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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:317-323

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:317-323.)
© 1997 American Heart Association, Inc.

Articles

Macrophages Isolated From Human Atherosclerotic Plaques Produce IL-8, and Oxysterols May Have a Regulatory Function for IL-8 Production

Yani Liu; Lillemor Mattsson Hulten; Olov Wiklund

the Wallenberg Laboratory for Cardiovascular Research, Goteborg University, Sweden.

Correspondence to Dr O. Wiklund, Wallenberg Laboratory, Sahlgren's Hospital, S-413 45 Goteborg, Sweden. E-mail wiklund@wlab.wall.gu.se.


*    Abstract
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 *Abstract
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 down arrowResults
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Oxysterols are biologically active molecules generated during oxidation of LDL. Several of these oxysterols were found in macrophage-derived foam cells from human atherosclerotic tissue (eg, 7-hydroxycholesterol, 7-ketocholesterol, 5-epoxycholesterol, and 25-hydroxycholesterol). A specific stimulation of interleukin-8 (IL-8) production by oxidized LDL (oxLDL) has been shown by other investigators. In foam cells from human atherosclerotic tissue, we found high levels of IL-8 (183.1 pg/106 cells) compared with monocytes (23.2 pg/106 cells) or monocyte-derived macrophages in culture (1.5 pg/106 cells). When monocytes and monocyte-derived macrophages, in vitro, were exposed to a series of different oxysterols, we found that all oxysterols tested had a tendency to stimulate IL-8 production but that 25-hydroxycholesterol was the most potent one. This stimulation of IL-8 production was time and dose dependent and could be blocked by cycloheximide. These results indicate that oxysterols in oxLDL may have a regulatory effect on IL-8 production. IL-8, a potent chemoattractant, may play a role in the recruitment of T lymphocytes and smooth muscle cells into the subendothelial space and may contribute to the formation of atherosclerotic lesions.


Key Words: atherosclerosis • foam cells • oxysterols • IL-8 • cell isolation


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Macrophage-derived foam cells are one of the hallmarks of the atherosclerotic lesion. Oxidized low-density lipoprotein (oxLDL) may contribute to lesion formation in many aspects. It has been shown that oxLDL stimulates macrophages to release IL-1 and IL-81 and induces monocyte chemotactic protein-1 production.2 Which component of oxLDL mediates the biological activities is still not well studied. One consequence of LDL oxidation is the formation of oxysterols.3 4 5 Several oxysterols, including 7{alpha}- and 7ß-hydroxycholesterol, 7-ketocholesterol, the {alpha} and ß isomers of cholestan-5,6-epoxy-3ß-ol, and 26-hydroxycholesterol,3 4 5 6 7 have been detected in the nanomolar range in normocholesterolemic human serum. Analysis of tissue from atherosclerotic human aortas has demonstrated the presence of 24-, 25-, and 26-hydroxycholesterol; 7{alpha}- and 7ß-hydroxycholesterol; 7-ketocholesterol; and a range of other minor sterol constituents.8 9 10 11 12 13 Oxysterols may contribute to the cytotoxicity of oxLDL and may play a role in vascular injury.12 14 Oxysterols can alter membrane permeability,15 16 are potent and specific inhibitors of cholesterol biosynthesis,17 and are capable of suppressing LDL receptor expression.18 Oxysterols such as 25-hydroxycholesterol and 7-ketocholesterol enhance the activity of acetyl CoA:cholesterol acyltransferase, resulting in augmented cholesterol esterification and increased cellular cholesterol ester, which directly contribute to foam cell formation.19 Oxysterols may also inhibit prostacyclin production by endothelial cells, which is suggested to enhance platelet adhesion to endothelial cells.20

The production of cytokines by macrophages in atherosclerotic plaques may be important in the initiation and amplification of inflammation in atherosclerosis. More and more data suggest that TNF-{alpha} and IL-1 are actively involved in the pathogenesis of atherosclerosis.21 22 23 24 25 IL-8, a multifunctional molecule that belongs to a C-X-C chemokine supergene family, is involved in the pathogenesis of a variety of diseases.26 27 IL-8 is chemotactic for T lymphocytes and neutrophils at picomolar and nanomolar concentrations, respectively.28 IL-8 may also increase the adhesiveness of monocytes to endothelial cells.29 It has been reported that IL-8 is probably chemotactic for vascular smooth muscle cells and may play a role in the migration of smooth muscle cells from media into intima of vessel walls.30 It has also been reported that IL-8 is angiogenic. Among the cells that produce IL-8, the macrophage is the major cell type.26 The capacity of macrophages in human atherosclerotic plaque to produce IL-8 is not yet well known.

