Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.

Previous studies have shown that cholesterol in atherosclerotic plaques is present in both intracellular and extracellular forms. In the current study, we investigated a mechanism for extracellular cholesterol accumulation and examined the capacity of this pool of cholesterol to be removed by cholesterol acceptors, a step in reverse cholesterol transport. Human monocyte-derived macrophages differentiated with macrophage-colony stimulating factor were incubated with acetylated LDL to allow cholesterol enrichment and processing. These macrophages were subsequently labeled with a monoclonal antibody that specifically detects ordered cholesterol arrays, revealing the presence of unesterified cholesterol-rich microdomains on the cell surfaces and in the extracellular matrix. Similar unesterified cholesterol-rich microdomains were present in human atherosclerotic plaques. Actin microfilaments functioned in microdomain deposition or maintenance, and Src family kinases regulated transfer of these microdomains from the cell surface onto the extracellular matrix. Mediators of reverse cholesterol transport, apolipoprotein A-I (apoA-I), and HDL were capable of removing these extracellular un-esterified cholesterol-rich microdomains. However, apoA-I removed the microdomains only when macrophages were present. ApoA-I removal of microdomains was blocked by glyburide and inhibitor of ATP-binding cassette transporter A1 (ABCA1) function. In summary, cultures of cholesterol-enriched human monocyte-derived macrophages generate extracellular unesterified cholesterol-rich microdomains, which can subsequently be removed by cholesterol acceptors and therefore potentially function in reverse cholesterol transport.


Culture of human monocyte-derived macrophages
Mononuclear cells were obtained from human donors by monocytopheresis and subsequently purifi ed using counterfl ow centrifugal elutriation as previously described ( 17 ). Monocytopheresis was carried out under a human subjects research protocol approved by a National Institutes of Health institutional review board. A different human donor was used for each experiment. The resulting elutriated human monocytes were diluted in RPMI 1640 medium containing 10% FBS and seeded onto 12-well CellBIND plates at a density of 2 × 10 5 cells/cm 2 . Cultures were incubated in a 37°C cell culture incubator with 5% CO 2 /95% air for 2 h and subsequently rinsed three times with RPMI 1640 to remove nonadherent cells. The remaining adherent cells were then differentiated with 50 ng/ml M-CSF and 25 ng/ml IL-10 in RPMI 1640 (hereafter referred to as "media") plus 10% FBS. IL-10 was included because it enhances the growth and differentiation of human monocytes cultured in M-CSF and creates a more homogenous macrophage phenotype ( 18 ). Cultures were washed and replaced with fresh media on day 6. The resulting monocyte-derived macrophages were used for experiments on day 7.

Immunostaining of cultured human monocyte-derived macrophages
Fixation, immunostaining, and microscopy were all performed with macrophages in their original CellBIND culture plates, and all steps were carried out at room temperature. Oneweek-old monocyte-derived macrophage cultures were rinsed three times in DPBS, fi xed for 10 min with 4% paraformaldehyde in DPBS, and then rinsed an additional three times in DPBS. Cells were then incubated 60 min with 5 µg/ml mouse anti-cholesterol microdomain MAb 58B1 IgM diluted in DPBS containing 0.1% BSA. Control staining was performed with an irrelevant purifi ed mouse anti-Clavibacter michiganense MAb (clone 9A1) IgM diluted in DPBS containing 0.1% BSA. Cultures were then rinsed three times (5 min each) in DPBS, followed by a 30-min incubation in 5 g/ml biotinylated goat anti-mouse IgM diluted in DPBS containing 0.1% BSA. After three rinses in DPBS (5 min each), cultures were incubated 30 min with 20 g/ml fl uorescein-avidin in 0.1 M sodium bicarbonate buffer with 0.15 M NaCl added (fi nal pH 8.2). Cultures were then rinsed three times with DPBS and mounted in Vectashield mounting medium with DAPI nuclear stain in preparation for digital imaging using an Olympus IX81 fl uorescence microscope. to plasma lipoproteins than they are to cellular lipid droplets ( 11 ), suggesting that extracellular lipid is not simply the released contents of dying macrophages. Data from these studies contribute to an alternative theory that extracellular cholesterol is a product of LDL that has been deposited within the extracellular matrix, taken up by cells, processed, and resecreted in the form of liposomes enriched in unesterifi ed cholesterol ( 12 ). It may be that both of these theories are correct and that multiple mechanisms contribute to the accumulation of extracellular cholesterol, underscoring the complexity of cholesterol deposition in atherosclerosis.
In this study, we sought to further investigate the theory that the extracellular lipid particles found in atherosclerotic lesions represent plasma lipoprotein that has been taken up by macrophages, processed, and effl uxed. Unesterifi ed cholesterol has previously been detected in atherosclerotic lesions using fi lipin, a fl uorescent polyene antibiotic that binds to the 3 ␤ -hydroxyl unit of single unesterifi ed cholesterol molecules ( 13 ). Because cholesterol is ubiquitously present in cell membranes, fi lipin cannot distinguish between the pool of cholesterol found in all cells, that which may be uniquely involved in reverse cholesterol transport or that which may represent pathologic accumulation. To address this diffi culty, we utilized a monoclonal antibody (MAb), 58B1, raised against cholesterol monohydrate crystals. This antibody has previously been reported to label ordered arrays of approximately 12 molecules of unesterifi ed cholesterol without recognizing individual cholesterol molecules ( 14,15 ) and has been successfully used to label cell surface microdomains of unesterifi ed cholesterol in human monocyte-derived macrophages differentiated using human serum. These macrophages are similar to monocyte-derived macrophages differentiated with FBS with added granulocyte macrophage-colony stimulating factor (GM-CSF) ( 16 ). Previous studies have shown that monocytes differentiated using FBS with added macrophage-colony stimulating factor (M-CSF) produce macrophages in culture that are more similar in shape and cell surface marker expression to those that predominate in atherosclerotic lesions than monocytes differentiated using FBS with added GM-CSF ( 16 ). We therefore conducted our experiments using cultures of these M-CSF-differentiated human monocytederived macrophages to more closely model cholesterol processing in vivo. Using these experimental tools, we tested the hypothesis that cholesterol-enriched macrophages produce extracellular accumulations of unesterifi ed cholesterol and also examined whether cholesterol acceptors can remove this pool of cholesterol, a step in reverse cholesterol transport.

