 I-VxLDL was only 25% compared with normal 125 I-VxLDL. This suggested that up to 75% degradation of 125 I-VxLDL may have been mediated through the LDLreceptor. Dissociation of 125 I-VxLDL degradation mediated by the LDL receptor and 125 I-VxLDL cell association was confirmed another way. The LDL receptor in human monocytemacrophages is down-regulated by cholesterol enrichment (22). Incubation of monocyte-macrophages with 50 g/ml AcLDL for 2 days increased macrophage cholesterol content about 2-fold (from 94 Ϯ 2 to 205 Ϯ 7 nmol/mg of cell protein). Cholesterol enrichment of the macrophages decreased 125 I-VxLDL degradation by 85% but only decreased 125 I-VxLDL cell association by 10% (Experiment II in Table II). DISCUSSION We have shown that LDL aggregated by vortexing or treatment with phospholipase C (and vortexed AcLDL) 2 induced and accumulated within SCC of human monocyte-derived macrophages. Uptake of aggregated LDL into SCC is a novel endocytosis pathway for this lipoprotein. Native nonaggregated LDL did not induce nor enter these compartments. Uptake into SCC was temperature-and actin-dependent. VxLDL that accumulated in SCC was degraded slowly compared with AcLDL. Though cell association of VxLDL was an active cellmediated process, nevertheless cell-associated VxLDL could be released from macrophages by trypsin. This confirmed the electron microscopic finding that VxLDL had accumulated within SCC rather than within phagocytic vacuoles.
Accumulation of aggregated lipoproteins in SCC may have been overlooked in previous studies. Often, cells are treated with protease to remove cell surface-associated lipoproteins that are considered to represent lipoproteins not actively taken up by the cells but rather specifically and nonspecifically bound to cell surfaces (8,23). However, our study shows that at least for human monocyte-macrophages, protease-releasable li-2 W.-Y. Zhang, P. M. Gaynor, and H. S. Kruth, unpublished data.

Role of LDL receptor in metabolism of 125 I-VxLDL
Two-week-old monocyte-macrophage or passaged skin fibroblast cultures were incubated for 5 h with 20 g/ml 125 I-lipoproteins as indicated. Lipoproteins were added to 1 ml of RPMI 1640 medium containing 0.2% fatty acid-free bovine serum albumin for monocytemacrophage cultures and to 1 ml of DMEM medium (also containing albumin) for fibroblast cultures. After incubations, the amount of TCAsoluble organic 125 I in media was measured to determine degraded 125 I-lipoprotein. Cultured cells were harvested, and the amount of cellassociated 125 I-lipoprotein was determined. In Experiment II, monocyte-macrophages were incubated in RPMI 1640 medium containing 10% human serum without or with 50 g/ml AcLDL for 2 days (to cholesterol-enrich the macrophages) before incubations with 125 I-Vx-LDL.

Condition
Cell-associated 125 I-lipoprotein  poproteins can actively accumulate within SCC. Previously, uptake of aggregated lipoproteins has been attributed to the process of phagocytosis. This was because uptake of aggregated lipoproteins was inhibited by cytochalasins and phagocytosis is an actin-dependent process that is inhibitable by cytochalasins (20). Actin dependence does not necessarily mean that phagocytosis is the uptake pathway. This is because, besides phagocytosis, other endocytotic pathways of macrophages such as sequestration into SCC and macropinocytosis are also actin-dependent (1,24). Electron microscopy was not used in many previous studies to confirm that aggregated lipoproteins entered macrophages by direct phagocytosis. Not only did cytochalasin D inhibit uptake of VxLDL into SCC, cytochalasin D also inhibited subsequent degradation of SCCbound VxLDL. Thus, it remains possible that VxLDL first entered SCC but then was transported from SCC into the cell cytoplasm by phagocytosis. However, we were not able to visualize by electron microscopy transport of VxLDL out of SCC, possibly because this process was slow and limited.