Since oxLDL has been shown in vitro to induce IL-8 production in macrophages, we wanted to explore whether IL-8 is synthesized in atherosclerotic lesions and whether oxysterols may regulate IL-8 expression by macrophages.


*    Methods
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 *Methods
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Materials
Reagents of analytical grade were used. Collagenase type IV, trypsin inhibitor, fraction V BSA, cycloheximide, and all the sterols (cholesterol; 7-ketocholesterol; 25-hydroxycholesterol; cholestane-3ß,5{alpha},6ß-triol; 5,6{alpha}-epoxycholesterol; 7ß-hydroxycholesterol; and 24-hydroxycholesterol) were purchased from Sigma Chemical Company. The sterols were found to be more than 95% pure, as analyzed by using thin-layer chromatography combined with flame ionization detection.31 The major contaminant was in all cases cholesterol. Dynabeads M-450 (uncoated); Dynabeads M-450 coated with anti-CD14 antibody (CD14 beads), anti-CD4 antibody (CD4 beads), and anti-CD8 antibody (CD8 beads); Pan-T beads; and the MPC were obtained from Dynal AS. Ficoll-Hypaque was purchased from Pharmacia. The Limulus amebocyte lysate assay kit was bought from Endosafe, Inc. Reagents for measuring LDH were purchased from Randox G.

Determination of Endotoxin
Precautions were taken to prevent endotoxin contamination during the cell separation and cell-culture procedures. Endotoxin contamination of all reagents, media, and sterols used in the studies was monitored with the chromogenic Limulus amebocyte lysate assay, using E coli 0112.B4 endotoxin supplied with the kit as a standard. The endotoxin level in the cell-culture media was <8 pg/mL, and there were no differences in control and sterol-containing media.

Isolation of Macrophages From Atherosclerotic Plaques
To study the cytokines produced by plaque macrophages, we used magnetic beads bound to anti-CD14 antibody (mouse IgG2a monoclonal antibody, RM052) to isolate macrophages from human atherosclerotic plaques. The anti-CD14 antibody binds to 57% of the human monocyte-derived macrophages.32 From human atherosclerotic aorta, 1x105 to 1x106 CD14-positive cells were recovered per gram of tissue. The efficiency of the immunomagnetic technique is about 80% for CD14-positive cells.32 As in vitro handling and culture might change the functional properties of the macrophages (for example, the adherence of monocytes to the plastic dishes could dramatically increase the cytokine production by these cells), we analyzed the cellular cytokines of macrophages directly after isolation from the tissue. Human atherosclerotic plaque samples of aorta, carotid, and femoral arteries were obtained from patients undergoing surgery for aortic aneurysm, intermittent claudication, or carotid stenosis. The fresh tissue samples were transferred to a test tube containing sterile Hanks' balanced salts without Ca2+ and Mg2+ (Seromed, Biochrom KG). The tissues were washed from contaminating red blood cells and then dissected from adventitia. From the appearance under the dissection microscope, the lesions were roughly classified as (1) fatty streaks, (2) fibrolipid lesions, or (3) complicated lesions.33 The intima-media was cut into 1-mm3 pieces and then incubated in a proteolytic solution containing 450 U/mL collagenase, 1 mg/mL trypsin inhibitor, and 4.8 mg/mL HEPES in Hanks' balanced salts in a siliconized bottle with a magnetic bar stirring slowly at 37°C. Free cells were collected every 10 to 15 minutes for up to 2 hours. Large fibers such as collagen and elastin were taken away by passing through a 100-mm mesh nylon filter and by incubating with 30 µL/mL uncoated magnetic beads for 10 minutes at 4°C. After washing, the cells were resuspended in RPMI 1640 supplemented with 1% BSA and incubated with 30 µL/mL CD14 beads (0.9 mg/mL) for 30 minutes at 4°C. CD14-positive cells were collected with an MPC. Unbound cells were washed away with Dulbecco's PBS containing 0.1% BSA. The CD14-positive cells were counted under a microscope. The viability of the cells was evaluated by trypan blue dye exclusion test,34 and >85% of the cells were determined to be viable in all cell preparations. Most of the CD14-positive cells were foam cells, as evidenced by their plump configuration and foamy cytoplasm, which stained positively with oil red O. Then the cells were lysed with lysing buffer (Tris buffer: 50 mmol/L, pH 7.4, and EDTA: 5 mmol/L). PMSF dissolved in DMSO, purchased from Sigma Chemical Company, was added freshly to the lysis buffer to a final concentration of 0.2 mmol/L to block proteinase activity. The cell lysates were stored at -20°C. Cell-associated cytokines were measured by ELISA within 2 months. Before determining the cytokine levels, the magnetic beads were taken away by an MPC. From eight buffy coats, human peripheral blood monocytes and monocyte-derived macrophages (cultured in vitro for 7 days) were prepared and treated the same way as the tissue macrophages (ie, collagenase treated for 30 minutes and isolated with beads) and used as controls.