Antibody labeling of extracellular unesterifi ed cholesterol-rich microdomains in cell culture
We incubated macrophage cell cultures with AcLDL (50 g/ml) for 1 day to enrich them with cholesterol. Serum was not included in the culture media at this point to prevent the introduction of potential cholesterol acceptors to our experimental system. Generation of extracellular unesterifi ed cholesterol-rich microdomains was then confi rmed using MAb 58B1. Fluorescence microscopy revealed antibody labeling of unesterifi ed cholesterol-rich microdomains on macrophage cell surfaces as well as in the extracellular spaces between cells (arrows in Fig. 1A, B ). The extracellular unesterifi ed cholesterol-rich microdomains appeared as spherical microparticles. The unesterifi ed cholesterol-rich microdomains did not represent AcLDL that had simply adhered to the culture plate, as slides coated with AcLDL did not label with MAb 58B1. Cultures incubated with AcLDL for 2 days ( Fig. 1B ) produced more extracellular unesterifi ed cholesterol-rich microdomain labeling than cultures incubated for only 1 day ( Fig. 1A ), suggesting that the generation of these micro-

Microscopic analysis
Cells were identifi ed using phase contrast microscopy or by locating DAPI-stained nuclei. The pattern and intensity of MAb 58B1 staining were then analyzed for cultures from each experimental parameter, and these data were compared with one another. MAb 58B1-labeling was considered cellular if it was located within cell membrane boundaries, as identifi ed on the corresponding phase contrast view. Labeling was considered extracellular if it was located outside the cell membrane boundaries seen on phase contrast view. Different planes of focus were visualized before acquiring images to confi rm that only a monolayer of cells was present, thereby ensuring that labeling seen outside cell membrane boundaries did not represent cellular labeling from cells lying in a different plane of focus.

Quantifi cation of cholesterol from macrophages
After incubations, human monocyte-derived macrophage cultures were rinsed three times in DPBS. Macrophages were then harvested from wells by scraping into 1 ml distilled water. Lipid was extracted from the resulting cell suspension using the Folch method ( 19 ), and quantities of esterifi ed and unesterifi ed cholesterol were determined using the method previously described by Gamble et al. ( 20 ). Protein quantifi cation was performed on an aliquot of cell lysate using the Lowry method ( 21 ) with BSA as a standard. Data are represented as means ± SEM. Means were determined from three culture wells for each data point, and statistical comparisons of means were made using the Student's t -test (unpaired). A P -value of р 0.05 was considered signifi cant.