Uptake into SCC is a process different from phagocytosis. Phagocytosis involves uptake of material into vacuoles that form from pinched-off regions of the plasma membrane (2). SCC form in part through invaginations of the plasma membrane that do not pinch off from the plasma membrane (1). The formed SCC can be delineated with the electron-dense stain ruthenium red that distinguishes intra-from extracellular membranes by staining only the latter. Hoff et al. (25,26) showed rapid degradation of aggregates of 4-hydroxynonenalmodified LDL taken up by mouse peritoneal macrophages through phagocytosis (verified by electron microscopy). On the other hand, these investigators reported that VxLDL showed delayed processing (i.e. degradation) by macrophages (Ref. 27; also shown in Ref. 23). The delayed processing of VxLDL was not due to a diminished capacity of lysosomal enzymes to degrade VxLDL (also shown in Ref. 28). They speculated that poor processing of VxLDL could have been due to the nature of the intracellular pathway taken by the VxLDL. Uptake of VxLDL into SCC is an intracellular pathway showing slow degradation that could explain these earlier findings.
Human monocyte-macrophages are capable of directly phagocytosing 0.5-3-m latex beads. 2 So what property of Vx-LDL targets this aggregate to SCC rather than to phagocytic vacuoles? VxLDL is multivalent with respect to its apolipoprotein B component, and valency of a ligand may target receptors to different endocytosis pathways. Tabas and co-workers (29, 30) described a unique endocytotic pathway for ␤ very low density lipoproteins (VLDL) in mouse peritoneal macrophages in which multi-valency of ligand was a factor determining the route of endocytosis. In this pathway, large ␤-VLDL enter mouse peritoneal macrophages through surface-connected tubules. Surface-connected tubules are plasma membrane invaginations that do not terminate in a labyrinth of interconnected compartments as do SCC. In contrast to the uptake of VxLDL into SCC in the present study, uptake of ␤-VLDL into surfaceconnected tubules is not inhibited by cytochalasin D. Also, uptake into these tubules occurs through the LDL receptor that binds the multivalent apolipoprotein E component of ␤-VLDL (30). Thus, uptake of ␤-VLDL by mouse peritoneal macrophages through the surface-connected tubule pathway and uptake of VxLDL by human monocyte-macrophages through the SCC pathway appear to be different but possibly related endocytic pathways.
It is possible that the specific macrophage uptake pathway taken by particles depends on the nature of the receptor that binds the particles. The receptor that mediated cell association of VxLDL was not the classical LDL receptor. Methylation of LDL, which prevents binding of LDL to the LDL receptor (21), did not block macrophage cell association of methylated 125 I-VxLDL, cholesterol accumulation by macrophages incubated with methylated VxLDL, or accumulation of methylated Vx-LDL within SCC. Similar to what Khoo et al. (11) found with mouse peritoneal macrophages, we observed that the LDL receptor did mediate much of the degradation of 125 I-VxLDL. Macrophage degradation of 125 I-VxLDL was partially inhibited by methylation of LDL and by macrophage cholesterol enrichment, a treatment previously shown to down-regulate the LDL receptor in macrophages (22). The results suggest that whereas uptake into SCC occurs independently of the LDL receptor, subsequent movement from SCC to lysosomes may depend in part on LDL receptor function.
It is conceivable that vortexing of LDL reveals a binding domain in LDL that is normally masked. Such a domain could then mediate LDL binding to some other receptor capable of uptake into SCC. In this regard, Gianturco et al. (31,32) have shown the existence of a macrophage receptor that recognizes a binding domain within apolipoprotein B-48 but which is not expressed by native LDL. Alternatively, vortexing of LDL could reveal a cryptic lipid ligand. In this regard, uptake of oxidized LDL appears to be mediated in part by a lipid moiety of the oxidized LDL (33). Trypsin treatment of the VxLDL in our study did prevent its uptake by monocyte-macrophages. 2 However, trypsin treatment also reversed the aggregation of Vx-LDL. Thus, it was not clear whether loss of a protein ligand or loss of multi-valency of aggregated VxLDL was responsible for the lack of macrophage uptake of the trypsin-treated VxLDL.
Uptake into SCC is a pathway that leads to cellular accumulation of at least two different types of aggregated LDL (e.g. vortexed and phospholipase C-treated LDL). In preliminary experiments, we have observed macrophage uptake into SCC for some lots of oxidized LDL, another form of LDL that can aggregate (27). Considering the role of SCC in the uptake of aggregated lipoproteins will be important in future studies. Furthermore, uptake of aggregated LDL by the non-LDL receptor pathway shown in this study could be important in cellular lipoprotein uptake in atherosclerotic lesions where activity of the classical LDL receptor is down-regulated (34).