In some of the cell preparations, after CD14-positive cells had been isolated, the cells were incubated with CD4 and CD8 beads or Pan-T beads to isolate T lymphocytes. The viability of the T lymphocytes was more than 90%. The cells were washed and lysed as described above.

Preparation of Human Peripheral Blood Monocytes
Mononuclear cells were isolated from buffy coats by Ficoll-Hypaque density gradient centrifugation and purified by adherence to plastic dishes.35 Mononuclear cells (1 to 2x106 cells per mL) in RPMI 1640 (Flow Laboratories) supplemented with 24 mmol/L NaHCO3, 10 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mmol/L sodium pyruvate, 4 mmol/L glutamine, and nonessential amino acid were seeded in six-well dishes. After 1 to 2 hours, nonadherent cells were washed away. The purity of the monocytes was about 95%, as measured by nonspecific esterase staining. For macrophage studies, the cells were incubated in RPMI 1640 supplemented with 10% human serum and 10% fetal calf serum for 7 days.

The adherent monocytes or day 7 macrophages were incubated in RPMI 1640 supplemented with 5 mg/mL human LDS with or without various amounts of the sterols in triplicate wells for 20 hours. The LDS was prepared by ultracentrifugation at d>1.21 g/mL. There were no lipids left in the preparation, as shown by gel electrophoresis. The sterols were dissolved in ethanol and added to the culture medium at a final concentration of 0.1% ethanol. The control incubations were carried out in medium containing 0.1% ethanol. After the incubation, media were collected and centrifuged for 5 minutes at 1000 rpm to get rid of free cells and cell debris. The cells were then washed twice with Dulbecco's PBS and lysed in lysis buffer as indicated above. An aliquot of media and cell lysate was taken for measuring LDH activity immediately; the rest was stored at -20°C for protein determination and cytokine measurement. The cytotoxic effect was evaluated by LDH leakage (%), which was calculated by LDH (U/L) in the media divided by LDH in the media plus cell-associated LDH. LDH activity was measured in a Cobas Bio autoanalyzer with reagents from Randox. The method for measuring LDH is an optimized method based on the direct, NADH2-coupled assay employing pyruvate as the substrate, according to the recommendation of the Scandinavian Committee on Enzymes. Absorbance was read at 340 nm.36

Study in THP-1 Cells
IL-8 production was also studied in THP-1 cells, a human leukemia cell line with distinct monocytic markers. Unstimulated THP-1 cells (2x106) at passages 4 through 8 were seeded in 6-well dishes in RPMI 1640 with 5 mg/mL LDFS and 5x10-5 mol/L ß-mercaptoethanol, supplemented with or without cholesterol or 25-hydroxycholesterol. The LDFS was prepared as was LDS (described above). Twenty hours later the media were collected and the cells and lysed as indicated above.