Staining of human aortic tissue sections
Human aortic tissue was obtained from autopsy of a 49-year-old individual who died of myocardial infarction (National Disease Research Interchange, Philadelphia, PA). The aortic tissue was snap frozen in liquid nitrogen and embedded in OCT media. Then 10 m frozen sections were prepared from regions with and without lipid deposits visible on gross inspection, placed onto gelatin-coated slides (SuperFrost Plus), and then stored at Ϫ 80°C until further processing.
For oil-red O staining, frozen tissue sections were thawed at room temperature and rehydrated with distilled water. Sections were fi xed for 10 min with 1.6% paraformaldehyde in DPBS followed by a single rinse in DPBS. Samples were then treated with 60% isopropyl alcohol for 2 min, stained for 20 min with 0.5% oil-red O, followed by an additional treatment with 60% isopropyl alcohol for 1 min. Sections were washed twice with distilled water and then treated with Mayer's hematoxylin for 5 min to stain nuclei blue. Following a 5 min rinse in distilled water, sections were mounted in glycerol-gelatin in preparation for imaging. All steps were carried out at room temperature.
For fl uorescence immunostaining, separate frozen tissue sections adjacent to sections stained with oil-red-O were thawed at room temperature and rehydrated with distilled water. Sections were then fi xed for 30 min with 4% paraformaldehyde in DPBS. After three rinses in DPBS, sections were blocked for 10 min with human FcR blocking reagent and for 15 min with avidin and biotin blocking solutions. Sections were then immunostained using the mouse anti-cholesterol array IgM MAb 58B1 or the irrelevant control antibody, purifi ed mouse anti-Clavibacter michiganense IgM MAb (clone 9A1), according to the protocol described above for cultured human monocyte-derived macrophages. All steps were carried out at room temperature, and digital imaging was performed using an Olympus IX81 fl uorescence microscope.

Fig. 1.
Incubation of human monocyte-derived macrophage cultures with AcLDL results in the generation of extracellular unesterifi ed cholesterol-rich microdomains. Human monocyte-derived macrophages differentiated in the presence of M-CSF for 1 week were incubated with AcLDL (50 g/ml) for 1 (A) or 2 days (B) or without AcLDL for 1 day (C). Staining with the anti-unesterifi ed cholesterol array Mab 58B1 (green in A-C) revealed extracellular unesterifi ed cholesterol-rich microdomains (arrows) in cultures incubated with AcLDL but not in cultures incubated without AcLDL. Cells were visualized using phase contrast microscopy (gray) and nuclei were stained using DAPI (blue). Control staining using an irrelevant Mab anti-Clavibacter michiganense clone 9A1 (green in D), following 1 day incubation with AcLDL revealed no staining. Bar = 25 m and applies to all.
Cultures incubated for 1 day with 50 g/ml AcLDL followed by incubation for a second day in media with 5 M nocodazole were stained with MAb 58B1. We observed that treatment with nocodazole, in contrast to treatment with cytochalasin D, did not reduce extracellular unesterifi ed cholesterol-rich microdomain labeling ( Fig. 3E, F ). Thus, microtubules do not contribute to the deposition of these microdomains onto the extracellular matrix.

Function of Src family kinase signaling pathway in generation of extracellular unesterifi ed cholesterol-rich microdomains
Members of the Src family of tyrosine kinases have been shown to function in the regulation of the actin cytoskeleton (25)(26)(27), and at least six are expressed in human monocyte-derived macrophages ( 28 ). We therefore hypothesized that the Src family kinase-signaling cascade could be involved in the regulation of extracellular unesterifi ed cholesterol deposition. SU6656 is a compound that has been shown to inhibit Src family kinase activity ( 29 ). Human monocyte-derived macrophage cultures were pretreated without or with 20 M SU6656 for 1 h. Cultures were then incubated for 2 days with 50 g/ml AcLDL without or with 20 M SU6656, depending on the pretreatment condition, domains occurs continuously from cholesterol-enriched macrophages. Labeling with MAb 58B1 was not detected in cultures incubated without AcLDL ( Fig. 1C ), and cultures incubated with AcLDL but stained with an irrelevant purifi ed mouse anti-Clavibacter michiganense antibody (MAb clone 9A1) similarly did not produce observable labeling ( Fig. 1D ).

Antibody labeling of unesterifi ed cholesterol-rich microdomains in human aortic tissue sections
We next sought to determine if the extracellular unesterifi ed cholesterol-rich microdomains identifi ed in cholesterol-enriched macrophage cultures are also present in vivo. Aortic tissue sections were obtained from the autopsy specimen of an individual who died of myocardial infarction, and adjacent sections from various regions of the aorta were stained with oil-red O, which labels esterifi ed cholesterol-containing oil-phase lipid droplets, or MAb 58B1, which labels only unesterifi ed cholesterol. The resulting patterns of labeling were compared to determine if there was a correlation between the prevalence of lipidenriched cells such as macrophage foam cells, as evidenced by staining with oil-red O, and the distribution of unesterifi ed cholesterol-rich microdomains, as evidenced by staining with MAb 58B1. Consistent with our hypothesis that foam cells produce these microdomains, regions that contained oil-red O-labeled foam cells also labeled with the anticholesterol microdomain antibody ( Fig. 2A, B ), whereas regions that lacked oil-red O-labeled foam cells (but showed only fi ne extracellular oil-red O staining previously attributed to extracellular deposits of LDL) ( 22 ) also lacked unesterifi ed cholesterol-rich microdomains ( Fig. 2C, D ).

Function of cytoskeleton in generation of extracellular unesterifi ed cholesterol-rich microdomains
We next sought to manipulate the cell cytoskeleton to determine if doing so would interfere with the production of extracellular unesterifi ed cholesterol-rich microdomains. Cytochalasin D has been shown to inhibit actin polymerization, thereby disrupting the microfi lament cytoskeleton ( 23 ). We incubated human monocyte-derived macrophage cultures with AcLDL (50 g/ml) for 1 day to allow cholesterol enrichment and processing. Cultures were then washed and incubated for a second day with media alone or media plus 4 g/ml cytochalasin D and then stained with MAb 58B1 to visualize unesterifi ed cholesterolrich microdomains. In cultures incubated with AcLDL followed by media alone, punctate labeling was visualized in the extracellular space, confi rming the presence of extracellular unesterifi ed cholesterol-rich microdomains ( Fig. 3A, B ). In contrast, cultures incubated with AcLDL followed by media plus cytochalasin D produced no labeling when stained with MAb 58B1 ( Fig. 3C, D ). This fi nding suggests an important function for actin microfi laments in either the deposition or maintenance of unesterifi ed cholesterol-rich microdomains in the extracellular matrix.
We similarly studied the effect of nocodazole, an inhibitor of microtubule formation ( 24 ), on the deposition of extracellular unesterifi ed cholesterol-rich microdomains. without or with 20 M SU6656 and then incubated for 24 h with 25 g/ml AcLDL without or with 20 M SU6656 depending on the pretreatment condition. Total cholesterol content in both treatment groups increased significantly above baseline ( P = 0.003) but did not differ signifi cantly between treatment conditions; cultures incubated with AcLDL alone contained an average of 279 ± 12 (mean ± SEM) nmol cholesterol/mg protein compared with cultures incubated with AcLDL and SU6656, which contained 305 ± 12 nmol/mg protein ( P = 0.193) ( Fig. 5 ).

Extracellular unesterifi ed cholesterol-rich microdomains can function in reverse cholesterol transport
Although we have shown that cholesterol-enriched macrophages can generate extracellular unesterifi ed cholesterol-rich microdomains, the function of these microdomains remains unclear. It has previously been suggested that extracellular accumulations of unesterifi ed and subsequently stained with MAb 58B1. Cultures incubated without SU6656 demonstrated labeling of unesterifi ed cholesterol-rich microdomains on cell surfaces and in the extracellular matrix ( Fig. 4A-C ), consistent with results above. Cultures incubated with SU6656, however, showed no extracellular labeling with MAb 58B1 and a concomitant increase in cell surface labeling ( Fig. 4D-F ) when compared with cultures incubated without SU6656.
To ensure that these results refl ect a regulatory function for Src family kinases in cholesterol processing rather than cholesterol uptake, we performed a quantifi cation assay to determine if SU6656 affects cholesterol enrichment. At baseline, human monocyte-derived macrophage cultures contained an average of 83 ± 0 (mean ± SEM) nmol cholesterol/mg protein. Cultures were then pretreated for 1 h Fig. 3. Actin microfi laments are involved in the deposition or retention of unesterifi ed cholesterol-rich microdomains in the extracellular matrix. One-week-old macrophage cultures were incubated for 1 day with AcLDL (50 g/ml), followed by incubation for a second day in media with no addition (A and B), media plus 4 g/ ml cytochalasin D to inhibit actin polymerization (C and D), or media plus 5 M nocodazole to inhibit microtubule formation (E and F). Unesterifi ed cholesterol-rich microdomains were labeled with MAb 58B1 (green), and cells were visualized using phase contrast microscopy (gray) and DAPI nuclear stain (blue). Images are also shown with MAb 58B1 staining alone (B, D, and F) for clarity. Bar = 25 m and applies to all. with MAb 58B1 (green), and cells were visualized using phase contrast microscopy (gray) and DAPI nuclear stain (blue). Phase contrast images (B and E) and unesterifi ed cholesterol-rich microdomain staining (C and F) are also shown separately for each condition for clarity. Bar = 25 m and applies to all. AcLDL on the fi rst day and no cholesterol acceptor on the second day contained an average of 78 ± 2 nmol unesterifi ed cholesterol and 2 ± 1 nmol esterifi ed cholesterol/mg protein. Incubation with AcLDL for 1 day produced cultures with an average of 285 ± 11 nmol unesterifi ed cholesterol and 178 ± 8 nmol esterifi ed cholesterol/mg protein, indicating that cholesterol enrichment results in the average net accumulation of 207 nmol unesterifi ed cholesterol and 176 nmol esterifi ed cholesterol/mg protein. Treatment with HDL reduced unesterifi ed cholesterol content by 34% ( P = 0.001) to 188 ± 6 nmol/mg protein, whereas esterifi ed cholesterol levels remained unchanged at 176 ± 6 nmol/mg protein ( P = 0.825). Similarly, treatment with apoA-I reduced unesterifi ed cholesterol by 42% ( P = 0.002) to 165 ± 11 nmol/mg protein, but esterifi ed cholesterol levels remained unchanged at 153 ± 10 nmol/mg protein ( P = 0.116). Although the cholesterol quantifi cation assay cannot distinguish between unesterifi ed cholesterol within cells, on the cell surface, or in the extracellular matrix, this data is consistent with the results of the MAb 58B1 immunostaining showing that unesterifi ed cholesterol-rich microdomains can be removed by the cholesterol acceptors, HDL and apoA-I.