Determination of Cytokines
IL-1ß, TNF-{alpha}, and IL-8 in the culture supernatants and cell lysates were quantified by commercial ELISA (R&D systems) using recombinant human IL-1ß, TNF-{alpha}, and IL-8 as standards. Monoclonal antibodies specific for IL-1ß, TNF-{alpha}, and IL-8, respectively, were coated onto polystyrene microtiter plates provided in the kit. Standards were diluted in cell-culture medium or in lysis buffer, and samples and standards were assayed in duplicate, strictly according to the manufacturer's instructions. The photometric measurement was performed at 450 nm on a microplate reader (Molecular Device Corporation). Sensitivity for TNF-{alpha} is 4.4 pg/mL; for IL-1ß, 0.3 pg/mL; and for IL-8, 3.0 pg/mL. Coefficient of variation of the ELISA measurement varied between 5% and 10%. Cell protein was analyzed in triplicate by the Bradford37 method (Bio-Rad protein assay), using gamma globulin as a standard. The level of cytokines was correlated with either cell protein or cell number.

Statistics
For group comparison between the levels of IL-8 in macrophages from human atherosclerotic plaques and human peripheral blood monocytes or human monocyte-derived macrophages, Wilcoxon's test was used.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
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Detection of IL-1ß, TNF-{alpha}, and IL-8 Proteins in Macrophages From Human Atherosclerotic Lesions
To study the cellular cytokine levels in plaque macrophages, we used anti-CD14 antibodies together with magnetic beads to isolate macrophages from human atherosclerotic plaques. Fig 1Down shows the CD14-positive macrophages, which were surrounded by magnetic beads. Those cells vary greatly in cell size and oil red O staining, and some cells have two nuclei, as shown in the figure. IL-8 in these macrophages was about 7 times higher than in freshly isolated human peripheral blood monocytes and more than 123 times higher than in human monocyte-derived macrophages (Table 1Down). We also determined IL-1ß and TNF-{alpha} in the cell lysates. The amount of cell lysate was too small to allow determination of both IL-1ß and TNF-{alpha}; therefore, only one of them was determined in most preparations. The levels of IL-1ß and TNF-{alpha} were variable; in many preparations they were low or undetectable. The level of IL-8 in tissue macrophages seemed to be related to the severity of the lesion, with the lowest level in cells derived from fatty streaks (Table 1Down).



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  		Figure 1. CD14-positive macrophages isolated from human atherosclerotic lesions. The cells were isolated from human atherosclerotic lesions by anti-CD14 magnetic beads after collagenase digestion of the tissue. They were collected by an MPC and then suspended in PBS. They were dried on a slide and then fixed in 4% formaldehyde/PBS. The cells were stained with oil red O and hematoxylin. The bar in the picture is 25 µm.


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  		Table 1. Expression of IL-8 Protein by CD14-Positive Macrophages Isolated From Human Atherosclerotic Plaque

We also tested whether the cells isolated from human atherosclerotic lesions could still respond to LPS. After the collagenase digestion, the cells were divided into portions and incubated with or without LPS (1 µg/mL) for 3 hours. After this incubation, the cells were washed and the CD14-positive cells isolated as before. In two cell preparations, the level of IL-8 in CD14-positive macrophages was 136 pg/106 cells and 159 pg/106 cells, while in LPS-stimulated CD14-positive macrophages the IL-8 levels were 1650 pg/106 cells and 1530 pg/106 cells.

In some cell preparations, after the isolation of the CD14-positive cells we used anti-CD4 and anti-CD8 or Pan-T magnetic beads to isolate T lymphocytes. We lysed the T lymphocytes and the rest of the cells (the remaining cells after CD14 and CD4/CD8 magnetic bead isolation) as we did with CD14-positive cells. Cell-associated IL-8, IL-1ß, and TNF-{alpha} in CD4- and CD8-positive T lymphocytes and in the rest of the cells from the tissue samples were very low or not detectable with the ELISA method (data not shown).