Removal of extracellular unesterifi ed cholesterol microdomains by apoA-I requires the presence of cells whereas removal by HDL does not
We next sought to determine if the removal of unesterifi ed cholesterol-rich microdomains from the extracellular space requires cellular function. Human monocytederived macrophage cultures were incubated with 50 g/ ml AcLDL for 1 day and then subjected to three freezethaw cycles ( Ϫ 80°C/37°C) to lyse and remove cells. The extracellular matrix left behind was then incubated with or without a cholesterol acceptor (apoA-I or HDL at 50 g/ ml) for a second day and stained with MAb 58B1. Removal of cells was confi rmed using phase contrast microscopy.
Cholesterol-enriched cultures that were freeze-thawed and then incubated without a cholesterol acceptor ( Fig. 7A, B ) or with apoA-I ( Fig. 7C, D ) retained extracellular microdomain labeling, confi rming that the freeze-thaw procedure in and of itself does not eliminate these microdomains and also showing that apoA-I requires the presence of cells to mediate removal of extracellular unesterifi ed cholesterolrich microdomains. In contrast, cholesterol-enriched cultures that were freeze-thawed and then treated with HDL produced no labeling when stained with MAb 58B1 ( Fig.  7E, F ). These results suggest that HDL, but not apoA-I, can remove extracellular unesterifi ed cholesterol-rich microdomains in the absence of cells.

Removal of extracellular unesterifi ed cholesterol-rich microdomains by apoA-I is inhibited by glyburide
Previous studies have shown that apoA-I must be phosholipidated through the action of the ATP-binding cassette protein, ABCA1, before it can act as a cholesterol acceptor ( 32 ). We therefore employed glyburide, an inhibitor of ABCA1 ( 32 ), to determine if the requirement of cells for apoA-I mediated removal of extracellular unesterifi ed cholesterol-rich microdomains can be attributed cholesterol-rich microdomains may be constituents of a reverse cholesterol transport pathway ( 12 ). We therefore hypothesized that the microdomains identifi ed using MAb 58B1 are capable of being removed by HDL and apoA-I, cholesterol acceptors known to function in reverse cholesterol transport. ApoA-I is an indirect cholesterol acceptor in that it mobilizes cholesterol only after the apoA-I complexes with phospholipid, the agent that binds cholesterol both in phospholipidated apoA-I and HDL ( 30,31 ). Human monocyte-derived macrophage cultures were incubated with 50 g/ml AcLDL for 1 day to allow cholesterol enrichment and generation of extracellular microdomains. Cultures were then washed and incubated in media without or with a cholesterol acceptor (HDL or apoA-I at 50 g/ml) for a second day. Additionally, these results were compared with cultures incubated without AcLDL on the fi rst day and without a cholesterol acceptor on the second day, which produced no labeling when stained with MAb 58B1 ( Fig. 6A, B ). Cholesterol-enriched macrophage cultures that were not treated with a cholesterol acceptor-produced cell surface and extracellular microdomain labeling with MAb 58B1 ( Fig. 6C, D ), consistent with results presented above. In contrast, cholesterol-enriched macrophages that were treated with the cholesterol acceptors, HDL ( Fig. 6E, F ) or apoA-I ( Fig. 6G, H ), showed reduced or no labeling with MAb 58B1 both on cell surfaces and in the extracellular matrix, suggesting that these cholesterol acceptors can indeed remove these unesterifi ed cholesterol-rich microdomains. We also observed that treatment of cholesterol-enriched macrophages with BSA did not reduce MAb 58B1-labeling (data not shown). This supports the idea that the capacity to remove these unesterifi ed cholesterol-rich microdomains is a feature unique to known mediators of the reverse cholesterol transport system.
To evaluate the effi cacy of cholesterol removal in this system, we also performed a cholesterol quantifi cation assay on cultures processed in the same conditions as described above ( Fig. 6, graph). Cultures that received no

DISCUSSION
In this study, we confi rmed our hypothesis that human monocyte-derived macrophages differentiated in the presence of M-CSF generate extracellular unesterifi ed cholesterol-rich microdomains when incubated with AcLDL. These microdomains were identifi ed using a Mab that labels ordered arrays of unesterifi ed cholesterol, MAb 58B1, and were capable of being removed by cholesterol acceptors known to function in reverse cholesterol transport.
In a previous study, experiments using MAb 58B1 were conducted using cholesterol-enriched cultures of monocyte-derived macrophages differentiated with human serum ( 15 ). These macrophages have a rounded "fried-egg" appearance and require inhibition of Acyl-CoA:cholesterol acyltransferase (ACAT), which blocks esterifi cation of cellular cholesterol, to induce the formation of plasma membrane unesterifi ed cholesterol-rich microdomains that could be visualized using the antibody. Although extracellular unesterifi ed cholesterol-rich microdomains were visualized in this earlier study, these extracellular domains were observed in association with only 10-20% of macrophages and depended on inhibition of ACAT activity to the need for phospholipidation by ABCA1 or if other cell functions are also necessary. Human monocytederived macrophage cultures were incubated with 50 g/ml AcLDL for 1 day to allow cholesterol enrichment and deposition of extracellular cholesterol-rich microdomains. Cultures were then washed and pretreated in media with glyburide (100 M) for 30 min followed by incubation for a second day with 100 M glyburide alone or 50 g/ml apoA-I plus 100 M glyburide. A third set of cultures was pretreated for 30 min in media without glyburide and subsequently incubated for a second day with 50 g/ml apoA-I alone. Cells were then stained with MAb 58B1 to determine the effect of glyburide on the capacity of apoA-I to remove extracellular unesterifi ed cholesterol-rich microdomains. As shown above, no extracellular cholesterolrich microdomain labeling was observed in cultures that received apoA-I alone ( Fig. 8A, B ). In contrast, cultures that received glyburide alone maintained extracellular unesterifi ed cholesterol labeling ( Fig. 8C, D ), confi rming that glyburide itself does not eliminate these microdomains. Cholesterol-enriched cultures treated with both apoA-I and glyburide ( Fig. 8E, F ) produced a MAb 58B1 labeling pattern and intensity that resembled cultures receiving glyburide alone. This result suggests that apoA-I mediated removal of extracellular microdomains is dependent on ABCA1 function, consistent with the known Fig. 6. Unesterifi ed cholesterol-rich microdomains can be removed by cholesterol acceptors. One-weekold macrophage cultures were incubated with media alone for 2 days (A and B) or incubated for 1 day with AcLDL (50 g/ml) followed by incubation for a second day with media alone (C and D), media plus 50 g/ ml HDL (E and F), or media plus 50 g/ml apoA-I (G and H). Cultures were stained for unesterifi ed cholesterol-rich microdomains using MAb 58B1 (green), and cells were visualized using phase contrast microscopy (gray) and DAPI nuclear stain (blue). Images are also shown with MAb 58B1 staining alone (B, D, F, and H) for clarity. Bar = 25 m and applies to all. Quantities of unesterifi ed and esterifi ed cholesterol were also determined, and the results are plotted as the mean ± SEM of triplicate cultures (graph). Statistical signifi cance ( P р 0.05) was determined using the Student's t -test (unpaired). macrophages may represent an important pathway for cholesterol effl ux. Because M-CSF-differentiated macrophages share a similar elongated phenotype and cell surface marker expression pattern to a subtype of macrophages found to predominate in atherosclerotic lesions ( 16 ), the cholesterol effl ux pathway studied here may be the same pathway utilized by macrophage foam cells in atherosclerotic lesions in vivo. Consistent with this idea, we observed that regions of human aortic tissue that were identifi ed by oil-red O staining to be enriched with cholesterol-enriched cells such as macrophage foam cells were also enriched with unesterifi ed cholesterol-rich microdomains that labeled with MAb 58B1. On the other hand, regions of aortic tissue that were relatively devoid of dis- ( 15 ). In the current study, we used cholesterol-enriched human monocyte-derived macrophages cultures differentiated in the presence of M-CSF and were able to visualize MAb 58B1-labeled cell surface and extracellular cholesterol-rich microdomains in association with almost all macrophages, without the need for ACAT inhibition. This may have been because the M-CSF-differentiated macrophages accumulated substantially more unesterifi ed cholesterol compared with the human serum-differentiated macrophages.