Influence of Oxysterols on the Production of IL-8
To investigate whether oxysterols could in any way influence the production of IL-8 by monocytes/macrophages, a panel of oxysterols was used. Freshly isolated adherent human peripheral blood monocytes were incubated with 5 µg/mL of various oxysterols, cholesterol, or control medium for 20 hours. Almost all oxysterols tested had a tendency to enhance the production of IL-8 (Fig 2Down); however, 25-hydroxycholesterol was the most potent. Cell-associated IL-8 in the monocytes stimulated with 25-hydroxycholesterol was 3-fold to 10-fold higher than in the control cells or cholesterol-treated cells. The absolute levels of IL-8 production varied among different donors, but the increase by treatment with 25-hydroxycholesterol was a very consistent finding. The oxysterols had a similar effect on macrophages, although the level of IL-8 was more than 20-fold lower in macrophages than in monocytes (Table 2Down).



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  		Figure 2. Effect of various oxysterols on the production of IL-8 by human monocytes. Adherent blood monocytes were incubated with 5 µg/mL of various oxysterols in triplicate in RPMI 1640 with 5 mg/mL LDS for 20 hours. The cells were then lysed with lysis buffer, and cellular IL-8 was determined in duplicate by ELISA. The results are represented as percentage of control (mean±SE) from four separate experiments. 7-ketochol indicates 7-ketocholesterol; 25-hydroxychol, 25-hydroxycholesterol; chol-3ß,5{alpha},6ß-triol, cholestane-3ß,5{alpha},6ß-triol; 5,6{alpha}-epoxychol, 5{alpha},6{alpha}-epoxycholestan-3ß-ol; 7ß-hydroxychol, 7ß-hydroxycholesterol; and 24-hydroxychol, 24-hydroxycholesterol.


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  		Table 2. Cellular IL-8 in Human Monocytes and Monocyte-Derived Macrophages Exposed to Different Oxysterols

This effect was not because of the trivial lymphocyte contamination in the cell preparation; when the T lymphocytes were depleted by anti-CD4/CD8 or Pan-T magnetic beads before the cells were seeded onto plastic dishes, we got similar results.

Enhanced IL-8 Production by 25-Hydroxycholesterol Is Dose and Time Dependent
Since 25-hydroxycholesterol was the most potent oxysterol to enhance IL-8 production, further studies were carried out on this oxysterol to study its effects as a regulator of IL-8 production.

Adherent human monocytes were incubated with 0.5 to 10 µg/mL of either cholesterol or 25-hydroxycholesterol for 20 hours. The effect on the production of IL-8 was found to be dose dependent (Fig 3ADown). At a concentration as low as 0.5 µg/mL, 25-hydroxycholesterol significantly enhanced IL-8 production (45 pg/µg protein) compared with IL-8 produced by cells incubated with 0.5 µg/mL cholesterol (35 pg/µg protein). The 25-hydroxycholesterol had a similar effect on THP-1 cells. At a concentration of 0.5 µg/mL, 25-hydroxycholesterol stimulated the THP-1 cells to produce 2.7-fold more IL-8 than in the control (Fig 3BDown).



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  		Figure 3. Dose-response study of 25-hydroxycholesterol on the production of IL-8 by human monocytes and THP-1 cells. A, Adherent blood monocytes were incubated with 0.5, 1, 2.5, 5, and 10 µg/mL cholesterol or 25-hydroxycholesterol (25-hydroxychol) in triplicate in RPMI 1640 with 5 mg/mL LDS for 20 hours. Cells were then lysed with lysis buffer, and cellular IL-8 was determined in duplicate by ELISA. The results are representative of three separate experiments. B, THP-1 cells at passages 4 through 8 were treated as the monocytes were, except the medium was RPMI 1640 with 5 mg/mL LDFS. Data are representative of two separate experiments.