Because we found that these unesterifi ed cholesterolrich microdomains could be removed by mediators of the reverse cholesterol transport system, the cholesterolprocessing pathway utilized by M-CSF-differentiated Fig. 7. Removal of extracellular unesterifi ed cholesterol-rich microdomains by apoA-I depends on the presence of cells, whereas removal by HDL does not. One-week-old macrophage cultures were incubated with AcLDL (50 g/ml) for 1 day to allow generation of extracellular unesterifi ed cholesterol-rich microdomains. Cultures were then subjected to three freeze-thaw cycles to lyse and remove cells, and the remaining extracellular matrix was incubated for 1 day with media alone (A and B), media with 50 g/ml apoA-I (C and D), or media with 50 g/ml HDL (E and F). Cultures were imaged using phase contrast microscopy (A, C, and E) to confi rm removal of cells, and extracellular unesterifi ed cholesterol-rich microdomains were labeled with MAb 58B1 (B, D, and F). Bar = 25 m and applies to all. Fig. 8. ApoA-I mediated removal of extracellular unesterifi ed cholesterol-rich microdomains is blocked by the ABCA1 inhibitor glyburide. One-week-old macrophage cultures were incubated with AcLDL (50 g/ml) for 1 day to allow generation of extracellular unesterifi ed cholesterol-rich microdomains. Cultures were then pretreated for 30 min without or with glyburide (100 M) to inhibit ABCA1 function before incubation for a second day with 50 g/ml apoA-I (A and B), 100 M glyburide (C and D), or both apoA-1 and glyburide (E and F). Unesterifi ed cholesterol-rich microdomains were labeled with MAb 58B1 (green), and cells were visualized using phase contrast microscopy (gray) and DAPI nuclear stain (blue). Images are also shown with MAb 58B1 staining alone (B, D, and F) for clarity. Bar = 25 m and applies to all. family kinase activity. Src family kinases are known to be involved in the regulation of the actin cytoskeleton (25)(26)(27), and thus, the effects of SU6656 on cholesterol processing may be partially explained by disruption of actin function. Indeed, cholesterol-enriched macrophages that were treated with SU6656 and those that were treated with cytochalasin D develop the same stellate morphology that distinguishes them from cholesterol-enriched macrophages that do not receive treatment. However, treatment with SU6656 and treatment with cytochalasin D showed different effects on development of cholesterolrich microdomains. Plasma membrane cholesterol-rich domains developed with SU6656 but were absent with cytochalasin D treatment. This suggests that SU6656 and cytochalasin D have different effects on cholesterol processing. It is possible that inhibition of Src family kinase activity does not affect the actin cytoskeleton by causing microfi lament depolymerization or that either SU6656 or cytochalasin D have other properties besides their effects on the actin cytoskeleton. For example, Src family kinases are known to be involved in the regulation of other cytoskeletal structures, including microtubules ( 35,36 ). We investigated the effect of disrupting tubulin polymerization using nocodazole, however, and found that it did not eliminate extracellular labeling or increase cell surface labeling of unesterifi ed cholesterol-rich microdomains when cultures were stained with MAb 58B1. The specifi c mechanism by which Src family kinases regulate cholesterol processing in M-CSF-differentiated macrophages therefore remains uncertain.
In addition to studying the processes behind the generation of extracellular cholesterol-rich microdomains, we also evaluated the potential for these microdomains to function in reverse cholesterol transport. Our results show that the cholesterol acceptors, apoA-I and HDL, can function in mobilization of unesterifi ed cholesterolrich microdomains from the extracellular matrix. Cholesterol quantifi cation assays revealed that treatment with apoA-I or HDL resulted in a signifi cant reduction in the amount of unesterifi ed but not esterifi ed cholesterol in cholesterol-enriched cultures. Cholesterol within macrophages has been shown to shift between esterifi ed and unesterifi ed forms due to its continuous hydrolysis and reesterifi cation ( 37 ). Thus, cholesterol removal from cells might be expected to reduce the cellular content of both esterifi ed and unesterifi ed cholesterol. The extracellular cholesterol microdomains identifi ed using MAb 58B1, however, may not undergo continuous hydrolysis and reesterifi cation. The ready mobilization of these extracellular deposits could therefore account for the isolated reduction in unesterifi ed cholesterol content in cholesterol-enriched cultures treated with a cholesterol acceptor.
We also found that apoA-I-mediated removal of extracellular unesterifi ed cholesterol requires the presence of cells, and in particular, the function of ABCA1 as suggested by glyburide inhibition of cholesterol removal. This result is consistent with previous studies regarding the mechanism of apoA-I-mediated removal of unesterifi ed ease and foam cells showed only minimal labeling with MAb 58B1.
Actin microfi laments were found to be necessary to maintain unesterifi ed cholesterol-rich microdomains in the extracellular matrix. Because extracellular staining with MAb 58B1 is observed in cell cultures incubated with AcLDL for 1 day, we did not anticipate the complete lack of extracellular microdomain labeling observed in cultures incubated with AcLDL for 1 day followed by treatment with cytochalasin D to disrupt actin polymerization. One potential explanation for this fi nding is that the unesterifi ed cholesterol-rich microdomains deposited by cells are constantly fl uxing through the extracellular matrix such that individual cholesterol-rich microdomains are only transiently located in the matrix. In this scenario, macrophages must continuously deposit cholesterol-rich microdomains onto the extracellular matrix for them to accumulate and label with MAb 58B1. We have shown, however, that extracellular microdomain labeling persists for at least 1 day after we remove cholesterol-enriched macrophages from culture plates with repeated freezethaw cycles. This suggests that extracellular microdomains deposited by macrophages can be mobilized by the macrophages either by being taken up by the macrophages or possibly solubilized by a macrophage-derived mediator of cholesterol effl ux. In regards to the latter, human monocyte-derived macrophages produce nascent apolipoprotein E-discoidal HDL particles that are capable of mobilizing cholesterol without addition of exogenous cholesterol acceptors ( 33,34 ). Thus, while deposition of microdomains onto the extracellular matrix may depend on actin microfi laments, it is possible that the reuptake or solubilization of extracellular cholesterol-rich microdomains can occur independently of actin microfi lament function. This proposed mechanism would account for the lack of extracellular microdomain labeling observed in the cytochalasin D experiment (i.e., cytochalasin D blocks deposition) as well as the persistence of extracellular microdomain labeling seen in the freeze-thaw experiment (i.e., macrophages are required to mobilize the deposited cholesterol).
Src family tyrosine kinases functioned in the macrophage deposition of unesterifi ed cholesterol-rich microdomains into the extracellular matrix. Cultures incubated with AcLDL in the presence of the Src family tyrosine kinase inhibitor, SU6656, showed increased unesterifi ed cholesterol-rich microdomain labeling on cell surfaces but no labeling in the extracellular matrix when compared with cultures incubated with AcLDL alone. Nevertheless, macrophage cultures incubated with AcLDL in the presence of SU6656 accumulated the same quantity of total cholesterol as cultures incubated with AcLDL alone. These fi ndings suggest that unesterifi ed cholesterol-rich microdomain deposition occurs through a two-step process in which macrophages fi rst accumulate unesterifi ed cholesterol in the form of cell surface microdomains through a mechanism involving actin microfi laments. These cell surface microdomains are subsequently deposited onto the extracellular matrix through a mechanism requiring Src cholesterol from cell surfaces that have shown that ABCA1 functions in the phospholipidation of apoA-I, transforming it into nascent HDL that can then remove cholesterol ( 32 ). In agreement with this proposed mechanism, HDL was capable of removing extracellular unesterifi ed cholesterol-rich microdomains even in the absence of cells and therefore without the need for phospholipidation by ABCA1, presumably because HDL is already rich in phospholipid.
Burgess et al. ( 38 ) have established the existence of phospholipid-containing sites in the macrophage extracellular matrix that are capable of binding apoA-I. These sites were found to promote cellular cholesterol effl ux through an ABCA1-dependent mechanism. Our cholesterol-rich extracellular sites do not appear to be identical with these phospholipid-rich extracellular sites. The extracellular phospholipid-rich sites underlie cultured macrophages, while the extracellular cholesterol-rich sites we describe occur in the extracellular matrix between macrophages. However, further study is required to determine the origins and possible functional relationships of these extracellular cholesterol and phospholipid lipid-containing sites.
Because unesterifi ed cholesterol-rich cell surface microdomains can function in reverse cholesterol transport, it remains curious as to what purpose the deposition of these microdomains onto the extracellular matrix serves. Our fi nding that extracellular microdomains can function in reverse cholesterol transport in the absence of cells suggests a potential explanation. Excessive accumulation of cholesterol has been shown to be toxic to macrophages in culture ( 8,9 ), consistent with the histological observation that lipid-laden areas of atherosclerotic plaques are often relatively acellular. It is therefore possible that cholesterolenriched macrophages process unesterifi ed cholesterol into an extracellular form to prevent buildup of toxic levels of cholesterol within the macrophage that under certain conditions can lead to macrophage apoptosis. Our other fi nding that extracellular unesterifi ed cholesterolrich microdomains are found within atherosclerotic lesions supports the idea that the reverse cholesterol transport system may not function effi ciently within atherosclerotic plaques.
In summary, we have shown that cholesterol-enriched human monocyte-derived macrophages in culture produce unesterifi ed cholesterol-rich cell surface microdomains. These microdomains transfer to the extracellular matrix through a mechanism regulated by Src family kinases. Both cell surface unesterifi ed cholesterol-rich microdomains and extracellular unesterifi ed cholesterol-rich microdomains are capable of removal by the cholesterol acceptors, apoA-I and HDL, and may therefore function in reverse cholesterol transport. ABCA1-generated cholesterol-containing microparticles released into the medium by cultured cells have been described ( 33,39,40 ). In future studies, it will be of interest to learn whether ABC family transporters function in the generation of the extracellular microparticles showing cholesterol-rich microdomains reported here.