Oxysterols in high concentrations are toxic to the cells. We measured LDH leakage as an indicator of cytotoxicity. When macrophages were incubated with 5 µg/mL oxysterols, no cytotoxic effect could be seen. Monocytes were more sensitive, and with doses of 25-hydroxycholesterol >2.5 µg/mL, there seemed to be a cytotoxic effect (Fig 4).Down



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  		Figure 4. Cytotoxic effect of various doses of 25-hydroxycholesterol (25-hydroxychol). Adherent blood monocytes were incubated with 0.5, 1, 2.5, 5, and 10 µg/mL cholesterol or 25-hydroxycholesterol in triplicate in RPMI 1640 with 5 mg/mL LDS for 20 hours. The media were collected and the cells lysed with lysis buffer. LDH in the media and cellular LDH were measured. LDH leakage (%) was calculated by LDH (U/L) in the media divided by LDH in the media plus cellular LDH. The results are representative of three separate experiments.

Noncytotoxic doses of cholesterol or 25-hydroxycholesterol (2 µg/mL) were incubated with the cells for 1, 4, 10, 20, and 48 hours. As shown in Fig 5Down, the production of IL-8 was time dependent. The 25-hydroxycholesterol–stimulated cells produced significantly more IL-8 (both cell-associated and secreted) when the incubation time was over 10 hours. This finding may indicate that the increased IL-8 as a result of 25-hydroxycholesterol stimulation is newly synthesized by the cells and not released from intracellular stores. The level of IL-8 decreased at 48 hours, probably because of degradation. Still, the IL-8 level was higher in the 25-hydroxycholesterol–treated cells than in the control cells. The IL-8 production could be totally inhibited by cycloheximide (Fig 6Down).



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  		Figure 5. Time-response study of 25-hydroxycholesterol (25-hydroxychol) on the production of IL-8 by human monocytes. Adherent blood monocytes were incubated with 2 µg/mL cholesterol or 25-hydroxycholesterol in duplicate in RPMI 1640 with 5 mg/mL LDS for 1, 4, 10, 20, and 48 hours. Cells were then lysed with lysis buffer, and IL-8 (cellular and in the media) was determined in duplicate by ELISA. The results are the mean±SE of four donors.



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  		Figure 6. Enhanced protein synthesis–dependent production of IL-8 by 25-hydroxycholesterol (25-hydroxychol). Adherent blood monocytes were incubated with 2 µg/mL cholesterol or 25-hydroxycholesterol in triplicate in RPMI 1640 with 5 mg/mL LDS for 20 hours in the presence or absence of cycloheximide (10 µg/mL). Cells were then lysed with lysis buffer. IL-8 in the cell lysates was determined in duplicate by ELISA. The results are the mean±SE of four donors.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
IL-8 belongs to a supergene family of chemokines and is chemotactic for neutrophils, lymphocytes, and endothelial cells, as well as smooth muscle cells. It may be involved in the recruitment of T lymphocytes from blood to the subendothelial space of arteries and in the migration of smooth muscle cells from the media into the intima of the vessel walls28 29 30 and thus may contribute to the pathogenesis of atherosclerosis. In macrophage-derived foam cells freshly isolated from atherosclerotic tissue, we find high levels of IL-8 compared with human peripheral blood monocytes or monocyte-derived macrophages in culture. The high level of IL-8 in the macrophages from human lesions is probably not due to in vitro handling, such as collagenase digestion, since control monocytes and macrophages were treated the same way as tissue macrophages. We used CD14 as a marker for the macrophages. In earlier studies we compared different antibodies and found that anti-CD14 gave the best recovery of cells from tissue.32 Still, the isolated cells probably represent a subpopulation of the macrophages present in arterial tissue. A selection of cells during preparation occurs due to both differential expression of CD14 and other reasons for cell loss. From the present study, it can be concluded that this subpopulation of macrophages contains high levels of IL-8. How these cells relate to the total macrophage population cannot at present be determined. The low level of IL-8 found in the crude remainder of the cell preparation might indicate that CD14-positive cells represent a more active fraction of macrophages. In studies by Koch et al38 using explants of aortic tissue, similar results to ours were found, with high levels of IL-8 in macrophages. In some of the cell preparations we also determined IL-1ß and TNF-{alpha}. The levels were found to be variable, which might indicate different degrees of inflammatory activation between different donors. Interestingly, in our study, when the macrophage-derived foam cells from human tissue were exposed to LPS, they were still capable of responding with increased production of IL-8. Thus the lipid-filled foam cells seem to have the capacity to respond to inflammatory activation. There are contradictory reports about whether tissue macrophages in inflammatory sites are capable of responding to LPS to produce IL-8. Koch et al39 reported that synovial tissue macrophages from patients with rheumatoid arthritis constitutively express and produce IL-8 but had no response to LPS, TNF, or IL-1. Seitz et al40 found that cells from synovial fluid of patients with rheumatoid arthritis were still capable of producing IL-8 when exposed to LPS.

A specific stimulation of IL-8 production by oxLDL has been shown by other investigators.1 Which component of oxLDL is responsible for the enhanced IL-8 production is unknown. Oxysterols, which are generated during oxidation of LDL, are one of the biologically active components in oxLDL. We found in this study that when human monocytes, macrophages, and THP-1 cells were exposed to a series of different oxysterols in vitro almost all the oxysterols tested had a tendency to stimulate IL-8 production but that 25-hydroxycholesterol was the most potent stimulator. It enhanced the IL-8 production up to 10 times. This effect was dose and time dependent and could be totally blocked by cycloheximide. This finding suggests that the enhanced IL-8 production may involve new protein synthesis.

Oxysterols have many important regulatory effects on macrophage function. Oxysterols can inhibit macrophage cholesterol biosynthesis17 and suppress LDL receptor expression18 ; they can also enhance cellular cholesterol esterification.19 We have shown that oxysterols may also regulate the expression of lipoprotein lipase in macrophages. In culture, 25-hydroxycholesterol and 7ß-hydroxycholesterol downregulated cellular mRNA as well as secreted enzyme activity in monocyte-derived macrophages.11 Oxysterols, including 25-hydroxycholesterol, have been shown to be formed during oxidation of LDL in vitro. 25-Hydroxycholesterol was found at a concentration of about 3.5 µg/mg LDL protein.5 41 The same oxysterols have also been detected in human atherosclerotic tissue42 and in macrophage-derived foam cells.11

How oxysterols play their regulatory role is not well known. Oxysterols have been shown to insert into biological membranes, resulting in changes of cell shape and interfering directly with membrane function.43 Permeability of cells to ions has been observed to be increased by cholesterol oxides such as 25-hydroxycholesterol.44 45 This increased permeability may trigger a signal-transduction pathway on the cell membrane that may have a regulatory effect on IL-8 production. The increased LDH leakage by oxysterols may be due partly to a cytotoxic effect of oxysterols and partly to the increased permeability of the cells by oxysterols. When oxysterols enter the cells, they can bind to oxysterol binding protein46 and/or cellular nucleic acid binding protein, a 19-kD 7 zinc finger DNA-binding protein.47 These proteins may mediate the regulatory actions of oxysterols.

In conclusion, we found in this study that macrophage-derived foam cells from human atherosclerotic lesions contain high levels of IL-8 compared with blood monocytes and monocyte-derived macrophages. These cells are probably still capable of responding to inflammatory stimuli. Oxysterols, which are cholesterol oxidation products, may have a regulatory effect on IL-8 production. IL-8, which is a potent chemoattractant, may play a role in the recruitment of T lymphocytes and smooth muscle cells into the subendothelial space. Thus, oxysterols together with IL-8 may contribute to the formation of atherosclerotic lesions.


*    Selected Abbreviations and Acronyms
 
IL = interleukin
LDFS = lipid-deficient sera from fetal calf sera
LDS = lipid-deficient serum
MPC = magnetic particle concentrator
oxLDL = oxidized LDL
TNF = tumor necrosis factor


*    Acknowledgments
 
This study was supported by grants from the Swedish Heart-Lung Foundation, the Swedish Medical Research Council (grant No. 4531), and the Medical Faculty, Goteborg University. For technical assistance and advice we thank Birgitta Rosengren.

Received September 28, 1995; revision received June 13, 1996;
*    References
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 *References
 

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